Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof

Definitions

• This invention relates to a novel form of medicament, novel formulations comprising the medicament and novel methods of treatment.
• Amphotericin is a polyene antibiotic which is active against both yeast-like and filamentous fungi, acting on sterols in the fungal cell membrane to cause an increase in permeability and leakage of cell constituents. Resistance to polyene antifungals in normally sensitive fungi, is virtually unknown. Orally delivered preparations of antifungals, such as amphotericin, are useful for the treatment of, inter alia, oral thrush and intestinal candidiasis and for the suppression of Candida in the GI tract which may, for example, be acting as a reservoir in recurrent vaginal candidiasis. Intravenous preparations of antifungals, such as amphotericin, are used for life- threatening systemic mycoses.
• Amphotericin by intravenous infusion is used for the treatment of systemic fungal infections and is active against most fungi and yeasts.
• Amphotericin is given intravenously for severe systemic fungal infections (e.g., aspergillosis, cryptococcal meningitis, disseminated cryptococcosis), and is used in immunocompromised (for example, AIDS or transplantation) patients who are at particular risk of severe, disseminated fungal-type infections.
• Amphotericin was first shown to have activity against the leishmaniasis protozoan infection in the late 1950s, and is used to treat visceral and mucocutaneous leishmaniasis. Again activity is probably due to amphotericin's affinity for sterols.
• amphotericin When given parenterally amphotericin is toxic and side effects are common. Close supervision is necessary and a test-dose is required.
• Typical side effects from parenteral administration include; anorexia, nausea and vomiting, diarrhoea, epigastric pain, febrile reactions, headache, muscle and joint pain, anaemia; renal function disturbance (including hypokalaemia and hypomagnesaemia), renal toxicity; cardiovascular toxicity (including arrhythmias), blood disorders; neurological disorders (including hearing loss, peripheral neuropathy); abnormal liver function, rash, possible anaphylactoid reaction, and pain and thrombophlebitis at injection site
• Lipid formulations of amphotericin are significantly less toxic but are expensive, for example, a 20 day dosage regime of AmBiSome® costs in the region of £2,000 (twelve thousand pounds sterling).
• Dextrins are known from European Patent Applications Nos 0 115 911 and 0 153 164 and are useful in peritoneal dialysis. When used in peritoneal dialysis, dextrins are generally administered into the peritoneal cavity as an aqueous solution.
• glucose polymer e.g. a dextrin polymer
• GP is a glucose polymer, or a mixture of glucose polymers, and optionally salts thereof;
• AF is an antifungal agent
• n is an integer from 1 to 12.
• antifungal infections such as Pneumocystis carinii are now thought possibly to be fungal infections, by the term antifungal used herein, it is intended to include antiprotozoal agents and anti-leishmaniasis agents.
• a pharmaceutical composition comprising an effective amount of a complex of an antifungal agent and a glucose polymer, or a mixture of glucose polymers and optionally salts thereof.
• the antifungal agent is preferably an antifungal agent which is capable of bonding to a glucose polymer. Therefore, preferentially, the antifungal agent may possess at least one free amine group.
• antifungal agents include polyene antibiotics, such as amphotericin and nystatin.
• the antifungal agent is amphotericin, e.g. amphotericin B.
• glucose polymers may be used, preferred glucose polymers or a mixture of glucose polymers, and optionally salts thereof, are those polymers described in European Patent Applications Nos 0 115 991 and 0 153 164.
• Salts include the anionic salts, e.g. sulphates.
• Glucose polymers which may hereinafter be referred to as dextrin, glucose dextrin or dextrin polymer are intended, on all occurrences, to include optionally salts thereof especially the sulphate.
• the dextrin sulphate optionally would contain at most two sulphate groups per unit. All references herein to dextrin sulphate, dextrin 2 sulphate, or D-2-S are within the scope of the aforementioned definition.
• Dextrin is a mixture of polymers of glucose and the glucose units may be substituted in one or more of the 2, 3 and 6 positions by sulphate groups.
• a dextrin sulphate of use in the present invention may have up to 2, 3 and 6 positions occupied by sulphate groups.
• a dextrin sulphate of use in the present invention may have up to two sulphate groups per glucose unit and preferred dextrin sulphates are those having about 1, or between 0.5 and 1.5, preferably up to 1.2, for example 1.1, sulphate groups per glucose unit. More preferably, the agent is the 2- or 6-sulphate of dextrin or a mixture thereof, most preferably dextrin-2-sulphate (D-2-S) that is dextrin wherein a substantial proportion of the sulphate groups are in the 2-position, preferably greater than 75%, more preferably greater than 90%, e.g. 94%.
• D-2-S dextrin-2-sulphate
• the glucose polymer comprises a glucose polymer mixture, said mixture including more than 15%) by weight of glucose polymers of D.P. greater than 12, more preferably, more than 50% by weight of glucose polymers of DP greater than 12.
• the mixture may contain from 50 to 90% by weight of glucose polymers of D.P. greater than 12. It is advantageous to use a mixture containing from 75 to 100%, preferably 90 to 100%, by weight of glucose polymers of D.P. greater than 12.
• the average molecular weight of the polymer mixture is preferably from 15,000 to 25,000, more preferably 18,000 to 22,000 (as determined by high pressure liquid chromatography). It is particularly desirable that the content of oligosaccharides in the glucose polymer mixture should be kept at a low level.
• the oligosaccharide content of the glucose polymer mixture should be no higher than 10% by weight, the mixture containing from 90 to 100%> by weight of glucose polymers of D.P.
• the preferred glucose polymer is a dextrin or a mixture of dextrins, and optionally salts thereof, e.g. poly ⁇ -1,4 glucose dextrin.
• the glucose polymer or mixtures thereof used in this invention can be produced by hydrolysis of starch in known manner, followed by treatment of the mixture of glucose polymers so obtained in order to remove some or all of the glucose polymers of lower molecular weight.
• a glucose polymer mixture is prepared by the process described in European Patent Application No. 0 115 911 and removal of lower molecular weight polymers is then effected by known fractionation techniques, such as solvent fractionation, or separation of the polymers with the aid of permeable membranes of appropriate cut-off characteristics.
• fractionation techniques employed are such as to remove mainly the lower molecular weight polymers. If desired, however, they may also be used to remove very high molecular weight polymers if this is found to be necessary in order to ensure that all of the final mixture of glucose polymers is sufficiently water- soluble for there to be no tendency for higher molecular weight polymers to precipitate from solution on standing.
• the pharmaceutical composition of the invention may be utilised in the treatment or alleviation of a wide range of yeast and yeast-like fungal infections including, candidiasis, such as Candida albicans.
• the pharmaceutical composition of the invention may also be advantageous in the treatment or alleviation of fungal and/or protozoal infections resulting from the Human Immunodeficiency Virus (HIV).
• HIV Human Immunodeficiency Virus
• Such . fungal infections include, for example, Pneumocystis carinii and Leishmania Spp.
• composition of the invention may be useful in the treatment of related diseases, such as Pneumocystis carinii pneumonia (PCP) and/or leishmaniases.
• the pharmaceutical formulation of the invention may be useful in the treatment or alleviation of protozoal infections.
• a method of treating a yeast, fungal or protozoal disease which comprises the administering of a therapeutically effective amount of a pharmaceutical composition as hereinbefore described to a patient suffering from such a disorder.
• a method of treating a Pneumocystis carinii infection e.g. Pneumocystis carinii pneumonia (PCP) which comprises administering a therapeutic amount of a pharmaceutical as hereinbefore described.
• PCP Pneumocystis carinii pneumonia
• the compounds and formulations of the invention are advantageous in that, inter alia, they are useful in the treatment or alleviation of yeast, fungal and/or protozoal infections in mammals. Furthermore, the compounds of the invention may target the reticuloendothelial system in mammals. It is a particular advantage of the compounds and formulations of the invention that they have improved solubility e.g. water solubility when compared to the free antifungal agent. Thus, it is an especially advantageous feature of the present invention that a polyene antibiotic therapy can be achieved with improved solubility in water.
• a polyene antibiotic derivative characterised in that the polyene antibiotic derivative has a solubility in water of from 0.1 mg/ml to 1 mg/ml, preferably 0.1 mg/ml to 0.5 mg/ml and especially 0.1 mg/ ml to 0.4 mg/ml.
• Amphotericin is known to be insoluble in water at room temperature and substantially neutral pH, i.e. pH 6 to 7.
• the term insoluble has been defined as 1 part requiring 10,000 parts of solvent to solubilise it. Thus a solubility of less than 100 ppm would be considered to be insoluble.
• a water soluble form of amphotericin e.g. amphotericin B.
• We especially provide a form of amphotericin which has a water solubility of at least 1000 ppm at room temperature and substantially neutral pH.
• an aqueous solution of an antifungal agent characterised in that the solution comprises from 0.1 mg/ml to 1 mg/ml, preferably 0.1 mg/ml to 0.5 mg/ml and especially 0.1 mg/ ml to 0.4 mg/ml.
• the antifungal agent is a dextrin amphotericin compound.
• the actual amount of antifungal agent, e.g. amphotericin, in the compound of the invention may vary, depending, inter alia, upon the degree of dicarboxylation of the dextrin.
• the antifungal agent content of the conjugate may be from 0.001 to 25% w/w, preferably from 0.1 to 20% w/w.
• the pharmaceutical ' composition may be administered in a variety of ways including but not limited to an intravenous infusion, injection or by way of intraperitoneal administration.
• the most preferred method of administration is by infusion.
• a composition for intraperitoneal administration containing a pharmaceutical composition as hereinbefore described.
• compositions of the present invention may be in the form of a solution, e.g. an aqueous solution, the concentration of the composition therein and the administered volume which is selected according to the nature of the treatment involved and the needs of the patient.
• the solution of composition may contain electrolytes and / or a glucose polymer in a manner similar to that described in UK Patent No. 2207050 which is incorporated herein by reference.
• the mode of use of the intravenous infusion solutions according to the invention is similar to that of known solutions.
• the pharmaceutical composition may be administered by inhalation.
• a method of administration is especially suitable in, for example, the treatment of Pneumocystis carinii infections and PCP in particular.
• the pharmaceutical composition can be administered by way of an inhaler, e.g. a metered dose inhaler or a dry powder inhaler, an insufflator or nebuliser, or any other conventionally known methods of administering inhalable medicaments.
• an inhaler e.g. a metered dose inhaler or a dry powder inhaler, an insufflator or nebuliser, or any other conventionally known methods of administering inhalable medicaments.
• the pharmaceutical composition When administered by way of inhalation the pharmaceutical composition may be in the form of a pressurised aerosol.
• a pharmaceutical formulation suitable for administration by way of a pressurised aerosol comprising a pharmaceutical composition as hereinbefore described in admixture with at least a suitable propellant and optionally with a surfactant or a mixture of surfactants.
• the propellant is preferably a non-CFC propellant, such as a hydrofluoroalkane (HFA). Any conventionally known HFA propellant may be used, however, HFAs which may be mentioned include a fluoroalkane such as a fluoromethane or a fluoroethane or a mixture of fmoroalkanes.
• Such fluoroalkanes include, but are not limited to, trichlorofluoromethane, diichlorodifluoromethane, 1,2-dichlorotetrafluorethane, trichlorotrifluoroethane and chloropentafluoroethane, HFA 227 or HFA 134 (1,1,1,2-tetrafluoroethane).
• the amount of propellant present may vary, but generally the pharmaceutical composition to propellant ratio will be from 1 to 300 to 1 to 5. Mixtures of propellants may be used, for example, a mixture of HFA 134 and HFA 227.
• the aerosol composition of the invention may be present as a solution or a suspension of the active ingredient with a propellant.
• the pressurised aerosol formulation of the invention may be administered in any conventionally known inhalation apparatus.
• the pharmaceutical composition may be administered as an inhalable dry powder formulation.
• the composition may be administered with or without an adjuvant, diluent or carrier.
• a pharmaceutical formulation suitable for administration by way of a dry powder inhaler comprising a pharmaceutical composition as hereinbefore described in admixture with a suitable adjuvant, diluent or carrier.
• Any conventionally used ingredients in dry powder formulations may be used, such as sugars, these include, but are not limited to, dextran and lactose, e.g. crystalline lactose.
• a carrier the pharmaceutical composition to carrier ratio is from 0.01:50 to 1:1.
• the dry powder formulation of the invention may be administered in any conventionally known inhalation apparatus.
• preferred apparatus are those commercially available as CLICKHALER (described in International Patent Application No. WO 92/00771) and/or TECHNOHALER (described in International Patent Application No. WO 93/16748).
• the inhalable formulation of the invention may be administered by way of a conventional nebuliser.
• a suitable nebuliser formulation consists of a sterile solution of the drugs in water optionally containing surfactant or a pharmaceutically acceptable co-solvent or a sterile solution containing finely divided suspended drug.
• the solution or suspension may be nebulised by an air jet, dropping onto an ultrasonic vibrating plate, forcing through small orifices or other known types of nebuliser, including unit-dose nebulisers such as Respimat (from Boehringer Ingelheim), AERxTM (from Aradigm), and AeroDose (from Aerogen).
• the pharmaceutical composition is preferably micronised.
• the particle size of the polymer may vary. However, it is preferred that the polymer will have a particle size of 10 microns or less.
• compositions of the present invention may be in the form of an aqueous solution or may be present as a dry powder for making up into a solution by a physician.
• the amount of the compound of the invention which is present in the composition may vary, and generally may be from 0.001 to 25%) w/w, preferably 0.1% to 20% w/w.
• the dosage of pharmaceutical composition administered to a patient may vary depending, inter alia, upon the nature and severity of the disorder being treated and the method of administration. Generally, the amount of the pharmaceutical composition administered is preferably in the range of from 0.1 mg to 500 mg from one to four times daily.
• the dextrin-amphotericin conjugate of the invention is advantageous, inter alia, in that it provides better targeting; reduced side effects; a variety of routes of administration (e.g. intraperitoneal, inhalational); reduced cost and lower amounts of active drug required.
• a method of manufacturing a compound of formula I as hereinbefore described which comprises reacting a dicarboxylated glucose polymer or a derivative thereof with an antifungal agent.
• the reaction may be carried out under conventional conditions known per se.
• methods conventionally used in peptide syntheses may be employed.
• a dicarboxylated e.g. succinoylated, dextrin, or a salt thereof may be derivatised prior to reaction with the antifungal agent.
• examples of such derivatives include, but are not limited to an acid halide, e.g. acid chloride; acid azide; mixed anhydride; or carbodiimide.
• a carbodiimide derivative is preferred, for example a carbodiimidazole derivative.
• the dicarboxylated glucose polymers and derivatives thereof are novel er se.
• a carbodiimidazole derivative of a dicarboxylated glucose polymer for use as an intermediate in the manufacture of a compound of formula I.
• dicarboxylated glucose polymer we mean a glucose polymer which has been reacted with a dicarboxylic acid. Any dicarboxylic acid may be used, thus the dicarboxylic acid may be of the general formula:
• n is an integer from 1 to 12
• the dicarboxylic acid may be an unsaturated dicarboxylic acid.
• dicarboxylic acids which may be mentioned include oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, maleic acid and fumaric acid.
• the saturated dicarboxylic acids are preferred and succinic acid the most preferred.
• Amphotericin B was conjugated to dextrin (0.9mol%) and dextrin sulphate (lmol%).
• the method for succinoylating dextrin and dextrin sulphate has been described elsewhere (PCT/GB00/02216).
• dextrin (lg) was dissolved in DMF (10ml).
• Succinic anhydride (62.5mg, 6.2xl0 "4 mol) was added to DMAP (28.5mg) to promote acylation reactions.
• the mixture was purged with nitrogen and poured rapidly on to diethyl ether and stored overnight.
• the ether was removed by filtration and remaining solids dissolved in the minimum amount of distilled water and dialysed before being freeze dried.
• amphotericin B conjugated to the polymers was determined by UV- VIS spectrometry and calibration curve. FTIR also confirmed the presence of amphotericin B. These reactions were repeated using dextrin and dextrin sulphate with higher degrees of succinoylation to increase the amphotericin B content. Dextrin was succinoylated to levels of 10 and 34.6mol% and dextrin sulphate succinoylated to levels of 14 and 24mol%.
• amphotericin B solutions in water (1, 0.5, 0.25, 0.125, 0.065 and 0.0313 mg/ml Amphotericin B equivalent) were compared with dextrin-Amphotericin B concentrations of 0.4, 0.16 and 0.1 mg/ml Amphotericin B equivalent and dextrin sulphate-Amphotericin B concentrations of 0.014 and 0.009 mg/ml Amphotericin B equivalent.
• the conjugates were found to be soluble in water at these concentrations. Amphotericin B is almost totally insoluble in water. In comparison the polymer conjugates were shown to have a ten fold increase in solubility when compared to . amphotericin B at the same concentrations.
• dextrin-Amphotericin B had a drug content of 0.006wt% and dextrin sulphate-Amphotericin B a drug content of 0.014 wt%.
• An adult rat was killed and blood quickly removed by cardiac puncture. Blood was placed in a lithium heparinised tube on ice. PBS was then added and the diluted blood centrifuged three times with the supernatant being removed each time.
• Dextrin-Amphotericin B and dextrin sulphate-Amphotericin B were dissolved in DMSO before being diluted to the appropriate concentration in buffer.
• Dextrin- amphotericin B concentrations were made in the range 0-2.5 ⁇ g/ml of Amphotericin B equivalent, dextrin sulphate-Amphotericin B in the range 0-14 ⁇ g/ml Amphotericin B equivalent, and Amphotericin B concentrations in the range 0-14 ⁇ g/ml.
• RBCs red blood cells
• Glutaraldehyde in PBS was added to fix the cells, followed by osmium tetroxide and cells were then dehydrated over time with increasing concentrations of ethanol.
• hexamethyl disilazine was added: the cells were then placed on a SEM platform using carbon cement and examined using SEM.
• concentrations used for the SEM were PBS (control), dextrin and dextrin sulphate (5mg/ml), Amphotericin B at EC50, dextrin-Amphotericin B at EC 50 (0.5mg/ml) and dextrin sulphate-Amphotericin B (Figure 18) at EC 50 (0.98mg/ml).
• Dextrin-Amphotericin B treated cells displayed no morphological abnormalities compared to the control cells ( Figure 29).
• Standard cell culture protocols were used to establish the cytotoxic profiles of dextrin-Amphotericin B, dextrin sulphate-Amphotericin B, dextrin and dextrin sulphate.
• the standard MTT assay is a rapid colormetric assay for assessing cell viablity. The basis of this assay is that viable cells are able to reduce the water soluble tetrazonium dye MTT, to a water insoluble coloured formazan salt.
• a cell growth curve was constructed for the B16F10 cells by titrating the absorbance against time (days). The growth curve confirmed that the cells used in each study were always in the exponential phase of cell growth.
• a 96-well microtitre plates were seeded with a suspension of 10 4 cells per well of B16F10 murine melanoma cells in RPMI 1640 media containing 10% foetal calf serum. The cells were allowed to incubate for 24 hours at 37°C/5 % CO 2 .
• Dextrin-Amphotericin B concentrations were made in the range of 0-2.5 ⁇ g/ml of Amphotericin B equivalent, and dextrin sulphate- Amphotericin B was in the range of 0-14 ⁇ g/ml Amphotericin B equivalent.
• the MTT assay assesses the ability of compounds to decrease cell viability.
• B16F10 cells were selected as they are a well characterised mammalian cell-line in which Amphotericin B is active, although to a lesser extent than fungal cells.
• Dextrin-Amphotericin B conjugate caused an initial decrease in cell viability to approximately 75% at a dose of 0.4 ⁇ g/ml (0.2 ⁇ g/ml Amphotericin B equivalent), which then remained at that level even at the highest concentration used (Figure 20).
• Dextrin sulphate-Amphotericin B was significantly more toxic to the B16F10 cells than equivalent quantities of Amphotericin B, with an IC 50 value of approximately 11 ⁇ g/ml Amphotericin B equivalent, which is approximately 8 times lower than Amphotericin B alone (Figure 21).

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