WO2011061747A1 - Antifungal and anti parasitic conjugates of amphotericin b derivatives - Google Patents

Abstract

This invention relates to conjugates of polysaccharides and active drug components and uses thereof in a variety of therapies, such as antifungal and antileishmanial therapies.

Description

ANTIFUNGAL AND ANTI PARAS ITIC CONJUGATES OF AMPHOTERICIN B DERIVATIVES

FIELD OF THE INVENTION

This invention relates to conjugates of polysaccharides and active drug components and uses thereof, e.g., in antifungal and antileishmanial therapy.

BACKGROUND OF THE INVENTION

Amphotericin B (AmB), a polyene antibiotic, is a standard drug for the treatment of fungal infections. However, AmB therapy is limited because of its negligible solubility in aqueous solutions, its toxicity and associated side effects.

In the past, numerous attempts to reduce the toxicity associated with the use of the drug, while maintaining its activity in vivo, have been reported. Conjugation of AmB to polysaccharides such as arabinogalactan (AG) has been demonstrated to only partially achieve the target activity-toxicity profile.

US patent no. 5,567,685 to Domb et al [1] discloses a process for the production of a stable water-soluble conjugate of a polysaccharide and a polyene antibiotic such as AmB. The method comprises (a) oxidation of the polysaccharide to a dialdehyde; (b) purification of the dialdehyde from various interfering anions and by-products; (c) conjugating the purified dialdehyde with the antibiotic; (d) reducing the imine bond to the more stable amine bond; and (e) purifying the conjugate.

US patent no. 6,011,008 also to Domb et al. [2], discloses a similar process for the conjugation of a polysaccharide to a non-polyene antibiotic.

Domb et al [3, 4] report on the preparation and characterization of a conjugate of AmB and AG. The AmB-AG conjugate was prepared by reacting the oxidized AG and the AmB at pH 11 for a period of time and under conditions permitting conjugation of the polysaccharide to the drug. No modification in the structure of the drug molecule was reported.

The conjugation of AmB to AG via tosylate or mesylate derivatives was investigated as a method for the conjugation of amino-containing drugs to polysaccharides [5].

International publication no. WO 2007/034495 [6] discloses a conjugate of a polysaccharide and an antibiotic which toxicity is further reduced by reacting each of the free aldehyde groups with a blocking group. US patent no. 7,115,576 to Sommermeyer [7] discloses water-soluble antibiotic- polysaccharide (starch derivatives) conjugates wherein the antibiotic is associated with the polysaccharide via an amide bond.

Conjugates of AmB with other polysaccharides have also been studied in the treatment against fungi and Leishmania [8]. Solubilized forms of AmB have also been studied [9].

REFERENCES

[1] US patent no. 5,567,685

[2] US patent no. 6,011,008

[3] "A Novel Injectible Water-Soluble Amphotericin B-Arabinogalactan Conjugate" Falk, R., Domb, A., and Polacheck, I., Antimicrob Agents Chemother., 1999 43(8): 1975-1981.

[4] "Synthesis and characterization of novel water soluble amphotericin B- arabinogalactan conjugates" Ehrenfreund-Kleinman, T., Azzam, T., Falk, R., Polacheck, I., Golenser J., and Domb, A., Biomaterials, 2002 23(5): 1327-1335.

[5] "Conjugation of arnino-containing drugs to polysaccharides by tosylation: amphotericin B-arabinogalactan conjugates" Ehrenfreund-Kleinman, T., Golenser J., and Domb, A., Biomaterials 2004 25(15): 3049-3057.

[6] WO 2007/034495

[7] US patent no. 7,115,576

[8] "Amphotericin B-Gum Arabic Conjugates: Synthesis, Toxicity, Bioavailability, and Activities against Leishmania and Fungi" Pharmaceutical Research 2007 24(5): 971- 980.

[9] "Use of amphotericin B for differentiating eukaryotic from prokaryotic activity in mixed microbial samples" J. Microbiol. Methods 1987 6: 257-264. SUMMARY OF THE INVENTION

It has been the aim of an extended research to develop antifungal drugs, which similarly to Amphotericin B (AmB) would be highly effective, but unlike AmB safe.

The conjugates of the invention are based on the conjugation of AmB derivatives, as defined herein, to highly water soluble polymers, e.g., polysaccharides. Unlike previously disclosed processes for the manufacture of conjugates of polysaccharides and AmB, the process leading to the formation of the new conjugates incorporates in situ formation of AmB derivatives (e.g., via saponification and/or reduction), which subsequently undergo conjugation to the polysaccharide by reductive amination. The conjugated product according to the invention demonstrates enhanced activity and safety as compared to the previously reported conjugates of polysaccharides and AmB. This class of novel conjugates is clearly a promising alternative to AmB.

In a first aspect of the present invention, there is provided a conjugate of a polymer and at least one drug moiety being an amphotericin B (AmB) derivative.

As used herein, the term "conjugate" refers to a compound constructed of a polymer, which, in some embodiments, may be a polysaccharide, as further disclosed, and at least one drug moiety which is chemically associated thereto. Any one conjugate of the invention may also be referred to as a composition of matter. In most general terms, a "composition of matter" similarly to a "conjugate", both used interchangably, refers to the association of the polymer and the at least one drug moiety, an association which produces, as further demonstarted below, properties which may be attributed to the composition (or conjugate) as a whole and not to any one of polymer and drug moiety (or moities) in their separate state.

The association/conjugation of the polymer and the at least one drug moiety may be via any chemical bonding, including covalent bonding, electrostatic interaction, acid base interaction, van der Waals interaction, etc. In some embodiments, the association is via covalent bonding of a nitrogen (N) or oxygen (O) atom of the drug moiety and a carbon (C) atom of the polymer. In some embodiments, the covalent association is via amine, imine or amide bonds.

The "polymer" with which the at least one drug moiety is associated (conjugated) is, as defined in the art, and typically of a molecular weight of at least 1,000 Daltons (Da), at least 10,000 Da, or in the range of 5,000 Da to 75,000 Da. The polymer employed may be a homopolymer, a copolymer, a terpolymer or any other higher polymer.

In some embodiments, the polymer is selected to permit covalent association with the at least one drug moiety. As such, a polymer in its native state may have one or more functional groups which permit such association or may be chemically modified to structurally attain one or more side groups or functionalities which are capable of chemical association with a drug moiety.

In some embodiments, the polymer is partially or fully oxidized to permit such covalent association with a plurality of drug moieties. In some embodiments, the polymer is at least about 5% oxidized, with the remaining polymer monomers remaining unoxidized (of original polymer structure and form). In other embodiments, the polymer is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or about 95% oxidized. In further embodiments, the polymer is between about 5% and 20% or between about 20% and 100% oxidized. In further embodiments, the polymer is between about 20% and 70% oxidized. In yet additional embodiments, the polymer is between about 50% and 100% oxidized.

In other embodiments, the polymer is a water-soluble or water-dispersible polysaccharide, which may or may not have a plurality of identical monomers (such as in the case of dextran) or different monomers (such as in the case of arabinogalactan). The polysaccharide may be natural or synthetic and may be branched or linear. The polysaccharide may be a chemically modified or a semi-synthetic polysaccharide, permitting association with the at least one drug moiety.

In some embodiments, the polysaccharide is selected from starch, glycogen, cellulose, dextran, pullulan, chitosan, arabinin, arabinogalactan, galactan, galactomannan, gelatin, pectin, amilo-pectin, glycan, poly-mannan, hyaluronic acid, guar gum and any other poly sugar or synthetic combination thereof.

In some embodiments, the polysaccharide is arabinogalactan (AG) or a modified form thereof, e.g., oxidized form.

Where the polymer is an oxidized polysaccharide, the polysaccharide may be partially or fully oxidized. In some embodiments, the polysaccharide is at least about 5% oxidized, namely having at least 5% of the saccharide monomers oxidized with the remaining saccharide monomers remaining unoxidized (of original structure and form). In other embodiments, the polysaccharide is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or about 95% oxidized. In further embodiments, the polysaccharide is between about 20% and 100% oxidized. In further embodiments, the polysaccharide is between about 20% and 70% oxidized. In yet additional embodiments, the polysaccharide is between about 50% and 100%) oxidized.

In other embodiments, at least one monomer (saccharide) of the polysaccharide is oxidized.

In some embodiments, the polysaccharide is oxidized AG (fully or partially oxidized). In some embdoeimnts, the AG is fully oxidized, namely all AG monomers are oxidized. In other embodiemnts, the AG is only partially oxidized, namely at least one of the AG monomers is oxidized with the remaining monomers unmodified.

In some embodiments, at least about 5% of the AG monomers are oxidized (with the remaining AG monomers unoxidized, namely of original structure and form). In other embodiments, the AG, as other polymers and polysaccharides, is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least about 99% oxidized. In further embodiments, the AG is between about 20% and 100% oxidized. In further embodiments, the AG is between about 20% and 70% oxidized. In yet additional embodiments, the AG is between about 50% and 100% oxidized. In still further embodiments, the AG is between about 50% and 70% oxidized.

For the sake of clarity, it should be noted that the oxidized AG, to any level of oxidztion, produced under the conditions of the invention or any alternative and equivalent set of oxidation conditions, is composed of one or more monomers of the general formula (I), which are capable of cojugation to at least one drug moiety via the aldehyde groups.

Thus, in the process for the preparation of a conjugate of oxidized AG and at least one drug moiety, the one or more oxidized monomers, each having aldehyde groups, i.e., -CiH(=0) and -C₂H(=0) as depicted in the monomer (I), are either converted upon reaction with the drug moiety to an imine, amine and/or reduced to the corresponding alcohol. The conjugate of the invention may comprise a mixture of monomers which differ in their bonding to the same or different drug moieties (DMs) and may also differ in the number of DMs which are associated to each monomer, namely whether each monomer is conjugated to one or more DMs or not at all. As stated hereinabove, the conjugate may additionally comprise unoxidized monomers. A conjugate of the invention is substantially free of aldehyde groups.

Therefore, a conjugate may be composed of:

(a) unoxidized monomers (i.e., monomers of the original polymer, e.g., polysaccharide) and/or monomers having no conjugation to a DM, with each of the aldehyde groups having been reduced to the corresponding hydroxyl (-OH) group, and

(b) a plurality of one or more of the following monomer types:

1. monomers being each conjugated to a single DM, via an imine bond, on either position, such monomers herein referred to as Ml and M2:

2. monomers being each conjugated to a single DM, via an amine bond, on either position, such monomers herein referred to as M3 and M4:

3. monomers being each conjugated to two DMs, one via an imine bond and the other via an amine bond, such monomers herein referred to as M5 and M6:

4. monomers being each conjugated to two DMs, each via an amine bond, such monomers herein referred to as M7

5. monomers being each conjugated to two DMs, each via an imine bond, such monomers herein referred to as monomers M8.

The monomers are bonded to each other as in the original unoxidized polymer (polysaccharide), forming the polymer backbone. The backbone presented for each of the above monomers is merely exemplary and may vary depending on the polymer employed, the position of the monomer and any existing substitution.

Thus, the conjugate of the invention being composed of such or similar monomers (2 or more) is a combination of any or all of such exemplary monomers.

The "drug moiety" (designated DM in the above structures) which is associated (conjugated or bonded) with said polymer, e.g., polysaccharide (having two or more monomer or saccharide units), is an amphotericin B (AmB) derivative, capable of binding (forming a bond) to at least one C atom of the polymer, e.g., polysaccharide, via at least one of its N or O atom. The drug moiety may be conjugated to the polymer, or presented in the final conjugate as a salt, e.g., wherein at least one of the acidic hydrogens may be replaced by a cation, optionally selected from Na⁺, K⁺, Cs⁺, Mg⁺², Ca⁺² and others.

As known in the art, AmB is a cyclic compound charcahtarized by a macrocyclic lactone and a series of conjugated E double bonds, having the structure:

As used in the structure above and in any other structure provided herein, the doted lines indicate a single bond with a specific stereochemistry of the carbon atom to which it refers.

In some embodiments, the drug moiety is an AmB derivative selected from saponified AmB and reduced AmB. In further embdoeimnts, the conjugate of a polysaccharide, e.g., AG, and an AmB derivative(s) comprises also unmodified AmB (not being a saponified and/or reduced AmB derivative). It should be noted that in the conjugates of the invention the drug moieties are not unmodified AmB (of the above structure) alone.

As demonstarted, the different AmB derivatives may be prepared separatly and isolated prior to conjugation or prepared in situ from AmB under conditions which permit their formation and subsequent conjugation to the polymer. Under acidic or basic hydrolysis conditions, as under ceratin reductive conditions, the lactone ring of the AmB molecule may undergo hydrolysis (saponification) to afford the bifunctional acyclic system of the formula (Dl):

In some embodiments, the drug moiety is a saponified derivative having the structure Dl, which is conjugated to the polymer, e.g., via the N atom of the amine group (optionally deprotonated upon conjugation).

Similarly, under reductive conditions, a reduced form of AmB is obtained, having the general formula (II):

wherein one of the indicated dotted double bonds (between carbons 19-20, 21- 22, 23-24, 25-26, 27-28, 29-30 and 31-32 as indicated above) are reduced.

In some embodiments, the reduced form is selected from derivatives of general formula (II) having a saturated C-C single bond between carbons 19-20 or 21-22 or 23- 24 or 25-26 or 27-28 or 29-30 or 31-32. The drug moieties of the general formula (II) having one saturated bond at either of the above positions are designated as follows: between carbon atoms 19-20- designated D2;

between carbon atoms 21-22- designated D3;

between carbon atoms 23-24- designated D4;

between carbon atoms 25-26- designated D5;

between carbon atoms 27-28- designated D6;

between carbon atoms 29-30- designated D7; and

between carbon atoms 31-32- designated D8.

The reduced form is conjugated to the polymer via the N atom of the amine group (optionally deprotonated upon conjugation).

In some embodiments, the drug moiety associated with the polymer is selected amongst reduced derivatives of the formula (II), designated D2 and D8:

In some other embodiments, the conjugate is of a polymer, as defined, and a reduced-saponified derivative of the general formula (III):

wherein one of the indicated double bonds (between carbons 19-20, 21-22, 23- 24, 25-26, 27-28, 29-30 and 31-32 as indicated above) of the saponified derivatives are reduced.

In some embodiments, the saponified and reduced form is selected from a saponified derivative of general formula (III) having a saturated C-C single bond between carbons 19-20 or 21-22 or 23-24 or 25-26 or 27-28 or 29-30 or 31-32. The derivatives of general formula (III) having one saturated bond at either of the above positions are designated as follows:

between carbon atoms 19-20- designated D9;

between carbon atoms 21-22- designated D10;

between carbon atoms 23-24- designated Dll; between carbon atoms 25-26- designated D12;

between carbon atoms 27-28- designated D13;

between carbon atoms 29-30- designated D14; and

between carbon atoms 31-32- designated D15.

The reduced-saponified form is conjugated to the polymer via the N atom of the amine group (optionally deprotonated upon conjugation).

In some embodiments, the reduced-saponified form is selected from D9 and D15. In other embodiments, the reduced-saponified form is D9. In still other embodiments, the reduced-saponified form is D15.

The conjugate of the invention is composed of a polymer, e.g., a polysaccharide, and a plurality of drug moieties selected from Dl, D2, D3, D4, D5, D6, D7, D8, D9, D10, Dll, D12, D13, D14 and D15, wherein each drug moiety is conjugated to the polymer as defined herein. In some embodiments, the conjugate is of a polymer and a plurality of a single type of drug moiety selected from Dl, D2, D8, D9 and D15. In some embodiments, the single drug moiety is Dl, D2, D8, D9 or D15.

In some embodiments, the conjugate is of a polymer and a plurality of Dl and a plurality of at least one further drug moiety selected from D2, D8, D9 and D15.

In some embodiments, the conjugate is of a polymer and a plurality of D2 and a plurality of at least one further drug selected from Dl, D8, D9 and D15.

In some embodiments, the conjugate is of a polymer and a plurality of D8 and a plurality of at least one further drug moiety selected from Dl, D2, D9 and D15.

In some embodiments, the conjugate is of a polymer and a plurality of D9 and a plurality of at least one further drug moiety selected from Dl, D2, D8 and D15.

In some embodiments, the conjugate is of a polymer and a plurality of D15 and a plurality of at least one further drug moiety selected from Dl, D2, D8 and D9.

In some embodiments, the conjugate is of a polymer and a plurality of Dl, D8 and D15. In further embodiments, the conjugate is of a polymer and a plurality of Dl, a plurality of D8 and a plurality of D15, at any ratio. The polymer is, in some embodiments, a polysaccharide such as AG. In further embodiments, the polysaccharide is oxidized AG.

Thus, the invention provides a conjugate of oxidized AG, as disclosed above, and a plurality of drug moieties Dl, D8 and D15; each of said moieties being associated with the oxidized AG via the amine group of the AmB derivative (via imine, amine or amide bond).

The conjugate of oxidized AG and a plurality of drug moieties Dl, D8 and D15 is herein identified as Conjugate 1 of the invention.

For the sake of providing further clarification as to the association between the polysaccharide and the DM, a partial non-limiting structure of a conjugate such as Conjugate 1 of the invention is presented below. It should be understood that the structure is provided for the sake of clarity alone and should not be taken in any way to be the accurate full structure of the conjugate or any section thereof. For the sake of clarification alone, the association of drug moieties Dl, D8 and D15 is demonstrated to occur via amine bonds. Association of one or more of the drug moieties may be through imine bonds, or amide bonds. Some of the polysaccharide monomers are shown oxidized (to the methylene hydroxides) while others are shown unoxidized. Although the partial structure demonstrates association of the polymer with drug moieties Dl, D8 and D15, other drug moieties, as well as AmB, as disclosed herein, may also be associated. Additionally, while the three drug moieties are shown to occupy a polymer section of 7 units, the distribution of the specifically shown drug moieties over the full polymer may be different.



While association between the polymer and each of the drug moieties is typically by forming a C_poiymer-Ndrug covalent bond (imine, amine or amide), association of the drug moiety and the polymer via Cpdymer-Odrug covalent bonds is also possible.

In some embodiments, where oxidized polysaccharides are used as polymers, as explained above, the association between the polysaccharide and the drug moiety may be through chemical transformation of the aldehyde groups. To achieve a conjugate in accordance with the invention, not all aldehyde moieties must be converted by conjugation with a drug moiety. In some embodiments, only at least 1% of the aldehyde groups undergo imination/amination with the drug moieties, namely the polymer may have only 1% of its active sites (aldehyde groups of the oxidized unconjugated monomers) bonded to drug moieties. In other embodiments, at least about 1% of the aldehyde groups available for reaction with the amine group of the AmB derivatives are reacted. In further embodiments, at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% and so forth up to 100% of the aldehyde groups of the oxidized AG are reacted with the drug moieties.

The conjugates of the invention may be prepared, as demonstrated, by employing a direct synthetic approach which involves reacting the components under basic conditions or a multi-stage approach which involves initial preparation and isolation of the drug moieties, followed by conjugation thereof to the polymer.

Thus, the invention provides, in another of its aspects, a process for the preparation of a conjugate according to the invention, said process comprising contacting a polymer, as defined herein, and AmB under conditions permitting in situ formation of saponified AmB and reduced AmB forms and subsequent conjugation of the saponified and/or reduced forms to said polymer.

In some embodiments, the polymer is a polysaccharide, as herein defined.

In other embodiments, the polysaccharide is an oxidized polysaccharide, which in some further embodiments is oxidized AG.

In some embodiments, the process of this aspect of the invention comprises contacting AmB and a polymer in an aqueous medium under basic conditions to induce saponification and reduction of AmB in situ and conjugation of said saponification and reduction products to said polymer.

In some embodiments, the saponification product is Dl. In other embodiments, the reduction products are any one or more of D2 through D15. In further embodiments, the reduction products are D8 and D15.

In some embodiments, the saponification and reduction products are obtained at a pH between 10 and 12. In some embodiments, said pH is greater than 11. In other embodiments, said pH is between 10 and 11. In further embodiments, said pH is about 11.

In further embodiments, the reaction is carried out at about pH 11 for a period of time sufficient to allow conversion of at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of said AmB to its saponified and/or reduced derivatives.

In some embodiments, the period of time is 1, 4, 8, 20, 24, 48 or 72 hours. In some further embodiments, said period of time is at least 20 hours, in some embodiments, at least 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 hours. In further embodiments, said period is at least 50 hours. In additional embodiments, said period is between 20 and 72 hours, between 48 and 72 hours, or between 50 and 72 hours. In yet further embodiments, said period of time is 24 hours or 48 hours.

In some embodiments, the reaction is carried out at a temperature between 4°C and 37°C. In further embodiments, the temperature is 4°C, 20°C, 25°C or 37°C. In yet further embodiments, the temperature is 20°C, 2 PC, 23°C, 24°C or 25°C.

In another aspect, the invention provides an alternative approach for the preparation of a conjugate according to the invention. In this approach, the drug moieties Dl through D15 to be associated with the polymer are first prepared by contacting a solution of AmB under conditions permitting formation of saponified and/or reduced AmB derivatives as disclosed. Once formed, the mixture containing the derivatives or each of the separate derivatives, optionally isolated and purified, may be conjugated to the polymer under the conditions of the preceding process.

In another aspect of the invention, there is provided a use of a conjugate according to the present invention for the preparation of a composition. In some embodiments, the composition is a pharmaceutical composition.

Also contemplated is a use of a conjugate of the invention in a method of therapeutic treatment of a subject, human or non-human. In a further aspect of the present invention, there is provided a pharmaceutical composition comprising a conjugate, as disclosed herein. In some embodiments, the composition is for the treatment of a disease or disorder selected from fungal diseases, parasitic associated diseases and disorders and leishmaniasis (cutaneous leishmaniasis, mucosal leishmaniasis and/or visceral leishmaniasis).

The disease or disorder to be treated is selected from or is one or more associated with Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Candida species, Rhodotorula species, Cryptococcus species, Sporothrix schenckil, Mucor species, Rhizopus species, Rhizomucor species, Absidia species, Tnchosporon species, Fusarium species, Paecilomyces species, Phailophora species, Phialemonium species, Exophiala species, Scedosporium species, Neoscytalidium species, Acremonium species, Alternaria species, Malassezia species and Aspergillus species. Fungal infections which may be treated are selected from aspergillosis, cryptococcosis, North American blastomycosis, invasive and systemic candidiasis, coccidioidomycosis, paracoccidioidomycosis, histoplasmosis, fusariosis, invasive trichosporonosis, phaeohyphomycosis, sporotrichosis, zygomycosis (mucormycosis) due to susceptible species of these genera.

The pharmaceutical composition of the invention comprises at least one conjugate as the active ingredient. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent or excipient which may be in a liquid, solid or semi-solid state. While the pharmaceutical composition typically facilitates administration of the active ingredient to the subject, the treatemnt as disclosed herein may be ensued by the administartion of the conjugate alone, as a carrier-free formulation. Whether via the use of a pre-made formulation or as a carrier-free formulation, the conjugate formulation may be administered according to any one of a variety of techniques of aciministering known in the art including, but not limited to oral, intranasal, injection, aerosol, parenteral and topical administrations.

The choice of carrier will be determined in part by the particular form of the conjugate, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. The following formulations are merely exemplary and are in no way limiting.

Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the extract dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents and pharmacologically compatible carriers. Lozenge forms can comprise the active ingredient in a different flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels and the like containing, in addition to the conjugate, such carriers as are known in the art.

Formulations containing one or more conjugates which are suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. The conjugate may be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, such as polyethyleneglycol 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants. Oils, which may be used in parenteral formulations, include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxy-ethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-P-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts and mixtures thereof.

Suitable preservatives and buffers may be used in such formulations. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use.

The requirements for effective pharmaceutical carriers for injectible compositions are well known to those of ordinary skill in the art. See Pharmaceutics and Pharmacy Practice, J.B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4^th ed., pages 622-630 (1986).

The compositions of the invention, those defined as antifungal compositions or anti leishmaniasis compositions, as well as compositions for generic therapeutic use may comprise one or more of the conjugates or the invention, optionally with one or more diluent, excipient or carrier, as disclosed. The compositions of the invention may further comprise at least one additional active ingredient. Such active ingredient may be selected from an antifungal drug and antileishmania drug, and any combination thereof. In some embodiments, a composition of the invention comprises, in free (unbound) form, at least one of AmB and/or any reduced and/or saponified derivatives thereof, as defined hereinabove, namely any one or more drugs selected from Dl through D15, in free form.

In some embodiments, a composition comprises a conjugate according to the invention and free AmB.

In some embodiments, a composition comprises a conjugate of the invention and free (unbound) saponified AmB (Dl). In further embodiments, a composition comprises a conjugate of the invention and free (unbound) reduced AmB, as defined. The reduced AmB may be any one or more of the drugs herein designated D2 through D15.

The invention also provides compositions comprising free AmB and at least one free (unbound) AmB derivative selected from Dl, D2, D8, D9 and D15.

In another aspect of the invention there is provided a combination of at least one conjugate according to the invention and at least one conjugate of AmB as disclosed in Refs. [1] and/or [2].

In another aspect of the present invention, there is provided a method of treatment of a disease or disorder, as disclosed herein, in a subject, said method comprising administering to said subject an effective amount of at least one conjugate according to the invention or a pharmaceutical composition comprising thereof.

A conjugate used according to the invention and the pharmaceutical composition comprising it are aimed at treating at least one fungal disease or disorder or leishmaniasis. The term "treatment as used herein refers to the administering of a therapeutic effective amount of a conjugate or a composition comprising it which is effective to ameliorate undesired symptoms associated with such a disease or disorder, to prevent the manifestation of symptoms before they occur, to slow down the progression of the disease or disorder, slow down the deterioration of symptoms, to enhance the onset of remission period, slow down the irreversible damage caused in the chronic stage of the disease or disorder, to delay the onset of said stage, to lessen the severity or cure the disease or disorder, to improve survival rate or more rapid recovery, or to prevent the disease or disorder form occurring or a combination of two or more of the above.

The term "effective amount or any lingual variation thereof, refers generally to a therapeutic or prophylactic amount of a conjugate, alone or in the form of a formulation, which is, when administered to a subject, human or non-human, is sufficient to reduce, prevent, delay and/or inhibit the onset or progression or worsening of an amyloidosis-associated disease or disorder; to reduce, relieve, and/or alleviate the severity, frequency, duration, susceptibility or probability of one or more undesirable symptom or condition associated with the disease or disorder and/or to hasten the recovery from one or more symptoms associated with the disease or disorder. The effective amount is typically determined according to methods known in the art.

In another aspect of the present invention, there is provided a compound herein designated D2, D3, D4, D5, D6, D7, D8, D9, D10, Dll, D12, D13, D14 or D15.

Also provided is a composition comprising a compound herein designated D2, D3, D4, D5, D6, D7, D8, D9, D10, Dll, D12, D13, D14 or D15. In some embodiments, the composition is a pharmaceutical composition, e.g., for use in a method of treatment of a fungal disease.

The invention also provides a kit or a commercial package comprising at least one conjugate of the invention contained in a vial or a receptacle of the kit, optionally at least one reconstituting fluid and instructions for use.

It should be noted that where various embodiments are described by using a given range, the range is given as such merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements. BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Figs. 1A-C are HPLC chromatograms of: Fig. 1A- untreated AmB; Fig. 1B- AmB treated under conditions employed in US patent no. 5,567,685; Fig. 1C- AmB treated under conditions of the present invention, showing the conversion to the AmB derivatives according to the invention.

Fig. 2 is the HPLC chromatogram of Dl.

Fig. 3 is the HPLC chromatogram of a reduced derivative according to the invention.

Fig. 4 is the MS-ESI spectrum of a reduced derivative according to the invention.

Fig. 5 is the HPLC chromatogram of a reduced and saponified derivative according to the invention.

Fig. 6 is the MS-ESI spectrum of a reduced and saponified derivative according to the invention.

Fig. 7 presents the results of a 28-day chronic dose toxicity study in LEW/S rats administered with the AG conjugate of the invention.

Fig. 8 presents the results of a study aimed at determining the survival of mice infected with C. albicans and treated with a conjugate of the invention.

DETAILED DESCRIPTION OF EMBODIMENTS

As disclosed above, in an effort to reduce the toxicity of AmB, including conjugates of AmB and various polymers, e.g., polysaccharide, the inventors have developed a unique conjugate which is based on AmB derivatives previously not conjugated.

Thus far, studies by the scientific community have been aimed at finding ways to increase water-solubility of AmB and also at reducing its toxicity to the subject being treated. These studies have concentrated only at conjugating intact unmodified AmB to water-soluble carriers such as polymers and polysaccharides. Acting under the assumption that the activity of the drug stems from hydrophobic physiochemical interactions with the ergosterol in the cell membrane of the fungal organism, the activity of AmB derivatives such as those disclosed herein has never been studied.

The conjugates of the invention, as will be further demonstrated hereinbelow, have exhibited greatly improved chemical and biological characteristics, even in comparison to AmB conjugates of the same polysaccharide. Clearly, such a successful use of AmB derivatives, which do not maintain the structure of the parent AmB, is clearly unexpected.

It is to be understood that the invention is not necessarily limited in any of its aspects to the details set forth in the following description or exemplified by the Examples, as these are provided only as non-limiting examples of the broad aspects of the invention. The invention is capable of other embodiments or of being practiced or carried out in various ways.

For the sake of demonstrating how the invention may be worked and utilized, a conjugate of oxidized arabinogalactan (AG) and the AmB derivatives is exemplified. As a person skilled in the art would appreciate, conjugates of other polymers or polysaccharides (oxidized or not) may be similarly prepared, mutatis mutandis.

PART A- Conjugates of the Invention

Example 1: Preparation of a conjugate

A. Method I- In situ preparation of AmB derivatives and conjugation Arabinogalactan (AG) oxidation

In a typical synthesis, AG (25 g, 156.25 mmol relative to a monosaccharide unit) was dissolved in 250 ml of double deionized water (DDW). Potassium periodate (35.93 g, 156.25 mmol) or sodium periodate (33.44g, 156.34 mmol) was added to the solution and stirred at room temperature in the dark for 2 h. The oxidized AG formed was purified from iodate (103), unreacted periodate (104^") and reaction by-products by either passing it through an anion-exchange column filled with Dowex-lx4 in the acetate form (Sigma, St. Louis, Mo.) or by adsorption to Dowex-lx4 resin. The clear solution containing oxidized AG was purified from acetic acid and low molecular weight polymers by ultrafiltration, employing "Minisette Casettes" with 5 kDa molecular weight cutoff (MWCO) and finally freeze-dried. The averaged yield over a number of preparations was 75±5%. The purified oxidized AG, at a concentration of 5.0 mg/ml, exhibited a degree of oxidation in the range of 62±3%, as determined by the hydroxyl amine hydrochloride method.

Drug conjugation to AG

Alternative 1:

In a typical experiment, to a 1,500 mL of 0.1 M borate buffer solution (pH 11±0.1), 15 g (93.75 mmol saccharide units) of purified oxidized AG were added (resulting in an oxidized AG solution of 10 mg/ml concentration). After full dissolution, 3.75 g of AmB (950 U/mg; Alpharma, Copenhagen, Denmark) were added (resulting in AmB concentration of 2.5 mg/ml). The solution became unclear and yellowish in color. The pH was adjusted several times during the first hour of the reaction stabilizing it on pH 11 ±0.1 until a clear yellow solution was attained. The solution was stirred at room temperature (between 20-25°C) in a light-protected container for 48 h (between 48 and 52 h) with pH adjustment during that period. The solution was next treated with 7.08 g (0.1875 mol) of sodium borohydride over a period of 18-22 h. The amine conjugate was purified by ultrafiltration as described below using ~12L of DDW followed by lyophilization.

Alternative 2:

In a typical scaling up synthesis procedure, purified oxidized AG was added to a 0.1 M borate buffer solution (pH 11±0.1) to reach a concentration of 40g/L. After full dissolution, AmB (950 U/mg; Alpharma, Copenhagen, Denmark) was added at a 1:4 w/w ratio to the AG content (i.e., a concentration of lOg/L AmB for 40g/L AG). The solution became unclear and yellowish in color. The pH was adjusted several times during the first hour of the reaction stabilizing it on pH 11±0.1 until a clear yellow solution was attained. The solution was stirred at room temperature between 20-25°C or at 37°C in a light-protected container for 24 or 48 h with periodic pH adjustment. The solution was next treated with sodium borohydride (added at a molar ratio of 2:1 relative to the sugar groups of the AG - over a period of 1 hour to 24 hours (e.g., 1 hour, 4 hours, 8 hours and 20 hours), at room temperature between 20-25°C or at 4°C. The amine conjugate was purified by ultrafiltration as described below using about 9- 12L of DDW followed by lyophilization. B. Method II- Preparing AmB derivatives first, followed by conjugation

In this approach, AmB was reacted under different reaction conditions (e.g., pH 11, at 4°C or room temperature, for a period of between 1 and 72 hours, between 1 and 24 hours, between 48 and 72 hours, specifically, for a period of lhour, 4 hours, 8 hours, 20 hours, 24 hours, 48 hours or 72 hours) to afford the individual AmB derivatives Dl- D6, as recited herein. The existence of these derivatives in the reaction soup existing prior to the conjugatiuon according to Method I above has been demonstrated (results not shown). It is noted that short periods of times such as 1 hour and low temperature such as 4°C were sufficient to produce Amb derivatives as confirmed by RP-HPLC and ESI-MS analysis (data not shown).

The results revealed that the AmB strucutre has undergone saponification and/or reduction during the conjugation process at the noted basic pH providing the acylic derivative and the reduced forms which have not been achieved in the past.

As Figs. 1A-C indicate, treating of the AmB under the conditions of the invention, namely at pH 11 , at room temperature and for a period of between 48 and 72 hours, resulted in the complete transformation of the AmB into its derivatives (Fig. 1C). Under the conditions of US patent no. 5,567,685 [1] no such transformation was observed (Fig. IB). Similarly, no transformation of AmB to its derivatives was observed under the conditions employed in [3].

Example 2: Preparation of individual AmB derivatives

Hydrolysis of AmB— Saponified acyclic derivative (Dl):

1 g of AmB was dissolved in 100 mL of 0.1 N NaOH (10 mg/mL). The solution was protected from light and kept under continuous stirring for 24h at 50°C. After the reaction was completed, the pH of the solution was reduced to 5-6 with 3N HCl to allow precipitation of the product. The precipitate was centrifuged and washed with DDW several times, followed by freeze drying.

The HPLC chromatogram of Dl is shown in Fig. 2.

Mass spectrum (MS-ESI) of a sample of Dl showed a molecular ion at 942.49 e^". The molecular weight was further confirmed by HR-MS (942.50346 e^"). Reduction of AmB - Reduced AmB (D8):

1 g of AmB was dissolved in 100 mL of a Borate Buffer 0.1 N pH 11 (10 mg/mL). The solution was protected from light and kept under continuous stirring, checking the pH to be stable at 11± 0.1 during the first hour. 2 molar excess (1.9 g) of NaBH-v were added to the solution. The solution was kept under continuous stirring for 24 h at 4°C. After the reaction was completed, the pH of the solution was reduced to 5-6 with 3N HC1 to allow precipitation of the product. The precipitate was centrifuged and washed with DDW several times, followed by freeze drying.

The HPLC chromatogram of D8 is shown in Fig. 3. The MS spectrum of D8 is shown in Fig. 4. The molecular weight of 926.51 e was confirmed by HR-MS (926.50271 e ).

Hydrolysis and reduction of AmB - Reduced and saponified acyclic derivative (D15):

1 g of AmB was dissolved in 400 mL of a Borate Buffer 0.1 N pHl l (2.5mg/mL). The solution was protected from light and kept under continuous stirring, checking the pH to be stable at 11± 0.1 during the first hour. The solution was kept protected from light and under continuous stirring for 48h. After this period, 2 molar excess (1.9 g) of NaB¾ were added to the solution. The solution was kept under continuous stirring for 20h at R.T. After the reaction was completed, the pH of the solution was reduced to 5-6 with 3N HC1 to allow precipitation of the product. The precipitate was centrifuged and washed with DDW several times, followed by freeze drying.

The HPLC chromatogram of D15 is shown in Fig. 5. The MS spectrum of D15 is shown in Fig. 6.

It should be pointed out at this stage that a study into the biological activity of each of the AmB derivatives Dl, D8 and D15, indicated that while each of the derivatives had some antifungal activity, their antifungal activity in comparison with AmB was reduced: each was less active and less hemolytic in comparison to AmB.

HPLC analysis The different AmB derivatives were determined by an HPLC (Hewlett-Packard, Waldbronn, Germany) system consisting of an HP 1100 pump, an HP 1050 auto- sampler with 200 μΐ loop, HP 1050 UV detector, coupled with HP ChemStation data analysis program (Agilent Technologies, Palo Alto, USA). The separation was performed on C18 RP analytical columns (LichroCartl 250-, Lichrospherl 100, 5 μπι), which was protected with a CI 8 RP guard column (LichroCartl 4-4, Lichrospherl 100, 5 1m).

AmB derivatives were determined employing an isocratic mobile phase composed of DDW: Acetonitrile (ACN): Acetic Acid (27:70:3) at a flow rate of 1.8 ml/min and a run time of 18 minutes, with UV detection at 406 nm.

The samples were prepared in DMSO at a concentration of 10 mg/ml and then diluted 1:40 with the mobile phase.

Mass spectrometry

Mass spectra were obtained with Orbi-trap (Thermo Finnigen). Data analysis was performed using bio works 3.3 package and database searches were performed against the NCBInr database using Mascot package (Matrix Science, England). Samples were dissolved in ACN: DDW solution.

Example 3: Conjugation of AmB derivatives with Arabinogalactan

The conjugation of oxidized AG with the AmB derivatives was conducted under the conditions disclosed above in an aquoues medium, at room temperature. The properties of the resultant conjugate were evaluate, and analyzed by NMR and HPLC- RP and have been found indentical to those of a conjugate obtained according to Method I.

Purification of the conjugate by cross flow ultra filtration

Purification of the prepared conjugation by ultrafiltration was completed from 1.5 L feed volume, with a concentration step combined with a diafiltration step. The temperature was kept at 22°C. The membrane pressure at the module inlet was 2 bar, the module outlet and the filtrate sides were not under pressure (0 bar as indicated by the manometer). The permeate flow returned to the feed container. The filtrate flow rate was 6 L /hour. Every 30 min., 3 L of DDW (double deionized water) were added. The ultra filtration process continued for ~2 h, washing the conjugate solution with total volume of -12 L of water for injection (WFI). The pH was monitored, starting from 11 and reducing it to a 7-7.5 value.

At the end of the experiment, the ultra filtration membranes were regenerated by flushing with pure DDW over a period of 30 min, sodium hydroxide IN for a period of 30 min, and finally with sodium hydroxide 0.1 N for a further period of 30 min. The membrane was stored in sodium hydroxide 0.1 N.

Example 4: Characterization of AG conjugates according to the invention

A. Chemical characterization

Ή-NMR of the conjugate, namely that of AG and the AmB derivatives disclosed herein, prepared according to either of the two methods of the invention, provided an indication as to the presence of the oxidized polysaccharide as well as to the presence of AmB derivatives, thus confirming the presence of the conjugate.

HPLC analysis of the conjugate provided evidence as to the existence of the conjugate as well as to small amounts of free amphotericin B reduced and saponified AmB derivatives. These results strongly support an earlier observation that some of the active AmB derivatives may be present in the conjugate as free entities. This is expected where imine bonds associate the drug moieties to the polysaccharide.

Molecular weight determination

The molecular weight (MW) of the conjugate, was determined by a high- pressure gel permeation chromatography (GPC) system, consisting of a Spectra Physics P1000 pump (Darmstadt, Germany) with a UV detector (Applied Bioscience 759A Absorbency UV detector) at 407 nm, or a refractive index (RI) detector, a Rheodyne (Coatati, CA) injection valve with a 20 μΐ loop, a Spectra Physics Data Jet integrator and a Breeze computer analyzer. Samples were eluted with 0.05 M sodium nitrate in DDW through a Shodex (SB-803HQ) column at a flow rate of 1 ml/min.

The molecular weights of the eluted samples were estimated with pullulan standards in the range of 1,300-112,000 Da. In fact, the molecular weight of the conjugate was determined to be between 22,000 and 27,000 Da. In some cases, the molecular weight was measured at between 22,000-27,000 Da. The averaged molecular weight was 26,200 Da.

Determination of degree of oxidation

Oxidized AG (0.1 g, 0.625 mmol) and AmB-AG conjugates (O.lg 0.32 mmol) were dissolved in 25 ml hydroxylamine hydrochloride solutions 0.25M at pH 4.0. The solutions were stirred for 3 h at room temperature and then titrated with a 0.1 M NaOH standard solution. The titration end point was calculated as known in the art.

While the oxidized AG demonstrated about 60% oxidation (aldehyde groups), after conjugation, no detectable levels of aldehydes were measured.

Periodate (IO₄ ) and iodate (IO₃ ) concentration determination in oxidized AG by iodometric method

Oxidized AG (20 mg, 0.125 mmol) was dissolved in 5 mL of DDW. The solution was shaken for 2 h at 37°C until full dissolution. 50 \ih of H₂S0₄ 0.01 N were added to pH 1-3, at which point 80 mg of potassium iodide (KI) were added. The solution was stirred for 15 min at room temperature. No color change (yellow-brown) was observed. In order to determinate traces of periodate (I0₄ ^") and iodate (I0₃ ^"), 2 mL of a starch solution (0.1 g per 20 mL boiled water) were added to the acidic AG solution. The solution was stirred for 15 min at room temperature. No color change (pale blue-blue) was observed. KI0₄ used as a control solution gave yellow-brown color.

These results indicate that the conjugate of the invention was substantially free of contaminants such as the oxidation reagents and ions associated therewith.

Solubility

The conjugates of the invention have exhibited high solubility in DDW, as well as in saline, both yielding clear solutions. In fact, and as will be further demonstrated below, the conjugates of the invention are capable of dissolving in water 8 times more the active material as compared with the conjugates of the art, in particular those of Refs. [1] and [3].

B. Biological characterization Antifungal activity according to broth dilution method

Candida albicans strain ATCC 90028 (The American Type Culture Collection, Manassas, Va) was used for susceptibility testing. Minimal inhibitory concentration (MIC) was determined by the broth microdilution method according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) formerly known as National Committee for Clinical Laboratory Standards (NCCLS) M27-A2 and M27-A3. This method involves the use of small volumes of broth dispensed in sterile, disposable, multiwell microtitation plates (96 U-shaped wells). Each well contained 0.1 ml of broth with the serially diluted drug. Briefly, twofold serial dilutions of drugs from stock solutions were prepared in RPMI 1640 broth medium (Lot 91k83061; Sigma, St. Louis, Mo.) buffered to a final pH of 7.0 with 0.165 M morpholine propanesulfonic acid (MOPS; 99.5%, Lot 091k5421, Sigma) and 1 M NaOH (Sigma) and were sterilized by filtration. A stock solution of 5 mg/ml was prepared in dimethyl sulfoxide (DMSO, 99.5% GC, lot 421672/1, Sigma) for free AmB or in water for conjugates. The final drug concentrations in the test ranged from 16 to 0.031 mg/L in a final volume of 0.1 ml.

The inoculum was prepared as follows:

(1) C. albicans was subcultured from sterile vials onto Sabouraud dextrose agar (Novamed, Jerusalem, Israel) and passaged to ensure purity and viability. The incubation temperature throughout was 35°C.

(2) The inoculum was prepared by picking five colonies of ~1 mm in diameter from a 24-hourold culture of C. albicans. The colonies were suspended in 5 mL of sterile 0.145-mol/L saline (8.5 g/L NaCl, Sigma).

(3) The resulting suspension was diluted after counting with RPMI 1640 broth medium, to 2 x 10³ cells per mL. The (twofold) inoculum was diluted 1:1 when the wells were inoculated and the desired final inoculum size was achieved (1 x 10 cells/mL).

Two wells containing drug-free medium and inoculum were used as controls. The inoculated plates were incubated at 35°C for 24h-48h. The growth in each well was then estimated visually. The MIC-0 was defined as the lowest drug concentration that resulted in complete inhibition of visible growth.

In vitro toxicity study (hemolysis) Sheep red blood cells (SRBCs) were suspended in phosphate-buffered saline (PBS pH 7.4, Oxoid LTD, Hampshire, UK; 5% [vol/vol]) PBS; 5% [v/v]) and were washed twice in the same buffer by centrifugation (1,800 xg for 10 min). The hemolysis reaction was conducted in glass tubes containing 0.1 ml of the serially diluted drug and 0.9 ml of SRBCs. The tubes were centrifuged after 1 h of incubation at 37°C in a water bath and the results were recorded visually.

Acute Toxicity in animal model

Male albino ICR mice weighing ~30 g each were injected through the tail vein with various doses of a control AmB (Fungizone, Squibb, Middlesex, UK) and a conjugate according to the invention. Each dosage form was administered intravenously as single bolus injections of 0.1 ml of the same dose every 10 min to a group of 10 mice until death was observed. The control AmB formulation as well as the formulation containing a conjugate of the invention were prepared in 5% dextrose (Fungizone) or saline (conjugate) and were filter-sterilized prior to injection through a sterile 0.2-mm- pore-size cellulose acetate filter (Schleicher & Schuell, Dassel, Germany). The survival of mice that received the maximal tolerated dose (MTD) was monitored for 8 days.

Repeated Toxicity in animal model

Male LEW/S rats weighting -150 g were injected through the tail vein with three doses (10, 20 and 30 mg/kg) of a control and conjugate formulations. Each dosage form was administered intravenously as single bolus injections of 0.1 ml of the same dose every 10 min to a group of 5 rats. The dose was given continuously for 28 days.

The control AmB and conjugate formulations were prepared in saline and were filter-sterilized prior to injection through a sterile 0.2-mm-pore-size cellulose acetate filter (Schleicher & Schuell, Dassel, Germany). The survival of rats was monitored for additional 7 days.

Systemic candidiasis: Therapeutic efficacy study

For the efficacy studies, C. albicans ATCC 90028 was used to induce systemic murine candidiasis. Yeast inocula were injected into the tail veins of male albino BALB/cICR mice (weight, 20 30 ± 3 g) by administration of a single bolus of a 0.2-ml suspension in PBS. The inocula range was from 10⁴ to 10⁶ yeast cells per mouse from a 24-h culture for C. albicans on SDA at 30°C. With these inocula systemic infections are regularly produced in mice and they cause total killing within 10 to 20 days. The appropriate inoculum for each experiment was experimentally determined. The yeast concentration was determined by counting with a hemacytometer. The viable count was measured as the number of CFU on SDA plates after 24 to 48 h of incubation at 30°C. In a typical experiment 5 ^χ 10⁴ yeast cells per mouse was used.

Infected mice were treated with a control AmB-DOC (Deoxycholate) formulation (Fungizone, Squibb, Middlesex, UK) and a conjugate of the invention at various doses. Ten mice were used for each treatment and maintained in separate cages. The treatment started 24 h after the initiation of the infection by intravenous injection of a daily single bolus (0.2 ml) of the conjugate for 5 consecutive days. In addition, a control group of 10 infected mice was treated with dextrose 5% instead of the conjugate. The number of surviving animals in each group was recorded daily over a period of 30 days. The experiment included 40 mice - 10 mice in control group, 10 mice Fungizone-treated and 20 mice in a conjugate treatment group with 2 various doses.

The results of the biological tests are presented in Part B herein next.

PART B- Improved activity in comparison to known AmB conjugates

In order to asses the biological activity and toxicity of the conjugates of the invention and in order to determine whether the conjugates of the invention are better alternatives to AmB or any one of its known conjugates in, e.g., the treatment of fungal diseases and disorders, an extensive comparative study comparing the conjugates of the invention to the AG-AmB conjugates of [1] has been carried out and is summarized below. The AG-AmB conjugate of [1] is herein reffered to as the "1996 conjugate".

Differences in the preparation

As discussed hereinabove and demonstrated e.g., in Figs. 1A-C, the process employed for the manufacture of the 1996 conjugate resulted in the conjugation of AmB and not in the conjugation of such AmB derivatives such as Dl, D8 and D15, as disclosed herein.

The process employed for the preparation of the conjugates reported in [3] also differed greatly. While pH 11 was also used, carrying out the conjugation at a relatively high temperature for a period of time did not permit initial transformation of the AmB in situ, but rather hastened the conjugation process. However, once the hydrolysis- reductive process was allowed to occur first, at a pH between 10 and 12, and at room temperature, conjugation with the AmB derivatives, now in the embryonic soup, could take place.

Table 1 below summarizes the differences between the process of the invention and the process leading to the formation of the 1996 conjugate. For the sake of clarity, the process for the preparation of the 1996 conjugate is reproduced herein.

The 1996 process: Arabinogalactan Oxidation

Arabinogalactan (AG, 25 g, 156.25 mmol relative to a monosaccharide unit) was dissolved in 500 mL of double deionized water (DDW) in a concentration of 50 mg/mL. To the clear solution, potassium periodate (KI0₄, 31.9 g, 138.4 mmol) was added and the temperature was maintained below 30°C. The reaction mixture was stirred at room temperature for 2 hours protected from light. The obtained oxidized AG was purified from traces of iodate (I0₃ ^"), unreacted periodate (I0₄ ^~) and by products by passing the oxidized AG through an anion exchange column filled with Dowex 1x4 in the acetate form. The solution of the oxidized AG was loaded into Dowex column which color changed from light to dark brown as the product passed through it.

Purified AG exhibited a degree of oxidation of 42% as determined by the hydroxyl amine hydrochloride method. The yield was 19 g, calculated to overall 76% yield.

The 1996 process: AmB-AG conjugation

Fractions containing oxidized product were collected (760 mL) and the volume of the clear solution with oxidized AG was reduced (by lyophilization) to 290 mL. DDW was added (180 mL) to a final concentration of 40 mg/mL. To 375 mL of the oxidized AG solution (15 g, 93.75 mmol relative to the monosaccharide units) 375 mL of 0.2 M borate buffer solution (pH 9±0.1) were added and the solution was allowed to stir at 37°C. To the clear solution 3.75 g AmB (950 U/mg; Alpharma, Copenhagen, Denmark) were added (20% w/w ratio), with a final concentration of 5 mg/mL of AmB in the solution. The mixture became unclear and was stirred at 37°C protected from light for a period of 16 h. The pH of the reaction solution was maintained at 9 during the reaction. At the end of the reaction the solution was still not clear. After 16 h, an unclear yellow solution was obtained. The temperature of the solution was reduced to 4°C. In order to obtain a more stable amine derivative (reduced AmB-AG), 2.97 g (78.5 mmol) of sodium borohydride were added to the conjugation reaction (twice the amount of the aldehyde present in the polymer, calculated by the titration test) which continued for another 30 min at 4°C while constant stirring. The amine conjugate was purified by dialysis through a 12,000 molecular weight cutoff dialysis tube against deionized water (5L x 4) for 30 h at 4°C. The unclear solution was then centrifuged and the supernatant (which was still not clear after centrifugation) was freeze-dried and stored at 4°C in the dry form.

Following oxidation of the AG according to the 1996 process, purification involves the use of Dowex resin, which implies that small impurities may be present in the oxidized product prior to conjugation, as evidenced mostly by the presence of small peaks in the GPC-RI spectrum of the material. In the process of the invention, the oxidized product was purified by ultra filtration to a product of high purity which is only then used for conjugation.

In the 1996 process, the pH used for the conjugation was 9. At this pH, maintaining a clear solution, dissolution of high AmB concentrations was not possible. In contrast, the process of the present invention, being carried out at a higher pH of between 10 and 12, typically at pH 11, and at a lower reaction temperature, permitted dissolution of larger quantities of AmB which seems to have been possible only upon transformation to its more soluble derivatives, namely the saponification and the reduction products, prior to the conjugation to the polysaccharide.

Table 2 presents a comparison of a select group of physical characteristics of the conjugate produced according to the 1996 protocol and the conjugate of the present invention.

Table 2: The physical attributes of the conjugate of the invention and the 1996 conjugate. The solubility of the compound was 140 mg/ml in water. The solution obtained was not clear, but no solids were observed in the flask also. The solution in a concentration of 10 mg/ml still remained unclear. Solution is clear at the highest concentrations. ³Analyzed with Size Exclusion Shodex SB803 HQ column, using sodium nitrate 0.05 M as eluent. Molecular weight calibration curve was constructed based on pullulans standards. Detection- Refractive Index. Mw - weight average molecular weight, Mn - number average molecular weight, P - polydispersity (Mw/Mn). ⁴ Analyzed with C₁₈ reverse phase column, using gradient eluent ACN:Sulfate Buffer pH 5.0. Detection UV at 406 nm.

As Table 2 indicates even though both lyophilized conjugates contained AmB or derivatives thereof, as noted by the yellow color of the products, it could be easily observed that the color of the 1996 product was to a greater extent lighter in color than that of the conjugate of the invention, providing an indication to the lower load of AmB in the 1996 product. In other words, in the conjugate of the invention a greater load of AmB-derivatives could be assumed. This greater load was possible not only due to the better solubilization of the drug moieties but also the more efficient conjugation process. This observation was confirmed also by spectroscopic analysis of both conjugates.

As stated, the conjugate of the invention showed higher solubility in water as well as in saline, forming in both media clear solutions. The solubility of the 1996 conjugate was much poorer. The drug moieties employed in the conjugate of the invention were 8 times more soluble as measured in an aqueous medium as compared to the solubility of AmB itself, in the 1996 conjugate.

The molecular weights of the two conjugates are similar, differing only by about 5-6 kDa.

Although both conjugates were purified to remove low molecular weight materials, about 0.5% of free AmB (or derivatives) could still be detected in the conjugates. The calculations of AG-AmB conjugate treatment dose were based on drug amounts (g). 100 mg of AG-AmB conjugate prepared according to the invention contained 19.5 mg of the AmB derivatives (associated with the polysaccharide), while 0.5 mg of AmB was not conjugated. On the contrary, only 2.5 mg of the conjugated AmB was detected in 100 mg of the conjugate prepared by 1996 process. 0.5 mg of the free, unassociated AmB was still found in the 1996 conjugate. Therefore, larger quantities of the AG-AmB conjugate prepared by the 1996 process would be required to achieve the same AmB dose effect in comparison with a conjugate of the invention. Therefore, use of the 1996 process would produce conjugates rich in unbound drug per dose. For example, 20 mg portion of the drug moieties in the conjugate of the invention contained -0.4 mg (2% based on total AmB) of the unbound drug, while the same amount in the 1996 conjugate contained ~ 3.4 mg (17% based on total AmB) of unconjugated AmB. This would expose patients to unnecessary native AmB drug with its inherent toxicity.

The biological activity of the conjugates was evaluated by the susceptibility (MIC) and toxicity tests (hemolysis and MTD) and the therapeutic efficacy study.

The results presented in Table 3 indicate that both the 1996 conjugate and the conjugate of the invention were active in vitro against Candida albicans as demonstrated by the susceptibility test. The 1996 conjugate exhibited lower MIC values because of its increased toxicity.

The in vitro toxicity, as measured by the hemolytic effect of a conjugate according to the invention, was much lower than that of the 1996 conjugate (hemolysis at a concentration higher than 10 mg/ml compared to a concentration lower than 1.5 mg/ml, respectively). Both conjugates exhibited reduced toxicity in comparison to free AmB (10 μg/mL), however, the toxicity of the conjugate of the invention was the lowest.

The results of the in vivo studies attest to a profound difference between the conjugates. The acute toxicity test confirmed that for the 1996 conjugate the maximal tolerated dose (MTD) was 20 mg/Kg as compared to 60 mg/Kg for the conjugate of the invention, which means- a 3-fold lower acute toxicity in the conjugate of the invention and 15- fold lower toxicity when compared to the commercial AmB - Fungizone.

The chronic toxicity studies confirmed these results. When the conjugate of the invention was injected in doses of 10, 20 and 30 mg/Kg/day to LEW/S rats during 28 days (Fig. 7), the survival population was of 80% for the highest dose (30 mg/Kg/day) and 100% for the lower doses (10 and 20 mg/Kg/day). The same experiment performed with the commercial AmB showed a similar survival at the 1 mg/Kg/day dose. The therapeutic efficacy of the conjugate of the invention was further studied in a murine candidiasis model. The results indicate that 100% of the mice treated with the conjugate of the invention at a concentration of 5 mg/kg/day survived the whole period of the experiment (30 days), while the control mice, which were treated with 5% dextrose, died within 17 days. The survival rate of mice treated with Fungizone or the 1996 conjugate at a concentration of 1 mg/kg/day was about 50%. The results are summarized in the Fig 8.

The conjugation of the AmB derivatives as disclosed herein to a polysaccharide such as AG dramatically altered the drug distribution by converting the drug moieties from drugs having a very extensive disposition and high clearance (CI) to a conjugate with a relatively small volume of distribution (Vss) and very low CI (Table 4). The steady state volume of distribution and total body clearance were respectively 20 and 10-fold lower than the corresponding values of the parent drugs. Consequently, the area under the curve (AUC) of the conjugate was significantly higher than that of the parent drugs, implying high drug concentration in the central compartment.

Table 4: AmB and AmB-AG conjugate model-independent pharmacokinetic analysis following IV administration of Fungizone® (1 mg/kg) and a conjugate (20 mg/kg) according to the invention, respectively.

Comparative Toxicities

The Conjugate of the invention:

Mice: Male albino ICR mice, weighing ~ 30g (5 weeks old).

The mice were divided randomly into 5 groups, 4 animals in each group in a separate cage.

Dosage Form: The dosage forms contained 20% w/w of the conjugates of the invention.

The formulations were prepared in sterile saline solution and were sterilized prior to injection by filter through a sterile 0.2^m-pore-size cellulose acetate filter (Schleicher & Schuell, Dassel, Germany). The dosage forms were blindly injected to the animals. Each group of 4 mice received a different dose (increasing dose) of the conjugated drug.

Acute toxicity study: Each dosage form was administered intravenously through the tail vein of the mouse, as single bolus injections of 0.2 ml of the same dose with intervals of 10 min between the injections, during that period the behavior of the mice was examined. The survival of mice and the assessment of gross toxicity were monitored through daily observation of physical condition and changes in body weight for 8 days post injections. At the 8^th day the livers and kidneys of the survived mice were extracted and preserved in formalin for histopathological examination if required.

The 1996 Conjugate:

Mice: ICR male, weight ~ 30g.

The mice were divided randomly into 6 groups, 4 animals in each group in a separate cage.

Dosage form: The dosage forms contained AmB.

Each group received a different treatment (increasing dose).

The volume of the administrated dose was 0.2 ml.

Acute toxicity study: The acute toxicity was evaluated by intravenously (IV) injection through the tail vein of ICR male mice. The dosage form was administered intravenously as single bolus injection of 0.2 ml, with intervals of 10 min between the injections, during that period the behavior of the mice was examined (Table 5). The survival of mice and the assessment of gross toxicity were monitored through daily observation of physical condition and changes in body weight for 8 days post injections. At the 8^th day the livers and kidneys of the survived mice were extracted and preserved in formalin for histopathological examination if required.

As the results presented in Table 5 indicate, the maximal tolerated dose (MTD) determined for the conjugate of the art was quite lower than that of the conjugate of the invention, attesting to the reduced toxicity associated with the new conjugate.

The in vitro antifungal activities of AmB- AG conjugates against Candida albicans were determined using the broth microdilution method, according to the CLSI (Clinical Laboratories Standard Institute) recommendations. This method involveed the use of small volumes of broth dispensed in sterile plastic 96 U-shaped wells microtitration trays. Each well contained 0.1 ml of broth with the serially diluted drug. Briefly, twofold serial dilutions of drugs from stock solutions were prepared in RPMI 1640 broth medium (Lot 125K3551; Sigma, St. Louis, Mo.) buffered to a final pH of 7.0 with 0.165 M morpholine propanesulfonic acid (MOPS; 99.5% Lot 091K5421 Sigma) and 1 M NaOH (Sigma) and were sterilized by filtration through 0.2^m-pore- size cellulose acetate filter (Schleicher & Schuell, Dassel, Germany).

A stock solution of 5 mg free AmB (Alpharma; Lot A1960561) was prepared in 1 ml dimethyl sulfoxide (DMSO, Fluka, 99.5% GC, lot 421672/1) to yield 5 mg/ml AMB in DMSO. The conjugates were diluted in DDW (5 mg AmB in 5 ml DDW, results in 1 mg/ml). Then, further dilutions to 50 μg/ml were done in RPMI for the free AmB and conjugates and further serial dilutions in RPMI, resulting in final drug concentrations that ranged from 25 to 0.003 μg/ml in a final volume of 0.1 ml.

Candida albicans ATCC strain number 90028 (The American Type Culture Collection, Manassas, Va) was used as a reference strain for the susceptibility testing The yeast inoculum was prepared from a 24-h culture of on Sabouraud dextrose agar (SDA) plates (Novamed, Jerusalem, Israel) and tested to ensure purity and viability. Several colonies were immersed in saline solution to yield a yeast suspension of 5xl0⁶ yeasts per ml, and a further dilution in RPMI 1640 broth medium was done to yield a final inoculum concentration of 2x10³ yeast cells per ml, as measured by counting with a hemacytometer.

The microdilution wells, which contained 0.1 ml of the serially diluted drug, were inoculated with 0.1 ml of the resulting suspension. The final inoculum concentrati *on after dilution with the drug suspensi *on was 103 yeast cells per ml. Two wells containing drug-free medium and inoculum were used as controls. The inoculated plates were incubated at 35°C for 24-48 h. The growth in each well was then estimated visually. The MIC was defined as the lowest drug concentration that resulted in complete inhibition of visible growth.

The minimum inhibitory concentration (MIC) values of two conjugates: Conjugate 1 of the invention and the 1996 conjugate- as well as of the free-AmB are summarized in Table 6. Results presented are values of two independent repeats of this test, exhibiting similar results.

As the 1996 conjugate did not dissolve well in DDW, it was filtered through a 0.2^m-pore-size cellulose acetate sterile filter (Schleicher & Schuell, Dassel, Germany); the MIC values were the same as with the original non-filtered conjugate.

Table 6: Susceptibility of Candida albicans [mg L^" ]. Final concentrations of AmB in the test were: 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.195, 0.097, 0.049, 0.024, 0.012 mg L-¹.

In vitro activity of the conjugates against Leishmania

To evaluate the in vitro effect of AmB and the conjugates of the invention, samples were added to promastigote cultures of L. donovani. The leishmanicidal effect of the conjugates was compared to the AmB alone. The ED₅₀ of AmB was 0.03 g/mL. The ED₅₀ of the conjugates was 0.78 μg/mL for the amine form (namely the conjugate wherein the drug moieties are conjugated to the polymer via an amine bond, as detailed hereinabove) synthesized at the lab scale, and 0.6 μg/mL at the pilot scale. The ED₅₀ for both the lab and pilot scale products was 1.6 μg/mL.

Claims

CLAIMS:

1. A conjugate of a polymer and at least one drug moiety being an amphotericin B (AmB) derivative.

2. The conjugate according to claim 1, wherein said polymer is associated with said at least one drug moiety via a covalent bond.

3. The conjugate according to claim 2, wherein said covalent bond is one or more of amine, imine and amide bonds.

4. The conjugate according to any one of the preceding claims, wherein said polymer has a molecular weight of at least 1 ,000 Daltons (Da).

5. The conjugate according to claim 4, wherein said molecular weight is at least 10,000 Da.

6. The conjugate according to claim 4, wherein said molecular weight is in the range of 5,000 Da to 75,000 Da.

7. The conjugate according to any one of the preceding claims, wherein said polymer is a polysaccharide.

8. The conjugate according to claim 7, wherein said polysaccharide is selected from starch, glycogen, cellulose, dextran, pullulan, chitosan, arabinogalactan, galactan, galactomannan, gelatin, pectin, glycan, hyaluronic acid and guar gum.

9. The conjugate according to claim 7 or 8, wherein said polysaccharide is arabinogalactan (AG) or a modified form thereof.

10. The conjugate according to claim 9, wherein said modified form of AG is oxidized AG.

11. The conjugate according to claim 10, wherein said oxidized AG comprises a plurality of monomers optionally associated with at least one drug moiety via an imine, amine or amide bond.

12. The conjugate according to claim 1, wherein said AmB derivative is selected from saponified AmB and reduced AmB.

13. The conjugate according to claim 12, wherein said saponified AmB is of formula Dl:

14. The conjugate accondng to claim 12, wherein said reduced AmB is selected amongst compounds of formula (II):

wherein one of the dotted double bonds is reduced.

15. The conjugate according to claim 14, the compound of general formula (II) having a saturated C-C bond between carbons 19-20 or 21-22 or 23-24 or 25-26 or 27- 28 or 29-30 or 31-32.

16. The conjugate according to claim 15, wherein the compound of general formula (II) is selected from D2, D3, D4, D5, D6, D7 and D8, as defined in the specification.

17. The conjugate according to claim 1, wherein the drug moiety associated with the polymer is selected from D2 and D8:

18. The conjugate according to claim 12, wherein said reduced AmB is selected amongst compounds of formula (III):

wherein one of the dotted double bonds is reduced.

19. The conjugate according to claim 18, the compound of general formula (III) having a saturated C-C bond between carbons 19-20 or 21-22 or 23-24 or 25-26 or 27- 28 or 29-30 or 31-32.

20. The conjugate according to claim 19, wherein the compound of general formula (III) is selected from D9, DIO, Dll, D12, D13, D14, and D15, as defined in the specification.

21. The conjugate according to claim 1, wherein the drug moiety associated with the polymer is selected from D9 and D15:

22. The conjugate according to claim 1, being of a polymer and a plurality of AmB derivatives selected from Dl, D2, D3, D4, D5, D6, D7, D8, D9, DIO, Dll, D12, D13, D14 and D15, as defined in the specification.

23. The conjugate according to claim 22, being of a polymer and a plurality of a single drug moiety selected from Dl, D2, D8, D9 and D15, as defined in the specification.

24. The conjugate according to claim 1, being of a polymer and a plurality of Dl and a plurality of at least one further drug moiety selected from D2, D8, D9 and D15, as defined in the specification.

25. The conjugate according to claim 1, being of a polymer and a plurality of D2 and a plurality of at least one further drug selected from Dl, D8, D9 and D15, as defined in the specification.

26. The conjugate according to claim 1, being of a polymer and a plurality of D8 and a plurality of at least one further drug moiety selected from Dl, D2, D9 and D15, as defined in the specification.

27. The conjugate according to claim 1, being of a polymer and a plurality of D9 and a plurality of at least one further drug moiety selected from Dl, D2, D8 and D15, as defined in the specification.

28. The conjugate according to claim 1, being of a polymer and a plurality of D15 and a plurality of at least one further drug moiety selected from Dl, D2, D8 and D9, as defined in the specification.

29. The conjugate according to claim 1, being of a polymer and a plurality of Dl, D8 and D15, as defined in the specification.

30. The conjugate according to claim 1, being of a polymer and a plurality of Dl, a plurality of D8 and a plurality of D15, as defined in the specification, at any ratio.

31. The conjugate according to any one of claims 21-30, wherein the polymer is a polysaccharide.

32. The conjugate according to claim 31 , wherein said polysaccharide is AG.

33. The conjugate according to claim 32, wherein said AG is oxidized AG.

34. A conjugate of oxidized AG and a plurality of drug moieties selected from Dl, D8 and D15, as defined in the specification.

35. Use of a conjugate according to any one of claims 1 to 34 for the preparation of a composition.

36. The use according to claim 35, wherein said composition is a pharmaceutical composition.

37. Use of a conjugate according to any one of claims 1 to 34 in a method of therapeutic treatment of a subject.

38. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 34.

39. The composition according to claim 38, for the treatment of a disease or disorder.

40. The composition according to claim 39, wherein said disease or disorder is selected amongst fungal diseases, parasitic associated diseases and disorders and leishmaniasis (cutaneous leishmaniasis, mucosal leishmaniasis and/or visceral leishmaniasis).

41. The composition according to claim 40, wherein said disease or disorder is associated with Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Candida species, Rhodotorula species, Cryptococcus species, Sporothrix schenckil, Mucor species, Rhizopus species, Rhizomucor species, Absidia species, Trichosporon species, Fusarium species, Paecilomyces species, Phailophora species, Phialemonium species, Exophiala species, Scedosporium species, Neoscytalidium species, Acremonium species, Alternaria species, Malassezia species, Aspergillus species, aspergillosis, cryptococcosis, North American blastomycosis, invasive and systemic candidiasis, coccidioidomycosis, paracoccidioidomycosis, histoplasmosis, fusariosis, invasive trichosporonosis, phaeohyphomycosis, sporotrichosis, zygomycosis (mucormycosis) due to susceptible species of these genera.

42. The composition according to any one of claims 38-41, further comprising at least one additional active ingredient selected from an antifungal drug and antileishmania drug, and any combination thereof.

43. The composition according to any one of claims 38-42 further comprising, in free (unbound) form, at least one of AmB and any reduced and/or saponified derivatives thereof.

44. The composition according to claim 43, wherein said AmB derivative is one or more of Dl through D15, as defined in the specification.

45. A method of treatment of a disease or disorder, in a subject, said method comprising administering to said subject an effective amount of at least one conjugate according to any one of claims 1 to 34.

46. The method according to claim 45, wherein said disease or disorder is selected amongst fungal diseases, parasitic associated diseases and disorders and leishmaniasis (cutaneous leishmaniasis, mucosal leishmaniasis and/or visceral leishmaniasis).

47. The method according to claim 46, wherein said disease or disorder is associated with Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Candida species, Rhodotorula species, Cryptococcus species, Sporothrix schenckil, Mucor species, Rhizopus species, Rhizomucor species, Absidia species, Trichosporon species, Fusarium species, Paecilomyces species, Phailophora species, Phialemonium species, Exophiala species, Scedosporium species, Neoscytalidium species, Acremonium species, Alternaria species, Malassezia species, Aspergillus species, aspergillosis, cryptococcosis, North American blastomycosis, invasive and systemic candidiasis, coccidioidomycosis, paracoccidioidomycosis, histoplasmosis, fusariosis, invasive trichosporonosis, phaeohyphomycosis, sporotrichosis, zygomycosis (mucormycosis) due to susceptible species of these genera.

48. The method according to any one of claims 45 to 47, wherein said conjugate is administered together with at least one additional active ingredient selected from an antifungal drug and antileishmania drug, and any combination thereof.

49. A method of treatment of a fungal disease or disorder, in a subject, said method comprising administering to said subject an effective amount of at least one conjugate according to any one of claims 1 to 34.

50. A method of treatment of a parasitic-associated diseases and disorders, in a subject, said method comprising administering to said subject an effective amount of at least one conjugate according to any one of claims 1 to 34.

51. A method of treatment of leishmaniasis, in a subject, said method comprising administering to said subject an effective amount of at least one conjugate according to any one of claims 1 to 34.

52. An antifungal formulation comprising at least one conjugate according to any one of claims 1 to 34.

53. An antileishmaniasis formulation comprising at least one conjugate according to any one of claims 1 to 34.

54. A compound selected from compounds herein designated D2, D3, D4, D5, D6, D7, D8, D9, D10, Dll, D12, D13, D14 and D15, as defined in the specification.

55. A composition comprising a compound selected from compounds herein designated D2, D3, D4, D5, D6, D7, D8, D9, D10, Dll, D12, D13, D14 and D15, as defined in the specification.

56. A composition of matter comprising a polymer and at least one drug moiety being an amphotericin B (AmB) derivative.

57. A conjugate as herein described in the examples and darwaings.