WO1992006709A1 - Composition lipidique utilisee comme agents immunogenes dans la prevention ou le traitement d'infections bacteriennes a gram negatif - Google Patents

Composition lipidique utilisee comme agents immunogenes dans la prevention ou le traitement d'infections bacteriennes a gram negatif Download PDF

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WO1992006709A1
WO1992006709A1 PCT/US1991/007507 US9107507W WO9206709A1 WO 1992006709 A1 WO1992006709 A1 WO 1992006709A1 US 9107507 W US9107507 W US 9107507W WO 9206709 A1 WO9206709 A1 WO 9206709A1
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lipid
human
gram
group
composition according
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PCT/US1991/007507
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English (en)
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Carl L. Alving
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Alving Carl L
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Priority to JP4500694A priority Critical patent/JPH06502424A/ja
Priority to AU89287/91A priority patent/AU667028B2/en
Publication of WO1992006709A1 publication Critical patent/WO1992006709A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • 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—Liposomes
    • 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
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/5555—Muramyl dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to encapsulated high- concentration lipid A compositions and human monoclonal antibodies as immunotherapeutic agents to prevent or treat Gram-negative bacterial infections.
  • Gram-negative bacteria are ubiquitous in the environment, and are present in the air, water, food, and are present in particularly high concentrations in. the intestines.
  • the surface of the skin is coated with Gram- negative bacteria and many skin infections are caused by these organisms. Because of their widespread distribution these bacteria cause opportunistic infections in association with other afflictions.
  • sepsis is the most common cause of death in hospitals, and is particularly associated with conditions such as cancer, traumatic injury, and burns.
  • a major cause of death in association with burns on the skin is infection and sepsis caused by Gram-negative Pseudomonas organisms.
  • Gram-negative infections are caused by Gram-negative organisms. Individuals who are weakened by chronic diseases, or who have massive traumatic or burn injuries, or who have penetrating injuries of the intestines, are particularly at risk for developing life-threatening Gram-negative sepsis. Gram-negative infections that cause diarrhea are one of the major causes of death in infants.
  • the normal treatment of Gram-negative sepsis is by the administration of antibiotics that are active against sensitive organisms. The widespread use of such antibiotics has led to the continuous emergence of resistant strains of organisms and this has limited the effectiveness of antibiotic treatment.
  • lipid A The structure of lipid A has been completely defined for most Gram-negative bacteria, and it has been totally synthesized. It consists of a backbone of (B-l,6)-linked D-glucosamine d saccharide which carries phosphate residues in the positions 1 and 4'. Amidated or esterified long-chain fatty acids (generally D-3-hydroxy and/or acyloxy fatty acids) are present in each of the possible sites in the glucosamine moieties. Minor differences in structures of lipid A occur in molecules derived from various bacteria.
  • so called “native" lipid A i.e., unmodified lipid A derived by isolation from Gram-negative bacteria usually consists of a mixture of molecules having the same basic structure as the "complete" lipid A described above but with differing degrees of phosphorylation or different numbers or structures of fatty acids.
  • the monophosphoryl lipid A which has reduced toxicity, lacks the phosphate residue at position 1.
  • a further useful approach to immunotherapy of Gram- negative bacteria would be in the development of a vaccine (i.e., immunoprophylaxis) against lipopolysaccharide via its lipid A component.
  • a vaccine i.e., immunoprophylaxis
  • lipopolysaccharide via its lipid A component.
  • a major theoretical and practical impediment to this approach is posed by the extremely high level of toxicity of lipopolysaccharide.
  • a synonym for lipopolysaccharide is the term "endotoxin", and all of the endotoxic activity of lipopolysaccharide is caused by the lipid A component.
  • Lipopolysaccharide has literally dozens of biological activities when it is studied with in vitro biological systems or when it is injected .in vivo. Many of these activities are associated with toxicity and are responsible for the adverse reactions that commonly observed in the course of Gram-negative bacterial infections and sepsis. Among these activities are included: pyrogenicity, neutropenia, thrombocytopenia, hypotension and shock, shock lung, renal failure, cachexia, and death. All of these toxic effects.are caused by the lipid A component of lipopolysaccharide.
  • compositions which contain high concentrations of shielded or "hidden” lipid A which permits the slow release of lipid A therefrom thereby avoiding the deleterious and lethal results caused by the presence of high concentration of neat lipid A in the body.
  • lipid A-containing component wherein the lipid A is sequestered, embedded or "hidden” in pharmaceutically-acceptable encapsulating-delivery material, said material being capable of (a) physically sequestering, "hiding” or shielding hydrophobic fatty acid portion of the lipid A from its aqueous environment, thereby preventing said portion from expressing its toxic properties in the host-mammal and (b) slowly releasing the lipid A from said material at a dose level within the range of 185 to 2,200 micrograms.
  • the lipid A can be native lipid A, monophosphoryl lipid A or diphosphoryl lipid A.
  • the encapsulating slow- releasing delivery materials or devices can be a liposome, biocompatible-biodegradable polymer, microcapsules, or microspheres, mechanical slow drug- releasing devices and combination thereof.
  • Proteus Shigella, Vibrio, meningococcus and gonococcus.
  • it is a further object of this invention is to provide human antibodies in the form of "hyperimmune" polyclonal antiserum or a human monoclonal antibody reactive with Gram-negative bacteria and providing effective passive prophylaxis against or therapeutic treatment of sepsis caused by Gram-negative bacteria, said monoclonal antibody produced by a self- reproducing carrier cell containing genes that produce a protective human antibody.
  • antimmunogenic composition containing a very high concentration of lipid A encapsulated within slow drug release materials such as liposomes, biocompatible- biodegradable polymers, microcapsules, microspheres, slow-release devices, or combinations thereof, can be injected systemically, including intramuscularly or subcutaneously, into a mammalian host.
  • slow drug release materials such as liposomes, biocompatible- biodegradable polymers, microcapsules, microspheres, slow-release devices, or combinations thereof.
  • MPL monophosphoryl lipid A
  • the maximum safe intravenous dose of MPL was established as 100 ⁇ g of MPL per m 2 . Assuming a normal surface area of 1.7 to 1.8 ro 2 , the maximum safe dose of MPL is 170 to 180 ⁇ g. Applicants have discovered that dose levels of the lipid A in the range of 185 to 2,200 micrograms can be administered to mammalian-hosts without toxic effect and result in the production of extremely high titers of antibodies, such that the antibody titers are higher than can be obtained with lower doses of lipid A.
  • the level of antibody activity to lipid A obtained was proportional to the dose of liposomal lipid A administered during the original single initial immunization. Even when the same total amount of liposomal lipid A was given in divided doses in multiple injections at different times the resultant antibody activity did not reach the levels achieved when the entire liposomal lipid A dose was given at a single time.
  • Liposomes consist of bilayers of lipids, such as phospholipids and other lipids, in which the hydrophobic regions of the lipids are oriented towards each other and the hydrophilic regions of the lipids are oriented toward the aqueous phase in which the lipids are suspended.
  • the adjacent lipid molecules are held together by van der Waals forces and this results in the spontaneous formation of lipid spherules consisting of membranes containing lipid bilayers that surround internal aqueous spaces.
  • lipid A in included in the lipid bilayer When lipid A in included in the lipid bilayer it can be demonstrated that anti-lipid A antibodies that are known to have specificities against the lipid headgroup (diglucosamine diphosphate) are readily bound to the liposomal lipid A. This can be shown to cause agglutination or complement fixation, and complement activation can cause lysis of the lipid bilayer resulting in increased permeability of the liposome to marker compounds present in the internal aqueous spaces.
  • anti-lipid A antibodies that are known to have specificities against the lipid headgroup (diglucosamine diphosphate) are readily bound to the liposomal lipid A. This can be shown to cause agglutination or complement fixation, and complement activation can cause lysis of the lipid bilayer resulting in increased permeability of the liposome to marker compounds present in the internal aqueous spaces.
  • lipid A molecule is oriented in the expected manner with the hydrophilic portion oriented toward the aqueous medium and the hydrophobic (fatty acid) portion buried in the lipid bilayer. Similar types of studies can be used to demonstrate that anti-lipid A antibodies can bind to lipid A that has been absorbed to erythrocytes.
  • LAL Limulus amebocyte lysis
  • liposomes containing very high concentrations of lipid A can exhibit biological activities that are qualitatively different than liposomes containing lesser amounts of lipid A.
  • liposomal lipid A per se can have properties that are qualitatively different than those of nonliposomal lipid A.
  • monophosphoryl lipid A was included in liposomes that were employed as a candidate vaccine for human malaria (Plasmodium falciparum) . These liposomes also contained a recombinant antigen that was used for immunizing against the malaria.
  • the liposomal lipid A served as an adjuvant for enhancing the immunogenicity of the recombinant antigen and the liposomes themselves were also absorbed with another adjuvant, aluminum hydroxide (alum) .
  • the candidate vaccine was injected three times, for example at 0, 8-12, and 16-20 weeks, into five groups of human volunteers (5 volunteers were in group 1, 6 in groups 2-4, and 7 in group 5). Increasing doses were sequentially injected in order to test the safety of the vaccine but each group received exactly the same dose of alum.
  • Vaccine doses were as follows, based on dilution of the liposomes: group 1, 1:100; group 2, 1:10; group 3, 1:4;, group 4, 1:2; group 5, 1:1 (i.e., undiluted).
  • Group 5 received 2.2 mg of monophosphoryl lipid A during each intramuscular injection, and to our knowledge this is the highest dose of lipid A that has ever been purposely given to a human subject.
  • a liposomal anti-sepsis vaccine could be used either for immunoprophylaxis (as with other types of vaccines in their conventional usage forms) or as an immunotherapeutic agent in individuals who have already developed sepsis or individuals at risk for developing sepsis (e.g., patients subjected to traumatic injuries or to burns, or patients who are debilitated because of old age or chronic diseases such as cancer or diseases that might cause a prolonged bedridden state) .
  • the results indicate that the vaccine would be effective within two weeks after administration, but the extremely high levels achieved in group 5 suggest that the vaccine could be effective even at earlier times.
  • the vaccine could be used in conjunction with other therapeutic measures, such as correction of the underlying inciting condition (e.g., removal of abscess, removal of cancer, closing of perforated intestines, removal of infected intravenous catheters, replacement of infected heart valves, etc.), or with concurrent administration of antibiotics, steroids, anticancer drugs, or immunotherapeutic or any other therapeutic measures that would normally be expected to have therapeutic benefit in the absence of the liposomal anti-sepsis vaccine.
  • Another unexpected benefit from our anti-sepsis vaccine appeared to be that the high titers of antibody activity that were achieved at the highest initial dose of vaccine (group 5) could not be duplicated simply by giving multiple immunizations with a smaller dose of vaccine.
  • each group received a boosting immunization at either 8 weeks (groups 1-3) or 10 weeks (groups 4 and 5) that was identical to the original immunization dose. Therefore, after the second (boosting) immunization group 4 received a total dose of liposomal monophosphoryl lipid A in two injections that was identical to the dose of lipid A that was present in the single initial immunization dose of group 5. Despite this, when measured 2 weeks after the boosting immunization, group 4 achieved an IgG antibody level that was only slightly more than half as high as the level achieved 2 weeks after the initial immunization with the twice the antigen dose given to the volunteers in group 5.
  • liposomes containing lipid A are "hidden" from the aqueous environment. This result presumably could also be accomplished by other types of particles or strategies. For example, polymers of various types, microcapsules, and microspheres all would be expected to accomplish the same result.
  • liposomes are often used informally in the literature to describe various types of carrier systems for drugs and proteins, there is no universal agreement or any generally accepted national or international conventions on the meanings of such nomenclature. Liposomes are ordinarily understood to consist of lipid membranes that are capable of enclosing an internal aqueous space and the membranes may consist of a variety of types of lipids.
  • Liposomes used in accordance with this invention can be made by different methods.
  • the size of the liposomes varies depending on the method used for making them.
  • a liposome in solution is generally in the shape of a spherical vesicle having one or several concentric layers of lipid molecules each of which is represented by the formula XY wherein X is a lipophobic-hydrophilic moiety and Y is a lipophilic-hydrophobic moiety.
  • the concentric layers are arranged such that the hydrophilic moiety remains in contact with an aqueous phase.
  • the lipid molecules will, at a minimum, form a bilayer, known as a lamella, of the arrangement XY-YX.
  • liposomes within the present invention can be prepared in accordance with known laboratory techniques.
  • liposomes can be made by mixing together the lipids to be used, including lipid A, in a desired proportion in a container, e.g., a glass pear-shaped flask, having a volume ten times greater than the volume of the anticipated suspension of liposomes.
  • a container e.g., a glass pear-shaped flask
  • the solvent is removed at approximately 40°C under negative pressure.
  • the vacuum obtained from a filter pump aspirator attached to a water faucet may be used.
  • the solvent normally is removed within about 2 to 5 minutes.
  • the composition can be dried further in a desiccator under vacuum.
  • the dried lipids are generally discarded after about 1 week because of its tendency to deteriorate with time.
  • the dried lipids can be hydrated at approximately 30 mM phospholipid in sterile, pyrogen-free water by shaking until all the lipid film is off the glass.
  • the aqueous liposomes can be then separated into aliquot, each placed in a vaccine vial, lyophilized and sealed under vacuum.
  • liposomes can be prepared in accordance with other known laboratory procedures, e.g., the method of Bangham et al., J. Mol. Biol. 13:238-52 (1965) ; the method of Gregoriadis, "Liposomes” jLn DRUG CARRIERS IN BIOLOGY AND MEDICINE, pp. 287-341 (G. Gregoriadis ed. 1979) ; the method of Deamer and Uster, "Liposome Preparation: Methods and Mechanisms" in LIPOSOMES (M. Ostro ed. 1983); and the reverse-phase evaporation method described, for example, by Szoka, Jr.
  • lipids that have been used either alone or in combination with other lipids to construct liposomes are included phospholipids, glycolipids, glycophospholipids, diglycerides, triglycerides, sterols, steroids, terpenoids, free fatty acids, and lipoidal vitamins.
  • phospholipids include phospholipids, glycolipids, glycophospholipids, diglycerides, triglycerides, sterols, steroids, terpenoids, free fatty acids, and lipoidal vitamins.
  • Numerous liposomal constructs have been made that contain synthetic fatty acyl groups conjugated to proteins or other polymers. Liposomes may also be formed that use lipids to reconstitute insoluble hydrophobic proteins.
  • microcapsule and microsphere in the present specification refer to particulate constructs that are used as carriers for antigeni ⁇ molecules (such as lipid A) , and the molecules that form the constructs, whether they are liposomes, lipids, proteins, or various other types of polymers, provide a diffusion barrier to prevent immediate release of the antigenic material. Slow release of the antigenic material to the immune system is promoted by these constructs.
  • antigeni ⁇ molecules such as lipid A
  • liposomes, lipids, proteins, or various other types of polymers provide a diffusion barrier to prevent immediate release of the antigenic material. Slow release of the antigenic material to the immune system is promoted by these constructs.
  • the diffusion mechanisms of materials from polymers are multitudinous, including release from a reservoir surrounded by a polymeric film; release from a matrix in which the material to be released is distributed uniformly through the polymer; cleavage of the material from a polymer backbone; dissolution of the polymer by exposure to an environmental solvent; permeation by water leading to leakage due to osmotic swelling either by creating pores in the polymer or through preformed pores; and release of material by imposition of external magnetic fields.
  • FIG. 1 shows human IgG Responses to lipid A.
  • the IgG antibody levels to native lipid A were measured by ELISA and are shown as absorbance at 405 nm of a 1/100 dilution of the serum. Each line represents the mean of the values observed in the individual human sera in each group.
  • Each group was immunized with liposomes containing DMPC, DMPG, CHOL, lipid A and R32NS1. Prior to injection the liposomes were diluted, as indicated below, and mixed with alum as adjuvant.
  • Group V was immunized with liposomes containing 2.2 mg of monophosphoryl lipid A.
  • This vaccine was diluted 1/2, 1/4, 1/10, and 1/100, respectively for groups IV, III, II, and I prior to immunization.
  • Groups I, II, and III were immunized at 0 and 8 weeks; group IV was immunized at 0 and 10 weeks; and group V was immunized at 0 and 12 weeks.
  • the data demonstrate that a single injection with undiluted vaccine (group V at 0 weeks) resulted in a higher immune response 2 weeks later (and even 12 weeks later) than two injections of a 1/2 dilution (group IV) .
  • Figure 2 shows Individual IgG Responses to Lipid A.
  • Figure 3 shows Individual IgG Responses to Lipid A. This shows that when the preimmunization levels of antibodies to lipid A are subtracted in each volunteer serum, the group V individuals had the most consistent and the highest levels of antibodies to lipid A induced by immunization.
  • Figure 4 shows Individual Human IgG Responses to Lipid A Two Weeks Following a Boosting Immunization. The boosting injection times are described in the legend to Figure 1. Even after boosting immunization, group V outperformed all of the other groups in developing high individual antibody titers.
  • Figure 5 shows Individual IgM Response to Lipid A. This shows that when the preimmunization levels of antibodies to lipid A are subtracted in each volunteer serum, the individuals in groups II, III, IV, and V had more consistent and the higher levels of antibodies to lipid A induced by immunization compared to group I. The most consistently elevated levels, showing that all individuals developed high levels, of IgM antibodies were observed in group V.
  • Figure 6 shows Individual IgA Responses to Lipid A. This shows that individuals in groups II, III, IV, and V developed IgA antibodies to lipid A. The levels, particularly in groups IV and V, were more consistent and higher than in group I.
  • FIG. 7 shows IgG Response in Group I to Individual Liposome Constituents After Injection of Liposomes.
  • the liposomes used for injection consisted of dimyristoyl phosphatidylcholine (DMPC) , dimyristoyl phosphatidylglycerol (DMPG) , cholesterol (CHOL) , and monophosphoryl.
  • DMPC dimyristoyl phosphatidylcholine
  • DMPG dimyristoyl phosphatidylglycerol
  • cholesterol CHOL
  • monophosphoryl monophosphoryl.
  • lipid A Each of the components was individually tested by ELISA for the appearance of IgG antibodies against the purified individual component.
  • the individuals were injected with liposomes containing 0.022 mg of monophosphoryl lipid A, but the ELISA analysis was performed with purified native lipid A.
  • FIG. 8 shows IgG Responses in Group II to Individual Liposome Constituents After Injection of Liposomes.
  • the liposomes used for injection consisted of dimyristoyl phosphatidylcholine (DMPC) , dimyristoyl phosphatidylglycerol (DMPG) , cholesterol (CHOL) , and monophosphoryl lipid A.
  • DMPC dimyristoyl phosphatidylcholine
  • DMPG dimyristoyl phosphatidylglycerol
  • cholesterol CHOL
  • monophosphoryl lipid A monophosphoryl lipid A
  • the liposomes used for injection consisted of dimyristoyl phosphatidylcholine (DMPC) , dimyristoyl phosphatidylglycerol (DMPG) , cholesterol (CHOL) , and monophosphoryl lipid A.
  • DMPC dimyristoyl phosphatidylcholine
  • DMPG dimyristoyl phosphatidylglycerol
  • cholesterol CHOL
  • monophosphoryl lipid A monophosphoryl lipid A.
  • lipid A the individuals were injected with liposomes containing 0.55 mg of monophosphoryl lipid A, but the ELISA analysis was performed with purified native lipid A. The data are shown with preimmunization values, if any, subtracted from the postimmunization values (2 weeks after initial immunization) .
  • Each serum was diluted 1/100 for ELISA analysis. The predominant antibody activity in each case was developed against lipid A.
  • FIG. 10 IgG Responses in Group IV to Individual Liposome Constituents After Injection of Liposomes.
  • the liposomes used for injection consisted of dimyristoyl phospatidylcholine (DMPC) , dimyristoyl phosphatidylglycerol (DMPG) , cholesterol (CHOL) , and monophosphoryl lipid A.
  • DMPC dimyristoyl phospatidylcholine
  • DMPG dimyristoyl phosphatidylglycerol
  • cholesterol CHOL
  • monophosphoryl lipid A monophosphoryl lipid A.
  • Each of the components was individually tested by ELISA for the appearance of IgG antibodies against the purified individual component.
  • lipid A the individuals were injected with liposomes containing 1.1 mg of monophosphoryl lipid A, but the ELISA analysis was performed with purified native lipid A.
  • Liposome Constituents After Injection of Liposomes The liposomes used for injection consisted of dimyristoyl phosphatidylcholine (DMPC) , dimyristoyl phosphatidylglycerol (DMPG) , cholesterol (CHOL) , and monophosphoryl lipid A. Each of the components was individually tested by ELISA for the appearance of IgG antibodies against the purified individual components. In the case of lipid A, the individuals were injected with liposomes containing 2.2 mg of monophosphoryl lipid A, but the ELISA analysis was performed with purified native lipid A. The data are shown with preimmunization values, if any subtracted from the postimmunization values (2 weeks after initial immunization) . Each serum was diluted 1/100 for ELISA analysis. The predominant antibody activity in each case was developed against lipid A.
  • DMPC dimyristoyl phosphatidylcholine
  • DMPG dimyristoyl phosphatidylglyce
  • Figure 12 Complement-Mediated Immune Damage to Membranes Induced by Antisera From Individuals in Group V.
  • Complement-dependent lysis of liposomes leading to release of trapped glucose marker from inside the liposomes was measured with sera from each individual in group V taken at 0, 2 weeks and 14 weeks following immunization with liposomes containing DMPC, CHOL, DMPG, and monophosphoryl lipid A (2.2 mg) .
  • the complement- mediated lysis of liposomes containing or lacking native lipid A as an antigen was examined.
  • the dashed line indicates an arbitrary value of glucose release that we have utilized in the past to indicate the level at which a significant amount of immune damage has occurred to the liposomes compared to the background variability in the assay.
  • Liposomes for immunization were prepared from a mixture of dimyristoyl phosphatidylcholine (DMPC) , dimyristoyl phosphatidylglycerol (DMPG) , and cholesterol (Choi) in molar ratios of 1.8/0.2/1.5.
  • DMPC and DMPG were purchased from Avanti Polar Lipids, Pelham, AL. Choi was purchased from Sigma Chemical Co. (St. Louis, MO) and was recrystallized three times from ethanol before use.
  • Monophosphoryl lipid A (Ribi Immunochem Research Inc.) was incorporated in the liposomes at a concentration of 52.6 ⁇ g per ⁇ mole of phospholipid.
  • lipids were prepared as solutions in redistilled chloroform. Following the additions of the appropriate volumes of lipid solutions into round bottom flasks, they were rotary evaporated until all the solvent was evaporated and a thin film of lipid was left on the flask wall. The flask was further dried in a desiccator under high vacuum overnight. The lipids were then hydrated with sterile pyrogen free wate. (USP) so that a phospholipid concentration of approximately 40 mM was achieved. The hydrated lipids were then aliquotted into vaccine bottles and freeze dried. After lyophilization, the bottles were stoppered under vacuum and stored at 4 degrees C in the dark.
  • USP sterile pyrogen free wate.
  • SK & F 105154 (R32NS1) was injected into each bottle.
  • the phospholipid concentration in the bottle was 250 mM after the reconstitution with the R32NS1.
  • the bottles were hand shaken until all t 3 lipid was suspended.
  • the bottles were then washed with sterile PBS, pH 6.4 and centrifuged. After two more washes the liposomes were resuspended with PBS, pH 6.4. The liposomes were then dispensed, using aseptic technique, into vaccine bottles at 0.9 ml per bottle.
  • Alum concentrate (Rehsorptar, Lot #C17502, Armour Pharmaceuticals, Kankakee, IL) , diluted to the 1.2-1.8 mg Al/ml range with PBS, pH 6.4, was dispensed into each vaccine vial at a volume of 0.7 ml.
  • the phospholipid concentration of the final product was 55 mM.
  • the lipid A concentration was 2.9 mg/ml.
  • Enzyme-linked immunosorbent assays were performed utilizing lipid A from S. innesota R595 (List Biologies, Campbell, CA) as antigen. Wells of "U" bottom polystyrene microtitre plates (Immulon II; Dynatech Laboratories, Inc., Alexandria, VA) were coated with
  • Blocking buffer PBS with 10% heat-inactivated fetal calf serum
  • Blocking buffer was added to all wells at a volume of 100 ⁇ l and held at room temperature for 2 hours. Blocking buffer was removed by inversion of the plates followed by a sharp tap of the plate to remove all the well contents. Human sera were diluted 1:100 in blocking buffer and added to wells in triplicate at a volume of 50 ⁇ l per well.

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Abstract

L'invention se rapporte à la production d'anticorps agissant contre le lipide A, au moyen de dispositifs ou de substances encapsulants destinées à l'administration à libération lente de médicaments et contenant des concentrations de lipide A supérieures à celles qui pourraient être administrées sans danger à des humains en l'absence desdites substances ou dispositifs. Les anticorps agissant contre le lipide A peuvent être utilisés pour lier les anticorps au lipide A présent dans le lipopolysaccharide qui recouvre la surface des bactéries à Gram négatif.
PCT/US1991/007507 1990-10-22 1991-10-21 Composition lipidique utilisee comme agents immunogenes dans la prevention ou le traitement d'infections bacteriennes a gram negatif WO1992006709A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP4500694A JPH06502424A (ja) 1990-10-22 1991-10-21 グラム−陰性菌感染の予防又は処置のためのヒト抗体製造用免疫原剤としての封入高−濃度リピドａ組成物
AU89287/91A AU667028B2 (en) 1990-10-22 1991-10-21 Lipid A composition as immunogenic agents to prevent or treat gram-negative bacterial infections

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US60109090A 1990-10-22 1990-10-22
US601,090 1990-10-22

Publications (1)

Publication Number Publication Date
WO1992006709A1 true WO1992006709A1 (fr) 1992-04-30

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/007507 WO1992006709A1 (fr) 1990-10-22 1991-10-21 Composition lipidique utilisee comme agents immunogenes dans la prevention ou le traitement d'infections bacteriennes a gram negatif

Country Status (5)

Country Link
EP (1) EP0554364A4 (fr)
JP (1) JPH06502424A (fr)
AU (1) AU667028B2 (fr)
CA (1) CA2094588A1 (fr)
WO (1) WO1992006709A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0562020A1 (fr) * 1990-12-10 1993-09-29 EntreMed, Inc. Vaccin contre le cholesterol pour prevenir l'hypercholesterolemie et l'atherosclerose
US5753260A (en) * 1986-06-17 1998-05-19 Entremed, Inc. Vaccines against sterols
US6749831B1 (en) 1997-05-16 2004-06-15 Medical Defense Technology, Llc Vaccine against lipopolysaccharide core
WO2013007570A1 (fr) * 2011-07-12 2013-01-17 Centre National De La Recherche Scientifique Procédé de criblage grande vitesse de l'activité lipase et/ou d'inhibiteurs de lipase dans des échantillons biologiques et des milieux de culture

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4029762A (en) * 1971-11-17 1977-06-14 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Lipid A-preparation
EP0151128B1 (fr) * 1983-05-06 1991-07-03 Velos Group Anticorps monoclonaux reagissant avec un noyau d'endotoxine
US4918163A (en) * 1985-09-27 1990-04-17 Pfizer Inc. Monoclonal antibodies specific for lipid-A determinants of gram negative bacteria

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
A. BRAUDE, et al (eds.) "Infectious Diseases and Medical Microbiology", published 1986 by W.B. Saunders 6. (PA) see pages 51-60. *
Immunology Letters, Vol. 25, issued 1990, C.A. ALVING, et al "Liposomes containing Lipid A: A potent non toxic adjuvant for a human Malaria sporoziote vaccine", pages 275-289, see entire document. *
Infection and Immunity, Vol. 39, No. 3, issued March 1983, E.C. RICHARDSON et al., "Interactions of Lipid A and Liposome-associated Lipid-A with Limulus polyphamus Amoebocytes", pages 1385-1391, see entire document. *
See also references of EP0554364A4 *
The Journal of Immunology, Vol. 138, No. 8, issued 15 April 1987, J. DIJKSTRA et al. "Modulation of biological activity of bacterial endo toxin by incorporation in to lipolames", pages 2663-2670, see entire document. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5753260A (en) * 1986-06-17 1998-05-19 Entremed, Inc. Vaccines against sterols
US6224902B1 (en) 1986-06-17 2001-05-01 Entremed, Inc. Vaccines against sterols
EP0562020A1 (fr) * 1990-12-10 1993-09-29 EntreMed, Inc. Vaccin contre le cholesterol pour prevenir l'hypercholesterolemie et l'atherosclerose
EP0562020A4 (en) * 1990-12-10 1993-11-18 Alving, Carl L. A vaccine against cholesterol to prevent hypercholesterolemia and atherosclerosis
US6749831B1 (en) 1997-05-16 2004-06-15 Medical Defense Technology, Llc Vaccine against lipopolysaccharide core
WO2013007570A1 (fr) * 2011-07-12 2013-01-17 Centre National De La Recherche Scientifique Procédé de criblage grande vitesse de l'activité lipase et/ou d'inhibiteurs de lipase dans des échantillons biologiques et des milieux de culture

Also Published As

Publication number Publication date
JPH06502424A (ja) 1994-03-17
AU667028B2 (en) 1996-03-07
EP0554364A4 (fr) 1994-03-18
EP0554364A1 (fr) 1993-08-11
CA2094588A1 (fr) 1992-04-23
AU8928791A (en) 1992-05-20

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