EP0917473A1 - Conjugues lipidiques polaires covalents associes a des composes biologiquement actifs utilisables dans les onguents - Google Patents

Conjugues lipidiques polaires covalents associes a des composes biologiquement actifs utilisables dans les onguents

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Publication number
EP0917473A1
EP0917473A1 EP96925431A EP96925431A EP0917473A1 EP 0917473 A1 EP0917473 A1 EP 0917473A1 EP 96925431 A EP96925431 A EP 96925431A EP 96925431 A EP96925431 A EP 96925431A EP 0917473 A1 EP0917473 A1 EP 0917473A1
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EP
European Patent Office
Prior art keywords
pharmaceutical composition
drug
spacer
composition according
disease state
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP96925431A
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German (de)
English (en)
Inventor
Milton B. Yatvin
Michael H. B. Stowell
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Oregon Health Science University
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Oregon Health Science University
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Application filed by Oregon Health Science University filed Critical Oregon Health Science University
Publication of EP0917473A1 publication Critical patent/EP0917473A1/fr
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    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • 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
    • A61K47/54—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 compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A61K47/544—Phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders

Definitions

  • a major goal in the pharmacological arts has been the development of methods and compositions to facilitate the specific delivery of therapeutic and other agents to the appropriate cells and tissues that would benefit from such treatment, and the avoidance of the general physiological effects of the inappropriate delivery of such agents to other cells or tissues of the body.
  • One common example of the need for such specificity is in the field of antiproliferative agent therapy for the treatment of skin diseases and disorders, in which the amount of a variety of antiproliferative agents to be safely administered topically or locally to a patient is limited by their systemic cytotoxic effects.
  • the invention also provides polar lipid drug conjugates that target dermal, intradermal and infradermal structures in skin for delivery of therapeutic agents for the treatment of skin diseases and disorders.
  • a therapeutic agent it is desirable to increase the efficiency and specificity of administration of a therapeutic agent to the cells of the relevant tissues in a variety of pathological states. This is particularly important as relates to antiproliferative agents. Such agents typically have pleiotropic antibiotic and cytotoxic effects that damage or destroy uninvolved cells and tissues as well as cells and tissues comprising the pathological site.
  • an efficient delivery system which would enable the delivery of such drugs specifically to the diseased or affected tissues cells would increase the efficacy of treatment and reduce the associated "side effects" of such drug treatments, and also serve to reduce morbidity and mortality associated with clinical administration of such drugs.
  • An additional challenge in designing an appropriate drug delivery scheme is to include within the drug conjugate a functionality which could either accelerate or reduce the rate at which the drug is released upon arrival at the desired site. Such a functionality would be especially valuable if it allowed differential rates of drug release.
  • Medicinal salves and ointments for topical treatment purposes are known in the prior art for the treatment of a variety of pathological conditions.
  • a multitude of pathological and other conditions have been treated by topical application of many classes of compounds in a variety of carriers, such as salves and ointments.
  • carriers used in these conventional treatments are in no way specific for deposition of drugs, and suffer from non-specific deposition of the antiproliferative drug into both healthy and affected portions of the skin.
  • Appropriate concentrations of topically-applied antiproliferative drugs, for example, are currently limited by the escape of the active agent(s) into the systemic circulation, with deleterious effects on other tissues and organs.
  • An example of such a situation is the use of the drug methotrexate to treat psoriasis, where the amount of methotrexate that is capable of being topically applied is limited by hepato- and nephrotoxicity caused by systemic escape of the compound from the skin.
  • the present invention is directed to an improved method for delivering biologically-active compounds, particularly drugs including preferably antiproliferative, antibiotic, antimycotic, antiviral and antineoplastic drugs, to cells comprising skin in animals in vivo and in vitro.
  • This delivery system achieves specific delivery of such biologically-active compounds through conjugating the compounds with a polar lipid carrier.
  • This invention has the specific advantage of facilitating the entry of such compounds into cells via a polar lipid carrier, achieving effective intracellular concentration of such compounds more efficiently and with more specificity than conventional delivery systems.
  • the invention particularly provides pharmaceutical composition comprising the drug/polar lipid conjugates of the invention formulated with a medicinal ointment or salve for treatment of a variety of skin disorders.
  • compositions of matter comprising a biologically- active compound covalently linked to a polar lipid carrier molecule.
  • Preferred embodiments also comprise a spacer molecule having two linker functional groups, wherein the spacer has a first end and a second end and wherein the lipid is attached to the first end of the spacer through a first linker functional group and the biologically-active compound is attached to the second end of the spacer through a second linker functional group.
  • the biologically-active compound is a drug, most preferably an antiproliferative drug or agent, an antibiotic drug, an antiviral drug, an antineoplastic drug or a corticosteroid.
  • Preferred polar lipids include but are not limited to acyl- and acylated carnitine, sphingosine, ceramide, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine, cardiolipin and phosphatidic acid.
  • Preferred biologically-active compounds include antineoplastic and antiproliferative agents such as methotrexate, corticosteroids, antimycotics, antibiotics and antiviral compounds.
  • Pharmaceutical compositions comprising the drug/polar lipid conjugates of the invention formulated with a medicinal ointment or salve are also provided.
  • the invention also provides compositions of matter comprising a biologically-active compound covalently linked to a lipid, most preferably a polar lipid, carrier molecule via a spacer molecule wherein the spacer allows the biologically-active compound to act without being released at an intracellular site.
  • the first linker functional group attached to the first end of the spacer is characterized as "strong" and the second linker functional group attached to the second end of the spacer is characterized as
  • the spacer allows the facilitated hydrolytic release of the biologically-active compound at an intracellular site.
  • Other embodiments of the spacer facilitate the enzymatic release of the biologically-active compound at an intracellular site.
  • the spacer functional group is hydrolyzed by an enzymatic activity found in skin, preferably an esterase and most preferably an esterase having a differential expression and activity profile in different skin layers.
  • specific release of biologically-active compounds is achieved by enzymatic or chemical release of the biologically-active compound by intracellular cleavage of a cleavable linker moiety in cells infected by a pathogenic organism or otherwise expressing a disease state (for example, hyperplasia associated with a benign or malignant skin condition) via an enzymatic activity specific for such a pathogenic organism or disease state, or by extracellular cleavage of a cleavable linker moiety via an enzymatic activity specific for a pathogenic organism or disease state.
  • a disease state for example, hyperplasia associated with a benign or malignant skin condition
  • the invention also provides polar lipid drug conjugates that target dermal, intradermal and infradermal structures in skin for delivery of dierapeutic agents for the treatment of skin diseases and disorders.
  • the invention provides such conjugates comprising a spacer that allows facilitated hydrolytic or enzymatic release of the biologically-active compound at a dermal, intradermal or infradermal site in skin.
  • the spacer molecule is a peptide of formula (amino acid) n , wherein n is an integer between 2 and 25, preferably wherein the peptide comprises a polymer of one or more amino acids.
  • the biologically-active compound of the invention has a first functional linker group, and a lipid, most preferably a polar lipid, carrier has a second functional linker group, and the compound is covalently linked directly to the lipid carrier by a chemical bond between the first and second functional linker groups.
  • each of the first and second functional linker groups is a hydroxyl group, a primary or secondary amino group, a phosphate group or substituted derivatives thereof or a carboxylic acid group.
  • compositions of matter comprising a drug, most preferably an antiproliferative drug, an antineoplastic drug, an antibiotic, an antimycotic, or an antiviral drug, covalently linked to a polar lipid carrier molecule.
  • a drug most preferably an antiproliferative drug, an antineoplastic drug, an antibiotic, an antimycotic, or an antiviral drug, covalently linked to a polar lipid carrier molecule.
  • Preferred embodiments also comprise a spacer molecule having two linker functional groups, wherein the spacer has a first end and a second end and wherein the lipid is attached to the first end of the spacer through a first linker functional group and the drug is attached to the second end of the spacer through a second linker functional group.
  • Preferred embodiments of the invention are provided wherein the drug is an antiproliferative agent, such as methotrexate, an antiviral agent such as an antiherpetic agent, an antibiotic agent such as rifampicin or streptomycin, or an antimycotic such as econazole.
  • Preferred polar lipids include but are not limited to acyl- and acylated carnitine, sphingosine, ceramide, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine, cardiolipin and phosphatidic acid.
  • compositions comprising the drug/polar lipid conjugates of the invention formulated with a medicinal ointment or salve are also provided.
  • the invention also provides compositions of matter comprising an antiproliferative agent, an antineoplastic drug, an antibiotic, an antimycotic, or an antiviral drug, covalently linked to a polar lipid carrier molecule via a spacer molecule, wherein the spacer allows the drug to act without being released at an intracellular site.
  • the first linker functional group attached to the first end of the spacer is characterized as "strong” and the second linker functional group attached to the second end of the spacer is characterized as “weak”, with reference to the propensity of the covalent bonds between each end of the spacer molecule to be broken.
  • the spacer allows the facilitated hydrolytic release of an antiproliferative drug, an antineoplastic drug, an antibiotic, an antimycotic, or an antiviral drug, at an intracellular site.
  • Other embodiments of the spacer facilitate the enzymatic release of the antiproliferative, antineoplastic, antibiotic, antimycotic or antiviral drugs of the invention at an intracellular site.
  • the spacer functional group is hydrolyzed by an enzymatic activity found in skin, preferably an esterase and most preferably an esterase having a differential expression and activity profile in different skin layers.
  • specific release of the antiproliferative, antineoplastic, antibiotic, antimycotic or antiviral drugs of the invention is achieved by enzymatic or chemical release of these drugs by intracellular cleavage of a cleavable linker moiety in cells infected by a pathogenic organism or otherwise expressing a disease state (for example, hyperplasia associated with a benign or malignant skin condition) via an enzymatic activity specific for such a pathogenic organism or disease state, or by extracellular cleavage of a cleavable linker moiety via an enzymatic activity specific for a pathogenic organism or disease state.
  • a disease state for example, hyperplasia associated with a benign or malignant skin condition
  • the invention also provides polar lipid conjugates of of the antiproliferative, antineoplastic, antibiotic, antimycotic or an antiviral drugs of the invention that target dermal, intradermal and infradermal structures in skin for delivery of therapeutic agents for the treatment of skin diseases and disorders.
  • the invention provides such conjugates comprising a spacer that allows facilitated hydrolytic or enzymatic release of the of such antiproliferative, antineoplastic, antibiotic, antimycotic or an antiviral drugs at a dermal, intradermal or infradermal site in skin.
  • the spacer molecule is a peptide of formula (amino acid) n , wherein n is an integer between 2 and 25, preferably wherein the peptide comprises a polymer of one or more amino acids.
  • each of the first and second functional linker groups is a hydroxyl group, a primary or secondary amino group, a phosphate group or substituted derivatives thereof or a carboxylic acid group.
  • the drug is an antiproliferative agent, such as methotrexate, an antiviral agent such as an antiherpetic agent, an antibiotic agent such as rifampicin or streptomycin, or an antimycotic such as econazole.
  • Preferred polar lipids include but are not limited to acyl- and acylated carnitine, sphingosine, ceramide, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine, cardiolipin and phosphatidic acid.
  • Pharmaceutical compositions comprising the drug/polar lipid conjugates of the invention formulated with a medicinal ointment or salve are also provided.
  • the invention also provides compositions of matter comprising an antiproliferative drug, an antineoplastic drug, an antibiotic, an antimycotic, or an antiviral drug covalently linked to a polar lipid carrier molecule via a spacer molecule wherein the spacer allows the drug to act without being released at an intracellular site.
  • the first linker functional group attached to the first end of the spacer is characterized as "strong” and the second linker functional group attached to the second end of the spacer is characterized as "weak", with reference to the propensity of the covalent bonds between each end of the spacer molecule to be broken.
  • the spacer allows the facilitated hydrolytic release of the antiproliferative, antineoplastic, antibiotic, antimycotic or an antiviral drug at an intracellular site.
  • Other embodiments of the spacer facilitate the enzymatic release of a drug as provided by the invention at an intracellular site.
  • the spacer functional group is hydrolyzed by an enzymatic activity found in skin, preferably an esterase and most preferably an esterase having a differential expression and activity profile in different skin layers.
  • specific release of the antiproliferative, antineoplastic, antibiotic, antimycotic or antiviral drugs of the invention is achieved by enzymatic or chemical release of these drugs by intracellular cleavage of a cleavable linker moiety in cells infected by a pathogenic organism or otherwise expressing a disease state (for example, hype ⁇ lasia associated with a benign or malignant skin condition) via an enzymatic activity specific for such a pathogenic organism or disease state, or by extracellular cleavage of a cleavable linker moiety via an enzymatic activity specific for a pathogenic organism or disease state.
  • a disease state for example, hype ⁇ lasia associated with a benign or malignant skin condition
  • the spacer allows the facilitated enzymatic or hydrolytic release of the antiproliferative, antineoplastic, antibiotic, antimycotic or an antiviral drug at dermal, intradermal and infradermal structures in skin for delivery of therapeutic agents for the treatment of skin diseases and disorders.
  • the spacer molecule is a peptide of formula (amino acid) friendship, wherein n is an integer between 2 and 25, preferably wherein the peptide comprises a polymer of one or more amino acids.
  • a preferred embodiments of this aspect of the invention include compositions of matter that are N-methotrexate ceramide, methotrexate-glycylglycylglycylglycyl ceramide ester, methotrexate-(tri- ⁇ -hydroxypropionylester)-O x -ceramide ester, methotrexate-glycylglycylglycylglycylglycyl ceramide ester, methotrexate-aminohexanoyl) sphingosine amide, methotrexate-valinylvalinyl sphingosine amide and methotrexate- O x -ceramide ester.
  • compositions comprising the drug/polar lipid conjugates of the invention and any of a variety of emollients or other commonly encountered components of cremes, salves, poultices, lotions, gels or other substances well-known in the art for applying compounds to skin and other tissues.
  • Appropriate formulations of such compositions comprising the drug/ polar lipid conjugates of the invention will be apparent and within the skill of one of ordinary skill in this art to advantageously prepare in view of the instant disclosure.
  • the drug/lipid conjugates of the invention comprise a functionality recognized by an enzymatic activity, most preferably an esterase activity, that has a differential pattern of expression or activity in different skin layers.
  • specific release of the antiproliferative, antineoplastic, antibiotic, antimycotic or antiviral drugs of the invention is achieved by enzymatic or chemical release of these drugs by intracellular cleavage of a cleavable linker moiety in cells infected by a pathogenic organism or otherwise expressing a disease state (for example, hyperplasia associated with a benign or malignant skin condition) via an enzymatic activity specific for such a pathogenic organism or disease state, or by extracellular cleavage of a cleavable linker moiety via an enzymatic activity specific for a pathogenic organism or disease state.
  • a disease state for example, hyperplasia associated with a benign or malignant skin condition
  • the invention comprehends a polar lipid-drug conjugate wherein the polar lipid will selectively associate with certain biological membranes, and thereby facilitate entry of the drug into cells and cellular organelles.
  • the spacer component of the conjugates of the invention will preferably act to release the drug from the lipid, target the conjugate to the cell, or perform other functions to maximize the effectiveness of the drug.
  • the drug-lipid conjugates of the invention promote the intracellular entry of a variety of potentially useful drugs at pharmokinetic rates not currently attainable.
  • the range of targeted cell types is not limited per se by particular, limited biological properties of the cell (such as the number and type of specific receptor molecules expressed on the cell surface).
  • this method may target drugs to specific intracellular organelles and other intracellular compartments.
  • the compositions of matter of the invention incorporate a variable spacer region that may allow pharmacologically- relevant rates of drug release from polar lipid carrier molecules to be engineered into the compositions of the invention, thereby increasing their clinical efficacy and usefulness.
  • the conjugates of the invention can be combined with other drug delivery approaches to further increase specificity and to take advantage of useful advances in the art.
  • the conjugates of the invention can be topically applied to skin, and the layer of skin penetrated determined by the formulation used.
  • the amount and activity of the topically-applied drug can be modulated by release via cleavage, preferably hydrolytic cleavage, of the spacer moiety, most preferably by an enzymatic activity in skin that has a differential pattern of expression or activity in different skin layers.
  • Figure 1 depicts the synthetic scheme put forth in Example 1.
  • Figure 2 depicts the synthetic scheme put forth in Example 2.
  • FIG. 3 depicts the synthetic scheme put forth in Example 3.
  • FIG. 4 depicts the synthetic scheme put forth in Example 4.
  • Figure 5 depicts the synthetic scheme put forth in Example 5.
  • Figure 6 depicts the synthetic scheme put forth in Example 6.
  • Figure 7 depicts the synthetic scheme put forth in Example 7.
  • Figure 8 depicts the synthetic scheme put forth in Example 8.
  • FIGS 9A through 9D depict prodrugs tested as in Example 9.
  • Figure 10 depicts the synthetic scheme put forth in Example 10.
  • FIGS. 11 through 19 illustrate targeting of polar lipid-conjugated biologically active compounds to skin.
  • compositions of matter and methods for facilitating the entry into cells of biologically-active compounds are intended to encompass all naturally-occurring or synthetic compounds capable of eliciting a biological response or having an effect, either beneficial or cytotoxic, on biological systems, particularly cells and cellular organelles. These compounds are intended to include but are not limited to all varieties of drugs, particularly antiproliferative drugs and agents, antibacterial, fungicidal, anti-protozoal and antiviral drugs, antineoplastic drugs, and cytotoxic and cytostatic compounds.
  • compositions comprising the drug/polar lipid conjugates of the invention formulated with a medicinal ointment or salve are also provided.
  • medicinal ointment or salves are considered equivalent.
  • the term is intended to encompass any of a variety of salves and other topically or locally applied formulations known in the art, and specifically to encompass any of a variety of emollients or other commonly encountered components of cremes, salves, poultices, lotions, gels or other substances well-known in the art for applying compounds to skin and other tissues.
  • Appropriate formulations of such compositions comprising the drug/ polar lipid conjugates of the invention will be apparent and within the skill of one of ordinary skill in this art to advantageously prepare in view of the instant disclosure.
  • compositions of matter provided by the invention comprise the biologically-active compounds of the invention covalently linked to a polar lipid carrier.
  • a polar lipid carrier as defined herein is intended to mean any polar lipid having an affinity for, or capable of crossing, a biological membrane, including but not limited to sphingosine, ceramide, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine, cardiolipin, phosphatidic acid, sphingomyelin and other sphingolipids, as these terms are understood in the art (see, Lehninger, Biochemistry, 2d ed., Chapters 11 & 24, Worth Publishers: New York, 1975).
  • lipids such as acylated carnitine
  • conjugates of the invention see Small, 1986, “From alkanes to phospholipids," Handbook of Lipid Research: Physical Chemistry of Lipids, Volume 4, Chapters 4 and 12, Plenum Press: New York).
  • compositions of matter of the invention may be further comprised of a spacer moiety comprising a first end and a second end, each end of the spacer having a functional linking group.
  • a spacer moiety comprising a first end and a second end, each end of the spacer having a functional linking group.
  • spacer or “spacer moiety” is intended to encompass any chemical entity that links the biologically-active compound and the polar lipid.
  • Such spacer moieties may be designed to facilitate the attachment of the conjugates of the invention to a target cell, or to facilitate, influence, modulate or regulate the release of the biologically- active compound at the desired target site.
  • Spacers may also facilitate enzymatic release at certain intracellular sites.
  • Spacer groups include, but are not limited to aminohexanoic acid, polyglycine, poly amides, polyethylenes, and short functionalized polymers having a carbon backbone which is from one to about twelve carbon molecules in length.
  • Particularly preferred embodiments of such spacer moieties comprise peptides of formula (amino acid ⁇ , wherein n is an integer between 2 and 25 and the peptide is a polymer of one or more amino acids.
  • linker functional group is defined herein as any functional group for covalently binding the polar lipid carrier or biologically-active agent to the spacer group. These groups can be designated either “weak” or “strong” based on the stability of the covalent bond which the linker functional group will form between the spacer and either the polar lipid carrier or the biologically-active compound.
  • the weak functionalities include, but are not limited to phosphoramide, phosphoester, carbonate, amide, carboxyl-phosphoryl anhydride, ester and thioester.
  • the strong functionalities include, but are not limited to ether, thioether, amine, amide and ester.
  • the drug/polar lipid conjugates of the invention are preferably provided comprised of spacer moieties that impart differential release properties on the conjugates related to differential expression or activity of enzymatic activities in different layers of skin.
  • Biologically active agents such as antiproliferative, antiviral or antineoplastic drugs linked to polar lipids can be delivered to different layers and structures in the skin based on the distinct differences in lipid handling within the skin. Such different structures include dermal, intradermal and infradermal regions or sections of the skin. As shown herein in Example 1 1 , there are important differences in the delivery of a fluorescent marker in mouse skin, based on the polar lipid to which it was linked via an amide bond. In addition to distribution by lipid handling, differences in hydrolytic enzymes activity, such as esterases and peptidases, within the skin may be employed to convey specificity to drug distribution.
  • Nonspecific esterase activity increases in cells around the edge of many types of wounds (Dachun et al., 1992, Forensic Science International 53: 203-213). Although not produced by skin cells per se, esterases are known to increase in the skin due to influx of other cell types during illness. On example is chloroacetate esterase activity is increased in any condition causing chronic uticaria due to mast cell infiltration
  • monocytes and macrophages found in the skin in leprosy increase esterase activity (SivaSai et al., 1993, Int. J. Leprosy & Other Mycobacterial Diseases 61 : 259-269).
  • Other enzymatic activities are associated with skin.
  • Peptidases are found in high levels within skin tissues and the non-dermal cell types infiltrating the skin. Expression of both endo- and exo-peptidases and proteases varies with cell type and pathological condition.
  • Skin fibroblasts contain high levels of peptidases, not seen in other more highly differentiated layers of skin (Bou-Gharios et ai, 1995, Annals of Rheumatic Disease 5.4: 111-116). High levels of dipeptidyl dipeptidase IV are seen in precancerous dermatoses and basal cell carcinoma (Moehrle et ai, 1995, J. Cutaneous Pathology 22'- 241-247). Increased peptidase activity is associated with stress responses: exemplary are apoptotic skin cells produced after UV irradiation (so- called sunburn cells), which have high amino and endopeptidase activity: Brown et al. (1994, J.
  • the specificity of the cleavage of the linker moiety as provided by this invention is the result of the combination of particular linker moieties selected to be specifically cleaved inside an infected skin cell.
  • such specific cleavage is due to an chemical linkage which is labile within the infected cell due to conditions caused by or that result from infection of the cell with a particular pathogenic organism.
  • such specific cleavage is due to an enzymatic activity which is produced either by the pathogen itself or by the cell as the result of infection with said pathogen, wherein the linkage is enzymatically cleaved by the enzymatic activity.
  • Examples of such combinations resulting in specific release of an antimicrobial drug embodiment of the invention within infected cells include but are not limited to a urea-based linker for use against a pathogen which produces urease (e.g. , Mycobacteria spp. and B. pertussis); a peptide linker comprised of
  • n can be an integer from 1-5, for use against a pathogen that produces the protease ohgopeptidase A (e.g. , Salmonella spp.); a peptide comprised of from 3 to about 20 amino acids comprising the sequence — Pro-Xaa- Pro-, where Xaa is any amino acid, for use against a pathogen that produced proline peptidase (e.g. , Salmonella spp.); peptides comprising the dipeptide MetMet or LeuAla, or peptides comprising the amino acid sequence GSHLVEAL,
  • HIV-1 protease a specific protease termed HIV-1 protease; a peptide comprising the amino acid sequence: -Ala-Xaa ⁇ Cys Acm -Tyr-Cys-Arg-Ile-Pro- Ala-Cys Acm -Ile-Ala-GIy-Asp-Arg-Arg-Tyr-Gly-Thr-Cys AcS Ile-Tyr-Gln-GIy-Arg-Leu- T ⁇ -Ala-Phe-Cys Acm -Cys Acm -, wherein the pathogen expresses an enzymatic activity that specifically disables the endogenous antimicrobial peptide defensin (e.g.
  • (-Cys Acm -) represent cysteine residues having the sidechain sulfur atom protected by covalent linkage to an acetamidomethyl group (it will be recognized that embodiments of such peptides having alternative sulfur protecting groups are also within the scope of the disclosure herein) and Xaa is either absent or Asp (said peptides are also useful against a pathogen such as Legionella spp. producing a 39 kDa metal loprotease); hippurate esters that are hydrolyzed by pathogen-specific (e.g. , L.
  • pneumophila and Listeria spp. hydrolase nicotinic acid amides cleaved by nicotinamidases, pyrazinamides cleaved by pyrazinamidase; allolactose linkages cleaved by ⁇ - galactosidase; and allantoate linkages cleaved by allantoicase (e.g. , Mycobacterium spp.).
  • the present invention provides methods and compositions of matter for facilitating the entry of antiproliferative, antibiotic, antimycotic, antiviral and antineoplastic agents, drugs and compounds into dermal and epidermal cells, across mucosal membranes where appropriate, and distributed within skin tissue for efficient delivery of such compounds locally and topically for the treatment of animal, preferably human, diseases and pathological conditions.
  • the invention provides salves, ointments, poultices and other topically-applied embodiments of the drug/lipid conjugates of the invention for the treatment of a variety of skin diseases and disorders.
  • dermatitis further differentiated into contact, seborrheic, nummular, exfoliative stasis, and neurodermatitis.
  • Vesicular dermatitis commonly referred to as eczema, is separated from other dermatitis to reflect the chronic nature of the condition. All forms of dermatitis are characterized by superficial inflammation, vesiculation and localized edema. Accompanying these psoriasiform conditions is thickening of the epidermis showing both hyperkeratosis and parakeratosis. Topical corticosteroids are widely prescribed for these conditions, including the Potency class I drugs betamethasone dipropionate, clobetasol propionate, diflorasone diacetate and flucinolone.
  • the invention provides a conjugate, methotrexate ceramide ester (ME6C), that does not concentrate in either liver or kidney to the same extent as the free drug, and is therefore useful in for treatment of chronic psoriasis and related conditions.
  • M6C conjugate, methotrexate ceramide ester
  • Another skin condition conventionally treated by topical application of an antineoplastic agent is the treatment of precancerous lesions classified as actinic keratoses. These lesions respond to 5-fluorouracil (5-FU) topically applied in a propylene glycol carrier.
  • Topical application of 5-FU is limited by the toxicity of this compound in the systemic circulation.
  • Topical application can be improved by covalent, hydrolyzable linkage of 5-FU to polar lipids that is cleaved by an esterase or peptidase activity in skin.
  • Such compounds have the advantage of better penetration, longer retention and controlled release of active drug.
  • these drugs are delivered to specific areas of the skin in greater quantity and maintained for longer periods of time as a consequence of the lipid formulation. Release of active drug from these reservoirs is effectuated by the cleavage of hydrolyzable bonds between the drug and the lipid carrier.
  • a greater therapeutic index is thereby achieved, thus reducing the need for system antimycotics and reducing the risk of hepatic toxicity.
  • the characteristic accumulation of fluids in eczema, or other bullus conditions may be a limiting factor in the delivery of water soluble antimycotics.
  • Use of the lipid prodrugs to effectively deliver antifungals to deeper skin structures would improve treatment of eczema and yeast-exacerbated dermatitis.
  • the invention provides polar lipid/drug conjugates comprising corticosteroids, including but not limited to cortisone, cortisol, hydrocortisone, prednisone, fluorinated corticosteroids (such as fluocinalone and triamcinolone), dexamethasone, alcloethasone, fluoroandrenolide and mometasone.
  • corticosteroids including but not limited to cortisone, cortisol, hydrocortisone, prednisone, fluorinated corticosteroids (such as fluocinalone and triamcinolone), dexamethasone, alcloethasone, fluoroandrenolide and mometasone.
  • the invention provides polar lipid/drug conjugates comprising antimycotic compounds including but not limited to clotrimazole, ciclopirox, nystatin, econazole and myconixole.
  • the invention provides polar lipid/drug conjugates comprising antibiotics including but not limited to penicillin and drugs of the penicillin family of antimicrobial drugs, including but not limited to penicillin-G, penicillin-V, phenethicillin, ampicillin, amoxacillin, cyclacillin, bacampicillin, hetacillin, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, azlocillin, carbenicillin, mezlocillin, piperacillin, ticaricillin, and imipenim; cephalosporin and drugs of the cephalosporin family, including but not limited to cefadroxil, cefazolin, caphalexn, cephalothin, cephapirin, ce
  • the invention also provides polar lipid/drug conjugates of antiviral agents, including but not limited to reverse transcriptase inhibitors, protease inhibitors, antiherpetics such as acyclovir and gangcyclovir, azidothymidine, cytidine arabinoside, ribavirin, amantadine, iododeoxyuridine, poscarnet, trifluoridine, methizazone, vidarabine and levanisole.
  • antiviral agents including but not limited to reverse transcriptase inhibitors, protease inhibitors, antiherpetics such as acyclovir and gangcyclovir, azidothymidine, cytidine arabinoside, ribavirin, amantadine, iododeoxyuridine, poscarnet, trifluoridine, methizazone, vidarabine and levanisole.
  • the invention provides polar lipid/drug conjugates of antiproliferative and antineoplastic agents, including but not limited to methotrexate, doxarubicin, daunarubicin, actinomycin D, vinblastine, vincristine, colchicine and taxol.
  • the invention specifically provides methods for preparing and administering such antiproliferative compounds for use in treating pathological conditions in vivo.
  • Animals to be treated with polar lipid-antiproliferative agent conjugates using the methods of the invention are intended to include all vertebrate animals, preferably domesticated animals, such as cattle, horses, goats, sheep, fowl, fish, household pets, and others, as well as wild animals, and most preferably humans.
  • domesticated animals such as cattle, horses, goats, sheep, fowl, fish, household pets, and others
  • wild animals such as wild animals, and most preferably humans.
  • An antibiotic drug/polar lipid conjugate of the invention is prepared by conjugating a specifically-cleavable peptide to a polar lipid and an antibiotic drug as follows.
  • An derivatized polar lipid comprising unconjugated amino groups is reacted with a proteolytically-inert peptide in which the terminal amine and any of the constituent amino acid sidechain reactive amines are covered by tert- butoxycarbonyl (t-Boc) protecting groups in the presence of triphenyl phosphine as described by Kishimoto (1975, Chem. Phys. Lipids 15.: 33-36).
  • the peptide/polar lipid conjugate is then reacted in the presence of pyridine hydrofluoride as described by Matsuura et al.
  • HTV1 protease inhibitor (HTV1 protease inhibitor; compound 8) is conjugated to sphingosine as follows. Sphingosine is reacted with 1,3 t ⁇ (trimethylsilyl)urea as described by Verbloom et al. (1981, Synthesis 1032: 807-809) to give a trimethylsilyl derivative of sphingosine.
  • the sphingosine derivative is then conjugated with a specifically-cleavable peptide in which the terminal amine and any of the constituent amino acid sidechain reactive amines are covered by tert- butoxycarbonyl (t-Boc) protecting groups in the presence of diethylazo-dicarboxylate (DEAD) and triphenyl phosphine as described by Kishimoto (1975, Chem. Phys. Lipids 15.: 33-36).
  • the sphingosine/peptide conjugate is then reacted in the presence of pyridine hydrofluoride as described by Matsuura et al. (1976, J. Chem. Soc. Chem. Comm.
  • An antiviral compound (compound 8) is conjugated to ceramide via a polyglycine spacer as follows and as illustrated in Figure 3.
  • the amino terminus of polyglycine is protected by a t-Boc group.
  • Polyglycine is conjugated through its carboxy terminus to ceramide forming an ester linkage, as described in Anderson et ai, ibid.
  • the resulting compound is then conjugated through the amino terminus of the polyglycine residue.
  • the amino terminus of Compound 8 is also protected by a t-Boc protecting group. Conjugation with polyglycyl-sphingosine takes place between the amino terminus of the polyglycyl spacer moiety and the carboxy terminus of the HIV-1 protease inhibitor.
  • EXAMPLE 4 An antiviral compound is prepared wherein ceramide is first conjugated to a first end of an oligomeric 3-hydroxy propanoic acid spacer through an ester functional group, and wherein AZT is conjugated to a second end of said polyester spacer through a phosphodiester bond.
  • First a polyester spacer is obtained, having a carboxyl at a first end and a triphenylmethyl group esterified to a second end. This spacer is conjugated to ceramide at its first end through an ester functional linker group according to the method of Anderson et ai, ibid.
  • This compound is then conjugated through the second end of the spacer compound to AZT monophosphate by means of a phosphodiester bond according to the method of Baer (1955, Can. J. Biochem. Phys. 24 288).
  • the bond breakage between the spacer and the drug would be slow in the absence of a phosphohydrolase. This reaction scheme is illustrated in Figure 4.
  • EXAMPLE 5 An antiviral compound wherein phosphatidic acid, phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol or phosphatidylethanolamine is linked through a phosphoester linker functional group to the antiviral drug azidothymidine (AZT).
  • AZA antiviral drug azidothymidine
  • Phosphatidic acid, phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol or phosphatidyl ed anolamine is conjugated to AZT according to the method of Salord et al. (1986, Biochim. Biophys. Acta j£g£: 64-75). This reaction scheme is illustrated in Figure 5.
  • EXAMPLE 6 An antiviral compound is prepared wherein aminohexanoyl sphingosine is conjugated to AZT. Aminohexanoyl sphingosine is conjugated with AZT according to the method of Kishimoto (1975, Chem. Phys. Lipid 15: 33-36). This reaction scheme is illustrated in Figure 6 to yield aminohexanoyl sphingosine conjugated to AZT through a phosphoramide bond.
  • EXAMPLE 7 An antiviral compound consisting of ceramide conjugated to AZT- monophosphate is provided. Ceramide is reacted with AZT-monophosphate in the presence of dicyclohexylcarbodiimide as described in Smith and Khorana (1958, J. Amer. Chem. Soc. SQ: 1141) to yield ceramide conjugated through a phosphodiester bond to AZT-monophosphate. This reaction scheme is illustrated in Figure 7.
  • An antiviral compound is prepared wherein ceramide is conjugated through an ester functional group to a first end of a polyglycine spacer, and wherein AZT is conjugated through a phosphoester functional group to a second end of the polyglycine spacer.
  • Ceramide is first conjugated through an ester functional group to a first end of a polyglycine spacer (as described in Example 2).
  • the ceramide- polyglycine compound is then conjugated through a phosphoester bond to a second end of the polyglycine spacer to AZT monophosphate according to the method of Paul and Anderson, ibid. This reaction scheme is illustrated in Figure 8.
  • the effect of presenting a biologically active compound such as a drug to mammalian cells as a prodrug covalently linked to a polar lipid carrier moiety was determined as follows.
  • the antifolate drug methotrexate was conjugated with a variety of polar lipid carriers via organic spacer moieties having specific reactive functional groups.
  • FIG. 9 A through 9C A representative sample of such compounds is shown in Figures 9 A through 9C, wherein MC represents Mtx linked to sphingosine via an amide bond to a 6-aminohexanoic acid spacer, ME ⁇ C represents Mtx linked to sphingosine via an ester linkage to a 6-hydroxyhexanoic acid spacer, and MSC represents Mtx linked to sphingosine via a salicylic acid ester linkage to a 6-aminohexanoic acid spacer. Also studied was a conjugate of azidothymidine linked to sphingosine via an ester linkage to a 6-hydroxyhexanoic acid spacer (N-AZT-ceramide; Figure 9D).
  • the compounds were tested for their growth inhibitory effects on murine NIH 3T3 cells growing in cell culture. About one million such cells per PI 00 tissue culture plate were grown in DMEM media supplemented with 10% fetal calf serum (GIBCO, Grand island, NY) in the presence or absence of a growth-inhibitory equivalent of each prodrug. Cell numbers were determined after 70 hours growth in the presence or absence of the prodrug. In a second set of experiments was included in the growth media an amount of a brain homogenate containing an enzymatically-active esterase. The results from these experiments are shown in Table I. As can be seen from these data, the MC prodrug had no effect on the growth and survival of the cells.
  • Table II shows the results of drug uptake studies performed with the prodrug N-AZT-ceramide. Antiviral amounts of the prodrug conjugate were added to NIH 3T3 cell cultures, and the antiviral activity of the prodrug was found to be equivalent to the activity of free AZT. In addition, upon removal of the prodrug, intracellular retention of prodrug was found to be up to 15-fold higher than free AZT (Table II) over a 23h period.
  • An antiproliferative agent is prepared wherein the anti-proliferative drug methotrexate (Mtx) is conjugated to sphingosine via a 6-aminocaproic acid spacer.
  • This reaction scheme is illustrated in Figure 10.
  • the primary amino and hydroxyl groups of sphingosine are acylated by reaction with activated N- (methotrexate)aminocaproic acid overnight at 40-50°C, followed by base hydrolysis in 0.1N methanolic KOH.
  • the Mtx derivative of 6-aminocaproic acid is synthesized by activating the carboxylic acid moiety of Mtx and reacting with 6- aminocaproic acid for 2 days at 60-70 °C. This reaction is stopped under acidic conditions to liberate anhydrides that form under these conditions.
  • EXAMPLE 11 An in vivo mouse skin model system was used to demonstrate the use of embodiments of the polar lipid conjugates of the invention for introducing biologically-active compounds through the epidermal layer of the skin and into the underlying skin layers.
  • NBD 7-nitro-2-l,3-benzoxadiazol- 4-yl)-hexanoate
  • DMSO DMSO were applied to shaved mouse skin and allowed to penetrate the skin for 4 hours. After the 4 hour incubation, skin sections were excised and prepared for light or fluorescence microscopy, using standard histological techniques.
  • Figure 11 is a photomicrograph that illustrates hematoxylin-eosin (H&E) staining of mouse skin, observed by light microscopy under 100X magnification.
  • H&E hematoxylin-eosin
  • Figure 12 is a fluorescence photomicrograph that illustrates ceramide-NBD staining of mouse skin, observed by fluorescence microscopy under
  • Figure 13 is a fluorescence photomicrograph that illustrates phosphatidylcholine-NBD staining of mouse skin, observed by fluorescence microscopy under 100X magnification.
  • Figure 14 is a fluorescence photomicrograph that illustrates phosphatidylethanolamine-NBD staining of mouse skin, observed by fluorescence microscopy under 100X magnification
  • Figure 15 is a fluorescence photomicrograph that illustrates phosphatidylserine-NBD staining of mouse skin, observed by fluorescence microscopy under 100X magnification.
  • Each of these conjugates was observed to result in localized fluorescence in the outer layers of the skin.
  • Figure 16 is a fluorescence photomicrograph that illustrates l-R ⁇ 6[(7-nitro-2-
  • Figure 17 is a fluorescence photomicrograph that illustrates phosphatidylserine-NBD staining of mouse skin, observed by fluorescence microscopy under 100X magnification.
  • the compound used in Figure 17 differs from the phosphatidylserine-NBD conjugate shown in Figure 15 in that the NBD dye is conjugated to the polar lipid via a dodecanoyl moiety in the compound of Figure 17 and via a caproyl moiety in the compound of Figure 15.
  • the dodecanoyl-conjugated NBD compound of Figure 17 penetrated into the cornified layer, with only minimal penetration into the epidermis.
  • Figure 18 is a fluorescence photomicrograph that illustrates l-R(12 ⁇ (7-nitro- 2-l,3-benzoxadiazol-4-ethylamino) ⁇ dodecanoyl)-NBD (termed phospho-rac-(l- glycerol)-NBD(dodecanoyl)) staining of mouse skin, observed by fluorescence microscopy under 100X magnification.
  • This compound is related to the compound of Figure 16, but differs in the size of the acyl chain to which the fluorescent NBD label is conjugated; here, it is a dodecanoyl chain, while in Figure 16 it is a caproyl chain. Both the compound of Figure 16 and the instant compound were observed to penetrate the papillary dermis and the reticular dermis. In addition, the dodecanoyl-containing compound of Figure 18 was also observed to accumulate in hair follicles.
  • Figure 19 is a fluorescence photomicrograph that illustrates phosphatidylethanolamine-NBD staining of mouse skin, observed by fluorescence microscopy under 100X magnification.
  • the compound used in Figure 19 differs from the phosphatidylethanolamine-NBD conjugate shown in Figure 14 in that the NBD dye is conjugated to the polar lipid via a dodecanoyl moiety in the compound of Figure 19 and via a caproyl moiety in the compound of Figure 14.
  • the dodecanoyl-conjugated NBD compound of Figure 19 penetrated into the dermis.
  • Caproyl-conjugated phosphatidylethanolamine- NBD did not penetrate beyond the outermost layers of the epidermis, while dodecanoyl-conjugated phosphatidylethanolamine-NBD was observed to penetrate into the dermis.
  • caproyl-conjugated phosphatidylserine-NBD penetrated into the papillary dermis, while dodecanoyl- conjugated phosphatidylethanolamine-NBD did not penetrate past the cornified layer of the epidermis.
  • polar lipid composition is a determinant in the penetrating ability of the conjugates of the invention, and demonstrated that linkage to polar lipids produced increased penetration of the skin by non-penetrating compounds.
  • conjugates of the invention partitioned selectively in skin layers and cells, depending on the lipid carrier used in the conjugate.
  • conjugating antiproliferative compounds of the invention with polar lipids can be used to deliver drugs to specific areas of the skin in greater quantity and concentration than can currently be achieved, and that such drugs can be maintained in specific areas and cells in the skin for longer periods of time.
  • lipid-drug formulation release of active drug from these conjugates can be achieved by the use of hydrolyzable bonds between drug and carrier.
  • the specific partitioning of the conjugates of the invention achieved through the use of polar lipid conjugates, also permits a greater therapeutic index to be achieved.
  • These capacities of the antiproliferative drug conjugates of the invention have important applications to the delivery of medicinal compounds into the skin to treat a variety of pathological conditions. Medicinal salves and ointments for topical treatment pu ⁇ oses are known in the prior art for the treatment of a variety of pathological conditions, but they suffer from non-specific deposition of the antiproliferative drug into both healthy and affected portions of the skin.
  • topically-applied antiproliferative drugs are currently limited by the escape of the active agent(s) into the systemic circulation, with deleterious effects on other tissues and organs.
  • An example of such a situation is the use of the drug methotrexate to treat psoriasis, where the amount of methotrexate that is capable of being topically applied is limited by hepato- and nephrotoxicity caused by systemic escape of the compound from the skin.
  • methotrexate-containing embodiments of conjugates of the invention is that this compound does not concentrate in the liver or kidney to the same extent as free drug, even upon escape into the systemic circulation.
  • polar lipid-drug conjugates of the invention provides a means of increasing the dosages of such antifungal agents that can be topically applied.
  • Other uses of the conjugates of the invention include treatment of precancerous lesions with polar lipid conjugated 5-fluorouracil. The present invention therefore solves a problem common to treatment of a variety of pathological conditions in skin tissue with topically-applied salves, ointments or similar medicaments.

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Abstract

Cette invention se rapporte à un procédé visant à faciliter l'entrée de médicaments à l'intérieur de cellules et de tissus à des niveaux pharmacinétiquement utiles, ainsi qu'à un procédé d'apport ciblé de médicaments à des organites spécifiques de la cellule. Cette invention relative au ciblage de conjugués polaires lipidiques et de médicaments constitue un progrès par rapport aux autres procédés de ciblage de médicaments du fait que, conformément à ce procédé, les concentrations intracellulaires de médicaments peuvent atteindre des niveaux qui sont de très loin supérieurs à ceux obtenus par d'autres procédés. L'invention affine également le processus d'apport de médicaments en permettant de diriger des agents thérapeutiques vers certaines structures intracellulaires. Ce procédé convient aux médicaments antiprolifératifs, antibiotiques, antifongiques, antiviraux et antinéoplasiques, notamment lorsque ces médicaments sont utilisés en association à une multiplicité d'autres émollients et agents, et il peut servir à la fabrication de substances à activité topique telles que des onguents, destinées à permettre une introduction rapide et efficace de ces agents dans l'épiderme pour le traitement des dermatoses et autres troubles de la peau.
EP96925431A 1996-07-23 1996-07-23 Conjugues lipidiques polaires covalents associes a des composes biologiquement actifs utilisables dans les onguents Withdrawn EP0917473A1 (fr)

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WO2000033883A1 (fr) * 1998-12-04 2000-06-15 Oregon Health Sciences University Conjugues lipides polaires covalents avec medicaments antimicrobiens et antineoplasiques destines a cibler des sites biologiques proteges
US6464992B2 (en) * 2000-04-14 2002-10-15 University Of Kentucky Research Foundation Topical micronutrient delivery system and uses thereof
US7045543B2 (en) 2001-11-05 2006-05-16 Enzrel Inc. Covalent conjugates of biologically-active compounds with amino acids and amino acid derivatives for targeting to physiologically-protected sites
EA032840B8 (ru) * 2011-06-22 2020-06-18 Вайоми Терапеутикс Лимитед Пролекарства на основе конъюгатов противогрибковых агентов и их применение
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US5256641A (en) * 1990-11-01 1993-10-26 State Of Oregon Covalent polar lipid-peptide conjugates for immunological targeting
US5543390A (en) * 1990-11-01 1996-08-06 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education, Acting For And On Behalf Of The Oregon Health Sciences University Covalent microparticle-drug conjugates for biological targeting
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