Classifications

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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
• • A—HUMAN NECESSITIES
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/74—Synthetic polymeric materials
  • A61K31/765—Polymers containing oxygen
  • A61K31/78—Polymers containing oxygen of acrylic acid or derivatives thereof
• • A—HUMAN NECESSITIES
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  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P31/06—Antibacterial agents for tuberculosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
  • A61P31/08—Antibacterial agents for leprosy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P33/00—Antiparasitic agents
  • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
• • A—HUMAN NECESSITIES
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  • A61P33/00—Antiparasitic agents
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  • A61P33/06—Antimalarials
• • A—HUMAN NECESSITIES
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  • A61P33/10—Anthelmintics
  • A61P33/12—Schistosomicides
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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

• the present invention also provides a pharmaceutical preparation which comprises a complex of the present invention in admixture or conjunction with a pharmaceutically suitable carrier.
• the invention further provides a method of treating an infection by a pathogenic organism in a subject in need of such treatment, which comprises administering to the subject a therapeutically effective amount of a complex comprising a narrow molecular weight distribution polymer that includes units derived from an acrylic acid or a salt thereof, and a substance that has pharmacological activity against the pathogenic organism.
• the invention further provides a method of inducing an immune response to the pathogenic organism in a subject in need thereof, which comprises administering to the subject an effective amount of a complex comprising a narrow molecular weight distribution polymer that includes units derived from an acrylic acid or a salt thereof, and a substance that has pharmacological activity against the pathogenic organism.
• the invention further provides a method of inducing an immune response to a cancer in a subject in need thereof, which comprises administering to the subject an effective amount of a complex comprising a narrow molecular weight distribution polymer that includes units derived from an acrylic acid or a salt thereof, and a substance that has pharmacological activity against the cancer.
• the invention further provides a method of inducing an immune response to the pathogenic organism in a subject in need thereof, which comprises administering to the subject effective amounts of a narrow molecular weight distribution polymer that includes units derived from an acrylic acid or a salt thereof, and a substance that has pharmacological activity against the pathogenic organism.
• the invention further provides a method of inducing an immune response to a cancer in a subject in need thereof, which comprises administering to the subject effective amounts of a narrow molecular weight distribution polymer that includes units derived from an acrylic acid or a salt thereof, and a substance that has pharmacological activity against the cancer.
• ranges include but are not limited to ranges of from 80,000 to 4,000, 75,000 to 5,000, 65,000 to 10,000, 55,000 to 10,000, and 45,000 to 10,000. Further examples include the ranges from 50,000 to 4,000, for example, from 40,000 to 25,000. Molecular weights in the range of from 45,000 to 10,000 may be preferred.
• PMAA-Na polymethacrylic acid, sodium salt. Unless stated otherwise, it denotes a polymethacrylic acid, sodium salt prepared as described in Examples A1 to A4.
• complex is used herein to denote an association between a narrow molecular weight distribution polymer that includes units derived from an acrylic acid or a salt thereof and another substance as defined herein, the association between the components being primarily non-covalent, for example, involving any one or more of ionic, electrostatic and van der Waals forces. Although a complex according to the present invention predominantly involves non-covalent association between the components, there may nevertheless be some covalent bonding.
• Figure 1 shows red blood cell lysis after incubation of cells with PMAA-Na in RPMI.
• Figure 3 shows the lack of toxicity of PMAA-Na on human monocyte derived macrophages (Figure 3a) and on primary human peritoneal macrophages ( Figure 3b).
• Figure 4 shows release of MIP-1 ⁇ from human peritoneal macrophages by endotoxin free PMAA-Na.
• Human peritoneal cells from 3 donors (A, B and C) were cultured with endotoxin-free PMAA-Na (500 ⁇ g/ml) and culture supernatants harvested after 36 h were analysed for MIP-1 ⁇ . All reagents and the PMAA-Na contained ⁇ 0.06 endotoxin units/ml (EU/ml).
• Figure 4a shows the results obtained for donor A, Figure 4b for donor B and Figure 4c for donor C. There was a significant release of MIP-1 ⁇ in the presence of PMAA-Na.
• Figures 10a, 10b and 10c show the results of a 1 hour incubation with cells of donors A, B and C, respectively.
• Figures 11a, 11b and 11c show the results of a 6 hour incubation with cells of donors A, B and C, respectively.
• Figures 12a, 12b and 12c show the results of a 24 hour incubation with cells of donors A, B and C, respectively,
• amphotericin B-PMAA-Na complex being prepared as described in Example C.
• the degree of red cell lysis by the amphotericin B - PMAA-Na preparation ("drug") was determined after a 1 hour and 24 hour incubation for concentrations up to 1,000 ⁇ g/ml. The results obtained after a 1 hour incubation are shown as open squares, the results after a 24 hour incubation as closed circles.
• Figure 18 shows inhibition of intracellular Leishmania mexicana amastigote growth by amphotericin B-PMAA-Na complex in human monocyte derived macrophages.
• Figure 19 shows the corresponding inhibition using clinical grade amphotericin B or AmBiosome (liposomal amphotericin B, Gilead Sciences, Great Abingdon, Cambridge, UK).
• Figure 20 shows inhibition of intracellular Leishmania mexicana amastigote growth in human macrophages by amphotericin B -PMAA-Na complex compared to AmBiosome.
• Figures 21 and 22 show inhibition of intracellular Leishmania donovani amastigote growth by amphotericin B-PMAA-Na complex in human monocyte derived macrophages (four experiments, shown in Figures 21a to 21 d), by clinical grade amphotericin B ( Figure 22a) and by AmBiosome (Gilead Sciences, as above) ( Figure 22b).
• [ 3 H]-Thymidine (specific activity 20-30 Ci/mmol; Amersham Biosciences, UK) was added at 1 ⁇ Ci/well for a further 18 hours. The cells were then harvested and proliferation determined using a liquid scintillation counter. The results were expressed as the mean counts/minute (cpm) + sem.
• Figure 24a shows the results obtained for the PBMC proliferation after 6 days incubation of the cells of donor A with the tuberculin-PMAA-Na preparation.
• Unstimulated cells were used as the negative control and recombinant human interferon- ⁇ was used in the positive control.
• the plate was incubated for 24 hours at 37°C / 5% CO 2 .
• the manufacturer's instructions were then followed to develop the microplate and the number of positive spots for interferon- ⁇ counted. Each spot represented a single interferon- ⁇ secreting cell.
• Figure 31 shows the inhibition of various strains of Cryptococcus neoformans that have infected human monocyte derived macrophages.
• the inhibition was measured after using various concentrations of amphotericin B - PMAA-Na complex prepared as described in Example C (solid squares) for three days, with amphoteracin B (open circles) as a reference for comparison.
• the number of infected and uninfected macrophages, and the number of gram positive yeast CFU were counted.
• the results were expressed as the infection index, which was calculated as the average number of CFU/percentage of infected cells.
• the LD 50 values were also determined.
• a complex comprising an antigen, namely tuberculin purified protein derivative BP, and a methacrylic acid sodium salt homo-polymer (PMAA-Na) produced as described herein caused more T lymphocyte proliferation than did the tuberculin antigen on its own, and also increased interferon- ⁇ secretion from T lymphocytes more than did the tuberculin antigen alone.
• an antigen namely tuberculin purified protein derivative BP
• PMAA-Na methacrylic acid sodium salt homo-polymer
• the pathogens against which an immune response is to be induced are primarily those that replicate and/or persist intracellularly, in particular those that replicate and/or persist in tissue based macrophages and other antigen presenting cells, for example, dendritic cells.
• tissue based macrophages and other antigen presenting cells for example, dendritic cells.
• Mycobacterium tuberculosis atypical mycobacteria, and mycobacterium leprae.
• Members of the schistosoma family that cause Schistosomiasis for example, Schistosoma haematobium, Schistosoma mansoni, Schistosoma japonicum, Schistosoma intercalatum, and Schistosoma mekongi.
• Organisms that cause toxoplasmosis for example, Toxoplasma gondii.
• Organisms that cause Human African Trypanosomiasis for example, Trypanosoma brucei gambiense or Trypanosoma brucei gambiense.
• Organisms that cause HIV and HTLV infections for example HIV-1 and HIV-2 and HTLV-I and HTLV-II.
• substances that have pharmacological activity against pathogenic organisms include but are not limited to clotrimazole, flucytosine, fluconazole, griseofulvin, itraconazole, ketoconazole, miconazole, pyrazinamide, ciprofloxacin, rifampicin, thiacetazone, cycloserine, clofazimine, dapsone, rothionamide, metriphonate, oxamniquine, praziquantel, co-trimoxazole, pyrimethamine, sulfadoxine, spiramycin, melarsoprol, nifurtimox, amodiaquine, chloroquine, mefloquine, primaquine, proguanil, quinine, zidovudine, efavirenz, indinavir, ribavirin, vidarabine, levamisole, and acyclovir.
• a treatment for use according to this aspect of the present invention it is not necessary for a treatment to be fully successful, i.e., to cure the subject or to completely eliminate the organism as the immune response to the organism that is being generated will provide an effective "second line" of defence. Any residual organisms will be eliminated by the effector cytotoxic T-lymphocyte responses generated because they will be therapeutically directed against any persisting organisms in the remaining chronically infected cells. What is necessary is that some organisms are killed, thereby releasing antigens.
• One of the advantages of the present invention is that even if the treatment does not completely eliminate the organism, the induction of effector cytotoxic T-lymphocyte responses will provide protective vaccine based responses against future re-infection. Pharmacological treatment, therapeutic vaccination and protective vaccination are therefore achieved.
• a complex of the invention that comprises a narrow molecular weight distribution polymer that includes units derived from an acrylic acid or a salt thereof and a substance that has pharmacological activity against cancer, especially a cytotoxic agent that kills or partially kills otherwise disrupts the transformed, i.e., cancerous cells.
• the killing or disruption of the cells results in the release of antigenic material some of which will be "seen” by macrophages and antigen presenting cells as non-self antigens.
• the presence of the polymer potentiates the immune response to the antigen(s) because the dendritic cells that are recruited into this altered microenvironment take up and process the antigen(s) released.
• the dendritic cells then initiate the CD4+ T cell activation that promotes the generation of effector cytotoxic T- lymphocyte responses.
• Some of these responses will be therapeutically directed against other cancer cells and will therefore provide protective vaccine based responses against the recurrence of the cancer and the growth of any remaining micrometastasis.
• These responses will be greatest for those cancers in which large numbers of macrophages and antigen presenting cells are present. This is especially so in lymphomas and leukaemias. Pharmacological treatment, therapeutic vaccination and protective vaccination are therefore achieved.
• Pathogenic organisms substances that have pharmacological activity against a pathogenic organism, substances that have pharmacological activity against a cancer, and antigens and immunogens are, for example, as described above.
• a narrow molecular weight distribution polymer that includes units derived from an acrylic acid or a salt thereof is to be co-administered with a substance (i), (ii) or (iiii) as defined above instead of being administered in the form of a complex with the substance
• the polymer and the substance (i), (ii) or (iii) may be formulated in the same pharmaceutical preparation or they may be formulated in separate preparations, in each case in admixture with a pharmaceutically suitable carrier. If in separate preparations, one may be administered before the other, but it may be preferable to administer the two preparations substantially simultaneously.
• the immune response that promotes healing and parasite clearance in leishmaniasis is dominated by an interferon gamma mediated Th-1 response.
• macrophages ingest leishmania efficiently, they are not activated by the ingestion of the organism; consequently pro-inflammatory chemokines, pro- inflammatory cytokines and interferon- ⁇ are not released.
• dendritic cells take up leishmania parasites, mature and then promote the development of cellular immune responses.
• infected macrophages can be activated, killing of the parasite ensues. Therefore, two different processes, namely, antigen processing and cellular maturation/activation must be combined for an effective cellular vaccine response.
• the cellular immune response is significantly enhanced because of the close proximity of the antigen presenting cells, the release of Leishmania antigens by the killing activity of amphotericin B, and the simultaneous promotion of an appropriate and local Th1 cytokine/chemokine environment.
• a complex of the invention or a narrow molecular weight distribution polymer that includes units derived from an acrylic acid or a salt thereof may be "particulate associated".
• Such particulates may be produced by emulsion, homogenisation and spray drying processes, which are known in the art.
• emulsion, homogenisation and spray drying processes which are known in the art.
• Pharmaceutical compositions comprising such particulate associated complexes of the invention may be administered mucosally by a pulmonary or nasal route, by ingestion, or by a non-mucosal parenteral route, especially subcutaneously or intramuscularly.
• the polymer or the complex should remain in the circulating blood for an appropriate period of time.
• the period of time that is appropriate will depend on a number of factors including, in the case of a complex, the nature of the substance complexed to the polymer.
• the polymer or complex may remain in the circulation for several hours, for example, for up to 24 hours, for example, from about 4 to 6 hours up to about 24 hours.
• the groups R 3 which may be the same or different, are selected from the group consisting of C-i-Ce alkylene groups, preferably 1 ,2-alkylene, and C 6 -C 12 arylene groups, most preferably methylene, ethylene, 1,2-propylene and 1,3- propylene.
• all groups R 3 are the same, most preferably all are 1 ,2- ethylene or 1 ,2-propylene.
• L preferably comprises a C ⁇ -C ⁇ 8 alkylene or C 6 -C ⁇ 8 arylene group which may be substituted and/or interrupted with 1 or more heteroatoms.
• L comprises a group selected from the group consisting of C ⁇ -C 6 alkylene, C 6 -C ⁇ 2 arylene, C C ⁇ 2 oxyalkylene and C ⁇ -C 6 acyl.
• L comprises an alkylene group, it can be branched, linear or cyclical, substituted or unsubsfituted with one or more alkyl groups, and is preferably methylene, 1,2-ethylene, 1,2-propylene, 1 ,3-propylene, ter1 butylene, sec butylene, hexylene or octylene.
• L comprises an arylene group, it is preferably benzylene, tolylene or xylylene.
• a polymer may be a homopolymer incorporating unit (I) or may be a copolymer or block copolymer incorporating other polymeric, oligomeric or monomeric units, for example, a block copolymer comprising units (II) as described above.
• further polymeric units incorporated in a homopolymer or copolymer or block copolymer may comprise acrylic polymers, alkylene polymers, urethane polymers, amide polymers, polypeptides, polysaccharides and ester polymers.
• additional polymeric components comprise polyethylene glycol, polyaconitic acid or polyesters.
• R, R 1 , R 2 and R 3 , L, m and n are defined as above, R 4 , R 5 and R 6 are selected, independently, from the same groups as R, R 1 and R 2 , respectively;
• Q denotes a group that is not cleaved or is not substantially cleaved under the conditions used to produce the polymer; and
• p denotes an integer 1 or greater than 1.
• Q may be a targeting group, i.e. a group that targets the polymer to a cell type, eg macrophages, or to an organ eg the liver.
• Polymers produced by such a method may have biological properties analogous to some or all of the biological properties demonstrated by the PMAA-Na produced as described herein.
• the POMSu used as the precursor polymer in the production of PMAA-Na as described herein was produced as described in WO 01/18080 and in Example A4 herein by homogeneous polymerisation of methacryloxysuccinimide using an atom transfer radical polymerisation method, in particular a copper mediated method.
• Such a method for the production may be useful for the production of that and other precursor polymers for use according to the present invention, but the invention is not limited to such methods, nor to precursor polymers produced by such methods nor to polymers produced from such precursor polymers.
• a polymer including units derived from an acrylic acid or a salt thereof for use according to the invention may be produced by hydrolysis of a corresponding precursor polymer that has, in place of the hydrogen atom of the acrylate carboxylic acid in the units derived from an acrylic acid, a group that can be cleaved by hydrolysis to give the acid, for example, cleaved by mild hydrolysis.
• the polymer generally has a molecular weight as described above in the Definitions section.
• a polymer precursor having the desired polydispersity may be produced by atom transfer radical polymerisation method, for example, a copper mediated atom transfer radical polymerisation, or other methods including free radical polymerisation, may be used. Such methods are described in WO 01/18080 and in WO 03/059973. Examples are given below.
• the invention also provides a polymer including units derived from an acrylic acid or a salt thereof obtainable according to such methods. However, the invention is not limited to polymers produced by such methods. Any method suitable for producing a polymer that includes units comprising an acrylic acid atom, in particular having a polydispersity as described above, may be used.
• the hydrolysis of the polymer precursor in the presence of the antigen or immunogen or the substance that has pharmacological activity against a pathogenic organism or a cancer should generally be carried out under conditions such that the antigen, immunogen or substance is not substantially affected adversely, for example, the hydrolysis should generally be carried out under mild conditions.
• the conditions should be such that the antigen, immunogen or substance is not substantially decomposed, degraded or otherwise caused to lose relevant activity.
• a method of producing a complex of the invention by producing a polymer for use according to the invention, for example, by hydrolysing a polymer precursor, in the presence of the antigen or immunogen or the substance that has pharmacological activity against a pathogenic organism or a cancer, for example, as described above, is part of the present invention, as is a complex obtainable by such a method. It is considered that, in a complex of the invention, the association between the polymer and the substance that has pharmacological activity against a pathogenic organism or the antigen or the cancer is predominantly non-covalent (i.e., by ionic or Van der Waal's bonding), but that some molecules of the pharmacologically active substance or the antigen may be covalently linked to the polymer. The extent of covalent bonding may be dependent on the nature of the pharmacologically active substance or antigen and on the nature of the polymer.
• complex is used herein to denote an association between the polymer and the substance that has pharmacological activity against a pathogenic organism or the antigen or the cancer is predominantly non-covalent, but that may include some covalent linkages.
• the complexes of the invention are polyelectrolytes having acidic groups.
• the extent of deprotonafion may vary according to the environment of the complex.
• a complex of the invention may be in the form of a salt.
• a salt may be, for example, with a monovalent, bivalent, trivalent or quadrivalent counter ion.
• a monovalent counter ion may be, for example, an alkali metal ion, for example, a sodium or potassium ion, or an ammonium ion.
• a bivalent counter ion may be, for example, an alkaline earth metal ion, for example, a calcium or magnesium ion.
• Other counter ions include ions of transition metals, for example, iron, tin etc.
• More than one type of counter ion may be present and associated with the polymer.
• the number and proportion of salt-forming groups may be controlled, for example, by the use of appropriate polymer precursors, for example, as described above.
• the substance or agent that is to form a complex with the polymer should generally be capable of sustaining a positive charge to enable direct formation of the complex by means of non-covalent interactions including electrostatic forces due to opposing charges.
• the substance or agent used for complex formation may having basic, cationic or zwitterionic properties or may be adapted to have such properties.
• the polymer used in a complex of the invention or in conjunction with an antigen or immunogen, or a substance that has pharmacological activity against a pathogenic organism or a cancer is a polymethacrylic acid (PMAA) or a salt thereof, for example, a sodium salt, having a polydispersity of less than 1.4, preferably less than 1.2.
• the molecular weight of the polymer is generally as described in the Definitions section above. It may be advantageous to produce the polymer as described or substantially as described in the Examples herein, for example, to hydrolyse a precursor polymer, for example, a precursor polymer having an N-succinimide leaving group, using sodium hydroxide. It may be advantageous to form a complex with an antigen or immunogen, or with a substance that has pharmacological activity against a pathogenic organism or a cancer in situ during the production of the polymer.
• a polymer of the invention and a substance that has pharmacological activity against a pathogenic organism for example, against leishmaniasis, for example, amphotericin B
• a preparation may be administered before the other, but it is generally preferable to administer the two preparations substantially simulataneously.
• Example A1 Chemical synthesis: Preparation of methacrylic acid sodium salt homo-polymer: Poly(methacrylic acid, sodium salt) (PMAA-Na) 2_was prepared by the hydrolysis of poly(N-methacryloxysuccinimide) (PMOSu) 1 with sodium hydroxide (Scheme 1).
• Polymerisations were conducted at temperatures ranging from 80-130 °C to maintain solution homogeneity at methacryloxysuccinimide 3 to solvent weight ratios spanning 33-91 %.
• the preferred solvent was DMSO, but similar results were obtained with DMF.
• the weight ratio of monomer 3 to polar solvent (DMSO or DMF) was critical for the outcome of the polymerization. In DMSO at weight ratios less than 56% monomer 3 (e.g. 50 and 41%) resulted in lower yields of polymer (52 and 40% respectively). At weight concentrations higher than 60% monomer 3 in DMSO, the polymerisation solution solidified.
• the copper chelating legend used in these THF reactions was N, N, N', N", N"-pentamethyldiethylenetriamine (PMDETA). Additional precipitation reactions are listed in Table 3 with 2-bromo-2- methyl propionic acid (BMA) used as initiator.
• Example A3 Hydrolysis of PMOSu ⁇ to give PMAA-Na 2 (Scheme 1).
• the resulting solution was allowed to stir for 1 h at room temperature whereupon it was diluted to 105 mL with fresh water and dialysed against 5 L of water for 24 h using a Visking dialysis membrane (MWCO 7000, Medicell International). The 5 L of water was changed 6 times.
• the dialysed solution was filtered though a 0.2 ⁇ m filter and then freeze- dried to afford PMAA-Na 2 (0.56 g) as a solid product; Mw 26,100 Da, Mn 15,200 g/mol, Mw/Mn 1.7 (SEC 0.2 M aqueous NaNO 3 / 10% CH 3 CN, PMAA-Na standards)
• Example A6 Synthesis of PMOSu 1_ by free radical polymerization with 4,4'- azobisfcyanovaleric acid) followed by hydrolysis to give PMAA-Na 2.
• aqueous solutions of methacrylic acid sodium salt homo-polymers and co-polymers were treated to remove endotoxin using activated carbon or polymixin B columns.
• Endotoxin levels were determined using the limulus amebocyte lysate (LAL) assay and the compounds used in the experiments described contained ⁇ 0.06 endotoxin units/ml. This is the European Community standard for water for injection.
• Example D1 Human red blood cells:
• a 2% v/v solution of human red blood cells was prepared in RPMI 1640.
• a stock solution of PMAA-Na ("drug") was prepared in RPM1 1640.
• a 1% solution of Triton X-100 was used as a positive reference for 100% cell lysis.
• Dextran and poly-L- lysine were used as negative and positive controls respectively.
• An equal volume of the sample and red blood cells were aliquoted into a 96 well microtitre plate and incubated at 37°C. After 1 h and after 24 h, each sample was centrifuged (2,000g, 10 min) and the supernatant added to a 96 well microtitre plate. The absorbance was measured at 490 nm using a spectrophotometer.
• the degree of lysis was expressed as a percentage of the 100% lysis caused by Triton X-100. No significant toxicity was seen with the PMAA-Na using human red blood cells from 3 donors (A, B and C) up to a concentration of 2,000 ⁇ g/ml after 1 h or after 24 hours as shown in Figure 1.
• a 2% v/v solution of human whole blood was prepared from donor D in RPMI 1640.
• a stock solution of PMAA-Na ("drug") was prepared in RPM1 1640.
• a 1% solution of Triton X-100 was used as a positive reference for 100% cell lysis.
• Dextran and poly-L-lysine were used as negative and positive controls respectively.
• An equal volume of the sample and whole blood were aliquoted into a 96 well microtitre plate and incubated at 37°C. After 1 h, 6 h and after 24 h, each sample was centrifuged (500g, 10 min) and the supernatant added to a 96 well microtitre plate. The absorbance was measured at 490 nm using a spectrophotometer.
• the degree of lysis was expressed as a percentage of the 100% lysis caused by Triton X-100. No significant toxicity was seen with the PMAA-Na using human whole blood up to a concentration of 500 ⁇ g/ml after 1 h or after 6 h or after 24 hours as shown in Figure 2.
• the viability of the cell culture was expressed as a percentage of the viability of cells grown in the absence of any compound. Dextran and poly(L-lysine) were used as negative and positive controls respectively. PMAA-Na was not toxic to MDMs at the highest concentration of 2,000 ⁇ g/ml tested using the MTT assay and the Trypan blue assay as shown in Figure 3a.
• Human peritoneal cells were cultured in RPM1 1640 medium, 20 mM L-glutamine, 10% mixed donor human serum, 200 lU/ml penicillin and 200 ⁇ g/ml streptomycin and their density adjusted to 1 x 10 6 cells/ml. Media containing PMAA-Na was then added to the cells over the concentration range of 0 - 2,000 ⁇ g/ml. The cells were incubated for 71 h prior to the addition of MTT. The viability of the cells was expressed as a percentage of the viability of cells grown in the absence of any compound. Dextran and poly(L-lysine) were used as negative and positive controls respectively.
• PMAA-Na was not toxic to peritoneal macrophages up to 500 ⁇ g/ml. Between 500 ⁇ g/m and 2,000 ⁇ g/ml, a modest amount of toxicity was seen using the MTT assay and the Trypan blue assays as shown in Figure 3b.
• Peritoneal cells from patients on continuous ambulatory peritoneal dialysis were cultured with each compound and culture supernatants harvested after 36 h of incubation.
• Pro-inflammatory chemokines and pro-inflammatory cytokines were measured by EIA (R & D Systems).
• Example F1 Human peritoneal cells from 3 donors (A, B and C) were cultured with PMAA-Na (500 ⁇ g/ml) and culture supernatants harvested after 36 h were analysed for MIP- 1 ⁇ . All reagents and the PMAA-Na contained ⁇ 0.06 endotoxin units/ml (EU/ml) as determined using the Limulus amebocyte lysate assay (Pyrotell, Associates of Cape Cod, US). This is the European Community standard value for water for injection. There was a significant release of MIP-1 ⁇ in the presence of PMAA-Na as shown in Figure 4.
• EU/ml endotoxin units/ml
• Example F2 Human peritoneal cells from 2 donors (A and B) were cultured with PMAA-Na (500 ⁇ g/ml and 2,000 ⁇ g/ml) and culture supernatants harvested after 36 h were analysed for TNF- ⁇ . All reagents and the PMAA-Na contained ⁇ 0.06 endotoxin unit/ml. There was a significant release of TNF- ⁇ in the presence of PMAA-Na at both concentrations as shown in Figure 5.
• Example F3 Human peritoneal cells from 2 donors (A and B) were cultured with PMAA-Na (500 ⁇ g/ml and 2,000 ⁇ g/ml) and culture supernatants harvested after 36 h were analysed for TNF- ⁇ . All reagents and the PMAA-Na contained ⁇ 0.06 endotoxin unit/ml. There was a significant release of TNF- ⁇ in the presence of PMAA-Na at both concentrations as shown in Figure 5.
• Example F3 Human peritoneal cells from
• Human peritoneal cells were cultured with PMAA-Na (500 ⁇ g/ml) and culture supernatants harvested after 36 h were analysed for the pro-inflammatory chemokines MIP-1 ⁇ , MIP-1 ⁇ and IL-8, and for the pro-inflammatory cytokines TNF- ⁇ , IL-1 ⁇ and IL-6. All reagents and the PMAA-Na contained ⁇ 0.06 endotoxin unit/ml. The results with cells from up to 3 different human donors (A, B and C) are shown in Figure 6. The release of these chemokines and cytokines from tissue antigen presenting cells was at a level that would promote a pharmacological Th1 response in man but not high enough to cause significant adverse side-effects in man.
• Blood-derived monocyte derived macrophages were cultured with PMAA-Na at concentrations of up to 2,000 ⁇ g/ml. All reagents and the PMAA-Na contained ⁇ 0.06 endotoxin unit/ml. No release of MIP-1 ⁇ was seen.
• the results with cells from 3 different human donors are shown in Figure 7. There is therefore a differential immuno-modulatory effect of PMAA-Na on cells of blood monocyte origin compared to cells of macrophage origin and other antigen presenting cells (e.g., dendritic cells) from tissue body based compartments.
• antigen presenting cells e.g., dendritic cells
• Example H1 Red blood cells
• Example D1 The comparison was made between clinical grade amphotericin B and the amphotericin B - PMAA-Na preparation (both called "drug" in the Figure).
• Stock solutions of the compounds for testing were prepared in MGM. Human red cell lysis was determined after 1 hour ( Figure 10), 6 hour ( Figure 11) and 24 hour ( Figure 12) incubation in 3 different donors (A, B and C). No toxicity was seen with the amphotericin B - PMAA-Na preparation after a 1 hour incubation. After a 6 hour incubation, the toxicity of the amphotericin B - PMAA-Na preparation was considerably less than that of clinical grade amphotericin B. When the incubation time was increased to 24 h, the toxicity of the clinical grade amphotericin B increased to 100% but there was no further increase in the toxicity of the amphotericin B - PMAA-Na preparation.
• Example H2 Red blood cells
• Example H3 Peripheral blood mononuclear cells:
• PBMCs Peripheral blood mononuclear cells
• RPMI medium 10% mixed donor human serum
• 200 lU/ml penicillin and 200 ⁇ g/ml streptomycin at 1 x 10 5 cells and cultured in 96-well tissue culture plates at 37°C with 5% CO 2 .
• Media containing the amphotericin B - PMAA-Na preparation ("drug") was added to the cells over the concentration range of 0 - 70 ⁇ g/ml in a final volume of 100 ⁇ l/well.
• the cells were incubated for 1 day or 2 days or 6 days prior to the addition of MTT (5 mg/ml).
• the MTT solution was removed after 1 h and DMSO (100 ⁇ l) added to dissolve the MTT crystals.
• the optical density was measured at 550 nm using a plate reader (Molecular Devices, Wokingham, UK).
• the viability of the cell culture was expressed as a percentage of the viability of the cells grown in the absence of any compound. Dextran and poly(L-lysine) were used as negative and positive controls respectively.
• the compound was not toxic to peripheral blood mononuclear cells at the highest concentration of 70 ⁇ g/ml tested using the MTT assay and the Trypan blue assay after 1 day of culture, 2 days of culture or after 6 days of culture.
• Figure 14 shows the individual results from up to 3 representative human donors. The results from each donor are shown as line graphs in Figure 14.
• Example H4 Monocyte derived macrophages:
• the 50% lethal dose (LD 50 ) of clinical grade amphotericin B for Leishmania mexicana promastigotes was 0.14 ⁇ g/ml ( Figure 16 inset).
• the 90% lethal dose (LDgo) of clinical grade amphotericin B for Leishmania mexicana promastigotes was 1.49 ⁇ g/ml ( Figure 16 inset).
• the results from 3 experiments with the amphotericin B - PMAA-Na preparation are shown in Figure 16.
• the 50% lethal dose (LD 50 ) of the amphotericin B - PMAA-Na preparation for Leishmania mexicana promastigotes was 0.10 - 0.19 ⁇ g/ml ( Figure 16).
• the 90% lethal dose (LD 90 ) of amphotericin B - PMAA-Na preparation for Leishmania mexicana promastigotes was 1.02 - 1.49 ⁇ g/ml ( Figure 16).
• the activity of the amphotericin B - PMAA-Na preparation against Leishmania mexicana promastigotes was similar to that of clinical grade amphotericin B on a weight per weight basis.
• Leishmania donovania promastigotes were used for these experiments. They were maintained in Schneider's Growth Media 199 supplemented with 15% fetal calf serum (heat inactivated at 56°C for 1 hour) and gentamicin (1 mg/100 ml). The parasite concentration was adjusted to 2 x 10 6 parasites/ml. Doubling dilutions of the test compound were prepared at twice the desired final concentration in growth media. An equal volume of the drug and the parasite suspension were then mixed in a 96 well plate and incubated for 24 h at 26°C. At this time, MTT (5 mg/ml) was added and the plate incubated for a further 24 h at 26°C.
• the plate was then centrifuged (2000g, 5 min), the supernatant discarded and the pellet resuspended in 100 ⁇ l DMSO. Absorbance was measured at 570 nm using a spectrophotometer. The results were expressed as the percentage viability of the promastigotes with 100% viability being determined using the OD of the untreated, replicating promastigotes in the control wells.
• Leishmania mexicana amastigotes were used to infect human monocyte derived macrophages that were maintained in RPM1 1640 (Invitrogen) supplemented with 10% human serum (heat inactivated at 56°C for 1 hour) and 200 lU/ml penicillin and 200 ⁇ g/ml streptomycin.
• the cells were plated at 10 6 cells/ml into Lab-Tek Chamber Slides (Nunc) and incubated at 37°C/5% CO 2 for 3 days. The media was then aspirated from the chamber slides and an equal volume of fresh media added back in which the amastigote concentration had been adjusted to give a parasite ell infection ratio of 5:1.
• the cells were then incubated at 32°C for 20 h to enable infection of the macrophages to become established. Media was then aspirated from the chamber slides and replaced with doubling dilutions of the test compound. The cells were then incubated at 32°C for 72 h. After washing with PBS, the chambers were detached, the slides allowed to dry and then fixed in methanol. Each slide was then stained with Giemsa and examined microscopically. A total of 250 cells were counted to determine the number of infected and uninfected ceils.
• the infection index was calculated as the average number of parasites/cell multiplied by the percentage of infected cells.
• a 50% inhibition of intracellular Leishmania mexicana amastigote growth was achieved with 0.18 - 0.32 ⁇ g/ml of the amphotericin B - PMAA-Na preparation ( Figure 18) compared to 0.14 ⁇ g/ml of clinical grade amphotericin B ( Figure 19a) or 0.45 ⁇ g/ml of Ambisome ( Figure 19b).
• a 90% inhibition of intracellular Leishmania mexicana amastigote growth was achieved using 1.18 - 1.55 ⁇ g/ml of the amphotericin B - PMAA-Na preparation compared to 0.95 ⁇ g/ml of clinical grade amphotericin B or 3.89 ⁇ g/ml of Ambisome.
• Leishmania donovani amastigotes were used to infect human monocyte derived macrophages that were maintained in RPM1 1640 (Invitrogen) supplemented with 10% human serum (heat inactivated at 56°C for 1 hour) and 200 lU/ml penicillin and 200 ⁇ g/ml streptomycin.
• the cells were plated at 10 6 cells/ml into Lab-Tek Chamber Slides (Nunc) and incubated at 37°C/5% CO 2 for 3 days. The media was then aspirated from the chamber slides and an equal volume of fresh media added back in which the amastigote concentration had been adjusted to give a parasite ell infection ratio of 5:1.
• the cells were then incubated at 32°C for 20 h to enable infection of the macrophages to become established. Media was then aspirated from the chamber slides and replaced with doubling dilutions of the test compound. The cells were then incubated at 32°C for 72 h. After washing with PBS, the chambers were detached, the slides allowed to dry and then fixed in methanol. Each slide was then stained with Giemsa and examined microscopically. A total of 250 cells were counted to determine the number of infected and uninfected cells.
• a 90% inhibition of intracellular Leishmania donovani amastigote growth was achieved using 2.18 - 3.18 ⁇ g/ml of the amphotericin B - PMAA-Na preparation compared to 2.31 ⁇ g/ml of clinical grade amphotericin B or >8 ⁇ g/ml of Ambisome.
• the level of interferon- ⁇ release with the endotoxin free amphotericin B - PMAA-Na preparation would be sufficient to promote a pharmacological Th1 response in man but would not be high enough to cause significant adverse side-effects in man.
• PBMCs peripheral blood mononuclear cells
• All reagents and compounds contained ⁇ 0.06 endotoxin unit/ml as determined using the Limulus amebocyte lysate assay (Pyrotell,
• Example L3 ELISpot assay for Interferon- ⁇ secreting cells:
• Human PBMCs were isolated and adjusted to 2 x 10 5 cells/well in RPM1 1640 supplemented with 10% human serum, 200 ⁇ g/ml penicillin and 200 lU/ml streptomycin. All reagents and the compounds contained ⁇ 0.06 endotoxin unit/ml.
• the PBMCs and the compounds were mixed in the wells of an ELISpot PVDF- backed microplate coated with the human interferon- ⁇ monoclonal antibody (R&D Systems, UK). Unstimulated cells were used as the negative control and recombinant human interferon- ⁇ was used in the positive control. The plate was incubated for 24 hours at 37°C / 5% CO 2 .
• FIG. 25 shows that the tuberculin PPD - PMAA-Na preparation was significantly more effective than tuberculin antigen alone in stimulating interferon- ⁇ release from primary human T lymphocytes. This increase in interferon- ⁇ secretion from T lymphocytes was seen with 50 ⁇ g/ml of the tuberculin PPD - PMAA-Na preparation and with 100 ⁇ g/ml of the tuberculin PPD - PMAA-Na preparation.
• Leishmania donovani strain (MHOM/ET/67/L82) amastigotes were collected from the spleens of heavily infected donor Syrian Hamsters- esocr/fef ⁇ s auratus. An inoculum containing 7.5 - 10 x 10 7 amastigotes/mL was prepared.
• Female BALB/c mice (20 g) that were specified pathogen free were infected intravenously with 200 ⁇ L (equivalent to 1.5 - 2 x 10 7 amastigotes) on day 0. The end point of infection was evaluated by microscopic reading on day 14 to check for the level of infection in one mouse, and on day 21 post-infection of Giemsa stained liver impressions to determine the total parasite burden.
• the following compounds were administered intravenously according to the following regimens:- a) untreated control after infection. b) 0.5 mg/kg of blank liposome on days 14, 16 and 18 after infection. c) 8 mg/kg of PMAA-Na on days 14, 16 and 18 after infection. d) 1 mg/kg of clinical grade amphotericin B on days 14, 16 and 18 after infection. e) 0.5 mg/kg of AmBisome on days 14, 16 and 18 after infection.
• the carrier solution for the intravenous injections of PMAA-Na and for amphotericin B - PMAA-Na preparation was 5% dextrose. All drug preparations were filtered using a 0.2 ⁇ L syringe filter. The intravenous volume of injection was 200 ⁇ L per injection.
• mice were weighed before and after treatment as a measure of toxicity and the percentage weight change noted.
• the parasite burden was determined microscopically from methanol fixed liver impression slides stained with 10% Giemsa. The number of amastigotes/500 liver cells were counted microscopically and the results expressed as a percentage of the untreated control.
• Figure 26 shows that the blank liposomes and the PMAA-Na had no anti- leishmanial activity. There was significant killing activity (P ⁇ 0.0001) by clinical grade amphotericin B, Ambisome, amphotericin B - PMAA-Na preparation at 1 mg/kg, and amphotericin B - PMAA-Na preparation at 2 mg/kg of Leishmania donovani amastigotes compared to the controls. The anti-leishmanial activities of the clinical grade amphotericin B, Ambisome, and the amphotericin B - PMAA-Na (2 mg/kg) were not significantly different from each other (P ⁇ 0.05). These results therefore show that the amphotericin B - PMAA-Na preparation was as effective as Ambisome in this animal model of visceral leishmaniasis.
• PMAA-Na constructs of varying molecular weight -
• the polymerisation conditions were altered as described in Example A to create several polymers with molecular weights that ranged from 19 kD to 37 kD.
• the molecular weight and the polydispersity index were established using gel permeation chromatography (GPC).
• the toxicity of PMAA-Na constructs of different molecular weights for primary human cells was established using an MTT assay.
• the PMAA-Na constructs did not cause red blood cell lysis at a concentration of 2 mg/ml after a 1 h, 5 h and a 24 h incubation, see Figure 27.
• they were not toxic to peripheral blood mononuclear cells at 2 mg/ml after incubation for 1 day and for 2 days, see Figure 28.
• Example O Stability of amphotericin B - PMAA-Na complex during storage:
• Lyophilised amphotericin B - PMAA-Na was stored at 4°C under argon for 4 months. It was also solubilised in 5% dextrose at a concentration of 1 mg/ml and stored at 4°C for 7 months under argon. At the end of this time, the stability of the amphotericin B - PMAA-Na complex was determined by incubating it with human red blood cells. Previous experiments have shown that the amphotericin B - PMAA-Na complex is much less toxic than amphotericin B. This assay was therefore repeated after storage of the complex for several months. The experiment was carried out as previously described. Freshly prepared, clinical grade amphotericin B was used as a reference for the comparison.
• Figure 29 shows that the amphotericin B - PMAA-Na complex was not toxic to red blood cells after storage. The complex was therefore stable when it was stored as a lyophilised powder for 4 months, and also when it was stored in 5% dextrose at 4°C for 7 months.
• Cryptococcus strains were subcultured in YNB supplemented with 10% human serum for 48 h in the presence of 5% CO 2 to allow them to form a capsule.
• Peritoneal human macrophages or monocyte derived macrophages ( ⁇ 750,000) were plated onto chamber slides (Lab-Tek, USA) and allowed to adhere for 24 h.
• Yeast cells were spun down, washed, and resuspended in RPMI supplemented with 10% human serum and 2% glucose; the latter facilitates the growth of the yeast. This suspension was added at 15 times the concentration of the macrophages and the infection allowed to proceed for ⁇ 21 h at 37°C in a humidified chamber with 5% CO 2 .
• Figure 32 (i) and (ii) show the results for Cryptococcus neoformans var neoformans
• Figure 32 (iii) and (iv) show the results for Cryptococcus neoformans var gatti when these organisms have infected human monocyte derived macrophages.
• the LD50 for amphotericin B and for amphotericin B - PMAA-Na were similar in all of the 8 experiments performed.
• the LD 50 for amphotericin B was 0.9 - 1.4 ⁇ g/ml and that for amphotericin B - PMAA-Na was similar at 1.6 - 2.7 ⁇ g/ml.
• the LD 50 for amphotericin B was 0.06 - 1.0 ⁇ g/ml and that for amphotericin B - PMAA-Na was similar at 0.1 - 0.5 ⁇ g/ml. Therefore, the complex is as active against cryptococci as amphotericin B alone.
• Figure 33 (i) shows the results for Candida albicans and Figure 33 (ii) shows the results for Candida glabrata.
• the LD S0 for amphotericin B (0.9 - 1.8 ⁇ g/ml) and for amphotericin B - PMAA-Na (1.6 - 2.3 ⁇ g/ml) are similar. Therefore, the complex is as active against Candida sp. as amphotericin B alone.

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