JPS63502117A - Controlled release liposome delivery system - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Liposomes loaded with 1. Main league to division The present invention provides a liposome-based system and a controlled rapid 1, Abra, R-M-1 et al., Res Commun Chem’Pa th and Pharm.

37:199 (1982).

2, Fukunaga, M. + et al., Endocrinology, 11 5(2) Near 57 (1985).

3. Greenwood+ F, C, et al., Biochem J, 89: 114 (1963).

4, Jackson, A, J, + Drug Metab and Disp os, 9(6):535 (1981).

5, McParlane, A.S., Nature, 182:53 ( 195B).

6, 5zoka, F,, Jr, Proc Nat Acad Sci, USA+ 75:41943, Noriyoshi Sato Liposome delivery systems are used to deliver drugs and peptide hormones.

A variety of pharmaceutically active compounds have been proposed. Compounds administered parenterally For this purpose, liposomes provide controlled release of “encapsulants” over long periods of time. , and reduce toxic potential by limiting plasma peak levels of free compounds in the bloodstream. It has the power to suppress side effects. These combined advantages make it possible to use fewer compounds. can be administered more frequently and at higher dosage levels, and This increases the convenience of treatment.

One route of liposome administration that has been widely proposed for parenteral administration is intravenous administration. (IV) There is an injection. Liposomes administered by this route generally have intrareticular cleared by the skin system (RES), and as a result, liposomes tend to concentrate in organs such as the liver, pancreas, and lungs, which are rich in RES cells. Li The ability to target posomes to RES-rich tissues in a somewhat specific manner is 1, for example, in the liver. , is advantageous for the treatment of diseases of the pancreas or lungs. This method can be used in two ways, for example, Patent application “Liposome/Anthraquinone Drug Compositions and Methods”’ (1985 Patent Application No. 806084, filed on December 6, 2008). This patent issue The application includes an innovation for the treatment of liver tumors with doxorubicin entrapped in liposomes. Discloses improved treatments. but.

Particularly when the compound to be administered is to be applied to a site other than RES-rich tissues. However, when long-term release into the bloodstream is required, IV administration is often not used. Limited.

Intramuscular (IM) or subcutaneous (SQ) administration of compounds entrapped in liposomes also . Proposed. This method ensures that as long as liposomes are present at the injection site, RE Rapid uptake and removal (clearance) by S is unlikely. It has the advantage that it cannot be removed. Liposomes fixed at the injection site are then captured released compounds into the bloodstream over an extended period of time. As an example, British Patent Application No. 2.050.287 describes how to deliver insulin from the SQ injection site. Liposome systems intended for slow release are disclosed. very recently , a system for the release of liposome-encapsulated calcitonin from the site of IN administration. Proposed (Fukunaga).

In IM or SO release systems of the type described above, liposomes from the injection site It would be desirable to be able to control the rate of release of captured compounds. However, note Prior art attempts to understand and control the variables that affect the rate of drug release from the injection site Attempts have had limited success. One of the reasons for the difficulty of these studies at the injection site due to factors that affect the release of entrapped compounds from liposomes. to isolate factors that influence the rate of degradation and/or release of liposomes in This is the problem. For example, if liposome-permeable drugs are studied, The rate of drug release into the bloodstream is largely independent of the stability of the liposomes at the site of injection. It is possible that there is a relationship between

In one study reported to date, lipolysis from the IM injection site in the release of insulin trapped in the somes. Some liposome variables The effects of this were investigated (Jackson). The summary of the results is as follows. Yes: (a) Smaller liposomes (0.15-0.7 microns) are larger more quickly by lymph than by small liposomes (0.3-2.0 microns) and (b) as the total amount of liposomes injected decreases; Absorption of liposomes from the injection site is slower. Both of these results are quadratic Not predicting things. In other words, I? From liposomes administered to one site, liposomes How can we control the rate at which non-mechanical substances (e.g. peptides) are released? I have not predicted whether it will happen. Additionally, for use in human therapy , how large liposomes can be prepared in sterile form. is also not clear.

4. Hikariban■Next currency Therefore, the rate of release of liposome-impermeable compounds from the IM or SQ site can be determined by 1. It is a general aspect of the invention to provide a system and method for controlling in selected ways. purpose.

Another object of the present invention is to package the product in a sterile form suitable for IM or SQ administration to humans. Easy to prepare. The objective is to provide such a system.

Yet another object of the invention is the parenteral delivery of hormones with controlled release rates. used for. By providing stable liposome/peptide hormone compositions, Ru.

The present invention selectively increases the release rate of liposome-impermeable compounds into the bloodstream. Including methods. The compound is present in a liposomal suspension containing the compound in an entrapping form. It is administered by forming a liquid and injecting this suspension into the IM or SQ site. Ru.

The rate of release of a substance from an injection site is determined by the average size of the liposomes injected at that site. 2 selectively by increasing the total injection amount of liposomal lipids and liposomal lipids. capsule The substances that have been converted into

Insulin, growth hormone, calcitonin, interferon, interleukin It can be a peptide such as Kin-2.

The peptide is advantageously released into the bloodstream at a controlled rate over several days.

Also the rate of release of substances encapsulated in liposomes. Altering the lipid composition of liposomes can be controlled by Negatively, such as phosphatidylglycerol (PG) charged phospholipids.

Acts to decrease release rate, cholesterol acts to increase rate do.

In one embodiment, liposomes are prepared by the following method: first, A suspension of small liposomes encapsulating the compound is prepared and the compound is extracted from the suspension. Uncapsulated compounds are removed, the suspension is filter sterilized, and the desired Prepare this suspension aseptically until the average size and concentration of liposomes are Add empty liposomes. Small liposomes are smaller than about 0.3 microns. is suitable.

Empty liposomes are preferably 0.5 microns or larger.

The present invention also includes: Impermeabilization of liposomes into the bloodstream from the IM or SQ injection site and liposomal compositions for administration of the compound. This composition traps substances liposomes containing in the form of a liposome in which the average size of the two particles is less than about 0.3 microns; , and the half-life of elimination of such substances from the injection site is between about 1 and 14 days. having an effective amount of empty liposomes to increase the desired half-life. liposome Contains an aqueous suspension of. The size, lipid composition, and relative amounts of empty liposomes are The injection site is selected to deliver the encapsulated material at the desired release rate. .

In yet another aspect, the invention includes liposome/calcitonin (CT) compositions. . The composition contains at least about 0.2 mole percent alpha-tocopherol. a sterile liposome suspension having at least Contains CT entrapped in liposomes at a concentration of 0.1 to 1 μ/mfl. its composition The substance contains ferrioxamine in a molar excess over the amount of ferric iron present in the suspension. The presence may provide further stabilization.

These and other objects and features of the invention are explained in the following detailed description of the invention. It will become clearer if you read it together with the diagram.

Brief description of the drawing Figure 1 shows a liposome composition containing both an empty lipid and a peptide-encapsulating lipid. Used in the preparation of things. A flowchart of the processing method; Figure 2 shows the kinetics of liposomal tracer lipid release from the IM injection site. Kinetics (circled) and secreted by the injected animal. Indicates tracer accumulation (triangle); Figure 3 shows the tracer release data (dashed line) of Figure 2 and the smaller size liposomes. Similar tracer release data (solid line) from liposomal compositions with It is a semi-log plot; Figure 4 shows encapsulated radioactive calcitonin (CT) from the IM injection site. ) release kinetics (circles, marks), accumulation of hormones secreted by animals (three) squares), and release of free CT from the injection site (squares); and.

FIG. 5 is a semi-log plot of the CT emission data from FIG.

Look around! 40 [(sacred place) A. Liposomes in the composition are formed from standard vesicle-forming lipids. The lipid is 9 Usually neutral and negatively charged phospholipids, and steroids such as cholesterol. Including rolls. The following considerations are usually taken into account when selecting a lipid: (a) the desired adhesiveness; bosome size and ease of liposome size adjustment; and (b) liposome size. rate of lipid and hormone release from the injection site.

Typically, the major lipid component in liposomes is phosphatidylcholine (pc). be. PCs are available with different acyl chain groups with different chain lengths and degrees of saturation. or isolated or synthesized by known methods. In general, it is very boring. Unsized PCs are more easily resized. Especially for filter sterilization applications. The liposomes must be sized to approximately less than 0.3 microns. Sometimes it is. Methods used for size adjustment and liposomes for sterilization by filtration is stated below. The saturating effect of acyl chains is due to the fact that liposomes are administered intravenously The acyl chains of phospholipids, although they do not seem to affect the rate of drug release, The composition is also. Rate of removal of liposomal lipids and entrapped compounds from the injection site can have an impact on One preferred PC is egg PC (EPC), which is derived from egg lipids. ), which includes a mixture of saturated and unsaturated acyl chain groups.

The experiments conducted in accordance with the present invention and reported in Examples indicate a matter. In other words, negatively charged phospholipids can be used alone or in combination with PC. Compared to liposomes generated from sterol mixtures, lipids and entrapped IM injection site significantly increases the rate of removal of the compound from the IM injection site.

This study involved liposomes produced using two selected molar ratios of PG; Phosphatidylserine (ps) and phosphatidylinosinol (PI) Other negatively charged phospholipids such as phospholipids could also be used. observed P The effect of G9 is due in part to charged lipids that block naturally occurring liposome association. It seems to be related to quality ability. As reported in Example ■, 1 micron pore size Liposomes are prepared after sizing by extrusion through a polycarbonate membrane. When measuring the size, the liposome containing PG has a stable size of approximately 1 micron. It has a lot of features. In contrast, liposomes containing only PC have a diameter of approximately 3 to 5 microns. It is shown to have a particle size between. As seen below, and the present invention According to one important embodiment of the method, the larger liposome size Shows longer drug release at the injection site.

The fact shown in Examples ■ and ■ also applies to negatively charged phospholipids. liposome Increases the release of intact lipids and drugs by a size-independent mechanism Show what you get.

Simply put, lipoproteins containing pure PG (plus a small amount of α-tocopherol) For somesomes, similar products containing only 5 or 10 mole percent of PG ’ lipid tracer and radiolabeled capsules than the liposomes of size peptide.

It has been shown to be quickly removed. It has various acyl chain components.

Negatively charged phospholipids are available. One preferred lipid is egg PG (EPG ), which contains a mixture of saturated and unsaturated acyl chain moieties.

Added cholesterol to drug and lipid release (from IM injection site) The impact of in general.

Cholesterol is known to increase liposome stability; This increases the time required to remove lipids and trapped components from the IM site. It can be expected that

PC and cholesterol in a molar ratio of approximately 6:4 as reported in Example V. When releasing the encapsulated peptide, liposomes containing It was shown that the time period was approximately 20% longer. However, this is true for liposomes containing PG. Even with the addition of sterols, lipids or encapsulated peptides remain at the injection site. There was little change in release rate.

B. Sufudan The lipid component of liposomes is known to promote peroxidation and free radical reactions. It is. These reactions promote liposome degradation. This problem is caused by Laquinone/Liposomal Compositions and Methods'' It has been stated. Briefly, the application includes lipid peroxidative and free Radical damage can damage the lipids and trapped substances in liposomes/drug compositions. It has been reported that both drug components deteriorate. Active against lipids and drug components The degree of damage caused by free radicals is greater for lipophilic substances such as α-tocopherol (α-lower). When free radical scavengers are included in the vesicle-forming lipids, they are significantly reduced. It was a little. Interestingly, the significant decrease in lipid damage and drug modification was due to the first-order observed in cases. That is, the lipid/drug composition is below α-.

and in the presence of both a water-soluble iron-specific chelator such as ferrioxamine. observed when prescribed in

Since ferrioxamine can strongly chelate to iron ions at 6-coordinate sites, its The compound is in the aqueous phase of the liposome suspension.

It is likely to act to prevent iron-catalyzed peroxidation. The effects of the two protective agents are Together, they suggest the first order of things. In other words, the iron-catalyzed peroxidation reaction that occurs in the aqueous phase, Both free radical reactions (enhanced in the lipid phase) can cause peroxidative damage to lipids. make a significant contribution to This suggests that.

The lipophilic free radical scavenger used in this composition is:

Preferably α-T or a pharmaceutically acceptable analog or ester thereof (e.g. alpha-tocopherol succinate). Other free radical scavenging Agents include butylated hydroxytoluene (BIIT), propyl gallate (Au gustin) + and their pharmaceutically acceptable salts and analogs. be done. Additional agents can be administered parenterally to humans at effective levels in liposomes. Also a lipophilic free radical scavenger.

It can be used at effective levels within liposomes. This free radical scavenger is Typical lipid components used to prepare liposomes by conventional methods include. The preferred concentration of protective compound is based on the total lipid component of the liposome. Between about 0.2 and 2 mole percent. However, higher concentrations of compounds (especially α- or its succinate analogs) are compatible with the stability of the liposomes; Accepted by the pharmaceutical system.

Water-soluble iron-specific chelators are natural and synthetic trihydroxamic acid chelators. Selected from Las. This chelating agent.

with a very high binding constant (on the order of 10”) for the second ion, and compared to valent cations (e.g. calcium and magnesium cations) It is characterized by having a relatively low binding constant. trich of various natural origins Doloxamic acid is known. They include ferrichrome, ferrichrome A, and compounds in the ferrichrome class such as albomycin; ferrioxa the ferrioxamine class of compounds, including amines and ferrioxamines; Included are the zalamine class of compounds.

One desirable chelating agent is ferrioxamine B.

This ferrioxamine B also includes ferrioxamine, desferrioxamine, Desferrioxamine B and Desferal”. The compound exhibits exceptional iron-binding affinity and can be used in humans for the treatment of iron storage diseases and pre-iron therapy. It has been demonstrated to be safe for parenteral use.

The above chelating agents were prepared in three compositions at concentrations that provided a molar excess of ferric iron in the liposome suspension. Exist in things. Typically, the aqueous medium used to prepare liposomes is contains at least about 1 to 2 μH of ferric iron, and 100 μJ or more of ferric iron. May contain iron. Aqueous media containing iron up to about 20 μH have a sharpness of about 50 μH. A concentration of the agent is preferred.

The chelating agent is preferably added to the vesicle-forming lipids during liposome formation. , thereby during liposome preparation.

Lipids are protected from drug-promoted oxidative damage to lipids. chelating agent water A method for preparing liposomes by adding a liquid solution is described below. here Understand the first order of things.

That is, only liposomal suspensions formed in this manner contain the chelating agent to a large extent. and in encapsulated form (i.e., the aqueous internal liposome region). Included in both areas).

That's what I understand. Alternatively, the chelating agent can be placed in suspension after liposome formation. may be contained in

C-Kinsheng Compounds entrapped in liposomes are liposome-impermeable drugs or peptides. be. The rate of diffusion out of the liposome is determined by the collapse of the liposome at the IM injection site. It is substantially higher than the speed. The reagent may be a lipophilic drug or hormone, or can be a water-soluble drug or peptide. The lipophilic drugs or hormones mentioned above are , the oil/water distribution is significantly tilted toward the liposome bilayer, and the water-soluble The drug or peptide could diffuse (through the liposome bilayer). ), it is possible to diffuse slowly. Spreads freely outside liposomes Water-soluble drugs that can be dispersed and have a half-life of less than several hours are specifically excluded from the present invention. .

Peptide hormones and immunostimulatory agents are one of the important compounds used in the present invention. It's a good thing. Typical peptide hormones include insulin and growth hormone. , and calcitonin (CT), which improve blood levels of calcium. control. Interferon and interleukin 2 are substitutes for immune activators. It is a table. According to the present invention, nine selected peptide compounds can be synthesized in a slow and controlled manner. It is possible to release the drug into the bloodstream at a rapid rate over a period of several hours to several days. the However, the large fluctuations in blood peptide levels that are characteristic of free hormone administration are avoided. avoided.

Additionally, and according to one discovery in the present invention, the peptide can be used at relatively low concentrations. If present, carrier liposomes provide safety for the peptide during storage. quality can be significantly enhanced. The experiment reported in Example ■ shows that the stability of free OCT is .

When the CT concentration increases from 0.010 mg/ml to 1.0 mg/d, it increases several times Show that you have been strengthened. The drug is added to liposomes at a relatively high concentration (e.g. 1 mg/ml). cells, but with a relatively low concentration of the predominant phase (e.g. CT present at a state of 0.010 μ/m) is seen at high concentrations of free hormone. It shows a stabilizing effect similar to that of . Therefore, according to the present invention, it is more stable and can be stored at higher concentrations. and delivered. In that state, the hormone is in a microenvironment with a high concentration. This provides good stability during storage. Water-soluble peptides like CT The preparation of liposomes encapsulating 3 selected internal volume concentrations was next investigated. ing.

Steroid hormones and anti-inflammatory agents are other important classes useful in the present invention. . Typical steroids include hydrocortisone, estradiol, and Includes tosterone. Liposomes enclosing entrapped steroids contain the compound are easily formulated by incorporating them into vesicle-forming lipids. steroid The rate of release from the IM or SQ injection site depends on drug distribution and injection. It is controlled by the stability of the liposomes at the site and their migration from the injection site.

1M or other materials suitable for delivery of slow release liposomes from SQ sites. Compounds such as antibiotics such as amphotericin B and cyclosporine immunosuppressants, and antitumor agents such as doxorubicin. 1985 Joint application patent application No. 781.395 “Amhotella "Syn B/Liposome Composition" includes (total Novel formulations containing a high molar ratio of amphoteracin B (up to 20 mole percent of lipids) Posomal compositions are disclosed. Especially for the delivery of antibiotics or antineoplastic agents. One advantageous feature of the invention is to select the drug release site and highest drug concentration. It is an ability.

(Margin below) D A variety of methods can be used to prepare liposomes. There are these. certain preference In a new method, multilamellar vesicles (MLVs) of heterogeneous size are prepared. This person In the method, lipophilic and, if appropriate, lipophilic drug mixtures are Thin lipid films are formed by dissolving in organic solvents or solvent systems and drying under vacuum or inert gas. to form. , if necessary, the membrane is soaked in a suitable solvent such as tert-butanol. Re-dissolve and then lyophilize to form a more homogeneous powder that is more easily hydrated. A lipid mixture may be formed. This membrane is coated with a hydration medium and by stirring Hydration typically takes 15 to 60 minutes. The size distribution of the obtained MLV is By hydrating the lipids under more vigorous agitation conditions, they are transformed to smaller sizes. can be made into

For forming liposomes with encapsulated water-soluble compounds such as peptide hormones. If the compound is present in the internal volume of the liposomes in the final liposome suspension, Dissolve in the hydration medium at the desired concentration. Therefore, in 9 cases, for example, an internal space of about 1■/ml To form liposomes in which CT is encapsulated at intermediate concentrations.

The hydration medium contains 1 μ/ml CT. This hydration medium is about 10-50mM It may also be formulated to contain iron-specific chelating agents at preferred concentrations between. fruit Example 1 describes a method for producing a suspension of CT-encapsulated MLVs.

Another advantageous method for producing liposomes is described by Zoka (1978). This is the reverse phase evaporation method described. Here, vesicle formation in organic solvents or solvent systems The lipid solution should be encapsulated in a relative volume compatible with the water-in-oil emulsion. Add to an aqueous solution of the material. This mixture is then emulsified and the organic phase removed using reverse phase fat. Prepare a quality gel. When resuspended in an aqueous solution, this gel generally forms back-evaporation vesicles ( It forms a suspension of relatively large oligolamellar vesicles called REV). child The method of gives efficiency. Therefore, expensive drugs, smokes or peptides such as peptide hormones Useful for encapsulating compound compounds.

Regardless of the method used, liposome preparation is preferably a sterile liposome suspension. It is carried out under such conditions. This means that conventional sterilization techniques are not used during this method. It is done by being present.

E, liposome size.

Even if the initial liposome preparation can be performed under sterile conditions'.

It is generally necessary to treat the preparation to remove free drug; Such treatments generally involve methods that are difficult to perform aseptically. subordinate Therefore, before these liposomes can be used for parenteral injection, the final Sterilization required.

An excellent method for sterilizing liposomes, including liposomes containing heat-sensitive encapsulating materials. The only method that can be used to sterilize the system is 1 normal depth filter, typically is by filtration using a 0.22 micron filter. This method is First, reduce the size of the liposomes to about 0.2-0.3 microns or smaller. Only when well-tuned can it be implemented on a high throughput and practical basis.

There are several techniques that are useful for reducing liposomes to this size range. Ru. The liposome suspension can be sonicated in a bath or with a probe. When sonicated at either side, small lamellae with a size smaller than about 0.05 microns are formed. A significant size reduction occurs to the vehicle (SUV). Homogenization is another method, The cutting energy fragments large liposomes into smaller ones. typical In a standard homogenization method, pass the MLV through a standard emulsion homogenizer to liposome size, typically between about 0.1 and 0.5 microns. observed and recirculated until In both methods the particle size distribution is typically 2 can be monitored using laser beam particle size identification methods.

Extruding liposomes through a small-pore polycarbonate membrane is an effective way to reduce size to a relatively well-defined size distribution ( Szoka 1978). Typically, the desired liposome size distribution is achieved The suspension is circulated through the membrane several times until the successively smaller and smaller liposomes The liposome size can be gradually reduced by extrusion through a membrane with large pores.

F, play 14j■ Shihiki λhu Even the most efficient encapsulation methods, such as the REV method mentioned above.

The maximum encapsulation efficiency for water-soluble compounds is about 50%. Therefore, early stage liposo suspension of the compound liberated (unencapsulated) 50% or more of the compound. ) including in the form. The above-mentioned method typically has an encapsulation efficiency of between about 10 and 20%. The liposome suspension formed by the MLV method has a large amount of unencapsulated material. Even more. The study reported in Example V is based on free (unencapsulated) for compounds encapsulated in liposomes, ranging from hours to days. indicates that it is removed from the injection site more than 1 hour earlier compared to IM injection site.

Therefore, to minimize the effects of rapid drug release from the injection site, It is important to remove any free drug that may be present in the composition.

There are several methods useful for removing unencapsulated compounds from liposomal suspensions. There are some. One simple method involves removing liposomes in suspension by high-speed centrifugation. Pellet, leaving free compound and very small liposomes in the supernatant.

Another method involves concentrating the suspension by ultrafiltration and transferring the concentrated liposomes to the drug. including resuspension in an alternative medium that does not contain. Or even bigger Gel filtration can be used to separate liposome particles from solute molecules.

Yet another method of removing free compounds is ion exchange.

Use molecular sieves or affinity chromatography. here, 'f1 binds to the separated form of the compound but not to the entrapped form of the compound 9 that specifically binds to resins or unencapsulated compounds, such as antibiotics. The liposome suspension is passed through a column containing a carrier to which binding molecules such as the like are attached. this The method is by selecting the resin appropriately.

It can also be effective in removing free pyrogens.

Following treatment to remove free drug, liposome suspensions were administered at 1M or Make the desired concentration for use in SQ administration.

This means that the liposomes are concentrated by 2 e.g. centrifugation or ultrafiltration. If necessary, the liposomes can be resuspended in an appropriate volume of injection medium or The suspension may be concentrated if the agent removal step increases the total volume of the suspension. may be included. This suspension is then sterilized by filtration as described above.

G, -9 points: ≦ Yo ground side work Figure 1 shows 9 liposomes of the present invention 1, liposome processing suitable for preparing compositions. It is a work process diagram. The working steps on the left side of the diagram represent liposols with entrapped compounds. The process of forming a sterile suspension of the microorganism is illustrated. As detailed above. These processes 9 First prepare a liposomal suspension containing a selected entrapment concentration of the compound. include. This suspension is then reduced to 0.2-0.3, taking into account the final sterile filtration. or smaller. After removing any uncaptured drug, this material The right column of Figure 1 shows the parallel steps used to prepare empty liposomes. represents. The principle of preparing empty liposomes is that the material encapsulated in liposomes is , the rate of release from the IM or SQ injection site is determined by (a) lipid composition, (b) average lipid Posome size.

or (C) selectively by changing the total lipid content of the injected liposomes. It is based on the discovery that changes can be made. These parameters and compound release rates The relationship between the two is detailed in Section (1) and Examples (2) to (2) below. Here, the top If the majority of liposomes injected into the site do not contain the entrapped compound It is sufficient to note that even these effects are achieved. Sunawa Therefore, the effects of lipid composition, size, and amount on the rate of compound release are liposomes/liposomes that similarly affect both liposomes and compound-containing liposomes. It appears to depend on somatic interactions.

Advantages of empty liposomes as a method to vary lipid composition, size, and amount is that they can be prepared in sterile form without the need for a final sterile filtration step. be. mainly.

Free compounds? A of the liposome preparation due to the need to remove it from the suspension. It is recalled that sterility is compromised.

such that the compound is not present or does not result in significant amounts of free compound. A final sterile filtration step by forming liposomes in the presence of the amount of compound can be avoided. Empty liposomes do not need to be reduced in size for filtration. By adding relatively large liposomes, the rate of compound release can be significantly improved. An important advantage is that larger size-related effects can be achieved.

Considering the steps shown on the right side of Figure 1, empty liposomes can be processed as needed. can remove smaller liposomes. This is 1, for example, a larger This is done by fixing the liposomes and aseptically removing the unfixed material. obtain. Alternatively, these liposomes can be filtered without further processing. 2 selected average lipid compositions and sizes by direct addition to sterilized liposomes. These liposomes can be used to prepare final compositions with was added to the filter-sterilized liposomes under sterile conditions. Its size can be adjusted by extruding it through a polycarbonate membrane of .

Accordingly, the processing method described above (a) has a selected concentration of captured compound, and (b) frees and (C) contain little or no compound of the selected The release rate is the average liposome molecule that releases the entrapped compound from the injection site. allows the preparation of sterile liposomal compositions with specific sizes, concentrations, and compositions.

2. Another layer” 1111 A, Hiroshi f1 One advantageous feature of the invention is that the peptide molecule is encapsulated in liposomes. The aim is to increase the stability of pharmacological compounds such as Mon. Especially for CT As has been found, such compounds may be more stable in concentrated form. In cases where the compound is encapsulated in liposomes, it remains stable in suspension. It can be stored in a fixed form. In the suspension.

Although this compound has high local concentrations, the total concentration is relatively low. For example, Example According to studies reported in ■, even in the presence of carrier proteins, free OCT 1 mg/mlO is substantially more stable than 0.01 mg/d. Internal capacity thick The concentration is 1 μ/mfl, but the suspension concentration is 0.01 mg/d, and the peptide is released. When encapsulated within posomes, this peptide exhibits stability properties at high concentrations of CT. .

The stability of liposomes is determined by the presence of lipophilic free radical scavengers 2 such as α-T (both Improvement can be achieved by containing it at a molar ratio of about 0.2. Furthermore, the stability of one composition is , free iron-specific chelators such as ferrioxamine in liposomal suspensions. This is achieved by adding a molar excess over the amount of ferric iron to remove free CT. It may be processed and sterile filtered.

The resulting composition comprises (a) liposomes containing at least about 0.2 mol% α-T; and (b) within the liposome at a concentration of at least about 1 μm/ml. Contains captured calcitonin.

Preferably, the free CT concentration is less than about 10 mole percent of the encapsulated peptide.

Peptide hormones such as CT can be stabilized by being encapsulated in liposomes. The discovery that it can be interpreted and preserved in the h-shape.

Applied to various other peptides and proteins. For example, this method stabilizes the enzyme. It can be used for storage in diluted form.

Perform clearance (removal) rates of lipids and encapsulated compounds from the IM injection site Examples ■～■ were investigated as reported.

The objectives of these studies are twofold: (1) lipid clearance from the injection site and (2) determining the relationship between peptide clearance; and (2) peptide release rate. The purpose of this study is to investigate the variables of liposomes that affect the binding of liposomes. These studies was performed using CT as a peptide compound, but in order to be released into the bloodstream, This finding applies to any water-soluble compound where liposome disruption is required. It is recognized that it can be applied.

To track the rate of liposomal lipid clearance from the 1M injection site, - As a sirolipid! 51-labeled phosphatidylethanolamine (''I- Liposomes containing PH) were injected into the limbs of laboratory animals. The arrangement of tracer lipids is , measured at a minimum of eight time points over a test period of at least 70 hours. note Reduction of tracer from the injection site (circles) and accumulation of excreted tracer (three circles) square mark) is shown in Figure 2. The upper racer clearance data from the injection site is − When plotted as a semi-log function, it becomes a straight line plot as shown in Figure 3. and from this the lipid clearance half-life can be calculated. This laboratory animal was injected IM. The clearance of radiolabeled CT from liposomes was similarly as follows: . Obtained from a typical experiment. Tracer reduction from the injection site (circled) and A plot of the cumulative amount of excreted tracer (triangles) is shown in FIG. As shown in Figure 5. Clearing from the injection site using a semi-logarithmic plot of CT clearance Lance half-life was calculated. The linear relationship between the retained logarithm of CT and time is This shows that lipids are removed from the injection site with first-order kinetics. injection site The rapid release of free OCT into the bloodstream is also shown in this figure.

The effect of liposome size on the clearance ratio of lipids and CT is shown in Example ①. It is clear from the data. The data is '.

Liposomes range in size from 0.2 microns to about 5 microns, and larger compared with Kina liposomes (showing a 60-100% longer lipid clearance rate). It is something that The data of Example ■ is based on small liposomes (about 0.2 microns). Clearance of lipid tracer from larger unlabeled liposomes (approximately 1 mi We have shown that chlorine) can be increased by adding it to injected liposomes. It shows. The latter result suggests that lipid clearance is related to average liposome size. and in accordance with certain aspects of the present invention.

Release of smaller liposomes by adding larger empty liposomes This indicates that the lipid clearance forms the basis for controlling the production characteristics. .

The effect of liposome dosage on lipid clearance is also reported in Example ■ . As discussed there, the half-life of lipid clearance depends on the size or can be increased by more than 2-fold with increasing lipid dosage, regardless of composition or composition. . A similar doubling of the clearance rate with increasing dose indicates that Seen on capsular CT (Example ■).

Interestingly, lipid and Comparing the release rates of OCT and CT (Example V), it was found that OCT cleared from the injection site. The clearance is about twice as fast as the corresponding lipid tracer. This discovery , the liposomes are destabilized and release their encapsulated contents primarily at the injection site. and lipid clearance from the site was different. A slower mechanism This suggests that it will be treated as such.

The effect of lipid composition on the clearance of lipids and CT from the IM site was also investigated. It was. As discussed above and reported in Examples ■ and V, the negatively charged Phospholipids 9 When PG, for example, is added to liposomes, lipids from the IM site and The clearance rate of the encapsulated CT is significantly increased. As noted in Example■ , the PG effect is partially due to the reduced effects seen in PG-containing liposomes. may be associated with liposome aggregation.

Cholesterol, at a molar ratio of about 40%, absorbs lipids from PG-containing liposomes. from neutral PC liposomes, although it had little effect on Significant stabilizing effect on OCT release - i.e. longer clearance halving Period-1 was shown. CTs are liposomes with the same size and lipid composition characteristics. The liposomal lipids were removed approximately twice as fast as the lipid tracer. The mechanism that acts on encapsulated compounds is different. removed from the injection site by a slower mechanism. It was confirmed that

The above results indicate that the release of encapsulated compounds from liposomes at the injection site can be selectively controlled by changes in the size, dosage, and lipid composition of the membrane. This shows that In particular, when carrying out the method of the invention, encapsulation from the injection site The rate of compound release is determined by the average size and total amount of liposomes injected at the site. controlled by In some aspects of the invention, the average size and lipid content is greater than A compound of interest is encapsulated in a large empty liposome. smaller filtration It is selectively increased by adding it to cultured liposomes.

According to the invention, either compound-containing liposomes or empty liposomes , or even longer release times by adding cholesterol to both. It can be achieved. In order to release the encapsulated compound more quickly, more negatively charged lipids can be used. Liposomes can be formulated to progressively contain substances such as PG.

3. Beat 193 (company) Marusato The liposome compositions of the present invention can be administered parenterally with various liposome-impermeable compounds. It is useful to give One important application is peptide hormone or immune promoting used to administer drugs into the bloodstream in a controlled manner over several days . This composition can selectively alter the half-life of peptide release and can release at a selected period of time. At that time, peptides are not frequently given without the rapid fluctuations seen when using injections of free peptide. can be obtained. Moreover, it is more effective than using previously proposed liposome preparations. A high degree of control can be achieved.

Insulin and CT are examples of peptides that are routinely administered in free form today. It is. Both of these peptides.

is easily incorporated into the liposomal compositions of the present invention and can be easily incorporated into the liposomal compositions by 1M injection of the compositions. can be supplied over several days. The proportion of hormone supply is determined according to the method of the present invention. By using the average liposome size, amount, and composition selected by controlled by For some peptides, e.g. CT, the liposome composition An added benefit is that increased stability is achieved, which makes this material The material can be stored in highly diluted form for long periods of time.

The following examples describe specific embodiments of the construction and use of the present invention. 9 and is not intended to limit the invention in any way.

(Margin below) Su3Jli Mouth: 1 line, 1 tear Egg phosphatidylcholine (EPC) and egg phosphatidylglycerol are Supplied by Avanti Lipid (Birmingham, AL). child These lipids were determined to be approximately 99% pure by thin layer chromatography. Ta. Cholesterol (CI) and α-tocopherol (α-T) are each NuChek Prep.

Inc. (Ilijan, MN) and Sigma Chemical Co. , (St. Louis, 11o), the purity of both is about 99% or It was more than that. Salmon calcitonin (sCT) is a Provided by r-maceuticals+Kankaki-, IL, sCT was performed using IZ using I251 by the chloramine T method (McFarland). ST-radiolabeled.

Multilamellar vesicles (MLVs) containing one of the lipid compositions A-E shown in Table 1 were prepared. It was done. The molar ratios of lipid components in the four vesicle preparations are shown in Table I. The values in the table are , forming various vesicle preparations. The micromolar ratio of each lipid component used in represent.

In a study measuring the rate of liposomal lipid clearance from the intramuscular injection site, Vesicle-forming lipids are also radioiodinated derivatives of phosphatidylethanol-amine ( 125T-PE)) included a racer. to form this tracer , p-hydroxybenzamide of egg phosphatidylethanolamine (EPE) was synthesized as described by Abra and this compound was Iodine-labeled with &:”’I. The specific radioactivity was 3°85×10 bcpm per nanomole of PEI. You Labeled lipids can be used for injections ranging from 0.2 to 10 micromol total lipids. Uptake was performed at 10' cpm.

B 94 5 1 C495401 E 59 40 1 To form MLVs, the lipid composition in chloroform stock solution is transferred to a test tube or Mixed in a round bottom flask. Remove chloroform with rotary evaporator , this lipid mixture was dissolved in L-butanol. Then pour the butanol solution into Freeze in lye ice/acetone and lyophilize overnight. Convert dry lipids into a number of phosphorus Hydrate with acid buffered saline (PBS), pH 7.4.

In studies dealing with CT/MLV, the hydration buffer should be It contained T, and sCT.

The lipid film was hydrated by stirring for approximately 15 minutes at room temperature.

MLV suspensions with non-uniform sizes ranging from approximately 0.2 to 10 microns Formed. The vesicle preparation was transferred to a polycarbonate membrane with a selected pore size. The size was adjusted by extruding through. All preparations were 1.0 micron Initial vesicle cells (before aggregation) of approximately 1 micron are extruded through a polycarbonate membrane. The vesicles containing the molecule were prepared. When forming vesicles of smaller size, the size The pore size-adjusted vesicles are then added to membranes with 0.4 micron and 0.2 micron pore sizes. extruded through successively through vesicles were produced.

MLV containing encapsulated sCT by washing three times with PBS.

Non-liposome-associated free sCT was removed. These preparations contain 7 Limulus Amoeba degradation product analysis (aemachem, Inc., St. Louis, M. The pyrogenic factor test was conducted by O.

1 treatment ±1 Characteristics of liposome sCT Composition B MLV containing 1.0 μ/ml of encapsulated sCT was prepared as in Example I. did. These vesicles were sequentially passed through 1.0 and 0.4 polycarbonate filters. Free sCT was removed by extrusion through and washing three times in PBS. these The liposomes were diluted to a final concentration of 0.010 mg/ml sCT, and 0.4 Sterilize by passing through a standard sterile filter 5 m deep and and separated into sterile Nunc vials. Free in PBS containing 0.5% BSA sCT of 0.010 mg/d or 1. , Omg/d. The liquid was also separated into vials. Liposome-encapsulated sCT preparations and free s The CT preparations are designated as L-sCT and F-sCT, respectively, in Table 1. .

The stability of F-sCT and L-sCT preparations was determined by heating the vials at 4°C. Incubate either at room temperature (approximately 24°C), and 37°C, and place The biological activity of sCT was analyzed after incubation for a certain period of time and then tested. trial The material was 0.5% Triton-X obtained from Sigma (St. Louis, MO). Inside) let's have customers.

40ml/J and 120ml/ml of PBS containing 0.5% BSA Diluted. An alicon of frozen standard F-5CT was treated similarly.

Each sample was tested in 8 rats, 4 of which received 10 ml of sCT in the throat. (40 mU/ml solution at 0, 25’m) and 30 mU (0, 25’ m , 25d (120 mU/mA solution) was administered subcutaneously. Control rats had 0.2 5m1! of PBS/BSA was administered. Exactly 60 minutes after injection.

Blood samples were taken from each rat. These blood samples are then coagulated to produce serum samples. It was collected by reduced separation. Calcium level in serum is determined by Sigma Che using a kit supplied by Mical Co., (St. Louis, MO) It was determined by one-dye binding assay. The low calcium activity of these samples is due to the 60 minutes later.

was determined from the standard curve. Results are average values of 4 rats for each data value and is given in Table 1H.

(Margin below) Inside-”- Storm formation + temperature time activity )　T'1lT)　TERr)　Talso) The first three rows are the diluted and concentrated solutions of F-sCT and the diluted solutions of L-sCT. This is a comparison of the stability of 128 days at 4°C. Obviously, dilution F -sCT loses most of its activity during this period, but L-sCT does not.

What is the liposome stabilizing effect?

It is likely that there is a locally high concentration of 5CT (approximately 1 ■/volume) within the encapsulation space of the liposome. ). As shown in the fourth row, the encapsulated sCT in the diluted solution is small. (Both are stable for 215 days.

A similar liposome protective effect is seen with sCT incubated at room temperature.

During an incubation period of 43 days, a diluted solution of F-sCT lost approximately its activity. concentrated F-sCT preparations and diluted L-sCT preparations showed no deactivation. Didn't show it. The latter two preparations have approximately the same stability, indicating that the latter two preparations have approximately the same stability at room temperature. Data for 8 days of incubation and 7 days of incubation at 37°C It is clear from the data.

Exercising IL IM Note・5 Clearance from @tachi The studies reported here demonstrate that injections from intramuscular (IM) injection sites in laboratory rats The ratio of liposomal lipid clearance was investigated, and the clearance ratio was measured by radioactive lipid tracer. One of the lipid compositions A-D of Example I MLV with 0.2. 2. or selected doses of 10 micromole lipids. The animals were injected with a given dose.

aSD rats weighing 95-110 g were lightly anesthetized with ether.

Inject 20 μl containing 0.2 micromoles lipid into the forelimb.

Alternatively, 100 μl containing 2-10 micromoles lipid was injected into each rat's hind paw. did. Sixteen rats were used in each experiment. Two of the rats were placed in a metabolic cage. , excretion of radiolabel in urine and feces was monitored. Exam period starts from 70 hours Eight time points were established for each experiment over 6 days. These eight points in time are As is clear from Figures 1 and 2 for typical experiments, the amount of fat released from the IM site The semi-KOJI of quality clearance is the semi-logarithm of residual radioactivity versus time after injection. (Figure 2). At each time point, 2 rats was anesthetized for blood sampling. 2 ml of blood was collected and the radioactivity of 1251 was measured. I counted. These rats were then asphyxiated in a CO2 chamber and injected with The limbs were dissected and radioactivity was counted. Urine and feces were collected from two animals in metabolic cages during the experiment. collected from animals. These two animals were also killed, but this would be the last time.

And also the thyroid, lungs and heart to count.

The liver, lungs, stomach, intestines, and reproductive organs were dissected. The remaining body was disassembled before counting. Ta. Measure radioactivity levels at the end of the experimental period. Residues in these animals The placement of the lipid tracer was determined.

The results of a typical experiment are shown in Figure 2. The upper curve (circle) is the injection site (cavity) represents the radioactivity retained in the lipid and is expressed as a percentage of the initial lipid dose administered. ing. Vertical lines of error represent the width between two animals at each time point. seems obvious In addition, the half-life of clearance from the injection site is between approximately 40 and 50 hours; By plotting the data semi-logarithmically as shown in Figure 3 (discussed in the next section), It is determined that

Cumulative radioactivity levels measured in urine and feces are represented by black triangles in Figure 9. . The total excreted radioactivity at the end of the 70-hour test period in this experiment was approximately 72% of the initial dose administered. The remaining counts are limbs (approximately 5%) and torso (approximately 20%). %).

and organs (approximately 3%). Localized in organs.

Of the remaining radioactivity of approximately 3%, approximately 55% is in the intestine, approximately 34% in the liver, and lungs. , less than about 5% are found in each of the trachea, kidneys, stomach, heart, lungs, and reproductive organs. It will be done.

Jitsufunemasu■ Liposome size 1 for IM tJ ・o 2 clearance and retention of tracer lipids at the liposome injection site teeth.

As measured by the half-life of tracer clearance from the site, liposomes investigated as a function of muscle size, composition, and lipid dosage. Clearance half-life decreased radioactivity in the injected paw of male rats as described in Example ■. .

Calculated by tracking over a period of at least 70 hours. Each experiment? A curve similar to the black circle in FIG. 2 was obtained. This curve shows 1 hour of injection. It shows that the radioactivity of the site decreases.

The upper dotted line in Figure 3 is the same data presented in a semi-log plot as a function of 1 hour. shows. From this plot, the lipid clearance half-life from the injection site is easily determined. can be determined. In certain embodiments, the half-life (at which point 50% of the lipids are retained) ) is 48 hours. Half-life data are presented as semi-log plots for each experiment. It is determined from the slope of the curve and is shown in the table ■ below.

The liposome sizes are shown in Table 1.

The pore size of the polycarbonate membrane used to size the MLV and the actual It is represented by the average size of the MLV at that time. The average size of MLV is Ni Standardized latex as measured by comp particle size analyzer (Model 200) Calibrated with Subies.

In this way, the injected MLV in the first row, for example, is 1 micron polycarbonate. The measured average The particle size of the sample was approximately 3.2 microns. Extruded size and measured size The discrepancy in Is probably reflects liposome aggregation.

When the liposome concentration is high, only the MLVs of compositions A and E are produced. There is a tendency to These MLVs do not contain negatively charged lipids (PG).

The dose administered to rats is 20μ per injection! Or 100μ! Ma around Measured by icromol lipids. The values shown in the dosage column of the table are for each injection. The total dose of

Table■ Nα Composition Size (μ ribs Dose per injection T-1/2 extrusion value Measured value (μM/μ2) - The data in Rolow's 1st to 3rd columns indicate the lipid concentration from the IM site. Figure 2 represents the effect of total lipid dose on rearance. As is clear, the injected fat As the mass increases from about 0.2 micromolar to 10 micromolar, the clearance The half-life of is increased by about 2 times. The same effect was observed in the abdomen V (5th and 6th rows) and 9 Composition B. and the MLV of composition C (columns 8 and 9).

The half-life data indicate that the rate of lipid clearance is large and depends on the size of the bosome. It also shows that there is a significant increase in This effect applies to columns 4, 5, and 7. It is clear from the column and the 8th column. Both of these are 0.2 microns and 1.0 microns, respectively. Comparing the clearance percentage of MLV extruded through a 0 micron filter This is what I did. Large liposomes reduce clearance half-life by approximately 60-100% only increased. Relatively large clearance rates observed in hLVs of Composition A is measured, at least in part, by laser particle size analysis. These ML This may reflect that the VO size is relatively large. Figure 2 shows a semi-log plot.

From this figure, the half-life in the first column (dotted line) and the half-life in the fourth column (solid line) are calculated. Ta.

Column 1 to Column 4 and Column 7 (0.2/20 of dose level).

and when comparing column 3 with columns 6 and 9 (dose level 10/100) , showed a significant effect of negatively charged phospholipids (PGs) on the clearance rate. However, the presence of cholesterol has little effect. as suggested above , the effect of PG was observed, at least in part, with PG-containing liposomes. This may be due to the small size. However, for EPG MLV The measured half-lives (column 10) of either 5 mol% or 10 mol% PG The liposome size compared to the containing V (column 6 or column 9, respectively) The results show a strong effect of PG concentration that does not appear to be related to PG concentration.

Sukun Ll1 Liposome size, In′, and sCT clearance from 1M injection site and encapsulated in liposomes from the intramuscular (IM) injection site of the liposome group. The clearance rate of the sCT was calculated using the same procedure as described in Example ①. Determined by measuring the retention of "J-sCT" at the injection site.Example MLV having one of lipid composition yJA, B, D and E as described in 1. ．． Animals were injected with selected doses of 2 or 10 micromoles lipid. this Approximately 0.5 μg of sCT is encapsulated in their MLV, and the ratio of tzsH-CI is The radioactivity was approximately 10.5 μg at 10' cpm. Free sCT is The somoid suspension was removed by centrifugation. Trial for 70 hours to 6 days after injection Pairs of animals were killed at intervals over the experimental period, and the injected The retention of radioactive sCT in the limbs and numerous organs was described in Example ■ above. It was measured as follows. Two of the injected animals had urine and feces during the study period. The excretion of sCT was monitored.

The results of a typical experiment are shown in FIG. The upper curve (circle mark) is at the injection site. ”Represents the retention of J-sCT; the middle curve (triangle mark) represents the cumulative radioactivity excreted. As can be seen, the initially injected sCT of ca. 70-80% is excreted after 70 hours of this experiment. F-sCT fat to bloodstream Quality clearance is indicated by the lower curve (square mark).

When the sCT retention data is plotted in semi-logarithmic form as shown in Figure 5, the result is becomes a straight line plot. From this plot.

Clearance half-life can be determined. In the example shown, the half-life is approximately 8 hours. Ru. Measured in this way for the four lipid compositions and two lipid doses investigated. The half-life data provided are given in Table ■ below. In all studies.

The MLV was pre-extruded through a 1.0 pore polycarbonate membrane. Therefore, the size is adjusted, but the small pores are not pushed through the small membrane. won.

Table■ Sato amount received dosage fi T-1/2 (p mole/vol) The data in columns 1 to 3 and the data in columns 6 to 7 are from the IM site. Figure 3 shows the effect of total lipid dose on sCT clearance. tracer lipid Similar to what was observed for the clearance of 0.2 to 10 micromol fat As total lipid dosage increases across a range of quality, the clearance half-life of sCT decreases. It increased 2-3 times.

These data demonstrate a significant effect of lipid composition on the clearance rate of sCT. It also represents The presence of PG in the MLV (compositions B and D) makes the tracer Similar to what was found for lipids, two PG-free MLVs at equal doses The clearance ratio was much smaller than that of (compositions A and E).

Additionally, adding cholesterol to PG-free lipids increases the clearance of sCT. The percentage increased significantly. This compares the 2nd and 6th columns, and the 3rd and 7th columns. It is clear then. Here, the second and sixth columns are for the dose 2/100, and the third Column and column 7 relate to the dose 1tlo/100.

The clearance half-lives of preparations administered 1M and SO were similar. It must be noted that there is.

For example, preparations with IM half-lives of clearance of 20.6 hours and 9.7 hours. The SQ half-lives of the products are 19.4 hours and 8.3 hours, respectively.

Table V below shows some of the sCT clearance percentages in Table 1 for lipid tracers. This is a comparison of the corresponding ratios in Table 2 and ■. Interestingly, all lipids For compositions and doses for which comparative data are available, sCT rates are - about half of the total. This discovery suggests that liposomes are destabilized and their encapsulated components is released primarily at the injection site, and lipid clearance from the injection site differs. . This suggests that it is handled by a slower mechanism.

(Prefectural Kinpaku , Table-N- s] 1 川■ Liposomal EPC spliced by sCT method from small MLV: α- T is 99 = 1, and reverse evaporation vesicles (REV) containing encapsulated “J-sCT” are targeted. Prepared according to the standard REV procedure (Szoka). These vesicles are 0.4μ Extruded through 0.2 μm and 0.2 μm polycarbonate filters and encapsulated. sCT that has not been removed can be cleaned by centrifugation and washing 3 times in PBS. cells were removed from the suspension. The preparation is then filtered through a 0.22 micron sterile filter. It was filtered through.

Empty MLVs of the same composition were prepared and 1 μm It was extruded through a filter. Mixing REV with MLV in a 1:500 molar ratio, Each 1M injection contains approximately IX10' cpm of +251- sCT and about 0.5ug of sCT. Mixture of liposomes before injection The mixture was allowed to stand overnight for aggregation. The liposome mixture or or REV was injected subcutaneously into rats. Clearance of 1251-radiolabel from the injection site The lance was as described in Example ■.

Determined by semi-log plot. For REV only and empty ML in REV Half-life data when V is added is shown in Table ■.

once the table Danso beans Types of liposomes Yakuho 1 Main ingredient... (u mole/100 u i! ) As can be seen, when adding an empty MLV to a 0.2 micron REV, the RE The half-life of OCT clearance increased by more than 5-fold from V. The third column in the table is , giving the half-life of CT clearance for OCT release from MLV alone (implemented As in example ■). Comparing the second and third columns, the peptide-containing liposomes Size, along with the size and/or amount of empty liposomes.

This site is shown to influence the rate of peptide release from this site.

Preferred embodiments of the invention are described herein.

It will be obvious that various changes and modifications may be made without departing from the invention. Ru. In particular, one uses liposomal delivery of CT to illustrate the general principles of the invention. However, similar release characteristics have been observed for other liposome-impermeable compounds. It is clear to those skilled in the art that

Fine weather (hhis,)” Time (hs5.) Time closed (hF, s,) international search report Attachment to Form PCT ISA/210 Part I.

INT, CL, BOIJ 13102; B52B S/16,9102, 9104

Claims (14)

[Claims] 1. forming a suspension of liposomes containing liposome-impermeable compounds in entrapped form; , administering the compound by injecting the suspension into an intramuscular or subcutaneous site. selectively increases the rate of release of the compound from the injection site into the bloodstream when law, selectively increasing the average size of liposomes injected into the site; and selectively increasing the amount of liposomal lipid injected into the site; How to include. 2. These compounds include calcitonin, growth hormone, insulin, and interferonin. and interleukin-2, the claimed peptide is selected from the group consisting of The method described in item 1 of the scope. 3. The hormone is calcitonin, and the hormone is present in small amounts in the suspension of liposomes. Claim 2 is encapsulated at a concentration of at least about 0.10 to 1.0 mg/ml. The method described in section. 4. For the liposome, the amount of negatively charged phospholipids is reduced to The claim further comprises increasing the rate of release of said compound from the injection site. The method described in item 1 of the scope. 5. The average size of the liposomes containing the entrapped compound is about 0.2 microns. Empty liposomes with an average size larger than approximately 0.5 microns By increasing the amount of added liposomes, the average size of the injected liposomes can be increased. 2. The method of claim 1, wherein: is selectively increased. 6. Liposome-impermeable compounds from intramuscular or subcutaneous injection sites into the bloodstream A liposome composition used when administering, Lithium containing the compound in entrapped form and having an average particle size of less than about 0.3 microns. Aqueous suspension of posomes and clearing of the compound from such injection sites in an amount effective to extend the half-life of the drug to a desired half-life of about 1 to 14 days. A composition containing a quantity of empty liposomes in suspension. 7. Claim: the empty liposome has an average particle size greater than about 0.5 microns. The composition according to item 6. 8. These compounds include calcitonin, insulin, growth hormone, and interferonin. and interleukin-2, the claimed peptide is selected from the group consisting of The composition according to item 6. 9. The hormone is calcitonin, and the hormone concentration within the encapsulation space of the liposome is 9. The composition of claim 8, wherein the composition has a concentration of at least about 1 mg/ml. 10. The liposome contains alpha-tocopherol in an amount of at least about 0.2 mol%. A composition according to claim 6, comprising: 11. A liposomal calcitonin composition comprising at least about 0.2 mol% α. - a sterile aqueous suspension of liposomes containing tocopherol, and entrapped in the liposome, and the concentration within the encapsulated space of the liposome is at least about 1 m A composition containing calcitonin, which is g/ml. 12. The liposomes contain between about 5 and 100 mole percent phosphatidylglycerol. 12. The composition according to claim 11, comprising: 13. the liposomes have an average size of at least about 0.3 microns; When the composition is injected into the intramuscular or subcutaneous site, calcito is released into the bloodstream. further comprising empty liposomes in an amount effective to increase the half-life of nin release; A composition according to claim 11. 14. the empty liposomes have an average size of at least about 0.5 microns; A composition according to claim 13.