US20110195940A1 - Protease Inhibitors Having Enhanced Features - Google Patents

Protease Inhibitors Having Enhanced Features Download PDF

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US20110195940A1
US20110195940A1 US13/119,079 US200913119079A US2011195940A1 US 20110195940 A1 US20110195940 A1 US 20110195940A1 US 200913119079 A US200913119079 A US 200913119079A US 2011195940 A1 US2011195940 A1 US 2011195940A1
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protease inhibitor
hiv
atazanavir
hiv protease
darunavir
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C. Simone Jude-Fishburn
Laurie A. Vander Veen
Timothy A. Riley
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Nektar Therapeutics
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4402—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 2, e.g. pheniramine, bisacodyl
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4433—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with oxygen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

  • 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.
  • HIV human immunodeficiency virus
  • 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/ ⁇ L but before a level of 200/ ⁇ L. Hammer et al. (2006) JAMA 296(7):827-843.
  • 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.
  • 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.
  • 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.
  • 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.
  • CYP-450 cytochrome P-450
  • a protease inhibitor itself has been used and has shown demonstrated efficacy in clinical studies.
  • 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.
  • the dose of ritonavir administered in “boosted” protease inhibitor-based regimens is generally considered subtherapeutic.
  • Moyle et al. 2001) HIV Medicine 2:105-113.
  • a 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.
  • 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.
  • 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.
  • 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.
  • a 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.
  • a 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.
  • a potent protease inhibitor e.g., an HIV protease inhibitor or a hepatitis virus protease inhibitor such as a HCV protease inhibitor
  • the potent protease inhibitor is administered in a CYP3A4-competent biological system.
  • a 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.
  • FIGS. 1A-1E provide five graphs showing p24 levels in response to PI (protease inhibitor) and PEG oligo -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 Ravitonavir
  • FIG. 1E Choicago Sky Blue
  • FIGS. 2A-2D are graphs comparing the metabolic stability of PEG oligo -protease inhibitor conjugates and protease inhibitor molecules following incubation with cryopreserved human hepatocytes, as further described in Example 2. Values were obtained using 3 ⁇ M test compound concentration.
  • FIG. 2A Alazanavir, di-mPEG 3,5,7 -atazanavir
  • FIG. 2B Darunavir, mPEG 3,5,7 -N-darunavir
  • FIG. 2C Tepranavir, mPEG 3,5,7 -amide-tipranavir
  • FIG. 2D (Saquinavir, PEG 3,5,7 N-Saquinavir).
  • FIGS. 3A-3E are graphs showing the stability of PEG oligo -protease inhibitor conjugates and protease inhibitor molecules expressed as values (percent compound remaining) normalized to the 30 minute timepoint.
  • FIG. 3A Alignazanuvir, 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. 3A Alignazanuvir, di-mPEG3-atazanavir, mono-mPEG3-atazanavir
  • FIG. 3B Darunavir, mPEG-3-N-darunavir,
  • 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 ⁇ M test compound concentration.
  • FIGS. 4A-4F are graphs showing the metabolic stability of PEG-protease inhibitors following incubation with CYP3A4- or CYP2D6-expressing BactosomesTM, 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. 4A 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
  • FIG. 4E CYP3A4 Bactosome Stability, Saquinavir, mPEG3-NHCO-saquinavir, mPEG5-NHCO-saquinavir, mPEG7-NHCO-saquinavir
  • 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.
  • 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.
  • an oligomer is non-peptidic when it has less than 35% (by weight) of amino acid residues.
  • 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 polyethylene glycol
  • polyethylene glycol is meant to encompass any water-soluble poly(ethylene oxide).
  • 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.
  • PEG oligomers for use in the present invention will comprise one of the two following structures: “—(CH 2 CH 2 O) n —” or “—(CH 2 CH 2 O) n-1 CH 2 CH 2 —,” depending upon whether the terminal oxygen(s) has been displaced, e.g., during a synthetic transformation.
  • 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.
  • 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.
  • 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.
  • the degree of first pass metabolism 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.
  • 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.
  • substituted alkyl or “substituted C q-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, C 1-7 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, butyl, t-butyl, and so forth), C 1-7 alkoxy, C 1-7 acyloxy, C 3-7 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 C 1 -C 20 alkyl (e.g., methoxy, ethoxy, propyloxy, benzyl, etc.), preferably C 1 -C 7 .
  • “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.
  • 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.
  • halo F, Cl, Br, I
  • hydroxy hydroxy
  • cyano nitro
  • alkyl e.g., C 1-6 alkyl
  • alkoxy e.g., C 1-6 alkoxy
  • benzyloxy carboxy, aryl, and so forth.
  • 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.
  • an “alkyl” moiety generally refers to a monovalent radical (e.g., CH 3 —CH 2 —)
  • 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., —CH 2 —CH 2 —), which is equivalent to the term “alkylene.”
  • 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.
  • biological system is a collection of living cells and includes both a collection of living cells as well as 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.
  • the present disclosure is directed to the unexpected advantages and features of the subject protease inhibitor conjugates, and methods related thereto.
  • 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.
  • 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.
  • 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)
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • a potent HIV protease inhibitor acts via the same pharmacologic mechanism as known HIV protease inhibitors.
  • 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.
  • a potent hepatitis virus protease inhibitor acts via the same pharmacologic mechanism as known hepatitis virus protease inhibitors.
  • 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
  • the 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.
  • substantially unchanged or “stable” it is possible to determine whether an protease inhibitor is substantially unchanged (or stable) by performing the following test.
  • 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.
  • a potent protease inhibitor e.g., a potent HIV protease inhibitor or a potent hepatitis virus protease inhibitor such as a potent H
  • 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.
  • 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.”
  • 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:
  • 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.
  • 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).
  • the potent HIV protease inhibitor may possess one or more geometric isomers.
  • 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.
  • a potent HIV protease inhibitor may have a targeting agent, tag, or transporter attached thereto, prior to or after covalent attachment of an oligomer.
  • 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.
  • a phospholipid e.g., distearoylphosphatidylethanolamine or “DSPE,” dipalmitoylphosphatidylethanolamine or “DPPE,” and so forth
  • DPPE dipalmitoylphosphatidylethanolamine
  • 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).
  • each oligomer is a co-oligomer of two monomers or, more preferably, is a homo-oligomer.
  • 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; ⁇ -hydroxy acid, such as lactic acid or glycolic acid; phosphazene, oxazoline, amino acids, carbohydrates such as monosaccharides, saccharide or mannitol; and N-acryloylmorpholine.
  • 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
  • ⁇ -hydroxy acid such as lactic acid or
  • Preferred monomer types include alkylene oxide, olefinic alcohol, hydroxyalkyl methacrylamide or methacrylate, N-acryloylmorpholine, and ⁇ -hydroxy acid.
  • 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.
  • the oligomer is a homo-oligomer of ethylene oxide.
  • the terminus (or termini) of the oligomer that is not covalently attached to a small molecule is capped to render it unreactive.
  • 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
  • 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.
  • 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 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.
  • 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.
  • 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.
  • the oligomer (and the corresponding conjugate) contains 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or monomers in series.
  • the oligomer (and the corresponding conjugate) possesses 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 monomers in series.
  • 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.
  • 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.
  • 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.
  • the composition containing an activated form of the water-soluble, non-peptidic oligomer be monodispersed.
  • the composition will possess a bimodal distribution centering around any two of the above numbers of monomers.
  • 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.
  • each peak possesses a MW/Mn value of 1.0000.
  • 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.
  • 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
  • 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
  • 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
  • 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.
  • a urea linkage such as this, R oligomer —NH—(C ⁇ O)—NH—R′ drug , is considered to have a chain length of 3 atoms (— N H— C (O)— N H—).
  • the linkage does not comprise further spacer groups.
  • 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.
  • 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)—, —CH 2 —C(O)O—, —CH 2 —OC(O)—, —C(O)O—CH 2 —, —OC(O)—CH 2 —, —O—, —NH—, —S—, C(O)—NH, NH—C(O)—NH, O—C(O)—NH, —C(S)—, —CH 2 —, —CH 2 —CH 2 —, —CH 2 —CH 2 —CH 2 —, —CH 2 —CH 2 —CH 2 —, —CH 2 —CH 2 —CH 2 —, —O—CH 2
  • 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.
  • 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′).
  • 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.
  • 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.
  • the oligomer will have the following structure: CH 3 O—(CH 2 —CH 2 —O) n —(CH 2 ) 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.
  • the carbon atom alpha to the —C(O)H moiety can optionally be substituted with alkyl.
  • the terminus of the water-soluble, non-peptidic oligomer not bearing a functional group is capped to render it unreactive.
  • 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.”
  • 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.
  • nucleophilic groups that may be present in either the oligomer or the small molecule include hydroxyl, amine, hydrazine (—NHNH 2 ), hydrazide (—C(O)NHNH 2 ), 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.
  • 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.
  • 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.
  • 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.
  • esters include imide esters, of the general form —(CO)O—N[(CO)—] 2 ; for example, N-hydroxysuccinimidyl (NHS) esters or N-hydroxyphthalimidyl esters.
  • 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. include groups of the form —(CH 2 ) 2-3 C( ⁇ 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.
  • electrophilic groups include succinimidyl carbonate, maleimide, benzotriazole carbonate, glycidyl ether, imidazoyl carbonate, p-nitrophenyl carbonate, acrylate, tresylate, aldehyde, and orthopyridyl disulfide.
  • 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.
  • 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.
  • Isocyanates 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
  • 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).
  • electrophilic functional groups include electrophilic double bonds to which nucleophilic groups, such as thiols, can be added, to form, for example, thioether bonds.
  • nucleophilic groups such as thiols
  • 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.
  • the protease inhibitor may not have a functional group suited for conjugation.
  • 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).
  • 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.
  • a coupling reagent such as dicyclohexylcarbodiimide or “DCC”
  • 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.
  • a conjugate of a small molecule protease inhibitor bearing an amine group it is possible to prepare a conjugate of a small molecule protease inhibitor bearing an amine group.
  • 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., NaCNBH 3 ) is added.
  • a suitable reducing agent e.g., NaCNBH 3
  • 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).
  • a coupling reagent e.g., DCC
  • Exemplary conjugates of small molecule protease inhibitors which may be “potent” or not) which can still have usefulness as having anti-HIV activity include:
  • X is a spacer moiety and POLY is a water-soluble oligomer.
  • Approaches for preparing the above compound are described in the Examples.
  • Anti-HIV activity can be tested as described in the Experimental.
  • 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-1 3B stock (10 4.7 50% tissue culture infection doses per ml) can be diluted 100-fold and incubated with MT-4 cells at 4 ⁇ 10 5 cells per ml for one hour at 37° C.
  • 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.
  • 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 EC 50 represents the concentration of compound that gave 50% inhibition of the cytopathic effect.
  • the CCIC 50 is the concentration of compound which gives a 50% cytotoxic effect.
  • 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.
  • a pharmaceutical excipient e.g., a pharmaceutical excipient
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • parenteral formulations in the substantially dry form typically as a lyophilizate or precipitate, which can be in the form of a powder or cake
  • formulations prepared for injection which are typically liquid and requires the step of reconstituting the dry form of parenteral formulation.
  • 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.
  • compositions intended for parenteral administration can take the form of nonaqueous solutions, suspensions, or emulsions, each typically being sterile.
  • 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.
  • parenteral formulations described herein can also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents.
  • 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.
  • 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.
  • 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.
  • 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.
  • parenteral includes subcutaneous, intravenous, intra-arterial, intraperitoneal, intracardiac, intrathecal, and intramuscular injection, as well as infusion injections.
  • any route suited for delivery of the potent protease inhibitor to the biological system can be used.
  • 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.
  • 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.
  • the potent protease inhibitor e.g., a HIV protease inhibitor or a hepatitis virus protease inhibitor such as a HCV protease inhibitor
  • a potent protease inhibitor therapy e.g., a potent HIV protease inhibitor therapy or a potent hepatitis virus protease inhibitor therapy.
  • 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.
  • 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.
  • one or more other active agents may also be administered
  • a potent protease inhibitor therapy 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.
  • a CYP3A4 inhibitor e.g., ritonavir
  • co-administration of the CYP3A4 inhibitor would take place prior to, simultaneously with, or after administration of the protease inhibitor.
  • 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.
  • 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.
  • 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.
  • 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
  • a CYP3A4-competent biological system 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.
  • a biological system such as a cell culture
  • an excess of ritonavir relative functional cytochrome P450 3A4 enzymes
  • a biological system such as an individual infected with HIV and/or HCV
  • the biological system can be an in vitro cellular system as well as a human.
  • oligomers i.e., polymers
  • 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.
  • 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.
  • 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.
  • the methods described herein are expected to reduce the complexities of protease inhibitor-based therapies.
  • HPLC method had the following parameters: column, Betasil C18, 5- ⁇ m (100 ⁇ 2.1 mm); flow, 0.5 mL/min; gradient, 0-23 min, 20% acetonitrile/0.1% TFA in water/0.1% TFA to 100% acetonitrile/0.1% TFA; detection, 230 nm.
  • t R refers to the retention time.
  • TPTU O-(1,2-Dihydro-2-oxo-1-pyridyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate
  • DIPEA N,N′-Diisopropylethylamine
  • DSC N,N′-Disuccinimidyl carbonate.
  • Methoxycarbonyl-L-tert-Leucine (3) (1.37 gm, 7.24 mmol) was dissolved in anhydrous ethyl acetate (21 mL). To the clear solution was added N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) (1.12 gm, 5.82 mmol, 1.1 equivalents). The suspension was stirred under nitrogen at room temperature. After ten minutes added HOBT (1.08 gm, 7.97 mmol, 1.1 equivalents), followed by 4-methyl-morpholine (1.35 mL, 12.32 mmol, 1.7 equivalents).
  • EDC N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
  • Methoxycarbonyl-L-tert-Leucine hydrazine (6) (1.35 gm, 6.65 mmol) was taken up in i-PrOH (60 mL) and then added pyridyl benzaldehyde (7) (1.22 gm, 6.65 mmol).
  • the yellow reaction mixture was heated to reflux (85° C.). After approximately two hours, TLC and HPLC showed the reaction was complete. The heat was removed and the thick yellow mixture was cooled to 0° C. The solvent was removed under reduced pressure. The yellow residue was taken up in DCM (250 mL) and partitioned with water. The aqueous layer was extracted with DCM (4 ⁇ 50 mL).
  • Hydrazone (8) (1.10 gm, 2.98 mmol) was dissolved in anhydrous THF (30 mL). Then added solid NaCNBH 3 (0.40 gm, 5.97 mmol, 2.0 equivalents) all at once, followed by slow addition via syringe of PTSA (p-toluene sulfonic acid) (1.13 gm, 5.97 mmol, 2.0 equivalents) in THF (15 mL). There was bubbling observed during the PTSA addition. The cloudy mixture was heated to reflux (70° C.). After approximately 40 h, the cloudy reaction mixture was concentrated under reduced pressure and the white residue partitioned with DCM (30 mL) and water (50 mL).
  • PTSA p-toluene sulfonic acid
  • the Cbz-azaketone (11) (0.84 gm, 1.26 mmol) was taken up in diethyl ether (15 mL) and cooled to 0° C.
  • LTBA Lithium tri-tert-butoxy-aluminum hydride
  • the light-yellow suspension was stirred under nitrogen at 0° C. After one hour at 0° C., the cloudy yellow mixture was stored overnight at ⁇ 20° C.
  • the reaction mixture was quenched with water (0.9 mL), at 0° C. The solvent was removed under reduced pressure.
  • the light-yellow reaction mixture was diluted with dichloromethane (60 mL), transferred to a separatory funnel, and partitioned with deionized water (100 mL). The aqueous layer was extracted with dichloromethane (4 ⁇ 80 mL). The combined organics were washed with water, saturated sodium bicarbonate, and saturated sodium chloride. The dried organic layer was filtered, concentrated under reduced pressure and dried overnight under high vacuum, to give 2.79 g (75%) of mPEG 3 -SC-carbonate as a light yellow oil.
  • L-tert-Leucine (1) (0.43 g, 3.27 mmol) and deionized water (12 mL). The solution was stirred for 30 min until clear, followed by the addition of solid sodium bicarbonate (1.27 g, 15.0 mmol, 4.6 equivalents). The cloudy solution was stirred at room temperature, under nitrogen.
  • mPEG 3 -SC-carbonate (15) (1.24 g, 4.09 mmol, 1.25 equiv.) was taken up in deionized water (12 mL) and this solution was added all at once to the basic L-tert-Leucine solution.
  • the cloudy light-yellow reaction mixture was stirred at room temperature, under nitrogen. After approximately 20 h, the clear mixture was cooled to 0° C., and carefully acidified with 2 N HCl to pH 1 (20 mL). The acidic mixture was transferred to a separatory funnel and partitioned with dichloromethane (50 mL) and additional water (50 mL). The aqueous layer was extracted with dichloromethane (4 ⁇ 50 mL). The combined organic layers were washed with water and saturated sodium chloride, and dried over sodium sulfate.
  • the mPEG 3 -tert-Leucine reagent (16) (0.34 gm, 1.06 mmol, 3.0 equivalents) was taken up in anhydrous dichloromethane (3.0 mL) and cooled to 0° C.
  • TPTU (0.31 gm, 1.06 mmol, 3.0 equivalents) was added all at once, and the solution was stirred under nitrogen at 0° C.
  • the amino aza-isostere (13) (0.19 gm, 0.35 mmol) was taken up in anhydrous dichloromethane (3.0 mL) and diisopropylethylamine (0.37 mL, 2.13 mmol, 6.0 equivalents).
  • the synthesis of the bis-aryl hydrazine (9) is described above and represents an approach for preparing an intermediate useful for the preparing the atazanavir “core.”
  • the synthesis began with reaction of the chiral amino acid, L-tert-Leucine (1), with methyl chloroformate (2), to give methoxycarbonyl-L-tert-Leucine (3).
  • the methoxycarbonyl-L-tert-Leucine moiety also establishes the correct stereochemistry of the t-butyl group.
  • Reaction of (3) with tert-butylcarbazate gave the methoxycarbonyl-L-tert-Leucine-Boc protected hydrazine (5).
  • mPEG 5 -OH were obtained from India Sai CRO. 5-Trifluoromethyl-2-pyridinesulfonyl chloride was purchased from Toronto research chemicals (North York, ON, Canada). DCM was distilled from CaH 2 . Tetrahydrofuran (THF), ether, Ethyl acetate, and other organic solvents were used as they purchased.
  • Cupper(I) bromide DMS (7.2 g, 35.1 mmol) was dissolved in THF (43 mL) and the solution was cooled to ⁇ 35° C. Phenylethyl magnesium chloride (1M, 35.1 mL, 35.1 mmol) was added dropwise in ten minutes. The Mg-cupper reagent was kept at ⁇ 30 to ⁇ 10° C. over 20 minutes before it was cooled down to ⁇ 78° C. and above aldehdye (2.54 g, 11.7 mmol) in THF (20 mL) was added dropwise during 15 minutes.
  • the substrate (32) (7.28 g, 14.05 mmol) and MgBr 2 .OEt 2 (4.0 g, 15.5 mmol) were added.
  • the flask was protected in dry N 2 and THF (68 mL) was added.
  • the solution was cooled down to ⁇ 78° C. in acetone/dry ice bath before KHMDS (0.5 M, 42.1 mL, 21.08 mmol) was dropwise added in ten minutes.
  • KHMDS 0.5 M, 42.1 mL, 21.08 mmol
  • the above mixture was kept at ⁇ 78° C. for 30 minutes before acetyl chloride (1.50 mL, 21.08 mmol) was added in five minutes.
  • the reaction mixture was warm up gradually during the overnight reaction.
  • distilled DCM 50 mL was added following by addition of Ti(OPr) 4 (982 ⁇ L, 3.35 mmol) and TiCl 4 (1.03 mL, 9.41 mmol) in order.
  • the mixture was cooled down to ⁇ 78° C. in acetone/dry-ice bath and a mixture of substrate (33) (5.86 g, 10.5 mmol) in DCM (16 mL) was dropwise added in ten minutes.
  • the reaction was kept at this temperature for 5 min before DIPEA (2.37 mL, 13.6 mmol) was added slowly in 5 min.
  • the reaction was warm up to 0° C. and kept in 30 minutes.
  • the product (34) was solidified (3.48 g, 38% yield) after high vacuo.
  • the starting material mixture also has been recovery (5.43 g, 47%). Since this product is a diasteromer mixture, the 1 H NMR cannot be read and recorded.
  • the starting material (34) (3.23 g, 3.66 mmol) was dissolved in THF (91 mL). The solution was cooled down to 0° C. in ice-water bath before KOBu t (1M, 4.21 mL, 4.21 mmol) was added. The reaction was kept at this temperature for 25 minutes and quenched with NH 4 Cl aqueous solution (200 mL). EtOAc (200 mL) was added and the separated aqueous phase was extracted with EtOAc (50 mL ⁇ 2). The combined organic phase was washed with brine (100 mL ⁇ 2) and dried over Na 2 SO 4 . It was concentrated and the product mixture was performed DCC/DMAP lactonization without purification.
  • the DCC/DMAP lactonization was applied based on the amount of free acid in the product mixture (36).
  • the design was based on the hplc-UV detector in diluted solution (0.02 M).
  • the DCC (6 eq of remaining free acid) and DMAP (25% of DCC) was added at ambient temperature. In general, this lactonization was accomplished in one hour and DCM was evaporated.
  • the product residue was loaded on the Biotage column (40M, 15-48% EtOAc/Hex in 16 CV).
  • the collected product (37) (1.82 g with 94% purity) and product mixture (858 mg, 59% purity) was obtained after high vacuo (84% total yield).
  • mPEG 1 -4-nitrophenyl carbonate In a 25-mL flask, 2-methoxyethanol (56 ⁇ L, 0.705 mmol) was added in DCM (5 mL). p-Nitrophenyl-chloroformate (44) (128 mg, 0.635 mmol) and TEA (147 ⁇ L, 1.06 mmol) was added. The reaction was kept at ambient temperature for 30 minutes. The DCM solution was concentrated to 3 mL in order to complete this reaction in next two hours. The reaction was stopped by addition of NH 4 Cl (100 mL) and the product was extracted with DCM (30 mL ⁇ 3). The combined DCM solution was dried over Na 2 SO 4 and concentrated under the vacuo. The product (38) was used after high vacuo drying 10 minutes without further purification. RP-HPLC (betasil C18, 0.5 mL/min, 30-100% ACN in 10 minutes) 5.14 minutes.
  • mPEG 0 -4-nitrophenyl carbonate Methanol (10 eq), 4-nitropheyl chloroformate (1.1 eq), and TEA (1.5 eq).
  • RP-HPLC betasil C18, 0.5 mL/min, 30-100% ACN in 10 minutes) 4.84 minutes.
  • mPEG 1 -OCONH-core 39a: The product after phthalimide deprotecton (0.352 mmol) was dissolved in DCM (3 mL). The vacuo dried mPEG 1 -p-nitrophenyl-carbonate (0.635 mmol) was transferred to the above solution with DCM (6 mL in total). TEA (147 ⁇ L, 1.05 mmol) was added and the reaction was kept at room temperature for 20 hours. After the reaction was completed, it was quenched with NH 4 Cl aqueous solution and extracted with DCM (30 mL ⁇ 3). The combined organic phase was dried over Na 2 SO 4 .
  • mPEG 0 -OCONH-core-NH 2 (40a): The substrate (39a) mPEG 0 -OCONH-core-NBn 2 (197.2 mg, 0.305 mmol) was dissolved in EtOAc (6.0 mL) and MeOH (6.0 mL) mixture solution. The solution vial was bubbling N 2 for exchange at lease 15 minutes before catalyst addition. Stop stirring, and the Pd/C catalyst (39 mg, 10wt % ⁇ 2) was added slowly. The system was evacuated and recharged with hydrogen gas ( ⁇ 50 psi) three times (stop stirring during vacuo). The hydrogenolysis was then kept at room temperature under 50 psi for 24 hrs to complete.
  • mPEG 1 -OCONH-core-NH 2 (40b): RP-HPLC (betasil C18, 0.5 mL/min, 20-600% ACN in 10 minutes) 6.89+7.18 minutes; LC-MS (ESI, MH + ) 511.3.
  • mPEG 3 -OCONH-core-NH 2 (40c): RP-HPLC (betasil C18, 0.5 mL/min, 20-60% ACN in 10 minutes) 7.20+7.43 minutes; LC-MS (ESI, MH + ) 599.3.
  • mPEG 5 -OCONH-core-NH 2 (40d): RP-HPLC (betasil C18, 0.5 mL/min, 30-100% ACN in 10 minutes) 4.05+4.29 minutes; LC-MS (ESI, MH + ) 687.4.
  • mPEG 3 -OCONH-Tipranavir-2 (42c): The free amine mPEG 3 -OCONH-core-NH 2 (40c) (67.3 mg, 0.112 mmol) was dissolved in DCM (3.0 mL) under N 2 protection. After dissolving, the solution was cool down in an ice-water bath and sulphonyl chloride (27 mg, 0.112 mmol) was added. Pyridine (18 ⁇ L, 0.224 mmol) was then added and the reaction was kept at 0° C. for 30 minutes. Methyl amine (2M, 500 ⁇ L, 1.0 mmol) was added and the reaction was kept at this temperature for three hours.
  • Protease inhibitors are used to treat HIV infection by preventing viral assembly and maturation through the inhibition of HIV-1 protease activity.
  • Biochemical assays were performed to evaluate the potential of PEG-PI conjugates to inhibit HIV-1 protease activity, relative to their respective PI parent molecules.
  • Activity assays were performed at using the SensoLyte 520 HIV-Protease Assay Kit (Anaspec Inc., San Jose, Calif.) and recombinant HIV-1 protease.
  • Protease activity was monitored by the formation of a fluorescent reporter product generated during HIV-1 protease-mediated digestion of a quenched, fluorimetric substrate containing the p17/p24 Pr gag cleavage site. The results are summarized in Table 1.
  • IC 50 data reveal that PEG-modified PI compounds generally retained the ability to inhibit HIV-1 protease activity at levels comparable to that observed with their respective parent molecules and within nanomolar range IC 50 values that confirm significant inhibition of the purified HIV-1 protease.
  • CEM-SS cells infected with the RF strain of HIV-1 were treated with test compounds for six days, and then cell viability was monitored using the tetrazolium dye XTT.
  • Infection studies were performed and EC 50 values were calculated as the protease inhibitor concentration leading to 50% reduction in cell death compared to virus-infected cells without protease inhibitor.
  • TC 50 values were calculated as the protease inhibitor concentration leading to 50% cell death in the absence of viral infection.
  • a value for Therapeutic Index (TI) was calculated as TC 50 /EC 50 .
  • HIV-1 protease is generated in the cytoplasm of infected host cells, therefore protease inhibitors must cross cell membranes to reach their target and inhibit viral maturation.
  • PEG conjugation led to a decrease in activity in the cell-based CEM-SS assay.
  • CEM-SS cells were treated for 2, 8, or 24 hours with various concentrations of saquinavir or mPEG7-NHCO-Saquinavir, washed free from drug, then infected with HIV-1 RF in the absence of test compounds. Cell toxicity was monitored six days after infection using XTT reagent.
  • test compounds were added to cells 0, 24, 48, or 72 h after HIV-1 infection, and cell viability was measured six days post-infection.
  • entry inhibitor Chicago Sky Blue
  • nucleoside reverse transcriptase inhibitor azidothymidine (AZT)
  • AZA azidothymidine
  • nevirapine a non-nucleoside reverse transcriptase inhibitor
  • ritonavir the protease inhibitor
  • saquinavir demonstrated significant antiviral activity regardless of the time it was added to cells, albeit with reduced potency when addition was delayed to 72 hours post-infection (Table 4).
  • the mPEG7-Saquinavir conjugate yielded similar EC 50 values at all exposure times, although was generally less potent than saquinavir.
  • AZT and nevirapine demonstrated significant antiviral activity when added 24 hours after HIV exposure that decreased with each day the addition of compounds was delayed. In contrast, Chicago Sky Blue was efficacious only when added at the time of HIV-infection and had no effect when added at later times.
  • the cytoprotective effect against HIV of the PEG-protease inhibitor conjugates was evaluated during a single round of high multiplicity of infection (“MOI”) HIV-1 infection in CEM-SS cells.
  • CEM-SS cells were incubated for one hour with a single high MOI dose of HIV-1 RF .
  • Cells were washed to remove free virus, then test compounds were added to cells at 0, 2, 4, 6, 8, 16, or 24 hours after HIV-1 infection.
  • test compounds were added to cells at 0, 2, 4, 6, 8, 16, or 24 hours after HIV-1 infection.
  • cell supernatants were analyzed at 30 hours post-infection for levels of the HIV capsid protein p24 ( FIG. 1 ).
  • Saquinavir and mPEG7-NHCO-Saquinavir demonstrated similarly potent antiviral activity at 0.1 ⁇ M when added up to 16 hours post-infection, and at 1 ⁇ M when added up to 24 hours post-infection, as evidenced by reduced p24 expression in comparison to virus-alone controls.
  • Chicago Sky Blue did not affect p24 levels when added to cells at any time after HIV-infection, even at 50 ⁇ g/mL.
  • Nevirapine inhibited viral replication at 1 ⁇ M when added to cells up to four hours post-infection, and at 10 ⁇ M when added up to eight hours post-infection.
  • Ritonavir also exhibited antiviral activity at 0.1 and 1 ⁇ M up to six and 24 hours post-infection, respectively.
  • the infectivity of virions collected from cells exposed to 0.1 or 1 ⁇ M SQV at 8 and 16 hours post-infection was reduced by at least 90% when compared to virus-only control cells. Additionally, virus produced by cells exposed to 1 ⁇ M saquinavir at 24 hours post-infection exhibited nearly 90% reduction in infectivity compared to virus-only controls. Cells treated with 0.1 ⁇ M mPEG7-NHCO-SQV at 8 or 16 hours post-infection reduced virus infectivity to 63 and 99.9% of virus-alone controls, respectively, while 1 ⁇ M mPEG7-NHCO-aquinavire treatment reduced infectivity ⁇ 90% when added up to 24 hours post-infection.
  • PBMCs peripheral blood mononuclear cells obtained from a commercial source were purified following centrifugation using a Ficoll-Hypaque density gradient. Viable cells were induced to proliferate in the presence of PHA-P and recombinant human IL-2 for 72 hours.
  • TTP tritiated thymidine triphosphate
  • Saquinavir and mPEG5-NHCO— and mPEG7-NHCO-Saquinavir conjugates were protective against HIV-1, yielding EC 50 values of 0.009, 0.02, and 0.12 ⁇ M, respectively (Table 6).
  • the test compounds did not alter viability of PBMCs at test concentrations up to 1 ⁇ M.
  • TI was calculated as TC 50 /EC 50 .
  • Mononuclear cells were isolated from fresh human peripheral blood cells by Ficoll-Hypaque density gradient by centrifugation at 600 g for 30 minutes. Banded peripheral blood mononuclear cells (PBMCs) were aspirated from the resulting interface and washed with phosphate buffered saline by low speed centrifugation. Viable cells were diluted to 4 ⁇ 106 cells/mL in Hanks Balanced Salt Solution with 10% heat-inactivated human AB serum, then 100 ⁇ L of the cell suspension was plated to individual wells of a 96-well flat-bottom plate.
  • PBMCs Banded peripheral blood mononuclear cells
  • DPBS Dulbecco's phosphate buffered saline
  • MOI multiplicity of infection
  • HIV-1 replication was quantified by ELISA measurement of cell-free HIV-1 viral capsid protein (p24) in the tissue culture supernatant of treated monocyte-macrophage monolayers.
  • ELISA to quantify p24 was performed according to manufacturer's instructions (Perkin Elmer, Waltham, Mass.). The quantity of free HIV-1 p24 antigen in the sample was determined by comparing its absorbance at 490 nm to that of control standards.
  • PEG-atazanavir displayed increased potency compared with unconjugated atazanavir, AZT protected against HIV-1 infection with an EC 50 value of 0.001 ⁇ M, Test compounds were not toxic to human monocytes at test concentrations up to 1 ⁇ M. A TI was calculated as described above using TC 50 values derived from cell toxicity data in response to test compounds in the absence of viral infection.
  • CYP450 inhibition assays identify and quantify the extent that a test compound inhibits key cytochrome P450 enzymes in liver microsomes. Inhibition studies are performed by incubating multiple concentrations of test compound (0.1-25 ⁇ M) with individual CYP isoform-specific probe substrates in buffer (0.1 M phosphate buffer, pH 7.4) containing human liver microsomes (0.1 mg/mL). The final concentration of organic solvent in all incubations is 0.25%. Samples are incubated at 37° C., and NADPH (1 mM final concentration) is added to initiate the reaction. Following five minutes incubation, the reactions are terminated by the addition of methanol containing internal standard then centrifuged at 2500 rpm for 20 minutes at 4° C.
  • Standard in vitro probe substrates for CYP enzymes include: CYP1A2, Phenacetin, 7-ethoxyresorufin; CYP2A6, Coumarin; CYP2B6, Bupropion; CYP2C8, Paclitaxel; CYP2C9, Diclofenac, Tolbutamide, S-Warfarin; CYP2C19, S-Mephenyloin, Omeprazole; CYP2D6, Bufurolol, Dextromethorphan; CYP2E1, Chlorzoxazone; CYP3A4, Midazolam, Testosterone.
  • CYP3A4 CYP3A4 IC50 ( ⁇ M) IC50 ( ⁇ M) Compound Testosterone Midazolam Atazanavir (repeated tests) 2.5 11.0 1.43 1.10 di-mPEG3-Atazanavir 23.4 >50.0 di-mPEG5-Atazanavir >50.0 >50.0 di-mPEG7-Atazanavir >50.0 >50.0 mono-mPEG1-Atazanavir 10.4 2.56 mono-mPEG3-Atazanavir 12.8 2.85 mono-mPEG5-Atazanavir >25 9.35 mono-mPEG6-Atazanavir >25 16.7 mono-mPEG7-Atazanavir >25 20.1 Darunavir (repeated tests) 0.9 0.9 0.648 0.682 mPEG3-N-Darunavir 3.1 1.2
  • CYP2C9 IC50 Compound Tolbutamide Atazanavir 10.3 mono-mPEG1-Atazanavir >25 mono-mPEG3-Atazanavir 24.6 mono-mPEG5-Atazanavir 23.7 mono-mPEG6-Atazanavir >25 mono-mPEG7-Atazanavir >25
  • CYP2D6 IC50 ( ⁇ M) Compound Dextromethorphan Atazanavir 14.9 mono-mPEG1-Atazanavir 18.3 mono-mPEG3-Atazanavir >25 mono-mPEG5-Atazanavir 22.2 mono-mPEG6-Atazanavir >25 mono-mPEG7-Atazanavir >25
  • CYP1A2 IC50 ( ⁇ M) Compound Phenacetin Atazanavir 20.6 mono-mPEG1-Atazanavir >25 mono-mPEG3-Atazanavir >25 mono-mPEG5-Atazanavir >25 mono-mPEG6-Atazanavir >25 mono-mPEG7-Atazanavir >25
  • the human liver microsome-mediated biotransformation of PEG-PI compounds was determined by monitoring the loss of the substrate as a function of time. (Table 10). Test compounds (3 ⁇ M final concentration) were preincubated with pooled (male and female) human liver microsomes (0.5 mg/mL final protein concentration) in buffer (0.1 M phosphate buffer, pH 7.4) at 37° C. Reactions were initiated by the addition of NADPH (1 mM final concentration), and allowed to continue for 0, 5, 15, 30, and 45 minutes. Reactions are terminated by the addition of 50 ⁇ L methanol containing internal standard then centrifuged at 2500 rpm for 20 minutes at 4° C. Sample supernatants are analyzed by liquid chromatography-tandem mass spectrometry.
  • Metabolism of PI conjugates was investigated by monitoring the levels of unmetabolized parent compound following incubation with cryopreserved human or monkey hepatocytes.
  • the use of whole hepatocytes rather than microsomes requires that the drugs cross the cell membrane to interact with intracellularly-localized metabolic enzymes, and thus provides the most relevant in vitro system for understanding overall metabolic stability of these compounds.
  • the results of these hepatocyte stability studies are provided in Tables 11A and 11B.
  • Test compounds (0.1 ⁇ M or 3 ⁇ M, as indicated) are incubated with cryopreserved human hepatocytes at a cell density of 0.5 ⁇ 10 6 viable cells/mL. The final DMSO concentration in the incubation is 0.25%. Duplicate samples (50 ⁇ L) are removed from the incubation mixture at various timepoints up to four hours (for 0.1 ⁇ M test compound concentration), or one or two hours (for 3 ⁇ M test compound concentration), and the reaction is quenched by the addition of methanol containing internal standard (100 ⁇ L). The samples are centrifuged (2500 rpm at 4° C. for 20 min), and the supernatants at each time point pooled for cassette analysis by LC-MS/MS.
  • Optimized assay conditions utilized to observe metabolism of the non-PEG protease inhibitors comprise a four-hour incubation with human hepatocytes at a concentration of 0.1 ⁇ M test compound.
  • Table 11A and FIG. 3 demonstrate that PEG conjugation greatly reduced the rate of metabolism of PI molecules in human hepatocytes in comparison with non-PEG parent, with the exception of mPEG3-N-darunavir.
  • Table 11B demonstrates that stability in monkey hepatocytes is somewhat different from that in human hepatoctyes, suggesting that cynomolgus monkeys would not be a representative species for examining the activity of these molecules in order to predict their disposition in humans.
  • Table 11C demonstrates that stability in rat hepatocytes is somewhat different from that in human hepatoctyes. There appears to be a clear PEG-length dependent trend towards increased stability in rat hepatocytes.
  • Table 11D demonstrates that stability in dog hepatocytes is somewhat different from that in human hepatoctyes, suggesting that beagle dog would not be a representative species for examining the activity of these molecules in order to predict their metabolism in humans.
  • Timecourse analyses of test molecules (5 ⁇ M) with CYP2D6- or CYP3A4-expressing BactosomesTM are performed in buffer (0.1 M phosphate, pH 7.4) in a final reaction volume of 25 ⁇ L. Samples are preincubated at 37° C., and each reaction is initiated by the addition of NADPH (1 mM final concentration). The final concentration of organic solvent is maintained at 0.25%. Reactions are terminated by the addition of methanol (50 ⁇ L) containing internal standard at 0, 5, 15, 30, and 45 minutes. Samples are centrifuged at 2500 rpm for 20 minutes at 4° C. to precipitate protein, and the supernatant is analyzed by liquid chromatography-tandem mass spectrometry.
  • the di-mPEG3-Atazanavir derivative was metabolized to a similar level as ATZ in both CYP3A4- and CYP2D6-expressing BactosomesTM.
  • Darunavir conjugates were metabolized more rapidly than non-PEG parent in both isoform-specific BactosomesTM, with a clear correlation between increased PEG length and decreased half-life.
  • Saquinavir conjugates were also more rapidly metabolized in CYP2D6-expressing BactosomesTM compared to saquinavir, however there was an inverse relationship between PEG length and improved metabolism.
  • Parent and PEG-derivatives of saquinavir were rapidly metabolized by CYP3A4-BactosomesTM, and there was no differentiation in half-life. See Table 12 and FIGS. 4A-F .
  • PEGylated protease inhibitors were evaluated for their propensity to be bound by proteins in normal human plasma or in human liver microsome preparations using equilibrium dialysis analysis.
  • the protein-free and microsome-containing solutions were then added to either side of an equilibrium dialysis system and allowed to reach equilibrium at 37° C.
  • the concentration of compound on both sides of the membrane at equilibrium was quantified by LC-MS/MS, and the fraction of compound that remains unbound (F u ) is calculated.
  • test compound solutions (5 ⁇ M) are prepared in buffer and species-specific plasma (final DMSO concentration 0.5%).
  • the plasma-containing solution is added to one side of the membrane of an equilibrium dialysis system, and the plasma-free is added to the other side. Dialysis was allowed to reach equilibrium by incubation for two hours at 37° C. The concentration of compound on both sides of the membrane was measured using LC-MS/MS.
  • PEGylation of protease inhibitors demonstrated significantly reduced binding to proteins in human, rat, dog, mouse, and monkey plasma in comparison to non-PEGylated parent molecules, as demonstrated by increased free fraction (F u ). There was very little binding of both PEG-protease inhibitors and non-PEG protease inhibitors to proteins present in human liver microsomal preparations. Data presented in Tables 15A and 15B.
  • Freshly separated human PBMCs from a single donor were suspended in DPBS at 4 ⁇ 10 6 cells/mL and incubated in a 75 cm 2 cell culture flask for 90 minutes at 37° C., 5% CO 2 . Non-adherent cells were removed by washing with DPBS.
  • the infected cells were washed and resuspended at 1 ⁇ 106 cells/mL in complete medium, then 100 ⁇ L of the cell suspension was aliquoted to individual wells of a 96-well plate. Compound diluted in complete medium was added to cells.
  • HIV-1 replication was quantified by measuring HIV-1 reverse transcriptase activity in cell-free supernatants using a standard radioactive tritiated thymidine triphosphate (TTP) polymerization incorporation assay.
  • TTP radioactive tritiated thymidine triphosphate
  • 1 ⁇ L of TTP (1 Ci/mL) was combined with 4 ⁇ L of dH 2 O, 2.5 ⁇ L of poly rA and oligo dT (0.5 mg/mL and 1.7 Units/mL, respectively), and 2.5 ⁇ L reaction buffer (125 ⁇ L 1 mol/L EGTA, 125 ⁇ L dH 2 O, 125 ⁇ L 20% Triton X-100, 50 ⁇ L 1 mol/L Tris, pH 7.4, 50 ⁇ L 1 mol/L DTT, 40 ⁇ L mol/L MgCl 2 ), then 10 ⁇ l, of the resulting mixture was combined with 15 ⁇ L, of virus-containing supernatant in a round-
  • Test compound-induced cytotoxicity was evaluated by monitoring the reduction of the tetrazolium dye XTT in microtiter plates containing compound-treated cells in the absence of HIV-1 virus.
  • XTT solution was prepared at 0.15 mg/mL in phosphate buffered saline.
  • Phenazine methosulfate (PMS) solution was prepared at 0.15 mg/mL in phosphate buffered saline.
  • a XTT/PMS stock solution was prepared immediately before use by combining 40 uL of PMS per mL of XTT solution.
  • CEM-SS cells were passaged in T-75 flasks in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, 100 Units/mL penicillin and 100 ⁇ g/mL streptomycin.
  • RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, 100 Units/mL penicillin and 100 ⁇ g/mL streptomycin.
  • cells were split 1:2 to assure exponential growth at the time of the assay. Cells were counted using a hemocytometer, and viability measured by Trypan Blue dye exclusion. Cell viability was greater than 95% in all assays.
  • CEM-SS cells were resuspended at 5 ⁇ 10 4 cells/mL in medium, and 100 ⁇ L of the cell suspension was aliquoted to individual wells of a 96-well microtiter plate containing 100 ⁇ L 2 ⁇ concentration test compound. Plates were incubated for 7 days at 37° C., 5% CO 2 until cytotoxicity evaluation.
  • Test compound-induced cytotoxicity was evaluated by monitoring the reduction of the tetrazolium dye XTT to a soluble formazan product in microtiter plates containing compound-treated cells in the absence of HIV-1 virus.
  • XTT solution was prepared at 0.15 mg/mL in phosphate buffered saline.
  • Phenazine methosulfate (PMS) solution was prepared at 0.15 mg/mL in phosphate buffered saline.
  • a XTT/PMS stock solution was prepared immediately before use by combining 40 uL of PMS per mL of XTT solution.
  • CEM-SS cells (2.5 ⁇ 103 cells/well) were cultured in the presence or absence of test compound for 7 days at 37 C, 5% CO2 in a 96-well microtiter plate. On day 6 of the incubation, 1 uCi of [methyl-3H]-thymidine for DNA synthesis, [5-3H]-uridine for RNA synthesis, or [3,4,5-3H]-leucine for protein synthesis was added to compound-treated cells, and the plate was further incubated for 16 hours. The cells were transferred to a glass fiber filtermat using an Inotech cell harvester. The filtermats were washed with distilled water then sealed in a bag with scintillation fluid. Incorporated radioactivity was measured using a Wallac scintillation counter.
  • Mononuclear cells were isolated from fresh human peripheral blood cells by Ficoll-Hypaque density gradient by centrifugation at 600 g for 30 minutes. Banded peripheral blood mononuclear cells (PBMCs) were aspirated from the resulting interface and washed with phosphate buffered saline by low speed centrifugation. Cell viability was evaluated by Trypan Blue dye exclusion.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs were centrifuged and resuspended in growth medium (RPMI 1640 with 15% fetal bovine serum, 2 mmol/L L-glutamine, 3.6 ng/mL recombinant human interleukin (IL)-2, 100 Units/mL penicillin and 100 ⁇ g/mL streptomycin to induce proliferation.
  • RPMI 1640 with 15% fetal bovine serum, 2 mmol/L L-glutamine, 3.6 ng/mL recombinant human interleukin (IL)-2, 100 Units/mL penicillin and 100 ⁇ g/mL streptomycin to induce proliferation.
  • IL human interleukin
  • Assays were initiated with PBMCs that had been induced to proliferate for 72 hours.
  • PHA-P stimulated PBMCs from three donors were pooled and resuspended at 1 ⁇ 10 6 cells/mL and 50 ⁇ L of the cell suspension were aliquoted to individual wells of a 96-well microtiter plate. Test compound was then added to cells to the desired final concentration. Plates were incubated at 37° C., 5% CO 2 for 7 days prior to cytotoxicity evaluation. XTT measurement for cytotoxicity.
  • Test compound-induced cytotoxicity was evaluated by monitoring the reduction of the tetrazolium dye XTT to a soluble formazan product in microtiter plates containing compound-treated cells in the absence of HIV-1 virus.
  • XTT solution was prepared at 0.15 mg/mL in phosphate buffered saline.
  • Phenazine methosulfate (PMS) solution was prepared at 0.15 mg/mL in phosphate buffered saline.
  • a XTT/PMS stock solution was prepared immediately before use by combining 40 uL of PMS per mL of XTT solution.
  • PBMCs PBMCs (5 ⁇ 104 cells/well) were cultured in the presence or absence of test compound for 7 days at 37 C, 5% CO2 in a 96-well microtiter plate. On day 6 of the incubation, 1 uCi of [methyl-3H]-thymidine for DNA synthesis, [5-3H]-uridine for RNA synthesis, or [3,4,5-3H]-leucine for protein synthesis was added to compound-treated cells, and the plate was further incubated for 16 hours. The cells were transferred to a glass fiber filtermat using an Inotech cell harvester. The filtermats were washed with distilled water then sealed in a bag with scintillation fluid. Incorporated radioactivity was measured using a Wallac scintillation counter.
  • mono-mPEG3-atazanavir displayed comparable or lower toxicity than atazanavir, as evident from a higher TC 50 value, determined by four different assay methods.
  • One of these methods (XTT) produced a similar result in PBMC cells, while the other assay methods in PBMCs displayed slightly lower TC 50 values.
  • XTT XTT
  • mPEGn-O-Darunavir conjugates demonstrated a similar pattern to mono-mPEG3-atazanavir; thus in CEM-SS cells they displayed higher TC 50 values by all experimental readouts, with the exception of mPEG5-O-Darunavir in the Thymidine Incorporation assay.
  • mPEG-O-Darunavir conjugates displayed an improved toxicity profile compared with darunavir using all experimental approaches tried, while mPEG-3-O-Darunavir had comparable TC50 values to darunavir.
  • the mPEG-O-Darunavir conjugates are likely to have good activity and therapeutic indices in antiviral use against HIV.
  • the mPEG-N-Darunavir conjugates displayed somewhat lower TC 50 values in PBMC cells than darunavir.
  • the ion channel blocking profile of compounds NKT-10315, NKT-10404, NKT-10496, NKT-10497 were characterized on hERG current recorded from a human cell line stably expressing hERG (HEK-293). Currents were measured using the whole-cell variant of the patch clamp method. Glass pipettes were produced with tip openings of 1 to 2 gm for K current recordings. Pipette tip resistance was approximately 1.0 to 2.0 M ⁇ when filled with K internal solutions (130 mM KCl, 5 mM MgCl 2 , 10 mM HEPES, 5 mM EGTA, 5 mM ATP-Na 2 , pH7.2). An Axopatch 1-B amplifier (Axon Instruments, Foster City, Calif.) was used for whole-cell voltage clamping. Voltage clamp pulses were controlled by pClamp software (ver 9.2, Axon Instruments).

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