HK1108567A - Compositions and methods for the prevention and control of insulin-induced hypoglycemia - Google Patents

Description

Compositions and methods for preventing and controlling insulin-induced hypoglycemia

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional patent application No. 60/584,449 (filed on 6/29 of 2004), which is incorporated herein by reference in its entirety.

Technical Field

[0002] The present invention relates to the fields of biology, medicine and medicine. In particular, the present invention relates to compositions and methods of using compositions to control blood glucose levels.

Background

[0003] Insulin is produced by the beta cells of the Islets of Langerhans (Pancreatic Islets), and glucagon is produced by the alpha cells of the Islets of Langerhans. One of the main effects of insulin is to lower blood glucose by inhibiting hepatic glucose output and stimulating peripheral glucose uptake. In some patients with diabetes (diabetes mellitis), endogenous insulin levels may be low or undetectable. In cases where circulating insulin levels are low or ineffective, exogenous insulin is often administered to reduce hyperglycemia. Glucagon generally has effects that are opposite to those of insulin, including, primarily, increasing hepatic glucose output and thereby increasing blood glucose levels. Glucagon levels tend to increase when blood glucose levels drop to abnormally low levels, particularly in patients who utilize exogenous insulin.

[0004] Current goals of diabetes treatment (diabetes management) include near-normal blood glucose levels in order to delay or prevent microvascular complications; to achieve this goal, insulin potentiation therapy (intensive therapy) is often required. In an effort to achieve this goal, physicians have encountered a problem with the greatly increased frequency and severity of hypoglycemia in their diabetic patients.

[0005] Hypoglycemia (hypoglycemia) is characterized by low blood glucose levels, which lead to autonomic and adrenergic symptoms, as well as hypoglycemic neurological symptoms; these symptoms are typically encountered as a result of inadvertent overdosing of insulin. Hypoglycemia is currently defined as blood glucose < 70mg/dl, e.g., above 50 or 60 mg/dl. Frequent recurrence of hypoglycemia may be associated with hypoglycemia unconsciousness, which may further contribute to the development of hypoglycemia, which is sometimes severe. Thus, efforts to apply insulin to normal glucose levels may cause patients to develop hypoglycemia of varying frequency and severity. Hypoglycemia and lack of awareness of the presence of hypoglycemia are serious complications of insulin therapy, which occurs with higher frequency and severity when a diabetic suffers from impaired adverse-regulatory (anti-insulin) responses. Generally, one of the major counter-regulatory hormones that responds to hypoglycemia is glucagon. It is not uncommon for the glucagon response to acute hypoglycemia to be impaired or lost in patients with advanced type I and type II diabetes.

Summary of The Invention

[0006] There is a need for new methods of treating diabetes and new insulin and glucagon formulations that reduce the risk of hypoglycemia induced by insulin treatment. The present invention fulfills this need and others.

[0007] One aspect of the present invention provides a pharmaceutical composition comprising both insulin and glucagon in amounts such that, upon administration to a diabetic patient, not only therapeutically effective control of diabetes is achieved, but also hypoglycemia is prevented. Formulations may include, for example, formulations suitable for injection, including subcutaneous (s.c.) administration, formulations suitable for oral administration, formulations suitable for transdermal administration, formulations suitable for ocular administration, and formulations suitable for inhalation. In some embodiments, the composition is formulated for subcutaneous administration and contains sufficient glucagon for administration of 5 to about 20ng/kg/min of glucagon (e.g., the efficacy is: more than 5 to 20ng glucagon per minute per kilogram per person). In one embodiment, 5 to about 20ng/kg/min of glucagon is administered 1-20 units of insulin. The ratio of administration may be, for example, 1 administration for 1 hour. In a preferred embodiment, the composition is suitable for administering more than 5 to about 20ng/kg/min (nanograms/kg/min) of glucagon per 1-2 units of insulin administered. It will be appreciated that in some embodiments, the glucagon and insulin may be held in separate containers and not administered simultaneously, but with the appropriate ratio between glucagon and insulin maintained. In one embodiment, the separate containers are contained in a single device suitable for administering glucagon and insulin, e.g., subcutaneously; in another embodiment, two devices, one for each reagent, may be used.

[0008] In another aspect, methods are provided for treating diabetes in a human or other mammal without or with a substantially reduced risk of inducing hypoglycemia. In one embodiment, a composition comprising insulin and glucagon is administered to a patient prior to the onset of mild, moderate or severe hypoglycemic symptoms. In some embodiments, the methods of the invention are practiced to prevent nocturnal hypoglycemia in type I diabetics who are receiving insulin therapy, including insulin potentiation. The method comprises co-administration of insulin and glucagon, wherein the insulin is administered in an amount therapeutically effective to control diabetes and the glucagon is administered in an amount therapeutically effective to prevent hypoglycemia, and preferably wherein the insulin and glucagon are administered simultaneously with each other, or contemporaneously with each other, i.e., within about 4 hours of each other (e.g., when conventional insulin, LISPRO insulin, and ASPART insulin are used) or within about 6 to 12 hours of each other (e.g., when long acting insulin is used), and, in any event, prior to the occurrence of clinically observable hypoglycemia. In one embodiment, the glucagon is administered prior to the administration of insulin. In another embodiment, insulin is administered prior to the administration of glucagon. In one embodiment, the method comprises maintaining blood glucose levels above 70mg/dL and below 180mg/dL by co-administering insulin and glucagon to the patient. In another embodiment, the method comprises subcutaneously administering glucagon in an amount of about 6 to 18ng/kg glucagon per minute. In one embodiment, 1-20 or 2-20 units of insulin is administered to a diabetic patient receiving subcutaneously administered glucagon in an amount of 6 to 18ng/kg/min. In another embodiment, the method comprises subcutaneously administering about 8 to 12ng/kg of glucagon per minute. In one embodiment, 0.1 to 2 or 2-20 units of insulin is administered to a diabetic patient receiving subcutaneously administered glucagon in an amount of 8 to 12ng/kg/min. In another embodiment, the glucagon is administered by a means other than intravenously or subcutaneously and in a dose equal to the subcutaneous dose as described above.

[0009] In another aspect, the present invention provides a method of maintaining blood glucose levels in a range that is neither hyperglycemic nor hypoglycemic. These methods comprise co-administration (co-administration) of insulin and glucagon.

[0010] In another aspect, glucagon formulations and modified glucagon are provided that are suitable for co-administration with insulin in accordance with the methods of the present invention.

[0011] In another aspect, a kit for preventing hypoglycemia is provided. In one embodiment, the kit preferably includes instructions for insulin, glucagon, and an appropriate combination thereof to be administered simultaneously.

[0012] In another aspect, a kit comprises insulin, a long acting form of glucagon, and instructions for use.

[0013] In some aspects, methods of restoring or preventing loss of consciousness or sensitivity to hypoglycemia are provided. The method comprises administering to the patient an amount of glucagon over a period of time sufficient to prevent or restore the patient's consciousness to hypoglycemia. In one embodiment, the patient is administered insulin concurrently with the glucagon.

[0014] In one aspect, a pharmaceutical formulation is provided comprising an amount of insulin effective to control diabetes and an amount of glucagon effective to prevent hypoglycemia in a human or other mammal. The pharmaceutical formulation is formulated for subcutaneous administration, and the ratio of insulin to glucagon is typically about 1 unit of insulin to over 40 milliunits to 200 milliunits of glucagon. In some embodiments, the amount of glucagon is between about 50 and 100 milliunits. In some embodiments, the glucagon is a long-acting form of glucagon. In some embodiments, the long acting form of glucagon contains iodine. In some embodiments, the long acting form of glucagon contains zinc. In some embodiments, the long acting form of glucagon also contains protamine.

[0015] In another aspect, methods of treating diabetes in a human or other mammal without inducing hypoglycemia are provided. The method comprises administering insulin in an amount therapeutically effective to control diabetes. The amount of insulin may be between 0.5 and 20 units of insulin. The methods further comprise administering glucagon in a time and amount therapeutically effective to prevent hypoglycemia. Glucagon can be administered subcutaneously in amounts of from 5 or more to less than or equal to 100ng/kg patient/minute of desired glucagon effectiveness. In some embodiments, the amount of glucagon administered is from 6ng/kg patient/minute to 18ng/kg patient/minute. In some embodiments, the glucagon is a glucagon with an extended duration of action. In some embodiments, the glucagon is contained in a liposome formulation. In some embodiments, the glucagon is contained in a microsphere. In some embodiments, a formulation comprising both insulin and glucagon is administered. In some embodiments, the insulin and glucagon are contained in a pump that controls the administration of the drug to the patient. In some embodiments, the glucagon is administered simultaneously with insulin. In some embodiments, the ratio of glucagon to insulin is about 40 or more to 200 milliunits of glucagon to 1 unit of insulin. In some embodiments, 2 units of insulin is administered. In some embodiments, 10 units of insulin is administered and between 30ng/kg/min. and 90ng/kg/min. glucagon is administered subcutaneously.

[0016] In another aspect, a kit for administering a hypoglycemic preventing amount of glucagon and insulin is provided. The kit comprises glucagon and insulin. The ratio of glucagon to insulin is 1-20 units of insulin to 32-480 milliunits of glucagon. The kit also includes a means for subcutaneous administration of glucagon and instructions for administering insulin and glucagon such that the glucagon prevents a hypoglycemic event. In some embodiments, the concentration of glucagon when fully dissolved in the glycerol solution is above 500 micrograms per ml, but below 2000 micrograms per ml. In some embodiments, the ratio of glucagon to insulin is 1-3 units of insulin to 32-96 milliunits of glucagon. In some embodiments, the device for subcutaneous administration of glucagon is a pump configured to deliver about 6 to 20ng/kg/min of glucagon.

[0017] In another aspect, a combination of glucagon and insulin for the preparation of a medicament for the treatment of diabetes is provided. The glucagon is applied in an amount sufficient to prevent the onset of hypoglycemia, wherein the ratio of glucagon to insulin is greater than 40 micrograms and less than 500 micrograms of glucagon to 1-20 units of insulin. In some embodiments, the amount is sufficient to prevent the onset of hypoglycemic unawareness. In some embodiments, the amount of insulin is between 1 and 20 units and the amount of glucagon is between 41 and 200 milliunits. In some embodiments, the ratio of insulin to glucagon is 1 to 3 units of insulin to 40 or more to less than or equal to about 96 milliunits of glucagon. In some embodiments, the glucagon also contains protamine.

Brief Description of Drawings

[0018] FIG. 1 is a graph illustrating idealized pharmacokinetics of a mixture of conventional and intermediate insulin (Lente or NPH).

[0019] FIG. 2 is a graph illustrating the insulin profile of a hypothetical patient, as described in section A (i) of example 1, showing a very simple, flat line graph (base level established by GLARGINE (LANTUS)) incorporating several peaks at positions corresponding to prandial LISPRO (HUMALOG) insulin injections.

[0020] Figure 3 is a schematic diagram of a drug delivery pump designed to practice an embodiment described in example 2.

[0021] Figure 4 is a schematic diagram of a drug delivery pump designed to practice an embodiment described in example 2.

[0022] Figure 5 is a schematic diagram of a drug delivery pump designed to practice an embodiment described in example 2.

[0023] Figure 6 illustrates the effect of molecular weight and lipophilicity on the rate of transdermal transport in the case of permeation (upper and lower grey curves representing more or less lipophilic species, respectively) or in the case of TRANSFEROME ® -mediated penetration (black lines and dots). Interspersed black dots represent commercial drugs in transdermal patches.

[0024] Figure 7 is a graph illustrating insulin and glucagon profiles for an exemplary patient, as described in example 3, showing a very simple, flat line graph (basal insulin and glucagon infusions) with several peaks (corresponding to prandial insulin and glucagon infusions) for both drugs, corresponding to when insulin and glucagon are administered in a mixed formulation.

[0025] Figure 8 is a graph illustrating the effect of continuous infusion of glucagon on the mean glucagon level, as described in example 7.

[0026] Figure 9 is a graph illustrating the effect of continuous infusion of glucagon on average glucose levels, as described in example 7.

[0027] Figure 10 is a graph comparing the effect of continuous glucagon infusion at 12ng/kg/min on average glucose levels and average glucagon levels, as described in example 7.

[0028] Figure 11 is a graph comparing the effect of continuous infusion of glucagon at 16ng/kg/min on average glucose levels and average glucagon levels, as described in example 7.

[0029] Figure 12 is a graph comparing the effect of different doses of glucagon (0, 8 and 16ng/kg/min. glucagon) on increasing insulin levels (from about 1 to about 2.7 units). The graph illustrates that low doses of glucagon are able to prevent insulin-induced hypoglycemia, as described in example 8.

[0030] Figure 13 is a graph showing the effect of low doses of glucagon on blood glucose levels and how it can prevent hypoglycemia, as described in example 8.

Detailed Description

[0031] Methods and compositions are provided that can prevent or significantly reduce the frequency and severity of hypoglycemia in insulin-treated diabetic patients (including types 1 and 2). In one aspect, the methods and compositions can be used to treat diabetes while regulating glucose levels above a low blood glucose level. The present methods and compositions are useful for supplementing or restoring abnormally low glucagon responses that often accompany insulin administration, thereby preventing hypoglycemia.

[0032] One problem complicating the prevention of hypoglycemia is that repeated hypoglycemic events can cause a loss of consciousness of hypoglycemia, and thus, despite being initially detected by the patient, the patient's ability to recognize hypoglycemic symptoms can be compromised or lost over time. Thus, compositions and methods that can prevent or reverse loss of consciousness due to hypoglycemia are desirable. One way in which this can be achieved is by administering glucagon, or other agent that raises blood glucose levels as described herein, at a relatively low dose over a period of time during which insulin is active, in order to prevent the onset of moderate hypoglycemia. This may also be used to reverse or prevent loss of consciousness of hypoglycemia.

[0033] In one aspect, the present invention provides pharmaceutical formulations comprised of two hormones, insulin and glucagon, combined in molar ratios to optimize glycemic control and attenuate the incidence of or prevent hypoglycemia. In another aspect, methods and compositions are provided for the simultaneous, but separate, administration of insulin and glucagon to achieve this benefit. Although the simultaneous administration of these two hormones with seemingly counteracting activities may not have a beneficial effect traditionally seen, some embodiments of the invention are partly predicated on the recognition that such administration achieves the beneficial effect of preventing hypoglycemia without reducing the glucose-modulating beneficial effect provided by insulin, due to the buffering or attenuating effect of glucagon. In some embodiments, a small amount of glucagon is administered continuously to a patient who is, has, or will receive insulin. Thus, the use of hyperglycemic agents, such as glucagon, is contemplated to prevent the occurrence of hypoglycemia and its associated symptoms due to the administration of insulin. In some embodiments, a hyperglycemic agent, such as glucagon, is used to prevent the onset of iatrogenic hypoglycemia.

[0034] Thus, some embodiments provide a method of controlling diabetes while reducing the risk of hypoglycemia by administering insulin and glucagon simultaneously to a diabetic patient. In one embodiment, a method is provided for preventing hypoglycemia in a diabetic patient receiving insulin treatment and not experiencing symptoms of hypoglycemia, comprising administering glucagon to the patient in an amount therapeutically effective to prevent hypoglycemia. In one embodiment, the glucagon and insulin are administered simultaneously. In one embodiment, glucagon is administered 10 minutes to several hours prior to administration of the additional insulin, more preferably 30 minutes to 60 minutes prior to administration of the additional insulin. In other embodiments, the glucagon is administered within about 1 minute to about 4 hours after the patient is administered insulin. In one embodiment, the prevention of hypoglycemia includes preventing symptoms associated with hypoglycemia from becoming apparent in a subject. In another embodiment, prevention of hypoglycemia is achieved by maintaining the subject's average blood glucose level above about 70mg/dL, or above about 50-60 mg/dL. Preferably, the subject's blood glucose level is maintained below about 140-200mg/dL and below at least about 350 mg/dL. Preferably, the subject's blood glucose level, and thus euglycemia, is maintained.

[0035] Any of a number of different forms of insulin, as well as any of a number of different routes of administration of insulin, including forms and routes approved by the FDA and in development, may be used in the methods and formulations of the present disclosure, as will be apparent to those skilled in the art upon consideration of the disclosure herein. Moreover, any of the currently available glucagon formulations may be equally applied in the present methods and formulations. However, since prior to the present disclosure glucagon was only administered parenterally to control hypoglycemia, the present disclosure provides novel glucagon derivatives, novel glucagon and glucagon derivative formulations, and methods of administering glucagon and glucagon derivatives, including delayed and/or prolonged action glucagon, that are particularly well suited to achieve the benefits provided by some embodiments of the present invention.

[0036] Although the precise dosage of insulin and glucagon will vary from patient to patient and will depend upon a variety of factors including, but not limited to, the age and sex of the patient, the type and severity of diabetes, the past history of the patient, including hypoglycemic and hyperglycemic episodes, the type of insulin and glucagon employed, and the like, the dosage for any patient can be determined by one skilled in the art having regard to the disclosure herein. The beneficial effects of some embodiments are generally achieved by administering insulin and glucagon in a ratio of about 1 unit of insulin to about 0.02-40 milliunits (0.02 to 40 micrograms) of glucagon when the glucagon is administered intravenously. When the term is typically used in the treatment of diabetes, 1 unit of insulin is defined as, for example, about 34.2 micrograms to about 40 micrograms. The amount of insulin may also be measured in International Units (IU). 1 unit of glucagon corresponds to 1 microgram of glucagon. In one embodiment, when glucagon is administered intravenously, and insulin is administered subcutaneously, the ratio is 1 unit of insulin to 0.2 to 4.0 milliunits (0.2 to 4.0 micrograms) of glucagon. When glucagon is to be administered subcutaneously, 1 unit of insulin may be administered, the amount of glucagon administered being 40 or more to 200 milliunits per unit of insulin administered to a 100kg human, e.g., 40 to 200 milliunits per hour. In another embodiment, 48 to 150mU, 50 to 120mU, or 80 to 100mU of glucagon is administered per unit of insulin. In a preferred embodiment, the glucagon is administered subcutaneously in a ratio of about 1 unit of insulin to more than 5 to about 20ng/kg glucagon in an amount administered per minute during the insulin dose effect. In one embodiment, 1 unit of insulin is administered and the rate of glucagon administration is 8-12ng/kg/min. In combination with 1 unit of insulin, a standard dose can be established, for example, for 1 hour for the treatment of a 100kg human. As will be appreciated by those skilled in the art, this dose may be used for basal insulin rates. When it is desired to reach a postprandial insulin level, the amount of glucagon in the dose will increase. In some embodiments, the glucagon is administered subcutaneously in an amount of between 5ng/kg/min. More preferably, the amount administered is between about 8 and 16ng/kg/min. Of course, one skilled in the art will appreciate that other equivalent dosages (i.e., the same effective amount administered by alternative methods) may also be administered in view of the teachings herein. As understood by those skilled in the art, and as will be shown in more detail below, this dose may be adjusted to correspond to the amount of glucagon required to prevent hypoglycemia without inducing hyperglycemia. Thus, in some embodiments, the amount of glucagon administered, even subcutaneously administered, is below a value of 5-20ng/kg/min, which dosage value is described as effective in preventing insulin-induced hypoglycemia. As will be appreciated by those skilled in the art, while the present disclosure focuses on type 1 diabetes, similar methods and compositions can be used for type 2 diabetes. In general, the amount of glucagon can be increased several fold over the amounts disclosed herein for type 1 diabetes. For example, type 2 diabetes requires 1.5 to 5 times more glucagon, preferably, 2 to 3 times more glucagon than type 1 diabetes is included.

[0037] Any currently available form of insulin, including but not limited to recombinant human soluble (conventional) insulin, human insulin analogs, animal insulins, insulins derived from, e.g., bovine, porcine and other species, and delayed release forms of insulin, including intermediate and long acting insulins, may be used in the compositions and methods disclosed herein. Moreover, any of the presently used routes of administration, as well as newer routes in development, may be employed, including, but not limited to, subcutaneous, intramuscular, and intravenous injection, as well as oral, buccal, nasal, transdermal, sublingual, and pulmonary airway administration. Typical dosages and ranges of dosages of insulin administered to control diabetes are known in the art and are applicable to the methods and compositions of some embodiments.

[0038] For example, prandial short-acting insulins, such as regular insulin and its derivatives LISPRO, ASPART and GLULISINE, are well known in the art and are commonly used to treat diabetes. This insulin may be used to illustrate the present embodiment in a manner suitable for use with other forms of insulin, including but not limited to NPH, LENTE, SEMI-LENTE, DETEMIR, ULTRA-LENTE, and GLARGINE (LANTUS), as well as pre-mixed preparations of regular and long-acting insulin. In this illustration, the molecular weights of all three meal-time short acting insulins are considered similar, LISPRO 5808, ASPART 5825.8, GLULISINE 5823, regular insulin 5807. The molecular weight of glucagon is believed to be 3483.

[0039] In type 1 diabetes, the typical range of prandial insulin injections may deviate from the mean by about two standard deviations, such that the insulin dosage range is 2-20 units. More than 95% of type 1 diabetic patients are given prandial insulin within this dosage range. The three prandial insulins all reach peak plasma concentrations within 1-2 hours after subcutaneous administration and have a shelf life of about 5 hours.

[0040] Currently, hypoglycemia is treated by a single parenteral injection of about a 1mg (1 unit) dose of glucagon; it has been determined that this dose is an overdose of the dose actually needed to control hypoglycemia. When glucagon is administered subcutaneously or intramuscularly, serum glucagon peaks within 1 hour and its action can last for several hours. However, the currently marketed forms of glucagon appear to be unstable for extended periods of time in liquid forms, either isolated or in vivo, and in one embodiment, the present invention provides new pharmaceutical formulations of glucagon that are more stable, and new methods of using the stable forms of glucagon that are currently available but not widely used.

[0041] Based in part on the different times and durations of action of prandial insulin and subcutaneously administered glucagon at which peak serum levels are reached, it has been found that there is a mismatch between subcutaneous insulin and glucagon pharmacokinetics. One embodiment of the present invention provides long acting glucagon formulations and derivatives thereof that can be used to correct this mismatch, which is desired or beneficial to the patient. As used herein, "long-acting" glucagon refers to glucagon having a longer half-life than standard glucagon, including natural extracts and synthetic glucagon produced by rDNA.

[0042] To provide the dose of glucagon required to achieve a duration of effect similar to that of prandial insulin, a dose approximating the basal replacement dose may be employed. Typical basal glucagon replacement doses by intravenous infusion (ivifusion) are 0.5-0.75 ng/kg/min; a broader range of glucagon infusions can be assumed, from as low as 0.10 to 5.00ng/kg/min (more often 0.10 to 3.00ng/kg/min.), to be effective, depending on the patient, insulin dosage, method of administration (e.g., intravenous versus subcutaneous), and other factors. For example, in subcutaneous administration, the inventors have found that the amount of glucagon administered can be higher, as the bioavailability of glucagon administered by subcutaneous infusion can be as low as 10%, and for bolus (bolus) subcutaneous administration can be as low as about 35%. Thus, the dose will be increased or decreased accordingly in order to obtain a therapeutic effect equivalent to that of glucagon administered at a rate of 5 to 20ng/kg/min. To match the PK of insulin, the rate of infusion of these glucagons may continue for a period of time ranging from 150 minutes to 300 minutes. In some embodiments, the infusion time may last for more than 6 hours, e.g., 6-7, 7-10, 10-15, 20-24 hours, or longer. The maximum and minimum times can then be multiplied by the rate of substitution to give the total dose/kg. If it is assumed that a typical type 1 diabetic patient has a body weight in the range of 50 to 100kg and that the high and low dose range of subcutaneous prandial insulin injections is between 2 units and 20 units, the ratio of insulin/glucagon can be calculated as shown in table 1 below (showing the proportion administered subcutaneously). In one embodiment, the amount of glucagon administered to the patient in a delayed or extended release form is determined using the same calculation as described above, taking into account that lower levels of glucagon are initially available and higher levels of glucagon are subsequently available. While the amount of glucagon released at any point in time may not be precisely known, sufficient glucagon is released from the administered formulation per unit time such that, for embodiments in which glucagon is administered intravenously, an amount of about 0.1 to 5.0ng/kg/min, on average, is released to the patient. In one embodiment, 0.5, 2, 3 or 4 times glucagon can be released at any given time unit, depending on the patient, the type of diabetes being treated, and the mode of administration.

[0043] In some embodiments, the glucagon is administered subcutaneously in an amount of between about 5.0 above to about 30ng/kg/min, between about 6 to 25ng/kg/min, between 6 to 20ng/kg/min, or between about 8.0 to 12.0ng/kg/min, as shown in figure 1.

TABLE 1

Insulin (subcutaneous)/glucagon (subcutaneous) weight ratio (ng/ng) and inverse ratio (% terms (terms))
					Patient's weight	50kg	50kg	100kg	100kg
Duration of glucagon administration	150 minutes	300 minutes	150 minutes	300 minutes
					2U insulin/6 ng/kg/min glucagon	1.8[56.3％]	0.9[112.5％]	0.9[112.5％]	0.4[225％]
20U insulin/12 ng/kg/min glucagon	8.9[11.3％]	4.4[22.5％]	4.4[22.5％]	2.2[45％]
					2U insulin/18 ng/kg/min glucagon	0.6[168.8％]	0.3[337.5％]	0.3[337.5％]	0.1[675％]
20U insulin/6 ng/kg/min glucagon	17.8[5.6％]	8.9[11.3％]	8.9[11.3％]	4.4[22.5％]
					2U insulin/12 ng/kg/min glucagon	0.9[112.5％]	0.4[225％]	0.4[225％]	0.2[450％]
Glucagon 20U/18ng/kg/min	5.9[16.9％]	3.0[33.8％]	3.0[33.8％]	1.5[67.5％]

Interpretation of the entries in the table:

2U insulin/6 ng/kg/min glucagon: refers to a total of 2 units of insulin administered during the indicated infusion period, and glucagon administered at a rate of 6ng/kg/min during the infusion period. The two numbers given in the table to a 50kg person over 150 minutes are the weight ratio of insulin to glucagon (absolute), and the weight ratio of glucagon to insulin expressed as a percentage (e.g., 1.8-80000 ng (2 units of insulin)/750 ng (50 x 150 x 6 glucagon); 56.3-1/1.8-inversely proportional).

[0044] In table 1, the amount of glucagon administered is in the range of 5.6-675% of the amount of insulin administered (by weight); however, for many patients, glucagon is administered at less than 225% by weight of insulin, or is present in the composition at less than 225% by weight of insulin. As will be appreciated by those skilled in the art, the amount of glucagon administered can vary depending on a number of factors. Thus, the percentage range of glucagon to insulin ratio may vary between 5.6 to 675%, e.g., above 188% to below 675%. In one embodiment, the amount of glucagon administered is expressed as a ratio to the amount of insulin administered; for example, the amount of glucagon administered may be 5.6% to 11.3% of the amount of insulin administered. In some embodiments, the amount of glucagon administered is 112% to 225% of the amount of insulin administered. As shown in example 7 below, these amounts of glucagon can induce elevated blood glucose levels close to hyperglycemia, so a lower dose range may be sufficient to prevent hypoglycemia without the risk of hyperglycemia. The lower range or dosage may be, for example, 0.09% to 188% by weight of insulin. As understood by those skilled in the art, the weight of a patient may vary from an infant, e.g., 2kg, to an adult, e.g., 150kg to 200kg or more.

[0045] In other embodiments, the amount of glucagon administered can be described as an amount of glucagon by weight or activity that is independent of the amount of insulin administered; for example, in one embodiment, the amount of glucagon administered is 360-900 mg over a 9 hour period. In one embodiment, the glucagon is administered in a range of 5ng/kg/min. In one embodiment, the amount is about 8 to about 16ng/kg/min, or about 12ng/kg/min. In some embodiments, a much lower value may also be sufficient, depending on the situation and manner of administration.

[0046] In one embodiment, hypoglycemia is a blood glucose level below about 50-60mg/dL, typically below about 70 mg/dL; hyperglycemia is blood glucose levels above about 140 to 200 mg/dL. In one embodiment, excessive hyperglycemia is defined as blood glucose levels above 350 mg/dL. The ratio of glucagon to insulin and the respective amounts are set in accordance with the present method to effectively maintain the blood glucose level between a low blood glucose level and a high blood glucose level. In another embodiment, the blood glucose level is maintained between a low blood glucose level and an excessively high blood glucose level. In another embodiment, the blood glucose level is maintained between a high blood glucose level and an excessively high blood glucose level. As will be appreciated by those skilled in the art, this level need not be observed precisely, and slight decreases or excesses of these ranges are permissible. In one embodiment, the dose to be administered to the patient is therapeutically equivalent to a dose of 0.5 to 0.75ng/kg/min. administered intravenously, or is therapeutically equivalent to a dose of 5 or more to about 20ng/kg/min. administered subcutaneously, i.e., 8-16ng/kg/min. glucagon is administered subcutaneously. In some embodiments, the same amount of glucagon (or effective glucagon to insulin ratio) is used even when an agent other than insulin is used to reduce or control blood glucose levels. Thus, in some embodiments, the methods of the invention may be practiced with agents other than insulin, in accordance with the present disclosure, as coadministered with glucagon, a hypoglycemic formulation is contemplated. Similarly, in some embodiments, a hyperglycemic formulation other than glucagon is used to prevent the occurrence of hypoglycemia in diabetes mellitus treated with insulin. In some embodiments, neither insulin nor glucagon is used, and the diabetic is administered a hyperglycemic agent (an agent that causes an increase in blood glucose levels) and a hypoglycemic agent (an agent that causes a decrease in blood glucose levels) simultaneously.

[0047] As will be appreciated by those skilled in the art in view of this disclosure, the amount of glucagon or insulin administered to a patient may vary depending on the manner of administration. For example, the amount of glucagon to be added (or the ratio of insulin to glucagon) can be described in terms of an amount administered intravenously (as in PCT publication No. WO2004/060387, which is incorporated herein by reference). This amount can vary widely depending on how the glucagon is to be administered, e.g., subcutaneously or by inhalation. For simplicity, the amount of glucagon required to achieve an equivalent result can be described as the "equivalent dose". For example, a "subcutaneous equivalent dose of 10 ng/kg/min" is the amount required to achieve the same results as obtained when 10ng/kg/min is administered subcutaneously to a patient. A "subcutaneous equivalent dose, for intravenous administration" is an amount of glucagon administered intravenously that is required so that the resulting amount of glucose or glucagon in the blood is the same as the amount of glucose or glucagon in the blood that would result from subcutaneous administration of the amount of glucagon. Thus, in the latter words, the first administration describes how much of the administered dose will be the same as the dose to which the other administration is applied, and the second administration is the administration that is actually applied. The intravenously equivalent dose of glucagon administered subcutaneously is typically greater than the amount described for intravenous administration, as can be seen by comparing the above tables to table 1 in PCT publication No. WO 2004/060387. For example, in some patients, one unit of intravenous delivery is 0.1ng/kg/min, while in these patients, the subcutaneous delivery to achieve the same effect may be 8ng/kg/min. Furthermore, in the clinical trial described in the examples below, the amount of glucagon required to be administered to induce hyperglycemia in insulin-treated diabetes is in the 8-16ng/kg/min range for some patients (although lower doses appear to be effective), and thus, in some patients, the amount of glucagon required to prevent hypoglycemia alone will be below this range. Given this disclosure, one skilled in the art will be able to determine the appropriate amount in each case.

[0048] As understood by those skilled in the art, the "amount of glucagon administered" need not be the amount of glucagon actually entering the patient's bloodstream. Conversely, for example, subcutaneous administration of 9ng/kg/min. glucagon to a patient means that a known starting amount of starting solution is established, from which 9ng/kg/min. glucagon is administered to the patient. Less glucagon enters the patient if there is loss or degradation of glucagon prior to administration, and the effective therapeutic amount is even lower if there is loss or degradation of glucagon as it enters the patient's bloodstream and tissues. As will be appreciated by those skilled in the art, in this case, the actual amount of glucagon active and entering the patient's circulation will be less than 9ng/kg/min.

[0049] Equivalent dosages for various methods or modes of administration can be determined by one skilled in the art given the present disclosure. This equivalent dose may also vary depending on inter-or intra-patient variability and drug bioavailability. For example, if it is assumed that intravenously administered glucagon is 100% bioavailable, some glucagon formulations administered via subcutaneous bolus may have a bioavailability of about 35%, while the same glucagon formulation via continuous subcutaneous infusion may have a bioavailability of 10%, as shown by the patient data in the examples below. In addition, differences between the methods of administration of insulin and glucagon are also contemplated and determined by the methods and examples provided herein. One way in which this can be determined is by various tests for insulin, glucose and glucagon in patients receiving glucagon administration by various routes, for example, as set forth in example 7.

[0050] Pharmaceutical compositions for use in many embodiments of the invention include those useful in traditional methods of controlling diabetes and treating hypoglycemia. These conventional methods, as the phrase applies to conventional methods herein, include FDA approved methods, methods in development, and methods described in Diagnosis and Management of Type II Diabetes (PCI press, 5 th edition) by s.v. edelman and r.r.henry, the entire contents of which are incorporated herein by reference, and chapters 7 and 8 of which are of particular relevance to the present invention. As used herein, a pharmaceutical formulation or pharmaceutical composition may contain a pharmaceutically acceptable excipient, diluent or carrier. The phrase "pharmaceutically acceptable" means that the carrier, diluent or excipient is compatible with the other ingredients of the formulation and the administration device, and not deleterious to the recipient thereof. Pharmaceutically acceptable excipients are well known in the art. See, for example, Remington: the Science and Practice of Pharmacy (19The part, 1995.Gennavo. ed.).

[0051] In one embodiment, the glucagon or similar substance is administered in a buffer. Suitable buffers are those that maintain the pH of the mixture in the range of about 6.0 to about 9.0, but do not interfere with the function of glucagon. Examples of buffers include, but are not limited to, Goode's buffer, HEPES, Tris, ammonium acetate, sodium acetate, Bis-Tris, phosphate, citrate, arginine, histidine, and Tris acetate. The selection of one or more suitable buffers is within the skill of one of ordinary skill in the art.

[0052] The control of diabetes by insulin therapy, and the control of hypoglycemia by glucagon therapy, may involve parenteral administration of insulin or glucagon. Parenteral administration can be carried out by subcutaneous or intramuscular injection using a syringe, preferably a pen syringe. This methodology may be applied to practice some embodiments of the present method, although as noted above, in some cases it is preferred to provide glucagon in a manner that ensures that its duration of action more closely matches that of the applied insulin, so that glucagon is present when the risk of hypoglycemia is greatest, typically a relatively long period after a meal, but still during the time that the administered insulin continues to exert its effect.

[0053] Where subcutaneous administration of insulin and glucagon is desired, the benefits of controlling diabetes and preventing hypoglycemia can be achieved using a variety of methods. In one such method, a glucagon is administered that acts for a shorter duration than the insulin within about 1 to 4 hours after the insulin is administered. This approach provides the benefits of: most hypoglycemic episodes begin hours after the patient's last meal, and many patients administer insulin shortly before the meal. Thus, the benefits of the methods of the present invention can be obtained by delivering glucagon hours after insulin administration, but before hypoglycemic symptoms occur. Some embodiments provide methods for controlling diabetes by continuous administration of insulin and long-acting glucagon and formulations thereof, while reducing the risk of inducing hypoglycemia. Thus, in one embodiment, compositions are provided that contain a long acting form of glucagon.

[0054] In general, the long-acting forms are also referred to as extended release, extended release or controlled release (or similar terms). In one embodiment, delayed action or slow action glucagon is a particular form of glucagon in extended or long-term release form. Delayed action glucagon is within the general classification of long acting glucagon, as delayed action or slow acting glucagon would allow glucagon activity to occur after a period of time following administration of the glucagon; however, delayed action glucagon is effective at lower amounts when initially administered, with increasing potency over time. The glucagon itself may be long-acting in nature, or it may be combined with other ingredients that allow it to be released over an extended amount of time.

[0055] In another embodiment, insulin and glucagon may be administered simultaneously, by parenteral delivery, typically by subcutaneous injection, of insulin and optionally glucagon. In this method, a glucagon with a longer duration of action is preferably used, or the glucagon is administered by a route that provides a longer duration of action, e.g., by continuous infusion, as exemplified in the examples below.

[0056] Such glucagon includes, but is not limited to, glucagon formulations and routes of administration described in U.S. patent application publication No. 2002114829 and U.S. patents 6,197,333 and 6,348,214, which describe liposomal formulations of glucagon that provide reduced dose effects and are long-acting; PCT patent publication No. WO0243566, which describes delivery of glucagon via a transdermal patch; us patent No.5,445,832, which describes long acting glucagon formulations in the form of polymeric microspheres; PCT patent publication No. WO0222154, which describes the dosing of sustained release glucagon for a duration of action in weeks; and U.S. Pat. No. 3,897,551 and the Great Britain Patent (Great Britain Patent No.)1,363,954, which describe extending the duration of glucagon by iodination (each of these patents is incorporated herein by reference in its entirety). In one embodiment, the glucagon is administered (e.g., with polyethylene glycol) as a sustained release formulation or depot formulation (depotformulation).

[0057] There are many techniques known to those skilled in the art for modifying the release and/or pharmacokinetic properties of a protein, including modifying the amino acid sequence corresponding to the site of metabolic inactivation of the protein. These techniques and compositions include "pegylation" or PEG modification of proteins (see, e.g., PCT patent publication nos. WO0232957, WO9831383 and WO9724440, european patent publication nos. EP0816381 and EP0442724, and U.S. patent publication nos. 2002/0115592; 5,234,903; and 6,284,727); other polymer encapsulants (encapsulations) are described in european patent publication No. EP 0684044); lipophilic modifications (see U.S. Pat. Nos. 5,359,030; 6,239,107; 5,869,602 and 2001/0016643; European patent publication No. EP 1264837; and PCT patent publication Nos. WO9808871 and WO 9943708; formulated as liposomes (see U.S. Pat. Nos. 6,348,214 and 6,197,333), serum albumin modifications (discussed in more detail below and in PCT publication Nos. WO02066511 and WO0246227 and U.S. Pat. No. 4,492,684), formulations in the form of emulsions, microspheres, microemulsions, nanocapsules (nanoencapsulations) and microbeads (see U.S. Pat. Nos. 4,492,684; 5,445,832; 6,191,105; 6,217,893; 5,643,604; 5,643,607; and 5,637,568), formulations comprising ligands (see PCT patent publication No. WO0222154), and iodination (see U.S. Pat. No. 3,897,551).

[0058] In one embodiment, iodination methods are employed that increase half-life (as described in U.S. Pat. No. 3,897,551, see form I3G). Iodinated glucagon has a prolonged activity (measured as elevated glucose levels) of 1 to 3 hours, depending on the degree of iodination. In one embodiment, LISPRO insulin and I3glucagon can be mixed such that the modified glucagon is present at about 1.5% by weight of the insulin in the mixture (keeping the concentration of insulin per ml constant in the LISPRO formulation). Because modified glucagon has a longer lasting effect, a smaller ratio of glucagon to insulin weight would be required to prevent hypoglycemia in some patients.

[0059] Another form of long acting glucagon is a zinc-protamine-glucagon formulation. Examples of such zinc-protamine-glucagon formulations are known in the art (see, e.g., Kaind et al, VerhDtsch Ges Inn Med.1972; 78: 1099-, horm res.1998; 50(2): 94-8; all documents are incorporated herein by reference in their entirety.

[0060] Furthermore, Pichler et al (Wien Klin Wochenschr 19: 91 (2): 49-51(1979)) demonstrated that glucagon in the form of zinc-protamine had the greatest effect up to 3 hours after actual administration of the drug and showed a decrease in activity only after the 4 th hour. Thus, the effective half-life of zinc-protamine-glucagon is in this few hours range.

[0061] In one embodiment, glucagon binds to zinc without protamine, as described in Tardinget al, (European Journal of Pharmacology, 7: 206-210(1969), incorporated herein by reference in its entirety). This also forms a long acting form of glucagon. In one embodiment, the mixture comprises a zinc to glucagon ratio of 1 to 2.

[0062] In one embodiment, zinc-protamine-glucagon is prepared in a manner similar to that for zinc-protamine-insulin, except that glucagon is substituted for insulin. In one embodiment, zinc-glucagon and zinc-protamine-glucagon are prepared as described in Tarding et al (European Journal of pharmacy 7: 206-210 (1969)). For example, zinc-glucagon can be prepared by suspending lyophilized zinc-glucagon crystals in zinc acetate buffer to a final concentration of 1mg glucagon/ml, 0.05mg zinc/ml. Alternatively, zinc-protamine-glucagon can be prepared by suspending lyophilized zinc-glucagon crystals in a zinc acetate buffer containing protamine to a final concentration of 2mg glucagon/ml, 0.15mg zinc/ml and 0.5mg protamine/ml.

[0063] Another example of an agent that may be included with glucagon in a composition useful in the present method includes protamine sulfate, as described in U.S. Pat. No. 6,703,365 in combination with GLP-1 (published 3.3.4.2004, issued to Galloway et al). Among these, GLP-1 combinations exhibit increased half-life and also exhibit increased shelf life. Any glucagon that exhibits an increased half-life can be useful in the present methods and compositions.

[0064] Another means by which the half-life of a protein can be extended is by the use of "serum binders", such as albumin can be coupled to glucagon via a linker. In one embodiment, the glucagon contains a moiety that links itself to albumin in vivo. Alternatively, the glucagon may be modified so that it is capable of being linked to a linker, which then allows the glucagon molecule to be linked to a protein, such as albumin, in vivo. Thus, the modified glucagon can be administered directly to the patient where it subsequently binds to the albumin of the host, which in turn causes an extension of the useful life span of the glucagon in the system. This approach has been described for another purpose of GLP-1 in U.S. patent publication No. 20030108568, which is published on day 6/12 2003, to Bridon et al, and for a variety of other proteins (see, e.g., U.S. patent No. 6,277,863, published on day 8/21/2001, to Krantz et al, 6,500,918, published on day 12/31/2002, to Ezrin et al, 6,107,489, published on day 8/22/2000, to Krantz et al, 6,329,336, published on day 11/2001, to Bridonet al, and 6,103,233, published on day 8/15/2000, to poulty et al, all of which are incorporated herein by reference in their entirety). In one embodiment, the binding between glucagon and albumin occurs with the aid of biotin and avidin or streptavidin. In another embodiment, glucagon can be linked to other proteins through the use of maleimide and thio groups. Glucagon can be linked to any suitable protein, not just albumin.

[0065] In one embodiment, the extended release form of glucagon is a gel or fibril form of glucagon. They may be prepared as described in Gratzer and Davies (European j. biochem., 11: 37-42(1969), incorporated herein by reference in its entirety).

[0066] Other forms of extended release glucagon are also contemplated for use in the methods of the invention. The extended release preparation may be prepared by complexing or adsorbing glucagon with a polymer. Controlled delivery can be achieved by selecting appropriate macromolecules and macromolecule concentrations and methods of incorporation to control release. For example, a diffusion control system may be applied. Examples of such materials include particles of polymeric materials such as polyesters, polyamino acids, polyvinylpyrrolidone, methylcellulose, carboxymethylcellulose, hydrogels, polylactic acid, or ethylene vinyl acetate copolymers. Alternatively, instead of incorporating the compound with these polymer particles, it is possible to entrap the compound of some embodiments in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules, respectively; or entrapping the compounds of some embodiments in a colloidal drug delivery system, such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules, or macroemulsions. These teachings are disclosed in Remington's Pharmaceutical Sciences (1980). In some embodiments, the dissolution rate of a drug is primarily controlled by the dissolution of the shell encapsulating the drug.

[0067] In one embodiment, an osmotic pump system is used to provide extended release of glucagon such that the rate of release of the drug is controlled by the influx of water through the semipermeable membrane into the reservoir containing the osmotic agent. In another embodiment, the release of glucagon is controlled using an ion exchange resin.

[0068] In one embodiment, the glucagon in sustained release form is glucagon precursor (preproglucagon) [ Lund, et al, proc.natl.acad.sci.u.s.a.79: 345-349(1982)]. This polypeptide is then processed to form a pro-glucagon which is then cleaved into glucagon and a second polypeptide (Patzelt, C., et al., Nature, 282: 260-266 (1979)). Thus, by providing for the administration of this form of a glucagon precursor-or pro-glucagon, one is able to delay the onset of glucagon activity.

[0069] In one embodiment, glucagon is administered in the form of: so that substantially no active glucagon is initially released, followed by a small release of glucagon over time. This form of medication is useful when an extended period of time will occur between ingestion of the medication and the desired effect is one towards the end of the extended period, for example, at night. The form may be intrinsic to the glucagon protein, i.e., the semi-synthetic glucagon variant itself, due to components associated with the protein, due to the formulation of the glucagon administered, due to the route and method of administration of the glucagon, and for other reasons described herein.

[0070] In some cases, glucagon with low activity is desirable. Since it can be difficult to release precise small volume amounts, particularly over long periods of time, in some cases a glucagon composition containing a glucagon activity reducing component may be required to enable administration of larger volumes of sample. Alternatively, glucagon variants or mutants with lower activity levels can be used to achieve this result. The term "glucagon" may include wild-type glucagon and variants or derivatives of glucagon.

[0071] In some embodiments, a combination of an insulin component and a slow release form of glucagon provided by the present invention is employed in the methods of the present invention. Those skilled in the art will appreciate that the combination may be achieved by a single formulation or multiple formulations and a single device or multiple devices that administer the insulin component or glucagon component.

[0072] In one embodiment, parenteral administration is performed by use of an infusion pump. Various insulin pumps are available and are all suitable for delivery of insulin and glucagon components (as well as for delivery of insulin, while glucagon is delivered by another route, such as transdermal). These pumps include, for example, but are not limited to, the pumps sold by Medtronic (e.g., Mini Med), Animas Corporation, Disetronic, and Dana. Glucagon is optionally administered with insulin, and a glucagon with a short duration of action may be used, so that the glucagon can be administered when needed. In a preferred embodiment, the glucagon is administered subcutaneously in an amount between about 5ng/kg/min. or more and 30ng/kg/min. or between about 5.0ng/kg/min. or more and 25ng/kg/min. or between about 8.0ng/kg/min. and 20ng/kg/min. or between about 8.0ng/kg/min. and 12.0ng/kg/min. Lower amounts of glucagon (0.1-5 or 2-5ng/kg/min.) may be administered subcutaneously to prevent hypoglycemia in some patients. In one embodiment, the dose will prevent hypoglycemia without causing excessive hyperglycemia. Hyperglycemia is a higher than normal range of blood glucose. Glucagon can increase blood glucose above that which would be achieved without the administration of exogenous glucagon, and in a preferred embodiment, is administered in a dose that still protects against hypoglycemia and only minimally increases blood glucose levels above that which would be maintained if the patient were not subjected to hypoglycemia.

[0073] Thus, in one embodiment, the present invention provides a new drug delivery device, a pump suitable for delivering insulin for diabetes control and glucagon for hypoglycemia control in a human, i.e., the pump contains both insulin and glucagon. The pump may comprise a reservoir containing both insulin and glucagon. In another embodiment, the pump contains insulin and glucagon in two separately controlled reservoirs. There is provided a method of controlling diabetes to reduce the risk of hypoglycemia in a human patient, the method comprising administering insulin and glucagon to the diabetic patient using the pump of one of the above embodiments.

[0074] In another method, insulin or glucagon or both are provided in a powder or liquid formulation suitable for nasal or pulmonary spray administration or ocular administration. A variety of such formulations are known for insulin and glucagon, and the present disclosure provides methods of using these known formulations to administer each formulation independently, as well as the corresponding formulations of embodiments containing insulin and glucagon, to control diabetes while reducing the risk of inducing hypoglycemia.

[0075] Methods and formulations for nasal, pulmonary or ocular administration include the following: PCT patent publications WO0182874 and WO0282981, which describe sprayed insulin and glucagon; european patent publication EP1224929 and us patent No. 6,004,574, in which inhaled insulin with melezitose diluent is described; us patent No.5,942,242, which describes insulin formulations and glucagon formulations suitable for nasal administration; us patent No.5,661,130, which describes a formulation suitable for administration via the ocular, nasal and nasolacrimal ducts or the inhalation route; U.S. patent No.5,693,608, which describes formulations and methods for nasal administration of insulin and glucagon; U.S. patent No.5,482,006, which describes formulations and methods for the administration of insulin and glucagon via the nasal mucosa and other mucosa; us patent No.5,397,771, which describes methods and formulations for transmucosal administration of insulin and glucagon; U.S. Pat. No.5,283,236, which describes formulations and methods for ocular administration of insulin and glucagon; european patent publication EP0272097, which describes a formulation for nasal administration of glucagon. In addition to these formulations, methods of delivering these described formulations are also contemplated.

[0076] In one embodiment, compositions and methods are provided for controlling diabetes by administering insulin and glucagon while reducing the risk of inducing hypoglycemia, wherein one or both of insulin and glucagon are administered transdermally, e.g., via a patch, optionally an iontophoretic patch, or transmucosally, such as the buccal mucosa. The manufacture and use of transdermal delivery devices is well known in the art (see, for example, U.S. Pat. Nos. 4,943,435 and 4,893,174; and patent publication No. US 2001033858). Transdermal delivery of glucagon has been discussed above, as well as patent publications describing transdermal formulations of glucagon, and U.S. patent No.5,707,641 describes methods and formulations for transdermal delivery of insulin.

[0077] In addition, some embodiments of the invention may be practiced by oral administration of insulin and/or glucagon in amounts effective for the treatment described herein and their equivalents. Formulations and methods for oral administration of insulin, and formulations and methods for oral administration of glucagon, including those described in PCT patent publication No. WO 9703688.

[0078] The insulin and/or glucagon used in the present methods and formulations may be supplemented with, or substituted with, compounds and compositions having similar activity or action. For example, glucagon may be substituted with a mimetic of glucagon or a glucagon variant.

[0079] Insulin may be replaced or supplemented with hypoglycemic agents including, but not limited to, Insulin Sensitizers, DPP IV inhibitors and GLP1 analogs, Insulin secretagogues, including, but not limited to, sulfonylureas such as acetohexamide (dylmelor), chlorpropamide (telin, diabine), azinam (tolazamide), tolbutamide (tolbutamide), glimepiride (rimary), glipizide (glicotrol XL), glibenclamide (diabetamide, miconase), micronized glibenclamide (glyase, PRESTAB); meglitinides such as nateglinide (STARLIX) and repaglinide (PRANDIN); gastric Inhibitory Polypeptide (GIP); glucagon-like peptide (GLP) -1; moroxydine (morphililinoananide) BTS 67582; a phosphodiesterase inhibitor; and succinate derivatives; an insulin receptor agonist; insulin-sensitizing biguanides such as metformin (glycophage), Thiazolidinediones (TZD) such as troglitazone (REZULIN), pioglitazone (ACTOS) and roziglitazone (avandia); GL262570, a non-TZD peroxisome proliferator-activated receptor gamma (PPAR γ) agonist; alpha-glucosidase inhibitors such as glucoronidase (acarbose PRECOSE) and miglitol (GLYSET); combination agents such as Glucovance (glyburide and glyburide); tyrosine phosphatase inhibitors such as vanadium, PTP-1B inhibitors, and AMPK activators including 5-glibenclamide-4-carboxamide ribonucleoside (AICAR); and other agents such as Exendin (Exendin-4) and dextrin (SYMLIN ® (praminide acetate)), D-Chiro-Inositol (D-Chiro-Inositol), altered peptide ligands (NBI-6024), angiox DB complex, GABA-inhibitory melanocortin, glucose lowering agents (ALT-4037), aerodosis (aerogen), insulin mimetics, insulin-like growth factor-I alone or in complex with BP3(SOMATOKLINE), metoclopramide hydrochloride (metoclopramide HCL) (Emitasol/425), motillode/erythromycin analogues, and GAG mimetics.

[0080] In one embodiment, variants of glucagon are contemplated. Such variants may include one or a number of amino acid changes, for example, from one amino acid to all amino acids may be changed relative to the native human glucagon sequence, so long as the resulting variant functions as desired herein. In one embodiment, the helical volume of the C-terminus (HELIX content) is modulated, and local agonists are combined, so as to provide multiple "basal" inputs. In one embodiment, transient PEG modifications at the N-terminus to control activation may be supplemented with the introduction of mutations at the C-terminus for control of affinity. Examples of such variant glucagon molecules, and the resulting properties and activities thereof, are available in the art. For example, Sturm et al, (J.Med.chem.41: 2693-. In one embodiment, the variant glucagon has Lys substitutions at positions 17 and 18 and Glu substitutions at position 21, resulting in a variant with 500% binding affinity and 700% relative potency. In one embodiment, the variant glucagon amide has a Lys substitution only at position 17, resulting in a variant with 220% binding affinity and 230% potency. In another embodiment, a variant glucagon amide has an Nle substitution at position 17, a Lys substitution at position 18, and a Glu substitution at position 21, resulting in a variant with 150% binding affinity and 300% potency. In another embodiment, the variant glucagon molecule exhibits low binding capacity or low activity. Thus, for example, a glucagon amide variant with a lysine at position 18 may be used, as it has only 36% of normal binding affinity and only 12% of normal potency. Another example is a glucagon variant with Phe at position 18, which has only 4.7% normal binding affinity and 0.9% relative potency.

[0081] In one embodiment, the only pharmaceutically active ingredients in the formulation are insulin and glucagon. In one embodiment, the pharmaceutical composition (e.g., including both insulin and glucagon) is not formulated as an aerosol and/or does not contain troglitazone hydrochloride (and may not contain any thiazolidinediones). In one embodiment, the formulation is not administered orally and/or not administered nasally. In one embodiment, the pharmaceutically active ingredient of the formulation is administered transdermally, but not via a patch. For example, the active ingredient may be administered by applying a cream.

[0082] As discussed above, the effective administration of both low doses of glucagon and insulin may help prevent insulin-induced hypoglycemic events. Prevention of these events can have a number of beneficial consequences.

[0083] Repeated mild to moderate hypoglycemic events can cause a loss of hypoglycemic awareness in the subject. Thus, the subject may no longer be able to perceive that he or she is actually experiencing a hypoglycemic event, which increases the risk of more hypoglycemic events occurring. Thus, the above compositions and methods can be optimized to reduce the risk of this occurring. The above ratios of insulin to glucagon and the stated amount of glucagon used to prevent hypoglycemia are sufficient to treat such conditions, and the methods of the invention include methods of adjusting the amount, mode of delivery, or formulation administered to a particular patient to achieve the desired therapeutic effect. In some embodiments, a combination of glucagon and insulin is administered to a patient to prevent loss of hypoglycemic consciousness in the subject. In other embodiments, the glucagon and insulin are administered in order to restore hypoglycemic awareness to the subject. This can be achieved by administering an amount of glucagon to reduce or prevent additional hypoglycemic episodes. The amount may vary, for example, 8-16ng/kg/min.

[0084] As will be appreciated by those skilled in the art, these therapies and compositions are useful not only for people with diabetes, but also for people who employ insulin or other low blood glucose inducing agents.

[0085] As will be appreciated by those skilled in the art, prevention is not required for every mild or moderate hypoglycemic episode. The amount or percentage of events inhibited may vary depending on the particular situation and subject, and may include 2-5%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-99 or 99% inhibition of all mild/moderate hypoglycemic events. Furthermore, there is no need to prevent hypoglycemia in every case, which can be delayed in some embodiments. Any amount of delay may be useful, for example, delays of 1-10, 10-30, 30-60, 60-120, 120-300, 300-600 minutes or more. Optionally, the delay is additional 1-20%, 20-60%, 60-100%, 100-200%, or 200% to 10 times. In addition, not all patients need to have their sensitivity to hypoglycemia restored or maintained, e.g., loss can be restored or prevented by 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-99%, 99-100%. "Hypoglycemic sensitivity" or "Hypoglycemic unconsciousness" may be based on the ability of an individual to perceive the occurrence of a Hypoglycemic event. For example, hypoglycemic unconsciousness may be the inability to detect 1-20%, 20-40%, 40-50%, 50-70%, or more percent of hypoglycemic events (e.g., glucose levels in the blood falling below 50 mg/dL). Alternatively, the specific symptoms of hypoglycemia that are not detectable may also be used to determine the unconsciousness of hypoglycemia and how successfully it has been treated. Signs and symptoms include, for example, tremors, dizziness, sweating, hunger, headache, irritability, pale skin color, sudden mood or behavior, inflexibility or cramping (clumsy movements), difficulty concentrating, confusion, and sensation of prickling around the mouth. A description of the various possible hypoglycemic categories is found in "Defining and Reporting hyperglycemic in Diabetes" Diabetes Care, 28: 1245-1249(2005), which is incorporated herein by reference in its entirety. In particular, symptomatic, asymptomatic and possibly symptomatic hypoglycemia includes plasma glucose levels less than or equal to 70 mg/dl. As described herein, in some cases, a lower blood glucose level may be used as the threshold.

Reagent kit

[0086] In some embodiments, the compositions described herein are provided in kit form. In one embodiment, the kit comprises a vial of glucagon, a vial of insulin, an administration device such as a syringe or pump, and instructions for administering the glucagon and insulin. In some embodiments, the glucagon and insulin are pre-mixed in a single vial. In other embodiments, the insulin and glucagon are pre-mixed in a syringe. The specific instructions will vary depending on the intended use of the kit, e.g., nighttime control of hypoglycemia or other uses. Given the disclosure and the particular use envisioned for the kit, one skilled in the art will be able to determine the instructions for use. In one embodiment, the specification will describe the methods disclosed herein.

[0087] An exemplary kit contains glucagon and one or more of the following packaged together: (1) insulin; (2) a solution (e.g., excipient) to resuspend or dilute the glucagon (3) a device to administer glucagon and/or insulin; and (4) instructions. In one embodiment, the device (3) contains glucagon and/or contains insulin.

[0088] The kit may comprise glucagon in powder form in a sterile vial with a standard septum seal (septum seal). In one embodiment, the vial contains a mixture of 1mg of lyophilized glucagon, 49ng lactose and hydrochloric acid to adjust the pH (glucagon is soluble above pH 3 or below pH 9.5). The kit also had a pre-filled glycerol syringe containing 12mg/ml of glycerol in a mixture of water and hydrochloric acid. The second container contains a 1mg/mL insulin solution, which can be stored in a syringe in liquid form. The kit also has instructions directing the user to inject 1mL of diluent into the vial in a pre-filled glycerol syringe. The instructions then instruct the user to collect an amount of the glucagon/glycerol solution into a syringe containing insulin. This amount will vary depending on the intended application and the particular user, and can be determined by a physician.

[0089] In one embodiment, the volume of glucagon collected in the syringe is 0-5% of the volume of insulin to be injected. The kit may include tables and/or graphs to allow for ease of use and customization to determine what amount or ratio should be used for each user and situation.

[0090] The full dose in the insulin syringe can then be injected (children are typically given 50% of the standard dose, so the kit can be modified). In one embodiment, the insulin syringe and the glycerol syringe are one and the same, in which case the initial amount of glucagon is reduced to maintain the proper ratio of glucagon and insulin injected. In another embodiment, the insulin, glucagon, and glycerol are premixed in the kit. The description is therefore adapted to the particular embodiment employed. In one embodiment, the kit includes a glucagon kit, an insulin kit, and instructions for how to combine the two kits. As will be appreciated by one of skill in the art, any of the compositions or methods discussed above may be included in a kit as a component or instructions. Thus, for example, multiple methods of administration, multiple insulin or glucagon compositions, and multiple buffers or solvents can be used in the kit. In one embodiment, a device that administers insulin rapidly is combined with a device that administers glucagon more slowly. In one embodiment, the kit comprises only one form of glucagon and a set of instructions.

[0091] In one embodiment, the instructions may instruct the user to subcutaneously administer glucagon in an amount of more than 5 up to 20ng/kg/min, e.g., 6 to 16ng/kg/min. The kit may include a device for subcutaneous administration. In one embodiment, the glucagon unit is a 36 microgram sized dose, which is the dose used once per hour, for a 50kg human. In another embodiment, the glucagon unit is a dose of 36 to 96 micrograms in size for a 100kg human, which is the dose used once per hour. These subcutaneous values are sufficient to develop hyperglycemia in some diabetic patients; thus, in some embodiments, less glucagon is required, e.g., similar to an intravenous amount, 0.1-5ng/kg/min. In some embodiments, the kit may include instructions regarding the dosage for the age, weight, and sex of the individual. In one embodiment, the instructions include information about the dosage to be taken for future behavior such as sleeping, eating (e.g., what type of food and amount of food), sedentary, or athletic. As will be appreciated by those skilled in the art, an intravenous equivalent dose or a subcutaneous equivalent dose may also be employed if the glucagon is to be administered in another manner. In some embodiments, the kit contains a unit dose of glucagon to be added with insulin. For example, the unit dose may be about 50 or 100 micrograms (or milliunits), which is sufficient to protect a 100kg subject from hypoglycemia over a 1 hour period. Unit doses can be prepared for 1, 2, 3,4, 5,6, 7,8, 9,10, 11, 12, 14, 16, 18, 20, 22, 24 or more hours or days. Smaller doses for smaller people or for fewer hours are also contemplated.

Unit dose of glucagon

[0092] In one embodiment, rather than providing a mixture of glucagon and insulin, a unit dose of glucagon is provided alone so that it can be readily administered to a subject in need thereof to prevent insulin-induced hypoglycemia. Since the ratio of glucagon to insulin can be low, the amount of glucagon in a unit dose can also be low. As will be appreciated by those skilled in the art, the actual amount of glucagon in each dose will depend on the identity of the individual, the likely activity that the individual will be or has performed, how the dose is to be administered, and the form of glucagon. Thus, the dosages described below are only representative of some of the possible dosages. In light of this disclosure, the skilled artisan can determine the dosage.

[0093] The unit dosage forms of glucagon contain discrete amounts of glucagon for administration, and may be in the form of tablets, capsules, or powders, in a container such as a bottle or ampoule, a cartridge, a syringe, an inhaler, a transdermal patch, or other container or package. For subcutaneous administration, the unit dose of glucagon may be, for example, 0.036mg to 0.4mg for a 100kg human, which dose should be effective for at least 1 hour. Preferably, the unit dose is between about 40 micrograms and 300 micrograms, more preferably, between about 50 micrograms and 100 micrograms. Of course, these values may be adjusted depending on the size of the average person being treated and the duration of hypoglycemia prevention required per unit dose. For example, for slow release formulations, it is particularly advantageous to have sufficient glucagon to be released over 2-3, 3-5, 5-8 or more hours. Thus, in some cases, larger doses are possible. Lower doses, even subcutaneous administration, may also be used to ensure that hyperglycemic doses do not even occur transiently.

[0094] The unit dosage form contains sufficient glucagon for a single administration of glucagon, as described herein. Unit doses may be designed for specific events. For example, they may be designed to be applied before or after administration of insulin. Alternatively, they may be designed to be administered for certain behaviors such as eating or exercising or sleeping. Since the amount of glucagon to be administered depends on various factors of the patient, such as lifestyle and weight, the unit dose can be expressed as a universal unit, making it easy to adjust the dose. In addition, how the units are administered can also vary the amount of glucagon placed in each unit dose. These universal unit doses are in fact unit doses divided into smaller individual portions. Thus, 50kg of an individual may take 5 of these universal unit doses, while 100kg of an individual may take 10. This enables larger amounts to be tailored for glucagon uptake. Of course, lower amounts of glucagon may also be used if lower levels of elevated blood glucose are satisfactory, e.g., similar dosages as administered intravenously. Thus, even if administered subcutaneously, the unit dose may be, for example, between 0.036mg and 0.2 mg.

[0095] In a related aspect, a pharmaceutical preparation of glucagon in daily unit dose form is provided. Daily unit dosage forms include sufficient glucagon for a day, including where the glucagon is administered multiple times during the day, as described herein. For example, glucagon can be administered multiple times (e.g., 2 or 3 or more times) after a meal or meals of the day (see, e.g., example 1 and/or administration to prevent nocturnal hypoglycemia; or continuously via, e.g., a transdermal patch or pump). For subcutaneous administration, 960 to 4800 micrograms of glucagon may be administered within 1 day. In one embodiment, the glucagon is in a slow release form, which is administered once. In another embodiment, it is in the form of, for example, 6 pills, 1 for every 4 hours.

[0096] In a related aspect, a multiple dose form glucagon drug preparation is provided. Multiple dosage forms of glucagon may contain sufficient doses to be administered for 1, 2, 3,4, 5, or 6 days, 1 week, or even more than 1 week. In one embodiment, for example, 0.02 to 0.036mg to 1mg of glucagon is provided in a container with instructions (e.g., a label) that the glucagon should be administered in separate doses over a period of 1 day or more.

[0097] In one embodiment, a daily dose or multiple doses of glucagon are prepared by resuspending the powder in a liquid vehicle, and a portion of the resulting solution can be administered at each administration on that day (or, in the case of some multiple dose forms, on several days).

[0098] In addition to glucagon, the unit dosage form, daily dosage form, or multiple dosage forms may contain other ingredients such as excipients, buffers, stabilizers, carriers, and the like. And other pharmaceutically active agents. In one embodiment, the unit dose comprises insulin or an insulin secretagogue, as described above.

[0099] Multiple doses of glucagon (e.g., multiple daily doses) may be packaged together in a box, foam wrap, or in other known forms.

[0100] In general, dosage forms will be labeled or will be accompanied by instructions for administration of the appropriate dosage. For example, the daily dosage form may be marked to indicate the number and/or weight or volume of a unit dose in the container. Dosage forms may also be labeled or otherwise indicate the age of the patient for whom the preparation is intended. For example, dosage forms may be indicated for adults, children over 15 years of age, children over 10 years of age, children over 5 years of age, and the like.

[0101] In one embodiment, any of the above glucagon doses may comprise sustained release glucagon. In such embodiments, the dosage is appropriately adjusted to maintain blood glucose levels within a desired range, as described herein.

Glucagon solution

[0102] Administration of low doses of glucagon (e.g., below 0.01U) using formulations prepared according to conventional methods can be inconvenient (e.g., to obtain a solution of about 1 mg/ml). Thus, in some embodiments, lower concentration glucagon solutions are prepared and/or administered. As used herein, administration includes self-administration (whether by injection, infusion by pump, or other method) and administration by another person. In various embodiments, the glucagon is administered in a glucagon solution at a concentration of about 0.25mg/ml or less, e.g., about 0.2mg/ml or less, about 0.1mg/ml or less, about 0.05mg/ml or less, about 0.01mg/ml or less, or even about 0.005mg/ml or less. Such amounts are suitable for intravenous administration and equivalent dosages may be established for other methods of administration. For example, if the method of administration is subcutaneous injection, the concentration of glucagon may be higher, being at least about 0.01mg/ml, or 0.05mg/ml, or 0.2mg/ml, 0.5mg/ml, or between about 0.5mg/ml and 2mg/ml glucagon. In some embodiments, these doses are combined with a device, such as a pump, capable of administering the doses in low amounts over an extended period of time.

[0103] The glucagon may be resuspended in any pharmaceutically acceptable carrier, diluent or excipient.

[0104] In one embodiment, the pharmaceutically acceptable glucagon formulation, when administered intravenously, contains a concentration of less than about 0.25mg/ml, less than about 0.2mg/ml, less than about 0.1mg/ml, less than about 0.05mg/ml, less than about 0.01mg/ml, or even less than about 0.005mg/ml, or an equivalent dose below that used for other methods of administration. For example, for compositions intended for subcutaneous administration, pharmaceutically acceptable glucagon formulations contain between 0.5 and 2mg/ml glucagon.

[0105] In a related embodiment, a method is provided for preparing a glucagon formulation for therapeutic use, the method comprising adding an aqueous solution to a composition comprising glucagon (such as, but not limited to, a single unit dose, daily dose, or multiple doses of glucagon as described above) in an amount to yield a solution comprising a glucagon concentration of less than about 0.25mg/ml, less than about 0.2mg/ml, less than about 0.1mg/ml, less than about 0.05mg/ml, less than about 0.1mg/ml, or even less than about 0.05mg/ml, for intravenous administration. For other methods of administration, such as subcutaneous administration, concentrations 2 to 10 times higher (or higher) may be used. The solution may contain other agents, which may be pharmaceutically active and/or inactive.

[0106] In one embodiment, the glucagon solution is loaded into, or contained in, a device for delivery to a patient. In some embodiments, the device contains at least about 0.1ml, at least about 0.2ml, at least about 0.3ml, at least about 0.4ml, at least about 0.5ml, or more than 0.5ml of glucagon solution.

[0107] In a related aspect, further steps are provided for administration or self-administration to a subject of the class of diabetic patients. In one embodiment, the subject does not exhibit hypoglycemic symptoms. In one embodiment, the human is an adult. In one embodiment, the human is a human over 10 years of age, optionally over 15 years of age or older. In one embodiment, the subject does not have a gastric disorder.

[0108] In one embodiment, any of the above glucagon solutions may comprise a sustained release glucagon. In such embodiments, the dosage is suitably adjusted, as described herein, in order to maintain the blood glucose level within the desired range described herein.

[0109] The methods and compositions disclosed herein can be used to treat human patients as well as other mammals (e.g., rats, mice, pigs, non-human primates, and other animals). In some embodiments, the human patient is a child or adolescent; in one embodiment, the human patient is an adult. In some embodiments, the patient is a type I diabetic patient. In one embodiment, the patient is a type II diabetic patient. In one embodiment, the patient is a fragile type I or type II diabetic patient. In one embodiment, the non-human mammal is an animal model for diabetes studies, such as Zecker diabetic obese (ZDF) rats and db/db mice.

[0110] While many of the examples and many of the descriptions provided herein specifically relate to subcutaneous administration of low dose glucagon, other aspects are also disclosed. For example, while subcutaneous doses described herein are generally above 5ng/kg/min. and below 20ng/kg/min, other doses are contemplated for the various embodiments described herein. For example, dosages from 0.1 to 5ng/kg/min for intravenous or subcutaneous administration, particularly in combination with long acting forms of glucagon (such as zinc protamine), kits, and unit dosages are contemplated for use in dosages of from above 5 to 20 or 30ng/kg/min. Furthermore, as noted below, subcutaneous glucagon at doses of 4ng/kg/min or less may also be effective in preventing or delaying hypoglycemia. Thus, in some embodiments, the dose administered subcutaneously to prevent or delay hypoglycemia is about 0.1-5, 1-2, 2-3, 3-4, 4-5, 5 or more, 5-6, 6-8, 8-12, 12-16, 16-20, or 20-30ng/kg/min. Corresponding amounts of unit dose, other doses administered or kits are also contemplated. For example, for a 100kg human, a 1 hour unit dose of glucagon administered at 4ng/kg/min will contain 24 micrograms of glucagon, and a 1 hour pharmaceutical composition will contain 24 micrograms of glucagon and 1-3 units of insulin. As will be understood by one of skill in the art given this disclosure, any disclosed dose may be converted to a 1 hour unit dose or other aspect described herein. This may depend on the dose (e.g., 0.1 to 30ng/kg/min. or 6 to 16ng/kg/min.), the presence and amount of insulin (e.g., 0 or 2-20 units), the size of the patient (e.g., 3-200kg), and the number of hours of desired effect (e.g., 0.5-24 or more).

[0111] The following examples describe exemplary embodiments of the invention and are not intended to be limiting in any way.

Example 1

Parenteral co-administration of glucagon and insulin for the control of diabetes and prevention of hypoglycemia

[0112] Glucagon currently available in the north american market is rDNA-derived human glucagon produced by Eli Lilly & Co or Bedford Labs (Novo). There are 4 brands known: GlucoganDiagnostic Kit (Lilly); glucoglan emery Kit (Lilly); the Glucogan Emergenecy Kit for Low Blood Sugar (Lilly); and glucAGEN (Bedford labs).

[0113] Novo produces glucagon under its own name in regions outside north america. Novo produces its glucagon in yeast, Lilly produces its glucagon in e. The following examples illustrate the practice of some of the methods using these commercially available glucagon and insulin administered via various routes.

[0114] Glucagon produced by Lilly, is typically provided in the form of a kit. The glucagon in the kit is in the form of a powder placed in a sterile vial with a standard rubber sealed neck. The vial contains a mixture of 1mg of lyophilized glucagon, 49mg lactose, and hydrochloric acid to adjust the pH (glucagon, soluble at pH below 3 or above 9.5). The patient injected 1ml of diluent into the vial from a pre-filled syringe containing 12mg/ml of glycerol in a mixture of water and hydrochloric acid. The vial was shaken until the solution became clear. The fluid is withdrawn into the syringe and the full dose is injected (children typically administer 50% of the standard dose).

[0115] Glucagon is administered parenterally by subcutaneous, intramuscular, and intravenous routes, and thus, as known to those skilled in the art, its pharmacokinetic properties are different. The maximum plasma concentration is reached about 20 minutes after subcutaneous administration. The half-life in vivo ranges from 8 to 18 minutes. A peak plasma concentration of about 8ng/ml is reached about 20 minutes after administration, and elevated glucose levels can be maintained for about 1 half hour after administration and increase almost immediately after administration. Patients with insulin-induced coma typically regain consciousness within 15 minutes of glucagon administration. When parenteral glucagon is used to treat hypoglycemia, treatment is primarily achieved by increasing serum glucose availability through increased glucose output by the liver (glycogen to glucose, and new glucose formation through gluconeogenesis).

[0116] There are a variety of insulin dosing regimens that are used. The regimen employed depends upon whether the disease being treated is type 1 or type 2 diabetes, and depends upon a number of factors specific to the individual being treated. It is normal medical practice to replace insulin with a combination of parenterally (usually subcutaneously) administered insulin as follows: fast acting/short acting insulin (lispro (humalog) or aspart (novolog)), slow acting/short acting insulin (regular human insulin), intermediate acting insulin (NPH or LENTE), long acting insulin (ULTRALENTE), basic (bristol Myers squibb), and 24 hour peak-free sustained insulin (GLARGINE (lantus) and DETEMIR).

[0117] The dosing regimen (dosage regimes) can be quite complex. For example, a typical twice-a-day dosing regimen may include the administration of short and medium acting insulins before the breakfast and dinner. Thus, the insulin curve has several peaks, which roughly correspond to the predicted postprandial glucose output, and provides basal insulin levels throughout any 24 hour period. This is illustrated in figure 1, which shows an ideal pharmacokinetic profile for a mixture of normal insulin and intermediate insulin. The curve shows the effect of a twice daily insulin regimen: regular insulin (solid line) and mean insulin LENTE or NPH (dashed line) given twice a day before breakfast and dinner gave insulin peaks after injection, and relatively constant insulin baseline levels throughout that day after injection of mean insulin.

[0118] Insulin levels may vary significantly from individual to individual, and even within the same individual, depending on various factors such as the location and depth of injection, local blood flow, total volume and type of insulin injection, and other factors known to those skilled in the art. Thus, in the subcutaneous absorption of insulin, there can be significant inter-and intra-patient variability, which increases the likelihood of changes in serum glucose, including the likelihood of hypoglycemia.

[0119] With the appearance of rapid acting insulin and long acting insulin glargine (lantus) with no or little peaks; DETEMIR is also a long-acting insulin in development; in addition, ULTRALENTE is a long-acting insulin, but it tends to have some peak effects (in most patients), making it possible to control insulin levels (and thus blood glucose levels) more accurately. The basic methodology is to replace basal insulin and prandial insulin by the combined use of insulin preparations with different onset rates and duration of action. This may involve the use of separately administered insulins with different onset, (e.g. GLARGINE and LISPRO) or the use of various pre-mixed formulations (e.g. 70/30-70% NPH in combination with 30% normal insulin) for which formulations are commercially available.

[0120] The point in time at which glucagon is administered is before, during, or immediately before the period during which insulin action is least adversely affected, e.g., when significant insulin action persists in the absence of sufficient serum glucose available. Thus, insulin-induced hypoglycemia can occur at any time when the circulating insulin level and glucose level do not match (insulin action is relatively excessive for available glucose).

Parenteral administration of insulin

(i) GLARGINE/LISPRO/ASPART/GLULISINE insulins

[0121] For this illustrative example, the patients (all patients mentioned herein are in phantom except for the patients mentioned in the examples describing the real clinical trial; they are coincidental with any resemblance to the real patient) were adult males aged 50 years, weighing 75kg, having a blood volume of 5L, suffering from type II diabetes and treated with insulin (without concomitant oral combination therapy). He has used insulin for up to 10 years and his glucagon response to hypoglycemia is minimal. His insulin regimen included basal insulin replacement at bedtime with 20 unit dose levels of subcutaneous injection of GLARGINE (LANTUS) in addition to prandial insulin injections of LISPRO (HUMALOG), ASPART (NOVOLOG), GLULISINE (APIDRA) at 5 to 10 units (depending on the amount of carbohydrate consumed) at mealtimes.

[0122] His insulin profile was quite simple, with a flat line of fluctuation (basal levels established by GLARGINE (LANTUS)) with several peaks at positions corresponding to prandial LISPRO (HUMALOG), ASPART (NOVOLOG), GLULISINE (APIDRA) insulin injections. This insulin profile is shown in figure 2. In this patient, the risk of hypoglycemia typically occurs between 2 hours and 5 hours after a meal. During this period, glucagon administration is most effective and effective in preventing the possibility of hypoglycemic events. In non-diabetic patients, glucagon generally decreases (in response to increased glucose levels) after a carbohydrate meal, and then recovers as the glucose levels return to normal levels. In type 1 diabetic patients (and type 2 diabetic patients who have been on for 5 years or more), glucagon response to low serum glucose is limited. Thus, if insulin causes the serum glucose level to drop well below the basal level, hypoglycemia will occur.

[0123] Hypoglycemic symptoms are typically observed in diabetic patients with glucose levels below 50mg/dl, or sometimes below 40 mg/dl. In normal individuals, glucagon release will increase (about 40pg/ml or higher, for example about 60pg/ml or higher) before glucose levels fall to this low level, and prevent the onset of hypoglycemic symptoms. However, glucagon production (or regulation) is insufficient in many insulin-treated diabetic patients. Thus, administration of glucagon to achieve this level during the susceptible phase will prevent or attenuate the severity of hypoglycemic episodes.

[0124] In some embodiments, the subcutaneous dose of glucagon required to provide prophylaxis is about 6-20ng/kg/min for basal insulin levels, proportionally higher amounts for higher levels of insulin.

[0125] Thus, according to the present method, 41 to 90 micrograms of glucagon is administered subcutaneously to the patient after a meal. Two similar doses of glucagon were administered once an hour for an additional 2 hours. Thus, 3 doses are formed at 2, 3,4 hours, providing up to 3 hours of protection against hypoglycemia, which begins 2 hours after a meal. With formulations comprising insulin and glucagon, a lower percentage (5.6%) of glucagon can be used, since the risk and extent of hypoglycemia is (partially) insulin dose-dependent. In this example, although the glucagon concentration of the dose administered 2 hours after meal will fall back to approximately the basal level after 1 hour, the rise in blood glucose caused by this dose will last for more than 1 hour, which provides time for the 2 nd dose to work. The same pharmacokinetics apply for the 3 rd dose of glucagon. In alternative embodiments, two doses, or even one dose, of glucagon may be administered.

[0126] Although this example employs a simple basal and prandial insulin model, it will be understood by those skilled in the art that it is applicable to all currently practiced dosage regimens. The timing (and frequency) of glucagon injections can be adjusted to match the period of the patient's greatest susceptibility to hypoglycemia, i.e., when insulin action and glucose availability are the least matched.

(ii) NPH/human insulin

[0127] A typical diabetic patient is an adult male, 63 years old, weighing 75kg, suffering from type 2 diabetes for 18 years and using combined insulin therapy (without concomitant oral anti-diabetic therapy). Heretofore, he typically applied oral antidiabetic drugs, including glibenclamide and glipizide, but when his serum glucose level continued to be above 250mg/dl, the drugs were deactivated and insulin application commenced. He will have applied one or the other insulin for more than 10 years and will develop the following signs: background retinopathy, mild renal impairment with 1.9mg/dl of serum creatinine and creatinine clearance of 60ml/min, mild proteinuria, bilateral peripheral symmetric neuropathy of both feet, and exertional angina. His insulin dosing regimen typically involved split-mixed regiment (split-mixed regiment) of subcutaneous NPH insulin, 20 units before breakfast and 15 units before dinner, with the aim of providing a basal insulin distribution throughout the day, plus a moderate postprandial distribution of insulin after lunch and dinner (and overnight). Furthermore, before the meal he injected between 6 and 10 units of normal insulin (depending on the pre-meal serum glucose level and the meal size and carbohydrate content).

[0128] His insulin profile is similar to that shown in figure 1, with less rapid peaks and slower decay due to prandial injections of normal insulin, and slow-onset and delayed-decay effects due to twice-daily intermediate-acting NPH insulin. Typically, his fasting glucose levels are well controlled in the 90-130mg/dl range, but his 1-2 hours post-prandial glucose levels are less than ideal, usually in the 180-240mg/dl range. The glycosylated hemoglobin was increased by 7.9% (normal range 4-6%). Efforts to increase their prandial regular insulin doses for breakfast or dinner to reduce postprandial glucose levels are often accompanied by frequent intermittent hypoglycemia, of mild to moderate intensity, usually occurring 1-2 hours before lunch or hours after dinner. These hypoglycemic events can be quite severe and are associated with symptoms of sweating, tremors, nausea and headache, particularly when eating late. He never had insulin-induced coma, but once coma occurred he did not avail himself to increase his insulin dose. He is concerned that he may lose his driver's license if this happens, or that he is afraid of losing work as a night attendant. Since the patient has a longer history of diabetes and has significant complications, it is likely that he will exhibit impaired glucose back-regulation to hypoglycemia, particularly indicative of a weakened or absent glucagon response.

[0129] In this patient, the risk of hypoglycemia is typically maximal 3 to 5 hours after a meal (late postprandial hypoglycemia), when circulating insulin levels still increase above fasting levels, but glucose availability (absorbed from the stomach and produced from the liver) is minimal. During this time, glucagon administration prior to the onset of hypoglycemia is the most potent and effective in preventing the possibility of hypoglycemic episodes by increasing the availability of circulating glucose. In non-diabetic individuals, both insulin and glucagon following a meal are tightly regulated to balance glucose production and utilization to maintain euglycemia. Once the insulin effect becomes apparent, the level of glucagon will increase to counteract the underlying hypoglycemia.

[0130] According to one embodiment of the method, 36-96 milliunits of Glucagon are administered subcutaneously (optionally using the Eli Lilly's Glucagon emery rogenckit as described above) to the patient 2 to 3 hours after each meal in order to prevent the development of hypoglycemia. In one embodiment, glucagon is added when glucose detection indicates that the glucose level is approaching a low blood glucose level. As mentioned above, this administration provides the required protection within 3 to 5 hours.

[0131] Although this illustrative example employs a simple basal and prandial insulin model, it will be understood by those skilled in the art that it is applicable to virtually all currently employed insulin drug dosage regimens. The glucagon injection is optimally timed and varied depending on the insulin therapy applied, but is designed to achieve sustained glucagon levels over the expected period of relatively unopposed insulin action.

[0132] In the presently hypothetical example, this situation tends to occur at several times during the day. For example, hypoglycemia tends to occur when the injected normal insulin absorption "tail" is combined with peak insulin availability from the intermediate-acting NPH. This occurs hours after breakfast, when serum glucose availability (primarily from intestinal absorption and liver production) is minimal or reduced. Similar situations also often occur before lunch, at bedtime and at midnight. Thus, for all insulin therapies, the timing of glucagon injection can vary depending on the pharmacological characteristics and timing of the insulin applied.

[0133] To counteract the glucose-increasing potential of glucagon administered, the dose of insulin acting during hypoglycemia may be increased slightly to maintain euglycemia. However, in the particular case described above, the increased availability of glucagon provides a buffering or slowing effect on the excessive glucose lowering effect of insulin and weakens or prevents hypoglycemia.

B. Insulin administration by pump

[0134] In this example, the patient described in example 1.A.i administered the required insulin using a pump. The patient's insulin pump is programmed to provide a continuous, rapid-acting flow of insulin (e.g., LISPRO or ASPART) in place of the glargine (lantus) once daily administration of basal insulin in example 1. A.i. In this example, the patient is injected with ASPART at the time of a meal in a dosage of 5 to 10 units, depending on the pre-meal glucose level and the amount of carbohydrates and calories consumed. Then, according to the present method, the patient is injected with glucagon 2 hours after meal (e.g., using the GLUCANGEN product of Bedford Lab) and this dose is repeated every hour for another 2 hours. This amount may be about 36 to 96 micrograms of glucagon delivered subcutaneously. Glucagon is administered subcutaneously. This administration provided a 2 to 5 hour protection against hypoglycemia as described in example 1, A.i. One skilled in the art will appreciate that glucagon may also be administered via a pump. In an alternative embodiment, the glucagon amount is increased proportionally per unit of insulin; thus, 36 to 96 micrograms per unit of insulin is applied.

C. Transdermal administration of insulin [ including patches and topical creams]

[0135] In this example, the patient is a 62 year old, wasting type 2 diabetic, with 6 years of illness. Initially, a two-day treatment with 20mg of glyburide was applied followed by a two-day treatment with 1 g of metformin, but fasting and postprandial blood glucose levels were consistently in the range of 200-350 mg/dl. The doctor advises him to apply insulin. The oral antidiabetic drug is discontinued and GLARGINE (LANTUS) insulin 15 units is administered at bedtime to provide its basal insulin replacement needs for one day. Postprandial insulin is administered via a transdermal patch to provide 2-6 units of rapid acting insulin (patches applied in increments of 2 units; although patches are referred to in this example, one skilled in the art will appreciate that this embodiment may be practiced with a substantially similar methodology and that insulin or glucagon may be delivered transdermally by other means, such as a cream or lotion). Alternatively, he was advised to replace once-a-day GLARGINE with 24 hour basal insulin instead of patch. The basal insulin replacement patch contains a single formulation of insulin designed to provide stable sustained absorption and low sustained serum insulin levels throughout the day. As fasting plasma glucose continued to rise, his physician gradually increased his GLARGINE insulin dose by 24 units and transdermal patch dose by 4-10 units over 6 months. With increasing GLARGINE and transdermal insulin doses, fasting glucose levels ranged from 70-110mg/dl and 1-2 hours postprandial glucose levels ranged from 130-180mg/dl within 3 months.

[0136] The patient applied the rapid acting insulin patch 30-60 minutes prior to meals. This timing arrangement is selected so that the absorption of food is consistent with the kinetics and action of insulin patch absorption. As indicated above, this patient has near-normal glycemic control, but he begins to suffer from early morning hypoglycemia, which typically occurs at 1 or 2 am. At these times, this hypothetical patient frequently experiences confusion, irritability, and sometimes anxiety. Several finger-stick glucose readings taken during this event revealed blood glucose values of 35-40mg/dl, which resolved rapidly after juice intake. In an effort to control the onset of hyperglycemia, his physician gradually reduced the nighttime dose of GLARGINE, but this was associated with worsening glycemic control and substantially increased preprandial glucose levels.

[0137] According to some embodiments of the method, to restore near normal blood glucose but prevent early morning hypoglycemic symptoms, the physician increases back 24 units of GLARGINE insulin at bedtime and prescribes subcutaneous administration of Glucagon in an amount of 18ng/kg/min for protection purposes (Bedford Labs Glucagon Product) immediately after injection of GLARGINE at 23:00 o' clock. The timing of glucagon administration depends essentially on the rate of absorption, which is rapid, reaching peak levels within 15-30 minutes, and on the duration of action, which is about 2-3 hours. In one embodiment, plasma glucagon is maintained near the "high normal basal level" during this period and prevents non-insulin resistant effects from GLARGINE insulin or delayed effects of the evening (pre-evening meal) patch. For example, insulin levels above 120-160mg/dl are contemplated. In an alternative embodiment, the low end of normal blood glucose levels is set as the target of blood glucose levels. As mentioned above, this treatment provided the desired protection against hypoglycemia for about 3 hours after the GLARGINE injection. With the addition of bedtime glucagon with his diabetes therapy, early morning hypoglycemic events should be resolved and near normal blood glucose maintained throughout the day. As will be appreciated by those skilled in the art, subcutaneous or intravenous equivalent doses of glucagon may also be administered in the same manner as insulin (e.g., transdermal administration via a patch or cream).

D. Inhaled insulin including pulmonary, buccal, nasal and sublingual]

[0138] This example is similar to example 1.A.i, except that the patient administers insulin by inhalation instead of subcutaneous injection. It will be understood by those skilled in the art that similar methods apply when insulin is administered buccally, nasally or sublingually according to these methods, although equivalent doses will be applied. The patient will continue to administer his basal insulin requirement via GLARGINE (LANTUS), or he will administer the basal insulin requirement using an insulin inhaler. The patient will administer his prandial insulin needs (equivalent to between 5 and 10 units administered subcutaneously) using his insulin inhaler (either pulmonary, nasal, buccal or sublingual).

[0139] According to these methods, the patient is administered 45 micrograms of Glucagon subcutaneously two hours after a meal, optionally using the Lilly's Glucagon Kit, and two more doses once an hour thereafter. He will administer glucagon subcutaneously. This will provide the required protection against hypoglycemia for 2 to 5 hours as described in example 1. A.i. It will be appreciated by those skilled in the art that glucagon may also be administered by inhalation in equivalent doses. It will be appreciated by those skilled in the art that the precise amount of glucagon administered can be varied and determined for different amounts of insulin by the method shown in example 8 below.

Example 2

Pump for sitting on the seat for supplying pancreas beneficialBlood glucose co-administered with insulin for controlling diabetes and preventing hypoglycemia

[0140] In one method, insulin may be administered by a pump. Many suitable pumps for use in the present process are commercially available (or soon to be available) in the U.S. market and elsewhere. This includes, but is not limited to:

ANIMAS (IR-1000 and IR-1200)

·DELTEC(Cozmo pump)

DISETRONIC (H-TRONplus and D-TRONplus)

LIFESCAN & DEBIOTECH (MEMS Insulin Pump, in development)

·MEDTRONIC MINIMED(PARADIGM Insulin Pump and 508 Insulin Pump)

MEDTRONIC MINIMED (2007 Implantable Insulin Pump System (EU only))

[0141] When both insulin and glucagon are to be administered by a pump (from separate reservoirs), several configurations may be employed in the practice of the present method. A typical configuration is:

(1) a single device with a single pump and two reservoirs with each drug (dual reservoir pump, see for example us patent 5,474,552) delivered through two separate lines that are merged prior to intubation.

(2) A single device with a single pump and two reservoirs with each drug delivered through two separate lines, each line being separately cannulated.

(3) A single device with two independent pumps and two reservoirs with each drug delivered through 2 lines that are combined prior to intubation.

(4) A single device with two independent pumps and two reservoirs with each drug delivered through two separate lines, each line being separately cannulated.

(5) Two devices, each with a single pump and a reservoir, with each drug delivered through 2 separate lines that are merged prior to intubation; and

(6) two devices, each with a single pump and a reservoir, with each drug delivered through 2 separate lines that are separately cannulated.

[0142] Those skilled in the art will appreciate that other configurations are possible, and practice of the present embodiments is not limited to the devices and configurations of the devices enumerated above. For example, an implantable pump can be used in much the same way as is obtained with an external pump.

[0143] Embodiment (1) above minimizes the trauma of the cannula to the patient, reduces cost, simplifies infusion, and minimizes complexity. With this embodiment, a single pump can be programmed to deliver the appropriate volume from each reservoir, each containing a different concentration of one of the two hormones. An example of such a pump is provided herein.

[0144] In a typical insulin pump, the internal pump structure typically contains an electromagnetically driven impulse pump having a solenoid-operated piston arranged for reciprocating movement in a cylinder to draw a drug from an internal reservoir (reservoir) and deliver the drug through a delivery line and then to the patient via a cannula or micro-tube.

[0145] Since the delivery line used with an insulin pump is typically half a meter in length and 1/10 mm in lumen diameter (dead volume (void volume) of 1/10 ml or about 10 IU of insulin), the time delay between when the new drug reaches the body and when the pump begins infusing the drug can be quite long (about half a day).

[0146] To reduce this delay, one embodiment provides a pump that is shorter in length and/or has a very small internal lumen diameter, which greatly reduces the lag time for drug switching. This embodiment also provides a peristaltic type pump acting on both delivery lines.

[0147] One embodiment provides a system comprising a pump and a set of 4 valves, two immediately before the pump and two immediately after the pump, which controls what drugs are pumped when the pair is active. In one embodiment, the two lines are merged where the intubation is performed, thereby eliminating delay or (dead volume) time. The additional space required for electrically actuated microvalves is minimal, adds little bulk or expense, and can be assembled using commercially available devices. Other possibilities of applying less than 4 valves are described below.

[0148] In one embodiment, an economical pump System suitable for use in some methods is a micropump known as MEMS (Micro-Electro-Mechanical System), which is being developed by Debiotech corporation for diabetes, under the trade name Chronojet. In a single device, two such micropumps are employed, adding little volume and only minimal expense to existing designs. As noted above, the two delivery lines may merge (in this regard, the two drugs come into direct contact) at their junction with the cannula, or at their junction with a similar microneedle device used to pierce the skin and deliver the drugs.

[0149] In one embodiment, a single, divided (dual lumen) line is used instead of two physically separate lines. This method has the advantage that the patient only needs to deliver via one flexible wire instead of two. Alternatively, two standard lines physically adhered together along their length may also be applied to achieve the same advantages, according to some embodiments of the present method.

[0150] Thus, in one embodiment, the pump is a currently applied insulin pump modified to have two drug reservoirs instead of one, each administered independently by a single (or two) pump and a single control system to control the amount and relative timing of administration of the two drugs. As noted above, in some embodiments, the device contains 1, 2, or 4 valves and suitable connecting piping. Schematic diagrams of apparatus to which some methods according to the invention may be applied are shown in figures 3,4 and 5. In fig. 3, the insulin reservoir (1) and glucagon reservoir (2) have lines in fluid communication with the pump (3) and then proceed through 4 valves (6) and (7) via the subchamber delivery line (4) to the cannula (5). When valve (6) is open and valve (7) is closed, only insulin is pumped in. When valve (6) is closed and valve (7) is open, only glucagon is pumped. In this way, a single pump may be used to deliver insulin or glucagon to a patient simultaneously or separately, while at the same time mixing of the two substances is minimized by virtue of delivery through a chambered delivery line in which the liquids are mixed only at the cannula, i.e., the delivery point. In fig. 4, the insulin reservoir (1) and glucagon reservoir (2) have lines in fluid communication with the pump (3) and then proceed through 2 valves (6) and (7) via the subchamber delivery line (4) to the cannula (5). These valves are two-way valves that allow either the insulin or glucagon paths to open-but only allow one path to open at a time. A small mixing of the two liquids will occur in a short section of the line through the pump, but this will be an insignificant volume compared to the typical pumped volume. In this way, insulin and glucagon can be delivered to the patient with minimal mixing and dead space in the line. The advantage of this arrangement compared to the arrangement disclosed in fig. 3 is that only two valves need to be operated. In fig. 5, the construction is the same as that disclosed in fig. 4, but in this case the two valves (6) and (7) are combined into a single device with a single transmission mechanism. In this way, the device remains as cheap and simple as possible, with only one valve and one pump being necessary to obtain the desired result.

[0151] Setting the pump to deliver basal levels of insulin, and manual intervention to administer prandial insulin as needed, is a common practice for pumping insulin to a patient.

[0152] In one embodiment, the delivery of glucagon is administered automatically over a suitable period (e.g., continuously between hours 3 and 5 following manual instruction of the delivery of prandial insulin). The control logic required to generate such a sequence of events may be programmed into the pump.

A. Insulin administration by pump

[0153] This example illustrates how some of the methods of the present invention can be practiced using pump-based administration. A typical hypothetical patient is an adult male, age 35, weighing 75kg, who, starting from age 15, suffers from type 1 diabetes and, starting from a diagnosis, is treated with insulin. He had previously received many different insulin therapies, but blood glucose was not optimally controlled. In the last 5 years, he started to develop clear background retinopathy, mild renal insufficiency and hypertension, and he worried that these complications would continue to progress rapidly if he could not improve glycemic control from the current 7.8% glycated hemoglobin level. In a more recent period, he applied ULTRALENTE 22 units at bedtime and LISPRO insulin 4-8 units immediately before each meal and snack. The dose of ULTRALENTE has been adjusted to provide basal replacement of insulin, while the dose of LISPRO at meal time may vary depending on the prevailing pre-injection serum glucose and the total calories and carbohydrate content of each meal.

[0154] Despite self-monitoring of capillary blood glucose 4-6 times daily using a glucometer, his glycemic control is often erratic, ranging from high values of 200mg/dl, to occasional hypoglycemia. In the last 1 year, he had 3 severe hypoglycemic episodes with coma or near coma, two of which occurred at work and one after a handball race. In all of these cases he needs the help of others and after exercise the carer is required to inject him intramuscularly with glucagon.

[0155] When using an insulin pump therapy, short acting insulin is typically applied because the pump provides a continuous supply to stimulate basal insulin over a long period of time. In this example, the patient applied ASPART (NOVOLOG) as his insulin selection. The PUMP, reservoir and control device (in this illustrative example, MEDTRONIC MINIMED PARADIGM INSULIN PUMP) may be attached at various points around the patient's body (e.g., most commonly on the belt) and connected by flexible plastic tubing to a microcannula that has been inserted into the abdomen, thigh or arm by him (women tend to place the infusion site in the lower abdomen, while men generally choose the upper abdomen).

[0156] The patient has programmed the device to deliver 1 IU of insulin every 50 minutes (20 units per day). When the patient eats during the day, he programs the device to release an amount of insulin (between 3 and 8 units) that is appropriate for the meal (pre-meal glucose, total calories and carbohydrate content). He does this by pressing an appropriate button on the device (or, if applicable, a remote control device) to select the size of the insulin bolus required.

[0157] After application of the programmable insulin infusion pump, the patient was able to achieve significantly improved glycemic control with pre-prandial glucose levels ranging from 70-110mg/dl, post-prandial (1-2 hours) levels ranging from 120-160mg/dl, and 6.4% glycated hemoglobin. However, he is continuously afflicted with frequent mild-to-moderate hypoglycemic events, which he is usually unaware of until he measures his index glucose. Many of these low glucose values are in the range of 30-40 mg/dl. His wife and friends tell him that he is sometimes behaving abnormally, but is improved by ingesting food or juice.

[0158] Due to long-term type 1 diabetes and frequent and often unrecognized hypoglycemia, this patient has significant impairment of glucose counterregulation, accompanied by a deficiency of glucagon and a significantly weakened epinephrine response to hypoglycemia. I.e. he is unable to mount an effective response to abnormal hypoglycaemia and the consequent danger that it may cause. Moreover, he has a hypoglycemic unconsciousness, which frequently accompanies recurrent hypoglycemia, and increases the risk of developing severe hypoglycemia. When his blood sugar is dangerously low, he is unconscious because his body has defective mechanisms to recognize hypoglycemia. This is a common phenomenon in long-term diabetics and it is most common when these individuals attempt to achieve normal or near-normal glycemic control. Since he is concerned that his hypoglycemic events increase in frequency and severity and he often cannot recognize them, he is seriously considering "relaxing" the glycemic control to alleviate hypoglycemia. He knows that this may have the detrimental consequences of increased microvascular complications, but he feels the greater and more urgent risk of severe hypoglycemia.

[0159] This fictitious patient has endeavored to achieve the best possible glycemic control based on the knowledge that the risk of developing microvascular disease is reduced as long as his day-wide blood glucose reaches non-diabetic levels. Although he applies the most advanced and flexible form of insulin delivery system currently available and has achieved significantly improved glycemic control towards the proposed target, he is plagued by frequent and potentially dangerous hypoglycemic episodes. To alleviate this condition, and also to allow him to maintain the same level of glycemic control, the methods provided herein are applied, and in one embodiment, a second pump device is applied, which is the same as the first device, but with the glucagon cartridge replacing the insulin cartridge. The devices are separately cannulated and separately controlled for continuous subcutaneous infusion of glucagon when desired.

[0160] In one embodiment, the patient is instructed to practice as described below. After a meal, the patient injects prandial insulin (3-8 units) and the program for setting the glucagon pump is: about 162 μ g glucagon is administered subcutaneously over a 3 hour period, hourly and timed to begin administration 2-3 hours after prandial insulin administration. In one embodiment, the above instructions are contained in a kit with a composition for controlling hypoglycemia. In this example, unlike example 1.a.ii, the sustained release of glucagon produced a smoother curve with fewer peaks and a delay period compared to glucagon injection alone subcutaneously. The increased availability of glucagon during the period in which the patient is most susceptible to hypoglycemia greatly reduces the likelihood and severity of such events. To counteract the glucose-raising potential of subcutaneous glucagon, the dose of insulin infused may be increased to maintain euglycemia during glucagon administration. By administering glucagon in this way, the patient is provided with sufficient glucagon to act as a buffer or buffer to the effects of insulin that is not resistant, thereby preventing the risk of a hypoglycemic event. Thus, the administration of glucagon enables patients to maintain good glycemic control without the undue risk of frequent and severe hypoglycemia.

B. Parenteral administration of insulin

[0161] In one embodiment, the glucagon may be administered by a pump and the insulin administered parenterally, including by a pump or other subcutaneous route. In one embodiment, a pump suitable for administering insulin is also suitable for administering glucagon. Insulin can be administered parenterally as described in example 1. A.i. However, instead of injecting glucagon as described in example 1.A.i, the pump was programmed (or driven) to deliver glucagon continuously 2 to 5 hours after a meal. Within this 3 hour period, the total dose of glucagon released was about 121-324 milliunits, which was sufficient to provide protection against hypoglycemia.

C. Transdermal administration of insulin including patches and topical creams]

[0162] In one embodiment, insulin may be administered transdermally. According to example 1.C, the patient administers the insulin he needs by applying a transdermal patch (or cream). However, instead of administering glucagon parenterally as described in that example, the patient administered glucagon using an insulin pump (no insulin but glucagon) to prevent hypoglycemia in the early morning. Prior to bedtime, the patient programs the pump to deliver glucagon subcutaneously in amounts of 50-120 milliunits between 01:00 and 02:00 hours of their greatest susceptibility to hypoglycemia. By doing so, the patient is able to maintain normal blood glucose without the risk of hypoglycemia occurring during sleep using the method of the present embodiment.

D. Administration of insulin by inhalation [ including pulmonary, buccal, nasal and sublingual]

[0163] According to example 1.A.i, the patient administers insulin by inhalation rather than subcutaneous injection. It will be appreciated by those skilled in the art that a similar method may be used when insulin is administered buccally, nasally or sublingually. The patient will either continue to be administered the basal requirement by GLARGINE (LANTUS) or the basal insulin requirement using an insulin inhaler. The patient will administer his prandial insulin requirement (equivalent to 5 to 10 units administered subcutaneously) using his insulin inhaler (pulmonary, buccal, nasal or sublingual). However, rather than injecting glucagon as described in example 1.A.i, the insulin pump (containing glucagon but not insulin) was programmed (or actuated) to deliver glucagon continuously subcutaneously 2 to 5 hours after a meal. The total dose of glucagon released over the 3 hour period was approximately 121-500 units, which was sufficient to provide protection against hypoglycemia during the most susceptible period of the patient.

Example 3

Co-administration of mixed glucagon and insulin by pump for diabetes control and prevention of hypoglycemia

[0164] Some embodiments of the methods of the invention may also be practiced using a pump-based administration of a mixture of insulin and glucagon. The present method provides protection against hypoglycemia, is proportional to the amount of insulin applied and establishes a delay. This also replaces the basal glucagon level throughout the day, especially after a meal, as this would be given the basal insulin by the pump. This embodiment can be implemented with standard pumps currently available and the pump described in example 2. One difference is that the insulin cartridge used will contain a mixture of insulin and glucagon (optionally modified release glucagon) with a glucagon component of between 41-96mU, which is administered every hour that will require protection (prevention of hypoglycemia). In some embodiments, the amount of glucagon administered subcutaneously may be from about 6ng/kg/min of protection desired to about 20ng/kg/min of protection desired, and in some embodiments, 12ng/kg/min.

[0165] The insulin/glucagon mixture is administered with the pump (for basal and prandial insulin). The resulting plasma concentration of glucagon (modified glucagon) will be plotted on the insulin curve, but with the impaired characteristics of the glucagon variant used. This is illustrated in fig. 7. This method will provide protection against hypoglycemia during the susceptible period as needed. In one embodiment, the pump is equipped with a glucose sensor (see U.S. patent No.5,474,552).

Example 4

Oral co-administration of glucagon and insulin for the control of diabetes and prevention of hypoglycemia

[0166] Oral delivery of macromolecules (e.g., proteins) is well known in the art. This typically involves enteral administration (see U.S. Pat. No.5,641,515). In one embodiment, glucagon is delivered orally using a similar method. In a typical regimen involving oral delivery of a mixture of insulin and glucagon, the patient takes an enteric-coated tablet containing insulin adapted to his meal up to 1 hour prior to eating. The insulin component is designed to take effect quickly once it begins to be released. The glucagon component is designed to be released later, in one embodiment, 2-3 hours later, than the insulin component. Optionally, modified glucagon with a long half-life can be used to ensure that glucagon levels increase over an extended period. In this way the glucagon will be correctly and properly timed so as to prevent hypoglycemia. In another embodiment, the patient administers insulin using any of the methods described herein and a bolus of glucagon as needed, e.g., with each meal, to prevent hypoglycemia.

Example 5

Compositions of glucagon suspensions and methods of characterizing them

[0167]This example describes a composition of one embodiment of the present invention that was found to be useful for the storage of glucagon, as well as a method of one embodiment of the present invention for verifying that a composition storing glucagon provides a desired degree of stability to glucagon. In an infusion pump cartridge, GlucaGen was administered^®Mixing in 5% mannitol in Water For Injection at concentrations of 200. mu.g/mL and 500. mu.g/mL. The solution was held at 30 ℃ for a set duration. After a set duration, the pH and percentage of residual glucagon were measured. The percentage of glucagon remaining was determined by HPLC. 200 ug/mL GlucaGen^®The results of (A) are shown in Table 2 below, 500. mu.g/mL GlucaGen^®The results of (a) are shown in fig. 3. The results and data for the 3-group experiments are expressed as mean ± mean standard deviation (s.e.m.).

TABLE 2

Point in time	pH	Residual glucagon% versus time 0
			The time is 03 hours, 6 hours, 9 hours and 24 hours	3.36±0.013.45±0.043.53±0.033.64±0.043.79±0.04	100.0±2.3101.1±1.595.2±4.399.1±5.389.3±0.7

TABLE 3

Point in time	pH	Residual glucagon% versus time 0
			The time is 03 hours, 6 hours, 9 hours and 24 hours	2.98±0.003.10±0.023.08±0.013.13±0.033.21±0.01	100.0±1.1100.2±1.699.4±0.9101.2±0.193.8±2.1

[0168] All solutions at the time of preparation were clear and colorless. The solution remained clear and colorless throughout the compatibility test. No distinct particles were visible. A significant amount of glucagon remains in the mannitol solution even at high concentrations and for all time periods tested, including 24 hours. In addition, higher concentrations of glucagon result in a higher relative percentage of glucagon remaining. Thus, it appears that this combination is sufficient to maintain glucagon in solution for a useful period of time.

Example 6

Compositions of glucagon suspensions and methods of characterizing them

[0169] This example illustrates the composition of an embodiment of the present invention and a method by which such compositions can be analyzed to determine the longevity of glucagon solutions undergoing activation, "wear" (wear) applications. Samples of Glucagon Infusate were prepared at 200. mu.g/mL and 500. mu.g/mL in 5% mannitol solution. About 3mL aliquots of each Glucagon infisate sample were added to separate cartridges three times. The tubes were inverted 10 times and 0.5-mL aliquots were removed from each tube at "Time 0(Time Zero)" for HPLC analysis. The filled cartridge was placed on a shaking platform (PlatformShaker) set at 50RPM and incubated at 30 ℃ for 24 hours. At 3,6, 9, 12 and 24 hours, the cartridges were removed from the incubator, inverted 10 times, and 0.5-mL aliquots were dispensed from each cartridge for HPLC analysis. Appearance and pH were also recorded at each time point.

[0170] All solutions at the time of preparation were clear and colorless. The solution remained clear and colorless throughout the stability test. No distinct particles were visible. Other results are shown in tables 4 and 5.

TABLE 4

Point in time	pH	Residual glucagon% versus time 0
			The time is 03 hours, 6 hours, 9 hours, 12 hours and 24 hours	3.47±0.053.45±0.033.47±0.033.50±0.003.50±0.033.53±0.01	100.0±2.785.7±4.283.6±2.682.7±2.975.5±6.048.9±1.0

TABLE 5

Point in time	pH	Residual glucagon% versus time 0
			The time is 03 hours, 6 hours, 9 hours, 12 hours and 24 hours	3.05±0.023.01±0.013.02±0.003.05±0.003.03±0.003.00±0.00	100.0±0.598.5±1.297.6±0.795.1±2.095.0±3.693.1±1.1

[0171]200 ug/mL GlucaGen in 5% mannitol with constant shaking at 50RPM^®Shows a loss of 25% of glucagon after 12 hours of storage at 30 ℃ and about 50% of glucagon after 24 hours of storage at 30 ℃. GlucaGen dissolved in 5% mannitol at 500. mu.g/mL^®Showed a loss of 7% over a 24 hour period. Thus, even with lower concentrations of glucagon, e.g., 200 μ g/mL, a substantial amount of glucagon remains in solution after an extended period of time. Moreover, it appears that higher glucagon concentrations result in a more stable formulation, even after 24 hours of active shaking of the formulation.

[0172] In one embodiment, the glucagon, or variant thereof, is stored at a high concentration. The high concentration may be, for example, 100. mu.g/ml or more. In one embodiment, the high concentration is 200 or more, 200-300, 300-400, 400-500, 500-600, 600-800, 800 or more μ g/mL of glucagon up to the saturation (or supersaturation) limit. In one embodiment, the less active form of glucagon is stored at a higher concentration. This makes the glucagon solution more stable and enables a larger volume of glucagon solution to be administered to the recipient.

Example 7

Very Low Dose (VLD) glucagon can be used to raise blood glucose in diabetic patients receiving insulin therapy

[0173] This example illustrates the efficacy of administering a low dose of glucagon to a patient via a particular route of administration in order to raise the patient's blood glucose level. This example describes a clinical trial in which glucagon levels required to increase glucagon and blood glucose levels are determined. As explained in more detail below, the results indicate that the methods of some embodiments of the present invention can be used to prevent insulin-induced hypoglycemia.

A Glucagon from 0.8 to 4ng/kg/min

[0174] At the night titration period, 6 patients with faithful type 1 diabetes were stabilized overnight at blood glucose levels of 90 to 120 mg/dl. Blood glucose levels stabilized in this 90 to 120mg/dL range were achieved using insulin doses that typically varied between 0.65 to 1.4 units/hour.

[0175] The following morning, these patients were administered glucagon subcutaneously via a continuous infusion pump, ranging from 0.8 to 4.0ng/kg/min (specific doses were 0.8, 1.6, 2.4, 3.2 and 4.0ng/kg/min) and their usual insulin doses. Each dose level within this range was infused for 2 to 3 hours. At this lower dose range of 0.8-4.0ng/kg/min. glucagon, there was no apparent consistent trend in patients' glucagon or glucose levels.

B. Glucagon from 8 to 16ng/kg/min

[0176] Following a night titration period in which blood glucose levels were maintained at 90 to 120mg/dl, 6 patients were given glucagon in the range of 8 to 16ng/kg/min. Glucagon is administered subcutaneously by a continuous infusion pump. The specific doses administered were 8, 12 and 16 ng/kg/min; each dose level was infused for 3 hours. Patients were maintained at their normal basal insulin level by continuous subcutaneous pump administration prior to glucagon administration.

[0177] A glucagon dose of 8ng/kg/min did cause a significant rise in glucose and glucagon, approximately doubling blood glucagon levels, and resulted in an increase in blood glucose levels of approximately 40%. Thus, a dose of 8ng/kg/min can raise the blood glucose levels in these patients. Moreover, even those lower ranges of detection, while insufficient for increasing blood glucose levels under these test conditions, are sufficient in preventing hypoglycemia induced by the administration of insulin in some patients.

[0178] Among 3 patients, subcutaneous infusion of 12ng/kg/min of glucagon for 9 hours resulted in an increase in plasma glucose levels, which were maintained for 6 to 8 hours, ranging from 100 to 200pg/ml (see fig. 8). The mean peak plasma level at dose was 185 pg/ml. The blood glucose level of the patient was also greatly increased (see fig. 9). This clearly demonstrates that sustained, very low glucagon doses can be administered to a patient, effective over an extended period of time, with the desired result of controllably elevating blood glucose levels.

[0179]2 patients were treated subcutaneously with glucagon at 16ng/kg/min for 9 hours. In both subjects, a 16ng/kg/min. dose was administered to achieve higher peak plasma glucagon levels than the levels obtained at the 12ng/kg/min. dose (see figure 8). In both subjects, peak levels were obtained during the 3 hours of initiation of infusion, which subsequently tended to decrease to levels seen with the 12ng/kg/min. At the 16ng/kg/min dose level, the mean peak plasma level was about 254 pg/mL. While glucagon levels decreased during the post-infusion fraction, glucose levels remained elevated throughout the infusion (see fig. 8-11). The glucose levels obtained at the 16ng/kg/min. dose were generally higher than those obtained at the 12ng/kg/min. lower dose (see fig. 9).

[0180] The subjects were generally maintained at their basal insulin rates throughout the fasting evaluation period. The insulin administered varies from patient to patient and over time, from 0.5 to 1.8 units of insulin per hour. Glucose levels began to rise as early as 30 minutes after the start of glucagon infusion. On average, this elevation is maintained throughout the glucagon infusion period.

[0181] These results indicate that very low doses of continuously administered glucagon can be used to increase blood glucose levels in patients receiving insulin treatment without experiencing hypoglycemia.

[0182] These data demonstrate that administration of glucagon at doses ranging from 8 to 16ng/kg/min results in an increase in plasma glucagon levels ranging from about 100 to 200+ pg/mL, and that these levels can be maintained over a significant period of time. The normal reference range for glucagon is 50 to 150pg/ml. Those skilled in the art will appreciate that these results indicate that low, sustained doses of glucagon can elevate blood glucose levels in diabetic patients, thus providing a means of preventing hypoglycemia in those patients. These results indicate the amount of glucagon required to induce increased blood glucose levels. Those skilled in the art will appreciate that other patients may be prevented from hypoglycemia with lower glucagon doses, but the 8ng/kg/min and higher doses exemplified herein are effective in preventing hypoglycemia. The actual minimum amount may vary from patient to patient, and the minimum amount for a particular patient may be readily determined by one skilled in the art given this disclosure.

[0183] Interestingly, in this example, VLD glucagon is used in amounts of 12 to 16ng/kg/min at levels similar to those seen in non-diabetic patients who responded to the symptoms of hypoglycemia induced by the test.

[0184] The following examples illustrate how these doses of glucagon prevent hypoglycemia, even when there is an insulin challenge (insulin challenge), i.e., there is a dose of insulin that would otherwise induce hypoglycemia in the patient.

Example 8

Prevention of insulin-induced hypoglycemia with glucagon

[0185] This example illustrates that insulin-induced hypoglycemia (for this example, blood glucose below 50 mg/dl) can be prevented using low doses of continuously administered glucagon. This study set up 3 observations (visit). The first observation involves increasing the amount of insulin administered to the subject without supplying any glucagon; monitoring a subject's blood glucose level. This first study identified the insulin challenge required to induce hypoglycemia in these patients. In the next two observations, the patient was given two different glucagon doses and the patient's blood glucose level was monitored to determine if glucagon prevented or delayed any hypoglycemia that would otherwise have been induced by insulin challenge. Thus, by comparing the glucose level determined in the first observation with the glucose levels in the latter two observations, it can be determined whether glucagon prevents hypoglycemia.

[0186] In a first observation, the basal insulin rate of each subject was titrated upward (increasing by 50% every 90 minutes (e.g., from a basal rate of 1.0 unit/hour to 1.5 units/hour, 90 minutes, to 2.0, 90 minutes, to 2.5)) to induce hypoglycemia (blood glucose below 50 gm/dl). The results for each patient are shown in fig. 12, with observation 1 represented by a square. The lower set of lines depicts the increase or decrease in insulin administered to the subject. Insulin administered returns to the basal infusion rate (and glucose administered) after 12: 30; thus, a peak after 12:30 is awaited. Blood glucose levels were allowed to vary between 50mg/dL and 350 mg/dL. Glucose levels below 50mg/dL are considered to be indicative of hypoglycemia.

[0187] During observations 2 and 3, subjects received multiple doses of continuous infusion of glucagon (beginning at 7:00 am and ending at about 4:00 pm) to determine whether the dose would prevent insulin-induced hypoglycemia or delay the time of the onset of hypoglycemia in the first observation. Since the effect on glucose levels is between about 60 minutes and 90 minutes, glucagon infusion begins 1 hour before the insulin infusion rate is gradually increased.

[0188] Two different doses of glucagon were determined, 8ng/kg/min (triangles) and 16ng/kg/min (circles). The results of these low dose sustained glucagon on blood glucose levels are shown in figure 12. As shown in figure 12, the 8ng/kg/min. dose of glucagon (triangle) maintained higher blood glucose levels in the patient compared to the test without glucagon administration. In addition, the control line (no glucagon, only increased insulin, squares) effectively terminated at 12: 30; after this point in the figure, no insulin is administered, and therefore the comparison should be between 7:00 and 12: 30. Hypoglycemia is delayed by about 2 hours.

[0189] As shown in figure 12, the 16ng/kg/min. dose (circle) maintained blood glucose levels well above control levels, even above 8ng/kg/min. (triangle) glucagon doses for a considerable period of time. In addition, while a dosage level of 8ng/kg/min is able to prevent hypoglycemia and maintain blood glucose levels above 50mg/dL, a 16ng/kg/min dosage maintains blood glucose levels near 100 mg/dL. This indicates the potency and dose dependence of these two ranges.

[0190] The lines in the lower portion of the graph (i.e., the triangle, square and circle labeled lines below the 50mg/dl line in fig. 12) represent steps in the infusion of insulin during the trial. Thus, the lower square-marked line represents insulin levels during observation 1, and the lower circular and triangular-marked lines represent insulin levels during observations 3 and 2, respectively.

[0191] In another patient, the effect of the 8ng/kg/min dose was not as pronounced as shown in fig. 12. Therefore, this patient requires higher doses or infusion rates to prevent hypoglycemia. This is confirmed by the 16ng/kg/min. dose data, which indicates a similar effect to that shown in the first patient in figure 12, i.e. an increased blood glucose level above 200 mg/dl.

[0192] In addition to the glucose levels detected above, the effects of lower levels of glucagon were also detected using a similar protocol. In another patient tested, the above experiment was repeated with a lower dose, 4.0ng/kg/min. glucagon and a slightly lower upper insulin limit (1.5 times the basal rate of insulin). The results are shown in FIG. 13. As can be seen in the figure, while the increase in BRI during the first observation (diamond) resulted in lower blood glucose levels (square), the administration of 4ng/kg/min glucagon in the second observation (asterisk) helped to maintain elevated blood glucose levels ("X") and delayed the decrease in blood glucose levels even as insulin levels increased (circle). In this patient, while increased levels of insulin would otherwise induce hypoglycemia, the presence of 4.0ng/kg/min glucagon is sufficient to prevent the occurrence of hypoglycemia.

[0193] In addition to the above-identified benefits of preventing hypoglycemia from administration of low doses of glucagon, there appears to be a prolonged benefit of this dosage schedule for subcutaneously administered glucagon. Even after glucagon ceased administration, blood glucose levels remained elevated (see the part after 4:00 pm in fig. 12). Thus, sustained administration of glucagon is not necessary to maintain elevated blood glucose levels for extended periods of time.

[0183] The appropriate amount of glucagon to be administered to a particular patient can be determined using the methodology above. For example, testing patients taking larger amounts of insulin, e.g., 2-10, 10-20, 20-30 or more units per hour, in this manner can be used to determine an appropriate dosing regimen for glucagon to prevent hypoglycemia. In the above examples, the amount of insulin is increased by a factor of 1.5 to about 2.5. In one embodiment, when the amount of insulin is increased, the amount of glucagon is proportionally increased, and it is then determined (by the methodology above) whether the new glucagon dosage is correct for preventing hypoglycemia. In this way, an appropriate glucagon dosage can be determined for a particular patient. Those skilled in the art will recognize that this relationship need not be 100% proportional, and will recognize that some degree of routine experimentation may be useful in optimizing the final values. In addition, the above embodiments can be used to determine a dosage schedule to understand which frequency of glucagon actually needs to be administered. In some embodiments, only a percentage of 99-90, 90-80, 80-60, 60-40, 40-20, 20-10, 10-1, or less, percent of time is administered, rather than being administered continuously.

[0194] As will be understood by those skilled in the art, using this methodology, variations in the method of administration (e.g., patch, inhalation, topical cream, intravenous, etc.) can also be measured. Furthermore, as will be understood by those skilled in the art, the methodology may be modified to determine the appropriate time to administer glucagon to the patient. For example, glucagon may be administered 1 hour before insulin levels are adjusted, and may be increased to 90 minutes or more or decreased to 45 minutes or less, e.g., to determine whether to maintain better control of blood glucose levels. As will be appreciated by those skilled in the art, the timing and method of administration can affect dosage requirements. Furthermore, the importance of various additives can also be determined in the methodology described above.

Example 9

Determining the amount of VLD glucagon to be used for preventing hypoglycemia

[0195] This example describes a clinical trial demonstrating that glucagon administered according to the methods of the invention would function as expected in maintaining a desired blood glucose level. The present method also provides means for determining the optimal ratio of insulin to glucagon to be administered to a particular patient in order to prevent insulin-induced hypoglycemia.

[0196] Subjects reported at night and ate a standard meal at about 17:00 to 18:00 hours. They are instructed to take their usual dose of insulin in bolus form by a pump based on carbohydrate dosing. At 20:00 hours, another pump (pump 2) was activated on the opposite side of the abdomen, infusing saline at the same rate as insulin infusion. From about 22:00 hours, subjects fasted and given their usual basal dose of insulin via CSII. Plasma glucose was checked with YSI every hour (or more frequently if needed) throughout the night. Plasma glucose is maintained between about 100 to 125mg/dl by adjusting the basal insulin rate, if necessary, using IV 5% glucose (if plasma glucose is reduced below 90 mg/dl) and IV insulin (if their plasma glucose is raised above 160 mg/dl).

[0197] From hours 07:00 to 08:00, baseline blood samples were drawn and plasma glucose, free fatty acids, ketone bodies, glucagon, and insulin levels were measured. At 08:00 hours, subjects received two normal basal doses of Insulin via an Insulin Pump (Insulin Pump) to induce controlled hypoglycemia. They received VLD glucagon on the opposite side of the abdomen via an Animas Pump (Animas Pump) 2. The glucagon doses administered were treated individually according to previous studies. In each individual subject, the dose selected was the highest dose that did not cause hyperglycemia (> 180 mg/dl).

[0198] Blood was drawn every 5 to 10 minutes from 08:00 to 12:00 hours and YSI plasma glucose was determined. Free fatty acid, ketone bodies, glucagon and insulin levels were also determined every 15-30 minutes from 08:00 to 10:22 hours. Although much higher doses than the basal insulin dose were received by CSII (continuous subcutaneous insulin infusion), the subject's plasma glucose level did not drop to a low blood glucose level (< 60mg/dl) due to the simultaneous continuous subcutaneous administration of glucagon, which negates the glucose lowering effect of the high basal insulin dose. If the subject's plasma glucose is found to begin to decrease and consistently decrease below 90mg/dl in 2 consecutive cases during the study, the sustained glucagon infusion dose is titrated upward 25% to counter the decrease in plasma glucose. On the other hand, if the subject's plasma glucose begins to increase and rises by more than 25% over 1 hour, the glucagon infusion dose is titrated down 25% to counteract the increase in blood glucose. Thus, the present trial demonstrates that a combination of small amounts of glucagon and insulin can prevent insulin-induced hypoglycemia. Both insulin and glucagon can be administered subcutaneously.

[0199] Various forms of glucagon formulations and methods of administration may also be detected by the above methodology. Thus, the optimal timing and delivery pattern of glucagon or glucagon mimetics or variants, or formulations thereof, can be determined. These methods may also be applied to detect the suitability of other hypoglycemic and hyperglycemic substances for the methods and compositions disclosed herein.

Example 10

Prevention of nocturnal hypoglycemia

[0200] This example illustrates a method for demonstrating that compositions, e.g., compositions of glucagon, glucagon variants, or formulations thereof, are effective in preventing dysesthesia-type insulin-induced nocturnal hypoglycemia in humans. In addition, it provides a method of detecting the effect of each glucagon or variant or preparation in preventing nocturnal hypoglycemia.

[0201] The subject received two regular doses of insulin via the first pump 2 hours (-18: 00 hours) after the standard meal to induce the onset of nocturnal hypoglycemia. At 22:00 hours, they received glucagon infusion via a second pump (SCI). Blood glucose levels were then monitored throughout the night. A sufficient dose of glucagon prevents the blood glucose level from dropping to a low blood glucose level. Both glucagon and insulin may be administered subcutaneously.

Example 11

Administration of low doses of glucagon to prevent loss of hypoglycemic consciousness

[0202] To prevent loss of hypoglycemic consciousness in a subject taking insulin, glucagon is administered subcutaneously from above 5 to about 16ng/kg/min to the subject to prevent blood glucose levels from entering an unfavorably low blood glucose level (e.g., below 50 mg/dL). To determine that the dose administered is sufficient, the blood glucose level may be measured at various time points during the test period in order to ensure that the subject's blood glucose level does not fall below a certain point (e.g., below 50 mg/dL). Such tests can also be used to optimize the glucagon dosage administered to a patient. The administration of glucagon is chronic, i.e. by prophylactic administration of low doses of glucagon, the patient will avoid hypoglycemia and thus not develop hypoglycemia unconsciousness due to repeated hypoglycemic events in prolonged insulin use.

Example 12

Administration of low doses of glucagon to restore consciousness of hypoglycemia

[0203] A patient suffering from a loss of consciousness to hypoglycemia can be identified by screening to determine if the patient's blood glucose level drops below 70mg/dl blood glucose or not. Once determined, an appropriate dose of glucagon, in one embodiment 8-16ng/kg/min, is then administered subcutaneously to the subject to prevent the blood glucose level from dropping below a certain level (e.g., below 50 mg/dL).

[0204] The blood glucose level may be taken at various points in time, and it is determined that the subject's blood glucose level has not decreased to the hypoglycemic point. These assays can also be used to optimize the amount of glucagon administered to a patient. By administering glucagon, the patient experiences fewer hypoglycemic events and his or her hypoglycemic awareness improves. The subject's awareness of hypoglycemia can be determined, as described above, by determining whether the subject is identifiable when his or her blood glucose level is below a certain point (e.g., 70 mg/dL).

[0205] Multiple doses, multiple dosage forms, and multiple methods of administration (administration) can also be assayed using the above methodology to determine whether the optimal dose, or formulation or method of administration, is optimal for preventing hypoglycemia.

Example 13

Transdermal administration of glucagon, in combination with insulin, for the control of diabetes and prevention of hypoglycemia [ including patches and topical creams ]

[0206] The use of transdermal patches to deliver therapeutic agents is becoming increasingly common. The patch provides a non-invasive and simple method of releasing some drugs into the bloodstream. Nicotine and hormone replacement therapy are perhaps the best known uses in the art. One of the characteristics of drug delivery from transdermal patches is that the delivery rate is typically constant and lasts for an extended period of time (as long as the patch is worn). This feature has proven beneficial in terms of pain control (FENTANYL) and nicotine replacement therapy where a flat profile over time is the ideal profile. This feature makes transdermal patches suitable for basal replacement of insulin or glucagon. See PCT patent publication No. WO0243566, which is incorporated herein by reference.

[0207] Quick-acting patches are also known. In U.S. patent 5,707,641, which is incorporated herein by reference, it is reported that proteins, particularly insulin, are delivered transdermally into the bloodstream for a period of well below 1 hour. The ability to deliver other proteins in the same manner and using similar formulations is also described. Glucagon can therefore also be administered in this manner.

[0208] Currently, Helix Biopharma from canada and IDEA in germany are developing insulin patches where phase II trials are in progress. The IDEA technique (transfermers ®) involves the transport of macromolecules such as peptides across the skin barrier. Figure 6 illustrates the effect of molecular weight and lipophilicity on transdermal transport rate at permeation (upper and lower grey curves represent more or less lipophilic substances, respectively) or at TRANSFEROME ® -mediated penetration (black lines and black dots). The grey circles represent commercial drugs in transdermal patches. Regardless of the technique employed, the ability to transport peptides efficiently transdermally has been demonstrated and is significant. In particular, both insulin and glucagon can be delivered transdermally, thereby providing some embodiments of the invention practiced using transdermal patches and similar devices.

[0209] A variety of possible patch configurations and matrices may be employed, the specific type being selected according to the specific manner of intended use. For example, two basal forms of insulin matrix may be used, one for basal insulin replacement and the other for prandial insulin replacement. Glucagon can be formulated to provide a postprandial glucagon or basal glucagon replacement that protects against hypoglycemia. In one embodiment, the invention may be practiced using a patch matrix containing a combination of these basic forms, either coexisting in the same matrix or separately in a sub-matrix.

[0210] Thus, in the practice of some embodiments, the following patch matrices are useful.

● matrix containing insulin for basal insulin replacement;

● matrix containing insulin for prandial insulin replacement;

● matrix containing glucagon for basal glucagon replacement;

● matrix containing glucagon for postprandial protection against hypoglycemia;

● contains a matrix of insulin for prandial insulin replacement and glucagon for postprandial protection against hypoglycemia;

● contains a matrix of insulin and glucagon for basal replacement of insulin and glucagon; and

● matrix consisting of 2 or more sub-matrices, each of which is one of the above matrices.

[0211] A topical cream may be an alternative to a patch.

[0212] For prandial insulin base, short acting insulin such as LISPRO (HUMALOG), ASPART (NOVOLOG) or GLULISINE (APIDRA) may be used. Depending on the speed of onset, prandial insulin matrices are typically applied at the time of a meal, or some time prior to a meal. A prandial insulin patch that minimizes onset time may be applied, so the patch is applied near mealtime. The time of onset depends on the insulin concentration and the nature of the formulation. For example, a simple insulin moist matrix has a slower onset of action than an insulin patch formulated according to U.S. Pat. No.5,707,641. Monomeric insulin acts faster and is more easily absorbed than larger clustered insulin molecules, because the size of the molecule affects the bioavailability obtained from transdermal patches.

[0213] Many different methods of regulating prandial insulin delivery may be employed. For example, patches of different concentrations may be applied over a fixed period of time, and the concentration selected by the patient depends on the amount of carbohydrate it consumes. The time of application may be fixed. The patch does not have to be exhausted when removed, i.e. it can deliver a fixed concentration during its use. In another example, a single concentration of a meal patch may be applied as follows. The time for wearing the patch varies depending on the amount of carbohydrate consumed. The patch need not be exhausted when removed, i.e., it can deliver a fixed concentration during use.

[0214] In another example, a meal patch containing a fixed dose of insulin may be used. An advantage of a self-depleting patch is that the patch itself is not removed with the risk of hypoglycemia. Such patches will be substantially exhausted upon removal and the infusion rate will be pre-loaded. In one embodiment, the prandial insulin patch is applied with a variable insulin concentration (compatible with the amount of carbohydrate consumed), a very fast onset of action (preferably immediate onset, but not more than one hour), is removed or inactivated (preferably from between 3 to 5 hours) after a fixed period of time, and is activated at the time of meal (or not more than one hour before meal). Depending on the speed of onset associated with the patch, the prandial glucagon patch is applied at the meal or some predetermined time after the meal. The speed of onset is determined by the concentration of glucagon used and the nature of the formulation used. For example, a simple glucagon wet matrix has a slower onset than a glucagon patch formulated according to the techniques described in U.S. patent 5,707,641.

[0215] In one embodiment, the glucagon patch is configured to provide peak output of glucagon at a time between 2 and 5 hours post-application. The patch is applied to the meal time. The amount of glucagon in the patch will be an amount sufficient to deliver to the patient an amount of subcutaneously administered glucagon equal to 5-30ng/kg/min.

[0216] Many different patch configurations may be employed. These structures include:

● A patch contains a single matrix or a set of sub-matrices in a single compartment;

● A patch containing 2 or more than 2 separate compartments, each compartment containing its own matrix or set of sub-matrices, the patch being activated or deactivated as a single unit; and

● A patch containing 2 or more than 2 separate compartments, each compartment containing its own matrix or set of sub-matrices, wherein each compartment is independently activated or deactivated.

[0217] Other patch configurations may be employed and practice of the invention is not limited to the configurations described above.

A. Transdermal administration of insulin including patches and topical creams]

(i) Transdermal administration of insulin including patches and topical creams]And insulin administration by subcutaneous injection

[0218] In this example, only prandial insulin and prandial glucagon are administered via a transdermal patch. This can be achieved in a number of ways, including: (i) use of a single matrix of mixed glucagon and insulin; (ii) using a single spacer with 2 sub-matrices, wherein one spacer contains insulin and the other spacer contains glucagon; (iii) applying a single patch containing 2 compartments, one compartment containing insulin and the other compartment containing glucagon, both compartments being simultaneously activated and deactivated; and (iv) the use of two separate patches (or two compartments in a single patch), one containing insulin and the other containing glucagon, each patch or compartment being independently activated or deactivated. Basal insulin is delivered parenterally by subcutaneous injection of a long acting insulin such as GLARGINE or ULTRALENTE as described in example 1. a.1. In one embodiment, method (i) is applied. If different matrices are used to achieve the desired pharmacokinetics, method (ii) or method (iii) may be used. Method (iv) can be applied if the insulin onset time and the glucagon onset time do not match.

[0219] In this illustrative example, the method (ii) described above is applied. The user activates the prandial patch at mealtime (or preferably, within one hour prior to mealtime), thereby activating both sub-matrices simultaneously. If a fixed concentration patch is used, the user removes the patch after a period of time, where the period of time is proportional to the amount of carbohydrate ingested. If a variable concentration patch is applied, the user removes the patch after a fixed time, typically between 1 and 3 hours after a meal. In one embodiment, a fixed concentration is applied. In such embodiments, the amount of glucagon administered may increase with the amount of insulin administered.

(ii) Transdermal administration of insulin including patches and topical creams]

[0220] In this example, both insulin and glucagon are administered transdermally. Two different types of patches (or independently activated compartments) may be applied. A patch (or compartment) contains a matrix designed to replace basal insulin for 24 hours. A single patch (or compartment) may be used containing both prandial insulin and glucagon in separate subunits with an onset time compatible with the application of the prandial patch (or activation of the prandial compartment) at (or near) mealtime.

[0221] In one embodiment, a single device is used containing 4 independently activatable intervals, one containing basal insulin which is activated and remains activated for 24 hours at the time of administration, and the other 3 intervals containing prandial insulin and glucagon, both present in separate subunits in the same interval, each interval being activated separately at mealtime and inactivated at some time after meal, the activation time being proportional to the amount of carbohydrate consumed.

[0222] At the beginning of a meal (or some time up to an hour before a meal), the patient activates one of these meal intervals [ e.g., by pulling out a sealed plastic seal between the patch and the skin ], which initiates transdermal delivery of insulin and glucagon. At some time thereafter, which is proportional to the amount of carbohydrate ingested, the mealtime interval is inactivated [ e.g., by replacing the barrier for the activation interval or removing the interval entirely from the patch end ]. The insulin preparation in the insulin sub-matrix is a short acting insulin and the patch is designed for rapid onset of action. The glucagon formulation in the glucagon subgasket is designed to reach an effective concentration in the bloodstream 1 to 3 hours after interval activation, thereby providing protection against hypoglycemia in the appropriate part of the circulation, as described in example 13. A.i.

[0223] In an alternative embodiment, a single device that allows for more than 3 meals per day can be easily designed to allow more than 3 meal intervals. As noted above, in an alternative embodiment, the meal time medication may be contained in a completely separate (and independently activated) meal time patch. In an alternative embodiment, the prandial patch may consist of separate insulin and glucagon compartments, such that each compartment may be independently activated and deactivated.

[0224] A single device containing separate and independently controlled insulin and glucagon compartments may contain 7 separate compartments, one for basal insulin, 3 for prandial insulin, and 3 for prandial glucagon. The base patch was designed to replace the base insulin (24 hours before being replaced). The insulin used may be any insulin suitable for transdermal delivery. The basal insulin interval may also optionally contain an amount of glucagon (mixed with insulin or in a submatrix) sufficient to provide basal glucagon during each 24 hour period. This will have the beneficial effect of providing protection against hypoglycemia throughout the day, particularly during sleep.

[0225] The amount of glucagon in the patch can be an amount sufficient to deliver to the patient glucagon equivalent to 5-30ng/kg/min. or more, administered subcutaneously, e.g., 6-20ng/kg/min. or more, and for another example 8-16ng/kg/min. or more. When more than 0-3 units of insulin are to be administered, a greater amount of glucagon may be present. For example, when 3-20 units of insulin are to be administered in 1 hour, a greater amount of glucagon may be present. Optionally, the same amount of glucagon is present regardless of the amount of insulin.

B. By passingAdministration of insulin by inhalation [ including pulmonary, buccal, nasal and sublingual]

[0226] In this example, insulin is delivered by inhalation, as described above. Inhalation may be a delivery mode that delivers only prandial insulin (basal insulin delivered parenterally), or all insulin applied by inhalation delivery. The patient administers an amount of insulin (in one or more activations) appropriate to his diet by inhalation. The patient may optionally increase insulin appropriately after a meal.

[0227] Glucagon was administered via a patch as described in example 13. A.i. The patch (or a set of glucagon compartments in a single patch) is adhered to the skin and the patch or (sub-compartments) is activated at mealtimes. The patch is designed to have a slow onset of action so that glucagon is only present in the body in effective amounts after 2 hours. The patch was worn for 4 hours prior to removal, with glucagon remaining in the body sufficient to provide protection against hypoglycemia during the desired 2-5 hours.

[0228] In one embodiment, the glucagon in the patch is a long-acting glucagon (e.g., an iodinated glucagon), as described above. Thus, in some embodiments, the patch may be worn for a shorter period of time while still ensuring that the protection provided by the modified glucagon is provided over a period of 2-5 hours.

[0229] In another embodiment, the user may administer glucagon using a transdermal cream that functions in a manner similar to a transdermal patch (an amount equivalent to at least about 8-16ng/kg/min. glucagon administered subcutaneously may be administered via a cream). Such a cream formulation may be different from the one used in the patch, but both perform essentially the same function. When administering glucagon using this method, it is advantageous to encapsulate the glucagon in liposomes or transfenomes ® to prevent the glucagon supply from drying on the skin and reducing bioavailability.

C. Parenteral administration of insulin

[0230] According to example 1.A.i, the insulin requirements of a patient are met by parenteral administration. Glucagon was administered via a patch as described in example 13. A.i. The patch (or a set of glucagon compartments in a single patch) is adhered to the skin and the patch or (sub-compartments) is activated at mealtimes. The patch is designed to have a slow onset of action so that glucagon is only present in the body in effective amounts after 2 hours. The patch was worn for 4 hours prior to removal, with glucagon remaining in the body sufficient to provide protection against hypoglycemia during the required 2-5 hours.

[0231] In one embodiment, the glucagon in the patch is a long-acting glucagon (e.g., iodinated glucagon), as described herein. The patch can thus be worn for a shorter period of time while still ensuring that the protection provided by the modified glucagon is provided over the required 2-5 hour period.

[0232] In an alternative embodiment, the user may administer glucagon using a transdermal cream that functions in a manner similar to a transdermal patch. Such a cream formulation may be different from the one used in the patch, but both perform essentially the same function. When administering glucagon using this method, it is advantageous to encapsulate the glucagon in liposomes or transfenomes ® to prevent the glucagon supply from drying on the skin and reducing bioavailability.

D. Insulin administration by pump

[0233] In this example, the patient's insulin requirements were administered by a pump as described in example 2. Glucagon was administered via a patch as described in example 13. A.i. The patch (or a set of glucagon compartments in a single patch) is adhered to the skin and the patch or (sub-compartments) is activated at meal time. The patch is designed to have a slow onset of action so that glucagon is present in the body in effective amounts only after 2 hours. The patch was worn for 4 hours prior to removal, with glucagon remaining in the body sufficient to provide protection against hypoglycemia during the required 2-5 hours.

[0234] In one embodiment, the glucagon in the patch is a long-acting glucagon (e.g., an iodinated glucagon). The patch can thus be worn for a shorter period of time while still ensuring that the protection conferred by the modified glucagon is provided over the required 2-5 hour period.

[0235] In an alternative embodiment, the user may apply the glucagon via a transdermal cream that acts in a manner similar to a transdermal patch. Such a cream formulation may be different from the one used in the patch, but both perform essentially the same function. When administering glucagon using this method, it is advantageous to encapsulate the glucagon in liposomes or transfersomes (transporters ®) to prevent the glucagon from drying on the skin and reducing bioavailability.

Example 14

Inhaled glucagon, co-administered with insulin for the control of diabetes and prevention of hypoglycemia [ including pulmonary, buccal, nasal and sublingual ]

[0236] Many dry powder inhalation technologies are currently under development, including: aradigm's AERx ®, Extera ® by inlale Therapeutics, AIR by Alkermes and Eli Lilly, InsulinTechnipheres (Mannkind/PDC), and Aerogen's and Disetronic's aerobes. In U.S. Pat. nos. 5,997,848; 6,131,567, respectively; 6,024,090, respectively; 5,970,973; 5,672,581, respectively; 5,660,166, respectively; 5,404,871; and 5,450,336, which describe methods and devices for delivering insulin into the alveoli where it is absorbed into the blood stream. The main difficulties that must be overcome to allow aerosol macromolecule delivery are: low system efficiency (bioavailability); low dose per inhalation (c.f. asthma); and poor dose reproducibility.

[0237] One relevant factor is efficiency (bioavailability). Bioavailability is primarily dependent on the size of the aerosol particles (most existing systems deliver only 10% -20% of the administered drug to the alveoli), and not on the nature of the administered drug. When the administered drug actually reaches the alveoli, its bioavailability is very high almost regardless of the drug studied. Since the technical problems (and protocols) associated with delivering insulin are similar to those associated with delivering glucagon, protocols capable of delivering insulin can be directly applied to macromolecules of similar size, such as glucagon. One embodiment provides a dry powder formulation prepared by mixing insulin and glucagon. The use of insulin-delivering inhalers is primarily aimed at providing rapid insulin delivery for prandial purposes. If desired, long-acting insulin may be delivered by inhalation.

[0238] Some embodiments may be practiced by applying the inhaler in a number of ways, including with insulin and glucagon in separate inhalers; with insulin and glucagon mixed in a fixed ratio in the inhaler; using a dual chamber inhaler, wherein insulin and glucagon are administered separately; a dual chamber inhaler is utilized in which insulin and glucagon are administered simultaneously. Since meal inhalers usually contain fast acting insulin, they are not suitable for delivering basal insulin (in the manner used by insulin pumps). If the prandial insulin and basal insulin are to be delivered by inhalation, separate pumps or chambers may be provided.

A. Administration of insulin via inhalation [ including pulmonary, buccal, nasal and sublingual]

[0239] At bedtime, hypothetical patients administered basal insulin by subcutaneous injection using ULTRALENTE in an amount of 20 units of dose level. Alternatively he may choose to administer the same drug by inhalation (at a dose that will provide 20 units of daily bioavailability). It may also be beneficial or desirable to administer the base dose via the inhaler at several times of the day, such as at meals other than bedtime. Since there is a slight delay (approximately 20 minutes) before insulin reaches a significant serum concentration when compared to subcutaneous delivery, the user will administer their meal-time insulin dosage about 20 minutes before the meal. He does this by dosing 25-50 units of insulin (assuming a bioavailability of about 20%) with a metered dose inhaler.

[0240] The inhaler may be variable dose (see U.S. Pat. Nos. 5,970,973; 5,672,581; 5,660,166; 5,404,871; and 5,450,336) or similar to currently used asthma devices that deliver a fixed and pre-set dose each time they are activated. Regardless of the form used, it is desirable to initiate the administration of insulin multiple times. By doing so, the patient can adjust his inhaled mass by "topping up" his dose at some time after the start of a meal, depending on the amount of carbohydrates he actually consumes rather than the amount he wishes to have a meal. Moreover, the more actuations used to administer insulin, the better the corresponding dose reliability (repeatability) since inhalation administration tends to vary from one actuation to another and there is an averaging or smoothing effect over multiple actuations of delivery.

[0241] To prevent the hypoglycemia associated with the use of inhaled insulin from occurring between 2 and 5 hours after a meal, a glucagon inhaler was used to administer a subcutaneous equivalent dose of 5 to 16ng/kg/min. In one embodiment, a different inhaler for each type of insulin and glucagon is employed. In one embodiment, a single inhaler with at least 2 drug chambers (for prandial insulin, glucagon and/or optionally basal insulin) and capable of independent activation is used.

B. Parenteral administration of insulin

[0242] According to example 1.A.i, the patient administers his basal insulin and prandial insulin parenterally. Since the risk of hypoglycemia associated with the use of LISPRO insulin typically occurs between 2 and 5 hours after a meal, a glucagon inhaler is used to administer a subcutaneous equivalent dose of 6 to 16ng/kg/min. between 2 and 5 hours after a meal (i.e., to obtain the same amount by inhalation as the effect of 6 to 16ng/kg/min. glucagon on blood glucose administered subcutaneously). Alternatively, in a glucagon inhaler, a modified long-acting glucagon (e.g., iodinated glucagon) with delayed onset is used and administered with prandial insulin at mealtime.

C. Insulin administration by pump

[0243] According to example 2.a., basal insulin and prandial insulin are delivered by a pump. The risk of hypoglycemia occurs after 2 to 3 hours, therefore, patients administer glucagon via an inhaler 2 hours after a meal. At 2 hours, 3 hours, 4 hours, he administered one spray (puff) from a metered dose inhaler, providing protection during the period of sensitization. The dose at each priming corresponds to a subcutaneous equivalent dose of glucagon from 5 to 16ng/kg/min. Alternatively, in a glucagon inhaler, a modified long-acting glucagon (e.g., an iodinated glucagon) with delayed onset is used and administered with prandial insulin at mealtime.

D. Transdermal administration of insulin including patches and topical creams]

[0244] According to example 3.a.ii, the patient administers insulin (basal insulin and prandial insulin) by transdermal patch or via topical cream. The risk of hypoglycemia occurs after 2-3 hours, therefore, patients administer glucagon via an inhaler 2 hours after a meal. At 2, 3 and 4 hours, he administered one spray from the metered dose inhaler, providing protection during the period of susceptibility. The dose at each priming corresponds to a subcutaneous equivalent dose of glucagon from 5 to 16ng/kg/min. Alternatively, in a glucagon inhaler, a modified long-acting glucagon (e.g., iodinated glucagon) with delayed onset is used and administered with prandial insulin at mealtime.

Example 5

Glucagon and insulin are mixed and co-administered parenterally for the control of diabetes and prevention of hypoglycemia

[0245] In example 1, insulin and glucagon were administered separately parenterally. In one embodiment, the two agents are administered simultaneously in admixture. Insulin and glucagon may be less mixed if there is any interaction between the two or degradation of either product. In non-diabetic patients, it is typically found that as postprandial insulin output of carbohydrates increases, the output of glucagon correspondingly increases (in effect, a return in output following an initial decrease in glucagon output due to an initial gut-induced increase in blood glucose following carbohydrate digestion). This pattern of insulin production, and consequently glucagon production, exhibits a relatively fixed relationship.

[0246] To ensure that the glucagon provides a protective effect over a desired period, the amount of the glucagon component in the mixture may be increased so that it appears at the desired concentration when needed (to prevent hypoglycemia between 2-5 hours post-prandial, at a subcutaneous equivalent dose of 5 to 30ng/kg/min. or more, preferably 8-16ng/kg/min. or more), or a delayed onset glucagon formulation may be used. In one embodiment, the glucagon formulation, is both delayed and sustained (e.g., delayed for 2 to 3 hours and released within about 3 hours). For example, any of the formulations described herein may be used.

[0247] In this example, iodination methods that increase half-life were used (as described in U.S. Pat. No. 3,897,551; see Table I3G). LISPRO insulin and I3Glucagon (I3 Glucagon) were mixed so that the modified Glucagon was present in the mixture at 1.5% by weight of insulin (in our LISPRO formulation, the concentration of insulin per ml was kept constant). Since modified glucagon has a longer lasting effect, a smaller ratio of glucagon to insulin weight will be required.

[0248] The hypothetical patient then administers 5 to 10 units (measured in terms of the insulin contained therein) of the insulin-glucagon formulation at mealtimes using standard methods. In doing so, he administers a subcutaneous equivalent dose of modified glucagon of 5 or more to 16ng/kg/min. This provides the same protective effect as described in example 1.a. in view of the longer acting time of the modified glucagon (assuming that the modified glucagon has e.g. a 2-fold effect on the glucose level compared to the standard glucagon). The glucagon so administered will be continuously effective for between 2 and 5 hours, as needed.

Example 16

Co-transdermal administration of mixed glucagon and insulin for the control of diabetes and prevention of hypoglycemia [ including patches and topical creams ]

[0249] In this example, both insulin and glucagon are administered transdermally. Prandial insulin and glucagon are mixed in the same matrix or cream. Two different types of patches (or independently activated compartments) may be applied. One patch (or interval) will contain a matrix designed to replace basal insulin over a 24 hour period. The patch may contain an amount of glucagon such that a subcutaneous equivalent dose of glucagon of more than 5 to 20ng/kg/min is delivered to the patient. Another patch (or independently controlled space) provides prandial glucagon and insulin in a separate matrix. The onset of action of glucagon and insulin are matched so that when the patch is activated, insulin reaches an effective plasma level very quickly, while glucagon reaches an effective level only after 2-3 hours. The patch is applied at meal time, and preferably not more than 1 hour before meal time.

[0250] In one embodiment, a single device is used containing 4 independently activatable intervals, 1 interval containing basal insulin which is activated at the time of administration and is in an activated state for 24 hours, and 3 other intervals containing prandial insulin and glucagon, both in the same matrix, each interval being independently activated at (or near) mealtime and deactivated at some time after meal, the activation time being proportional to the amount of carbohydrate consumed. At the beginning of a meal (or up to 1 hour before the meal), the patient activates one of these meal intervals (e.g., by pulling out a sealed plastic seal between the patch and the skin), which initiates transdermal infusion of the mixed insulin and glucagon. At some time thereafter, which is directly proportional to the amount of carbohydrate ingested, the mealtime interval is inactivated (e.g., by replacing the barrier for the activation interval, or removing the interval entirely from the end of the patch).

[0251] The insulin and glucagon combination in the time interval contains a short acting insulin and the patch is designed for a rapid onset of insulin action. The glucagon component is designed to reach an effective concentration in the bloodstream between 1 and 3 hours after activation of the interval, thus providing protection against hypoglycemia in the appropriate portion of the circulation.

[0252] In an alternative embodiment, a single device that allows for more than 3 meals per day may be used and contain more than 3 meal intervals. The base patch was designed to replace the base insulin (worn for 24 hours before being replaced). The insulin used may be any insulin suitable for transdermal delivery. It may be advantageous to use a medium acting insulin in preference to a short acting insulin so that any change in insulin absorption during the life of the patch will be minimized due to the relatively long life of the insulin involved. The basal insulin interval may also optionally contain an amount of glucagon (mixed) sufficient to provide basal glucagon during each 24 hour period. This will have the beneficial effect of providing protection against hypoglycemia throughout the day, particularly during sleep.

Example 17

Co-inhaled administration of mixed glucagon and insulin for diabetes control and prevention of hypoglycemia [ including pulmonary, buccal, nasal and sublingual delivery ]

[0253] The present embodiments provide pharmaceutical formulations and methods for delivering glucagon mixed with insulin via inhalation. In this example, a long acting glucagon (e.g., iodinated glucagon as described in U.S. patent No. 3,897,551, such as I2G, or zinc protamine glucagon) is mixed with LISPRO insulin and delivered by a typical insulin inhaler (e.g., as disclosed in U.S. patent No.5,970,973). Basal insulin can be delivered by subcutaneous injection, as described in example 1A, or by inhaler using standard methods. Glucagon may optionally be included in this formulation in a sustained release formulation if it is desired to provide a basal glucagon replacement.

[0254] The insulin powder applied is mixed with the modified glucagon so that the modified glucagon content is a subcutaneous equivalent dose of between 5 and above to 20ng/kg/min, more preferably between 8 to 16ng/kg/min, for 1-3 units of insulin applied. When a larger amount of insulin is used, a proportionally larger amount of glucagon may be used (although the amount of glucagon may remain the same regardless of the amount of insulin used). For this particular embodiment, the amounts used can be adjusted as desired based on the results of the foregoing examples. The patient will administer the combined insulin and glucagon at mealtimes to provide systemic insulin equivalent to between 5 and 10 units.

[0255] Although the present invention has been described in detail with reference to particular embodiments, those skilled in the art will recognize that modifications and improvements of the present invention are also within the scope and spirit of the invention, as set forth in the following claims. All publications and patent documents cited herein are incorporated by reference as if each individual publication or patent document were specifically and individually indicated to be incorporated by reference. Citation of publications and patent documents is not intended as an admission that any of the documents is pertinent prior art, nor does it constitute any admission as to the contents or timing thereof. The definitions provided herein govern definitions in the cited references or elsewhere. Having now described the invention by way of written description and examples, those skilled in the art will recognize that the invention can be practiced in a variety of embodiments, and that the foregoing description and examples are for purposes of illustration and not limitation of the following claims.

Claims (26)

1.A pharmaceutical formulation comprising:

insulin in an amount effective to control diabetes; and

glucagon in an amount effective to prevent hypoglycemia in a human or other mammal, wherein the pharmaceutical formulation is formulated for subcutaneous administration, and wherein the ratio of insulin to glucagon is about 1 unit of insulin to over 40 milliunits to 200 milliunits of glucagon.

2. The pharmaceutical composition of claim 1, wherein said glucagon is in an amount of between about 50 and 100 milliunits.

3. The pharmaceutical composition of claim 1, wherein said glucagon is a long-acting form of glucagon.

4. The pharmaceutical composition of claim 3, wherein said long-acting form of glucagon contains iodine.

5. The pharmaceutical composition of claim 3, wherein said long-acting form of glucagon contains zinc.

6. The pharmaceutical composition of claim 5, wherein said long-acting form of glucagon further comprises protamine.

7. A method of treating diabetes mellitus in humans and other mammals without inducing hypoglycemia, the method comprising:

administering insulin in an amount therapeutically effective for the control of diabetes, wherein the amount of insulin is between 0.5 and 20 units of insulin; and

administering glucagon for a time and in an amount therapeutically effective to prevent hypoglycemia, wherein said glucagon is administered subcutaneously, and wherein the amount of glucagon administered is a desired glucagon potency of from greater than 5ng to less than or equal to 100ng per minute per kg of patient.

8. The method of claim 7, wherein said amount of glucagon administered is a desired glucagon potency of between 6ng and 18ng or less per patient kg per minute.

9. The method of claim 7, wherein said glucagon is glucagon with an extended duration of action.

10. The method of claim 7, wherein said glucagon is contained in a liposome formulation.

11. The method of claim 7, wherein said glucagon is contained in a microsphere.

12. The method of claim 7, comprising administering a formulation comprising both insulin and glucagon.

13. The method of claim 7, wherein said insulin and glucagon are contained in a pump that controls administration of the drug to the patient.

14. The method of claim 13, wherein said glucagon is administered simultaneously with insulin.

15. The method of claim 14, wherein the ratio of glucagon to insulin is about 40 or more to 200 milliunits glucagon to 1 unit insulin.

16. The method of claim 15, wherein 2 units of insulin are administered.

17. The method of claim 7, wherein 10 units of insulin are administered and 30ng to 90ng glucagon is administered subcutaneously per kg of patient per minute.

18. A kit for administering glucagon and insulin for prevention of low blood glucose levels, the kit comprising:

glucagon;

insulin, wherein the ratio of glucagon to insulin is 1-20 units of insulin to 32-480 milliunits of glucagon;

a device for subcutaneous administration of glucagon; and

instructions for administering insulin and glucagon such that the glucagon prevents a hypoglycemic event.

19. The kit of claim 18, wherein the concentration of glucagon, when fully dissolved in the glycerol solution, is above 500 micrograms per ml, but below 2000 micrograms per ml.

20. The kit of claim 18, wherein the ratio of glucagon to insulin is 1-3 units of insulin to 32-96 milliunits of glucagon.

21. The kit of claim 18, wherein said means for subcutaneously administering glucagon is a pump configured to deliver about 6 to 20ng/kg/min of glucagon.

22. Use of a combination of glucagon and insulin in the preparation of a medicament for the treatment of diabetes, wherein the glucagon is used in an amount sufficient to prevent the onset of hypoglycemia, and wherein the ratio of glucagon to insulin is greater than 40 micrograms and less than 500 micrograms glucagon to 1-20 units of insulin.

23. The use of claim 22, wherein the amount is sufficient to prevent the onset of hypoglycemic involuntary.

24. The use of one of claims 22 and 23, wherein the amount of insulin is between 1 and 20 units and the amount of glucagon is between 41 and 200 milliunits.

25. The use of one of claims 22 and 23, wherein the ratio of insulin to glucagon is between 1 and 3 units and the amount of glucagon is between 40 and above to less than or equal to about 96 milliunits.

26. The use of one of claims 22-25, wherein said glucagon further comprises protamine.