Title:  Conjugate of biologically active compound and polar lipid conjugated to a microparticle for biological targeting

United States Patent:  6,339,060

Assignee:  Oregon Health & Science University (Portland, OR)

Filed:  May 16, 2000

Methods and reagents are provided for specifically targeting biologically active compounds such as antiviral and antimicrobial drugs, or prodrugs containing the biologically active compound to specific sites such as specific organelles in phagocytic mammalian cells. The biologically active compound or prodrug is linked to a microparticle with a linker that is non-specifically or specifically cleaved inside a phagocytic mammalian cell. Alternatively, the biologically active compound or prodrug is impregnated into a porous microparticle or coated on a nonporous microparticle, and then coated with a coating material that is non-specifically or specifically degraded inside a phagocytic mammalian cell. The prodrug contains the biologically active compound linked to a polar lipid such as ceramide with a specific linker such as a peptide that is specifically cleaved to activate the prodrug in a phagocytic mammalian cell infected with a microorganism. A microparticle linked antimicrobial drug or prodrug may be used for killing a microorganism infecting a phagocytic mammalian cell in vivo or in vitro.

SUMMARY OF THE INVENTION

The present invention is directed to an improved method for delivering biologically-active compounds to phagocytic cells and cellular organelles of such phagocytic cells in vivo and in vitro. This delivery system achieves such specific delivery of biologically-active compounds in inactive, prodrug form which are then specifically activated within a phagocytic cell, most preferably a phagocytic mammalian cell, infected with a microorganism, most preferably a pathological or disease-causing microorganism. In preferred embodiments, the inactive prodrugs of the invention are provided as conjugates between polar lipids and biologically-active compounds. In one preferred embodiment of the invention is provided a biologically-active compound in inactive, prodrug form that can be delivered to phagocytic cells through conjugating the compound with a microparticle via an cleavable linker moiety. Alternatively, specific delivery is achieved by impregnating a biologically-active compound in inactive, prodrug form into a porous microparticle which is then coated with a coating material. In an alternative embodiment, the delivery system comprises a nonporous microparticle wherein a biologically-active compound in inactive, prodrug form is made to coat the particle, and the particle is then further coated by a coating material. As used herein, these different embodiments of the microparticles of the invention are generically defined as "microparticle-conjugated" embodiments. In preferred embodiments of each aspect of the invention, the biologically-active compound in inactive, prodrug form is most preferably provided as a conjugate of the biologically-active compound with a polar lipid via a specific linker moiety that is specifically cleaved in a phagocytic cell, most preferably a phagocytic mammalian cell, infected with a microorganism, most preferably a pathological or disease-causing microorganism, wherein the inactivated prodrug form of the biologically-active compound is activated thereby.

In each case, non-specific release of the polar lipid-biologically-active compound conjugate is achieved by enzymatic or chemical release of the inactive, prodrug form of the biologically-active compound from the microparticle by cleavage of the cleavable linker moiety or the coating material in a phagocytic cells, followed by specific release of the biologically-active compound in particular phagocytic cells. In preferred embodiments, wherein the biologically-active compound in inactive, prodrug form is provided as a conjugate of the biologically-active compound with a polar lipid via a specific linker moiety, activation is specifically accomplished by chemical or enzymatic cleavage of a specific linker moiety between the biologically-active compound and the polar lipid. Most preferably, the biologically-active compound is inactive or has reduced activity in the form of a polar lipid conjugate, wherein the activity of the compound is restored or increased upon specific cleavage of the linker moiety in a particular phagocytic cell. In preferred embodiments, the specific linker moiety is enzymatically cleaved by an enzyme that is produced by a microorganism, most preferably a pathological or disease-causing microorganism or which is induced by infection by a microorganism, most preferably a pathological or disease-causing microorganism. In additional preferred embodiments, the specific linker moiety is chemically cleaved under physiological conditions that are specific for phagocytic cells infected with a microorganism, most preferably a pathological or disease-causing microorganism.

In addition, conjugation of the biologically-active compound with a polar lipid provides for targeting of the conjugate to specific subcellular organelles. This invention has the specific advantage of facilitating the delivery of such compounds to specific subcellular organelles via the polar lipid carrier, achieving effective intracellular concentrations of such compounds more efficiently and with more specificity than conventional delivery systems. Moreover, the targeted biologically-active compounds comprising the conjugates are specifically activated or their activity increased at the intracellular target by cleavage of the specific linker moiety and release of the biologically-active compound at the targeted intracellular site.

The specific delivery of biologically-active compounds to phagocytic cells, most preferably phagocytic mammalian cells, is achieved by the present invention by chemical or physical association of the inactive prodrug form of the biologically-active compounds with a microparticle. Specific intracellular accumulation and facilitated cell entry is mediated by the phagocytic uptake of microparticle-conjugated biologically active compounds by such cells. Preferred embodiments of phagocytic cellular targets include phagocytic hematopoietic cells, preferably macrophages and phagocytic neutrophils.

Particularly preferred targets of the microparticle-conjugated biologically active compounds of the invention are phagocytic cells, preferably macrophages and phagocytic neutrophils, and in particular such cells that are infected with any of a variety of microorganism, most preferably a pathological or disease-causing microorganism. For such cells, the embodiments of the microparticle-conjugated biologically active compounds of the invention are comprised of cleavable linker moieties whereby chemical or enzymatic cleavage of said linker moieties are non-specifically cleaved inside the cells, and in preferred embodiments, inside phagocytic cells, wherein specific activation of the inactive prodrug to the active form of the biologically-active compound is achieved specifically in infected cells. In preferred embodiments, the inactive prodrugs are provided as conjugates of the biologically-active compounds with a polar lipid moiety via a specific linker moiety, wherein the biologically-active compound is activated from the prodrug state in phagocytic cells infected with a microorganism, most preferably a pathological or disease-causing microorganism, via specific cleavage of the linker moiety forming the conjugate between the polar lipid and the biologically-active compound. This provides for the specific release of biologically-active compounds, such as antiviral and antimicrobial drugs, in such infected cells, preferably targeted to specific intracellular targets for more effective delivery of such drugs within an infected phagocytic cell. It is understood that all phagocytic cells will take up such antiviral and antimicrobial embodiments of the microparticle-conjugated biologically active compounds of the invention, and will cleave the cleavable linker so as to release the prodrug form of the biologically-active compound in all phagocytic cells. However, it is an advantageous feature of the microparticle-conjugated biologically active compounds of the invention that specific activation of the inactive prodrug form of the biologically-active compounds is achieved only in phagocytic cells infected with a microorganism, most preferably a pathological or disease-causing microorganism. Release of biologically-active forms of such antiviral and antimicrobial drugs is dependent on the presence of the infectious microorganism in the phagocytic cell, in preferred embodiments, by cleavage of the specific linker moiety comprising the polar lipid-biologically active compound conjugate.

In preferred embodiments of this aspect of the invention, the biologically active compounds of the invention linked to microparticles via the cleavable linker are covalently linked to a polar lipid moiety. Polar lipid moieties comprise one or a plurality of polar lipid molecules. Polar lipid conjugates of the invention are comprised of one or a plurality of polar lipid molecules covalently linked to a biologically-active compound via a specific linker moiety as described above. Such specific linker moieties are provided having two linker functional groups, wherein the linker has a first end and a second end and wherein the polar lipid moiety is attached to the first end of the linker through a first linker functional group and the biologically-active compound is attached to the second end of the linker through a second linker functional group. In these embodiments of the invention, the linker functional groups attached to the first end and second ends of the linker are characterized as "strong" , with reference to the propensity of the covalent bonds between each end of the linker molecule to be broken. In preferred embodiments of this aspect of the invention, the propensity of the covalent bonds between each of the ends of the linker molecule to be broken is low, that is, the polar lipid/biologically active compound conjugate is stable under intracellular physiological conditions in the absence of a chemical or enzymatic moiety specific for cellular infection by a microorganism, most preferably a pathological or disease-causing microorganism. In these embodiments, the specific linker moiety allows the biologically-active compound to accumulate and act at an intracellular site after being released from the microparticle only after having been released from the intracellular targeting polar lipid moiety.

In a particular embodiment of this aspect of the invention, the specific linker moiety is a peptide of formula (amino acid)_n, wherein n is an integer between 2 and 100, preferably wherein the peptide comprises a polymer of one or more amino acids.

In other embodiments of the compositions of matter of the invention, the biologically-active compound of the invention has a first functional linker group, and a polar lipid moiety has a second functional linker group, and the compound is directly covalently linked to the polar lipid moiety by a chemical bond between the first and second functional linker groups. In such embodiments, either the biologically-active compound or the polar lipid moiety comprises yet another functional linker group which is directly covalently linked to the cleavable linker moiety of the invention, which in turn is covalently linked to the microparticle. In preferred embodiments, each of the 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. In particular, in such embodiments the polar lipid/biologically active compound conjugate is preferably specifically cleaved in infected phagocytic mammalian cells. In these embodiments, the biologically-active compound is in an inactive, prodrug state when covalently linked to the polar lipid, which activity of the biologically active compound is restored or increased after the conjugate has been broken.

In the various aspects of the polar lipid conjugates of the invention, preferred polar lipids include but are not limited to acyl carnitine, acylated carnitine, sphingosine, ceramide, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine, cardiolipin and phosphatidic acid.

Preferred biologically active compounds comprising the polar lipid conjugates linked to the microparticles of the invention include antiviral and antimicrobial compounds, drugs, peptides, toxins and other antibiotic agents.

The invention also provides compositions of matter comprising a porous microparticle into which is impregnated an inactive, prodrug form of a biologically-active compound, the impregnated porous microparticle being further coated with a coating material. In this aspect of the invention, the coating material is non-specifically degraded by chemical or enzymatic means inside a cell, preferably a phagocytic mammalian cell, allowing the release of the inactive, prodrug form of the compound from the microparticle. In preferred embodiments, the coating material is a substrate for a protein having an enzymatic activity found in cells, preferably mammalian phagocytic cells. In additional preferred embodiments, the biologically-active compound in inactive, prodrug form is provided as a conjugate of the biologically-active compound with a polar lipid via a specific linker moiety. In such embodiments, activation of the inactive prodrug is specifically accomplished by chemical or enzymatic cleavage of a specific linker moiety between the biologically-active compound and the polar lipid. Most preferably, the biologically-active compound is inactive or has reduced activity in the form of a polar lipid conjugate, wherein the activity of the compound is restored or increased upon specific cleavage of the linker moiety in a particular phagocytic cell that is infected with a microorganism, preferably a pathological or disease-causing microorganism.

In preferred embodiments, specific release of the biologically-active compound in particular phagocytic cells that are infected with a microorganism, most preferably a pathological or disease-causing microorganism is achieved via specific cleavage of a specific linker moiety that forms the conjugate between the polar lipid and the biologically-active compound. In these preferred embodiments, cleavage of the specific linker moiety is achieved by chemical or enzymatic cleavage of the linker moiety between the biologically-active compound and the polar lipid. Preferably, the biologically-active compound is inactive or has reduced activity in the form of a polar lipid conjugate, wherein the activity of the compound is restored or increased upon specific cleavage of the linker moiety in a particular phagocytic cell. In preferred embodiments, the specific linker moiety is enzymatically cleaved by an enzyme that is produced by a microorganism, most preferably a pathological or disease-causing microorganism or which is induced by infection by a microorganism, most preferably a pathological or disease-causing microorganism. In additional preferred embodiments, the specific linker moiety is chemically cleaved under physiological conditions that are specific for phagocytic cells infected with a microorganism, most preferably a pathological or disease-causing microorganism.

Preferred biologically active compounds comprising the polar lipid conjugates used to impregnate such porous microparticles include antiviral and antimicrobial compounds, drugs, peptides, toxins and other antibiotic agents.

In these embodiments, the biologically active compounds of the invention impregnated within porous microparticles are covalently linked to a polar lipid moiety. Polar lipid moieties comprise one or a plurality of polar lipid molecules. Polar lipid conjugates of the invention are comprised of one or a plurality of polar lipid molecules covalently linked to a biologically-active compound via a specific linker moiety as described above. Such specific linker moieties are provided having two linker functional groups, wherein the linker has a first end and a second end and wherein the polar lipid moiety is attached to the first end of the linker through a first linker functional group and the biologically-active compound is attached to the second end of the linker through a second linker functional group. In these embodiments of the invention, the linker functional groups attached to the first end and second ends of the linker are characterized as "strong", with reference to the propensity of the covalent bonds between each end of the linker molecule to be broken. In these embodiments, the specific linker moiety allows the biologically-active compound to accumulate and act at an intracellular site after being released from the microparticle only after having been released from the intracellular targeting polar lipid moiety. In these embodiments, the propensity of the covalent bonds between each of the ends of the linker molecule to be broken is low, that is, the polar lipid/biologically active compound conjugate is stable under intracellular physiological conditions in the absence of a chemical or enzymatic moiety specific for cellular infection by a microorganism, most preferably a pathological or disease-causing microorganism.

In a particular embodiment of this aspect of the invention, the specific linker moiety is a peptide of formula (amino acid)_n, wherein n is an integer between 2 and 100, preferably wherein the peptide comprises a polymer of one or more amino acids.

In other embodiments of the compositions of matter of the invention, the biologically-active compound of the invention has a first functional linker group, and a polar lipid moiety has a second functional linker group, and the compound is directly covalently linked to the polar lipid moiety by a chemical bond between the first and second functional linker groups. In such embodiments, either the biologically-active compound or the polar lipid moiety comprises yet another functional linker group which is directly covalently linked to the cleavable linker moiety of the invention, which in turn is covalently linked to the microparticle. In preferred embodiments, each of the 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. In particular, in such embodiments the polar lipid/biologically active compound conjugate is preferably specifically cleaved in infected phagocytic mammalian cells, wherein the activity of the biologically active compound in restored or increased after the conjugate has been broken.

In the various aspects of the polar lipid conjugates of the invention, preferred polar lipids include but are not limited to acyl carnitine, acylated carnitine, sphingosine, ceramide, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine, cardiolipin and phosphatidic acid.

The invention also provides compositions of matter comprising a nonporous microparticle onto which is coated an inactive, prodrug form of a biologically-active compound, the non-porous microparticle being further coated with a coating material. In this aspect of the invention, the coating material is non-specifically degraded inside a cell, preferably a phagocytic mammalian cell, allowing the release of the inactive, prodrug form of the compound from the microparticle. In preferred embodiments, the coating material is a substrate for a protein having an enzymatic activity found in cells, preferably mammalian phagocytic cells. In additional preferred embodiments, the biologically-active compound in inactive, prodrug form is provided as a conjugate of the biologically-active compound with a polar lipid via a specific linker moiety. In such embodiments, activation of the inactive prodrug is specifically accomplished by chemical or enzymatic cleavage of a specific linker moiety between the biologically-active compound and the polar lipid. Most preferably, the biologically-active compound is inactive or has reduced activity in the form of a polar lipid conjugate, wherein the activity of the compound is restored or increased upon specific cleavage of the linker moiety in a particular phagocytic cell that is infected with a microorganism, preferably a pathological or disease-causing microorganism.

In preferred embodiments, specific release of the biologically-active compound in particular phagocytic cells that are infected with a microorganism, most preferably a pathological or disease-causing microorganism is achieved via specific cleavage of the linker moiety forming the conjugate between the polar lipid and the biologically-active compound. In preferred embodiments, cleavage of the specific linker moiety is achieved by chemical or enzymatic cleavage of the linker moiety between the biologically-active compound and the polar lipid. Preferably, the biologically-active compound is inactive or has reduced activity in the form of a polar lipid conjugate, wherein the activity of the compound is restored or increased upon specific cleavage of the linker moiety in a particular phagocytic cell. In preferred embodiments, the specific linker moiety is enzymatically cleaved by an enzyme that is produced by a microorganism, most preferably a pathological or disease-causing microorganism or which is induced by infection by a microorganism, most preferably a pathological or disease-causing microorganism. In additional preferred embodiments, the specific linker moiety is chemically cleaved under physiological conditions that are specific for phagocytic cells infected with a microorganism, most preferably a pathological or disease-causing microorganism.

In this aspect of the invention, the inactive, prodrug form of the biologically-active compound of the invention will be understood to dissolve from the surface of the microparticle upon enzymatic or chemical degradation of the coating material. Release of the biologically-active compound can be accomplished simply by mass action, i.e., whereby the compound dissolves from the surface of the nonporous microparticle into the surrounding cytoplasm within the cell.

Preferred biologically active compounds used to prepare the coated, non-porous microparticles of this aspect of the invention include antiviral and antimicrobial compounds, drugs, peptides, toxins and other antibiotic agents.

In preferred embodiments, the biologically active compounds of the invention coated onto nonporous microparticles are covalently linked to a polar lipid moiety. Polar lipid moieties comprise one or a plurality of polar lipid molecules. The polar lipid conjugates of the invention are comprised of one or a plurality of polar lipid molecules covalently linked to a biologically-active compound via a specific linker moiety as described above. Such specific linker moieties are provided having two linker functional groups, wherein the linker has a first end and a second end and wherein the polar lipid moiety is attached to the first end of the linker through a first linker functional group and the biologically-active compound is attached to the second end of the linker through a second linker functional group. In these embodiments of the invention, the linker functional groups attached to the first end and second ends of the linker is characterized as "strong", with reference to the propensity of the covalent bonds between each end of the linker molecule to be broken. In preferred embodiments of this aspect of the invention, the specific linker moiety allows the biologically-active compound to act at an intracellular site after being released from the microparticle only after having been released from the intracellular targeting polar lipid moiety. In these embodiments, the propensity of the covalent bonds between each of the ends of the linker molecule to be broken is low, that is, the polar lipid/biologically active compound conjugate is stable under intracellular physiological conditions in the absence of a chemical or enzymatic moiety specific for cellular infection by a microorganism, most preferably a pathological or disease-causing microorganism.

In a particular embodiment of this aspect of the invention, the specific linker moiety is a peptide of formula (amino acid)_n, wherein n is an integer between 2 and 100, preferably wherein the peptide comprises a polymer of one or more amino acids.

In other embodiments of the compositions of matter of the invention, the biologically-active compound of the invention has a first functional linker group, and a polar lipid moiety has a second functional linker group, and the compound is directly covalently linked to the polar lipid moiety by a chemical bond between the first and second functional linker groups. In such embodiments the polar lipid/biologically-active conjugate is the inactive, prodrug form of the biologically-active compound. Said conjugate is impregnated into porous microparticles or coats non-porous microparticles as described above. In preferred embodiments, each of the 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. In particular, in such embodiments the polar lipid/biologically active compound conjugate is preferably specifically cleaved in infected phagocytic mammalian cells. In these embodiments, the biologically-active compound is in an inactive, prodrug state when covalently linked to the polar lipid, which activity of the biologically active compound is restored or increased after the conjugate has been broken.

In the various aspects of the polar lipid conjugates of the invention, preferred polar lipids include but are not limited to acyl carnitine, acylated carnitine, sphingosine, ceramide, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine, cardiolipin and phosphatidic acid.

The invention also provides compositions of matter comprising a biologically-active compound in an inactive, prodrug form, wherein the prodrug is linked to a microparticle via a cleavable linker moiety. The cleavable linker moieties of the invention comprise two linker functional groups, wherein the cleavable linker moiety has a first end and a second end. The microparticle is attached to the first end of the cleavable linker moiety through a first linker functional group and the inactive, prodrug form of the biologically-active compound is attached to the second end of the cleavable linker moiety through a second linker functional group. The cleavable linker moieties of the invention are non-specifically cleaved inside a cell, preferably a phagocytic mammalian cell. In this aspect of the microparticles of the invention, the biologically active compound is provided in a non-biologically active form, wherein the compound is not activated merely by release from the microparticle. Rather, in this aspect of the microparticles of the invention, the biologically-active compound is specifically activated in a cell, preferably a phagocytic mammalian cell, that is infected with a microorganism, most preferably a pathological or disease-causing microorganism. In preferred embodiments, the biologically active compound is specifically activated by an enzymatic activity produced by a microorganism, most preferably a pathological or disease-causing microorganism or which is induced by infection by a microorganism, most preferably a pathological or disease-causing microorganism. In additional preferred embodiments, the biologically active compound is specifically activated by a chemical reaction under physiological conditions that are specific for phagocytic cells infected with a microorganism, most preferably a pathological or disease-causing microorganism.

In this aspect of the invention are also provided embodiments wherein the biologically active compound is covalently linked to a polar lipid moiety. Polar lipid moieties comprise one or a plurality of polar lipid molecules. Polar lipid conjugates of the invention are comprised of one or a plurality of polar lipid molecules covalently linked to a biologically-active compound. In preferred embodiments, activation of the biologically active compound as described above is achieved by specific cleavage of a covalent bond between the biologically active compound and a polar lipid moiety, or by specific cleavage of a specific linker moiety that comprises the conjugate between the polar lipid and the biologically-active compound.

In preferred embodiments of the invention, the biologically-active compound is a peptide. In other preferred embodiments, the biologically-active compound is a drug, most preferably an antiviral or antimicrobial drug. Preferred polar lipids include but are not limited to acyl carnitine, acylated carnitine, sphingosine, ceramide, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine, cardiolipin and phosphatidic acid.

Additional preferred embodiments of the microparticle-conjugated biologically active compounds of the invention also comprise a specific linker moiety wherein activation of the biologically active compound is achieved by specific cleavage of the linker moiety in a cell, preferably a phagocytic cell, infected with a microorganism, most preferably a pathological or disease-causing microorganism.

In a particular embodiment of this aspect of the invention, the specific linker moiety is a peptide of formula (amino acid)_n, wherein n is an integer between 2 and 100, preferably wherein the peptide comprises a polymer of one or more amino acids.

In other embodiments of the compositions of matter of the invention, the biologically-active compound of the invention has a first functional linker group, and a polar lipid moiety has a second functional linker group, and the compound is directly covalently linked to the polar lipid moiety by a chemical bond between the first and second functional linker groups. In such embodiments, either the biologically-active compound or the polar lipid moiety comprises yet another functional linker group which is directly covalently linked to a non-specific cleavable linker moiety of the invention, which in turn is covalently linked to the microparticle. In preferred embodiments, each of the 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. In particular, in such embodiments the polar lipid/biologically active compound conjugate is preferably specifically cleaved in infected phagocytic mammalian cells. In these embodiments, the biologically-active compound is in an inactive, prodrug state when covalently linked to the polar lipid, which activity of the biologically active compound is restored or increased after the conjugate has been broken.

In specific aspects of the invention provided herein are microparticles comprising a drug. In preferred embodiments, the drug is an antiviral or antimicrobial drug.

As disclosed herein, the invention comprehends a microparticle and a polar lipid/biologically-active compound conjugate, preferably comprising a drug, more preferably comprising an antiviral or antimicrobial drug, wherein the conjugate is further covalently linked to a microparticle, impregnated within a microparticle, or coating a microparticle, wherein the microparticles are specifically taken up by cells, preferably phagocytic mammalian cells, and wherein the conjugates of the invention are non-specifically released inside the cell. In preferred embodiments, the conjugates of the invention further comprise a specific linker moiety. The specific linker moiety of the conjugates of the invention preferably releases the drug from the lipid, targets the conjugate to a subcellular organelle, incorporate the drug into a viral envelope, or perform other functions to maximize the effectiveness of the drug, wherein the drug is in an inactive or reduced activity form until it is specifically released from the conjugate in a cell infected with a microorganism, most preferably a pathological or disease-causing microorganism. In other preferred embodiments, the biologically-active compound and the polar lipid are directly linked, preferably covalently linked, and the compound is restored from an inactive, prodrug form to the activity of the biologically-active compound by cleavage of the polar lipid from the biologically-active compound. In yet other preferred embodiments, the biologically-active compound is directly linked to the microparticle, or impregnated within the microparticle, or coats the microparticle, and is specifically restored from an inactive, prodrug form to the activity of the biologically-active compound in a phagocytic cell infected by a microorganism, most preferably a pathological or disease-causing microorganism.

It will be recognized that heterogenous preparations of said microparticles of the invention, comprising either different microparticles conjugated, impregnated or coated with different biologically-active compounds, or one particular species conjugated, impregnated or coated with different biologically-active compounds of the invention, explicitly fall within the scope of the invention disclosed and claimed herein. Said preparations will be understood to comprise a multiplicity of the biologically-active compounds of the invention, preferably provided in an inactive, prodrug form.

The microparticle-drug conjugates of this invention have numerous advantages. First, the drug-microparticle conjugates are specifically taken up by cells, particularly phagocytic mammalian cells. Also, drugs, preferably antiviral and antimicrobial drugs comprising the drug-microparticle conjugates of the invention, are linked to the microparticle by a cleavable linker moiety that is non-specifically cleaved upon entry into phagocytic cells. More importantly, the drugs, preferably antiviral and antimicrobial drugs, are also preferably conjugated with a polar lipid, most preferably via a specific linker moiety. In this form, the drugs have reduced or inhibited biological activity, which activity is restored upon chemical or enzymatic cleavage of the specific linker moiety in appropriate phagocytic cells, for example, phagocytic cells infected with a microorganisms, preferably a pathological or disease-causing microorganism. Third, the drug-polar lipid conjugates of the invention will promote the intracellular targeting of a variety of potentially useful antiviral or antimicrobial drugs at pharmacokinetic rates not currently attainable. In this aspect, the range of targeted subcellular organelles is not limited per se by, for example, any particular, limited biological properties of the subcellular organelle such as the number and type of specific receptor molecules expressed by the organelle. In contrast to traditional attempts to simply target drugs to specific cells, this method may target drugs to specific intracellular organelles and other intracellular compartments. Fourth, the compositions of matter of the invention incorporate polar lipid/drug conjugates comprising a variable specific linker region that may allow pharmacologically-relevant rates of drug release from polar lipid moieties to be engineered into the compositions of the invention, thereby increasing their clinical efficacy and usefulness. Thus, time-dependent drug release and specific drug release in cells expressing the appropriate degradative enzymes are uniquely available using the microparticle-drug-lipid conjugates of the invention. Fifth, 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. One example of antiviral therapy would involve incorporating the conjugates of the invention into the viral envelope, thereby directly modifying its lipid composition and influencing viral infectivity. Finally, the prodrug-microparticle conjugates of the invention specifically encompass prodrugs which are biologically inactive unless and until pathogen infection-specific chemical or enzymatic cleavage converts such prodrugs into an active drug form inside a phagocytic mammalian cell.

Thus, the invention also provides a method of killing a microorganism infecting a mammalian cell. This method comprises contacting an infected phagocytic mammalian cells with the compositions of matter of the invention. The invention also provides a method for treating a microbial infection in a human wherein the infecting microbe is present inside a phagocytic cell in the human, the method comprising administering a therapeutically effective amount of the compositions of matter of the invention to the human in a pharmaceutically acceptable carrier. Thus, the invention also provides pharmaceutical compositions comprising the compositions of matter of the invention in a pharmaceutically acceptable carrier.

Thus, in a first aspect the invention provides compositions of matter for targeting biologically active compounds to phagocytic cells. In a second aspect, the invention provides compositions of matter and methods for the specific release of biologically active compounds inside phagocytic cells. The invention in yet a third aspect provides methods and compositions for intracellular delivery of targeted biologically active compounds to phagocytic cells. The invention also provides for organelle-specific intracellular targeting of biologically active compounds, specifically to phagolysosomes and other subcellular structures including but not limited to the endoplasmic reticulum, the Golgi apparatus, mitochondria and the nucleus. In this aspect of the invention are also provided compositions and methods for organelle specific intracellular targeting using polar lipid moiety-linked compounds. In each of these aspects is provided methods and compounds for introducing biologically active compounds into phagocytic mammalian cells wherein the unconjugated compound would not otherwise enter said phagocytic cell. In this aspect is included the introduction of said biologically active compounds in chemical embodiments that would not otherwise enter the cell, for example, as phosphorylated embodiments. In yet another aspect is provided methods and compositions for the specific coordinate targeting of more than one biologically active compound to a specific cell type, that is, phagocytic mammalian cells. In another aspect, the invention provides reagents and compositions for introduction and specific release of antiviral or antimicrobial drugs and other biologically-active compounds into cells infected by a pathological microorganism. In a final aspect, the invention provides methods and reagents for delayed, sustained or controlled intracellular release of biologically active compounds conjugated to a micropartricle, or impregnated within a coated, porous microparticle, or coated onto a nonporous microparticle, wherein the degradation of either the coating, the cleavable linker, the specific linker moiety, the microparticle or any of these activity control points provides said delayed, sustained or controlled intracellular release of the biologically active compound of the invention.

Claim 1 of 73 Claims

What is claimed is:

1. A composition ofmaner comprising prodrugs of one or a multiplicity of biologically-active compounds, a microparticle and a cleavable linker moiety comprising two linker fulnctional groups, wherein the cleavable linker moiety has a first end and a second end and wherein the microparticle is attached to the first end of the linker moiety through a first linker functional group and each of the prodrugs are attached to the second end of the linker moiety through a second linker functional group, and wherein the cleavable linker moiety is non-specifically cleaved inside a phagocytic mammalian cell, and wherein each of the prodrugs further comprises a polar lipid moiety comprised of one or a plurality of polar lipid molecules and a specific linker moiety, wherein the specific linker moiety is covalently linked to the prodrug and the polar lipid moiety, wherein the specific linker moiety is specifically cleaved in a phagocytic mammalian cell infected with a microorganism and is specifically activated thereby.

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