Protease inhibitors having enhanced features   

This application claims the benefit of priority to the following: (i) U.S. Provisional Application No. 61/192,417 filed Sep. 17, 2008, (ii) U.S. Provisional Application No. 61/192,439 filed Sep. 17, 2008, (iii) U.S. Provisional Application No. 61/198,934 filed Nov. 12, 2008, and (iv) U.S. Provisional Application No. 61/192,459 filed Sep. 17, 2008, the disclosures all of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention provides (among other things) protease inhibitor compounds having enhanced features, along with methods for administering such compounds. The subject compounds can be administered without concomitant administration of a CYP3A4 inhibitor, have increased therapeutic index and/or increased potency, and are low-resistance inducing in nature. The methods and active agents described herein relate to and/or have applications in (among others) the fields of pharmacotherapy, physiology, organic chemistry and polymer chemistry.

Since the first cases of acquired immunodeficiency syndrome (AIDS) were reported in 1981, infection with human immunodeficiency virus (HIV) has grown to pandemic proportions, resulting in an estimated 65 million infections and 25 million deaths. See Aug. 11, 2006, MMWR 55(31):841-844 (Center for Disease Control and Prevention). Protease inhibitors represent an important class of compounds used to treat individuals infected with HIV, although these compounds can also treat individuals suffering from other viral infections (e.g., Hepatitis C).

With respect to HIV, protease inhibitors act to inhibit the HIV viral proteases that are necessary for the proteolytic cleavage of the gag and gag/pol fusion polypeptides necessary for the generation of infective viral particles. Thus, by inhibiting this proteolytic cleavage, protease inhibitors diminish the ability of larger HIV-fusion polypeptide precursors to generate the mature form of protein necessary for effective viral replication. McQuade et al. (1990) Science 247(4941):454-456.

Protease inhibitor-based therapy is acknowledged as an initial treatment for patients presenting symptomatic HIV disease and in non-symptomatic patients after the CD4 cell count is below 350/μL but before a level of 200/μL. Hammer et al. (2006) JAMA 296(7):827-843. In such cases, a protease inhibitor-based regimen will include a protease inhibitor (typically boosted with ritonavir) along with a combination of two nucleoside (or nucleotide) reverse transcriptase inhibitors. Id.

These conventional HIV protease inhibitors, as well as other protease inhibitors, have relatively low potency and/or relatively low (or narrow) therapeutic index.

HIV and other protease inhibitors having a relatively high potency and/or relatively high (or wide) therapeutic index would represent an improvement over conventional HIV protease inhibitors.

Moreover, although protease inhibitors serve an important role in treating patients suffering from HIV as well as hepatitis virues (e.g., hepatitis C virus), their use has been hampered by challenges associated with (among other things) limited oral bioavailability and lack of patient compliance due to the frequency of dosing and tolerability issues. Zeldin et al. (2004) J. Antimicrob. Chemother. 53:4-9. The lack of patient compliance, in turn, may lead to the development of resistant viral strains among patients treated with single PI regimens. Id.

In order to prevent or overcome resistance, concomitant administration with ritonavir—an inhibitor of cytochrome P-450 (CYP-450) and a protease inhibitor itself—has been used and has shown demonstrated efficacy in clinical studies. See Rathburn et al. (2002) Ann. Pharmacother. 36:702-706, Moyle et al. (2001) HIV Medicine 2:105-113, Flexner (2000) Ann. Rev. Pharm. Tox. 40:649-674, and Yu et al. (2000) Expert Opin. Pharmacother. 1:1331-1342. Interestingly, the dose of ritonavir administered in “boosted” protease inhibitor-based regimens is generally considered subtherapeutic. See Moyle et al. (2001) HIV Medicine 2:105-113.

Finally, sustained treatment with HIV protease inhibitors, however, has been found to lead to the generation of resistant HIV strains, which no longer respond to the protease inhibitor therapy. Such resistance is believed to be a consequence of mutations arising during viral replication that eventually lead to amino acid changes, which alter the binding interaction of the viral protease with the protease inhibitor and thus render the drug ineffective at preventing viral replication. In view of the chronic nature of HIV infection, the generation of resistant strains over the course of long-term therapy is particularly troubling.

The present disclosure seeks to address these and other needs in the art.

SUMMARY

In one or more aspects, a method is provided, the method comprising administering a HIV protease inhibitor conjugate to an individual infected with HIV, wherein the HIV protease inhibitor conjugate has an increased therapeutic index and/or increased potency. Preferably, the administering step has a dose (on a molar basis) of an HIV protease inhibitor conjugate that is both (i) different than (and preferably lower than) the corresponding HIV protease inhibitor in unconjugated form, and (ii) retains at least the same (or substantially the same) HIV protease inhibitor activity on a molar basis in a suitable model or patient.

In one or more embodiments, the administering step has a dose (on a molar basis) of an HIV protease inhibitor conjugate that provides greater HIV protease inhibitor activity on a molar basis in a suitable model or patient than the same dose (on a molar basis) of the corresponding HIV protease inhibitor in unconjugated form.

In yet another aspect, provided is a method comprising increasing the potency of a small drug molecule such as an HIV protease inhibitor by covalently attaching a water-soluble oligomer to the small drug molecule.

In one or more aspects, a compound is provided, the compound comprising a small drug molecule covalently attached to a water-soluble oligomer, wherein the potency of the small drug molecule covalently attached to the water-soluble oligomer has a greater potency than the small drug molecule in unconjugated form.

In yet a further aspect, a method is provided, the method comprising administering a potent protease inhibitor (e.g., an HIV protease inhibitor or a hepatitis virus protease inhibitor such as a HCV protease inhibitor) therapy to an individual infected with a virus, wherein said potent protease inhibitor therapy does not include co-administration of a CYP3A4 inhibitor.

In one or more related embodiments, the potent protease inhibitor is administered in a CYP3A4-competent biological system.

In one or more additional aspects, a method is provided, the method comprising administering an HIV protease inhibitor conjugate as a protease inhibitor monotherapy to a biological system infected with HIV, wherein: (i) in a biological model in which the HIV protease inhibitor conjugate is periodically added in a given molar amount over time, and in the same biological model in which a corresponding HIV protease inhibitor in unconjugated form is periodically added over time in the same given molar amount, the biological model in which the corresponding HIV protease inhibitor in unconjugated form is added is more likely to exihibit HIV protease resistance, and (ii) the HIV protease inhibitor conjugate retains at least the same (or substantially the same) HIV protease inhibitor activity on a molar basis in a suitable model or patient.

These and other objects, aspects, embodiments and features of the invention will become more fully apparent when read in conjunction with the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E provide five graphs showing p24 levels in response to PI (protease inhibitor) and PEGoligo-PI addition at various times after high MOI HIV-1 infection, as further described in Example 1. CEM-SS cells were infected with HIV-1 for one hour, and then washed free from excess virus. Test compounds were added at various times after infection, and p24 levels were measured by ELISA at 30 hours post-infection. Values are derived from a single experiment performed in triplicate. FIG. 1A (Saquinavir, SQV); FIG. 1B (mPEG7-NHCO-Saquinavir); FIG. 1C (Nevirapine); FIG. 1D (Ritonavir); FIG. 1E (Chicago Sky Blue).

FIGS. 2A-2D are graphs comparing the metabolic stability of PEGoligo-protease inhibitor conjugates and protease inhibitor molecules following incubation with cryopreserved human hepatocytes, as further described in Example 2. Values were obtained using 3 μM test compound concentration. FIG. 2A (Atazanavir, di-mPEG3,5,7-atazanavir); FIG. 2B (Darunavir, mPEG3,5,7-N-darunavir); FIG. 2C (Tipranavir, mPEG3,5,7-amide-tipranavir); FIG. 2D (Saquinavir, PEG3,5,7N-Saquinavir).

FIGS. 3A-3E are graphs showing the stability of PEGoligo-protease inhibitor conjugates and protease inhibitor molecules expressed as values (percent compound remaining) normalized to the 30 minute timepoint. FIG. 3A (Atazanuvir, di-mPEG3-atazanavir, mono-mPEG3-atazanavir); FIG. 3B (Darunavir, mPEG-3-N-darunavir, mPEG-5-N-darunavir, mPEG7-O-darunavir); FIG. 3C (Darunavir, mPEG-3-O-darunavir, mPEG-5-O-darunavir, mPEG-7-O-darunavir; FIG. 3D (Saquinavir, mPEG3-NHCO-saquinavir, mPEG5-NHCO-saquinavir, mPEG7-NHCO-saquinavir); and FIG. 3E (Tipranavir, mPEGO-OCO—NH-tipranavir, mPEG1-OCO—NH-tipranavir, mPEG3-OCO—NH-tipranavir, and mPEG5-OCO—NH-tipranavir) as further described in Example 2. Values obtained using 1 μM test compound concentration.

FIGS. 4A-4F are graphs showing the metabolic stability of PEG-protease inhibitors following incubation with CYP3A4- or CYP2D6-expressing Bactosomes™, as further described in Example 2 FIG. 4A (CYP3A4 Bactosome Stability, Atazanavir, di-mPEG3-atazanavir); FIG. 4B (CYP2D6 Bactosome Stability, Atazanavir, di-mPEG3-atazanavir); FIG. 4C (CYP3A4 Bactosome Stability, Darunavir, mPEG-3-N-darunavir, mPEG-5-N-darunavir, mPEG7-O-darunavir); FIG. 4D (CYP2D6 Bactosome Stability, Darunavir, mPEG-3-N-darunavir, mPEG-5-N-darunavir, mPEG7-O-darunavir), FIG. 4E (CYP3A4 Bactosome Stability, Saquinavir, mPEG3-NHCO-saquinavir, mPEG5-NHCO-saquinavir, mPEG7-NHCO-saquinavir); and FIG. 4F (CYP2D6 Bactosome Stability, Saquinavir, mPEG3-NHCO-saquinavir, mPEG5-NHCO-saquinavir, mPEG7-NHCO-saquinavir).

DETAILED DESCRIPTION

As used in this specification, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

In describing and claiming the present invention, the following terminology will be used in accordance with the definitions described below.

“Water soluble, non-peptidic oligomer” indicates an oligomer that is at least 35% (by weight) soluble, preferably greater than 70% (by weight), and more preferably greater than 95% (by weight) soluble, in water at room temperature. Typically, an unfiltered aqueous preparation of a “water-soluble” oligomer transmits at least 75%, more preferably at least 95%, of the amount of light transmitted by the same solution after filtering. It is most preferred, however, that the water-soluble oligomer is at least 95% (by weight) soluble in water or completely soluble in water. With respect to being “non-peptidic,” an oligomer is non-peptidic when it has less than 35% (by weight) of amino acid residues.

An “oligomer” is a molecule possessing from about 2 to about 50 monomers, preferably from about 2 to about 30 monomers. The architecture of an oligomer can vary. Specific oligomers for use in the invention include those having a variety of geometries such as linear, branched, or forked, to be described in greater detail below.

“PEG” or “polyethylene glycol,” as used herein, is meant to encompass any water-soluble poly(ethylene oxide). Unless otherwise indicated, a “PEG oligomer” (also called an oligoethylene glycol) is one in which substantially all (and more preferably all) monomeric subunits are ethylene oxide subunits. The oligomer may, however, contain distinct end capping moieties or functional groups, e.g., for conjugation. Typically, PEG oligomers for use in the present invention will comprise one of the two following structures: “—(CH2CH2O)n—” or “—(CH2CH2O)n-1CH2CH2—,” depending upon whether the terminal oxygen(s) has been displaced, e.g., during a synthetic transformation. For PEG oligomers, “n” varies from about 2 to 50, preferably from about 2 to about 30, and the terminal groups and architecture of the overall PEG can vary. When PEG further comprises a functional group, A, for linking to, e.g., a small molecule drug, the functional group when covalently attached to a PEG oligomer does not result in formation of (i) an oxygen-oxygen bond (—O—O—, a peroxide linkage), or (ii) a nitrogen-oxygen bond (N—O, O—N).

A “reduced rate of metabolism” in reference to the present invention, refers to a measurable reduction in the rate of metabolism of a water-soluble oligomer-small molecule drug conjugate as compared to rate of metabolism of the small molecule drug not attached to the water-soluble oligomer (i.e., the small molecule drug itself) or a reference standard material. In the special case of “reduced first pass rate of metabolism,” the same “reduced rate of metabolism” is required except that the small molecule drug (or reference standard material) and the corresponding conjugate are administered orally. Orally administered drugs are absorbed from the gastro-intestinal tract into the portal circulation and must pass through the liver prior to reaching the systemic circulation. Because the liver is the primary site of drug metabolism or biotransformation, a substantial amount of drug can be metabolized before it ever reaches the systemic circulation. The degree of first pass metabolism, and thus, any reduction thereof, can be measured by a number of different approaches. For instance, animal blood samples can be collected at timed intervals and the plasma or serum analyzed by liquid chromatography/mass spectrometry for metabolite levels. Other techniques for measuring a “reduced rate of metabolism” associated with the first pass metabolism and other metabolic processes are known in the art, described herein and/or in the relevant literature, and/or can be determined by one of ordinary skill in the art. Preferably, a conjugate of the invention can provide a reduced rate of metabolism reduction satisfying at least one of the following values: at least about 30%; at least about 40%; at least about 50%; at least about 60%; at least about 70%; at least about 80%; and at least about 90%. A compound (such as a small molecule drug or conjugate thereof) that is “orally bioavailable” is one that preferably possesses a bioavailability when administered orally of greater than 25%, and preferably greater than 70%, where a compound\'s bioavailability is the fraction of administered drug that reaches the systemic circulation in unmetabolized form.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to 20 atoms in length. Such hydrocarbon chains are preferably but not necessarily saturated and may be branched or straight chain, although typically straight chain is preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl, and the like. As used herein, “alkyl” includes cycloalkyl when three or more carbon atoms are referenced. An “alkenyl” group is an alkyl of 2 to 20 carbon atoms with at least one carbon-carbon double bond.

The terms “substituted alkyl” or “substituted Cq-r alkyl” where q and r are integers identifying the range of carbon atoms contained in the alkyl group, denotes the above alkyl groups that are substituted by one, two or three halo (e.g., F, Cl, Br, I), trifluoromethyl, hydroxy, C1-7 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, butyl, t-butyl, and so forth), C1-7 alkoxy, C1-7 acyloxy, C3-7 heterocyclic, amino, phenoxy, nitro, carboxy, carboxy, acyl, cyano. The substituted alkyl groups may be substituted once, twice or three times with the same or with different substituents.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbon atoms, and may be straight chain or branched, as exemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl. “Lower alkenyl” refers to a lower alkyl group of 2 to 6 carbon atoms having at least one carbon-carbon double bond.

“Non-interfering substituents” are those groups that, when present in a molecule, are typically non-reactive with other functional groups contained within the molecule.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substituted alkyl, preferably C1-C20 alkyl (e.g., methoxy, ethoxy, propyloxy, benzyl, etc.), preferably C1-C7.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” refers to component that can be included in the compositions of the invention in order to provide for a composition that has an advantage (e.g., more suited for administration to a patient) over a composition lacking the component and that is recognized as not causing significant adverse toxicological effects to a patient.

The term “aryl” means an aromatic group having up to 14 carbon atoms. Aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl, naphthacenyl, and the like. “Substituted phenyl” and “substituted aryl” denote a phenyl group and aryl group, respectively, substituted with one, two, three, four or five (e.g. 1-2, 1-3 or 1-4 substituents) chosen from halo (F, Cl, Br, I), hydroxy, hydroxy, cyano, nitro, alkyl (e.g., C1-6 alkyl), alkoxy (e.g., C1-6 alkoxy), benzyloxy, carboxy, aryl, and so forth.

An “aromatic-containing moiety” is a collection of atoms containing at least aryl and optionally one or more atoms. Suitable aromatic-containing moieties are described herein.

For simplicity, chemical moieties are defined and referred to throughout primarily as univalent chemical moieties (e.g., alkyl, aryl, etc.). Nevertheless, such terms are also used to convey corresponding multivalent moieties under the appropriate structural circumstances clear to those skilled in the art. For example, while an “alkyl” moiety generally refers to a monovalent radical (e.g., CH3—CH2—), in certain circumstances a bivalent linking moiety can be “alkyl,” in which case those skilled in the art will understand the alkyl to be a divalent radical (e.g., —CH2—CH2—), which is equivalent to the term “alkylene.” (Similarly, in circumstances in which a divalent moiety is required and is stated as being “aryl,” those skilled in the art will understand that the term “aryl” refers to the corresponding divalent moiety, arylene). All atoms are understood to have their normal number of valences for bond formation (i.e., 4 for carbon, 3 for N, 2 for O, and 2, 4, or 6 for S, depending on the oxidation state of the S).

“Pharmacologically effective amount,” “physiologically effective amount,” and “therapeutically effective amount” are used interchangeably herein to mean the amount of a water-soluble oligomer-small molecule drug conjugate present in a composition that is needed to provide a threshold level of active agent and/or conjugate in the bloodstream or in the target tissue. The precise amount will depend upon numerous factors, e.g., the particular active agent, the components and physical characteristics of the composition, intended patient population, patient considerations, and the like, and can readily be determined by one skilled in the art, based upon the information provided herein and available in the relevant literature.

The term “biological system” is a collection of living cells and includes both a collection of living cells as well as living organisms. With respect to living organisms, the biological system includes mammalian individuals such as a patient, which refers to a living organism suffering from or prone to a condition that can be prevented or treated following the methods described herein.

“Optional” or “optionally” means that the subsequently described circumstance may but need not necessarily occur, so that the description includes instances where the circumstance occurs and instances where it does not.

As indicated above, the present disclosure is directed to the unexpected advantages and features of the subject protease inhibitor conjugates, and methods related thereto.

In a first aspect, the disclosure is directed to (among other things) a method comprising administering an HIV protease inhibitor conjugate to an individual infected with HIV, wherein the potent HIV protease has an increased therapeutic index and/or increased potency. Preferably, the administering step has a dose (on a molar basis) of an HIV protease inhibitor conjugate that: (a) is both (i) different than (and preferably lower than) the corresponding HIV protease inhibitor in unconjugated form, and (ii) retains at least the same (or substantially the same) HIV protease inhibitor activity on a molar basis in a suitable model or patient; and/or (b) provides greater HIV protease inhibitor activity on a molar basis in a suitable model or patient than the same dose (on a molar basis) of the corresponding HIV protease inhibitor in unconjugated form.

HIV proteases such as atazanavir may have an amphiphilic pocket close to the protease binding site. Current protease inhibitors bind to the binding site in a manner that does not engage the amphiphilic pocket specifically. Without wishing to be found by theory, conjugation of a flexible water-soluble oligomer to the protease inhibitor enables (relevant bonding patterns that lead to) higher affinity interaction between the protease inhibitor and the HIV protease. This is believed to lead to higher potency.

Further, toxicity effects mediated through interaction with other targets are not significantly altered. As such, the therapeutic index, defined as the ratio of toxic EC50 (TC50) to efficacious EC50, is thus increased. Examples of such HIV protease inhibitor conjugates include conjugates of atazanavir, darunavir and tipranavir, wherein such conjugates, as well as methods for their synthesis, are described herein and in PCT/US2008/003354 (WO2008/112289).

In accordance with one or more of the methods described herein, the potent HIV protease inhibitor must be administered. Any route suited for delivery of the potent HIV protease inhibitor to the biological system (e.g., individual) can be used. If, for example, the biological system is a cell culture, administration can simply involve adding, via a pipette or dropper (for example), an aliquot of liquid containing the potent HIV protease inhibitor. To the extent that the biological system is an individual infected with HIV, administering the potent HIV protease inhibitor can take place via oral administration, but other modes of administration are also contemplated, such as pulmonary, nasal, buccal, rectal, sublingual, transdermal, and parenteral.

In a second aspect, the present disclosure is directed to (among other things) a method comprising administering a potent protease inhibitor therapy to an individual infected with, wherein said potent protease inhibitor therapy does not include co-administration of a CYP3A4 inhibitor.

With respect to an HIV protease inhibitor, an active agent is an HIV protease inhibitor if it has inhibitory activity against the HIV viral proteases that are necessary for the proteolytic cleavage of the gag and gag/pol fusion polypeptides necessary for the generation of infective viral particles. Several active agents that act by this mechanism have been approved; such compounds include saquinavir, ritonavir, indinavir, amprenavir, lopinavir, atazanavir, fosamprenavir, tipranavir and darunavir. Assays known to those of ordinary skill in the art can be used to determine whether any given active agent is an HIV protease inhibitor (e.g., an HIV-1 protease inhibitor).

With regard to one or more embodiments, however, the protease inhibitor is not simply a conventional protease inhibitor. Rather, the protease inhibitor must qualify as a potent protease inhibitor, when indicated as such herein, such that concomitant administration with a CYP3A4 inhibitor is not required to effect protease inhibition in the biological system of interest.

As an initial matter, a potent HIV protease inhibitor acts via the same pharmacologic mechanism as known HIV protease inhibitors. Thus, while a potent HIV protease inhibitor shares the same mechanism of action as saquinavir, ritonavir, indinavir, amprenavir, lopinavir, atazanavir, fosamprenavir, tipranavir and darunavir, the potent HIV inhibitor for use in this particular aspect of the invention is generally not selected from the group consisting of saquinavir, ritonavir, indinavir, amprenavir, lopinavir, atazanavir, fosamprenavir, tipranavir and darunavir. Similarly, a potent hepatitis virus protease inhibitor (such as a HCV protease inhibitor) acts via the same pharmacologic mechanism as known hepatitis virus protease inhibitors. Thus, for example, while a potent HCV protease inhibitor shares the same mechanism of action as NS3 protease inhibitors (e.g., telaprevir, boceprevir and ITMN-191), the potent HIV inhibitor for use in the subject aspect of the present invention is generally not selected from the group consisting of telaprevir, boceprevir and ITMN-191.

A potent protease inhibitor (e.g., a potent HIV protease inhibitor or a potent hepatitis virus protease inhibitor such as a potent HCV protease inhibitor), however, is a protease inhibitor that remains substantially unchanged in (or is stable throughout) a four-hour in vitro hepatocyte stability assay. It is preferred that the potent protease inhibitor not only remains substantially unchanged in (or is stable throughout) a four-hour in vitro hepatocyte stability assay, but also (at equilibrium) substantially exists (that is, greater than 50% by weight exists) outside the hepatocyte cytoplasm. In the instances where a potent protease inhibitor does (at equilibrium) substantially exist inside the hepatocyte cytoplasm, the potent protease inhibitor is not substantially metabolized by cytochrome P450 3A4. While not wishing to be bound by theory, it is believed that an active agent having protease inhibitor activity is potent if it avoids degradation via cytochrome P450 3A4 enzyme-containing hepatocytes. Avoidance of degradation by cytochrome P450 3A4 enzyme-containing hepatocytes can occur following administration by, for example, the protease inhibitor localizing (at equilibrium) outside cytochrome P450 3A4 enzyme-containing cytoplasm of hepatocytes, or, if the protease inhibitor does substantially penetrate into the cytochrome P450 3A4 enzyme-containing cytoplasm of hepatocytes, the protease inhibitor is not substantially metabolized by the cytochrome P450 3A4 enzyme.

A protease inhibitor remains unchanged (or is stable) when the number and type of atoms and type of bond between those atoms making up the protease inhibitor at the beginning of the four-hour in vitro hepatocyte stability assay are the same at the end of the four hour in vitro hepatocyte stability assay. With respect to being “substantially unchanged,” (or “stable”) it is possible to determine whether an protease inhibitor is substantially unchanged (or stable) by performing the following test. First, it is necessary to perform the following calculation: {[(amount of the unchanged amount of the protease inhibitor following the four hour in vitro hepatocyte stability assay) subtracted from (amount of the beginning amount of the protease inhibitor introduced to the four hour in vitro hepatocyte stability assay)] divided by (amount of the beginning amount of the protease inhibitor introduced to the four-hour in vitro hepatocyte stability assay)) multiplied by 100 to provide a % value. If the % value result falls within one or more of the following ranges, then the protease inhibitor is “substantially unchanged” (or stable) and is a potent protease inhibitor (e.g., a potent HIV protease inhibitor or a potent hepatitis virus protease inhibitor such as a potent HCV protease inhibitor): less than 25% of the beginning amount is changed; less than 20% of the beginning amount is changed; less than 15% of the beginning amount is changed; less than 10% of the beginning is changed; less than 8% of the beginning amount is changed; less than 6% of the beginning amount is changed; less than 5% of the beginning amount is changed; less than 4% of the beginning amount is changed; less than 3% of the beginning amount is changed; less than 2% of the beginning amount is changed; and less than 1% of the beginning amount is changed. In some circumstances, there may be no detectable change.

In yet a further aspect, provided herein (among other things) is a method comprising administering an HIV protease inhibitor conjugate as a protease inhibitor monotherapy to a biological system infected with HIV, wherein in a biological model in which the HIV protease inhibitor conjugate is periodically added in a given molar amount over time, and in the same biological model in which a corresponding HIV protease inhibitor in unconjugated form is periodically added over time in the same given molar amount, (i) the biological model in which the corresponding HIV protease inhibitor is in unconjugated form is more likely to exhibit HIV protease resistance, and (ii) the conjugate retains at least the same (or substantially the same) HIV protease inhibitor activity on a molar basis in a suitable model or patient.

Examples of HIV protease inhibitor conjugates include conjugates of atazanavir, darunavir and tipranavir, wherein such conjugates, as well as methods for their synthesis, are described herein and in PCT/US2008/003354. Examples of HIV protease inhibitor conjugates believed to be useful in one or more of the methods described herein are herein referred to as “potent HIV protease inhibitor.”

In one or more embodiments, structures of HIV protease inhibitors are provided. These structures are preferably, although not necessarily, potent protease inhibitors. Exemplary HIV protease inhibitors include those of the following formula:

Mono-mPEGn-Atazanavir (n=1, 3, 5, 6, 7);

mPEGn-N-Darunavir [wherein mPEGn is —(CH2CH2O)n—CH3 and n=3, 5, 7];

mPEGn-O-Darunavir (wherein n=3, 5, 7);

mPEGn-NHCO-Saquinavir [wherein each mPEGn is —(CH2CH2O)n—CH3 and n=5 or 7]; and

Di-mPEG3-Atazanavir [wherein each mPEGn is —(CH2CH2O)n—CH3 and n=3]. Although the foregoing HIV protease inhibitors are nonlimiting examples of potent HIV protease inhibitors, any HIV protease inhibitor that qualifies as a “potent HIV protease inhibitor” can be used in one or more embodiments of the invention.

Exemplary potent HIV protease inhibitors are mono-mPEG3-atazanavir, mPEGn-N-darunavir (wherein n is 5 or 7), mPEGn-O-darunavir (wherein n is 3 or 5), mPEGn-NHCO-saquinavir (wherein n is 5 or 7), and di-mPEG3-atazanavir. Preferred potent HIV protease inhibitors include mono-mPEG3-atazanavir, mPEGn-N-darunavir (wherein n is 5 or 7), mPEGn-O-darunavir (wherein n is 3 or 5). Such potent HIV protease inhibitors, as well as methods for their synthesis, are described herein and in PCT/US2008/003354.

Candidates that may qualify as a potent HCV protease inhibitor in accordance with the definitions provided herein are described in U.S. provisional application entitled “Oligomer-Protease Inhibitor Conjugates,” filed on Sep. 17, 2008 (Applicant\'s reference number: SHE0216.PRO) and assigned U.S. Provisional Patent Application No. 61/192,438.

An HIV protease inhibitor will generally have a molecular weight of less than 1000 Da. Exemplary molecular weights include molecular weights of: less than about 950; less than about 900; less than about 850; less than about 800; less than about 750; less than about 700; less than about 650; less than about 600; less than about 550; less than about 500; less than about 450; less than about 400; less than about 350; and less than about 300.

An HIV protease inhibitor, if chiral, may be in a racemic mixture, or an optically active form, for example, a single optically active enantiomer, or any combination or ratio of enantiomers (i.e., scalemic mixture). In addition, the potent HIV protease inhibitor may possess one or more geometric isomers. With respect to geometric isomers, a composition can comprise a single geometric isomer or a mixture of two or more geometric isomers. A potent HIV protease inhibitor for use in the present invention can be in its customary active form, or may possess some degree of modification. For example, a potent HIV protease inhibitor may have a targeting agent, tag, or transporter attached thereto, prior to or after covalent attachment of an oligomer. Alternatively, the potent HIV protease inhibitor may possess a lipophilic moiety attached thereto, such as a phospholipid (e.g., distearoylphosphatidylethanolamine or “DSPE,” dipalmitoylphosphatidylethanolamine or “DPPE,” and so forth) or a small fatty acid. In some instances, however, it is preferred that the potent HIV protease inhibitor does not include attachment to a lipophilic moiety.

The water-soluble, non-peptidic oligomer typically comprises one or more monomers serially attached to form a chain of monomers. The oligomer can be formed from a single monomer type (i.e., is homo-oligomeric) or two or three monomer types (i.e., is co-oligomeric). Preferably, each oligomer is a co-oligomer of two monomers or, more preferably, is a homo-oligomer.

Accordingly, each oligomer is composed of up to three different monomer types selected from the group consisting of: alkylene oxide, such as ethylene oxide or propylene oxide; olefinic alcohol, such as vinyl alcohol, 1-propenol or 2-propenol; vinyl pyrrolidone; hydroxyalkyl methacrylamide or hydroxyalkyl methacrylate, where alkyl is preferably methyl; α-hydroxy acid, such as lactic acid or glycolic acid; phosphazene, oxazoline, amino acids, carbohydrates such as monosaccharides, saccharide or mannitol; and N-acryloylmorpholine. Preferred monomer types include alkylene oxide, olefinic alcohol, hydroxyalkyl methacrylamide or methacrylate, N-acryloylmorpholine, and α-hydroxy acid. Preferably, each oligomer is, independently, a co-oligomer of two monomer types selected from this group, or, more preferably, is a homo-oligomer of one monomer type selected from this group.

The two monomer types in a co-oligomer may be of the same monomer type, for example, two alkylene oxides, such as ethylene oxide and propylene oxide. Preferably, the oligomer is a homo-oligomer of ethylene oxide. Usually, although not necessarily, the terminus (or termini) of the oligomer that is not covalently attached to a small molecule is capped to render it unreactive. Alternatively, the terminus may include a reactive group. When the terminus is a reactive group, the reactive group is either selected such that it is unreactive under the conditions of formation of the final oligomer or during covalent attachment of the oligomer to a small molecule drug, or it is protected as necessary. One common end-functional group is hydroxyl or —OH, particularly for oligoethylene oxides.

A water-soluble, non-peptidic oligomer (e.g., “POLY” in the conjugate formula)

) as described herein can have any of a number of different geometries. For example, the water-soluble, non-peptidic oligomer can be linear, branched, or forked. Most typically, the water-soluble, non-peptidic oligomer is linear or is branched, for example, having one branch point. Although much of the discussion herein is focused upon poly(ethylene oxide) as an illustrative oligomer, the discussion and structures presented herein can be readily extended to encompass any of the water-soluble, non-peptidic oligomers described above.

The molecular weight of the water-soluble, non-peptidic oligomer, excluding the linker portion, is generally relatively low. Exemplary values of the molecular weight of the water-soluble polymer include: below about 1500 Daltons; below about 1450 Daltons; below about 1400 Daltons; below about 1350 Daltons; below about 1300 Daltons; below about 1250 Daltons; below about 1200 Daltons; below about 1150 Daltons; below about 1100 Daltons; below about 1050 Daltons; below about 1000 Daltons; below about 950 Daltons; below about 900 Daltons; below about 850 Daltons; below about 800 Daltons; below about 750 Daltons; below about 700 Daltons; below about 650 Daltons; below about 600 Daltons; below about 550 Daltons; below about 500 Daltons; below about 450 Daltons; below about 400 Daltons; below about 350 Daltons; below about 300 Daltons; below about 250 Daltons; below about 200 Daltons; below about 150 Daltons; and below about 100 Daltons.

Exemplary ranges of molecular weights of the water-soluble, non-peptidic oligomer (excluding the linker) include: from about 100 to about 1400 Daltons; from about 100 to about 1200 Daltons; from about 100 to about 800 Daltons; from about 100 to about 500 Daltons; from about 100 to about 400 Daltons; from about 200 to about 500 Daltons; from about 200 to about 400 Daltons; from about 75 to 1000 Daltons; and from about 75 to about 750 Daltons.

Preferably, the number of monomers in the water-soluble, non-peptidic oligomer falls within one or more of the following ranges (end points for each range provided are inclusive): between about 1 and about 30; between about 1 and about 25; between about 1 and about 20; between about 1 and about 15; between about 1 and about 12; between about 1 and about 10. In certain instances, the number of monomers in series in the oligomer (and the corresponding conjugate) is one of 1, 2, 3, 4, 5, 6, 7, or 8. In additional embodiments, the oligomer (and the corresponding conjugate) contains 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or monomers in series. In yet further embodiments, the oligomer (and the corresponding conjugate) possesses 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 monomers in series. Thus, for example, when the water-soluble, non-peptidic polymer includes CH3—(OCH2CH2)n—, “n” is an integer that can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, and can fall within one or more of the following ranges: between about 1 and about 25; between about 1 and about 20; between about 1 and about 15; between about 1 and about 12; between about 1 and about 10.

When the water-soluble, non-peptidic oligomer has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 monomers, these values correspond to a methoxy end-capped oligo(ethylene oxide) having a molecular weights of about 75, 119, 163, 207, 251, 295, 339, 383, 427, and 471 Daltons, respectively. When the oligomer has 11, 12, 13, 14, or 15 monomers, these values correspond to methoxy end-capped oligo(ethylene oxide) having molecular weights corresponding to about 515, 559, 603, 647, and 691 Daltons, respectively.

When the water-soluble, non-peptidic oligomer is attached to the small molecule protease inhibitor (in contrast to the step-wise addition of one or more monomers to effectively “grow” the oligomer onto the small molecule protease inhibitor), it is preferred that the composition containing an activated form of the water-soluble, non-peptidic oligomer be monodispersed. In those instances, however, where a bimodal composition is employed, the composition will possess a bimodal distribution centering around any two of the above numbers of monomers. Ideally, the polydispersity index of each peak in the bimodal distribution, Mw/Mn, is 1.01 or less, and even more preferably, is 1.001 or less, and even more preferably is 1.0005 or less. Most preferably, each peak possesses a MW/Mn value of 1.0000. For instance, a bimodal oligomer may have any one of the following exemplary combinations of monomer subunits: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, and so forth; 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, and so forth; 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, and so forth; 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, and so forth; 5-6, 5-7, 5-8, 5-9, 5-10, and so forth; 6-7, 6-8, 6-9, 6-10, and so forth; 7-8, 7-9, 7-10, and so forth; and 8-9, 8-10, and so forth.

In some instances, the composition containing an activated form of the water-soluble, non-peptidic oligomer will be trimodal or even tetramodal, possessing a range of monomers units as previously described. Oligomer compositions possessing a well-defined mixture of oligomers (i.e., being bimodal, trimodal, tetramodal, and so forth) can be prepared by mixing purified monodisperse oligomers to obtain a desired profile of oligomers (a mixture of two oligomers differing only in the number of monomers is bimodal; a mixture of three oligomers differing only in the number of monomers is trimodal; a mixture of four oligomers differing only in the number of monomers is tetramodal), or alternatively, can be obtained from column chromatography of a polydisperse oligomer by recovering the “center cut”, to obtain a mixture of oligomers in a desired and defined molecular weight range.

It is preferred that the water-soluble, non-peptidic oligomer is obtained from a composition that is preferably unimolecular or monodisperse. That is, the oligomers in the composition possess the same discrete molecular weight value rather than a distribution of molecular weights. Some monodisperse oligomers can be purchased from commercial sources such as those available from Sigma-Aldrich, or alternatively, can be prepared directly from commercially available starting materials such as Sigma-Aldrich. Water-soluble, non-peptidic oligomers can be prepared as described in Chen Y., Baker, G. L., J. Org. Chem., 6870-6873 (1999), WO 02/098949, and U.S. Patent Application Publication 2005/0136031.

The linker or linkage (through which the water-soluble, non-peptidic polymer is attached) at least includes a covalent bond, and often includes one or more atoms such as an oxygen, two atoms, or a number of atoms. A linker is typically but is not necessarily linear in nature. The linkage, “X” (in

), is a stable linkage, and is preferably also enzymatically stable. Preferably, the linkage “X” is one having a chain length of less than about 12 atoms, and preferably less than about 10 atoms, and even more preferably less than about 8 atoms and even more preferably less than about 5 atoms, whereby length is meant the number of atoms in a single chain, not counting substituents. For instance, a urea linkage such as this, Roligomer—NH—(C═O)—NH—R′drug, is considered to have a chain length of 3 atoms (—NH—C(O)—NH—). In selected embodiments, the linkage does not comprise further spacer groups.

In some instances, the linker “X” comprises an ether, amide, urethane, amine, thioether, urea, or a carbon-carbon bond. Functional groups such as those discussed below, and illustrated in the examples, are typically used for forming the linkages. The linkage may less preferably also comprise (or be adjacent to or flanked by) spacer groups. Spacers are most useful in instances where the bioactivity of the conjugate is significantly reduced due to the positioning of the oligomer relatively close to the residue of the small molecule drug, wherein a spacer can serve to increase the distance between oligomer and the residue of the small molecule drug.

More specifically, in selected embodiments, a spacer moiety, X, may be any of the following: “—” (i.e., a covalent bond, that may be stable or degradable, between the residue of the small molecule protease inhibitor and the water-soluble, non-peptidic oligomer), —C(O)O—, —OC(O)—, —CH2—C(O)O—, —CH2—OC(O)—, —C(O)O—CH2—, —OC(O)—CH2—, —O—, —NH—, —S—, C(O)—NH, NH—C(O)—NH, O—C(O)—NH, —C(S)—, —CH2—, —CH2—CH2—, —CH2—CH2—CH2—, —CH2—CH2—CH2—CH2—, —O—CH2—, —CH2—O—, —O—CH2—CH2—, —CH2—O—CH2—, —CH2—CH2—O—, —O—CH2—CH2—CH2—, —CH2—O—CH2—CH2—, —CH2—CH2—O—CH2—, —CH2—CH2—CH2—O—, —O—CH2—CH2—CH2—CH2—, —CH2—O—CH2—CH2—CH2—, —CH2—CH2—O—CH2—CH2—, —CH2—CH2—CH2—O—CH2—, —CH2—CH2—CH2—CH2—O—, —C(O)—NH—CH2—, —C(O)—NH—CH2—CH2—, —CH2—C(O)—NH—CH2—, —CH2—CH2—C(O)—NH—, —C(O)—NH—CH2—CH2—CH2—, —CH2—C(O)—NH—CH2—CH2—, —CH2—CH2—C(O)—NH—CH2—, —CH2—CH2—CH2—C(O)—NH—, —C(O)—NH—CH2—CH2—CH2—CH2—, —CH2—C(O)—NH—CH2—CH2—CH2—, —CH2—CH2—C(O)—NH—CH2—CH2—, —CH2—CH2—CH2—C(O)—NH—CH2—, —CH2—CH2—CH2—C(O)—NH—CH2—CH2—, —CH2—CH2—CH2—CH2—C(O)—NH —, —NH—C(O)—CH2—, —CH2—NH—C(O)—CH2—, —CH2—CH2—NH—C(O)—CH2—, —NH—C(O)—CH2—CH2—, —CH2—NH—C(O)—CH2—CH2, —CH2—CH2—NH—C(O)—CH2—CH2, —C(O)—NH—CH2—, —C(O)—NH—CH2—CH2—, —O—C(O)—NH—CH2—, —O—C(O)—NH—CH2—CH2—, —NH—CH2—, —NH—CH2—CH2—, —CH2—NH—CH2—, —CH2—CH2—NH—CH2—, —C(O)—CH2—, —C(O)—CH2—CH2—, —CH2—C(O)—CH2—, —CH2—CH2—C(O)—CH2—, —CH2—CH2—C(O)—CH2—CH2—, —CH2—CH2—C(O)—, —CH2—CH2—CH2—C(O)—NH—CH2—CH2—NH—, —CH2—CH2—CH2—C(O)—NH—CH2—CH2—NH—C(O)—, —CH2—CH2—CH2—C(O)—NH—CH2—CH2—NH—C(O)—CH2—, bivalent cycloalkyl group, —N(R6)—, R6 is H or an organic radical selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl.

For purposes of the present invention, however, a group of atoms is not considered a spacer moiety when it is immediately adjacent to an oligomer segment, and the group of atoms is the same as a monomer of the oligomer such that the group would represent a mere extension of the oligomer chain.

The linkage “X” connecting the water-soluble, non-peptidic oligomer within the conjugate is typically formed by reaction of a functional group on a terminus of the oligomer (or one or more monomers when it is desired to “grow” the oligomer onto the protease inhibitor) with a corresponding functional group within the protease inhibitor. Illustrative reactions are described briefly below. For example, an amino group on an oligomer may be reacted with a carboxylic acid or an activated carboxylic acid derivative on the small molecule, or vice versa, to produce an amide linkage. Alternatively, reaction of an amine on an oligomer with an activated carbonate (e.g. succinimidyl or benzotriazyl carbonate) on the drug, or vice versa, forms a carbamate linkage. Reaction of an amine on an oligomer with an isocyanate (R—N═C═O) on a drug, or vice versa, forms a urea linkage (R—NH—(C═O)—NH—R′). Further, reaction of an alcohol (alkoxide) group on an oligomer with an alkyl halide, or halide group within a drug, or vice versa, forms an ether linkage. In yet another coupling approach, a small molecule having an aldehyde function is coupled to an oligomer amino group by reductive amination, resulting in formation of a secondary amine linkage between the oligomer and the small molecule.

A particularly preferred water-soluble, non-peptidic oligomer is an oligomer bearing an aldehyde functional group. In this regard, the oligomer will have the following structure: CH3O—(CH2—CH2—O)n—(CH2)p—C(O)H, wherein (n) is one of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 and (p) is one of 1, 2, 3, 4, 5, 6 and 7. Preferred (n) values include 3, 5 and 7 and preferred (p) values 2, 3 and 4. In addition, the carbon atom alpha to the —C(O)H moiety can optionally be substituted with alkyl.

Typically, the terminus of the water-soluble, non-peptidic oligomer not bearing a functional group is capped to render it unreactive. When the oligomer does include a further functional group at a terminus other than that intended for formation of a conjugate, that group is either selected such that it is unreactive under the conditions of formation of the linkage “X,” or it is protected during the formation of the linkage “X.”

As stated above, the water-soluble, non-peptidic oligomer includes at least one functional group prior to conjugation. The functional group typically comprises an electrophilic or nucleophilic group for covalent attachment to a small molecule, depending upon the reactive group contained within or introduced into the small molecule. Examples of nucleophilic groups that may be present in either the oligomer or the small molecule include hydroxyl, amine, hydrazine (—NHNH2), hydrazide (—C(O)NHNH2), and thiol. Preferred nucleophiles include amine, hydrazine, hydrazide, and thiol, particularly amine. Most small molecule drugs for covalent attachment to an oligomer will possess a free hydroxyl, amino, thio, aldehyde, ketone, or carboxyl group.

Examples of electrophilic functional groups that may be present in either the oligomer or the small molecule include carboxylic acid, carboxylic ester, particularly imide esters, orthoester, carbonate, isocyanate, isothiocyanate, aldehyde, ketone, thione, alkenyl, acrylate, methacrylate, acrylamide, sulfone, maleimide, disulfide, iodo, epoxy, sulfonate, thiosulfonate, silane, alkoxysilane, and halosilane. More specific examples of these groups include succinimidyl ester or carbonate, imidazoyl ester or carbonate, benzotriazole ester or carbonate, vinyl sulfone, chloroethylsulfone, vinylpyridine, pyridyl disulfide, iodoacetamide, glyoxal, dione, mesylate, tosylate, and tresylate (2,2,2-trifluoroethanesulfonate).

Also included are sulfur analogs of several of these groups, such as thione, thione hydrate, thioketal, is 2-thiazolidine thione, etc., as well as hydrates or protected derivatives of any of the above moieties (e.g. aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, ketal, thioketal, thioacetal).

An “activated derivative” of a carboxylic acid refers to a carboxylic acid derivative which reacts readily with nucleophiles, generally much more readily than the underivatized carboxylic acid. Activated carboxylic acids include, for example, acid halides (such as acid chlorides), anhydrides, carbonates, and esters. Such esters include imide esters, of the general form —(CO)O—N[(CO)—]2; for example, N-hydroxysuccinimidyl (NHS) esters or N-hydroxyphthalimidyl esters. Also preferred are imidazolyl esters and benzotriazole esters. Particularly preferred are activated propionic acid or butanoic acid esters, as described in co-owned U.S. Pat. No. 5,672,662. These include groups of the form —(CH2)2-3C(═O)O-Q, where Q is preferably selected from N-succinimide, N-sulfosuccinimide, N-phthalimide, N-glutarimide, N-tetrahydrophthalimide, N-norbornene-2,3-dicarboximide, benzotriazole, 7-azabenzotriazole, and imidazole.

Other preferred electrophilic groups include succinimidyl carbonate, maleimide, benzotriazole carbonate, glycidyl ether, imidazoyl carbonate, p-nitrophenyl carbonate, acrylate, tresylate, aldehyde, and orthopyridyl disulfide.

These electrophilic groups are subject to reaction with nucleophiles, e.g. hydroxy, thio, or amino groups, to produce various bond types. Preferred for the present invention are reactions which favor formation of a hydrolytically stable linkage. For example, carboxylic acids and activated derivatives thereof, which include orthoesters, succinimidyl esters, imidazolyl esters, and benzotriazole esters, react with the above types of nucleophiles to form esters, thioesters, and amides, respectively, of which amides are the most hydrolytically stable. Carbonates, including succinimidyl, imidazolyl, and benzotriazole carbonates, react with amino groups to form carbamates. Isocyanates (R—N═C═O) react with hydroxyl or amino groups to form, respectively, carbamate (RNH—C(O)—OR′) or urea (RNH—C(O)—NHR′) linkages. Aldehydes, ketones, glyoxals, diones and their hydrates or alcohol adducts (i.e. aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, and ketal) are preferably reacted with amines, followed by reduction of the resulting imine, if desired, to provide an amine linkage (reductive amination).

Several of the electrophilic functional groups include electrophilic double bonds to which nucleophilic groups, such as thiols, can be added, to form, for example, thioether bonds. These groups include maleimides, vinyl sulfones, vinyl pyridine, acrylates, methacrylates, and acrylamides. Other groups comprise leaving groups that can be displaced by a nucleophile; these include chloroethyl sulfone, pyridyl disulfides (which include a cleavable S—S bond), iodoacetamide, mesylate, tosylate, thiosulfonate, and tresylate. Epoxides react by ring opening by a nucleophile, to form, for example, an ether or amine bond. Reactions involving complementary reactive groups such as those noted above on the oligomer and the small molecule are utilized to prepare the conjugates of the invention.

In some instances the protease inhibitor may not have a functional group suited for conjugation. In this instance, it is possible to modify the “original” protease inhibitor so that it does have the desired functional group. For example, if the protease inhibitor has an amide group, but an amine group is desired, it is possible to modify the amide group to an amine group by way of a Hofmann rearrangement, Curtius rearrangement (once the amide is converted to an azide) or Lossen rearrangement (once amide is concerted to hydroxamide followed by treatment with tolyene-2-sulfonyl chloride/base).

It is possible to prepare a conjugate of small molecule protease inhibitor bearing a carboxyl group wherein the carboxyl group-bearing small molecule protease inhibitor is coupled to an amino-terminated oligomeric ethylene glycol, to provide a conjugate having an amide group covalently linking the small molecule protease inhibitor agonist to the oligomer. This can be performed, for example, by combining the carboxyl group-bearing small molecule protease inhibitor with the amino-terminated oligomeric ethylene glycol in the presence of a coupling reagent, (such as dicyclohexylcarbodiimide or “DCC”) in an anhydrous organic solvent.

Further, it is possible to prepare a conjugate of a small molecule protease inhibitor bearing a hydroxyl group wherein the hydroxyl group-bearing small molecule protease inhibitor is coupled to an oligomeric ethylene glycol halide to result in an ether (—O—) linked small molecule conjugate. This can be performed, for example, by using sodium hydride to deprotonate the hydroxyl group followed by reaction with a halide-terminated oligomeric ethylene glycol.

In another example, it is possible to prepare a conjugate of a small molecule protease inhibitor bearing a ketone group by first reducing the ketone group to form the corresponding hydroxyl group. Thereafter, the small molecule protease inhibitor now bearing a hydroxyl group can be coupled as described herein.

In still another instance, it is possible to prepare a conjugate of a small molecule protease inhibitor bearing an amine group. In one approach, the amine group-bearing small molecule protease inhibitor and an aldehyde-bearing oligomer are dissolved in a suitable buffer after which a suitable reducing agent (e.g., NaCNBH3) is added. Following reduction, the result is an amine linkage formed between the amine group of the amine group-containing small molecule protease inhibitor and the carbonyl carbon of the aldehyde-bearing oligomer.

In another approach for preparing a conjugate of a small molecule protease inhibitor bearing an amine group, a carboxylic acid-bearing oligomer and the amine group-bearing small molecule protease inhibitor are combined, typically in the presence of a coupling reagent (e.g., DCC). The result is an amide linkage formed between the amine group of the amine group-containing small molecule protease inhibitor and the carbonyl of the carboxylic acid-bearing oligomer.

Exemplary conjugates of small molecule protease inhibitors (which may be “potent” or not) which can still have usefulness as having anti-HIV activity include:

wherein, X is a spacer moiety and POLY is a water-soluble oligomer. Approaches for preparing the above compound are described in the Examples.

To determine whether the small molecule protease inhibitor or the conjugate of a small molecule protease inhibitor and a water-soluble non-peptidic polymer has anti-HIV activity, it is possible to test such compounds. Anti-HIV activity can be tested as described in the Experimental. In addition, Anti-HIV activity can be tested in a human T-cell line by, for example, the method disclosed in Kempf et al. (1991) Antimicrob. Agents Chemother. 35(11):2209-2214, HIV-13B stock (104.7 50% tissue culture infection doses per ml) can be diluted 100-fold and incubated with MT-4 cells at 4×105 cells per ml for one hour at 37° C. (multiplicity of infection, 0.001 50% tissue culture infective dose per cell). The resulting culture is then washed twice, resuspended to 105 cells per ml of medium, seeded in a volume of 1% dimethyl sulfoxide solution of compound in a series of half-log-unit dilutions in medium in triplicate. The virus control culture can be treated in an identical manner, except that no compound is added to the medium. The cell control is incubated in the absence of compound or virus. Optical density (OD) is then measured at day 5 by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in a colorimetric assay. See Pauwels et al. (1988) J. Virol Methods 20:309-321. Virus and control OD values are averaged over six determinations. Percent inhibition of HIV cytopathic effect (CPE) is calculated by the following formula: [(average OD−virus control OD/(cell control OD−virus control OD)]×100. Cytotoxicity is determined by the incubation in duplicate with serial dilutions of compound in the absence of virus. Percent cytotoxicity is determined according to the following formula: (average OD/cell control OD)×100. The EC50 represents the concentration of compound that gave 50% inhibition of the cytopathic effect. The CCIC50 is the concentration of compound which gives a 50% cytotoxic effect. It is noted that when conjugation of the water-soluble, non-peptidic oligomer occurs at the hydroxyl group located at 26 position of saquinavir, no anti-HIV activity is measured. See Table 1. While not wishing to be bound by theory, it appears that the availability of this hydroxyl group is required for activity (a “binding hydroxyl group”). As a consequence, it is preferred in some embodiments that the conjugate lacks attachment of the water-soluble, non-peptidic oligomer at a binding hydroxyl group. A “binding hydroxyl group” for any given protease inhibitor can be determined by one of ordinary skill in the art by, for example, experimental testing and/or by comparing the structure of the protease inhibitor of interest with the structure of saquinavir and determining which hydroxyl group in the protease inhibitor corresponds to the “binding hydroxyl group” at position 26 in saquinavir.

The present invention also includes pharmaceutical preparations comprising an HIV protease inhibitor (whether “potent” or not) in combination with a pharmaceutical excipient. Generally, the conjugate itself will be in a solid form (e.g., a precipitate), which can be combined with a suitable pharmaceutical excipient that can be in either solid or liquid form.

Exemplary excipients include, without limitation, those selected from the group consisting of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

The preparation may also include an antimicrobial agent for preventing or deterring microbial growth. Nonlimiting examples of antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the conjugate or other components of the preparation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

A surfactant may be present as an excipient. Exemplary surfactants include: polysorbates, such as “Tween 20” and “Tween 80,” and pluronics such as F68 and F88 (both of which are available from BASF, Mount Olive, N.J.); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; and chelating agents, such as EDTA, zinc and other such suitable cations.

Acids or bases may be present as an excipient in the preparation. Nonlimiting examples of acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.

The amount of the conjugate in the composition will vary depending on a number of factors, but will optimally be a therapeutically effective dose when the composition is stored in a unit dose container. A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the conjugate in order to determine which amount produces a clinically desired endpoint.

The amount of any individual excipient in the composition will vary depending on the activity of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects.

Generally, however, the excipient will be present in the composition in an amount of about 1% to about 99% by weight, preferably from about 5%-98% by weight, more preferably from about 15-95% by weight of the excipient, with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipients are described in “Remington: The Science & Practice of Pharmacy”, 19th ed., Williams & Williams, (1995), the “Physician\'s Desk Reference”, 52nd ed., Medical Economics, Montvale, N.J. (1998), and Kibbe, A. H., Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical compositions can take any number of forms and the invention is not limited in this regard. Exemplary preparations are most preferably in a form suitable for oral administration such as a tablet, caplet, capsule, gel cap, troche, dispersion, suspension, solution, elixir, syrup, lozenge, transdermal patch, spray, suppository, and powder.

Oral dosage forms are preferred for those conjugates that are orally active, and include tablets, caplets, capsules, gel caps, suspensions, solutions, elixirs, and syrups, and can also comprise a plurality of granules, beads, powders or pellets that are optionally encapsulated. Such dosage forms are prepared using conventional methods known to those in the field of pharmaceutical formulation and described in the pertinent texts.

Tablets and caplets, for example, can be manufactured using standard tablet processing procedures and equipment. Direct compression and granulation techniques are preferred when preparing tablets or caplets containing the conjugates described herein. In addition to the conjugate, the tablets and caplets will generally contain inactive, pharmaceutically acceptable carrier materials such as binders, lubricants, disintegrants, fillers, stabilizers, surfactants, coloring agents, and the like. Binders are used to impart cohesive qualities to a tablet, and thus ensure that the tablet remains intact. Suitable binder materials include, but are not limited to, starch (including corn starch and pregelatinized starch), gelatin, sugars (including sucrose, glucose, dextrose and lactose), polyethylene glycol, waxes, and natural and synthetic gums, e.g., acacia sodium alginate, polyvinylpyrrolidone, cellulosic polymers (including hydroxypropyl cellulose, hydroxypropyl methylcellulose, methyl cellulose, microcrystalline cellulose, ethyl cellulose, hydroxyethyl cellulose, and the like), and Veegum. Lubricants are used to facilitate tablet manufacture, promoting powder flow and preventing particle capping (i.e., particle breakage) when pressure is relieved. Useful lubricants are magnesium stearate, calcium stearate, and stearic acid. Disintegrants are used to facilitate disintegration of the tablet, and are generally starches, clays, celluloses, algins, gums, or crosslinked polymers. Fillers include, for example, materials such as silicon dioxide, titanium dioxide, alumina, talc, kaolin, powdered cellulose, and microcrystalline cellulose, as well as soluble materials such as mannitol, urea, sucrose, lactose, dextrose, sodium chloride, and sorbitol. Stabilizers, as well known in the art, are used to inhibit or retard drug decomposition reactions that include, by way of example, oxidative reactions.

Capsules are also preferred oral dosage forms, in which case the conjugate-containing composition can be encapsulated in the form of a liquid or gel (e.g., in the case of a gel cap) or solid (including particulates such as granules, beads, powders or pellets). Suitable capsules include hard and soft capsules, and are generally made of gelatin, starch, or a cellulosic material. Two-piece hard gelatin capsules are preferably sealed, such as with gelatin bands or the like.

Included are parenteral formulations in the substantially dry form (typically as a lyophilizate or precipitate, which can be in the form of a powder or cake), as well as formulations prepared for injection, which are typically liquid and requires the step of reconstituting the dry form of parenteral formulation. Examples of suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate-buffered saline, Ringer\'s solution, saline, sterile water, deionized water, and combinations thereof.

In some cases, compositions intended for parenteral administration can take the form of nonaqueous solutions, suspensions, or emulsions, each typically being sterile. Examples of nonaqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.

The parenteral formulations described herein can also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. The formulations are rendered sterile by incorporation of a sterilizing agent, filtration through a bacteria-retaining filter, irradiation, or heat.

The conjugate can also be administered through the skin using conventional transdermal patch or other transdermal delivery system, wherein the conjugate is contained within a laminated structure that serves as a drug delivery device to be affixed to the skin. In such a structure, the conjugate is contained in a layer, or “reservoir,” underlying an upper backing layer. The laminated structure can contain a single reservoir, or it can contain multiple reservoirs.

The conjugate can also be formulated into a suppository for rectal administration. With respect to suppositories, the conjugate is mixed with a suppository base material which is (e.g., an excipient that remains solid at room temperature but softens, melts or dissolves at body temperature) such as coca butter (theobroma oil), polyethylene glycols, glycerinated gelatin, fatty acids, and combinations thereof. Suppositories can be prepared by, for example, performing the following steps (not necessarily in the order presented): melting the suppository base material to form a melt; incorporating the conjugate (either before or after melting of the suppository base material); pouring the melt into a mold; cooling the melt (e.g., placing the melt-containing mold in a room temperature environment) to thereby form suppositories; and removing the suppositories from the mold.

The disclosure also provides a method for administering a conjugate as provided herein to a patient suffering from a condition that is responsive to treatment with the conjugate. As previously mentioned, in one aspect, the method comprises administering a potent HIV protease inhibitor. The mode of administration can be oral, but other modes of administration are also contemplated, such as pulmonary, nasal, buccal, rectal, sublingual, transdermal, and parenteral. As used herein, the term “parenteral” includes subcutaneous, intravenous, intra-arterial, intraperitoneal, intracardiac, intrathecal, and intramuscular injection, as well as infusion injections.

Depending upon the subject method, any route suited for delivery of the potent protease inhibitor to the biological system (e.g., individual) can be used. If, for example, the biological system is a cell culture, administration can simply involve adding, via a pipette or dropper (for example), an aliquot of liquid containing the potent protease inhibitor. To the extent that the biological system is an individual infected with a virus, administering the potent protease inhibitor can take place via oral administration, but other modes of administration are also contemplated, such as pulmonary, nasal, buccal, rectal, sublingual, transdermal, and parenteral, wherein an individual infected with HIV is administered a potent HIV protease inhibitor and an individual infected with HCV is administered a potent HCV protease inhibitor.

In some instances the potent protease inhibitor (e.g., a HIV protease inhibitor or a hepatitis virus protease inhibitor such as a HCV protease inhibitor) is administered to an individual as part of a potent protease inhibitor therapy (e.g., a potent HIV protease inhibitor therapy or a potent hepatitis virus protease inhibitor therapy). By potent protease inhibitor therapy is meant a regimen in which at least one protease inhibitor is administered to effect some measure of protease inhibition in a biological system (e.g., an individual infected with HIV or an individual infected with HCV). Such a protease inhibitor therapy may also include one or more other drugs such as (i) a pharmacoenhancer of the potent protease inhibitor, (ii) a drug to allieviate a side effect of a potent HIV protease inhibitor, and/or (iii) a means to effect some measure of HIV protease inhibition in the biological system. It is also recognized, however, that one or more other active agents may also be administered to the biological system for reasons other than to effect protease inhibition; in which case, such other active agent(s) are not considered to be a part the potent HIV protease inhibitor therapy.

In one or more particular embodiments in which a potent protease inhibitor therapy is being administered to a biological system (e.g., individual), it is preferred that said potent protease inhibitor therapy does not include the co-administration of a CYP3A4 inhibitor. Conventionally, co-administration of the CYP3A4 inhibitor (e.g., ritonavir) would take place prior to, simultaneously with, or after administration of the protease inhibitor. In one or more embodiments of the present invention, however, the potent protease inhibitor therapy does not include such co-administration of a CYP3A4 inhibitor (ritonavir).

Ritonavir or other CYP3A4 inhibitors are often included as pharmacoenhancers in conventional protease inhibitor therapy to effectively supply a “boosting” strategy. The “boosting” strategy is believed to increase the exposure of the protease inhibitor by leveraging the CYP3A4 inhitor\'s ability to inhibit cytochrome P-450 3A4-mediated metabolism of the protease inhibitor.

Although any CYP3A4 inhibitor can theoretically be used to inhibit cytochrome P450 3A4-mediated metabolism (including cytochrome P-450 3A4-mediated metabolism of the HIV protease inhibitor), the conventional approach has been the co-administration of ritonavir, which, in addition to its protease inhibitory activity, is a CYP3A inhibitor.

In one or more embodiments of the invention, provided is a method comprising administering a potent protease inhibitor (e.g., a potent HIV protease inhibitor or a potent hepatitis virus protease inhibitor such as a potent HCV protease inhibitor) to a CYP3A4-competent biological system.

By a CYP3A4-competent biological system is meant a cytochrome P450 3A4-containing biological system—containing a plurality of functional cytochrome P450 3A4 enzymes—in which a majority (i.e., greater than 50%) of that plurality are functioning and not inhibited by a CYP3A4 inhibitor. Thus, for example, a biological system (such as a cell culture) in which an excess of ritonavir (relative functional cytochrome P450 3A4 enzymes) was administered would not be considered a cytochrome P450 3A4-competent biological system. Also, a biological system (such as an individual infected with HIV and/or HCV) would not be considered a CYP3A4-competent biological system, for example, if the individual were taking ritonavir at 200 mg a day (either via a divided dose of 100 mg twice a day or 200 mg once a day). The biological system can be an in vitro cellular system as well as a human.

In instances where parenteral administration is utilized, it may be necessary to employ somewhat bigger oligomers (i.e., polymers) than those described previously, with molecular weights ranging from about 500 to 30K Daltons (e.g., having molecular weights of about 500, 1000, 2000, 2500, 3000, 5000, 7500, 10000, 15000, 20000, 25000, 30000 or even more).

The method of administering may be used to treat any condition that can be remedied or prevented by administration of the particular conjugate. Those of ordinary skill in the art appreciate which conditions a specific conjugate can effectively treat. The actual dose to be administered will vary depend upon the age, weight, and general condition of the subject as well as the severity of the condition being treated, the judgment of the health care professional, and conjugate being administered. Therapeutically effective amounts are known to those skilled in the art and/or are described in the pertinent reference texts and literature and/or can be determined experimentally. Generally, a therapeutically effective amount is an amount within one or more of the following ranges: from 0.001 mg/day to 10000 mg/day; from 0.01 mg/day to 7500 mg/day; from 0.10 mg/day to 5000 mg/day; from 1 mg/day to 4000 mg/day; and from 10 mg/day to 2000 mg/day.

The unit dosage of any given potent HIV protease inhibitor (again, preferably provided as part of a pharmaceutical preparation) can be administered in a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth. The specific dosing schedule will be known by those of ordinary skill in the art or can be determined experimentally using routine methods. Exemplary dosing schedules include, without limitation, administration five times a day, four times a day, three times a day, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Once the clinical endpoint has been achieved, dosing of the composition is halted.

One advantage of administering the potent HIV protease inhibitor is that doing so provides HIV protease activity in a biological system without the need to co-administer a 3YP3A4 inhibitor, thereby reducing the complexities inherent with coordinating the administration of two agents to achieve relatively high and efficient HIV protease inhibition in a biological system. Such a benefit has utility in simplifying in vitro and in vivo assays. In addition, in a patient suffering from HIV, the methods described herein are expected to reduce the complexities of protease inhibitor-based therapies.

All articles, books, patents, patent publications and other publications referenced herein are incorporated by reference in their entireties. In the event of an inconsistency between the teachings of this specification and the art incorporated by reference, the meaning of the teachings in this specification shall prevail.

It is to be understood that while the invention has been described in conjunction with certain preferred and specific embodiments, the foregoing description as well as the examples that follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

All chemical reagents referred to in the appended examples are commercially available unless otherwise indicated. The preparation of PEG-mers is described in, for example, U.S. Patent Application Publication No. 2005/0136031.

The nomenclature used in the following examples corresponds to the following chemical structures.

Mono-mPEGn-Atazanavir (n=1, 3, 5, 6, 7)

Di-mPEGn-Atazanavir

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