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EP1390053A2 - Hcg formulation - Google Patents

Hcg formulation

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Publication number
EP1390053A2
EP1390053A2 EP02764353A EP02764353A EP1390053A2 EP 1390053 A2 EP1390053 A2 EP 1390053A2 EP 02764353 A EP02764353 A EP 02764353A EP 02764353 A EP02764353 A EP 02764353A EP 1390053 A2 EP1390053 A2 EP 1390053A2
Authority
EP
European Patent Office
Prior art keywords
hcg
muramyl
protein
composition
beta
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02764353A
Other languages
German (de)
French (fr)
Inventor
Hernan F. Acevedo
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP1390053A2 publication Critical patent/EP1390053A2/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/24—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/113—Multiple emulsions, e.g. oil-in-water-in-oil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers

Definitions

  • This invention relates generally to a pharmaceutical formulation of water-soluble, biologically active proteins and polypeptides like hCG hormone which are useful for treating various diseases.
  • virus-induced, immune, and malignant diseases are preferred diseases to be treated by the composition of this invention.
  • More specifically diseases originated by a virus like human immunodeficiency virus (FAN) which causes acquired immunodeficiency syndrome (AIDS) and associated cancers in humans are contemplated.
  • FAN human immunodeficiency virus
  • AIDS acquired immunodeficiency syndrome
  • BACKGROUND INFORMATION AND DISCUSSION OF RELATED ART Human chorionic gonadotropin (hCG) belongs to a family of glycoprotein hormones, which includes luteinizing hormone (lutropin, LH), follitropin (FSH), and thyrotropin (TSH).
  • Each of these hormones is composed of two dissimilar, noncovalently bound subunits, alpha and beta.
  • the hormones share a common alpha subunit, while the beta subunits differ in length and amino acid sequence and unique to each hormone.
  • the most closely hormones are hCG and LH, which are 85% identical, except for an amino acid sequence at the carboxy terminus of hCG.
  • hCG and LH act through a common receptor.
  • hCG and related hormones are conventionally believed to play a ⁇ ole rn ⁇ f ⁇ r ⁇ alT ⁇ uman physiology-r
  • hCG is well established as pregnancy hormone which plays an important role in reproductive physiology during gestation.
  • An extensive background information on hCG is provided in a review article by the present inventor (see Hernan F. Acevedo "Human chorionic gonadotropin (hCG): The hormone of life and death, A review” as incorporated by reference).
  • hCG hormone has been proposed by others as useful for HIV/AIDS and cancer therapy. See for example U.S. Patent Nos. 5,700,781; 5,811,390; 5,851,997; 5,997,871; 5,877,148; and 5,677,275 or original publications by Bourinbaiar AS, Nagorny R. Effect of human chorionic gonadotropin (hCG) on reverse transcriptase activity in HIN-1 infected lymphocytes and monocytes. FEMS Microbiol Lett. 1992 Sep l;75(l):27-30 and Bourinbaiar AS, ⁇ agorny R.
  • hCG human chorionic gonadotropin
  • hCG can have opposi effect and may instead promote carcinogenesis, thus effectively teaching away from the usefulness of hCG as anticancer or antiviral agent. See for example Simonart et al. (Simonat T, Hermans P, Van Vooren JP, Meuris S. Paradoxical pro-Kaposi's sarcoma activity of preparations of human chorionic gonadotropin. Blood. 1999 Jul l;94(l):376-7).
  • Harris teaches sustained-release form of hCG provided as a transdermal hCG patch, after the fashion of the DURAGESICTM fentanyl patch so that transdermal delivery of proteins like hCG is accomplished iontophoretically or electroosmotically, i.e., under the influence of an electric field.
  • Another sustained-release form of hCG contemplated by Harris is an implantable hCG delivery system.
  • the NORPLANTTM levonorgestrol implant a representative type of device in this category it employs passive release of hCG through a non-biodegradable, rate-limiting membrane element composed, for instance, of a hydrogel or a microporous polymer.
  • Harris incorporates a pump function to administer the hormone.
  • the pumping action is osmotically driven or patient-activated.
  • Harris taught various alternative modes of hCG administration.
  • contemplated modes of delivery are through specific sustained-release drug delivery devices, i.e., transdermal skin patches and different types of implants or pumps. No other forms of hCG delivery are taught as reliable and practical modes of drug delivery.
  • hCG humanamyl peptides
  • muramyl peptides are mostly used as vaccine adjuvants to enhance the immune reaction against the antigen, i.e., hCG, present in the vaccine preparation (see for example U.S. Patent Nos. 5,840,313 or 5,876,724) and thus this combination would effectively render hCG inactive or may even exacerbate the course of the disease.
  • hCG variants with muramyl peptides
  • these vaccines contain exclusively either beta chain of hCG or carboxyterminal peptides of beta chain of hCG, and not whole, dimer hCG (see for example U.S. Patent Nos. 4,313,871; 4,256,629; 4,310,455; 6,146,633; 6,143,305; 6,096,318; 6,039,948;5,891,992; 5,817,753; 5,698,201 ;5,106,619; 5,006,334; 4,855,285; 4,791,062; 4,767,842; and 4,762,913).
  • beta chain hCG or peptides thereof is mainly due to a concern that vaccination with dimer or whole hCG will cause undesirable cross-reaction against related glycoprotein hormones such as LH, FSH, and TSH with ensuing deleterious consequences to the host. So far no prior art references exist in which hCG dimer is combined with muramyl peptides.
  • the invention also contemplates methods of administering to a subject an amount of hCG in a formulation that is clinically more effective in treating or preventing a viral infection from which the subject suffers or is about to suffer.
  • methods are provided for treating cancer in subject animals such as humans by administering to said subjects the instant composition in a dose effective to eliminate or prevent growth of said cancer in said animals.
  • AIDS Kaposi's sarcoma (both endemic and HIV-associated types), multiple myeloma, lymphoma, melanoma and other types of carcinomas and leukemia.
  • compositions are advantageously formed by solutions which can be administered parenterally, especially subcutaneously, intramuscularly, intravenously, or by perfusion.
  • the composition of the invention can be equally and advantageously administered orally either alone or in a galenica form ensuring their protection, for example, encapsulated in capsule or microsphere or liposomes enabling the gastric barrier to be crossed.
  • the present invention is based on the discovery that, in spite of the fact that muramy dipeptides (MDPs) generally used as a component of vaccine preparations to induce immunity against antigenic determinant of a vaccine, these muramyl peptide compounds are actually capable of synergizing rather than antagonizing hCG activity in vivo. Therefore, in spite of the teaching in the art referred to above, muramyl peptide compounds in combinatior with hCG are useful in the treatment, prophylaxis or management of HIV infection and other life-threatening conditions like cancer.
  • MDPs muramy dipeptides
  • sustained delivery devices denotes specific devices like transdermal patches, subcutaneous implants, and pumps containing the drug.
  • slow release formulations denotes forms of drug delivery other than sustained delivery devices and comprises hCG formulations that have not been taught in the prior art relating to formulations of hCG.
  • adjuvant by definition is a substance that is incorporated into or injected simultaneously with an antigen.
  • the adjuvants potentiate non-specifically the ensuing immune response against antigen.
  • a principal purpose for employment of an immunotherapeutic adjuvant is to achieve more durable humoral or cell-mediated immunity of a high level by employing lower levels of an antigen with fewer numbers of doses than could be achieved by administering the equivalent aqueous antigen.
  • adjuvants are used in combination with non-living agents (in place of living microorganisms), for the preparation o vaccines.
  • Adjuvants may also increase the effective immune response against low or nonimmunogenic tumor cells or cells infected with intracellular agents that are already present in the body and are not adequately checked by naturally elicited immune responses.
  • emulsif ⁇ er denotes nonionic surface-active compounds derived from alkylene oxide and/or hexahydric alcohols and/or higher natural fatty acids such as esters or ester-ethers.
  • CFA complete Freund's Adjuvant
  • CFA is comprised of a mineral oil, an emulsifying/stabilizing agent such as Arlacel A, and killed mycobacteria such as Mycobacterium tuberculosis. Aqueous antigen/protein solutions are mixed with these components to create a water-in-oil emulsion.
  • CFA causes severe side effects, however, including pain, abscess formation, and fever, which prevent its use in either human or veterinary vaccines. The side effects are primarily due to the patient's reactions to the mycobacterial component of CFA.
  • IFA Incomplete Freund's Adjuvant
  • Muramyl dipeptide represents the minimal unit of the mycobacterial cell wall complex that generates the adjuvant activity observed with CFA.
  • Many synthetic analogues of MDP have been generated that exhibit a wide range of adjuvant potency and side effects.
  • Three analogues that may be especially useful as vaccine adjuvants are threonyl derivatives of MDP, n-butyl derivatives of MDP, and lipophilic derivatives of muramyl tripeptide (see U.S. Patent No. 5,709,879). These compounds effectively stimulate humoral and cell-mediated immunity and exhibit low levels of toxicity.
  • This muramyl peptide has a phospholipid tail that allow association of the hydrophobic portion of the molecule with a lipid environment while the muramyl peptide portion associates with the aqueous environment.
  • the MTP-PE itself can act as an emulsifying agent to generate stable oil- in-water emulsions.
  • a muramyl peptide has a clear meaning to those skilled in the art. In particular, it refers to a compound containing one or more sugar residues, at least one of the sugar residues, which will often be a muramic acid residue, being substituted with at least one or more (and usually two or more) amino acid residues.
  • Muramyl peptide compounds may be peptidoglycans which are capable of enhancing the cellular antigenicity response in mammals and which are prototype muramyl dipeptide (MDP) or analogues or derivatives thereof.
  • a group of muramyl peptide compounds have previously been disclosed as useful in treating or preventing progressive septicaemia and septic shock, as having immunopotentiating anti-bacterial activities.
  • Muramyl peptides are however are effective onl in immunocompetent hosts.
  • For representative muramyl peptides see for example U.S. Patem Nos. 5,506,204 and 5,534,492, incorporated herein by reference.
  • composition of a muramyl peptide compound in combination with water-soluble biologically active protein like hCG is provided.
  • hCG other therapeutic agents are contemplated including but no limited to insulin, glucagons, calcitonin, atrial naturetic peptide, secretin, cholecystokinin, thyrotrophin releasing thymopentin, adrenocorticotripic hormone, growth hormone releasing factor, enkephalin, oxytocin, vasopressin and luteinizing hormone releasing hormone.
  • compositions can also additionally formulated into a matrix material selected from the group consisting of chitosan, algin, saturated polyglycolysed glyceride, glycerol palmitostearate, saturated C12 to C22 fatty acid esters of poly alcohol, glyceryl and polyethylene glycol behenate, polyethylene oxide, and ammonium glycyrrhizinate among many others.
  • a matrix material selected from the group consisting of chitosan, algin, saturated polyglycolysed glyceride, glycerol palmitostearate, saturated C12 to C22 fatty acid esters of poly alcohol, glyceryl and polyethylene glycol behenate, polyethylene oxide, and ammonium glycyrrhizinate among many others.
  • Adjuvant is selected from the group consisting of monophosphoryl lipid A, lipid A, keyhole himpet hemocyanin, histidine-tag, alum, Freund's adjuvant, beta-gal, palmitic acid, saponin, lipopolysaccharide, BCG cell wall skeleton, trehalose monomycolate, trehalose dimycolate, lipid X, isoprinosine, lithosperman (A, B or C), and muramyl dipeptide (MDP) lipid conjugate.
  • the lipid can be saturated or unsaturated phospholipid or a glycolipid.
  • Preferred lipids are selected from the group consisting of 1 ,2-dimyristoylphosphatidylcholine 1,2-dipalmitoylphosphatidylcholine, 1,2-dimyristoylphosphatidylglycerol, cholesterol and combinations thereof.
  • Examples of solid lipids suitable for the preparation of the instant composition are triglycerides composed of natural, even-numbered and unbranched fatty acids with chain lengths in the C10-C18 range, or microcrystalline glycerol triesters of saturated, even- numbered and unbranched fatty acids of natural origin such as tricaprin, trilaurin, trimyristin, tripalmi in, and tristearin.
  • any lipid component or mixture of lipid components which provides a solid phase at room temperature (25 °C.) when measured in bulk is suitable for the lipid core.
  • the preferred phospholipids which can be used for making instant composition are natural phospholipids such as soybean lecithin, egg lecithin, phosphatidylglycerol, phosphatidylinositol, phosphatidyl-ethanolamine, phosphatidic acid, sphingomyelin, diphosphatidylglycerol, phosphatidylserine, phosphatidyl-choline, cardiolipin, etc.; synthetic phospholipids such as dimyristoylphosphatidylcholine, dimyristoyl-phosphatidylglycerol, distearoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, monophosphoryl lipid A, diphosphoryl lipid A, etc.; and hydrogenated or partially hydrogenated lecithins and phospholipids.
  • natural phospholipids such as soybean lecithin, egg lecithin, phosphatidylglycerol, phosphat
  • Non-natural surfactants and detergents optionally can be incorporated into composition of the invention in desired amounts.
  • surfactants or “detergents” include a wide variety of manmade molecules which form micelles in aqueous solution and contain both lipophilic and hydrophilic domains.
  • emulsifying agents comprise sorbitan esters, polyoxyethylene sorbitan mono-, di-, or triesters, polyoxyethylene fatty acids, polyoxyethylene fatty acid ethers, and combinations thereof.
  • non- natural surfactants include but are not limited to polysorbates (“TWEEN” or sorbitan monooleate), sodium dodecylsulfate (SDS), polyethoxylated castor oil (“CREMOPHOR”), NP-40, and numerous other synthetic molecules.
  • TWEEN polysorbates
  • SDS sodium dodecylsulfate
  • CREMOPHOR polyethoxylated castor oil
  • NP-40 polyethoxylated castor oil
  • DDA dimethyldioctadecyl ammonium bromide
  • POP-POE linear polyoxypropylene-polyoxyethylene block polymers
  • Emulsifiers also comprise Arlacel A or Arlacel 80 or Span 80 (so-called mannide monooleates). These surfactants can be used at various doses. For example, while preferred range of Arlacel A or Arlacel 80 or Span 80 in instant composition is between about 2 to 15% by weight, the range of Tween 80 for example would be 0.2 to 4% by weight. In general, surfactants can comprise less than 30% of the total weight of composition, more preferably less than 25%, still more preferably less than 10%, and most preferably less than 5%.
  • MDP Functional equivalents of MDP include but are not limited to muramyl di- or tripeptides like N-acetyl-glucosaminyl-N-acetyl-muramyl-L-alanyl-D-isoglutamine (GMDP), N-acetyl-D-glucosaminyl(beta- 1 -4)-N-acetyl-muramyl-L-alanyl-D-isoglutamine, N-acetyl- glucosaminyl-N-acetyl-muramyl-L-alanyl-D-glutamic acid (GMDP-A), muramyl dipeptide- phosphatidyl-ethanolamine, muramyltripeptide phosphatidylethanolamine, muramyl tripeptide phosphatidyl ethanol amine, CGP11637 (nor-MDP), alpha(N-acetyl-muramyl-L- alanyl-D
  • aminoacyl residue can be any of aminoacyl moiety selected from the group consisting of alanyl, valyl, leucyl, isoleucyl, .alpha.-aminobutyryl, threonyl, methionyl, cysteinyl, glutamyl, isoglutamyl, glutaminyl, isoglutaminyl, aspartyl, phenylalanyl, tyrosyl, tryptophanyl, lysyl, ornithinyl, arginyl, histidyl, asparinginyl, prolyl, hydroxypropyl, seryl, and glycyl groups.
  • muramyl peptide derivatives include those wherein an L-threonyl residue is substituted for the L-alanyl residue of muramyl peptide group. It will additionally be appreciated that it is also possible to use any other adjuvant muramyl peptides carrying substituents in positions 1, 4 or 6 of the saccharide group, provided they have the same favorable effects as the preferred muramyl peptides mentioned above. Without limiting to above derivatives of muramyl peptides those disclosed, for example, in U.S. Patent Nos.
  • compositions can comprise N-cyclohexanoyl arginine; a mixture of N-cyclohexanoyltyrosine and N-cyclohexanoylleucine; a mixture of N- phenylsulfonylvaline, N-phenylsulfonylleucine, N-phenylsulfonylphenylalanine, N- phenylsulfonyllysine, and N-phenylsulfonylarginine; and a mixture of N-benzoylvaline, N- benzoylleucine, N-benzoylphenylalanine, N-benzoyllysine, N-benzoylarginine, and a stabilizer like sodium 2-cyclohexylbutyrate.
  • composition can also comprise a microsphere.
  • a microsphere Polymer matrices for forming microspheres are well known in the art. For example, semipermeable microspheres containing enzymes, hormones, vaccines, and other biologicals are disclosed in U.S. Patent No. 5,643,605. Another U.S. Patent No.
  • 5,075,109 discloses a method of potentiating an immune response by administering a mixture of at least two populations of microspheres containing bioactive agents such that one of the microsphere populations is sized between about 1 to 10 microns.
  • U.S. Patent No. 4,293,539 discloses a controlled release formulation of an active ingredient in a copolymer derived from about 60 to 95 weight percent lactic acid and about 40 to about 4 weight percent gly colic acid.
  • U.S. Patent No. 4,919,929 discloses the administration of an antigenic substance in a shaped structure of a biocompatible matrix material.
  • 4,767,628 discloses a composition comprising an active, acid stable polypeptide and a polylactide, which when placed in an aqueous physiological environment release the polypeptide at an approximately constant rate in an essentially monophasic manner.
  • U.S. Patent No. 4,962,091 discloses a microsuspension of water soluble macromolecular polypeptides in a polylactide matrix.
  • U.S. Patent Nos. 4,849,228 and 4,728,721 disclose a biodegradable, high molecular weight polymer characterized in that the content of water-soluble low molecular weight compounds, as calculated on the assumption that such compounds are monobasic acids, is less than 0.01 mole per 100 grams of high molecular weight polymer.
  • Patent Nos. 4,902,515 and 4,719,246 disclose polylactide compositions containing segments of poly(R-lactide) interlocked with segments of poly(S-lactide).
  • U.S. Patent No. 4,990,336 discloses a multiphasic sustained release system comprising allergen extract encapsulated in microspheres of bioerodible encapsulating polymer which permits a sustained, multiphasic release of the allergen.
  • This system includes a first portion of allergen extract that upon injection is capable of being released in a manner whereby initial allergenicity is minimized to producing a mild local reaction similar to that normally observed with low doses of conventional allergen administration, and secondary portions of allergen extract that provide a substantially higher level of allergen extract in doses that could provide a serious reaction in the patient, but for the release of the first portion of allergen extract.
  • U.S. Patent No. 4,897,268 discloses a microcapsule delivery system wherein the ingredients are encapsulated in biodegradable copolymer excipients of varying mole ratios, such that delivery of the ingredients occurs at a constant rate over a prolonged period of time.
  • Microspheres of the invention also comprise liposomes.
  • Compositions suitable to form liposomes are well known in the technical literature which is abundant in this field.
  • Preferred lipid compositions are those which bring into play phospholipids, such as phosphatidylcholine (a derivative of fatty acids comprising from 12 to 20 carbon atoms) (particularly from 16 to 20 carbon atoms), phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidic acid.
  • phospholipids such as phosphatidylcholine (a derivative of fatty acids comprising from 12 to 20 carbon atoms) (particularly from 16 to 20 carbon atoms), phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidic acid.
  • phospholipids such as phosphatidyl
  • the liposomes are formed from mixtures containing the last mentioned phospholipids in a ratio of 7 volumes of DSPC or phosphatidylcholine (PC) to 1 to 10, preferably 3, volumes of PS.
  • the biological activities of the liposomes containing the derivatives of the invention are manifested as well when the liposomes are in the unilamellar or plurilamellar form.
  • the particles of liposomes have sizes greater than or equal to 0.1 micron, for example, comprised between 1 and 10 microns.
  • the compound of the invention is useful as a modulator of unspecific antimicrobial resistance for systemic enhancement of immune response and unspecific immunity.
  • the slow release hCG is contemplated for use in both RNA and DNA viruses. These would include, but not be limited to, Hepatitis viruses, Herpes viruses, flu viruses and Raft Valley Fever viruses. [0045] It will be apparent to one of ordinary skill in the art that the precise amounts of active ingredients needed to produce a given effect will vary with the particular compounds, and with the size, age, and condition of the subject to be treated. These amounts can easily be determined using routine methods known to those of ordinary skill in the art. [0046] The adjuvant compositions and vaccines of the invention are generally administered by injection. However, one can devise easier modes of administration like oral administration.
  • composition of the present invention can be used directly for purposes of clinical therapy and prophylaxis it can have the form of an oral preparation or a parenteral preparation.
  • parenteral includes subcutaneous, intravenous, epidural, irrigation, intramuscular, release pumps, or infusion.
  • the composition can be also administered intra-articularly, intrasynovially, intrathecally, periostally, intratumorally, peritumorally, intralesionally, perilesionally, sublingually, buccally, transdermally, topically or by inhalation. It also can be administered as a dressing for a wound or lesion. However, oral administration of the composition is especially preferred.
  • composition of the present invention can be used alone or in combination with pharmaceutically acceptable carriers to form pharmaceutical preparations such as capsules, pills, lozenges, tablets, dragees, sachets, tea bags, granules, powders, coated tablets, sugar coated tablets, wafers, sugar cubes, gels, hydrogels such as particles of a hydrophilic-hygroscopic polysaccharide, foams, suppositories, inhalants, juices, shakes, chewing gum, tooth paste, dentifrice, mouthwash, candies, and emulsions.
  • pharmaceutical preparations such as capsules, pills, lozenges, tablets, dragees, sachets, tea bags, granules, powders, coated tablets, sugar coated tablets, wafers, sugar cubes, gels, hydrogels such as particles of a hydrophilic-hygroscopic polysaccharide, foams, suppositories, inhalants, juices, shakes, chewing gum,
  • Suitable pharmaceutical carriers include, for example, fillers such as lactose, sucrose, mannitol, glucose, starch, sorbitol, glycine, calcium phosphate and microcrystalline cellulose; binders such as starch, casein, gelatin, acacia, glucose, sucrose, sorbitol, mannitol, tragacanth, hydroxypropylcellulose, hydroxypropoxymethylcellulose, carboxymethylcellulose, 2-methyl-5-vinylpyridine/methyl methacrylate/ethylacrylate copolymer, polyvinylpyrrolidone and sodium alginate; alginate gel; lubricants such as stearic acid, hardened oil, magnesium stearate, calcium stearate, polyoxyethylene monostearate, talc, silicon oxide and polyethylene glycol; disintegrators such as potato starch, and starch containing a surfactant or the like; facilitators like magnesium sulphate; and humectants such as sodium
  • composition of the present invention can also be administered in the form of liposomes.
  • liposomes or artificial lipid vesicles are generally derived from phospholipids or other lipid substances. Additionally they can contain muramyl peptide, a metabolizable oil, and optionally an additional emulsifying agent. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium.
  • a typical process for making a liposome preparation comprised of liposomes that contain an encapsulated composition of the invention comprises hydrating a lipid or liposome formulation with a solution of a material to be encapsulated; providing a plurality of portions of a dry lipid or dry liposome formulation; hydrating each of said plurality of portions with a solution comprising said material to be encapsulated; and combining each of said plurality of portions to form a single liposome preparation, thereby forming a liposome preparation comprising liposomes that contain said encapsulated material.
  • Any non-toxic, physiologically-acceptable and metabolizable lipid capable of forming liposomes can be used.
  • compositions in liposome form can contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients, and the like.
  • the preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic.
  • Methods to form liposomes are well known in the art. For example, cochleates containing biologically relevant molecule component, a negatively charged lipid component, and a divalent cation component.
  • the cochleate has an extended shelf life, even in a desiccated state.
  • the cochleate can be ingested.
  • the dosage to be used will depend of course on the nature and severity of the disease to be treated, the mode of administration and the compound form used.
  • a suitable dosage is from about 100 IU to about 20,000 IU, administered in a single dose or in separate doses. Repeated admimstration may conveniently be effected one to four times per day or in less frequent form, e.g., once every three days, once a week, or once every two weeks, or once a month or once every three months.
  • Indicated unit dosage forms include from about 100 IU to about 10,000 IU of compound of the invention in situations of repeated administration, preferably in about 1,000 to about 5,000 IU per injection.
  • compositions of the present invention can be employed in a variety of other ways unrelated to therapeutic indication.
  • the composition can be employed bot as labeled and unlabeled reagent in various immunoassays, bioassays, and the like, for the detection of hCG or antibodies to hCG.
  • Suitable labels include radioisotopes, enzymes, fluorescent molecules, chemiluminescent labels, enzyme substrates or co-factors, enzyme inhibitors, particles, dyes, and the like.
  • labeled reagents may be used in a variety of we] known assays, such as radioimmunoassays, enzyme immunoassays, e.g., ELISA, fluorescent immunoassays, and the like.
  • the invention also embraces the concept of using the instant composition along wit drugs or compounds commonly used in the prior art for various disease categories.
  • the following categories and/or members of the following categories are active agents belonging to: adrenocortical steroid; adrenocortical suppressant; aldosterone antagonist; amino acid; anabolic; androgen; anti-AIDS drugs; anthelmintic; anti- acne agent; anti-adrenergic; anti-allergic; anti-amebic; anti-androgen; anti-anemic; anti- anginal; anti-arthritic; anti-asthmatic; anti-atherosclerotic; antibacterial; anticholelithic; anticholelithogenic; anticholinergic; anticoagulant; anticoccidal; antidiabetic; antidiarrheal; antidiuretic; antidote; anti-estrogen; antifibrinolytic; antifungal; antiglaucoma agent;
  • the compounds useful in the invention may be delivered in the form of drug cocktails.
  • a cocktail is a mixture of any one of the above compounds combined with the composition of the invention. The mixture does not need to be mixed physically and drugs can be administered separately, sequentially, or at the same time.
  • Antineoplastic drugs include but are not limited to: Acivicin; Aclarubicin; Acodazole Hydrochloride; AcrQnine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar
  • anti-neoplastic compounds include: 20-epi-l,25 dihydroxyvitamin D3; 5- ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; atrsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing mo ⁇ hogenetic protein- 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-
  • hCG water-soluble proteins and peptides
  • a protein is a growth hormone or a somatotropin, e.g., human growth hormone (HGH), bovine growth hormone (BGH or BST), porcine growth hormone (PGH or PST), their analogues and derivatives and epidermal growth factor (FGF) and its analogues.
  • HGH human growth hormone
  • BGH or BST bovine growth hormone
  • PST porcine growth hormone
  • FGF epidermal growth factor
  • Other contemplated proteins are interleukins, interleukin receptors and interleukin receptor agonists, chemokines, and interferons.
  • protein can be selected from the group consisting of blood cell growth stimulating factors and precursors and erythropoeitin (EPO) and its analogues.
  • Other equally desirable protein are parathyroid hormone (PTH), selenoprotein P, cystatin B and its liver thiol protease inhibitor analogues, endotoxin neutralizing protein, lymphocyte migration inhibitory factor (LIF), mast cell growth factor (MGF), megakaryocyte simulatory factor (MGDF), granulocyte macrophage colony stimulating factor (GM-CSF), genofibrate, alpha calcitonin, beta calcitonin, tumor necrosis factor (TNF), tumor invasion inhibiting factors, TGF-beta-type cytokines, transacting regulatory proteins (TAT's) of HIV and other retroviruses, protease inhibitors, and BPC 157, fat reducing hormones, and analogues and variants of these proteins.
  • PTH parathyroid hormone
  • SMF mast cell growth factor
  • cytokines are known and are equally suitable for the composition of the invention.
  • polypeptides are contemplated such as G-CSF, M-CSF, LIF, inhibin A, inhibin B, activin A, activin B, NAP-1, MCP-1, MIP- lalpha, MlP-lbeta, MIP-2, SISbeta, SISdelta, SISepsilon, PF4, PBP, gammaIP-10, MGSA, aFGF, bFGF, KGF, PDGF-A, PDGF-B, PD-ECGF, INS, IGF-I, IGF-II, NGF-beta, GRO/MGSA, PF4, PBP/CTAP/.beta.TG, IP-10, KC, 9E3, MCAF, ACT-2/PAT 744/G26, LD-78/ PAT 464, RANTES, G26, 1309, JE, TCA3, ICAM-1, ICAM-2, LFA-1, LFA
  • GDF-1 TGF- beta family
  • PKC-delta protein kinase C delta 1385 M13945 2713- 69042
  • Interleukin 12 beta chain Z22703 63-325 T-lymphocyte activated protein M86671 652-963 Activm type I receptor M31042 285-606 Vascular endothelial growth Z31663 847-1130 Neuronal-cadhe ⁇ n (N-cadhenn) factor Transcription factor TF II D M31131 1212-
  • CD 30L receptor (Lymphocyte Bax, Bcl-2 heterodime ⁇ zation 1728
  • Leukemia inhibitory factor L27105 2175-2400 binding protein 1 X06381 63-366 like protein
  • shwannomm XRCC1 DNA-repair protein, affecting hgation Angiotensin-converting enzyme Nucleobindm M96823 8C
  • PAX-8 (paired box protem PAX sulfated glycoprotem-2 S69336 832-108'
  • X57487 680-1011 associated gamma receptor accessory facto CamK IV, Ca2/calmodul ⁇ n- L12721 404-709 1 X58995 1269- protein (AF-1) 1608 Epidermal growth factor receptor Transc ⁇ ptional enhancer factor dependent protein kinase IV L21671 1562-1873 S74227 934-123: (catalytic chain) kinase substrate EPS8 (TEF-1)
  • Nm23-M2 nucleoside Lymphocyte-specific tyrosine- L- diphosphate M12056 1205- type) beta-3 subunit
  • MAPKK6 MAP kinase kinase 2 1) inhibitor family, candidate tumo X97052 375-711 M26283 647-884 suppressor gene 6(dual specificity) (MKK6) Zinc finger X-chromosomal snoN, ski-related oncogene DNA polymerase alpha catalytic protem U36203 671-100 ⁇ D 17384 563-908 M32309 2153- Homeo Box protem 7 1 (Hox- subunit (pi 80) 2554 7 1) Caspase-3, Nedd2 cysteme (ZFX) X14759 740-992 protease WTI, Wilms tumor protein, Neuronal cell surface protein F3
  • Bcl-xL apoptosis regulator (bcl-x Rad6 homologue; murine HR6B ERCC-1; DNA excision repair L35049 641-906 Relaxin Z27088 51-365 X07414 189-484 long); BcI-2 family member
  • Fasl Fas antigen ligand
  • protease upstream regulator of related generalized ICE X60671 1571-
  • X92411 542-807 M57999 3122- protem homologue xeroderma Thrombopoietin L34169 3417 pigmentosum group C (XPC) 652-954 factor) (TFDB5) repair Transforming growth factor beta Tumor necrosis factor receptor 1; complementing protein M13177 772-1075 M59378 1961- Integrm beta Y00769 1990- Granulocyte colony- stimulating 2376 2320 M13926 86-377 TNFR-1
  • MmRad52 yeast DNA repair factor (G-CSF) PDGFRa; platelet-derived growth protein Neuroleukin Ml 4220 M84607 474-803
  • L03529 762-1154 membrane- IGFR II; insulin-like growth (thrombin) receptor M27959 2469- factor PTPRG; protein-tyrosme 2705 U04710 707-1060
  • CD 4 receptor T cell activation colony stimulating factor 1 (CSF- D29951 2553- X04836 1652- 1) 2830 1877 receptor) Fibroblast growth factor 9 antigene) B-myb proto-oncogene, myb- D38258 91-379
  • Interleukin 6 B cell 1376 Interferon regulatory factor 2 differentiation oncogene (IRF 2)
  • Androgen receptor X53779 receptor NF-IB protein transcription 2189-2491 Glutamate receptor, tonotropic factor
  • Bone morphogenetic protein 4 D10651 506-786
  • D10217 3966- factor Transferrin receptor protein p90, 4209 Tissue plasminogen activator X57349 654-1023
  • NMD A2 A epsilon 1 J03520 622-1020 CD71
  • CD7 antigen D 10329 28- Romeo Box protein 4 2 (Rox-4 2)
  • TNF 55 tumor necrosis factor 1 receptor J04115 951-1238 X57796 656-1022 D 16250 1454- factor AP-1 component) (55kd) 1837 Se ⁇ ne protease inhibitor
  • MTI1 DnaJ-like heat-shock 354-602 Mast cell protease (MMCP) - 4 protein Homeo Box protein 1.1 (Hox- M55617 634-992
  • L13968 1052-1292 tumor suppressor gene; cell pathway member Interleukin-converting enzyme cycle p58/GTA; galactosyltransferase (TCE) regulator
  • TCE galactosyltransferase
  • Etkl (Mek4, HEK) tyrosine- Statl, signal transducer and 1749 protem activator Jnk activating kinase 1, (JNKK1,
  • Retinoic acid receptor RXR- VRL Von Hippel-Lindau tumor polarity gamma U12570 885-1111 U24160 1205-
  • U34960 515-834 TDAG51 couples TCR signaling c-Fos proto-oncogene; I-kB (I-kappa B) alpha chain to transcription
  • CTCF Transcription factor
  • FAF1 Fas-associated protein zmc Cytotoxic cell protease 2 (B10) factor
  • U51037 1625- X12822 439-686 Transcription factor CTCF (11 2598 FAF1, Fas-associated protein zmc Cytotoxic cell protease 2 (B10) factor
  • F ⁇ zzled-3 Drosophila tissue family member receptor. 894 X66032 874-1236 protein-4 (IGFBP-4)
  • HMG-14 non histone Gaddl53 growth arrest and protein-1 (IGFBP-1) chromosomal DNA- IGFBP-2; Insulin-like growth
  • X63190 1702- X74351 447-669 Z11974 807-1197 2040 group A correcting protein Rab-2 ras-related protein Vav; GDP-GTP exchange factor; Integrin alpha 2 (CD49b) X95403 232-505 X64361 1083- X75427 1595- Gluthathione S-transferase (theta 1351 1976 X98055 14-298 proto-oncogene Growth/diffferentiation factor 2 typel); phase II conjugation PAX-6 (paired box protein) X77113 939-1329 enzyme X63963 1081- (GDF-2) Zyxin; LIM domain protein; 1325 Insulin-like growth factor binding alpha- 1812 Stat5a, mammary gland factor Retinoid X receptor interacting actimn binding protem Z48538 2269-2628 U09419 1388- Met protooncogene Y00671 Hek2 murine homologue, Mdk5 16
  • Cytoskeletal epidermal keratin inflammatory protein- 1 alpha (homeodian transcription factor) (14 receptor, MIP-1 alpha-R, PLC gamma, phosphohpase C M13806 108-469 RANTES-R
  • Glucocorticoid receptor form A gamma R-ras protein, closely related to X13358 1527- Stromelysm-3 matrix ras 1816
  • Folhstatin Z29532 764- factor LCR-1, CXCR-4, CXC(SDF-l) 1053 GATA binding transcription D87747 584-867 Cyclin F (S/G2/M-spec ⁇ f ⁇ c) factor chemokine receptor 4, HIV
  • Rab-3b ras related protein CD31 (Platelet endothelial cell X62700 482-756
  • composition of this invention can be prepared in a variety of ways well known to those of skill in the art.
  • hydrophile-lipophile values required for various applications are as follows: 3 to 6 water-in oil emulsifier (e.g., Arlacel+Squalene); 7 to 9 wetting agent; 8 to 13 oil-in-water emulsifier; 13 to 15 detergent; 15 to 18 solubilizer (e.g., aluminum or magnesium stearate).
  • solubilizer e.g., aluminum or magnesium stearate
  • Analogous systems have been developed for anionic or cationic emulsifiers, but they are less useful than those for nonionic emulsifiers.
  • Hydrophilic- lipophilic balance numbers have been published for many nonionic. For this particular use an oil-in-water emulsion that has about 0.5 to 55% of an oil, about 0.1 to 15% of an emulsifier, about 0.05 to 5% of a nonionic surfactant, about 0.00001 to 10% of a therapeutic agent, and an aqueous continuous phase is most suitable.
  • the emulsifier in the composition is a phospholipid compound or a mixture of phospholipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin.
  • emulsifier is Arlacel A.
  • Surfactant is usually selected from the group consisting of fatty alcohols, polyethylene gly col esters of fatty acids, polyethoxylated fatty acid esters, polyethoxylated fatty alcohol ethers, an alkylene oxide condensate of an organic compound having one or more hydroxyl groups, and polyethoxylated alkylphenyl ethers.
  • Surfactant can be a natural biologically compatible surfactant such as lecithin or a pharmaceutically acceptable non-natural surfactant such as TWEEN-80.
  • Suitable natural components relating to lecithin are for example EPICURON 120 (Lucas Meyer, Germany) which is a mixture of about 70% of phosphatidylcholine, 12% phosphatidylethanolamine and about 15% other phospholipids; OVOTHIN 160 (Lucas Meyer, Germany) which is a mixture comprising about
  • phospholipid mixture 60%o phosphatidylcholine, 18%) phosphatidylethanolamine and 12%o other phospholipids; a purified phospholipid mixture-LIPOID E-75 or LIPOID E-80 (Lipoid, Germany) which is a phospholipid mixture comprising about 80% phosphatidylcholine, 8% phosphatidylethanolamine, 3.6% non-polar lipids and about 2% sphingomyelin. Purified egg yolk phospholipids, soybean oil phospholipids or other purified phospholipid mixtures are useful as this component. This listing is representative and not limiting, as other phospholipid materials which are known to those skilled in the art can be used.
  • surfactants are polyethylene glycol esters of fatty acids from sources such as castor oil
  • EULFOR polyethoxylated fatty acid, e.g., stearic acid
  • SIMULSOL M-53 polyethoxylated fatty acid, e.g., stearic acid
  • NONIDET polyethoxylated isooctylphenol/formaldehyde polymer
  • TYLOXAPOL polyethoxylated isooctylphenol/formaldehyde polymer
  • TYLOXAPOL polyoxyethylene fatty alcohol ethers
  • BTIJ polyoxyethylene nonphenyl ethers
  • TRITON N polyoxyethylene isooctylphenyl ethers
  • the emulsion may be formed and stabilized in the substantial absence of one or more cosurfactants selected from the group consisting of an unhalogenated aliphatic C3-C6 alcohol, a free fatty acid, a mono- or di-glyceride, a polyglycerol fatty acid ester, or a lysophosphatidyl choline.
  • cosurfactants selected from the group consisting of an unhalogenated aliphatic C3-C6 alcohol, a free fatty acid, a mono- or di-glyceride, a polyglycerol fatty acid ester, or a lysophosphatidyl choline.
  • cosurfactants selected from the group consisting of an unhalogenated aliphatic C3-C6 alcohol, a free fatty acid, a mono- or di-glyceride, a polyglycerol fatty acid ester, or a lysophosphatidyl choline.
  • alternative composition is equally
  • composition is prepared immediately prior to administration or refrigerated at 4°C.
  • Composition 2 will for example contain 5.0% TETRONIC 1501, 10% Squalane, 0.4%
  • Thr-MDP N-acetylmuramyl-L-threonyl-D-isoglutamine
  • concentration 500 ug/ml
  • the concentrate is then mixed with a 2x concentration solution of antigen (hCG in saline, 1 mg/mL) to form the formulation of the invention.
  • hCG is preferably present in an amount of about 0.00001 to 10%> by weight of the composition, preferably about 0.0001 to 5% and most preferably 0.001 to 1%.
  • the pH should be in a range which is suitable for the stability of hCG.
  • the continuous phase of the composition is aqueous, and can additionally contain salts, sugars, antioxidants, preservatives, microbicides, buffers, osmoticants, cryoprotectants, and other pharmaceutically useful additives or solutes.
  • Typical preservatives include Thimerosal, chlorbutanol, and methyl, ethyl, propyl or butyl
  • Typical osmotic pressure regulators include glycerol and mannitol, with glycerol being preferred.
  • the preferred oil phase antioxidant is alpha-tocopherol or alpha-tocopherol succinate.
  • the aqueous phase may also include an antioxidant of a polyamine carboxylic acid such as ethylene diamfno tetraacetic acid, or a pharmaceutically acceptable salt thereof.
  • the instant composition can consist of two parts. Part I is a N-Acetylmuramyl-L-
  • Threonyl-D-Isoglutamine Thr-MDP
  • Part II consists of phosphate buffered saline containing a final concentration of 5% Squalane,
  • mice are injected with soluble hCG mixed with alum according to the manufacturer's manual (Pierce
  • mice model is indicative that equivalent experiments or treatments will similarly induce clinical responses in humans, domesticated, or agricultural animals.
  • the amount of formulation and hCG useful to produce the desired clinical response can be determined empirically, without undue experimentation, by routine procedures well known to those of ordinary skill in the art.
  • those of ordinary skill in the art can determine a minimum level of such a mixture for administration to a human, domesticated, or agricultural animal in order to obtain an effective response, and thereby induce desired clinical outcome.
  • such a mixture will be injected by any one of a number of standard procedures, but particularly preferred is an subcutaneous or intramuscular injection at a location which will allow the emulsion to remain in a stable form for a period of several days or several weeks.
  • the hCG aqueous suspension is added in an oily phase at room temperature while simultaneously homogenizing with a homogenizer. The addition of the aqueous phase is stopped when the ratio of 5-6 parts of oil to 3-5 parts of hCG suspension is reached.
  • the oily phase contains following: Marcol 52 (white paraffmic Esso oil)--93.6%>;
  • the compounds of the oily phase can be separately heated to 110° C in the autoclave or sterile filtered as a mixture.
  • the stability of emulsion is determined by (1) pipetting drops directly after emulsifying onto a water surface, whereby the drops do not spread, but remain intact; and (2) storage at 37°C for 4 weeks does not lead to formation of any aqueous phase.
  • the final concentrations of the thus prepared emulsion contains about 10%> of hCG by weight and the preparation is used in a dosage of 0.5 ml per subject intramuscularly or subcutaneously.
  • composition comprising dimer hCG can contain two emulsifiers at the same time to provide better stability.
  • primary emulsifier is selected from the group consisting of polyoxypropylene- polyoxyethylene block copolymers, glycerol monooleate, glycerol dioleate, sorbitan sesquioleate, Brij 93 polyoxyethylene alcohol, sorbitan monooleate and mixtures thereof and secondary emulsifier is selected from the group consisting of polyoxyethylene fatty acid esters, Pluronic F68 ethylene oxide, egg lecithin and mixtures thereof.
  • the oil fraction in this composition is a triglyceride selected from the group consisting of tricaproin, tricaprylin, tripalmitin and mixtures thereof.
  • Other suitable oils include vegetable and animal oils.
  • Suitable vegetable oils include; olive, safflower, sesame and soybean and suitable animal oils include those containing glycerol esters.
  • Preferred mineral oils include: No. 40 white oil,
  • Carnation light oil and Klearol light oil Mixtures of these oils can be equally used.
  • the aqueous hCG solution in an amount of about 1 to about 50 volume percent, preferably about 2 to about 40 volume percent; mineral oil in an amount of about 8 to about 58 volume percent, preferably about 14 to about 25 volume percent; and primary emulsifying agent in an amount of about 2 to about 30 volume percent, preferably about 5 to about 15 volume percent are mixed.
  • the aqueous hCG solution is preferably slowly added to the rapidly stirring oil phase and mixed, such as by use of a magnetic stirrer, for about 15 minutes to about 60 minutes and preferably about 25 minutes to about 35 minutes.
  • the mixed primary emulsion is then subjected to high shear emulsification providing shear rates of about 100,000 to about 5,000,000 and preferably about 500,000 to about 1,000,000 per second such as by using a microfluidizer at a pressure drop of about 1000 to about 10,000 psi, preferably about 1000 to about 3000 psi, and most preferably about 1800 to about 2000 psi. More complete information regarding the microfluidizer is set forth in U.S.
  • the microfluidizer provides high sheer rates of short duration, a fraction of a second, without denaturation of proteins, such as hCG.
  • the primary emulsion is filtered, such as by using a 5 micron hydrophilic poly vinylidene difluoride filter (Duropore, Millipore
  • Albumin can be added to the hCG solution prior to emulsification in amounts of about
  • the primary emulsion of aqueous hCG in mineral or fixed oil to be suitable for preparation of the liquid multiple emulsion of this invention should result in primary emulsion droplet diameters in the range of less than 5, and preferably less than 3, microns.
  • a water-in-oil-in- water multiple emulsion consisting of 50% v/v of an outer aqueous phase of saline with 0.25% copolymer P123 is prepared.
  • the other 50%) v/v is dispersed water-in-oil phase consisting of 72%) saline, 18% Squalene, 2% Span 80 and 32 mg copolymer L310 and 0.5 mg TNP-HEA per 0.5 ml of emulsion.
  • the emulsion contains lmg of hCG in 0.5 ml emulsion.
  • the microscopic appearance of the emulsion is 1.0 to 20 micron diameter particles of water-in-oil emulsion.
  • Other water-in-oil emulsifiers equally suitable for this composition are SF 1328, Arlacel P135, DC 3225C, DC 5200, Abil EM-90, and Abil
  • Liposomes containing hCG are prepared according to the standard procedures well known in the art (see for example U.S. Patent No . 6, 110,492) . Following components can be employed: DLPC dilauroyl phosphatidyl-choline; DMPC dimyristoyl phosphatidyl-choline;
  • multilamellar liposomes are made from a mixture of DMPC:DMPG:CHOL:LA in a molar ratio of
  • the lipid A is included as an adjuvant.
  • the lipid mixture is rotary evaporated to a dry thin film at approximately 40 °C in vacuo from a chloroform solution in a pear-shaped flask. To ensure complete removal of the organic solvent, the flask is then dried under very low vacuum (about 0.05 mm Hg) overnight in a desiccator at room temperature. After drying, the lipids are carefully swollen in deionized, sterile pyrogen-free water by vortexing. The resulting suspension is frozen at -55 ° C lyophilized at -20°C overnight and 0°C -10 ° C the following day using the Virtis Unitop 800SL Freeze Mobile.
  • the lyophilized lipids are then reconstituted in the presence of the substance, i.e., hCG, to be encapsulated to obtain multilamellar liposomes containing this substance.
  • a suitable reconstituting buffer is phosphate-buffered saline (PBS) or Tris-glycine/NaCl.
  • PBS phosphate-buffered saline
  • Tris-glycine/NaCl Tris-glycine/NaCl.
  • the liposomal phospholipid concentration in the reconstituting buffer is 10-200 mM.
  • Unencapsulated hCG may be removed by washing the liposomes three times with 0.15 M NaCl at 27000xg for 10 minutes at 10°C.
  • the resulting liposomes are suspended either in 0.15 M NaCl or an appropriate isotonic buffer to reach a final phospholipid concentration of 10-200 mM.
  • the wash step may be omitted, leaving both unencapsulated and encapsulated hCG in the preparation.
  • This example allows coupling of hCG to MDP.
  • This procedure calls for quenching the first reaction with a thiol compound.
  • the reaction is carried out in 2-[N- morpholino] ethane sulfonic acid (MES) (pH 4.5-5.0).
  • MES 2-[N- morpholino] ethane sulfonic acid
  • MDP (10 mg) lyophilized from water resuspended in MES (0.5 ml) (pH 4.5-5.0) and 0.5 ml EDC (0.5 mg or about 2 mM) dissolved in MES (pH 4.5-5.0) are combined and reacted for 15 minutes at room temperature.
  • 2- mercaptoethanol final concentration of 20 mM is added to quench the EDC and separated by centrifugation.
  • the reaction mixture is washed once with MES and resuspended in 0.5 ml
  • MES pH 4.5-5.0
  • the hCG dissolved in MES is added to the activated MDP at a molar ratio of about 2:1.
  • the PH of the reaction is slowly increased over a 15 minute period to 8.5 by the addition of MES (0.5M pH 8.5) and reacted for 2 hours at room temperature.
  • the concentration of hCG added to MDP was calculated from quantitative analysis of MDP terminal carboxy group and expressed as mole per mg MDP.
  • the reaction is quenched by adding hydroxylamine to a final concentration of 10 mM. This method of quenching hydrolyzed any unreacted MDP activation sites and results in regeneration of the original carboxyls.
  • composition of this invention can also be used as a cream which upon topical application can penetrate across the skin.
  • An exemplary topical composition can contain following: 2% glycerol sorbitan oleostearate (Arlacel 481); 6% polyethoxylated fatty acids
  • mice of C3H background (H2k/k, Harlan Sprague Dawley) are used in these studies. Animals are maintained according to "Guide for the Care and Use of Laboratory
  • HOPE2 The tumor cell line HOPE2 is maintained by serial passage in vitro. Tumors are initiated in syngeneic C3H mice by subcutaneous injection of 150,000 in vitro passaged cells. Tumors are measured in 2 perpendicular directions at biweekly intervals. Each treatment group is compared to a control group that did not receive therapy. Therapy began 10 days after inoculation of HOPE2 cells, when a majority of the tumors are palpable (approx. 50-75 mm 3 ). Therapy is initiated by injecting mice with soluble hCG protein in MDP mixture or Alum adjuvants (subcutaneously in a total volume of 0.2 ml). Shortly before inoculation, hCG is mixed for 60 seconds MDP in
  • HBSS Hanks Balanced Salt Solution
  • the present invention can be characterized as a pharmaceutical formulation comprising a dimer hCG and a muramyl peptide.
  • the invention can be characterized as a therapeutic composition, comprising: (a) a therapeutically effective amount of dimer hCG in association with liposomes; and (b) an oil-in-water emulsion comprising an oil surrounding said liposomes, said emulsion being present in an amount sufficient to increase therapeutic response relative to that of hCG and liposomes in the absence of said emulsion.
  • the invention provides a method of treating HIV infection in a subject in need thereof, comprising administering an effective amount of hCG to said subject, said hCG being administered with a oil-in-water emulsion, and said emulsion is present in an amount sufficient to increase a clinical response relative to that of said hCG in the absence of said emulsion.
  • the invention further provides a method of treating cancer in a subject in need thereof, comprising administering an effective amount of hCG to said subject, said hCG being administered with a oil-in-water emulsion, and said emulsion is present in an amount sufficient to increase a clinical response relative to that of said hCG in the absence of said emulsion.
  • the invention still further provides a therapeutic composition which comprises an oil-in-water emulsion having a continuous water phase and a discontinuous oil phase and containing at least 0.01% by weight of dimer hCG.
  • the invention also provides a method for preparing a pharmaceutical formulation, which method comprises admixing, by supplying, an aqueous suspension comprising hCG and at least one oily ingredient and at least one emulsifier in a sufficient quantity to provide said formulation as an oil-in-water emulsion.
  • the invention still further provides a therapeutic composition which comprises an oil-in- water emulsion and at least one natural or recombinant water-soluble biologically active protein and optionally at least one muramyl peptide.
  • the invention also provides a kit for extemporaneous preparation of a pharmaceutical composition, said kit comprising: (1) a first container containing an emulsion, where said emulsion comprises: squalane or squalene; Arlacel, and an aqueous solution; and (2) a second container containing therapeutically effective amount of dimer hCG in an aqueous solution or as a powder, where the concentrations of the components in each container are selected such that combination of the contents of both containers produces a formulation comprising said Arlacel in an amount of between 1%> and 30%; squalane or squalene in an amount of between 1%> and 30%; muramyl peptide in an amount of between 0.0001% and 30%; and hCG in amount between 0.01% and

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Abstract

The invention relates to a pharmaceutical composition comprising a biologically active protein or a peptide formulated in water-in-oil emulsion with or without muramyl peptide. More specifically it relates to a preparation of human chorionic gonadotropin (hCG) and related hormones. Also provides are methods of making the composition; methods of using the same against viral infections, immune disorders, and cancer.

Description

HCG FORMULATION
TECHNICAL FIELD
[0001] This invention relates generally to a pharmaceutical formulation of water-soluble, biologically active proteins and polypeptides like hCG hormone which are useful for treating various diseases. In particular virus-induced, immune, and malignant diseases are preferred diseases to be treated by the composition of this invention. More specifically diseases originated by a virus like human immunodeficiency virus (FAN) which causes acquired immunodeficiency syndrome (AIDS) and associated cancers in humans are contemplated. BACKGROUND INFORMATION AND DISCUSSION OF RELATED ART [0002] Human chorionic gonadotropin (hCG) belongs to a family of glycoprotein hormones, which includes luteinizing hormone (lutropin, LH), follitropin (FSH), and thyrotropin (TSH). Each of these hormones is composed of two dissimilar, noncovalently bound subunits, alpha and beta. The hormones share a common alpha subunit, while the beta subunits differ in length and amino acid sequence and unique to each hormone. The most closely hormones are hCG and LH, which are 85% identical, except for an amino acid sequence at the carboxy terminus of hCG. hCG and LH act through a common receptor. hCG and related hormones are conventionally believed to play a^ole rn^δfϊrϊalTϊuman physiology-r For example, hCG is well established as pregnancy hormone which plays an important role in reproductive physiology during gestation. An extensive background information on hCG is provided in a review article by the present inventor (see Hernan F. Acevedo "Human chorionic gonadotropin (hCG): The hormone of life and death, A review" as incorporated by reference).
[0003] Recently, hCG hormone has been proposed by others as useful for HIV/AIDS and cancer therapy. See for example U.S. Patent Nos. 5,700,781; 5,811,390; 5,851,997; 5,997,871; 5,877,148; and 5,677,275 or original publications by Bourinbaiar AS, Nagorny R. Effect of human chorionic gonadotropin (hCG) on reverse transcriptase activity in HIN-1 infected lymphocytes and monocytes. FEMS Microbiol Lett. 1992 Sep l;75(l):27-30 and Bourinbaiar AS, Νagorny R. Inhibitory effect of human chorionic gonadotropin (hCG) on HIN-1 transmission from lymphocytes to trophoblasts. FEBS Lett. 1992 Aug 31 ;309(1). -82-4 the content of which is incorporated herein by reference. Some investigators, however, indicated that a contaminating factor found in commercial hCG preparations was responsible for anti-HIV and anti-cancer activity, refuting the idea that hCG can display antiviral and a cancer activity. See, for example, Lee-Huang S, Huang PL, Sun Y, Huang PL, Kung HF, Blithe DL, Chen HC. Lysozyme and RNases as anti-HIV components in beta-core preparations of human chorionic gonadotropin. Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2678-81; Sairam MR, Antaldy T. Debunking hCG. Nat Biotechnol. 1997 Nov (12):1228; Albini A, Paglieri I, Orengo G, Carlone S, Aluigi MG, DeMarchi R, Matteucci C Mantovani A, Carozzi F, Donini S, Benelli R. The beta-core fragment of human chorionic gonadotrophin inhibits growth of Kaposi's sarcoma-derived cells and a new immortalized Kaposi's sarcoma cell line. AIDS. 1997 May;l 1(6):713-21; Lunardi-Iskandar Y, Bryant JL, Blattner WA, Hung CL, Flamand L, Gill P, Hermans P, Birken S, Gallo RC. Effects of a urinary factor from women in early pregnancy on HIV-1, SIV and associated disease. Nat Med. 1998 Apr;4(4):428-34; Sipsas NV, Aroni K, Tsavaris N, Mavragani K, Paikos S, Kordossis T. AIDS-related cutaneous Kaposi's sarcoma: failure of treatment with human chorionic gonadotropin. J Chemother. 1999 Feb;ll(l):78-9; Masood R, McGarvey ME, Zheng T, Cai J, Arora N, Smith DL, Sloane N, Gill PS. Antineoplastic urinary protein inhibi Kaposi's sarcoma and angiogenesis in vitro and in vivo. Blood. 1999 Feb l;93(3):1038-44; Griffiths SJ, Adams DJ, Talbot SJ. Ribonuclease inhibits Kaposi's sarcoma. Nature. 1997 D< 11;390(6660):568; Darzynkiewicz Z. The butler did it: search for killer(s) of Kaposi's sarcoma cells in preparations of human chorionic gonadotropin. JNatl Cancer Inst. 1999 Jar 20;91 (2): 104-6; and Noonan D, Albini A. Anti-KS activity still a mystery. Nat Med. 1998 Jul;4(7):748 the abstracts of which are incorporated herein by reference. Due to confusing results several authors proposed that the urine from pregnant women and commercial preparations of crude clinical grade hCG contain various other factors with anti-Kaposi's sarcoma and anti-HIV activity. Researchers found in urine factors like eosinophil-derived neurotoxin (EDN), anti-neoplastic urinary protein (ANUP), inhibin, activin A, and angiostat among many others. Furthermore, some authors cautioned overtly that hCG can have opposi effect and may instead promote carcinogenesis, thus effectively teaching away from the usefulness of hCG as anticancer or antiviral agent. See for example Simonart et al. (Simonat T, Hermans P, Van Vooren JP, Meuris S. Paradoxical pro-Kaposi's sarcoma activity of preparations of human chorionic gonadotropin. Blood. 1999 Jul l;94(l):376-7). [0004] While there is still considerable controversy in the prior art as to whether hCG or some contaminating factor is responsible for antiviral/anti-cancer activity the use of this hormone or contaminating factor in commercial hCG preparation is inconvenient from the practical point of view. hCG as many other water-soluble glycoprotein hormones is rapidly metabolized in the body and thus its half-life is relatively short, which necessitates frequent injections of the hormone or requires very large loading doses of hCG, both of which are impractical and expensive. See U.S. Patent No. 5,700,781 "Method for treating Kaposi's sarcoma and HIV infections" issued to Harris and incorporated herein by reference. [0005] Modified forms of hCG delivery so-called "slow release" or "sustained release" forms of hCG have been proposed earlier but none have been successfully implemented since there is no specific enabling guidance with these particular forms of the hormone. See for example U.S. Patent No. 5,851,997 "Use of human chorionic gonadotropin as an immune- potentiating antiviral agent" issued to Harris. In this patent Harris cites various forms of slow release methods for hCG. Specifically, Harris teaches sustained-release form of hCG provided as a transdermal hCG patch, after the fashion of the DURAGESIC™ fentanyl patch so that transdermal delivery of proteins like hCG is accomplished iontophoretically or electroosmotically, i.e., under the influence of an electric field. Another sustained-release form of hCG contemplated by Harris is an implantable hCG delivery system. In the manner of the NORPLANT™ levonorgestrol implant, a representative type of device in this category it employs passive release of hCG through a non-biodegradable, rate-limiting membrane element composed, for instance, of a hydrogel or a microporous polymer. Another type of hCG implant contemplated by Harris incorporates a pump function to administer the hormone. The pumping action is osmotically driven or patient-activated. Thus, Harris taught various alternative modes of hCG administration. However, contemplated modes of delivery are through specific sustained-release drug delivery devices, i.e., transdermal skin patches and different types of implants or pumps. No other forms of hCG delivery are taught as reliable and practical modes of drug delivery.
[0006] It has been reported by these inventors and others that muramyl peptides alone can inhibit retroviral replication (Acevedo HF, Raikow RB, Acevedo HO, Delgado TF, Pardo M. Prevention of oncogenic viral infections in mice with CGP 11637, a synthetic muramyl dipeptide analog. Antimicrob Agents Chemother. 1985 Nov;28(5):589-96; Lazdins JK, Woods-Cook K, Walker M, Alteri E. The lipophilic muramyl peptide MTP-PE is a potent inhibitor of HIV replication in macrophages. AIDS Res Hum Retroviruses. 1990 Oct;6(10):1157-61; Masihi KN, Lange W, Rohde-Schulz B, Chedid L. Muramyl dipeptide inhibits replication of human immunodeficiency virus in vitro. AIDS Res Hum Retroviruses. 1990 Mar;6(3):393-9).
[0007] However, the combination of hCG with muramyl peptides is opposite to the current thinking in the field since muramyl peptides are mostly used as vaccine adjuvants to enhance the immune reaction against the antigen, i.e., hCG, present in the vaccine preparation (see for example U.S. Patent Nos. 5,840,313 or 5,876,724) and thus this combination would effectively render hCG inactive or may even exacerbate the course of the disease. In light of observations by others that certain muramyl peptides can actually enhance HIV replication the prospect that the clinical use of muramyl peptides may exacerbate HIV disease is especially troublesome (Schreck R, Bevec D, Dukor P, Baeuerle PA, Chedid L, Bahr GM. Selection of a muramyl peptide based on its lack of activation of nuclear factor-kappa B as a potential adjuvant for AIDS vaccines. Clin Exp Immunol. 1992 Nov;90(2): 188-93; Masihi KN, Lange W, Rohde-Schulz B. Exacerbation of human immunodeficiency virus infection in promonocytic cells by bacterial immunomodulators. J Acquir Immune Defic Syndr. 1990;3(3):200-5). To date, both MDP isolated from natural sources and synthetic MDP have been associated with significant toxicity when administered to mammals. This toxicity has significantly limited the use of MDP in clinics. Occasionally, though muramyl peptides are taught as useful when combined with biologically active proteins. For example, U.S. Patent No. 5,932,208 discloses compositions in which muramyl peptides are combined with some cytokines but the authors do not teach the combination with hCG.
[0008] However, the combination of hCG variants with muramyl peptides is known in the prior art mainly as a fertility or cancer vaccine. These vaccines contain exclusively either beta chain of hCG or carboxyterminal peptides of beta chain of hCG, and not whole, dimer hCG (see for example U.S. Patent Nos. 4,313,871; 4,256,629; 4,310,455; 6,146,633; 6,143,305; 6,096,318; 6,039,948;5,891,992; 5,817,753; 5,698,201 ;5,106,619; 5,006,334; 4,855,285; 4,791,062; 4,767,842; and 4,762,913). This selective preference for beta chain hCG or peptides thereof is mainly due to a concern that vaccination with dimer or whole hCG will cause undesirable cross-reaction against related glycoprotein hormones such as LH, FSH, and TSH with ensuing deleterious consequences to the host. So far no prior art references exist in which hCG dimer is combined with muramyl peptides.
[0009] Contrary to conventional wisdom prevailing in the prior art, the present inventor has surprisingly discovered that the combination of whole or dimer hCG molecule with muramyl peptides synergistically increases the anti-HIV and cancer activity of hCG in vivo and causes no harmful effect to the host. It was also a surprise that hCG formulated in oil-in-water emulsion with or without muramyl peptides had same potent clinical effect.
DISCLOSURE OF INVENTION
[0010] Accordingly, it is an object of the invention to provide novel composition that overcomes the above-identified and other deficiencies in the art. This invention provides the first successful reduction to practice of the use of specially formulated water-soluble hormone for the treatment of viral diseases and cancers. In light of these and other considerations, it is discovered by the present inventor that administration of hCG along with muramyl peptides has much better clinical effect or response than commercial, non-modified form of hCG available currently. It is also contemplated that hCG is formulated in oil-in-water emulsion with or without muramyl peptides. Other formulations like liposome encapsulated delivery are equally suitable. Accordingly, the present invention contemplates a composition comprising novel, therapeutic form of dimer hCG.
[0011] It is yet another object of the invention to provide methods for preparing composition which also overcomes the deficiencies in the art.
[0012] The invention also contemplates methods of administering to a subject an amount of hCG in a formulation that is clinically more effective in treating or preventing a viral infection from which the subject suffers or is about to suffer.
[0013] According to another aspect of the invention, methods are provided for treating cancer in subject animals such as humans by administering to said subjects the instant composition in a dose effective to eliminate or prevent growth of said cancer in said animals.
[0014] The preferred uses as envisaged by this inventor are the treatment of diseases like
AIDS, Kaposi's sarcoma (both endemic and HIV-associated types), multiple myeloma, lymphoma, melanoma and other types of carcinomas and leukemia.
[0015] Without implying any limitation, these pharmaceutical compositions are advantageously formed by solutions which can be administered parenterally, especially subcutaneously, intramuscularly, intravenously, or by perfusion. The composition of the invention can be equally and advantageously administered orally either alone or in a galenica form ensuring their protection, for example, encapsulated in capsule or microsphere or liposomes enabling the gastric barrier to be crossed. BEST MODE FOR CARRYING OUT THE INVENTION
[0016] The present invention is based on the discovery that, in spite of the fact that muramy dipeptides (MDPs) generally used as a component of vaccine preparations to induce immunity against antigenic determinant of a vaccine, these muramyl peptide compounds are actually capable of synergizing rather than antagonizing hCG activity in vivo. Therefore, in spite of the teaching in the art referred to above, muramyl peptide compounds in combinatior with hCG are useful in the treatment, prophylaxis or management of HIV infection and other life-threatening conditions like cancer.
[0017] The term "sustained delivery devices" as used hereinafter denotes specific devices like transdermal patches, subcutaneous implants, and pumps containing the drug. [0018] The term "slow release formulations" as used hereinafter denotes forms of drug delivery other than sustained delivery devices and comprises hCG formulations that have not been taught in the prior art relating to formulations of hCG.
[0019] The term "adjuvant" by definition is a substance that is incorporated into or injected simultaneously with an antigen. The adjuvants potentiate non-specifically the ensuing immune response against antigen. A principal purpose for employment of an immunotherapeutic adjuvant is to achieve more durable humoral or cell-mediated immunity of a high level by employing lower levels of an antigen with fewer numbers of doses than could be achieved by administering the equivalent aqueous antigen. Adjuvants are used in combination with non-living agents (in place of living microorganisms), for the preparation o vaccines. Adjuvants may also increase the effective immune response against low or nonimmunogenic tumor cells or cells infected with intracellular agents that are already present in the body and are not adequately checked by naturally elicited immune responses. [0020] The term "emulsifϊer" as used hereinafter denotes nonionic surface-active compounds derived from alkylene oxide and/or hexahydric alcohols and/or higher natural fatty acids such as esters or ester-ethers.
[0021] The term "complete Freund's Adjuvant (CFA)" denotes a powerful immunostimulatory agent that has been used successfully with many antigens on an experimental basis. CFA is comprised of a mineral oil, an emulsifying/stabilizing agent such as Arlacel A, and killed mycobacteria such as Mycobacterium tuberculosis. Aqueous antigen/protein solutions are mixed with these components to create a water-in-oil emulsion. CFA causes severe side effects, however, including pain, abscess formation, and fever, which prevent its use in either human or veterinary vaccines. The side effects are primarily due to the patient's reactions to the mycobacterial component of CFA. [0022] "Incomplete Freund's Adjuvant (IFA)" is similar to CFA without the bacterial component. While not approved for use in the United States, IFA has been useful for several types of vaccines in other countries. IFA has been used successfully in humans with influenza and polio vaccines and with several animal vaccines including rabies, canine distemper, and hoof-and-mouth disease. Experiments have shown that both the oil and emulsifier used in IFA can cause tumors in mice, indicating that an alternative adjuvant would be a better choice for human use.
[0023] Muramyl dipeptide (MDP) represents the minimal unit of the mycobacterial cell wall complex that generates the adjuvant activity observed with CFA. Many synthetic analogues of MDP have been generated that exhibit a wide range of adjuvant potency and side effects. Three analogues that may be especially useful as vaccine adjuvants are threonyl derivatives of MDP, n-butyl derivatives of MDP, and lipophilic derivatives of muramyl tripeptide (see U.S. Patent No. 5,709,879). These compounds effectively stimulate humoral and cell-mediated immunity and exhibit low levels of toxicity. This muramyl peptide has a phospholipid tail that allow association of the hydrophobic portion of the molecule with a lipid environment while the muramyl peptide portion associates with the aqueous environment. Thus the MTP-PE itself can act as an emulsifying agent to generate stable oil- in-water emulsions.
[0024] The term "a muramyl peptide" has a clear meaning to those skilled in the art. In particular, it refers to a compound containing one or more sugar residues, at least one of the sugar residues, which will often be a muramic acid residue, being substituted with at least one or more (and usually two or more) amino acid residues. Muramyl peptide compounds may be peptidoglycans which are capable of enhancing the cellular antigenicity response in mammals and which are prototype muramyl dipeptide (MDP) or analogues or derivatives thereof. [0025] A group of muramyl peptide compounds have previously been disclosed as useful in treating or preventing progressive septicaemia and septic shock, as having immunopotentiating anti-bacterial activities. Muramyl peptides are however are effective onl in immunocompetent hosts. For representative muramyl peptides see for example U.S. Patem Nos. 5,506,204 and 5,534,492, incorporated herein by reference.
[0026] A wide variety of analogues of prototype muramyl dipeptide are known, some of which have been proposed as treatments for the restoration of immune function or the nonspecific stimulation of the immune system. These analogues, and prototype MDP itself, are termed in general as "muramyl peptides".
[0027] According to a first aspect of the present invention, there is provided a composition of a muramyl peptide compound in combination with water-soluble biologically active protein like hCG.
[0028] Without limiting to hCG other therapeutic agents are contemplated including but no limited to insulin, glucagons, calcitonin, atrial naturetic peptide, secretin, cholecystokinin, thyrotrophin releasing thymopentin, adrenocorticotripic hormone, growth hormone releasing factor, enkephalin, oxytocin, vasopressin and luteinizing hormone releasing hormone. These compositions can also additionally formulated into a matrix material selected from the group consisting of chitosan, algin, saturated polyglycolysed glyceride, glycerol palmitostearate, saturated C12 to C22 fatty acid esters of poly alcohol, glyceryl and polyethylene glycol behenate, polyethylene oxide, and ammonium glycyrrhizinate among many others. [0029] Adjuvant is selected from the group consisting of monophosphoryl lipid A, lipid A, keyhole himpet hemocyanin, histidine-tag, alum, Freund's adjuvant, beta-gal, palmitic acid, saponin, lipopolysaccharide, BCG cell wall skeleton, trehalose monomycolate, trehalose dimycolate, lipid X, isoprinosine, lithosperman (A, B or C), and muramyl dipeptide (MDP) lipid conjugate. The lipid can be saturated or unsaturated phospholipid or a glycolipid. Preferred lipids are selected from the group consisting of 1 ,2-dimyristoylphosphatidylcholine 1,2-dipalmitoylphosphatidylcholine, 1,2-dimyristoylphosphatidylglycerol, cholesterol and combinations thereof.
[0030] Examples of solid lipids suitable for the preparation of the instant composition are triglycerides composed of natural, even-numbered and unbranched fatty acids with chain lengths in the C10-C18 range, or microcrystalline glycerol triesters of saturated, even- numbered and unbranched fatty acids of natural origin such as tricaprin, trilaurin, trimyristin, tripalmi in, and tristearin. In general, any lipid component or mixture of lipid components which provides a solid phase at room temperature (25 °C.) when measured in bulk is suitable for the lipid core.
[0031] The preferred phospholipids which can be used for making instant composition are natural phospholipids such as soybean lecithin, egg lecithin, phosphatidylglycerol, phosphatidylinositol, phosphatidyl-ethanolamine, phosphatidic acid, sphingomyelin, diphosphatidylglycerol, phosphatidylserine, phosphatidyl-choline, cardiolipin, etc.; synthetic phospholipids such as dimyristoylphosphatidylcholine, dimyristoyl-phosphatidylglycerol, distearoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, monophosphoryl lipid A, diphosphoryl lipid A, etc.; and hydrogenated or partially hydrogenated lecithins and phospholipids.
[0032] Non-natural surfactants and detergents optionally can be incorporated into composition of the invention in desired amounts. As used herein, the terms "surfactants" or "detergents" include a wide variety of manmade molecules which form micelles in aqueous solution and contain both lipophilic and hydrophilic domains. In general, emulsifying agents comprise sorbitan esters, polyoxyethylene sorbitan mono-, di-, or triesters, polyoxyethylene fatty acids, polyoxyethylene fatty acid ethers, and combinations thereof. Examples of non- natural surfactants include but are not limited to polysorbates ("TWEEN" or sorbitan monooleate), sodium dodecylsulfate (SDS), polyethoxylated castor oil ("CREMOPHOR"), NP-40, and numerous other synthetic molecules. In addition, several other synthetic surfactants, for example, dimethyldioctadecyl ammonium bromide (DDA) and certain linear polyoxypropylene-polyoxyethylene (POP-POE) block polymers (available commercially under the trademark PLURONIC) have been reported as having adjuvant activity. Among them poloxamer 401, Pluronic L62LF, Pluronic L101, Pluronic L64, PEG1000, Tetronic 1501, Tetronic 150R1, Tetronic 701, Tetronic 901, Tetronic 1301, Atmos 300, Tween 20, Tween 40, Tween 60, Tween 80, and Tetronic 130R1 are especially desirable. Emulsifiers also comprise Arlacel A or Arlacel 80 or Span 80 (so-called mannide monooleates). These surfactants can be used at various doses. For example, while preferred range of Arlacel A or Arlacel 80 or Span 80 in instant composition is between about 2 to 15% by weight, the range of Tween 80 for example would be 0.2 to 4% by weight. In general, surfactants can comprise less than 30% of the total weight of composition, more preferably less than 25%, still more preferably less than 10%, and most preferably less than 5%.
[0033] Functional equivalents of MDP include but are not limited to muramyl di- or tripeptides like N-acetyl-glucosaminyl-N-acetyl-muramyl-L-alanyl-D-isoglutamine (GMDP), N-acetyl-D-glucosaminyl(beta- 1 -4)-N-acetyl-muramyl-L-alanyl-D-isoglutamine, N-acetyl- glucosaminyl-N-acetyl-muramyl-L-alanyl-D-glutamic acid (GMDP-A), muramyl dipeptide- phosphatidyl-ethanolamine, muramyltripeptide phosphatidylethanolamine, muramyl tripeptide phosphatidyl ethanol amine, CGP11637 (nor-MDP), alpha(N-acetyl-muramyl-L- alanyl-D-isoglutamine), beta, gamma-dipalmitoyl-sn-glycerol, alpha(N-acetyl-muramyl-D- alanyl-D-isoglutamine), beta, gamma-dipalmitoyl-sn-glycerol, alpha(N-acetyl-muramyl-L- alanyl-D-isoglutaminyl-L-alanine), beta, gamma-dipalmitoyl-sn-glycerol, alpha(N-acetyl- muramyl-D-alanyl-D-isoglutaminyl-L-alanine), beta, gamma-dipalmitoyl-sn-glycerol, N- acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(l,2-dipalmitoyl-sn-glycero-3- hydroxyphosphoryloxy))ethylamide, glucoasminylmuramylpeptides, murametide, murabutide, muradimetide, myramistin, N-acetylmuramyl-L-threonyl-D-isoglutamine, N-acetylmuramyl- L-.alpha.-aminobutyryl-D-isoglutamine, 6-O-stearoyl-N-acetylmuramyl-L-.alpha.- aminobutyryl-D-isoglutamine, N-acetylmuramyl-L-valyl-D-isoglutamine, N-acetylmuramyl- L-alanyl-D-isoglutamine, N-acetyl-desmethylmuramyl-L-alanyl-D-isoglutamine, N- acetylmuramyl-L-alanyl-D-glutamine butyl ester, N-acetylmuramyl-L-seryl-D-isoglutamine, N-butyrylmuramyl-L- . alpha. -aminobutyryl-D-isoglutamine, N-acety lmuramyl-L-threonyl-D- isoglutamine, Bis(6-O-muramyl dipeptide)O-palmitoyltartronate, muramyl tripeptide phosphatidylethanolamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 ,2- dipalmitoyl-sn-g lycero-3 -(hydroxyphosphorloxy))ethylamide, N-acetyl-muramyl-L-alanyl-D- glutamine butyl ester, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-1,2- dipalmitoyl-sn-g lycero-3 -(hydroxyphosphoryloxy)ethylamide (MTP-PE), cholesteryl-MDP, beta-butyl glycoside of N-acetylmuramyl-L-alanyl-D-isoglutamine, 2-acetamido- 4,6-di-O- acetyl-2-deoxy-3-0-[(R)-l-(methoxycarbonyl)ethyl]-alpha-D-glucopyranose, (beta-butyl- MDP, MTPO-26, beta-cholesteryl-MDP), saponin (Taurosid I), N-acetylnor-muramyl-L-N- methylalanyl-D-isoglutamine octylamide, UDP-N-acetyl-muramyl-pentapeptide, L4-MDP- ONB, L-alanyl-gamma-D-glutamyl-meso-diaminopimelate, 1,6-anhydro muramyl dipeptide, N-acetylglucosamine-beta- 1 , 4-N-acetyl-muramyl-pentapeptide-pyrophosphoryl- undecaprenol, 3 -0-[N-acetylmuramyl-D-isoglutaminyl]- 1 ,2-di-O-palmitoyl-sn-glycerol, L- threonyl-MDP-GDP, L-allothreonyl-MDP-GDP, trehalose 6,6-diester, muramyl dipeptide, B30-muramyl dipeptide, and muramyl dipetide-lysine.
[0034] It is understood that aminoacyl residue can be any of aminoacyl moiety selected from the group consisting of alanyl, valyl, leucyl, isoleucyl, .alpha.-aminobutyryl, threonyl, methionyl, cysteinyl, glutamyl, isoglutamyl, glutaminyl, isoglutaminyl, aspartyl, phenylalanyl, tyrosyl, tryptophanyl, lysyl, ornithinyl, arginyl, histidyl, asparinginyl, prolyl, hydroxypropyl, seryl, and glycyl groups.
[0035] Other muramyl peptide derivatives include those wherein an L-threonyl residue is substituted for the L-alanyl residue of muramyl peptide group. It will additionally be appreciated that it is also possible to use any other adjuvant muramyl peptides carrying substituents in positions 1, 4 or 6 of the saccharide group, provided they have the same favorable effects as the preferred muramyl peptides mentioned above. Without limiting to above derivatives of muramyl peptides those disclosed, for example, in U.S. Patent Nos. 4,158,052; 4,220,637; 4,323,559; 4,323,560; 4,409,209; 4,423,038; 4,185,089; 4,406,889; 4,082,735; 4,082,736; 4,427,659; 4,461,761; 4,314,998; 4,101,536; 4,369,178; 5,075,287; 5,376,369; 5,264,431; and 5,709,879, incorporated herein by reference, are equally useful in this invention.
[0036] Other useful additives in the composition can comprise N-cyclohexanoyl arginine; a mixture of N-cyclohexanoyltyrosine and N-cyclohexanoylleucine; a mixture of N- phenylsulfonylvaline, N-phenylsulfonylleucine, N-phenylsulfonylphenylalanine, N- phenylsulfonyllysine, and N-phenylsulfonylarginine; and a mixture of N-benzoylvaline, N- benzoylleucine, N-benzoylphenylalanine, N-benzoyllysine, N-benzoylarginine, and a stabilizer like sodium 2-cyclohexylbutyrate.
[0037] Without limiting to above ingredients one skilled in the art can envision ovalbumin, cholera toxin, an acylated amino acid or a salt thereof, a poly amino acid comprising at least one acylated amino acid or a salt thereof, a sulfonated amino acid or a salt thereof, or any combination thereof. The composition can also comprise a microsphere. [0038] Polymer matrices for forming microspheres are well known in the art. For example, semipermeable microspheres containing enzymes, hormones, vaccines, and other biologicals are disclosed in U.S. Patent No. 5,643,605. Another U.S. Patent No. 5,075,109 discloses a method of potentiating an immune response by administering a mixture of at least two populations of microspheres containing bioactive agents such that one of the microsphere populations is sized between about 1 to 10 microns. U.S. Patent No. 4,293,539 discloses a controlled release formulation of an active ingredient in a copolymer derived from about 60 to 95 weight percent lactic acid and about 40 to about 4 weight percent gly colic acid. U.S. Patent No. 4,919,929 discloses the administration of an antigenic substance in a shaped structure of a biocompatible matrix material. U.S. Patent No. 4,767,628 discloses a composition comprising an active, acid stable polypeptide and a polylactide, which when placed in an aqueous physiological environment release the polypeptide at an approximately constant rate in an essentially monophasic manner. U.S. Patent No. 4,962,091 discloses a microsuspension of water soluble macromolecular polypeptides in a polylactide matrix. U.S. Patent Nos. 4,849,228 and 4,728,721 disclose a biodegradable, high molecular weight polymer characterized in that the content of water-soluble low molecular weight compounds, as calculated on the assumption that such compounds are monobasic acids, is less than 0.01 mole per 100 grams of high molecular weight polymer. U.S. Patent Nos. 4,902,515 and 4,719,246 disclose polylactide compositions containing segments of poly(R-lactide) interlocked with segments of poly(S-lactide). U.S. Patent No. 4,990,336 discloses a multiphasic sustained release system comprising allergen extract encapsulated in microspheres of bioerodible encapsulating polymer which permits a sustained, multiphasic release of the allergen. This system includes a first portion of allergen extract that upon injection is capable of being released in a manner whereby initial allergenicity is minimized to producing a mild local reaction similar to that normally observed with low doses of conventional allergen administration, and secondary portions of allergen extract that provide a substantially higher level of allergen extract in doses that could provide a serious reaction in the patient, but for the release of the first portion of allergen extract. U.S. Patent No. 4,897,268 discloses a microcapsule delivery system wherein the ingredients are encapsulated in biodegradable copolymer excipients of varying mole ratios, such that delivery of the ingredients occurs at a constant rate over a prolonged period of time. Thus, various means of making and using microspheres are known and can be easily adopted for the purposes of this invention.
[0039] Microspheres of the invention also comprise liposomes. Compositions suitable to form liposomes are well known in the technical literature which is abundant in this field. Preferred lipid compositions are those which bring into play phospholipids, such as phosphatidylcholine (a derivative of fatty acids comprising from 12 to 20 carbon atoms) (particularly from 16 to 20 carbon atoms), phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidic acid. These phospholipids can be used alone or in mixtures. It is more particularly advantageous to resort to mixtures of synthetic or natural distearyl-phosphatidylcholine (DSPC) or phosphatidylcholine and of phosphatidylserine (PS) or of phosphatidylglycerol (PG). Advantageously, the liposomes are formed from mixtures containing the last mentioned phospholipids in a ratio of 7 volumes of DSPC or phosphatidylcholine (PC) to 1 to 10, preferably 3, volumes of PS. The biological activities of the liposomes containing the derivatives of the invention are manifested as well when the liposomes are in the unilamellar or plurilamellar form. Preferably, the particles of liposomes have sizes greater than or equal to 0.1 micron, for example, comprised between 1 and 10 microns.
[0040] The compound of the invention is useful as a modulator of unspecific antimicrobial resistance for systemic enhancement of immune response and unspecific immunity. [0041] It is thus indicated e.g. in the curative treatment or in the supportive treatment (i.e. together with further specific or supportive forms of therapy) of conditions of decreased immune response, in particular conditions of decreased cellular and humoral immune response and conditions of decreased oversensitivity reactions of the delayed type, and further in the treatment of conditions generally in which a modulation of the immune response is desired.
[0042] It is in particular useful in the curative or supportive treatment of pathological conditions related to idiopathic immunodeficiencies or immunodeficiencies of the type encountered in geriatric patients or in patients with heavy burns or generalised infections. [0043] It is further useful in the curative or supportive treatment of viral infections such as disseminated herpes and disseminated varicella infections, HIV infection, and malignant tumors.
[0044] The slow release hCG is contemplated for use in both RNA and DNA viruses. These would include, but not be limited to, Hepatitis viruses, Herpes viruses, flu viruses and Raft Valley Fever viruses. [0045] It will be apparent to one of ordinary skill in the art that the precise amounts of active ingredients needed to produce a given effect will vary with the particular compounds, and with the size, age, and condition of the subject to be treated. These amounts can easily be determined using routine methods known to those of ordinary skill in the art. [0046] The adjuvant compositions and vaccines of the invention are generally administered by injection. However, one can devise easier modes of administration like oral administration. When the composition of the present invention can be used directly for purposes of clinical therapy and prophylaxis it can have the form of an oral preparation or a parenteral preparation. The term "parenteral" includes subcutaneous, intravenous, epidural, irrigation, intramuscular, release pumps, or infusion. Without limiting the composition can be also administered intra-articularly, intrasynovially, intrathecally, periostally, intratumorally, peritumorally, intralesionally, perilesionally, sublingually, buccally, transdermally, topically or by inhalation. It also can be administered as a dressing for a wound or lesion. However, oral administration of the composition is especially preferred. For oral use, the composition of the present invention can be used alone or in combination with pharmaceutically acceptable carriers to form pharmaceutical preparations such as capsules, pills, lozenges, tablets, dragees, sachets, tea bags, granules, powders, coated tablets, sugar coated tablets, wafers, sugar cubes, gels, hydrogels such as particles of a hydrophilic-hygroscopic polysaccharide, foams, suppositories, inhalants, juices, shakes, chewing gum, tooth paste, dentifrice, mouthwash, candies, and emulsions.
[0047] Suitable pharmaceutical carriers include, for example, fillers such as lactose, sucrose, mannitol, glucose, starch, sorbitol, glycine, calcium phosphate and microcrystalline cellulose; binders such as starch, casein, gelatin, acacia, glucose, sucrose, sorbitol, mannitol, tragacanth, hydroxypropylcellulose, hydroxypropoxymethylcellulose, carboxymethylcellulose, 2-methyl-5-vinylpyridine/methyl methacrylate/ethylacrylate copolymer, polyvinylpyrrolidone and sodium alginate; alginate gel; lubricants such as stearic acid, hardened oil, magnesium stearate, calcium stearate, polyoxyethylene monostearate, talc, silicon oxide and polyethylene glycol; disintegrators such as potato starch, and starch containing a surfactant or the like; facilitators like magnesium sulphate; and humectants such as sodium lauryl sulfate.
[0048] Composition of the present invention can also be administered in the form of liposomes. As is known in the art, liposomes or artificial lipid vesicles are generally derived from phospholipids or other lipid substances. Additionally they can contain muramyl peptide, a metabolizable oil, and optionally an additional emulsifying agent. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. A typical process for making a liposome preparation comprised of liposomes that contain an encapsulated composition of the invention, comprises hydrating a lipid or liposome formulation with a solution of a material to be encapsulated; providing a plurality of portions of a dry lipid or dry liposome formulation; hydrating each of said plurality of portions with a solution comprising said material to be encapsulated; and combining each of said plurality of portions to form a single liposome preparation, thereby forming a liposome preparation comprising liposomes that contain said encapsulated material. Any non-toxic, physiologically-acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are well known in the art. For example, cochleates containing biologically relevant molecule component, a negatively charged lipid component, and a divalent cation component. The cochleate has an extended shelf life, even in a desiccated state. Advantageously, the cochleate can be ingested.
[0049] For the above indications the dosage to be used will depend of course on the nature and severity of the disease to be treated, the mode of administration and the compound form used. For an average subject a suitable dosage is from about 100 IU to about 20,000 IU, administered in a single dose or in separate doses. Repeated admimstration may conveniently be effected one to four times per day or in less frequent form, e.g., once every three days, once a week, or once every two weeks, or once a month or once every three months. Indicated unit dosage forms include from about 100 IU to about 10,000 IU of compound of the invention in situations of repeated administration, preferably in about 1,000 to about 5,000 IU per injection. Used doses can go up to about 20,000 IU when a single administration for treatment is desired. Based on product description that are provided with commercial preparations of hCG those of skill in the art can easily convert IU units of hCG to weight units and vice versa. [0050] The compositions of the present invention can be employed in a variety of other ways unrelated to therapeutic indication. For example, the composition can be employed bot as labeled and unlabeled reagent in various immunoassays, bioassays, and the like, for the detection of hCG or antibodies to hCG. Suitable labels include radioisotopes, enzymes, fluorescent molecules, chemiluminescent labels, enzyme substrates or co-factors, enzyme inhibitors, particles, dyes, and the like. Such labeled reagents may be used in a variety of we] known assays, such as radioimmunoassays, enzyme immunoassays, e.g., ELISA, fluorescent immunoassays, and the like.
[0051] The invention also embraces the concept of using the instant composition along wit drugs or compounds commonly used in the prior art for various disease categories. Among the foregoing compounds, the following categories and/or members of the following categories are active agents belonging to: adrenocortical steroid; adrenocortical suppressant; aldosterone antagonist; amino acid; anabolic; androgen; anti-AIDS drugs; anthelmintic; anti- acne agent; anti-adrenergic; anti-allergic; anti-amebic; anti-androgen; anti-anemic; anti- anginal; anti-arthritic; anti-asthmatic; anti-atherosclerotic; antibacterial; anticholelithic; anticholelithogenic; anticholinergic; anticoagulant; anticoccidal; antidiabetic; antidiarrheal; antidiuretic; antidote; anti-estrogen; antifibrinolytic; antifungal; antiglaucoma agent; antihemophilic; antihemorrhagic; antihistamine; antihyperlipidemia; antihyperlipoproteinemic; antihypertensive; antihypotensive; anti-infective; anti-infective, topical; anti-inflammatory; antikeratinizing agent; antimalarial; antimicrobial; antimitotic; antimycotic, antineoplastic, antineutropenic, antiparasitic; antiperistaltic, antipneumocystic; antiproliferative; antiprostatic hypertrophy; antiprotozoal; antipruritic; antipsoriatic; antirheumatic; antischistosomal; antiseborrheic; antisecretory; antispasmodic; antithrombotic antitussive; anti-ulcerative; anti-urolithic; antiviral; appetite suppressant; benign prostatic hyperplasia therapy agent; bone resorption inhibitor; bronchodilator; carbonic anhydrase inhibitor; cardiac depressant; cardioprotectant; cardiotonic; cardiovascular agent; choleretic; cholinergic; cholinergic agonist; cholinesterase deactivator; coccidiostat; diagnostic aid; diuretic; ectoparasiticide; enzyme inhibitor; estrogen; fϊbrinolytic; free oxygen radical scavenger; glucocorticoid; gonad-stimulating principle; hair growth stimulant; hemostatic; hormone; hypocholesterolemic; hypoglycemic; hypolipidemic; hypotensive; immunizing agent; immunomodulator; immunoregulator; immunostimulant; immunosuppressant; impotence therapy adjunct; inhibitor; keratolytic; LHRH agonist; liver disorder treatment, luteolysin; mucolytic; mydriatic; nasal decongestant; neuromuscular blocking agent; non- hormonal sterol derivative; oxytocic; plasminogen activator; platelet activating factor antagonist; platelet aggregation inhibitor; potentiator; progestin; prostaglandin; prostate growth inhibitor; prothyrotropin; pulmonary surface; radioactive agent; regulator; relaxant; repartitioning agent; scabicide; sclerosing agent; selective adenosine Al antagonist; steroid; suppressant; symptomatic multiple sclerosis; synergist; thyroid hormone; thyroid inhibitor; thyromimetic; amyotrophic lateral sclerosis agents; Paget's disease agents; unstable angina agents; uricosuric; vasoconstrictor; vasodilator; vulnerary; wound healing agent; and xanthine oxidase inhibitor among many others.
[0052] The compounds useful in the invention may be delivered in the form of drug cocktails. A cocktail is a mixture of any one of the above compounds combined with the composition of the invention. The mixture does not need to be mixed physically and drugs can be administered separately, sequentially, or at the same time.
[0053] In addition to AIDS drugs of particular interest are anti-cancer drugs which can be administered along with the composition of the invention. Antineoplastic drugs include but are not limited to: Acivicin; Aclarubicin; Acodazole Hydrochloride; AcrQnine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflomithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized Oil I 131; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl; Interferon Alfa-n3; Interferon Beta- 1 a; Interferon Gamma- 1 b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safmgol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; and Zorubicin Hydrochloride.
[0054] Other anti-neoplastic compounds include: 20-epi-l,25 dihydroxyvitamin D3; 5- ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; atrsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing moφhogenetic protein- 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL- PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta- alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canaiypox IL-2; capecitabine; carboxamide-amino-triazole; carboxy amidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocannycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; fϊlgrastim; fmasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; irinotecan; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mfrimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A +myobacterium cell wall sk; mopidamol; multiple drug resistance genie inhibitor; multiple tumor suppressor 1 -based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone +pentazocine; napavin; naphteφin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocaφine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum- triamine complex; porfimer sodium; porfiromycin; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; puφurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone Bl; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1 ; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfmosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafαr; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thalidomide; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopuφurin; tirapazamine; titanocene dichloride; topotecan; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyφhostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; zinostatin stimalamer; and many others. [0055] It is also object of this invention to prepare formulations which instead of hCG would contain other biologically active water-soluble proteins and peptides. Such a protein is a growth hormone or a somatotropin, e.g., human growth hormone (HGH), bovine growth hormone (BGH or BST), porcine growth hormone (PGH or PST), their analogues and derivatives and epidermal growth factor (FGF) and its analogues. Other contemplated proteins are interleukins, interleukin receptors and interleukin receptor agonists, chemokines, and interferons. For example interferon alfa; interferon alfa-2a; interferon alfa-2b; interferon alfa-N 1; interferon alfa-n3; interferon beta; interferon beta-1 al; interferon beta- lb; interferon gamma- la; interferon gamma- lb; interferon omega; interferon tau; interleukin- 1; interleukin- 1 alpha; interleukin-1 beta; interleukin- 10; interleukin-11; interleukin- 12; interleukin- 12; interleukin- 15; interleukin-2; interleukin-3; interleukin-4; interleukin-5; interleukin-7; interleukin-8, MIP-1 alpha and beta, RANTES, among many others. Additionally, protein can be selected from the group consisting of blood cell growth stimulating factors and precursors and erythropoeitin (EPO) and its analogues. Other equally desirable protein are parathyroid hormone (PTH), selenoprotein P, cystatin B and its liver thiol protease inhibitor analogues, endotoxin neutralizing protein, lymphocyte migration inhibitory factor (LIF), mast cell growth factor (MGF), megakaryocyte simulatory factor (MGDF), granulocyte macrophage colony stimulating factor (GM-CSF), genofibrate, alpha calcitonin, beta calcitonin, tumor necrosis factor (TNF), tumor invasion inhibiting factors, TGF-beta-type cytokines, transacting regulatory proteins (TAT's) of HIV and other retroviruses, protease inhibitors, and BPC 157, fat reducing hormones, and analogues and variants of these proteins. Also contemplated are insulin, glucagon, gastrin, angiotensin, secretin, lactotropic hormone, thyrotropic hormone, melanocyte stimulating hormone, luteinizing hormone (LH), follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), thrombopoietin (TPO), luteinizing hormone stimulating hormone, human menopausal gonadotropin, vasopressin, oxytocin, protirelin, corticotropin, SOD, urokinase and lysozyme. One of skill in the art will recognize that other cytokines are known and are equally suitable for the composition of the invention. Without limiting to above listed biologically active proteins other polypeptides are contemplated such as G-CSF, M-CSF, LIF, inhibin A, inhibin B, activin A, activin B, NAP-1, MCP-1, MIP- lalpha, MlP-lbeta, MIP-2, SISbeta, SISdelta, SISepsilon, PF4, PBP, gammaIP-10, MGSA, aFGF, bFGF, KGF, PDGF-A, PDGF-B, PD-ECGF, INS, IGF-I, IGF-II, NGF-beta, GRO/MGSA, PF4, PBP/CTAP/.beta.TG, IP-10, KC, 9E3, MCAF, ACT-2/PAT 744/G26, LD-78/ PAT 464, RANTES, G26, 1309, JE, TCA3, ICAM-1, ICAM-2, LFA-1, LFA-3, CD72, CTAPIII, ENA-78, GRO, 1-309, PF-4, and LD-78.
[0056] Without limiting to above proteins other proteins of interest as encoded by genes listed in Table 1 are also contemplated to be used in the composition of the present invention: TABLE 1
Name; FRAGMENT GENBANK # LENGTH, bp (start-end)
MmRad51; yeast DNA repair antigen CD38 Flt3/Flk2 ligand protein Oncostatin M D31942 CD27; lymphocyte-specific NGF
DT3473 855-1199 1017-1360 L24495 596-846 Rad51 and E coli RecA CSA receptor L05630 841- receptor family member homologue 1165 Fibroblast growth factor receptor
Interleukin-8 receptor Heparin-binding EGF-like M28998 200-583
D17630 664-1022 growth Basic (b FGF-R) Catenin alpha D25281 L07264 258-673 Granulocyte colony - stimulatings 1276-1594 factor (Diphtheria toxin receptor) M58288 251-529 BST-1; lymphocyte differentiation Fms-related tyrosine kinase 3 factor receptor D31788 674-1014 U04807 46-418 Growth/ diffferentiation factor 1 M62301 2267- factor) neurofibromatosis type 2 2566 Intercellular adhesion molecule- 1 susceptibility protein
(GDF-1) (TGF- beta family) X52264 1053- Pιm-1 proto-oncogene PKC-delta, protein kinase C delta 1385 M13945 2713- 69042 1740- Interleukin- 1 receptor type II 2930 2011 X59769 882-1134 Egr-1 Zn-finger regulatory type Corticotropin releasing factor protein
GA binding protem beta-2 chain X72305 1411- M20157 399-753 M74517 613-931 1748 PKC-alpha, protein kinase C CD 40L receptor(TNF receptor receptor alpha M83312 417-754 Hepatocyte growth factor M25811 1566- family) X72307 641-965 1924 Fasl receptor (Fas antigen, Apo-1 (hepapoitein) type
M83649 416-736 Keratmocyte growth factor FGF- CD44 antigen M27129 antigen) 7 789-1141
Interleukin 12 (p40) beta chain Z22703 63-325 T-lymphocyte activated protein M86671 652-963 Activm type I receptor M31042 285-606 Vascular endothelial growth Z31663 847-1130 Neuronal-cadheπn (N-cadhenn) factor Transcription factor TF II D M31131 1212-
M95200 688-955 D01034 291-556 1409 (VEGF) ZO-1, Tight junction protein, ATP-dependent DNA hehcase II Interleukin 11 (adipogenesis dtscs- 70
U03421 196-475 D14340 3714- M38700 274-632 inhibitory factor) 4001 kDa subunit, thyroid Ku Interleukin 15 U14332 605- large family member, partially (p70/p80) 1057 homologous to a dlg-A tumor autoantigen p70 subunit, p70 Ku)
LIMK, LIM seπne/threonme suppressor in Drosophila G13, G-alpha-13 guanine kmase ERCC5 excision repair protein, nucleotide
U15159 1376- D16306 1336- M63660 2057- 1699 1639 2377
DAD-1, defender against cell DNA-repair protein regulatory protein death 1 complementing Transcription factor RelB
U83628 221-509 XP-G cells (XPG) M83380 1456-
CD 30L receptor (Lymphocyte Bax, Bcl-2 heterodimeπzation 1728
U25416 135-435 L22472 172-534 Vascular cell adhesion protein 1 activation antigene CD 30, Kι-1 partner and homologue M84487 984-1304 antigene) B7-2, T lymphocyte activation ERCC3 DNA repair hehcase,
Mast cell factor U44725 79- L25606 570-967 DNA- 417 antigen CD86, CD28 antigen S71186 1147-1444
C-C cheynokine receptor ligand repair protein complementing (Monocyte 2, B7-2 antigen, alternative XP-B
U56819 965-1262 CTLA4 cells (XPBC) chemoattractant protein 1 counter-receptor CRE-BP1, cAMP response receptor NF2, Merlin (moesm-ezrin- element (MCP-1RA) radixm- S76657 412-748
Leukemia inhibitory factor (LIF) L27105 2175-2400 binding protein 1 X06381 63-366 like protein), shwannomm, XRCC1 DNA-repair protein, affecting hgation Angiotensin-converting enzyme Nucleobindm M96823 8C
Nuclear hormone receptor ROR- L04946 850-1113 357 U53228 368-675 (ACE) (clone ACE 5 ) PAX-5 (B cell specific
ALPHA- 1 Clusteπn, complement lysis transcription
14-3-3 protein eta U57311 L08235 515-744 M97013 286-629 374-640 inhibitor, testosterone-repressed factor)
Prothymosin alpha X56135 prostate message 2, IFNgR2, interferon-gamma 186-455 apo poprotein J, receptor
PAX-8 (paired box protem PAX sulfated glycoprotem-2 S69336 832-108'
8) Adipocyte differentiation- second (beta) chain, interferon
X57487 680-1011 associated gamma receptor accessory facto CamK IV, Ca2/calmodulιn- L12721 404-709 1 X58995 1269- protein (AF-1) 1608 Epidermal growth factor receptor Transcπptional enhancer factor dependent protein kinase IV L21671 1562-1873 S74227 934-123: (catalytic chain) kinase substrate EPS8 (TEF-1)
ATP-dependent DNA hehcase II Jak3 tyrosine-protein kinase, Transcription factor NFAT 1, 80 Janus U02079 1601-
X66323 565-875 L33768 3123-3426 1910 kDa subunit, thyroid Ku kinase 3 isoform, alpha (p70/p80) Desmocolhn 2 L33779 DNA-bmding protem SATB1 autoantigen p80 subunit, p80 Ku) 1317-1691 U05252 1101- Ret proto-oncogene (Papillary Stat6, signal transducer and 1380 X67812 2359- activator CCHB3, calcium channel 2680 L47650 2057-2411 (voltage- thyroid carcinoma-encoded of transcription 6, IL-4 Stat, U20372 351-639 protein) STA6 gated, dihydropyπdine-sensitive
Nm23-M2, nucleoside Lymphocyte-specific tyrosine- L- diphosphate M12056 1205- type) beta-3 subunit)
X68193 80-454 1488 p57kφ2, cdk-inhibitor kιp2 kinase B, metastasis-reducing protein kinase LCK (cychn- protein, c-myc-i elated ERA-1 Protein (ERA-1-993) U20553 989-127: transcription M22115 723-1062 dependent kinase inhibitor 1 B) factor Homeo Box protem 2 1 (Hox- member of the p21CIPl Cdk
MAPKK6, MAP kinase kinase 2 1) inhibitor family, candidate tumo X97052 375-711 M26283 647-884 suppressor gene 6(dual specificity) (MKK6) Zinc finger X-chromosomal snoN, ski-related oncogene DNA polymerase alpha catalytic protem U36203 671-100< D 17384 563-908 M32309 2153- Homeo Box protem 7 1 (Hox- subunit (pi 80) 2554 7 1) Caspase-3, Nedd2 cysteme (ZFX) X14759 740-992 protease WTI, Wilms tumor protein, Neuronal cell surface protein F3
D28492 398-694 tumor X14943 1033- (positive regulator of M55512 1262- 1311 programmed 1563 GATA-3 transcription factor cell death ICH-1 homologue) suppressor X55123 858-112 PSD-95/SAP90A D50621 Tπstetraprolme M57422 262- YB1 DNA binding protem Dipeptidyl peptidase iv [Serotonin receptor type 2 kinase
X58384 61-294 (SHT2)] D30743 1816-
Fh-1 ets-related proto-oncogene Cyclin Dl (Gl/S-specific) 2159 X59421 267-623 S78355 1858-2205 Protein tyrosine phosphatase
RXR-beta cis-11 -retinoic acid Pur-alpha transcπptional D83966 1060- X66224 1225- activator; 1426 1477 U02098 1082- Jun-D; c-jun-related transcription receptor 1309 J05205 737-964 sequence-specific ssDNA- factor binding Integra alpha 7 L23423
C3H cytochrome P450; Cyplbl protein 2399-2713
X78445 295-593 Cdc2Sa; cdc2SMl; MPI1 (M- Gadd45; growth arrest and DNA- Ubiquitin-conjugating enzyme, phase L28177 144-434 yeast U27323 606-986 damage-inducible protein
X96859 51-392 mducer phosphatase 1) Bcl-xL apoptosis regulator (bcl-x Rad6 homologue; murine HR6B ERCC-1; DNA excision repair L35049 641-906 Relaxin Z27088 51-365 X07414 189-484 long); BcI-2 family member
Transcription factor LIM-1 protein N-myc proto-oncogene protein
Z27410 1673-1934 c-rel proto-oncogene X03919 3262- DNA topoisornerase I (Top I) X15842 1729- 3450
D10061 1051- 2064 cAMP-dependent protein kmase 1357 Inhibin alpha subunit type DNA topoisornerase II (Top II) X69618 810-1117 M20473 538-750
D 12513 520-870 Glutathione reductase I-beta regulatory chain GST Pi 1; glutathione S- X76341 115-377 IRFl; interferon regulatory factor transferase Insulin-like growth factor binding 1
D30687 62-369 X81581 474-719 M21065 1-233 Pi 1 , preadφocyte growth factor protein-3 (1GFBP-3) HSP86; heat shock 86kD protein Glutathione S-transferase A Cyclin A (G2/M-specιfϊc) M36830 255-551
J03958 54-311 Z26580 701-1009 LFA-1 alpha, tntegrin alpha L, Glutathione S-transferase Mu 1 Preproglucagon Z46845 M60778 1838-
J04696 13-263 172-531 2050 c-Abl proto-oncogene NF-kB p65, NF-kappa-B leukocyte adhesion glycoprotein
L10656 878-1145 M61909 101-363 LFA-1 alpha chain; antigen A-Raf proto-oncogene transcription factor p65 subunit; CD11A
M13071 1042- rel- (pi 80) 1320 related polypeptide APC; Adenomatous Polyposis c-Src proto-oncogene PKC-theta; protein kinase C theta Cob
M17031 452-758 D11091 658-957 M88127 4127- Retinoic acid binding protein II type 4476
M35523 276-571 VLA-3 alpha subunit protein cellular (CRABP-II) D13867 288-589 Cdc2Sb, cdc2SM2, MPI2 (M- Cyclin D2 (Gl/S-specific) NADPH-cytochrome P450 phase
M83749 781-1074 reductase S93521 1893-2200 Cyclin D3 (Gl/S-specific) D17571 326-605 mducer phosphatase 2)
U43844 484-790 Beta-protachykinin a P13-K pl l0; 5-Hydroxytryptamine receptor D17584 273-523 phosphatidylinositol 3- 1723 EBl APC-b ding protein mitochondπal matrix protein P kinase catalytic subunit U51196 607-834 c-Cbl proto-oncogene (Adaptoi RSP27, heat shock 27kD protein TANK, I-TRAF, TRAF family X57111 858-1 V. 1 U51907 135-437 protein)
U03560 245-500 member associated NF-kB Cdc25 phosphatase, guanine Csk, c-Src-kinase and negative activator X59868 942-12' U05247 645-984 Caspase-11; ICH-3 cysteine nucleotide releasing protein regulator U59463 352-686 Ezπn; Villin 2; NF-2 (merlin)
Fasl; Fas antigen ligand; protease, upstream regulator of related generalized ICE X60671 1571-
U06948 168-488 MLHI DNA mismatch repair 1812 lymphoproliferation disease gene U59883 1037- filament plasma membrane (gld) in mice 1278 associated protein MAPK; MAP kmase, p38 protein, MutL homologue Cyclin Bl (G2/M-specιfιc)
U10871 465-780 Insulin-like growth factor-IA X64713 1184- pl9mk4; cdk4 and cdk6 inhibitor X04480 183-406 1447
U19597 228-516 Cell surface glycoprotein MAC-1 Integrm alpha 6 X69902 26 Elf-1 Ets family transcription X07640 1892- 611 factor 2179 5-Hydroxytryptamιne (serotoni
U19617 1585- alpha subunit X72395 1422- 1902 N-ras proto-oncogene; 1711 CRAF1; TNF receptor (CD40 transforming receptor 3 U21050 1225- X13664 548-857 Homeobox protein HOXD-3 1466 G-protem X73573 141-362 receptor) associated factor, L-myc proto-oncogene protein Cyclin E (Gl/S-specific) TRAF- XI 3945 5287- X75888 799-114 related 5590 MAPKAPK-2, MAp kinase- SPI3; serpin; similar to human CD 18 antigen beta subunit X76850 719-987
U25844 915-1230 X14951 1366- activated protein kinase, proteinase inhibitor 6 (placental 1706 MAPKAP thrombin inhibitor) serine (leukocyte adhesion LFA-1) kinase 2 proteinase (CD3, Fra-2 (fos-related antigen 2) inhibitor PI 50, 95) X83971 617-844
RIP cell death protein; Fas/APO- c-Fgr proto-oncogene Cyclin Al (G2/M-specifιc) 1 X52191 1305- X84311 656-916
U25995 1945- 1538 DCC; netπn receptor, 2223 Integrm alpha 4 X53176 X85788 4193- (CD95) mteractor, contains death 2176-2449 4508 domain PKC-beta; protein kinase C beta- immunoglobulin gene
SLAP; src-like adapter protein, II superf mily Eck X53532 1712- member, former tumor
U29056 109-427 2089 suppressor receptor tyrosine kinase- type protein candidate associated HSP60; heat shock 60 kDa MHR23A; Rad23 UV excision Atm, ataxia telangiectasia murine protein 1 repair U43678 8989- X53584 1432- X92410 613-955 9170 1691 protein homologue; xerodemia repair L24755 2402-2676 Interleukin 10 M37897 175- complementing protein Uromodulm L33406 456 MHR23B; Rad23 UV excision 1809-2136 NF-kappa B binding subunit repair (nuclear
X92411 542-807 M57999 3122- protem homologue; xeroderma Thrombopoietin L34169 3417 pigmentosum group C (XPC) 652-954 factor) (TFDB5) repair Transforming growth factor beta Tumor necrosis factor receptor 1; complementing protein M13177 772-1075 M59378 1961- Integrm beta Y00769 1990- Granulocyte colony- stimulating 2376 2320 M13926 86-377 TNFR-1
MmRad52; yeast DNA repair factor (G-CSF) PDGFRa; platelet-derived growth protein Neuroleukin Ml 4220 M84607 474-803
Z32767 159-417 1110-1490 factor alpha-receptor Rad52 homologue Insulin-like growth factor-2 Interleukin-9 receptor Cyclin G-(G2/M-specifιc) M14951 46-328 M84746 795-1086
Z37110 300-619 (somatomedin A) iNOS 1 , nitric oxide synthase Prostaglandin E2 receptor EP4 Interleukin 1 beta M15131 M87039 3178- D13458 1146- 827-1225 3455 1442 c-myb proto-oncogene protein (inducible) subtype M16449 1212- Interferon alpha-beta receptor Interleukιn-5 receptor 1513 M89641 808-1120
D90205 1389- Tumor necrosis factor beta TNF- Activating transcription factor 4 1739 beta M94087 416-769 Epidermal growth factor (EGF) M16819 461-805 (mATF4) J00380 180-505 (Lymphotoxin-alpha) Beta2-RAR; retinoic acid Erythropoietin receptor Interleukin- 1 receptor receptor
J04843 1193-1377 M20658 2050- S56660 589-896 Insulin receptor J05149 653- 2410 beta-2 1011 CSF-1, M-CSF; colony Tie-2 proto-oncogene p53; tumor suppressor; DNA- stimulating S67051 1843-2179 K01700 1125- X05010 1268- IGF-I-R alpha; insulin-like 1517 1657 growth binding protein factor- 1 U00182 489-885 Cf2r; coagulation factor II Interleukm-4 receptor factor I receptor alpha subunit
L03529 762-1154 (membrane- IGFR II; insulin-like growth (thrombin) receptor M27959 2469- factor PTPRG; protein-tyrosme 2705 U04710 707-1060
L09562 1248-1504 bound form) receptor II, cation-independent phosphatase gamma fnterferon-gamma receptor mannose-6-P receptor; elevated DNA-binding protein SMBP2 M28233 1262- in
L10075 4790-5088 1550 Wilms's tumor cells Interleukin- 10 receptor Interleukin-7 receptor Stat3; APRF; acute phase
L12120 1762-2110 M29697 701-1104 response Interleukin-2 receptor gamma Gamma interferon induced U06922 1575- chain monokine 1910
L20048 1073-1313 M34815 42-323 factor U18542 1375- Transcription factor 1 for heat D17407 734-1079 1630 shock CdkS, cychn-dependent kinase 5 Endothehn b receptor [Ednrb] X61753 203-570 D29678 552-882
U32329 379-695 gene TGF-beta receptor type 1 Prepro-endothelm-3 CD40L, CD40 ligand X65453 D25540 1407-
U32330 703-1008 545-809 1629 Pre-platelet-deπved growth factor c-Fms proto-oncogene Kinesin like protein KIF 3B X04367 2336- (macrophage D26077 3519- 2677 X68932 2399- 3722 receptor 2686 Kinesin family protem KIF1A
CD 4 receptor (T cell activation colony stimulating factor 1 (CSF- D29951 2553- X04836 1652- 1) 2830 1877 receptor) Fibroblast growth factor 9 antigene) B-myb proto-oncogene, myb- D38258 91-379
Interleukin 7 X07962 241- related Neuional death protein 496 X70472 2109- D83698 627-805
Macrophage lnflamatory protein 2456 Syp, SR-PTP2, adaptor protein
X12531 25-359 protem B D84372 1229- Thrombomodulm XI 4432 Ear-2, v-erbA related proto- 1543 1082-1365 X76654 1065- tyrosine phosphatase
Interleukin 6 (B cell 1376 Interferon regulatory factor 2 differentiation oncogene (IRF 2)
X51975 1638- Tιe-1 tyrosine-protem kinase J03168 718-976 1898 X80764 1425- Lamimin receptor 1 J02870 factor) 1844 368-675
Androgen receptor X53779 receptor NF-IB protein (transcription 2189-2491 Glutamate receptor, tonotropic factor) Bone morphogenetic protein 4 D10651 506-786 D90176 452-791 X56848 1275- NMDA2B (epsilon 2) Jun-B, c-jun-related transcription 1513 Glutamate receptor, lonotropic J03236 514-740 (BMP-4) (TGF-beta family) D10217 3966- factor Transferrin receptor protein (p90, 4209 Tissue plasminogen activator X57349 654-1023 NMD A2 A (epsilon 1) J03520 622-1020 CD71) CD7 antigen D 10329 28- Romeo Box protein 4 2 (Rox-4 2)
Transforming growth factor beta 421 J03770 565-945 2 Transcription factor S-II Nur77 early response protein,
X57413 2227- D00926 518-767 J04113 825-1059 2541 (transcription elongation factor) thyroid hormone (TR3) receptor Glutamate receptor, tonotropic Basic Fibroblast growth factor (b- Ets-2 transcription factor X57497 1290- D 12482 290-620 J04103 917-1281 1657 FGF) c-Jun proto-oncogene AMPAI Bone morphogenetic protem (transcription
TNF 55, tumor necrosis factor 1 receptor J04115 951-1238 X57796 656-1022 D 16250 1454- factor AP-1 component) (55kd) 1837 Seπne protease inhibitor
Mdm2, pS3-regulatιng protem G-protein-coupled receptor homolog J6 X58876 1364- D17292 833-1115 J05609 581-855 NGF) Bcl- (19
KOI 759 642-901 L37296 1079-1375 M28698 194-500 Cdk4; cyclin-dependent kinase 4 XL and Bcl-2; promotes cell human)
L01640 230-616 death RAG-1; V(D)J recombination Acetylcholine receptor delta Jnk stress-activated protein M29475 2155- subunit kinase 2404
K02582 1400- L35236 795-1032 activating protein 1655 (SAPK) Interleukin-3 receptor MAPKKl; MAP kinase kinase 3 Cytoskeletal epidermal keratin M29855 1975-
L02526 1284-1583 (18 2254 (dual specificity) (MKK1) Ml 1686 473-773 K-fibroblast growth factor GABA-A transporter 4 human) M30642 309-577
L04662 960-1341 Nerve growth factor alpha (alpha- Octamer binding transcription GABA-A transporter 3 Mi 1434 294-494 factor
L04663 1010-1320 NGF) M34381 774-999 Vegfrl; Vascular endothelial Epidermal keratin (1 human) (Oct 3) growth M10937 326-683 Plasminogen activator inhibitor
L07297 1144-1541 Nicotinic acetylcholine receptor M33960 1096- factor receptor 1/Fms-related M14537 1226- 1344 tyrosine kinase 1 (Fltl) 1568 CD3 antigen, delta polypeptide Adrenergic receptor, beta 1 MDR1 ; P-glycoprotein; M33158 73-361
L 10084 404-772 multidrug Homeo Box protein 2.5 (Hox- Eph3 (Nuk) tyrosine-protein M14757 1500- 2.5) kinase 1886 M34857 11-277
L25890 2255-2491 resistance protein; efflux pump HSP84; heat shock 84kD protein receptor CD2 antigen M18934 M36829 342-736
MTI1; DnaJ-like heat-shock 354-602 Mast cell protease (MMCP) - 4 protein Homeo Box protein 1.1 (Hox- M55617 634-992
L16953 1059-1384 1.1) Erkl; extracellular signal- from mouse tumor M17192 466-723 regulated TTMP-3 tissue inhibitor of Fetal myosin alkali light chain M61177 115-373
L19622 274-592 M19436 205-504 kinase l; p44; Ert2 metalloproteinases-3 Interleukin 4 M25892 77- P13-K ρ85; phosphatidylinositol Insulin receptor substrate- 1 (IRS- 310 3-
1) Rb; ppl05; Retinoblastoma M60651 981-1260
L24563 1027-1304 M26391 2036- kinase regulatory subunit; YY1 (UCRBP) transcriptional 2296 phosphoprotein p85; PDGF factor susceptibility-associated protein signaling
L13968 1052-1292 (tumor suppressor gene; cell pathway member Interleukin-converting enzyme cycle p58/GTA; galactosyltransferase (TCE) regulator) M58633 1022-
L28095 30-269 Rsk; ribosomal protein S6 kinase 1284 Hepatoma transmembrane kinase M28489 1191- associated protein kinase (cdc2- L38847 927-1219 1436 related protein kinase) ligand Pletelet- derived growth factor (A Serine protease inhibitor 2 (spi-2) Voltage-gated sodium channel M29464 152-425 M64086 1499-
L36179 4179-4505 chain) (PDGF- A) 1754 M64429 1651- Cathepsm H U06119 325- MAPKK4, MAP kinase kinase 4, 2036 694 U18310 1380-
Etkl (Mek4, HEK) tyrosine- Statl, signal transducer and 1749 protem activator Jnk activating kinase 1, (JNKK1,
M68513 2681- U06924 1749- SEK1, MKK4) 2915 2104 Transcription factor LRG - 21 kinase receptor HEK of transcription U19118 618-966 RAG-2, V(D)J recombination p21/Cιpl/Wafl, cdk-inhibitor Interferon inducible protein 1
M64796 671-944 protein U19119 1342- activating protein U09507 9-403 1636 Collagenase type IV Cdk7, M015, cyclm-dependent A20 zinc finger protem,
M84324 696-1040 U11822 454-824 apoptosis Interleukιn-6 receptor beta chain, kinase 7 (homologue of Xenopus U19463 1952- M83336 1423- MO 15 cdk-activatmg kinase) 2293 1741 p27kιpl, Gl cyclin-Cdk protein inhibitor membrane glycoprotein gpl30 U 10440 270-454 pl8ιnk4, cdk4 and cdkβ inhibitor Alpha cardiac myosin heavy kinase inhibitor, p21 -related U19596 16-284 chain Gem, induced, immediate early I-kB (I-kappaB) beta
M76601 2094- U10551 220-471 U19799 419-778 2391 protein, Ras family member Dvl2, dιshevelled-2 tissue
Retinoic acid receptor RXR- VRL, Von Hippel-Lindau tumor polarity gamma U12570 885-1111 U24160 1205-
M84819 701-1082 suppressor protein 1578 Granulocyte-macrophage colony- Cek 5 receptor protein tyrosine piotem M85078 904-1289 U12983 1037- Nuclear factor related to P45 NF- stimulating factor receptor 1287 E2 GABA-A receptor alpha- 1 submit kinase ligand U20532 1429- M86566 1251- Glutathione peroxidase (plasma 1759 1606 U13705 766-1046 MSH2 DNA mismatch repair Endothelial ligand for L-selectm protem), selenoprotein U21011 2150- M93428 182-541 Integrm alpha 5 (CD51) 2490 (GLYCAM 1) U14135 2170- protem, MutS homologue 2 Integrm beta 7 subunit 2516 GapIII, GTPase-activating
M95633 2142- Ski proto-oncogene U14173 protem 2423 707-1037 U20238 328-644
DNAse I U00478 665- Ablphihn-I (abι-1) similar to Syk tyrosine-protein kinase
871 U17698 351-585 U25685 1235- Cortactin, protem tyrosine kinase HOXD3 1524
U03184 426-653 BAG-1, bcl-2 binding protem (activated p21cdc42Rs kinase substrate with (ack)) Adenosine A2M2 receptor U17162 17-334 pi 07, RBL1, Retinoblastoma
U05672 491-735 anti-cell death activity gene DNA hgase I U04674 She transforming adaptor protein, U27177 1973- 1678-2054 U15784 1220- 2365 Adenosine AIM receptor 1451 product-related protem pi 07 (cell
U05671 302-673 Src homology 2 (SR2) protein, cycle regulator) Non-muscle myosin light chain 3 SRB- PMS2 DNA mismatch repair U28724 749-1013 U43205 2037- U60530 584-820 yeast PMSI homolog 2 2285 Cyclin C (Gl -specific) Limphotoxin receptor (TNFR gene frizzled homologue 3; U62638 714-986 U29173 1415- dishevelled receptor Mph-1 nuclear transcriptional 1668 MAPKK3; MAP kinase kinase 3 U63386 1621- family) U43187 1436- 1884
BRCA1; Breast/ovarian cancer 1742 repressor for hox genes U31625 5126- (dual specificity) (MKK3, Rad50; DNA repair protein 5430 MEK3) U66887 1383- susceptibility locus 1 product Myelobiastin, trypsin- 1707 Pml; Murine homologue of the chymotrypsin Fyn proto-oncogene; Src family U33626 1667- U43525 503-807 U70324 584-882 2064 related serine protease member leukemia-associated PML gene Zinc finger Kruppel type Zfp 92 c-myc proto-oncogene protein Transducin beta-2 subunit U47104 578-896 X01023 379-667
U34960 515-834 TDAG51; couples TCR signaling c-Fos proto-oncogene; I-kB (I-kappa B) alpha chain to transcription
U36277 541-823 U44088 729-1042 V00727 482-734 TRAIL; TNF-related apoptosis Fas (CD95) expression factor AP-1 component fos
U37522 981-1288 POU domain, class 2, associated cellular inducing ligand; Apo-2 ligand U43788 610-884 oncogene pl30; Retinoblastoma gene factor 1 Cathepsin L X06086 267- product- ALG-2; calcium binding protem 588
U36799 970-1321 U49112 527-861 Glutamate receptor channel related protem Rb2/pl30 (cell required for programmed cell subunit cycle death X04648 41-408 regulator) Unconventional myosin VI gamma
CACCC Box- binding protein U49739 3784- c-Fes proto-oncogene BKLF 4021 X12616 2342-
U36340 826-1065 Transcription factor CTCF (11 2598 FAF1, Fas-associated protein zmc Cytotoxic cell protease 2 (B10) factor, U51037 1625- X12822 439-686
U39643 423-681 1911 Homeo Box protem 3.1 (Hox- apoptosis activator fingers) 3.1) Zinc finger transcription factor Transcription factor C 1 X07439 449-722 RU49 U53925 3895- Homeo Box protein 24 (Hox-
U41671 1229- 4227 2 4) 1591 Madrl; mSmadl, Mothers against X13721 1949- GTBP; G/T-mismatch binding U58992 238-476 2284 U42190 1477- dpp protein (Mad) murine Fos-B; c-fos-related protein fos B 1769 homologue; TGF-beta signaling X14897 920-1278 protein; MSH6. proteιn-1 (bsp-1), candidate Plasminogen activator inhibitor-2
PLC beta; phospholipase C beta tumor XI 6490 674-978 3 suppressor gene c-ErbA oncogene; thyroid
U43144 1933- Bcl-W apoptosis regulator, Bcl-2 hormone 2271 U59746 153-368 X51983 400-675
Fπzzled-3; Drosophila tissue family member receptor. 894 X66032 874-1236 protein-4 (IGFBP-4)
Vimentin X51438 868- Chop 10; murine homologue of Insulin-like growth factor binding
1096 X67083 17-332 X81579 27-256
HMG-14 non histone Gaddl53 (growth arrest and protein-1 (IGFBP-1) chromosomal DNA- IGFBP-2; Insulin-like growth
X53476 643-1017 damage-inducible protein) factor protein PD-1 possible cell death inducer; X81580 449-817
Macrophage infl amatory protein Ig binding protein 2; autocrine 2 X67914 1481- and/or
X53798 14-352 1734 paracrine growth promoter alpha (MIP 2 alpha) gene superfamily member Insulin-like growth factor binding Bone morphogenetic protein 7 Inhibin beta A subunit (TGF beta X81583 461-824 X56906 670-971 X69619 1064- protein-5 (IGFBP-5) (BMP-7) (osteogenic protein 1) 1304 Insulin-like growth factor binding Transcription factor SP1P family) X81584 701-1039
X56959 866-1128 Vegfr2; KDR/flkl vascular protein-6 (IGFBP 6) (POUdomain transcription factor) X70842 1394- A-myb proto-oncogene; myb- Homeo Box protein 8 (Hox-8) 1721 related
X59252 826-1132 endothelial growth factor tyrosine X82327 1017- Fibroblast growth factor receptor kinase receptor 1334 4 Protease nexin 1 (PN-1) protein A
X59927 2446- X70296 746-985 Membrane type matrix 2820 MRE-binding transcription factor X83536 877-1101 Racl murine homologue X71327 552-916 matalloproteinase
X57277 425-651 Activator- 1 140 KD subunit Elk-1 ets-related proto-oncogene Transcription factor UBF X72711 4137- X87257 1498-
X60831 689993 4375 1680 Kinesin heavy chain (replication factor C 140KD) E2F-5 transcription factor
X61435 1898- DP-1 (DRTF-polipeptide 1) cell X86925 426-728 2182 X72310 925-1305 Lbx 1 transcription factor
CCAAT- Binding transcription cycle regulatory transcription X90829 1000-
X61800 904-1150 factor 1306 factor(C/EBP) 5-Hydroxytryptamine (serotonin) P-selectin (glycoprotein ligand-1) TIMP-2 tissue inhibitor of X72230 982-1314 X91144 1095- X62622 1236- receptor lc 1323 1468 Gelatinase B X72795 599- Transcription factor SEF2 metalloproteinases-2 954 X91753 755-1054 Ets-related protein PEA 3 XPAC; xeroderma pigmentosum Macrophage mannose receptor
X63190 1702- X74351 447-669 Z11974 807-1197 2040 group A correcting protein Rab-2 ras-related protein Vav; GDP-GTP exchange factor; Integrin alpha 2 (CD49b) X95403 232-505 X64361 1083- X75427 1595- Gluthathione S-transferase (theta 1351 1976 X98055 14-298 proto-oncogene Growth/diffferentiation factor 2 typel); phase II conjugation PAX-6 (paired box protein) X77113 939-1329 enzyme X63963 1081- (GDF-2) Zyxin; LIM domain protein; 1325 Insulin-like growth factor binding alpha- 1812 Stat5a, mammary gland factor Retinoid X receptor interacting actimn binding protem Z48538 2269-2628 U09419 1388- Met protooncogene Y00671 Hek2 murine homologue, Mdk5 1682 3646-3933 Z49086 1702-1930 protem (RIP 15) c-Kit proto-oncogene (mast/stem mouse developmental kinase, Cek 7 receptor protein tyrosine cell Eph - U14752 504-837
Y00864 2867- related tyrosine-protein kinase kinase ligand 3181 receptor C-C CKR-1, CCR-1, C-C growth factor receptor tyrosine D-Factor/LIF receptor chemokine kinase) D26177 2376- U29678 168-495 Transcription factor BARX1 2775 receptor type 1, macrophage
Y07960 723-973 Cytoskeletal epidermal keratin inflammatory protein- 1 alpha (homeodian transcription factor) (14 receptor, MIP-1 alpha-R, PLC gamma, phosphohpase C M13806 108-469 RANTES-R
X95346 180-516 human) Glucocorticoid receptor form A gamma R-ras protein, closely related to X13358 1527- Stromelysm-3 matrix ras 1816
Z12604 1463-1806 M21019 215-555 Mothers against DPP protem metalloproteιnase-11 (MMP-11) proto-oncogenes (mad 5-Hydroxytryptamme (serotonin) Prolactin receptor PRLR2 X83106 464-728
Z14224 530-774 M22959 1-328 homolog Smad 1, transforming receptor le beta Blk, B lymphocyte kinase, Src growth factor beta signaling 5-Hydroxytryptamine (serotonin) M30903 1307- protem)
Z15119 588-940 1672 Hck tyros e-protein kinase receptor 2c family member Y00487 1308- Low density lipoprotem receptor Macrophage inflammatory 1563
Z19521 1047-1324 protem 1 Photolysase/blue-hght receptor 5-Hydroxytryptamine (serotonin) M35590 119-445 AB000777 1418-
Z23107 460-817 beta (Act 2) 1737 receptor 7 Alpha-1 protease inhibitor 2 homologue c-Mpl, thrombopoietin receptor, M75716 625-969 Osp94 osmotic stress protein,
Z22649 1561-1772 GABA-A transporter 1 APG- mematopoietic growth factor Bone morphogenetic protein 8a D49482 1026- receptor M97017 788-1139 1266 superfamily member (BMP-8a)(TGF-beta family) l, hsp70-related DNA-polymerase delta catalytic Erythroid kruppel-hke Glucose regulated protem, 78kD,
Z21848 1256-1600 transcription D78645 167-411 subunit M97200 783-1171 Grp78
Folhstatin Z29532 764- factor LCR-1, CXCR-4, CXC(SDF-l) 1053 GATA binding transcription D87747 584-867 Cyclin F (S/G2/M-specιfιc) factor chemokine receptor 4, HIV
Z47766 2431-2708 M98339 81-379 coreceptor (fusm), G protein- Ets-related protein Sap 1A (GATA-4) coupled
Z26885 1267-1521 Growth factor receptor receptor LCR1 homologue, Net, ets related transcription M98547 1701- Glucose transporter- 1, factor, 2014 erythrocyte,
Z32815 1211-1595 Crk adaptor protem M23384 325-653 Int-3 proto-oncogene; NOTCH ATP-binding casette 8; ABC8; M12302 585-830 M80456 1846- U34920 1011- serine protease CCP I gene 2145 1319 (CTLA- family member; NOTCH4 homolog of Drosophilia white 1) c-Akt proto-oncogene; Rac- CDC42 GTP-binding protein; Cathepsin B Ml 4222 384- alpha; G25K 729
M94335 604-899 U37720 1675- Growth hormone receptor proteine kinase B (PKB) 1982 M33324 1924- Bak apoptosis regulator; Bcl-2 Etoposide induced p53 2240 Y13231 1509- responsive CD28 (receptor for B71) 1786 U41751 1041- M34563 544-774 family member 1296 Estrogen receptor M38651 PS-2; homologue of the (Et24) mRNA 742-1013 Alzheimer's Casein kinase II (alpha subunit) Monotype chemoattractant
U57324 437-783 U51866 1237- protein 3 disease gene 1517 S71251 201-491 BRCA2; Breast cancer TSG101 tumor susceptibility CD45 associated protein (CD 45- susceptibility protein ap,
U65594 649-922 U52945 446-713 U03856 620-898 locus 2 product Tumor suppressor maspin LSM-1) DNA ligase III U66058 U54705 251-507 Orphan receptor Ul 1688 2980-3205 FLIP-L, apoptosis inhibitor; 1686-1943 Caspase-7; Lice2; ICE-LAP3 FLICE- Cannabinoid receptor 1 (brain) U67321 1040- U97076 1476- U17985 1091- 1280 1811 1437 cysteine protease like inhibitory protein Dystrog can 1 U43512 BID; apoptic death agonist CamK II; Ca2+/calmodulin- 2267-2505
U75506 452-777 X63615 1951- G-protein coupled receptor WBP6; pSK-SRPKl; WW 2219 U46923 350-671 domain dependent protein kinase II (beta Urokinase type plasminogen
U92456 482-774 subunit) X02389 1301- binding protein 6 serine kinase Htk; Mdk2 mouse developmental 1538 for Z49085 2032-2365 activator SR splicing factors kinase; Eph-related tyrosine- CTLA-4 (immunoglobin Cyclin G2 (G2/M-specifιc) protein superfamily
U95826 408-688 kinase receptor X05719 246-519
Ungl; uracil-DNA glycosylase Glial cell line-derived member)
X99018 444-729 neurotrophic Myogenic factor 5 X56182
D49921 236-539 232-528 factor uPARl; urokinase plasminogen
Rab-3b ras related protein CD31 (Platelet endothelial cell X62700 482-756
Y14019 232-562 L06039 1172-1494 activator surface receptor (CD87)
Inhibitor of the RNA-activated adhesion molecule 1) Serine protease inhibitor 2.4 U28423 180-487 CD22 antigen LI 6928 X69832 621-927 protein kinase, 58-kDa 2314-2645 SRY-box containing gene 4
Golgi 4-transmembraπe spanning Gbx 2 L39970 1122- X70298 34-311 U34259 742-1060 1395 Bone morphogenetic protein 2 (BMP-2)(TGF-beta family) coupled receptor) NEX-1 [K02588]P-l-450, dioxm- AT motif-binding factor ATBF1 Brain factor 1 (Hfhbfl) mducible D26046 9807- U36760 1080-
M10021 3729- 10112 1318 4014 HMG-box transcription factor Split hand/foot gene cytochrome P450 [K02588] from U41626 92-303 Bcl-2, B cell lymphoma protein D49474 427-662 Sim transcription factor 2, testιs (MusSoxl7) U42554 2828-
M16506 2125- Ikaros DNA binding protein 3066 2367 L03547 627-890 Glial cells missing gene homolog apoptosis inhibitor Early B cell factor (EBF) U59876 727-1080 CD 14 antigen M34510 L12147 750-1026 (mGCMl) 667-931 Engaπled protein (En-1) Sp4 zinc finger transcription
Somatostatm receptor 2 homolog factor
M81832 47-310 L12703 1323-1554 U62522 1704- Dopamine receptor 4 Engrailed protein (En-2) 1929
U19880 907-1191 homolog Heat shock transcription factor 2 Cannabmoid receptor 2 L12705 1626-1895 X61754 1445-
U21681 910-1262 Transcription factor A10 1640 (macrophage, CB2) L21027 499-806 (HSF 2) Erf(Ets-related transcription Myocyte nuclear factor (MNF) RNA polymerase I termination factor) L26507 1203-1456 factor
U58533 1286- Basic domain/leucme zipper X83974 3222- 1613 L36435 872-1073 3433 5-Hydroxytryptamine (serotonin) transcription factor TTF-1 Z11597 1043-1355 Caudal type Homeobox 1 (Cdxl) Hepatocyte nuclear factor receptor lb M37163 1040- 3/forkhead
Tob antoprohferative factor, 1301 L35949 913-1232 interacts Butyrate response factor 1 homolog 8 (HFH-8)
D78382 540-876 M58566 768-22 SRY-box containing gene 3 wιth pl85erbB2 Brain specific transcription factor (Sox3) Gluthathione S-transferase S53744 1548-1754 X94125 212-443
J03752 185-428 NURR-1 Cot proto-oncogene D 13759 (microsomal) Brn-3 2 POU transcription factor 696-956 Adensine A3 receptor S68377 877-1237 HR21spA, protein involved in
L20331 182-382 Caudal type Homeobox 2 (Cdx2) DNA p55cdc, cell division control S74520 1085-1367 D49429 103-434 protein Erythroid transcription factor NF- double-strand break repair,
U05341 1061- E2 PW29, 1348 U01036 1-241 calcium-bindmg protein 20 Gut-specific Kruppel-hke factor MmLιml5, RecA-hke gene,
AP endonuclease, U 12273 U20344 1558- DMC1 1894-2150 1789 D64107 581-781 apuπnic/apynmidinic GKLF homologue, meiosis-specific endonuclease Kruppel-hke factor LKLF homologous recombination (Apex) U25096 898-1193 protein Mas proto-oncogene (G-protem Neuronal hehx-loop-helix protein ERp72 endoplasrmc reticulum J05186 1160-1470 1561 2286 protein; protein disulfide mRNA Dtk) isomerase- ST AM; signal transducing H-ras proto-oncogene; related protein adaptor transforming HMG1 -related VDJ U43900 576-811 Z50013 1307-1544 recombination molecule G-protein
S50213 2263-2531 ShcC adaptor; She-related; brain- ERBB-2 receptor (c-neu; HER2 signal binding protein U46854 246-601 L47239 16-266 Gli oncogene; zinc finger specific protein tyrosine kinase)
S65038 104-505 MmMrel la putative U58987 ERBB-3 receptor L47240 4- transcription factor 866-1204 243 Tiam-1 invasion inducing endo/exonuclease Placental ribonuclease inhibitor protein; PCNA; proliferating cell nuclear U22516 512-766
U05245 4329- X53068 53-320 (Angiogenin) 4628 antigen; processivity factor myosin I L00923 2578-
GDP-GTP exchanger-related Translin; recombination hotspot 2921 Sik; Src-related intestinal kinase X81464 205-431 Ca2+ binding protein, Cab45 U16805 1246- binding protein U45977 597-1082 1623 PA6 stromal protein; RAG1 gene murine ornithine decarboxylase
Lfc proto-oncogene U28495 X96618 442-749 M10624 865-1252 853-1150 activator Oxidative stress-induced protein Sky proto-oncogene (Tyro3; Rse; U40930 1248- U18342 1927-
Example 1.
[0057] The composition of this invention can be prepared in a variety of ways well known to those of skill in the art. In general, the hydrophile-lipophile values required for various applications are as follows: 3 to 6 water-in oil emulsifier (e.g., Arlacel+Squalene); 7 to 9 wetting agent; 8 to 13 oil-in-water emulsifier; 13 to 15 detergent; 15 to 18 solubilizer (e.g., aluminum or magnesium stearate). These values are widely quoted in the literature as guides to the selection of emulsifiers for particular purposes. They are designed for use with nonionic emulsifiers. Analogous systems have been developed for anionic or cationic emulsifiers, but they are less useful than those for nonionic emulsifiers. Hydrophilic- lipophilic balance numbers have been published for many nonionic. For this particular use an oil-in-water emulsion that has about 0.5 to 55% of an oil, about 0.1 to 15% of an emulsifier, about 0.05 to 5% of a nonionic surfactant, about 0.00001 to 10% of a therapeutic agent, and an aqueous continuous phase is most suitable. The emulsifier in the composition is a phospholipid compound or a mixture of phospholipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin. An example of emulsifier is Arlacel A. Surfactant is usually selected from the group consisting of fatty alcohols, polyethylene gly col esters of fatty acids, polyethoxylated fatty acid esters, polyethoxylated fatty alcohol ethers, an alkylene oxide condensate of an organic compound having one or more hydroxyl groups, and polyethoxylated alkylphenyl ethers. Surfactant can be a natural biologically compatible surfactant such as lecithin or a pharmaceutically acceptable non-natural surfactant such as TWEEN-80. Suitable natural components relating to lecithin are for example EPICURON 120 (Lucas Meyer, Germany) which is a mixture of about 70% of phosphatidylcholine, 12% phosphatidylethanolamine and about 15% other phospholipids; OVOTHIN 160 (Lucas Meyer, Germany) which is a mixture comprising about
60%o phosphatidylcholine, 18%) phosphatidylethanolamine and 12%o other phospholipids; a purified phospholipid mixture-LIPOID E-75 or LIPOID E-80 (Lipoid, Germany) which is a phospholipid mixture comprising about 80% phosphatidylcholine, 8% phosphatidylethanolamine, 3.6% non-polar lipids and about 2% sphingomyelin. Purified egg yolk phospholipids, soybean oil phospholipids or other purified phospholipid mixtures are useful as this component. This listing is representative and not limiting, as other phospholipid materials which are known to those skilled in the art can be used. Other examples of surfactants are polyethylene glycol esters of fatty acids from sources such as castor oil
(EMULFOR); polyethoxylated fatty acid, e.g., stearic acid (SIMULSOL M-53); NONIDET; polyethoxylated isooctylphenol/formaldehyde polymer (TYLOXAPOL); polyoxyethylene fatty alcohol ethers (BRIJ); polyoxyethylene nonphenyl ethers (TRITON N); and polyoxyethylene isooctylphenyl ethers (TRITON X) among many others. In some embodiments the emulsion may be formed and stabilized in the substantial absence of one or more cosurfactants selected from the group consisting of an unhalogenated aliphatic C3-C6 alcohol, a free fatty acid, a mono- or di-glyceride, a polyglycerol fatty acid ester, or a lysophosphatidyl choline. However, alternative composition is equally suitable and can be readily prepared by those of skill in the art, for example a composition can contain 5.0%)
PLURONIC, 10%) Squalene, 0.4% Tween-80, qs phosphate buffered saline (pH 7.4); the components are added to a test tube and vortex-mixed until a milky emulsion is obtained.
This composition is prepared immediately prior to administration or refrigerated at 4°C.
Composition 2 will for example contain 5.0% TETRONIC 1501, 10% Squalane, 0.4%
Tween-80, qs phosphate buffered saline (pH 7.4); To these compositions is then added solid
N-acetylmuramyl-L-threonyl-D-isoglutamine (Thr-MDP) to a concentration of 500 ug/ml, to form the "concentrate." The concentrate is then mixed with a 2x concentration solution of antigen (hCG in saline, 1 mg/mL) to form the formulation of the invention. The hCG is preferably present in an amount of about 0.00001 to 10%> by weight of the composition, preferably about 0.0001 to 5% and most preferably 0.001 to 1%. Also, the pH should be in a range which is suitable for the stability of hCG. The continuous phase of the composition is aqueous, and can additionally contain salts, sugars, antioxidants, preservatives, microbicides, buffers, osmoticants, cryoprotectants, and other pharmaceutically useful additives or solutes.
Typical preservatives include Thimerosal, chlorbutanol, and methyl, ethyl, propyl or butyl
• parabens. Typical osmotic pressure regulators include glycerol and mannitol, with glycerol being preferred. The preferred oil phase antioxidant is alpha-tocopherol or alpha-tocopherol succinate. The aqueous phase may also include an antioxidant of a polyamine carboxylic acid such as ethylene diamfno tetraacetic acid, or a pharmaceutically acceptable salt thereof.
Example 2.
[0058] The instant composition can consist of two parts. Part I is a N-Acetylmuramyl-L-
Threonyl-D-Isoglutamine (Thr-MDP), a derivative of mycobacterium cell wall component.
Part II consists of phosphate buffered saline containing a final concentration of 5% Squalane,
1.25%) pluronic and 0.2%) TWEEN 80 (vehicle). For practical purposes, desired quantity of hCG is mixed with microfluidized vehicle (part II) to obtain a homogeneous emulsion. MDP is then added and vortexed briefly. The MDP concentration in the mixture is varied to determine the optimum concentration for clinical response. As an adjuvant control, mice are injected with soluble hCG mixed with alum according to the manufacturer's manual (Pierce
Chemical, Rockford, 111.) or with Complete Freund's Adjuvant (CFA).
[0059] Those of ordinary skill in the art will recognize that such a mouse model is indicative that equivalent experiments or treatments will similarly induce clinical responses in humans, domesticated, or agricultural animals. The amount of formulation and hCG useful to produce the desired clinical response can be determined empirically, without undue experimentation, by routine procedures well known to those of ordinary skill in the art. Thus, if desired to minimize the side effects of treatment with such a mixture those of ordinary skill in the art can determine a minimum level of such a mixture for administration to a human, domesticated, or agricultural animal in order to obtain an effective response, and thereby induce desired clinical outcome. In normal use, such a mixture will be injected by any one of a number of standard procedures, but particularly preferred is an subcutaneous or intramuscular injection at a location which will allow the emulsion to remain in a stable form for a period of several days or several weeks.
Example 3.
[0060] The hCG aqueous suspension is added in an oily phase at room temperature while simultaneously homogenizing with a homogenizer. The addition of the aqueous phase is stopped when the ratio of 5-6 parts of oil to 3-5 parts of hCG suspension is reached.
Homogenizing is continued until the drop-size of the aqueous phase is about 0.05-0.5 microm. The oily phase contains following: Marcol 52 (white paraffmic Esso oil)--93.6%>;
Arlacel A or Arlacel 80 or Span 80 (mannide monooleate)~6.0%; Tween 80
(polyoxyethylene 20 sorbitan monooleate)~0.4%. The compounds of the oily phase can be separately heated to 110° C in the autoclave or sterile filtered as a mixture. The stability of emulsion is determined by (1) pipetting drops directly after emulsifying onto a water surface, whereby the drops do not spread, but remain intact; and (2) storage at 37°C for 4 weeks does not lead to formation of any aqueous phase. The final concentrations of the thus prepared emulsion contains about 10%> of hCG by weight and the preparation is used in a dosage of 0.5 ml per subject intramuscularly or subcutaneously.
Example 4.
[0061] Without limiting to examples above present composition comprising dimer hCG can contain two emulsifiers at the same time to provide better stability. In this composition primary emulsifier is selected from the group consisting of polyoxypropylene- polyoxyethylene block copolymers, glycerol monooleate, glycerol dioleate, sorbitan sesquioleate, Brij 93 polyoxyethylene alcohol, sorbitan monooleate and mixtures thereof and secondary emulsifier is selected from the group consisting of polyoxyethylene fatty acid esters, Pluronic F68 ethylene oxide, egg lecithin and mixtures thereof. The oil fraction in this composition is a triglyceride selected from the group consisting of tricaproin, tricaprylin, tripalmitin and mixtures thereof. Other suitable oils include vegetable and animal oils.
Suitable vegetable oils include; olive, safflower, sesame and soybean and suitable animal oils include those containing glycerol esters. Preferred mineral oils include: No. 40 white oil,
Carnation light oil and Klearol light oil. Mixtures of these oils can be equally used.
[0062] For a primary emulsion using mineral oil, the aqueous hCG solution in an amount of about 1 to about 50 volume percent, preferably about 2 to about 40 volume percent; mineral oil in an amount of about 8 to about 58 volume percent, preferably about 14 to about 25 volume percent; and primary emulsifying agent in an amount of about 2 to about 30 volume percent, preferably about 5 to about 15 volume percent are mixed. The aqueous hCG solution is preferably slowly added to the rapidly stirring oil phase and mixed, such as by use of a magnetic stirrer, for about 15 minutes to about 60 minutes and preferably about 25 minutes to about 35 minutes. The mixed primary emulsion is then subjected to high shear emulsification providing shear rates of about 100,000 to about 5,000,000 and preferably about 500,000 to about 1,000,000 per second such as by using a microfluidizer at a pressure drop of about 1000 to about 10,000 psi, preferably about 1000 to about 3000 psi, and most preferably about 1800 to about 2000 psi. More complete information regarding the microfluidizer is set forth in U.S.
Pat. No. 4,533,254. The microfluidizer provides high sheer rates of short duration, a fraction of a second, without denaturation of proteins, such as hCG. The primary emulsion is filtered, such as by using a 5 micron hydrophilic poly vinylidene difluoride filter (Duropore, Millipore
Corp.). Albumin can be added to the hCG solution prior to emulsification in amounts of about
1 to about 5 g %>, preferably about 2 to about 3 g % to aid in stabilizing the multiple emulsion size distribution. The primary emulsion of aqueous hCG in mineral or fixed oil to be suitable for preparation of the liquid multiple emulsion of this invention, should result in primary emulsion droplet diameters in the range of less than 5, and preferably less than 3, microns.
Example 5.
[0063] A water-in-oil-in- water multiple emulsion consisting of 50% v/v of an outer aqueous phase of saline with 0.25% copolymer P123 is prepared. The other 50%) v/v is dispersed water-in-oil phase consisting of 72%) saline, 18% Squalene, 2% Span 80 and 32 mg copolymer L310 and 0.5 mg TNP-HEA per 0.5 ml of emulsion. The emulsion contains lmg of hCG in 0.5 ml emulsion. The microscopic appearance of the emulsion is 1.0 to 20 micron diameter particles of water-in-oil emulsion. Other water-in-oil emulsifiers equally suitable for this composition are SF 1328, Arlacel P135, DC 3225C, DC 5200, Abil EM-90, and Abil
WE-09. Those skilled in the art will recognize that there are several other water-in-oil emulsifiers could also be used in place of above named ingredients.
Example 6.
[0064] Liposomes containing hCG are prepared according to the standard procedures well known in the art (see for example U.S. Patent No . 6, 110,492) . Following components can be employed: DLPC dilauroyl phosphatidyl-choline; DMPC dimyristoyl phosphatidyl-choline;
DPPC dipalitoyl phosphatidyl-choline;DSPC distearoyl phosphatidyl-choline; DOPC dioleoyl phosphatidyl-choline; DLnPC dilinoleoyl phosphatidyl-choline; DMPG dimyristoyl phosphatidyl-glycerol; CHOL cholesterol; LA Lipid A. In a typical preparation, multilamellar liposomes are made from a mixture of DMPC:DMPG:CHOL:LA in a molar ratio of
9:1 :7.5:0.011. The lipid A is included as an adjuvant. The lipid mixture is rotary evaporated to a dry thin film at approximately 40 °C in vacuo from a chloroform solution in a pear-shaped flask. To ensure complete removal of the organic solvent, the flask is then dried under very low vacuum (about 0.05 mm Hg) overnight in a desiccator at room temperature. After drying, the lipids are carefully swollen in deionized, sterile pyrogen-free water by vortexing. The resulting suspension is frozen at -55°C lyophilized at -20°C overnight and 0°C -10°C the following day using the Virtis Unitop 800SL Freeze Mobile. The lyophilized lipids are then reconstituted in the presence of the substance, i.e., hCG, to be encapsulated to obtain multilamellar liposomes containing this substance. A suitable reconstituting buffer is phosphate-buffered saline (PBS) or Tris-glycine/NaCl. The liposomal phospholipid concentration in the reconstituting buffer is 10-200 mM. Unencapsulated hCG may be removed by washing the liposomes three times with 0.15 M NaCl at 27000xg for 10 minutes at 10°C. The resulting liposomes are suspended either in 0.15 M NaCl or an appropriate isotonic buffer to reach a final phospholipid concentration of 10-200 mM. Alternatively, the wash step may be omitted, leaving both unencapsulated and encapsulated hCG in the preparation.
Example 7.
[0065] This example allows coupling of hCG to MDP. This procedure calls for quenching the first reaction with a thiol compound. The reaction is carried out in 2-[N- morpholino] ethane sulfonic acid (MES) (pH 4.5-5.0). MDP (10 mg) lyophilized from water resuspended in MES (0.5 ml) (pH 4.5-5.0) and 0.5 ml EDC (0.5 mg or about 2 mM) dissolved in MES (pH 4.5-5.0) are combined and reacted for 15 minutes at room temperature. 2- mercaptoethanol (final concentration of 20 mM) is added to quench the EDC and separated by centrifugation. The reaction mixture is washed once with MES and resuspended in 0.5 ml
MES (pH 4.5-5.0). The hCG dissolved in MES is added to the activated MDP at a molar ratio of about 2:1. The PH of the reaction is slowly increased over a 15 minute period to 8.5 by the addition of MES (0.5M pH 8.5) and reacted for 2 hours at room temperature. The concentration of hCG added to MDP was calculated from quantitative analysis of MDP terminal carboxy group and expressed as mole per mg MDP. The reaction is quenched by adding hydroxylamine to a final concentration of 10 mM. This method of quenching hydrolyzed any unreacted MDP activation sites and results in regeneration of the original carboxyls. Other means of quenching involve adding 20-50 mM Tris, lysine, glycine, or ethanolamine. Other biological response modifiers such as IL2 are well known to those skilled in the art and could be used for this purpose in addition to hCG. Separation is achieved by centrifugation, a wash step, and resuspension in the buffer of choice.
Example 8.
[0066] The composition of this invention can also be used as a cream which upon topical application can penetrate across the skin. An exemplary topical composition can contain following: 2% glycerol sorbitan oleostearate (Arlacel 481); 6% polyethoxylated fatty acids
(Arlacel 989); 15% decyl oleate (Cetiol C); 8% branched-chain paraffin (Arlamol HD); 1% microwax; 1% MDP; 4%> glycerine; 0J%>magnesium sulfate heptahydrate; 0.2%> sodium salt of sorbic acid; 1% hCG; 0.1% diammonium hydrogenocitrate; citric acid (to adjust the pH to
4-5); and demineralized water to 100%.
Example 9.
[0067] Males and non pregnant females over the age of 18 are selected based on their having a serodiagnosis of HIV infection documented by Western Blot, with AIDS defining criteria, clinically symptomatic, and with a CD4 T lymphocyte count <300 cells/ul within 30 days prior to study entry. Standard clinical and laboratory parameters are used to evaluate efficacy. Clinical parameters include changes in opportunistic infections, changes in body weight, changes in gastrointestinal function, including stool consistency and frequency, changes in energy level, changes in appetite, physical strength and endurance, and an overall change in the quality of life. Laboratory parameters include changes in CD4 and CD8 lymphocyte numbers, selected hematology, blood chemistry and urinalysis, and, when available, changes in viral loads by measuring viral RNA by PCR.
[0068] The study results show that with the use of the formulated hCG according to the spirit of this invention HIV-positive patients improve clinically, with decreases in opportunistic infections, increases in body weight, changes in gastrointestinal function, including less severe diarrhea, increases in energy level, increases in appetite, increases in libido, improvements in physical strength and endurance, and an overall improvement in the quality of life. Laboratory parameters show improvements, with increases in CD4 and CD 8 lymphocyte numbers, improvements in hematology, blood chemistry, and urinalysis numbers, as well as decreases in viral loads by measuring viral RNA by PCR and decreases in infectivity when measured by TCID. Minor adverse reactions are noted upon administration of formulated hCG which are limited to minor fever, chills, headache, and muscle ache.
Example 10.
[0069] Female mice of C3H background (H2k/k, Harlan Sprague Dawley) are used in these studies. Animals are maintained according to "Guide for the Care and Use of Laboratory
Animals" (DHHS, NIH), and receive food and water ad libitum. The tumor cell line HOPE2 is maintained by serial passage in vitro. Tumors are initiated in syngeneic C3H mice by subcutaneous injection of 150,000 in vitro passaged cells. Tumors are measured in 2 perpendicular directions at biweekly intervals. Each treatment group is compared to a control group that did not receive therapy. Therapy began 10 days after inoculation of HOPE2 cells, when a majority of the tumors are palpable (approx. 50-75 mm3). Therapy is initiated by injecting mice with soluble hCG protein in MDP mixture or Alum adjuvants (subcutaneously in a total volume of 0.2 ml). Shortly before inoculation, hCG is mixed for 60 seconds MDP in
Hanks Balanced Salt Solution (HBSS) such that each mouse received a human equivalent of either 2,000 or 5,000 IU of hCG protein in 0.2 ml. Alum (Pierce Chemical Co.) is mixed with hCG, according to instructions by the manufacturer, such that each animal received an equivalent of 5,000 IU of hCG in 0.2 ml per mouse. In this example, 41 days after tumor cell inoculation only 5/8 and 4/8 of mice receiving a single injection of soluble hCG (2,000 IU or 5,000 IU respectively) display measurable tumors. In contrast, all of the mice injected with with hCG in Alum (8/8) show actively growing tumors. Additionally, significant inhibition of tumor growth is observed only in treatment groups injected with hCG in MDP as compared to control (untreated) or Alum treatment groups. Inhibition of tumor growth or increased tumor regression rates is not observable in mice receiving a single injection of hCG in Alum. [0070] Accordingly, the present invention can be characterized as a pharmaceutical formulation comprising a dimer hCG and a muramyl peptide. Alternatively, the invention can be characterized as a therapeutic composition, comprising: (a) a therapeutically effective amount of dimer hCG in association with liposomes; and (b) an oil-in-water emulsion comprising an oil surrounding said liposomes, said emulsion being present in an amount sufficient to increase therapeutic response relative to that of hCG and liposomes in the absence of said emulsion. Still further, the invention provides a method of treating HIV infection in a subject in need thereof, comprising administering an effective amount of hCG to said subject, said hCG being administered with a oil-in-water emulsion, and said emulsion is present in an amount sufficient to increase a clinical response relative to that of said hCG in the absence of said emulsion. The invention further provides a method of treating cancer in a subject in need thereof, comprising administering an effective amount of hCG to said subject, said hCG being administered with a oil-in-water emulsion, and said emulsion is present in an amount sufficient to increase a clinical response relative to that of said hCG in the absence of said emulsion. The invention still further provides a therapeutic composition which comprises an oil-in-water emulsion having a continuous water phase and a discontinuous oil phase and containing at least 0.01% by weight of dimer hCG. The invention also provides a method for preparing a pharmaceutical formulation, which method comprises admixing, by supplying, an aqueous suspension comprising hCG and at least one oily ingredient and at least one emulsifier in a sufficient quantity to provide said formulation as an oil-in-water emulsion. The invention still further provides a therapeutic composition which comprises an oil-in- water emulsion and at least one natural or recombinant water-soluble biologically active protein and optionally at least one muramyl peptide. The invention also provides a kit for extemporaneous preparation of a pharmaceutical composition, said kit comprising: (1) a first container containing an emulsion, where said emulsion comprises: squalane or squalene; Arlacel, and an aqueous solution; and (2) a second container containing therapeutically effective amount of dimer hCG in an aqueous solution or as a powder, where the concentrations of the components in each container are selected such that combination of the contents of both containers produces a formulation comprising said Arlacel in an amount of between 1%> and 30%; squalane or squalene in an amount of between 1%> and 30%; muramyl peptide in an amount of between 0.0001% and 30%; and hCG in amount between 0.01% and
99%.
[0071] While the present invention has been fully described above with particularity and detail in connection with what is presently deemed to be the most practical and preferred embodiment(s) of the invention, it will be apparent to those of ordinary skill in the art that many modifications thereof may be made without departing from the principles and concepts set forth herein. Accordingly, the proper scope of the present invention should be determined only by the broadest interpretation of the appended claims so as to encompass all such modifications as well as all relationships equivalent to those described in the specification.

Claims

CLAIMSWhat is claimed is:
1. A pharmaceutical formulation comprising a dimer hCG and a muramyl peptide.
2. The composition of claim 1 which further comprises an emulsifier.
3. The composition of claim 2 which further comprises a surfactant.
4. The composition of claim 3 which further comprises an oil.
5. The composition of claim 1 which comprises at least 0.01% by weight of dimer hCG.
6. A therapeutic composition, comprising: (a) a therapeutically effective amount of dimer hCG in association with liposomes; and (b) an oil-in-water emulsion comprising an oil surrounding said liposomes, said emulsion being present in an amount sufficient to increase therapeutic response relative to that of hCG and liposomes in the absence of said emulsion.
7. The composition of claim 6, wherein said oil is squalene.
8. The composition of claim 6, wherein said hCG comprises at least 0.1 % volume of said oil.
9. The composition of claim 6, wherein said composition further comprises an additional emulsifying agent.
10. The composition of claim 9, wherein said additional emulsifying agent is selected from the group consisting of sorbitan esters, polyoxyethylene sorbitan mono-, di-, or triesters, polyoxyethylene fatty acids, polyoxyethylene fatty acid ethers, and combinations thereof.
11. The composition of claim 6, wherein said composition further comprises an adjuvant.
12. The composition of claim 11, wherein said adjuvant is selected from the group consisting of N-acetyl-glucosaminyl-N-acetyl-muramyl-L-alanyl-D-isoglutamine (GMDP), N-acetyl- D-glucosaminyl(beta- 1 -4)-N-acetyl-muramyl-L-alanyl-D-isoglutamine, N-acetyl- glucosaminyl-N-acetyl-muramyl-L-alanyl-D-glutamic acid (GMDP-A), muramyl dipeptide-phosphatidyl-ethanolamine, muramyltripeptide phosphatidylethanolamine, muramyl tripeptide phosphatidyl ethanol amine, CGP 11637 (nor-MDP), alpha(N-acetyl- muramyl-L-alanyl-D-isoglutamine), beta, gamma-dipalmitoyl-sn-glycerol, alpha(N-acetyl- muramyl-D-alanyl-D-isoglutamine), beta, gamma-dipalmitoyl-sn-glycerol, alpha(N- acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine), beta, gamma-dipalmitoyl-sn- glycerol, alpha(N-acetyl-muramyl-D-alanyl-D-isoglutaminyl-L-alanine), beta, gamma- dipalmitoyl-sn-glycerol, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 ,2- dipalmitoyl-sn-g lycero-3 -(by droxyphosphoryloxy))ethy lamide, glucoasminylmuramylpeptides, murametide, murabutide, muradimetide, myramistin, N- acetylmuramyl-L-threonyl-D-isoglutamine, N-acetyhnuramyl-L-.alpha.-aminobutyryl-D- isoglutamine, 6-0-stearoyl-N-acetylmuramyl-L-.alpha.-aminobutyryl-D-isoglutamme, N- acetylmuramyl-L-valyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutamine, N- acetyl-desmethylmuramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D- glutamine butyl ester, N-acetylmuramyl-L-seryl-D-isoglutamine, N-butyrylmuramyl-L-
.alpha.-aminobutyryl-D-isoglutamine, N-acetylmuramyl-L-threonyl-D-isoglutamine,
Bis(6-0-muramyl dipeptide)0-palmitoyltartronate, muramyl tripeptide phosphatidylethanolamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 ,2- dipalmitoyl-sn-g lycero-3 -(hydroxyphosphorIoxy))etlιylamide, N-acetyl-muramyl-L- alanyl-D-glutamine butyl ester, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-
1,2-dipalmitoyl-sn-g lycero-3-(hydroxyphosphoryloxy)ethylamide (MTP-PE), cholesteryl-
MDP, beta-butyl glycoside of N-acetylmuramyl-L-alanyl-D-isoglutamine, 2-acetamido-
4,6-di-0-acetyl-2-deoxy-3-0-[(R)-l-(methoxycarbonyl)ethyl]-alpha-D-glucopyranose,
(beta-butyl-MDP, MTPO-26, beta-cholesteryl-MDP), saponin (Taurosid I), N-acetylnor- muramyl-L-N-methylalanyl-D-isoglutamine octylamide, UDP-N-acetyl-muramyl- pentapeptide, L4-MDP-ONB, L-alanyl-gamma-D-glutamyl-meso-diaminopimelate, 1,6- anhydro muramyl dipeptide, N-acetylglucosamine-beta-1, 4-N-acetyl-muramyl- pentapeptide-pyrophosphoryl-undecaprenol, 3-0- [N-acetylmuramyl-D-isoglutaminyl] -
1 ,2-di-O-palmitoyl-sn-glycerol, L-threonyl-MDP-GDP, L-allothreonyl-MDP-GDP, trehalose 6,6-diester, muramyl dipeptide, B30-muramyl dipeptide, and muramyl dipetide- lysine.
13. A method of treating HIV infection in a subject in need thereof, comprising administering an effective amount of hCG to said subject, said hCG being administered with a oil-in- water emulsion, and said emulsion is present in an amount sufficient to increase a clinical response relative to that of said hCG in the absence of said emulsion.
14. The method according to claim 13, wherein said emulsion further contains an effective amount of a muramyl peptide to be administered in an amount ranging from 1 to 500 microgramg/kg of body weight of the subject.
15. The method according to claim 13, wherein said hCG is provided in association with liposomes.
16. A method of treating cancer in a subject in need thereof, comprising administering an effective amount of hCG to said subject, said hCG being administered with a oil-in-water emulsion, and said emulsion is present in an amount sufficient to increase a clinical response relative to that of said hCG in the absence of said emulsion.
17. The method according to claim 16, wherein said emulsion further contains an effective amount of a muramyl peptide to be administered in an amount ranging from 1 to 500 microgram kg of body weight of the subject.
18. The method according to claim 16, wherein said hCG is provided in association with liposomes.
19. The method according to claiml6, wherein said hCG is administered intravenously, peritoneally, intrathecally, intramuscularly, subcutaneously, mtradermally, buccally, orally, intranasally, opthalmically or topically.
20. A therapeutic composition which comprises an oil-in-water emulsion having a continuous water phase and a discontinuous oil phase and containing at least 0.01%) by weight of dimer hCG.
21. The composition of claim 20 which further contains a therapeutically effective amount of muramyl peptide.
22. The composition of claim 20 wherein said oil phase comprises mineral oil, squalene, peanut oil, vegetable oil, or silicone oil.
23. The composition of claim 20 wherein said emulsion comprises surfactants selected from the group consisting of sorbitan esters, polyoxyethylene sorbitan mono-, di-, or triesters, polyoxyethylene fatty acids, polyoxyethylene fatty acid ethers, and combinations thereof.
24. The composition of claim 23 wherein said surfactants include polysorbates ("TWEEN" or sorbitan monooleate), sodium dodecylsulfate (SDS), polyethoxylated castor oil ("CREMOPHOR"), NP-40, dimethyldioctadecyl ammonium bromide (DDA), linear polyoxypropylene-polyoxyethylene (POP-POE) block polymers, poloxamer 401, Pluronic L62LF, Pluronic L101, Pluronic L64, PEG1000, Tetronic 1501, Tetronic 150R1, Tetronic 701, Tetronic 901, Tetronic 1301, Atmos 300, Tween 20, Tween 40, Tween 60, Tween 80, and Tetronic 130R1, Arlacel A, Arlacel 80, Span 80,and mannide monooleates.
25. The composition of claim 20 wherein the water phase is water per se, phosphate buffered saline, saline, Ringer's solution, or combination thereof.
26. A method for preparing a pharmaceutical formulation, which method comprises admixing, by supplying, an aqueous suspension comprising hCG and at least one oily ingredient and at least one emulsifier in a sufficient quantity to provide said formulation as an oil-in-water emulsion.
27. The method of claim 26, which further comprises the addition of a therapeutically effective amount of a muramyl peptide into said oil-in-water emulsion.
28. A therapeutic composition which comprises an oil-in-water emulsion and at least one natural or recombinant water-soluble biologically active protein and optionally at least one muramyl peptide.
29. The therapeutic composition of claim 28 wherein said biologically active protein is selected from the group consisting of somatotropin, human growth hormone (HGH), bovine growth hormone (BGH or BST), porcine growth hormone (PGH or PST), epidermal growth factor (FGF), interferon alfa; interferon alfa-2a; interferon alfa-2b; interferon alfa-N 1; interferon alfa-n3; interferon beta; interferon beta-1 al; interferon beta-lb; interferon gamma-la; interferon gamma-lb; interferon omega; interferon tau; interleukin- 1; interleukin- 1 alpha; interleukin- 1 beta; interleukin- 10; interleukin-11; interleukin- 12; interleukin- 12; interleukin-15; interleukin-2; interleukin-3; interleukin-4; interleukin-5; interleukin-7; interleukin-8, erythropoeitin (EPO), parathyroid hormone
(PTH), selenoprotein P, cystatin B, endotoxin neutralizing protein, megakaryocyte simulatory factor (MGDF), granulocyte macrophage colony stimulating factor (GM-CSF), cytokine synthesis inhibitory factor (CSIF), genofibrate, alpha calcitonin, beta calcitonin, tumor necrosis factor (TNF), lymphocyte migration inhibitory factor (LIF), tumor invasion inhibiting factors, transacting regulatory proteins (TAT's), protease inhibitors,
BPC 157, fat reducing hormones, insulin, glucagon, gastrin, angiotensin, secretin, lactotropic hormone, thyrotropic hormone, melanocyte stimulating hormone, luteinizing hormone (LH), follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), thrombopoietin (TPO), luteinizing hormone stimulating hormone, human menopausal gonadotropin, vasopressin, oxytocin, protirelin, corticotropin, SOD, urokinase, lysozyme,
G-CSF, M-CSF, LIF, inhibin A, inhibin B, activin A, activin B, NAP-1, MCP-1, MIP- lalpha, MlP-lbeta, MIP-2, SISbeta, SISdelta, SISepsilon, PF4, PBP, gammaIP-10,
MGSA, aFGF, bFGF, KGF, PDGF-A, PDGF-B, PD-ECGF, INS, IGF-I, IGF-II, NGF- beta, GRO/MGSA, PF4, PBP/CTAP/.beta.TG, IP-10, KC, 9E3, MCP-1/MCAF, MCAF,
ACT-2/PAT 744/G26, LD-78/ PAT 464, RANTES, G26, 1309, JE, TCA3, B7.1, B7.2,
ICAM-1, ICAM-2, LFA-1, LFA-3, CD72, CTAPIII, ENA-78, GRO, 1-309, PF-4, LD-78, eosinophil-derived neurotoxin (EDN), anti-neoplastic urinary protein (ANUP), ribonuclease, and angiostatin and analogues and variants thereof.
30. The therapeutic composition of claim 28 wherein the biologically active protein is selected from among those listed in Table 1.
31. A kit for extemporaneous preparation of a pharmaceutical composition, said kit comprising: (1) a first container containing an emulsion, where said emulsion comprises: squalane or squalene; Arlacel, and an aqueous solution; and (2) a second container containing therapeutically effective amount of dimer hCG in an aqueous solution or as a powder, where the concentrations of the components in each container are selected such that combination of the contents of both containers produces a formulation comprising said Arlacel in an amount of between 1% and 30%>; squalane or squalene in an amount of between 1% and 30%; muramyl peptide in an amount of between 0.0001%o and 30%>; and hCG in amount between 0.01% and 99%.
32. The kit of claim 31 further containing a therapeutically effective amount of muramyl peptide added either to the first or the second container.
33. The kit of claim 31 further containing a therapeutically effective amount of muramyl peptide enclosed in a separate container.
EP02764353A 2001-04-25 2002-04-25 Hcg formulation Withdrawn EP1390053A2 (en)

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