US 20070207952A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/0207952 A1 Silva et al. (43) Pub. Date: Sep. 6, 2007

(54) LONG ACTING BIOLOGICALLY ACTIVE Publication Classification CONUGATES (51) Int. Cl. (75) Inventors: Abelardo Silva, Ellicott City, MD A6II 38/16 (2006.01) (US); John E. Erickson, Potomac, MD A6II 38/10 (2006.01) (US); Michael Eissenstat, Frederick, C07K I4/47 (2006.01) MD (US); Elena Afonina, Frederick, C07K 7/08 (2006.01) MD (US): Sergei Gulnik, Frederick, (52) U.S. Cl...... 514/12: 530/326; 530/324; MD (US) 530/325; 514/13; 424/78.27 Correspondence Address: (57) ABSTRACT PROSKAUER ROSE LLP The invention provides biologically active compounds that 1001 PENNSYLVANIAAVE, N.W., may be reacted with macromolecules, such as albumin, to SUTE 4OO SOUTH form covalent linked complexes wherein the resulting com WASHINGTON, DC 20004 (US) plexes exhibit a desired biological activity in vivo. More specifically, the complexes are isolated complexes compris (73) Assignee: SEQUOIA PHARMACEUTICALS, ing a biologically active moiety covalently bound to a Gaithersburg, MD (US) linking group and a protein. The complexes are prepared by conjugating a biologically active moiety, for example, a (21) Appl. No.: 10/550,715 renin inhibitor or a viral fusion inhibitor peptide, with purified and isolated protein. The complexes have extended (22) PCT Filed: Mar. 24, 2004 lifetimes in the bloodstream as compared to the unconju gated molecule, and exhibit biological activity for extended (86) PCT No.: PCT/USO4/O8847 periods of time as compared to the unconjugated molecule. S 371(c)(1), The invention also provides anti-viral compounds that are inhibitors of viral infection and/or exhibit anti-fusiogenic (2), (4) Date: Aug. 16, 2006 properties. In particular, this invention provides compounds Related U.S. Application Data having inhibiting activity against viruses such as human immunodeficiency virus (HIV), respiratory syncytial virus (60) Provisional application No. 60/456,472, filed on Mar. (RSV), human parainfluenza virus (HPV), measles virus 24, 2003. Provisional application No. 60/456,952, (MeV), and simian immunodeficiency virus (SIV) and that filed on Mar. 25, 2003. Provisional application No. have extended duration of action for the treatment of viral 60/518,892, filed on Nov. 10, 2003. infections.

Patent Application Publication Sep. 6, 2007 Sheet 16 of 20 US 2007/0207952 A1

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Patent Application Publication Sep. 6, 2007 Sheet 18 of 20 US 2007/0207952 A1

SunO? Patent Application Publication Sep. 6, 2007 Sheet 19 of 20 US 2007/0207952 A1

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Patent Application Publication Sep. 6, 2007 Sheet 20 of 20 US 2007/0207952 A1

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LONG ACTING BIOLOGICALLY ACTIVE 0006 The majority of drugs are administered orally. CONUGATES Typically, the administered dosage requires that the drug be administered repetitively to maintain a therapeutic level and 0001. This application claims priority to provisional the rapid decrease in blood levels over time often results in applications 60/518,892, filed Nov. 10, 2003, Ser. No. initial levels that exceeds the desired therapeutic levels. 60/456,472, filed Mar. 24, 2003, and Ser. No. 60/456,952, Various technological approaches have been designed to filed Mar. 25, 2003, the contents of each of which are hereby avoid these problems, including the administration of bio incorporated by reference in their entireties. logically active agents by mechanical systems such as pumps, controlled release or slow release tablets and cap FIELD OF INVENTION Sules, depots and related technologies. 0002 The invention relates to biologically active com 0007. Therapeutic agents that are administered by injec pounds that may be used to react with proteins, such as tions encounter similar problems relating to their limited albumin, to form covalent linked complexes wherein the lifetime in vivo. Moreover, repetitive injections are incon resulting complexes exhibit a desired biological activity in venient and highly undesirable. Therefore, there is a need for vivo. More specifically, the complexes are isolated com new methods that allow for ease of administration of bio plexes comprising a biologically active moiety covalently logically active agents into the bloodstream and that main bound to a linking group and a protein. In one embodiment, tain effective levels of the therapeutic agents for an extended the protein is a blood protein such as albumin, or HSA. In period of time in vivo. another embodiment, the protein is recombinant HSA. The complexes are prepared by conjugating a biologically active HIV/AIDS moiety, for example, a renin inhibitor or a viral fusion 0008 Acquired immune deficiency syndrome (AIDS) is inhibitor peptide, with purified and isolated protein. The a fatal disease caused by infection with HIV-1. By the end complexes have extended lifetimes in the bloodstream as of 2002, over 42 million people will be infected with HIV-1 compared to the unconjugated molecule, and exhibit bio worldwide, and over 20 million individuals will have died of logical activity for extended periods of time as compared to HIV/AIDS. Drug resistant strains of HV are prevalent on the the unconjugated molecule. Optionally, the compounds and patient population. Current estimates are that up to 50% of complexes of the present invention are isolated and purified. drug-treated HIV-infected patients harbor a drug-resistant The invention also provides methods for achieving a desired strain of HIV. Transmission rates of dmg-resistant HIV are biological effect in vivo, comprising administering to the between 10-15% in the US alone. Estimates of reported bloodstream of a mammalian host the novel isolated com cases in the very near future also continue to rise dramati plexes of the present invention. cally. Consequently, there is a great need to develop drugs 0003. The invention also provides compounds, including and vaccines to combat AIDS. nucleosides, nucleoside analogs, nucleotides, nucleotide 0009. The AIDS virus was first identified in 1983. It has analogs, polypeptides, polypeptide derivatives, peptidomi been known by several names and acronyms. It was origi metic compounds and their bioconjugated forms that are nally the third known T-lymphocyte virus (HTLV-III), and it inhibitors of virus infections. The invention also provides has the capacity to replicate within cells of the immune methods for administering bioconjugated forms of these system, causing profound cell destruction and impairment of inhibitors having an extended duration of action for the immunity. The AIDS virus is a retrovirus, which is family of treatment of virus infections, including multidrug-resistant viruses that use reverse transcriptase during their replication. virus infections. This particular retrovirus is also known as lymphadenopa 0004. In particular, the invention provides compounds thy-associated virus (LAV), AIDS-related virus (ARV) and, and their bioconjugated forms that inhibit human immuno most recently, as human immunodeficiency virus (HIV). deficiency viruses (HIV), and methods for administering Viral Diversity: bioconjugated forms of inhibitors that provide a prolonged duration of action for the treatment of HIV infections, 0010 HIV is a member of the lentivirus family of retro including multidrug-resistant HIV infections (mdrHIV). viruses, which includes simian immunodeficiency virus (SIV), and numerous other retroviruses that cause immuno BACKGROUND OF THE INVENTION deficency diseases in mammals. Two distinct types of HIV have been described to date, namely HIV-1 and HIV-2, 0005 Certain active peptide and protein therapeutics although infection with HIV-1 is more common worldwide. useful for administration to a mammalian host exhibit poor The acronym HIV will be used herein to refer to all HIV-1 pharmacokinetic profiles, and are often rapidly metabolized viruses generically, unless otherwise noted. HIV-1 is further and cleared by the mammalian system before the peptide or divided into three groups: major (M), outlier (O), and new protein can bind to a specific target. Typically, once admin (N). Most HIV-1 isolates to date belong to one often distinct istered, the biologically active agents are susceptible to clades, or Subtypes, of the M group. The M group Subtypes enzyme degradation and clearance. As a consequence, cer are represented by the letters A-J. Subtype B is the most tain active agents must be administered more frequently common in the US and Europe. However, subtype C which result in undesired large fluctuations in the blood accounts for almost 50% of HIV worldwide, and is most plasma levels of the agent can lead to a variety of adverse common in Africa. All Subtypes are present in Africa, with side reactions and/or diminished efficacy. For an effective non-C clades tending to be cluster in distinct geographical therapeutic, the active agent must be able to be transported regions. Subtype identification is usually determined by to the active site or must be administered directly to the sequencing of the env gene, and comparison of the gp41 target site without significant loss of biological activity. sequences, which give a subtype "fingerprint. US 2007/0207952 A1 Sep. 6, 2007

Viral Life Cycle: undetectable levels eventually experience treatment failure (Grabar et al., 2000. Factors associated with clinical and 0011 HIV primarily infects CD4-bearing helper/inducer Virological failure in patients receiving a triple therapy T-cells, and can also infect other cells that express the CD4 including a protease inhibitor. Aids. 14:141-9: Wit et al., glycoprotein at the membrane Surface. Recent evidence has 1999. Outcome and predictors of failure of highly active shown that the co-localization of certain chemokine recep antiretroviral therapy: one-year follow-up of a cohort of tors at the cell surface is essential for efficient viral infection. human immunodeficiency virus type 1-infected persons. J HIV is cytopathic to CD4+ lymphocytes, and their numbers Infect Dis. 179:790-8). Moreover, 10 to 40% of antiviral steadily decline of over a period of years, resulting in a therapy-naive individuals infected with HIV-1 have persis severely compromised immune system. HIV infection can tent viral replication (plasma HIV RNA-500 copies/ml) also result in neurological deterioration and dementia. under HAART (Gulick et al., 1997. Treatment with indi Unless treated with effective chemotherapy, HIV infection is navir, Zidovudine, and lamivudine in adults with human almost always fatal, and leads to death from opportunistic immunodeficiency virus infection and prior antiretroviral infections, cancer or neurodegenerative disease. therapy. N Engl J Med. 337:734-9; Hammer et al. 1997. A 0012. The HIV-1 genome contains at least nine different controlled trial of two nucleoside analogues plus indinavir in genes. Ihe largest genes are gag (coding for structural persons with human immunodeficiency virus infection and proteins), pol (coding for the viral enzymes protease, CD4 cell counts of 200 per cubic millimeter or less. AIDS reverse transcriptase and integrase) and env (coding for the Clinical Trials Group 320 Study Team. N. Engl J Med. envelope glycoproteins). Homologues of the gag, pol and 337:725-33; Staszewski et al., 1999. Efavirenz plus zidovu env genes are found in all retroviruses dine and lamivudine, efavirenz, plus indinavir, and indinavir plus zidovudine and lamivudine in the treatment of HIV-1 0013 The gag and pol regions of the genome encode infection in adults. Study 006 Team. N Engl J Med. polycistronic messenger RNAS which are translated into 341:1865-73) possibly due to transmission of drug-resistant large polyprotein precursors. The viral polyproteins are HIV-1 variants (Wainberg, M. A., and G. Friedland. 1998. Subsequently cleaved into mature structural proteins and Public health implications of antiretroviral therapy and HIV enzymes by a viral-encoded protease that is, itself, a product drug resistance. JAMA. 27.9:1977-83). In addition, it is of the pol gene. The two Env proteins, gp120 and gp41, are evident that with these anti-HIV drugs only partial immu cleaved from a larger precursor (gp160) by a cellular nologic reconstitution is attained in patients with advanced enzyme. HIV-1 infection. 0014. Other HIV-1 gene products, e.g., Tat, Rev, Vpr and Drug Resistance: Nef intervene to regulate the virus life cycle. Nef also affects particle infectivity. The gene products Vif and Vpu 0016. The rapid emergence and spread of drug-resistant function in virus infectivity and virus particle maturation, mutant strains of HIV is rendering current drugs ineffective, respectively. The viral genome is flanked at each end by long and is one major cause of treatment failure. Recent estimates terminal repeat sequences (LTRs). The LTRs contain bind are that over 75% of drug-experienced patients in North ing sites for cellular proteins that are able to activate America harbor HIV that is resistant to one or more of the transcription and are also under the control of viral signals. 16 FDA-approved antiretroviral agents used in multi-drug The complex regulation of HIV allows the virus to establish cocktails. Drug-resistant HIV accounts for up to 12% of latency, then respond rapidly to various signals and synthe new infections. Drug-resistant HIV strains emerge in indi size high levels of viral proteins and virions, leading to the viduals who are infected with a wild type strain of HIV and production and release of large numbers of progeny virus, who are exposed to Suboptimal doses of one or more the subsequent destruction of the infected cell, and the antiretroviral agents (Burger, et al. Antivir. Ther., 1998). re-infection of large numbers of healthy CD4+ lymphocytes. There are three major classes of antiretroviral agents: nucleoside reverse transcriptase inhibitors (NRTIs), non Antiretroviral Agents: nucleoside reverse transcriptase inhibitors (NNRTIs), and 0.015 The field of antiretroviral chemotherapeutics protease inhibitors (PIs). The initial strain of drug resistant developed in response to the need for agents effective HIV that is selected depends on the particular drug regimen, against retroviruses, in particular EV. By the end of 2002, and often requires the replacement of one drug by another of sixteen antiretroviral agents were approved by the FDA for the same class. However, over time the continued selection treatment of HIV/AIDS. While there are many ways, in of new strains with multiple mutations often leads to class principle, in which an agent can exhibit anti-retroviral specific drug-resistance and, eventually, to complete treat activity, all of these agents inhibit either the viral reverse ment failure. Cross-resistance to drugs of the same class is transcriptase, or the viral protease. Highly active antiretro spreading at an alarmingly high rate (Erickson, et al. AIDS, viral therapy (HAART) refers to a variety of drug cock 13:S189, (1999); Gulnik, et al. Vitam. Horm., 58:213 tails, or combinations of three or more antiretroviral agents, (2000); Menendez-Arias, et al. Trends Pharmacol. Sci., that can potently suppress viral replication and prevent or 23:381 (2002)). delay the onset of AIDS (Mitsuya, H., and J. Erickson. 1999. Discovery and development of antiretroviral therapeutics for Drug Side Effects: HIV infection., p. 751-780. In T. C. Merigan and J. G. 0017 Based on the well-accepted theory that drug resis Bartlet and D. Bolognesi (ed.), Textbook of AIDS Medicine. tance emerges as a result of low level replication in the Williams & Wilkins, Baltimore). However, the ability to presence of Sub-optimal levels of a drug, it has become provide effective long-term antiretroviral therapy for HIV-1 common practice in antiretroviral therapy to prescribe the infection has had only partial success, since 40 to 50% of maximum tolerable dose of every drug in the cocktail. Since those who initially achieve favorable viral suppression to HIV is a chronic and incurable infection, the requirement for US 2007/0207952 A1 Sep. 6, 2007

daily dosing of antiretroviral drug cocktails at maximum including, for example, the entry of enveloped viruses into dosages results in very high peak drug levels. This practice cells, and the aberrant fusion of virus-infected cells with has led to an alarmingly high rate of life-threatening side healthy cells, leading to the formation of syncytia, and the effects due to the chronic toxicities of many of these drugs Subsequent clearance, or death, of the cells. Peptides and (for review see Tozser, et al. Ann. NY Acad. Sci. 946:145 small molecules are known to inhibit or otherwise disrupt (2001)). Some of the more serious side effects associated membrane fusion-associated events, including, for example, with HAART toxicity include liver problems, heart disease, inhibiting retroviral infection of target cells. and lipodystrophy (Chen, et al., J. Clin. Endocrinol. Metab., 87:4845 (2002); Holstein, et al. Exp. Clin. Endocrinol. 0021 Numerous polypeptides have been described which Diabetes 109:389 (2001)). The combination of resistance inhibit the HIV infection of CD4 cells by interfering with the and side effects result in poor adherence to drug regimens fusion reaction. Several of these so-called “anti-fusiogenic and, ultimately, to treatment failure rates of between 40-45% peptides' are derived from the native amino acid sequence (Wit, et al., J. Infect. Dis. 179:790 (1999); Fatkenheuer, et al. of either of the two helix-forming segments of gp41 (Jiang AIDS 11:F113 (1997); Lucas, et al. Ann. Intern. Med. et al. Curr. Pharmaceut. Design 8:563 (2002)). Polypeptides 131:81 (1999); Chen, et al., 41st Intl Conf Antimicrob. consisting of sequences from either the N- or C-helix Agents Chemother. Abstract I-1914 (2001)). Thus, a sub forming regions of gp41 exhibit antiviral activity in cell stantial number of patients currently taking HAART will culture assays. X-ray crystal structures and NMR solution Soon run out of therapeutic options. structures of various isolated, recombinant forms of HIV-1, HIV-2 and SIV fusion proteins show that they all form 0018. The long-term benefits of HAART are limited by trimers that with an anti-parallel, helical bundle-type fold. the dual problems of poor adherence and drug resistance. In The bundles consist of three sets of hairpins, each of which addition to these problems, the prohibitively high costs of is formed by the antiparallel association between the two drug have severely limited access of the global HIV-infected helix-forming segments from a single protein chain. The population to HAART. Thus, there is an urgent need for new hairpins are arranged in Such a way that the first, or therapeutics that are 1) effective against wild type and drug N-terminal, helical segments are associated in a trimeric resistant viruses, 2) safe and non-toxic, and, 3) relatively inner bundle, and the second, or C-terminal, segments inexpensive to produce or, at least, to deliver. These require interact with the grooves formed by two adjacent N-terminal ments pose formidable challenges when added to the con helices. Thus, the hairpin structure appears to be a conse ventional issues of potency, pharmacology, safety, and quence of assembly into the quaternary structure of the mechanism of drug action (De Clercq, Clin Microbiol Rev. trimer, as opposed to being the fundamental building block 10:674-93 (1997); Erickson et al., AIDS 13:S189-204 of the trimer. (1999)). 0022 Peptides from the C-terminal and N-terminal hep Fusion Inhibitors and their Limitations for Prolonged tad repeat regions, including DP178 (Wild, et al., Proc. Natl. Therapy: Acad. Sci. USA, 91:9770 (1994)), also known as T-20, C34 (Chan, et al. Proc. Natl. Acad. Sci. USA, 95:15613 (1998)), 0019. One attractive solution to the drug resistance prob and DP107 exhibit potent antiviral activity. lem is to develop drugs with different mechanisms of action than those currently on the market. There are many ways, in 0023 U.S. Pat. Nos. 6,013,263, 6,017,536 and 6,020,459 principle, in which an agent can exhibit anti-retroviral incorporated herein in their entirety, likewise disclose that activity in cell culture. Inhibitors of EV with novel mecha the 36 amino acid peptide DP178 corresponding to amino nisms of action have been reviewed by DeClerq, Curr Med acids 638 to 673 of gp41 from the HIV-1 isolate LAI (HIV-1 Chem. 8:1543-72 (2001). Among these compounds, LAI), and the 38 amino acid peptide DP107 corresponding polypeptide inhibitors of HIV fusion (“anti-fusiogenic pep to amino acids 558-595 of gp41 from the HIV-1 LAI, both tides’) have been shown to be effective in human clinical exhibit potent anti-HIV-1 activity. WO 00/06599 teaches the trials. HIV infects human lymphocytes and other cell types use of C34 to inactivate gp41, and thus, prevent or reduce bearing the membrane-bound CD4 glycoprotein and a HIV-1 entry into cells. They are postulated to bind to the chemokine receptor. The initial step in HIV infection of a trimeric coiled-coil, or core, structure of gp41 during the CD4-bearing cell is the recognition of the CD4 receptor by transient state, and thereby prevent binding of the endog the HIV gp120 envelope protein, which is non-covalently enous C-helices. T-20, a 34-residue peptide, has been shown associated with the viral membrane through the viral mem to effectively lower viral load in drug-experienced patients. brane-bound HW gp41 envelope protein. The gp41 protein, This validates gp41 as a promising target for the develop or “fusion protein', contains several “fusiogenic domains, ment of new anti-HIV drugs. Unfortunately, the therapeutic including a fusion peptide and two self-associating helix delivery of T-20 is limited by its peptidic nature. T-20 has a forming segments (the “N-helix' and “C-helix'). short half-life of 1.8 hours (Kilby, et al, Nat. Med., 4:1302 (1998)), and needs to be administered by subcutaneous 0020 Recognition and binding of gp120 protein to the injection, twice a day. Injection-site inflammation is a com CD4 and chemokine receptors triggers the unmasking of the mon side-effect reaction, and the drug formulation and fusiogenic domains, the insertion of gp41 into the cell manufacturing challenges result in high cost of treatment. membrane, and the self-association of the two helix-forming T-20 is also rendered ineffective through the selection of a segments into a “hairpin' structure. The formation of the number of single mutations that lead to drug resistance both hairpin structure of gp41 is believed to be an essential step in vitro and in vivo. in the fusion of the viral and cell membranes, and is a slow process, requiring up to 30 min to complete. Membrane 0024. A backup FI, T-1249, is in Phase II clinical trials. fusion events, while commonplace in normal cellular pro This compound is an even longer peptide than T-20. Its chief cesses, are also involved in a variety of disease states, advantage is that it is more potent and has a longer half-life US 2007/0207952 A1 Sep. 6, 2007

than T-20. However, T-1249 still suffers from the require advance in antiviral therapy. Inhibitors that are both resis ment for daily injection, and drug resistant mutants are tance-repellent and long-lasting are defined as broad spec readily selected using this drug. trum durable inhibitors. 0025. Like many polypeptides, both T-20 and T-1249 Serum Albumin as a Prodrug: must be administered intravenously or Subcutaneously, and, 0033. A doxorubicin-albumin conjugate has been dis both exhibit a short half-life in vivo, primarily due to rapid closed as an antineoplastic prodrug agent. (F. Kratz et al., J. serum clearance and peptidase and protease activity. These Med. Chem. 2000, 43, 1253-1256). However, the conjugate pharmacological limitations reduce the therapeutic effec was prepared with an acid sensitive linker that allows the tiveness of these agents, while at the same time resulting in drug to be released at the low pH values present in lysos a high cost of treatment. omes and indoSomes of tumor cells. The preparation of the 0026 C34, like T-20 and T-1249, also suffers from a short conjugate was designed to avoid the ex vivo synthesis and half-life in Vivo, primarily due to rapid serum clearance and characterization of drug albumin conjugate which was con peptidase and protease activity. This in, turn greatly reduces sidered to be costly. its effective anti-viral activity. SUMMARY OF THE INVENTION 0027. It can be generally assumed that many of the anti-fusiogenic polypeptides and peptidomimetics described 0034. The present invention relates to biologically active in the art will suffer from the same limitations as found with compounds that may be used to react with proteins to form T-20 and T-1249 to the extent that they are of a similar size, covalently linked complexes wherein the resulting com on the order of 30-40 amino acids. plexes are found to exhibit desirable biological activities in vivo. More specifically, the complexes are isolated com 0028 Current antiretroviral therapy is a trade-off plexes comprising a compound, Such as an antiviral com between the development of life-threatening side effects and pound and a linking group, and the blood component is a life-threatening drug resistance. There is, therefore, a gen protein Such as albumin. The present invention also provides eral need of a method for providing antiretroviral agents at methods for achieving a desired activity in vivo. Such as drug levels that will reduce the chronic toxicity of these agents without compromising their therapeutic effective anti-viral activity, comprising administering to the blood ness. As an example, there is a need for a method of stream of a mammalian host the novel isolated complexes of prolonging the half-life of peptides like C34 in vivo without the present invention. Substantially affecting its anti-fusiogenic activity. There is 0035) In one embodiment, a pharmaceutical composition also a need for developing an inhibitor that will be effective is provided that comprises a purified conjugate. Such as an in the treatment of drug-resistant HIV infections, particu anti-viral complex, according to the present invention as an larly infections due to viral strains that are resistant to T-20 active ingredient. Pharmaceutical compositions according to and T-1249. Further, there is a need to develop agents that the invention may optionally comprise 0.001%-100% of one can prevent, or retard, the emergence of drug resistant HIV or more conjugates. Such as anti-viral complexes, of this in the therapeutic setting of a wild type infection. invention. These pharmaceutical compositions may be administered or coadministered by various methods known 0029. There is also a need for a method of prolonging the in the art for administering biologically active agents to the half-life of reverse transcriptase inhibitors and protease bloodstream. In a preferred aspect of the invention, the inhibitors, for example, Such that these agents can be admin compositions may be administered by injection. In another istered in dosages that will be less toxic on a long term basis. preferred aspect, the compositions may be administered by Such methods are likely to result in less expensive therapies infusion. The composition may advantageously comprise a since cumulative drug quantities required per patient per buffered saline solution of the conjugate. year will be lower than for current therapies. 0036). In another embodiment, methods and compositions 0030. In view of the foregoing problems, there exists a are provided for delivery of isolated conjugated complexes need for inhibitors against drug resistant HIV strains. Fur comprising biologically active agents, particularly therapeu ther, there exists a need for inhibitors against drug resistant tic agents such as anti-viral agents, where the complexes HIV gp41. Further still, there exists a need for inhibitors of comprising the agents have an extended half-life in the HIV that can prevent or slow the emergence of drug resistant bloodstream as compared to non-conjugated agents. HIV strains in infected individuals. Inhibitors with the ability to inhibit drug resistant HWV strains, and to slow the 0037. The invention comprises using a biologically emergence of drug resistant strains in the setting of wild type active compound covalently attached or linked to a linking HIV infections, are defined as “resistance-repellent inhibi group, the linking group comprising at least one chemically tOrS. reactive moiety which is capable of forming covalent bonds with functionalities present on the protein. By preparing the 0031) There also exists a need for HIV fusion inhibitors isolated complexes before administration of the complexes with prolonged duration of action. Inhibitors with prolonged into the blood of the host, particularly the bloodstream of the in vivo half-lives that possess durable suppression of viral host, a biologically active complex is generated that main replication in vivo are defined as “long-lasting inhibitors. tain an effective therapeutic effect in the bloodstream for an 0032. It should be recognized that resistance-repellent extended period of time as compared to a non-conjugated inhibitors and long-lasting inhibitors each represent clear biologically active agent. and unique advantages in the treatment of HIV/AIDS. It 0038. In particular, the present invention provides for should also be recognized that the combination of these two resistance-repellent, long-lasting and broad spectrum properties in a single agent would represent a revolutionary durable inhibitors of HIV gp41 and HIV, their compositions, US 2007/0207952 A1 Sep. 6, 2007

methods of design, and uses thereof for treating drug group can be a moiety, such as a maleimide, which is resistant HIV and wt infections in both salvage therapy and reactive with a thiol group on a blood protein, including a first-line therapy modalities. mobile blood protein such as albumin. 0039. In one embodiment, the invention provides resis 0043. The invention also provides the compounds tance-repellent inhibitors of HIV gp41 that target wild type described above bound in a complex with wild type or drug and drug-resistant mutant gp41 proteins, and that have resistant mutant forms of HIV-1 gp41. antiviral activity against wild type and drug-resistant HIV 0044) The invention further provides pharmaceutical strains. In particular, these compounds are active against compositions, comprising an inhibitor as described above, wild type HIV strains that contain naturally-occurring poly together with a pharmaceutically acceptable additive, morphisms in the sequence of gp41, and that contain muta excipient, or diluent. The composition may further comprise tions that confer resistance to T-20 and/or T-1249. In one an additional HIV gp41 inhibitor and/or an HIV protease embodiment, these inhibitors are peptide sequences that are inhibitor and/or an HIV reverse transcriptase inhibitor. related to peptide sequences of the N and C-terminal helical repeat regions of gp41. 0045. The invention fulirther provides methods of treating a patient Suffering from HIV infection, comprising admin 0040. In another embodiment, this invention relates to the istering to the patient a pharmaceutical composition as design of broad spectrum durable (persistent) inhibitors of described above. HIV gp41 that target wild type and drug-resistant mutant gp41 proteins, and that have antiviral activity against wild 0046. In further embodiments, the present invention type and drug-resistant HIV strains. In particular, these relates to biologically active compounds that may be used to compounds are active against wild type HIV strains that react with proteins to form covalently linked complexes contain naturally-occurring polymorphisms in the sequence wherein the resulting complexes are found to exhibit renin of gp41, and that contain mutations that confer resistance to inhibition activities in vivo. More specifically, the com T-20 and/or T-1249. The design of broad spectrum durable plexes are isolated complexes comprising a renin inhibitor inhibitors of HIV gp41 relates to chemically reactive modi and a linking group, and the blood component is a protein fications of peptides exhibiting anti-viral and/or anti-fusio Such as albumin. The present invention also provides meth genic activity Such that the modified peptides can react with ods for inhibiting renin activity in Vivo comprising admin available functionalities on blood components to form stable istering to the bloodstream of a mammalian host the novel covalent bonds. In one embodiment of the invention, the isolated complexes of the present invention. modified peptides comprise a reactive group which is reac 0047. In one embodiment, a pharmaceutical composition tive with amino groups, hydroxyl groups, or thiol groups on is provided that comprises a purified renin inhibitor complex blood components to form stable is covalent bonds. In according to the present invention as an active ingredient. another embodiment of the invention, the reactive group can Pharmaceutical compositions according to the invention be a moiety, such as a maleimide, which is reactive with a may optionally comprise 0.001%-100% of one or more thiol group on a blood protein, including a mobile blood renin inhibitors complexes of this invention. These pharma protein Such as albumin. ceutical compositions may be administered or coadminis 0041 More specifically, the present invention provides tered by various methods known in the art for administering broad spectrum durable gp41 inhibitors which are capable of biologically active agents to the bloodstream. In a preferred reacting with thiol groups on a blood component, either in aspect of the invention, the compositions may be adminis vivo or ex vivo, to form a stable covalent bond. In addition, tered by injection. In another preferred aspect, the compo the complexes formed from the methods disclosed herein are sitions may be administered by infusion. in themselves more stable and are longer acting than the 0048. In another embodiment, methods and compositions un-modified compounds. These complexes formed from the are provided for delivery of isolated conjugated complexes present invention have an extended iii Vivo half-life when comprising biologically active agents, particularly therapeu compared with the corresponding un-modified compounds. tic agents such as renin inhibitors, where the complexes The complexes of the invention is stable toward hydrolytic comprising the agents have an extended half-life in the cleavage or degradation for a period of about 4 hours to bloodstream as compared to non-conjugated agents. about 120 days. 0049. The invention comprises using a biologically 0042. In a further embodiment, this invention relates to active compound covalently attached or linked to a linking the design of bioconjugated compositions of broad spectrum group, the linking group comprising at least one chemically durable inhibitors of HIV gp41 that are covalently linked to reactive moiety which is capable of forming covalent bonds a mobile blood protein Such as serum albumin in a manner with functionalities present on the protein. By preparing the such that the bioconjugated form of the inhibitor has anti isolated complexes before administration of the complexes viral activity against both wild type and drug-resistant HIV into the blood of the host, particularly the bloodstream of the strains. In particular, these compounds are active against host, a biologically active complex is generated that main wild type HIV strains that contain naturally-occurring poly tain an effective therapeutic effect in the bloodstream for an morphisms in the sequence of gp41, and that contain muta extended period of time as compared to a non-conjugated tions that confer resistance to T-20 and/or T-1249. In one biologically active agent. embodiment ofthis invention, the bioconjugates are formed using modified peptides that comprise a reactive group Definitions: which is reactive with amino groups, hydroxyl groups, or 0050. Unless otherwise stated, the following terms used thiol groups on blood components to form stable covalent in the specification and claims shall have the following bonds. In another embodiment of the invention, the reactive meanings for the purposes of this Application. US 2007/0207952 A1 Sep. 6, 2007

0051. A “complex' as used herein, is a compound com hydroxide and calcium hydroxide. Acceptable organic bases prising a biologically active agent Such as an anti-viral include ethanolamine, diethanolamine, triethanolamine, compound or a renin inhibitor, a linking group and a protein tromethamine, N-methylglucamine and the like. Such as albumin. 0.058 “Protected derivatives” means derivatives of 0.052 “Derivative” means a compound that is derived inhibitors in which a reactive site or sites are blocked with from Some other compound and usually maintains its gen protecting groups. Protected derivatives are useful in the eral structure. preparation of inlnbitors o anti-viral agents or in themselves 0053 “Isolated such as an isolated compound is a com may be active as inhibitors or anti-viral agents. A compre pound. Such as a naturally occurring compound Such as hensive list of Suitable protecting groups can be found in T. albumin, that is substantially separated from other compo W. Greene, Protecting Groups in Organic Synthesis, 3rd nents which accompany the compound in its natural state. edition, John Wiley & Sons, Inc. 1999. “Isolated as applied to a compound obtained from blood or 0059) “Therapeutically effective amount” means that blood plasma, means a compound, such as a particular amount which, when administered to an animal for treating biological component from blood protein or blood plasma, a disease, is sufficient to effect such treatment forthedisease. that is purified or isolated from other biological compounds or components in the blood or blood plasma before the 0060 “Treatment” or “treating means any administra compound is further conjugated with a biologically active tion of a compound of the present invention and includes: agent Such as an anti-viral agent or renin inhibitor or the like. 0061 (1) preventing the disease from occurring in an The isolated compound exists in a physical milieu distinct animal which may be predisposed to the disease but does not from that in which it occurs in nature and/or has been yet experience or display the pathology or symptomatology completely or partially separated or purified from other components in nature prior to Submitting the compound to a of the disease, reaction with the biologically active agent. The isolated 0062 (2) inhibiting the disease in an animal that is compounds or complex of the invention has the advantage of experiencing or displaying the pathology or symptomatol allowing more selective reaction or conjugation with the ogy of the disease (i.e., arresting irther development of the biologically active agents, such as an anti-viral agent or pathology and/or symptomatology), or renin inhibitor or the like, of the present invention with minimum interference from reactions with undesired com 0063 (3) ameliorating the disease in an animal that is ponents of the blood or blood plasma. experiencing or displaying the pathology or symptomatol ogy of the disease (i.e., reversing the pathology and/or 0054) “Linker as used herein, refers to a linking group symptomatology). which links or attaches a biologically active compound AV with a protein Pr, such as albumin, to form a covalently 0064 “Stable’: a conjugate is stable when it is not bound complex comprising the biologically active com cleaved prior to binding to a target, and where the macro pound, the linker, and the protein. molecular component of the conjugate, such as albumin, is not substantially degraded prior to target binding. The 0.055 “Pharmaceutically acceptable” means that which is macromolecule is not substantially degraded when, even useful in preparing a pharmaceutical composition that is though some protease cleavage may occur, the conjugate generally safe, non-toxic and neither biologically nor oth retains a molecular weight greater than about 50 kDa. The erwise undesirable and includes that which is acceptable for conjugate is considered intact when it retains a molecular veterinary use as well as human pharmaceutical use. weight of at least about 50 kDa. 0056 “Pharmaceutically acceptable salts' means salts of 0065 “Substantially retains’: a conjugate substantially inhibitors of the present invention which are pharmaceuti retains the activity ofthe pharmacologically active moiety cally acceptable, as defined above, and which possess the when its activity is at least about 10% of the non-conjugated desired pharmacological activity. Such salts include acid pharmacologically active moiety (and may be higher on a addition salts formed with inorganic acids such as hydro molar ratio). Typically the activity of the conjugate is 0.1 to chloric acid, hydrobromic acid, Sulfuric acid, nitric acid, 10 times the activity of the non-conjugated pharmacologi phosphoric acid, and the like; or with organic acids such as cally active, though further enhancements of activity may be acetic acid, propionic acid, hexanoic acid, heptanoic acid, observed. cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, Succinic acid, malic acid, maleic 0066 “Pharmacologically inert': with respect to a mac acid, fumaric acid, tartaric acid, citric acid, benzoic acid, romolecule used in a conjugate means that the molecule is o-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic non-toxic. The molecule may or may not have biological acid, methanesulfonic acid, ethanesulfonic acid, 1.2- activity distinct from that of conjugate, thought it typically ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benze does not. “No biological activity” means that administration nesulfonic acid, lauryl Sulfuric acid, gluconic acid, glutamic of non-conjugated carrier to Subject does not produce any acid, hydroxynaphthoic acid, salicylic acid, Stearic acid, Substantial perturbation in normal physiology of the Subject. muconic acid and the like. 0067 “Pseudo-peptides” or “peptide mimetics” or “pep 0057 Pharmaceutically acceptable salts also include base tidomimetics' means modified peptides that are structural addition salts which may be formed when acidic protons analogues of the peptide that are designed to mimic the present are capable of reacting with inorganic or organic structure, properties and activities of the peptide. The modi bases. Acceptable inorganic bases include Sodium hydrox fied peptides have improved biological and finctional activi ide, Sodium carbonate, potassium hydroxide, aluminum ties compared to the unmodified peptide due to their higher US 2007/0207952 A1 Sep. 6, 2007 level of resistance to enzymatic degradation while exhibiting bind to one or more blood proteins, such as HSA, preparing the same or improved biological activities. a conjugate in situ. This method has numerous drawbacks, including inability to control the composition and yield of 0068 For all peptides described herein, except where the conjugate with concomitant uncertainty regarding the specifically indicated otherwise, the peptide sequence will dosing of the conjugate. Moreover, many activated biologi be understood to indicate N- protected derivatives such as cally active molecules have limited aqueous solubility and N-acetyl compounds, and C-amide derivatives, as well as are chemically unstable, which not only makes handling and the free amino and free carboxy compounds. administration of the activated moiety problematic, but results in further uncertainty regarding in vivo reactivity and BRIEF DESCRIPTION OF THE DRAWINGS dosing. 0069 FIG. 1 shows fusion inhibitor peptides of the invention. 0077 Still other work has described preparation of fusion proteins containing HSA and a protein of interest. These 0070 FIG. 2 shows the pharmacolinetics of unconjugated methods are, of course, limited to conjugates of molecules (SPI-30014O) vs HSA-conjugated (SPI30014HSA) fusion that can be made by recombinant DNA methods. Also, the inhibitor peptide in Sprague-Dawley rats. site of attachment of the peptide or protein to the HSA is 0071 FIG. 3 shows the pharmacokinetics of unconju limited to either the C- or N-terminus of the HSA and the gated (SPI-70038Q) vs HSA-conjugated HIV (SPI nature of the attachment is necessarily via a peptide bond. 70038HSA) fusion inhibitor peptide in Sprague-Dawley 0078 Typically, the non-covalent binding or adsorption ratS. of a drug to a blood protein component is viewed as a 0072 FIG. 4 shows the pharmacokinetics of Reactive disadvantage to the extent that protein binding reduces the peptide (SPI-30014) vs HSA-peptide conjugates (SPI concentration of free drug available for pharmacological 30014HSA) in Sprague-Dawley rats. activity. However, the present inventors surprisingly have found that conjugates of peptide and non-peptide biologi 0073 FIG. 5 shows the pharmacokinetics of reactive cally active molecules linked to macromolecular moieties peptide (SPI-70038) vs HSA peptide conjugate (SPI that are prepared ex vivo and that carry non-labile linkers 70038HSA) in Sprague-Dawley rats provide valuable advantages over methods and composi tions that previously have been described. In particular, such DETAILED DESCRIPTION OF TEHE "cloaked’ compositions (where the macromolecule “cloaks' INVENTION the biologically active moiety) prepared ex vivo in which 0074 The invention provides conjugates, including puri biologically active molecules are covalently linked to HSA fied conjugates, of a biologically or pharmacologically have been found to have unexpectedly Superior pharmaco active moiety and a macromolecule, that have Superior logical and, in particular, pharmacokinetic properties, to pharmacological properties and that produce Sustained bio previously known compositions. logical activity when administered to a mammalian Subject. 0079 Specifically, the present inventors have found that In particular, the invention provides an isolated compound ex vivo conijugation of a biologically active moiety to a where a pharmacologically active moiety is covalently con macromolecule Such as HSA produces a highly soluble jugated to a pharmacologically inert macromolecular carrier, conjugate that can be purified and administered in tightly where the linkage between the pharmacologically active controlled dosage. The cloaked conjugate is biologically moiety and the carrier is stable in vivo, where the intact active as the conjugate, i.e. it does not act as a prodrug that compound Substantially retains the pharmacological activity releases the biologically active moiety from the conjugate of the pharmacologically active moiety, and where the active and cleavage of the conjugate is not required for biological half-life of the compound when administered to a mammal activity. Moreover, once administered to a subject the con is at least about twice that of the unconjugated pharmaco jugate has a Surprisingly long ini Vivo half-life, has excellent logically active moiety. The carrier advantageously is HSA tissue distribution and produces Sustained activity corre and the conjugates are used for methods of human therapy sponding to the activity of the biologically active moiety of and prophylaxis. the conjugate. In addition, assays using radiolabeled conju 0075 Previous work has described conjugates that con gate show that essentially all of the administered conjugate tain biologically active molecules linked to macromolecular can be accounted for in vivo following administration to the moieties. A significant body of work describes, for example, Subject, In comparison, in assays using radiolabeled active conjugates of the cytotoxic agents doxorubicin and meth moiety, where the conjugate presumably is formed in situ, up otrexate to human serum albumin (HSA). In these meth to 50% of the active moiety administered to the subject odologies the rationale has been to link the cytotoxic agent cannot be accounted for. In addition, chemical conjugation to the HSA via a labile linkage that is severed upon uptake between the biologically active moiety and the macromol of the conjugate at the desired site in vivo. Typically, the ecule permits variation in the length and nature of the linker. labile linkage is acid sensitive and is severed upon cellular 0080 Advantageously, the biologically active moiety and uptake into the acidic environment of the endosome. In this the macromolecule are linked in an approximately 1:1 ratio, manner, therefore, the conjugate was viewed in essence as a to avoid “haptenization of the biologically active moiety prodrug moiety that was required to be degraded to release and generation of an immune response to the conjugate. the biologically active molecule. Moreover, the biologically active moiety is advantageously 0076. Other work has described methods of injecting appended to a single site in the macromolecule. For “activated biologically active molecules into the blood example, selective linkage to the unusually reactive cysteine stream of subjects, where the activated moiety is assumed to 34 (C34) of HSA may be used. Methods for selective linkage US 2007/0207952 A1 Sep. 6, 2007 to C34 using, for example, a maleimide containing linker, nists, antiangiogenic peptides, opiods and anti-nociceptive are known in the art. Suitable linkers are commercially analogs for pain, antihypertensives such as renin inhibitors; available from, for example, Pierce (Rockford, Ill.). angiotensin receptor antagonists; 0081. In the event that more than one molecule of bio 0087 natriuretic peptide derivatives, antivirals such as logically active moiety is linked to the macromolecule, this interferons (including alpha and beta interferon for treatment is advantageously achieved via a “multivalent” linker that is of hepatitis C); cyanovirin derivatives, compounds for treat attached to a single point of the macromolecule. For ment of metabolic disorders such as insulin, bacterial and example, a linker can be appended to C34 of HSA that yeast extracellular virulence factors such as proteinases, permits attachment of a plurality of biologically active bacteriophage lysins, viral entry and fusion inhibitors (for moieties to the linker. Multivalent linkers are known in the viruses such as herpes viruses, such as HSV-2-genital her art and can contain, for example, a thiophilic group for pes, viral glycoprotein D-nectin-2 interaction, HCV-E 1.E2 reaction with C34 of HSA, and multiple nucleophilic (such glycoprotein interaction with CD81, LDL receptor and other as NH or OH) or electrophilic (such as activated ester) cell-specific and liver specific cofactors, malarial plasme groups that permit attachment of a plurality of biologically psins, Schistosomal aspartic proteinases, chaperones that active moieties to the linker. stabilize proteins 10 causing protein-misfolding diseases or drugs that downregulate production of these proteins and 0082) Advantageously the biologically active moiety is that may be used for treatment of diseases such as Alzhe relatively small in size compared to the macromolecule to imer's disease (for example secretase inhibitors). maximize the "cloaking effect of the macromolecule, such 0088 ACE-inhibitors, C- and B-adrenergic agonists ago as HSA. Although the skilled artisan will recognize that nists and antagonists, adrenocorticoids, hormones, aldose precise upper limits cannot be placed on the size of the reductase inhibitors, aldosterone antagonists, 5-Creductase biologically active moiety, it is believed that molecules with inhibitors, analgesics, anesthetics, anorexics, anthelmintics, molecular weights less than 50 kD. less than 10 kD, and antiacne agents, antiallergic agents, antialopecia agents, advantageously less than 7.5 kD or 5 kD can be used. antiamebic agents, antiandrogen agents, antianginal agents, 0.083 Methods of linking the biologically active moiety antiarrhythmic agents, antiarteriosclerotic agents, antiar to the macromolecule are known in the art and are discussed, thritic/antirheumatic agents, antiasthmatic agents, antibac for example, in WO00/76550, which is hereby incorporated terial agents, aminoglycoside antibiotics, ansamycins, anti by reference in its entirety. Such methods are also discussed biotics and antobacterials such as B-lactams, lincosamides, in more detail below with respect to conjugates of fusion macrollides, polypeptides, tetracyclines, 2,4-diaminopyrim inhibitor peptides and renin inhibitors. The skilled artisan idines, nitrofurans, quinolones and analogs, Sulfonamides, will recognize that the conjugation methods discussed for Sulfones, antibiotics, anticholelithogenic agents, anticholes the viral fusion inhibitors and renin inhibitors are generally teremic agents, anticholinergic agents, anticoagulant agents, applicable to a panoply of biologically active moieties, and anticonvulsant agents, antidepressant agents, hydrazides/ are merely illustrative of the present technology. Similarly, hydrazines, pyrrolidones, tetracyclics, antidiabetic agents, methods for purifying the conjugates (if necessary) are biguanides, hormones, Sulfonylurea derivatives, antidiar known in the art. For example, excess biologically active rheal agents, antiduretic agents, antidyskinetic, antieczem moiety can be removed by dialysis of the conjugate, which atic, antiemetic agents, antiepileptic agents, antiestrogen can be further purified by reversed-phase HPLC, ion agents, antifibrotic agents, antifiatulent agents, antifungal exchange chromatography, and/or size exclusion chroma agents, antiglaucoma agents, blood brain barrier peptides tography or the like. (BBB peptides), RGD peptides, glucagon-like peptides, antigonadotropin, antigout, antihemorrhagic and antihista 0084 Biologically Active Compounds minic agents; tricyclic antidepressants, antihypercholester 0085. The present invention encompasses a wide variety olemic, antihyperlipidemic, anthyperlipidemic and antihy of biologically and/or pharmacologically active moieties perlipoproteinemic agents, aryloxyalkanoic acid derivatives, that may be "cloaked using the methods described herein. bile acid sequesterants, HMG-CoA reductase inhibitors, In addition to the renin inhibitors and viral fusion inhibitors nicotinic acid derivatives, thyroid hormones/analogs, anti exemplified below, essentially any molecule for which hyperphosphatemic, antihypertensive agents, arylethanola enhanced pharmacological properties, and in particular, Sus mine derivatives, arloxypropanolamine derivatives, ben tained activity, are desirable, can be cloaked. Examples of Zothiadiazine derivatives, n-carboxyalkyl derivatives, groups that can be cloaked include peptide and non-peptide dihydropyridine derivatives, guanidine derivatives, hydra molecules. Specific examples include compounds having Zines/phthalazines, imidazole derivatives, quaternary metabolic effects, such as cholesterol lowering and blood ammonium compounds, quinazolinyl piperazine deriva pressure lowering compounds, compounds for treatment of tives, reserpine derivatives, Sulfonamide derivatives, anti neurological disorders (where the conjugate can be option hyperthyroid agents, antihypotensive agents, antihypothy ally administered directly into the CNS) including wound roid agents, anti-inflammatory agents, aminoarylcarboxylic healing agents, antibiotics (including anti-infectives), anti acid derivatives, arylacetic acid derivatives, arylbutyric acid oxidants, chemotherapeutic agents, anti-cancer agents, anti derivatives arylcarboxylic acids (including arylpropionic inflammatory agents, and antiproliferative drugs. Examples acid derivatives), pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, antileprotic, antileuke of these molecules are well known in the art and include, mic, antilipemic, antilipidemic, antimalarial, antimanic, merely for illustrative purposes: antimethemoglobinemic, antimigraine, antimycotic, antin 0.086 Inhibitors of matrix metalloproteinases (MMPs), auseant, antineoplastic and alkylating agents, antimetabo antagonists of the urokinase receptor, inhibitors of uroki lites, enzymes, androgens, antiadrenals, antiandrogens, anti nase, erb-2 receptor antagonists, TRAIL receptor antago estrogens, progestogens, uroprotective, antiosteoporosis US 2007/0207952 A1 Sep. 6, 2007 agents, antipagetic, antiparkinsonian, antiperistaltic, administered less frequently than presently available anti antipheochromocytoma, antipneumocystis, antiprostatic viral compounds. The compounds can be used, e.g., as a hypertrophy, antiprotozoal, antiprozoal, antipruritic, antip prophylactic against and/or treatment for infection of a Soriatic and antipsychotic agents, butyrophenes, phenothi number of viruses, including human immunodeficiency azines, thioxanthenes, antipyretic, antirheumatic, antirick virus (HIV), human respiratory syncytial virus (RSV), ettsial, antiseborreheic and antiseptic/disinfectant agents, human parainfluenza virus (HPV), measles virus (MeV) and antispasmodic,antisyphilitic, antithrombotic, antitubercular, simian immunodeficiency virus (SIV). antitumor, antitussive, antiulcerative, antiurolithic, antive nin, and antivertigo agents, purines/pyrimidinomes, anti 0091. The compounds of the invention achieve their anxiolytics, arylpiperazines, benzodiazepine derivatives, Sustained activity by covalent linkage to at least one blood carbamates, astringent, benzodiazepine antagonist, beta component, or to a variety of different blood components. blocker, bronchodilator, ephedrine derivatives, calcium This linkage can be carried out inl vivo or in vitro. When the channel blockers, arylalkylamines, dihydropyridine deriva linkage is carried out in vitro, the compound may optionally tives, piperazine derivatives, calcium regulators, calcium be further purified, by filtration for example, prior to admin Supplements, cancer chemotherapy agents, capillary pro istration to a patient. For peptide compounds composed of tectants, carbonic anhydrase inhibitors, cardiac depressants, naturally occurring amino acids, the covalent linkage can be cardiotonic, cathartic, cation-exchange resin, cck antago achieved either by chemical means, for example by using a nists, central stimulants, cerebral vasodilators, chelating Suitable cross-linking agent, or by preparation of a fusion agents, cholecystokinin antagonists, choleitholytic agents, protein with the blood component. For non-peptide com choleretic agents, cholinergic agents, cholinesterase inhibi pounds, or compounds containing non-naturally occurring tors, cholinesterase reactivators, cns stimulants, cognition amino acids, the linkage may be achieved by chemical activators, contraceptives, agents to control intraocular pres means. The skilled artisan will be aware of blood compo Sure, coronary vasodilators, cytoprotectants, dopamine nents that are suitable for use in the present invention. In a receptor antagonists, ectoparasiticides, emetics, enzymes, particular embodiment, the blood component is human digestive agents, mucolytic agents, penicillin inactivating serum albumin, and in another embodiment the blood com agents, proteolytic agents, enzyme inducers, estrogen ponent is a human or humanized antibody, antibody frag antagonists, gastric proton pump inhibitors, gastric Secretion ment or antibody derivative. The antibody, antibody frag inhibitors, C-glucosidase inhibitors, gonad-stimulating prin ment or antibody derivative may optionally be an antibody, ciples, gonadotrophic hormones, growth hormone inhibitor, antibody fragment or antibody derivative that specifically growth hormone releasing factor, growth stimulant, hema binds a blood component, such as human serum albumin. tinic, hemolydic, demostatic, heparin antagonist, hepatopro Anti-viral Compounds tectant, histamine h. Sub. 1-receptor antagonists, histamine H2-receptor antagonists, immunomodulators, immunosup 0092. The compounds of the present invention include pressants, inotrophic agents, keratolytic agents, lactation compounds having anti-viral activity that can be conjugated stimulating hormone, lipotrophic agents, mineralocorti to a blood component without a significant loss of anti-viral coids, minor tranquilizers, miotic agents, monoamine oxi activity. In the context of the present invention, a significant dase ihibitors, mucolytic agents, muscle relaxants, mydriatic loss of anti-viral activity refers to the situation where the agents, narcotic agents; narcotic antagonists, neuroleptic anti-viral activity of the conjugated compound is reduced to agents, neuromuscular blocking agents, neuroprotective the extent that the dosage of the compound must be agents, NMDA antagonists, nootropic agents, NSAID increased by at least a factor of 10 in molar terms in order agents, ovarian hormones, oxytocic agents, GP-41 peptides, to obtain suitable in vivo activity. insulinotropic peptides parasympathomimetic agents, pedi culicides, pepsin inhibitors, peripheral vasodilators, peristal 0093 Compounds suitable for use in the present inven tic stimulants, pigmentation agents, plasma Volumne tion include, but are not limited to: peptide inhibitors of viral expanders, potassium channel activators./openers, pressor fusion, nucleoside and nucleoside analog, non-nucleoside agents, progestogen, prolactin inhibitors, prostaglandin/ and non-nucleoside analog and nucleotide and nucleotide prostaglandin analogs, protease inhibitors, proton pump analog inhibitors of viral enzymes, inhibitors of viral pro inhibitors, reverse transcriptase inhibitors, scabicides, scle teases, and chemokine co-receptor blockers that inhibit viral rosing agents, sedative/hypnotic agents, serotonin receptor entry into cells. Examples of each of these compounds are agonists, serotonin receptor antagonists, serotonin uptake known in the art. inhibitors, skeletal muscle relaxants, Somatostatin analogs, spasmolytic agents, stool softeners, Succinylcholine syner 0094) Non-limiting representative compounds include, gists, sympathomimetics, thrombolytics, thyroid hormone, for example: thyroid inhibitors, thyrotrophic hormone, uricoSurics, 0095 nucleoside analogs, such cytosine-arabinoside, vasodilators, vasopressors, and vasoprotectants. adenine-arabinoside, iodoxyuridine and acyclovir: 0089 Antiviral Compounds and Complexes: 0096 nucleoside and nucleotide reverse transcriptase 0090 The present invention also provides anti-viral com inhibitors (NRTIs), such as AZT, ddI, ddC, d4T, 3TC, pounds that have prolonged or Sustained activity for the abacavir, tenofovir, emtricitabine, amdoxovir, dOTC, and treatment of viral disease. The invention also provides d4TMP; methods of treating viral diseases using these compounds. The compounds of the present invention have increased 0097 non-nucleoside reverse transcriptase inhibitors stability in vivo and a reduced Susceptibility to degradation, (NNRTIs), such as nevirapine, delaviridine, efavirenz; thio for example by peptidase or protease degradation. As a carboxanilide UC-781, capravirine, SJ-3366, DPC 083, and result, the compounds of the present invention may be TMC 125/R165335; US 2007/0207952 A1 Sep. 6, 2007

0.098 protease inhibitors (Pls), which include saquinavir, 0111. In addition, certain linking groups having func ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, ataza tional groups such as carboxylate, acid halide, azido, diazo, navir, mozenavir, tipranavir, and TMC-114; carbodiimide, anhydride, hydrazine, aldehydes, thiols, or amino group may be used to form amides, esters, imines, 0099 virus adsorption inhibitors, such as cosalane thioethers, disulfides, substituted anines, or the like. Other derivatives and cyanovirin-N co-receptor antagonists, for specific examples of functional groups that may be example, TAK-779 and AMD3100 employed include acyloxymethylketones, aziridines, diaz 0100 viral fusion inhibitors, for example pentafuside omethyl ketones, epoxides, iodo-, bromo- or chloroaceta T-20, betulinic acid, R170591, VP-14637, and NMS03 mides, C.-haloesters, chaloketones, Sulfoniums, chloroethyl Sulfides, O-alkylisoureas, alkyl halides, vinylsulfones, 0101 and viral uncoating inhibitors such as aZodicarbon acrylamides, vinylpyridines, organometallic compounds, amide. aryldisulfides, thiosulfonates, aldehydes, nitriles, C.-dike 0102). Other antiviral compounds that can be used include tones, C.-ketoamides, C.-ketoesters, diaminoketones, semi lamivudine, famciclovir, lobucavir and adefovir, Ribavirin, carbazones, and dihydrazides. integrase inhibitors (diketo acids), transcription inhibitors 0.112. The nature and type of compounds that may be (temacrazine, flavopiridol), viral uncoating inhibitors (ple selected as the linker depends on the type of reactions, the conaril); RNA replicase inhibitors (VP-32947); DNA poly relative reactivities, selectivities, reversibility and stability merase inhibitors (A-5021, L- and D-cyclohexenylguanine); characteristics that are desired among the anti-viral agents, bicyclic furopyrimidine analogues; cidofovir, neuramini the linker and the functional groups on albumin or the blood dase inhibitors (Zanamivir, oseltamivir, RWJ-270201); ade component. For example, certain reactions that form the fovir dipivoxil; N-glycosylation inhibitors (N-nonyl-deox conjugate complex arise from an alkylation reaction, a ynojirimycin); and, IMP dehydrogenase inhibitors and Michael type reaction, an addition-elimination reaction, an S-adenosylhomocysteine hydrolase inhibitors. addition to Sulfur, carbonyl, or cyano groups, or the forma 0103) For the treatment of HIV infection in particular, tion of a metal bond. compounds can be used that inhibit: 0113 Typically, the covalent bond that is formed from 0104 viral adsorption, through binding to the viral enve these reactions are stable during the active lifetime of the lope glycoprotein gp120 (polysulfates, polysulfonates, poly anti-viral agent. In one embodiment, the covalent bond that carboxylates, polyoxometalates, polynucleotides, and nega is formed in these complexes remain stable unless the tively charged albumins): biologically active subunit is intended to be released at the 0105 viral entry, through blockade of the viral corecep active site. tors CXCR4 (i.e., bicyclam (AMD3100) derivatives) and 0114. The linkers may comprise of compounds having CCR5 (i.e., TAK-779 derivatives): bifunctional or polyfunctional groups that are available for linking a protein such as albumin to multiple anti-viral 0106 virus-cell fusion, through binding to the viral enve agents or for linking multiple albumins to a single anti-viral lope glycoprotein gp41 (T-20. T-1249): agent. In a particular preferred embodiment, the linker 0107 viral assembly and disassembly, through NCp7 comprises polyfunctional groups that link a HSA to one or Zinc finger-targeted agents (2,2'-dithiobisbenzamides more anti-viral agents. In one embodiment, linking com (DIBAs), azadicarbonamide (ADA)); pounds as used herein include any compounds that can link the anti-viral agent to the protein in a single step. In another 0108 proviral DNA integration, through integrase inhibi embodiment, the linking compounds are linked to the anti tors such as 4-aryl-2,4-dioxobutanoic acid derivatives; and viral agent first to form a inhibitor-linker intermediate that 0109) viral MRNA transcription, through inhibitors of the can be further reacted with the protein. In another embodi transcription (transactivation) process (flavopiridol, fluoro ment, the linking compounds are reacted with the protein quinolones). first to form a protein-linker intermediate that can be further reacted with the anti-viral agent. In each of the above The Linkers L1 and L2: permutations, optionally, the linked compounds may be 0110) A variety of different linkers or linking groups L1 further purified and/or isolated before submitting to further and L2 may be used to link the blood component with the reactions to form the complex of Formula I or Formula II. anti-viral agent. The linking groups may be divalent or 0115 Non-exclusive examples of such polyfinctional polyvalent. For example, in the complex of Formula I, L1 compounds include compounds having at least one func may be n-valent where it is attached to Pr, and m-valent tional group selected from the group consisting of azido where it attaches to AV where mand n are integers as defined benzoyl hydrazide, N-4-(p-azidosalicylamino)butyl-3'-2'- above. Similarly, in the complex of Formula U, L2 may be pyridyldithio)propionamide), bis-sulfosuccinimidyl o-valent where it is attached to Prand p-valent where it is Suberate, dimethyl adipimidate, disuccinimidyl tartrate, attached to AV, where o and p are as defined above. Non N-y-maleimidobutyryloxysuccinimide ester, N-hydroxy sul exclusive examples of functional groups that may be present fosuccinimidyl-4-azidobenzoate, N-succinimidyl 4-azi in a linking group include compounds that have a hydroxyl dophenyl-1,3'-dithiopropionate, N-succinimidylA-io groups, such as N-hydroxysuccinimide, N-hydroxysulfos doacetylaminobenzoate, glutaraldehyde, and Succinimidyl luccinimide, and other compounds such as maleimide-ben Zoyl-succinimide, maleimido-butyryloxy Succinimide ester, 4-N-maleimidomethylcyclohexane-1-carboxylate. maleimidopropionic acid, N-hydroxysuccinimide, isocyan 0116. Any linker or linking group that is convenient for ate, thioester, thionocarboxylic acid ester, imino ester, car use and Subject to standard chemical transformations, or bodiimide, anhydride, or ester. linkers that form compounds that are physiologically accept US 2007/0207952 A1 Sep. 6, 2007

able at the desired dosages, and are stable in the bloodstream 0.124 Generally, there exists a very broad range of dif for the desired period of time, may be employed. The linking ferent methods available for the isolation of compounds group may be aliphatic, alicyclic, aromatic, heterocyclic, or from blood or blood plasma that provide a very broad range combinations thereof. Examples of groups that may be of final purities, and yields of the product. Albumin is the employed as a linking group include alkylenes, arylenes, main protein present in blood plasma, and may be extracted aralkylenes, cycloalkylenes, polyethers and the like. In a from blood, for examples as disclosed in JP 03/258 728, EP particular embodiment, polyfunctional polyethylene glycol 428 758, EP 452 753, and U.S. Pat. No. 6,638,740 and (PEG) and their derivatives may also be employed as references cited therein. Further examples of non-exclusive linkers. methods for the isolation of various compounds may be based on selective reversible precipitation, ion exchange 0117 The linking groups may have at least one atom in chromatography, protein affinity chromatography, hydro the linking chain, more preferably between 1 and 200 atoms phobic chromatography, thiophilic chromatography (J. in the chain, most preferably between 2 and 50 atoms in the Porath et al.; FEBS Letters, vol. 185, p.306, 1985; K. L. chain. The atoms in the chain can be linear or the chain can Knudsen et al. Analytical Biochemistry, Vol 201, p. 170, be part of one or more rings, each Substituted or unsubsti 1992), and various resin matrices (WO 96/00735; WO tuted, and the chain may include carbons or heteroatoms 96/09 116). Certain blood components of established purity selected from the group consisting of ON, P and S. The rings are commercially available. may be aliphatic, heterocylic, aromatic or heteroaromatic or mixtures thereof, each substituted or unsubstituted. In some Preparation of Linked Compounds AV-L1 and AV-L2: embodiments, amino acids or peptides or amino acids 0.125. In one embodiment, the linked compounds AV-L1 employed with mixtures of the above may be used as a or AV-L2 of the present invention may be prepared and used linking group. in the conjugation with albumin without further purification 0118. In one embodiment, L1 is absent and AV is attached and/or isolation. The purity of the linked compounds will directly to Pr. In another embodiment, L2 is absent and AV depend on the nature of the linker, the nature of AV. and the is attached directly to Pr. type of reaction and reaction conditions employed to attach AV to the linker. In another particular embodiment, the 0119). In another embodiment for the complex of Formula unpurified linked compounds are prepared and obtained with I, L1 is a linking group that is capable of linking more than a purity of at least 90%, preferably at least 95%, more one AV to one Pr, for example, where m is 2 or more. In one preferably at least 97%, and most preferably at least 98%. embodiment, m is 1, 2 or 3 and n is 1-30. In one preferred embodiment for the complex of Formula I, Pr is albuminand 0.126 In a particular embodiment, the present invention n is 1. In another particular embodiment, Pr is albumin, AV relates to methods for the preparation of the isolated linked is an anti-viral agent, and n is 2 - 25. compounds, that is, AV-L1 or AV-L2. In a preferred embodi ment, the isolated linked compounds AV-L1 and AVL2 are 0120 In another embodiment for the complex of Formula anti-viral agents that are attached to a linker. In one embodi II, L2 is a linking group that is capable of linking more than ment, the isolated linked compounds may be purified before one Pr to one AV, for example, in the case where o is 2 or conjugating with Pr. In another particular embodiment, the more. In one embodiment, Pr is albumin, AV is an anti-viral linked compounds AV-L1 or AV-L2 are isolated and purified agent, o is 1, 2 or 3 and p is 1-5. to a purity of at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% or 0121. In another embodiment, the linking group may be O. absent in cases where the inhibitor, Such as an anti-viral agent, can be reacted directly with a protein, optionally 0127. The linked compounds may be prepared using using a catalyst or coupling agent, Such that the complex that standard methods known in the art of chemical synthesis. is formed comprises only of the anti-viral agent that is The compounds may be purified using standard methods directly attached to the protein. An example of such a direct known in the art, Such as by column chromatography or coupling reaction is a mixed anhydride activated coupling HPLC to provide purified products suitable for in vivo reaction of a carboxylic acid followed by the coupling applications. The linked compounds may be further conju reaction of the intermediate mixed anhydride. gated with a protein, such as albumin to form the complex of Formulae I and II. The Protein Component Pr: Covalent Linkage to Blood Components 0122 Various blood components may be used to prepare the isolated complexes of the present invention. Naturally 0.128 Suitable blood components for use in the present occurring blood components include blood proteins, which invention are known in the art. Human serum albumin include red blood cells, and immunoglobulins, such as IgM (“HSA) is a predominant component of human blood and and IgG, serum albumin, transferrin, p90 and p38. In a is particularly Suited for use in the present invention. In preferred embodiment, the blood component or blood pro particular, HSA has an exposed surface cysteine residue that tein is albumin. More preferably, the albumin is a protein provides a reactive thiol moiety for covalent linkage of human serum albumin (HSA). anti-viral compounds to the protein. Activated linkers that are particularly Suited for linkage to thiols include unsatur 0123 The albumin used in the present invention may also ated cyclic imides such as maleimides, chalo esters, such be recombinant albumin. For example, the recombinant as C-iodo- and C-bromo acetates, and vinyl pyridine deriva human albumin may be produced by transforming a micro tive. Suitable activated linkers are commercially available organism with a nucleotide coding sequence encoding the from, for example, Pierce Chemical (Rockford, Ill.). Meth amino acid sequence of human serum albumin. ods for preparing Suitable activated compounds for linking US 2007/0207952 A1 Sep. 6, 2007

to HSA are known in art. See for example, U.S. Pat. No. 0.135 The antiviral compounds can be prepared using 5,612,034, which is incorporated herein in its entirety. synthetic methods that are well known to the skilled chemist. 0129. In one variation of the present invention, the linker For example, peptides can be prepared using well-known is specifically linked to the thiol group of cysteine 34, and techniques of Solid-phase peptide synthesis. See, for maybe formed via a nucleophilic reaction of the thiol group example, Solid Phase Peptide Synthesis, 2nd Ed., Pierce on an electrophilic group of the linker. Chemical Company, Rockford, Ill., (1984). Similarly, pep tides fragments may be synthesized and Subsequently com 0130 Moreover, the gene for HSA has been cloned, which permits the ready preparation of fusion proteins bined or linked together to form the desired sequence. containing HSA. These fusion proteins, which have thera 0.136. Other compounds may be prepared using methods peutic applications, include, but are not limited to a polypep known in the art, or by Straightforward variations on known tide, an antibody, or a peptide, or fragments and variants methods. thereof, filsed to a blood component. The fusion proteins exhibit extended shelf-life andlor extended or therapeutic Preparation of Linked Compounds Pr-L1 and Pr-L2: activity. Methods of making fusion proteins are known in the 0.137 For certain applications of the present invention, art. See, for example, WO01/79271 and WO01/79258, the the compounds as represented by Pr may be albumin, may contents of which are hereby incorporated by reference in be used as obtained from commercial sources without fur their entirety. The preparation of fusion proteins is useful for ther purification or isolation, to prepare the linked com preparing persistent derivatives of anti-viral peptides. pounds Pr-L1 and Pr-L2. In a particular embodiment, Pr is 0131) Another blood component that is suitable for link HSA. In another embodiment, the albumin may be further age to the anti-viral compounds is an immunoglobulin (“Ig) purified using various methods known in the art as disclosed molecule. Igs are persistent and are present in relatively high herein. concentration in the blood. For inz vitro coupling, Igs have 0.138. In one embodiment, the linked compounds Pr-L1 the advantage of being readily stable and readily isolated, and Pr-L2 may be prepared by treating a linker L1 or L2. and methods of making Ig conjugates are well known in the which may be derivatized or activated, with Pr. in a solution art. Moreover, Ig genes may readily be cloned and recom and monitoring the reaction mixture until the reaction is binant Ig and Ig fusion proteins prepared. Methods for substantially complete. In a particular preferred embodi obtaining fully human Igs are well known in the art. See for ment, Pr is a protein. In another preferred embodiment, the example, U.S. Pat. Nos. 5,969,108 and 6,300,064, the con tents of which are hereby incorporated by reference in their protein is HSA or recombinant HSA. entirety. In addition, phage display methods for selecting Igs 0.139. In another preferred embodiment, the linked com having a particularly desired binding activity, for example, pounds Pr-L1 or Pr-L2 obtained are substantially pure; that for binding to HSA, are well known in the art. See U.S. Pat. is, the linked compounds are obtained with a purity of at Nos. 5,885,793, 5,969,108 and 6,300,064. In the context of least 10%, preferably at least 30%, and more preferably at the present invention, an Ig refers to any suitable immuno least 50%. Where the Pr is HSA or recombinant HSA, globulin or immunogolobulin derivative known in the art, components that may be present with the linked compounds and includes, for example, whole IgG, IgM, Fab fragments, may comprise of unreacted HSA and various biological F(ab')2 fragments, and single chain FV fragments. components that are present in the HSA starting material. 0132). Other blood components suitable for use in the Preferably, the HSA or recombinant HSA is at least 10% present invention include transferrin, ferritin, steroid bind pure on a dry matter basis. ing proteins, thyroxin binding protein, and O-2-macroglo 0140. An excess of HSA or HSA related biologically bulin. materials present with the linked compounds will not sig 0133) For peptides, activated linkers may be coupled to nificantly interfere with the Subsequent conjugation step reactive side chain residues, such as lysine side chains. For with AV. In addition, the related biological materials and the example, a linker containing an active ester moiety and a conjugated complexes will also be pharmacologically safe maleimide moiety can be selectively reacted at the active for use in vivo. ester (such as an N-hydroxysuccinimidyl ester) via lysine side chains or at the N-terminus of the peptide. For non 0.141. However, in certain embodiments, the purity of the peptidyl anti-viral compounds, the skilled chemist readily linked compounds Pr-L1 or Pr-L2 may be at least 10% on a can recognize nucleophilic (or electrophilic) atoms or dry matter basis to enable the selective reaction of the groups on the compound that can selectively react with a compounds with AV without a significant amount of inter Suitable linking moiety, Such as an active ester. Suitable ferences or without the formation of undesirable by-products nucleophilic moieties include, but are not limited to, amino obtained from the conjugate reaction with other undesired and hydroxyl groups. For example, for nucleoside and blood components. However, the desired purity of Pr, such nucleotide analogs, hydroxyl groups can act as nucleophiles as HSA or recombinant HSA, for example, will depend on for the coupling reaction. For nucleotides, coupling also can the nature of the functional groups on Ih as well as the be achieved by formation of for example, phospho esters. functional groups employed on the linker. Typically, higher Similar strategies can be used in other anti-viral compounds, purities of HSA or recombinant HSA is required if the for example in protease inhibitors and other enzyme inhibi functional groups on the linker are more reactive and may tors, coupling can be achieved using nucleophilic groups form undesired by-products than functional groups on the that are distal from the enzyme active site. linker that are less reactive. 0134) Both natural and recombinant HSA and human Igs 0142. The albumin may be obtained from plasma or are commercially available and are suitable for use in the blood albumin from a host, purified to a desired level of present invention. purity, and linked with the linker. Purification of the albumin US 2007/0207952 A1 Sep. 6, 2007

from blood or blood plasma may be performed using well is from about 10 to 30. In yet another particular embodiment, established standard methods known in the art for the the ratio is from about 2 to 5. Where there is an excess of purification of albumin. Using purified blood albumin, the (AV)m-L1 in the product mixture, optionally, the conjugated isolated complexes of the present will comprise of a rela complex may be purified to a purity of at least 10%. In a tively homogeneous population of functionalized proteins. particular embodiment, the conjugated complex may be Preparation of the Complexes of Formula I or Formula II: purified to at least about 20% or at least about 30%. 0143. In one embodiment, the complexes of Formula I or 0149. In another embodiment, the complexes of Formula Formula II may be prepared by the conjugation of AV-L1 or I or Formula II may be prepared from a stoichiometric ratio of (AV)m-L1 with Pr or a stoichiometric ratio of AV with AV-L2 with Pr, the conjugation of Pr-L1 or Pr-L2 with AV. L2-(Pr)o, that is, in a 1:1 ratio. Optionally, the resulting or the conjugation of AV with Pr to form a complex wherein product from these preparations may be further purified to a the linker is absent. purity of at least 10%. In a particular embodiment, the 0144. In one embodiment, a solution of AV-L1 or AV-L2 conjugated complex may be purified to at least about 20% or is combined with Pr under conditions such that the conju to a purity of at least about 30%. In yet another particular gation reaction is deemed to be complete. In a particular embodiment, the 1:1 conjugated complex may be further embodiment, the linked compound is an anti-viral agent that purified to a purity of greater than about 90%. is attached to a linker, and the linked compound is added to 0150. In another embodiment, the conjugated cysteine an aqueous Solution of HSA. The resulting solution is present in albumin is reduced to the free cysteine prior to the incubated until the reaction is Substantially complete. reaction. 0145. In one embodiment, the AV-L1 or AV-L2 is com bined with an excess of HSA to ensure that the conjugation 0151 Optionally, the complex formed from the conjugate reaction proceeds selectively to a single site on the HSA. For reaction may be further purified prior to administration. example, the formation of AV-L1 on a single site on HSA 0152. In one embodiment, the complexes of Formula I or may permit ease of identification of a single complex of Formula II obtained from the conjugate reaction may be Formula I, for example, where n is 1. In one particular administered without further processing or purification since embodiment, the conjugate reaction of AV-L1 or AV-L2 with an excess of HSA or HSA related biologically materials HSA occurs on a single cysteine of HSA. Without being present with the complexes are pharmacologically safe for bound by any particular theory, for Some reactions, it is use in vivo. believed that the conjugate reaction may also occur initially with a cysteine —SH group to form a kinetic product that is 0153. In each of the above embodiments, AV is a peptide then rearranged to another amino acid finctional group. Such anti-viral agent and Pr is HSA or recombinant HSA. as a lysine, to form the thermodynamic product. 0154) In one embodiment, the isolated complex compris 0146 In another embodiment, the conjugate reaction may ing a protected or unprotected anti-viral agent with a linker form the complex of Formula I, for example, wherein more and albumin may be optionally further purified and then than one AV is linked to a single HSA to form the complex returned to the host. of Formula I; that is, wherein n is greater than 1. Optionally, O155 The complexes formed from the methods of the m may be greater than 1 if the linker L1 is a polyfunctional present invention may be tested in animal or human hosts linker that is capable of attaching more than one AV group. until the physiology, pharmacokinetics, and safety profiles In one embodiment, the complex of Formula I may be are well established over an extended period of time. Typi prepared by combining an excess of Pr relative to (AV)m- cally, the measured half-life of the complexes is about 5 to L1. Preferably, the ratio of Pr to (AV)m-L1 is about 50 to 7 days, more typically at least about 7 to 10 days, and 100. In another particular embodiment, the ratio is from preferably 15 to 20 days or more. In general, the duration is about 10 to 30. In yet another particular embodiment, the species dependent. For example, with human albumin, the ratio is from about 2 to 5. half life is about 17-19 days. Depending on the nature of the 0147 In one embodiment, Pr is added to (AV)m-L1 in a anti-viral agent, the linking group and the purity of the ratio of at least about 1.1:1, more preferably at least about albumin, the effective therapeutic concentration of the com 1.2:1, and most preferably at least about 1.4:1. In the case plexes may be at least 1 month or more. where Pr is albumin, the preferred ratios are based on the assumption that there is 0.7 free thiol per albumin. Prefer 0156 Half lives may be determined by serial measure ably, the resulting complex is formed as a 1:1 complex, since ments of whole blood, plasma or serum levels of the a Pr component such as albumin has only about 70% free complexes of Formula I or Formula II, the AV-L compounds, thiol functionality for conjugation. An excess of Pr. Such as the L-Pr compounds, or the AV compounds following label HSA or recombinant HSA is pharmacologically safe and ing of the complex or compounds with an isotope (e.g., 131I. may not require further purification. Where there is an 125I, Tc, Cr, 3H, etc . . . ) or fluorochrome and injection of excess of Pr in the product mixture, optionally, the conju a known quantity of labeled complex or compound intra gated complex may be purified to a purity of at least 10%. vascularly. Included are red blood cells (half life ca. 60 In a particular embodiment, the conjugated complex may be days), platelets (half life ca. 4-7 days), endothelial cells lining the blood vasculature, and long lived blood serum purified to at least about 20% or at least about 30%. proteins. Such as albumin, Steroid binding proteins, ferritin, 0148. In another embodiment, the complex of Formula I C.2-macroglobulin, transferrin, thyroxin binding protein, may be prepared by combining an excess of (AV)m-L1 immunoglobulins, especially IgG, etc. In addition to pre relative to Pr. Preferably, the ratio of (AV)m-L1 to Pr is ferred half lives, the subject components are preferably in about 50 to 100. In another particular embodiment, the ratio cell count or concentration sufficient to allow binding of US 2007/0207952 A1 Sep. 6, 2007 therapeutically useful amounts of the compound of the ing the fusion protein are placed into a suitable vector in present invention. For cellular long lived blood components, frame, and the vector is used to transform a suitable host cell counts of at least 2,000/ul and serum protein concen cell. The genes may be placed in either order (i.e. the trations of at least 1 ug/ml, usually at least about 0.01 mg/ml. anti-viral peptide may be placed at the N- or C-terminus of more usually at least about 1 mg/ml, are preferred. the fusion protein) and may be directly fused or separated by a linker peptide. Suitable linker peptides are known in the art 0157 However, where the nature of the complex is and include peptide sequences that have little secondary designed Such that the biologically active agent AV. Such as structure of their own and that are hydrophilic, for example, an anti-viral agent, is to be cleaved from the complex and linkers containing mixtures of glycine and serine residues. released into the host, the desired half life for the effective Methods for making fusion proteins of HSA are described in therapeutic concentration of the complex and/or the biologi WO01/79271 and WO01/9258, and similar methods can be cally active agent may vary from the measured half-life used for making fusion proteins with other blood compo above. The rate of release of the biologically active agent depends in part, on the Valency or the functionality on the nentS. biological agent which is to be released, the nature of the 0.163 The present invention particularly contemplates linking group, the purity and type of the protein, the com use of peptides that inhibit viral fuision with the cell position for administration, the manner of administration, membrane, and in particular contemplates peptides that and the like. Thus, various linking groups and biological inhibit fusion of HIV. Specific peptide inhibitors typically agents may be employed, where the environment of the contain up to about 51 amino acids, and contains a peptide blood, components of the blood, particularly enzymes, activ having the sequences as discosed herein. In particular, the ity in the liver, or other agent may result in the cleavage of peptides may contain the sequences shown in FIG. 1. These the linking group with release of the biological agent in the sequences are derived from sequences found in EHIV iso host at a desired rate. lates and, for peptides longer than the sequences shown in 0158. The isolated complexes of the present invention FIG. 1, for example, the remainder of the peptide sequence provides biological active compounds that have improved can be either N- or C-terminal to the sequence shown. Such pharmacokinetics, solubility, bioavailability, distribution, additional sequences can, for example, consist of, or can and/or immunogenicity characteristics as compared to the contain, the sequences that occur adjacent to the defined non-conjugated compounds. sequences in those HIV isolates. Administration of the Isolated Complexes of Formula I and 0159. Surprisingly, the complexes of Formula I and For Formula II: mula II, when prepared and used according to the methods of the present invention, provides an effective therapeutic 0164. In one embodiment, the administration of the iso concentration for a significantly longer time than the AV lated complex of the present invention may be accomplished component by itself. In addition, the complexes of the using a bolus, but may be introduced slowly over time by present invention provide improved solubility, distribution, transfusion using metered flow, or the like. pharmacokinetics, and result in decrease immunogenicity 0.165. The complex of the present invention may be when compared to the administration of the AV component administered in a physiologically acceptable medium, e.g. by itself. deionized water, phosphate buffered saline, Saline, mannitol, 0160 The present inventors surprisingly have found that aqueous glucose, alcohol, vegetable oil, or the like. A single administration to a subject of a conjugate that is prepared ex injection may be employed although more than one injection vivo from purified components (specifically HSA, linker and may be used, if desired. The complex may be administered an anti-viral agent) produces a remarkably efficient tissue by any convenient means, including Syringe, trocar, catheter, Vivo distribution of the conjugate compared to conjugates or the like. The particular manner of administration, will that are prepared by in vivo preparation of the conjugate by vary depending upon the amount to be administered, injection of an activated compound that binds in situ to whether a single bolus or continuous administration, or the endogenous albumin in the blood stream of the Subject. like. The administration may be intravascularly, where the Moreover, the present inventors have found that substan site of introduction is not critical to this invention, preferably tially all of the conjugate remains in circulation for hours or at a site where there is rapid blood flow, e.g. intravenously, even days following administration, compared to the dra peripheral or central vein. Other routes may find use where matic losses of compound that are observed when the the administration is coupled with slow release techniques or conjugate is prepared in vivo. This efficiency reduces the a protective matrix. number of times that the patient must be subjected to 0166 Surprisingly, it is noted that the administration of injection of active Substance, and also reduces the amount of the isolated complexes prepared by the methods of the anti-viral agent that must be given in a single administration. present invention, for example, from isolated blood protein, 0161 In the context of the present invention, a therapeu Such as albumin, results in anti-viral conjugate complexes tically effective amount of a composition is understood to that maintain an effective therapeutic effect in the blood mean an amount that, when administered to a Subject, stream for an extended period of time as compared to a produces a desired physiological effect to a degree that is non-conjugated anti-viral agents or as compared to com effective for treatment of a disease, condition, or syndrome plexes that are not prepared from isolated blood protein such in the patient, or that is effective in alleviating the symptoms as albumin. disease, condition, or syndrome. 0.167 In one embodiment, the present invention provides 0162 For preparation of fusion proteins containing the the compounds in the form of a pharmaceutically acceptable blood component and a peptide anti-viral, the genes encod salt. US 2007/0207952 A1 Sep. 6, 2007

0168 In another embodiment, the present invention pro the compound that it did not previously possess, and may vides the compounds present in a mixture of Stereoisomers. even positively affect the pharmacodynamics of the com In yet another embodiment, the present invention provides pound with respect to its therapeutic activity in the body. An the compounds as a single stereoisomer. example of a pharmacokinetic property that may be favor ably affected is the manner in which the compound is 0169. In yet another embodiment, the present invention transported across cell membranes, which in turn may provides pharmaceutical compositions comprising the com directly and positively affect the absorption, distribution, pound as an active ingredient. In yet another particular biotransformation and excretion of the compound. While the variation, the present invention provides pharmaceutical route of administration of the pharmaceutical composition is composition wherein the composition is a tablet or a solid important, and various anatomical, physiological and patho for administration as a depot. In another particular variation, logical factors can critically affect bioavailability, the solu the present invention provides the pharmaceutical compo bility of the compound is usually dependent upon the sition wherein the composition is a liquid formulation character of the particular salt form thereof, which is uti adapted for IV or subcutaneous administration. In yet lized. One of skill in the art will appreciate that an aqueous another particular variation, the present invention provides solution of the compound will provide the most rapid pharmaceutical composition wherein the composition is a absorption of the compound into the body of a subject being liquid formulation adapted for parenteral administration. treated, while lipid solutions and Suspensions, as well as 0170 It is noted in regard to all of the embodiments, and Solid dosage forms, will result in less rapid absorption of the any further embodiments, variations, or individual com compound. pounds described or claimed herein that all such embodi Peptides and Complexes: ments, variations, and/or individual compounds are intended to encompass all pharmaceutically acceptable salt forms 0.175. In one embodiment of the invention, there is pro whether in the form of a single stereoisomer or mixture of vided a peptide consisting of up to 51 amino acids compris Stereoisomers unless it is specifically specified otherwise. ing the sequence Similarly, when one or more potentially chiral centers are 0176) Y1-X X Y2-X X X Y3-X X X Y4 present in any of the embodiments, variations, and/or indi X X Y5-X X Y6, wherein: vidual compounds specified or claimed herein, both possible chiral centers are intended to be encompassed unless it is 0.177 the sequence is located at the N-terminal, C-termi specifically specified otherwise. nal or at an interior position of the peptide; 0171 Prodrug derivatives of compounds according to the 0.178 Y1 is selected from the group consisting of W, Y, present invention can be prepared by modifying Substituents F. H. L, N, Q, E, D, K, and R: of compounds of the present invention that are then con 0.179 Y2 is selected from the group consisting of W, Y, verted in vivo to a different substituent. It is noted that in F. H. L, N, Q, E, D, K, and R: many instances, the prodrugs themselves also fall within the Scope of the range of compounds according to the present 0180 Y3 is selected from the group consisting of L. V. L. invention. For example, prodrugs can be prepared by react A, S and T. ing a compound with a carbamylating agent (e.g., 1,1- acyloxyalkylcarbonochloridate, para-nitrophenyl carbonate, 0181 Y4 is selected from the group consisting of T. S., I, or the like) or an acylating agent. Further examples of K., N, H. R. Q., E and D; methods of making prodrugs are described in Saulnier et 0182 Y5 is selected from the group consisting of I. V. T. al.(1994), Bioorganic and Medicinal Chemistry Letters, Vol. K, L, N, Q, D, E, R and H: 4, p. 1985. 0183 Y6 is selected from the group consisting of any 0172 Protected derivatives of compounds of the present amino acid except P. G and C.; and, invention can also be made. Examples of techniques appli each X independently is any amino acid. cable to the creation of protecting groups and their removal 0184 can be found in T. W. Greene, Protecting Groups in Organic 0185. In another embodiment of the invention, there is Synthesis, 3rd edition, John Wiley & Sons, Inc. 1999. provided a peptide consisting of up to 51 amino acids comprising the sequence 0173 Compounds of the present invention may also be conveniently prepared, or formed during the process of the invention, as Solvates (e.g. hydrates). Hydrates of com pounds of the present invention may be conveniently pre pared by recrystallization from an aqueous/organic solvent 0187 Y1 is selected from the group consisting of W, Y, mixture, using organic Solvents such as dioxane, tetrahydro F. H. L, N, Q, E, D, K, and R: furan or methanol. 0188 Y2 is selected from the group consisting of W, Y, 0.174. A “pharmaceutically acceptable salt”, as used F. H. L, N, Q, E, D, K, and R: herein, is intended to encompass any compound according 0189 Y3 is selected from the group consisting of I. V. L. to the present invention that is utilized in the form of a salt A, S and T. thereof, especially where the salt confers on the compound improved pharmacokinetic properties as compared to the 0.190 Y4 is selected from the group consisting of T. S., I, free form of compound or a different salt form of the K., N, H. R. Q., E and D; compound. The pharmaceutically acceptable salt form may 0191 Y5 is selected from the group consisting of I. V. T. also initially confer desirable pharmacokinetic properties on K, L, N, Q, D, E, R and H: US 2007/0207952 A1 Sep. 6, 2007

0192 Y6 is selected from the group consisting of any 0218. In another embodiment of the invention, there is amino acid except P. G and C: provided a peptide consisting of up to 51 amino acids 0193 Y7 is selected from the group consisting of I, L. V. comprising the sequence N. Q. K. R. H. E. and D; and 0194) each X independently is any amino acid. 0.195. In another embodiment of the invention, there is provided a peptide consisting of up to 51 amino acids 0220 Y1 is selected from the group consisting of W, Y, comprising the sequence F. H. L, N, Q, E, D, K, and R: 0221, Y2 is selected from the group consisting of W, Y, F. H. L, N, Q, E, D, K, and R: 0197) Y1 is selected from the group consisting of W. Y. 0222 Y3 is selected from the group consisting of I. V. L. F. H. L, N, Q, E, D, K, and R: A, S and T. 0198 Y2 is selected from the group consisting of W. Y. 0223 Y4 is selected from the group consisting of T. S., I, F. H. L, N, Q, E, D, K, and R: K., N, H. R. Q., E and D; 0199 Y3 is selected from the group consisting of I. V. L. 0224 Y5 is selected from the group consisting of I. V. T. A, S and T. K, L, N, Q, D, E, R and H: 0200 Y4 is selected from the group consisting of T. S., I, 0225 YG is selected from the group consisting of any K., N, H. R. Q., E and D; amino acid except P. G and C: 0201 Y5 is selected from the group consisting of L. V. T. 0226 Y7 is selected from the group consisting of I. L. V. K, L, N, Q, D, E, R and H: N, Q, K, R, H, E and D: 0202 Y6 is selected from the group consisting of any 0227 Y8 is selected from the group consisting of Q. H. amino acid except P. G and C: R, N, E, D, K and P: 0203 Y7 is selected from the group consisting of I, L. V. 0228 Y9 is selected from the group consisting of Q. H. N, Q, K, R, H, E and D; N, E, D, K. R. L. and P: 0204 Y8 is selected from the group consisting of Q. H. 0229. Y 10 is selected from the group consisting of Q, H, RN, E, D, K and P; and N, E, D, K and R; and 0205) each X independently is any amino acid. 0230) each X independently is any amino acid. 0206. In another embodiment of the invention, there is provided a peptide consisting of up to 51 amino acids 0231. In another embodiment of the invention, there is comprising the sequence provided a peptide of up to 51 amino acids comprising the Sequence

0208 Y1 is selected from the group consisting of W. Y. F. H. L, N, Q, E, D, K, and R: 0233 Y1 is selected from the group consisting of W, Y, 0209 Y2 is selected from the group consisting of W. Y. F. H. L, N, Q, E, D, K, and R: F. H. L, N, Q, E, D, K, and R: 0234 Y2 is selected from the group consisting of W, Y, 0210 Y3 is selected from the group consisting of I. V. L. F. H. L, N, Q, E, D, K, and R: A, S and T. 0235 Y3 is selected from the group consisting of I. V. L. 0211 Y4 is selected from the group consisting of T. S., I, A, S and T. K., N, H. R. Q., E and D; 0236 Y4 is selected from the group consisting of T. S., I, 0212 Y5 is selected from the group consisting of I. V. T. K., N, H. R. Q., E and D; K, L, N, Q, D, E, R and H: 0237 Y5 is selected from the group consisting of I. V. T. 0213 Y6 is selected from the group consisting of any K, L, N, Q, D, E, R and H: amino acid except P. G and C: 0238 Y6 is selected from the group consisting of any 0214 Y7 is selected from the group consisting of I, L. V. amino acid except P. G and is C; N, Q, K, R, H, E and D: 0239 Y7 is selected from the group consisting of I. L. V. 0215 Y8 is selected from the group consisting of Q. H. N, Q, K, R, H, E and D: R, N, E, D, K and P: 0240 Y8 is selected from the group consisting of Q. H. 0216 Y9 is selected from the group consisting of Q. H. R, N, E, D, K and P: N, E, D, K, R, L, and P; and 0241 Y9 is selected from the group consisting of Q. H. 0217 each X independently is any amino acid. N, E, D, K, R, L, and P: US 2007/0207952 A1 Sep. 6, 2007

0242 Y10 is selected from the group consisting of Q, H, 0267 Y6 is selected from the group consisting of any N, E, D, K and R; amino acid except P. G and C: 0243 Y11 is selected from the group consisting of N. S. 0268 Y7 is selected from the group consisting of L. L. V. T. V. A and D; and N, Q, K, R, H, E and D: 0244) each X independently is any amino acid. 0269 Y8 is selected from the group consisting of Q. H. 0245. In another embodiment of the invention, there is R, N, E, D, K and P: provided a peptide of up to 51 amino acids comprising the 0270 Y9 is selected from the group consisting of Q. H. Sequence N, E, D, K, R, L, and P: 0271 Y10 is selected from the group consisting of Q, H, N, E, D, K and R; 0272 Y1 I is selected from the group consisting of N. S. 0247 Y1 is selected from the group consisting of W. Y. T, V, A and D; F. H. L, N, Q, E, D, K, and R: 0273 Y12 is selected from the group consisting of E. V. 0248 Y2 is selected from the group consisting of W. Y. K, G, R, Q, D., N, H., T and S: F. H. L, N, Q, E, D, K, and R: 0274 Y13 is selected from the group consisting of L., I, 0249 Y3 is selected from the group consisting of I. V. L. V. K and R; and A, S and T. 0275 each X independently is any amino acid. 0250 Y4 is selected from the group consisting of T. S., I, 0276. In another embodiment of the invention, there is K., N, H. R. Q., E and D; provided a peptide of up to 51 amino acids comprising the 0251 Y5 is selected from the group consisting of I. V. T. Sequence K, L, N, Q, D, E, R and H: 0252 YG is selected from the group consisting of any amino acid except P. G and C: 0253) Y7 is selected from the group consisting of I, L. V. 0278 Y1 is selected from the group consisting of W, Y, N, Q, K, R, H, E and D: F. H. L, N, Q, E, D, K, and R: 0254 Y8 is selected from the group consisting of Q. H. 0279 Y2 is selected from the group consisting of W, Y, R, N, E, D, K and P: F. H. L, N, Q, E, D, K, and R: 0255 Y9 is selected from the group consisting of Q. H. 0280 Y3 is selected from the group consisting of I. V. L. N, E, D, K, R, L, and P: A, S and T. 0256 Y10 is selected from the group consisting of Q, H, 0281 Y4 is selected from the group consisting of T. S. I. N, E, D, K and R; K., N, H. R. Q., E and D; 0257 Y11 is selected from the group consisting of N. S. 0282 Y5 is selected from the group consisting of I. V. T. T, V, A and D; K, L, N, Q, D, E, R and H: 0258 Y12 is selected from the group consisting of E. V. 0283 Y6 is selected from the group consisting of any K. G. R. Q, D. N. H., T and S; and amino acid except P. G and C: 0259 each X independently is any amino acid. 0284 Y7 is selected from the group consisting of I. L. V. N, Q, K, R, H, E and D: 0260. In another embodiment of the invention, there is provided a peptide of up to 51 amino acids comprising the 0285) Y8 is selected from the group consisting of Q. H. Sequence R, N, E, D, K and P: 0286 Y9 is selected from the group consisting of Q. H. N, E, D, K, R, L, and P: 0287 Y10 is selected from the group consisting of Q, H, 0262 Y1 is selected from the group consisting of W. Y. N, E, D, K and R; F. H. L, N, Q, E, D, K, and R: 0288 Y11 is selected from the group consisting of N. S. 0263 Y2 is selected from the group consisting of W. Y. T, V, A and D; F. H. L, N, Q, E, D, K, and R: 0289 Y12 is selected from the group consisting of E. V. 0264 Y3 is selected from the group consisting of L. V. L. K, G, R, Q, D., N, H., T and S: A, S and T. 0290 Y13 is selected from the group consisting of L., I, 0265 Y4 is selected from the group consisting of T. S. L. V, K and R; K., N, H. R. Q., E and D; 0291 Y14 is selected from the group consisting of L. S. 0266 Y5 is selected from the group consisting of I. V. T. M. Y. N, Q, E, D, K, and R; and K, L, N, Q, D, E, R and H: 0292 each X independently is any amino acid. US 2007/0207952 A1 Sep. 6, 2007

0293. In another embodiment of the invention, there is 0317 Y9 is selected from the group consisting of Q. H. provided a peptide consisting of up to 51 amino acids N, E, D, K, R, L, and P: comprising the sequence 0318 Y10 is selected from the group consisting of Q, H, N, E, D, K and R; 0319 Y11 is selected from the group consisting of N. S. T, V, A and D; 0295 Y2 is selected from the group consisting of W. Y. 0320 Y12 is selected from the group consisting of E. V. F. H. L, N, Q, E, D, K, and R: K, G, R, Q, D., N, H., T and S: 0296 Y3 is selected from the group consisting of I. V. L. 0321 Y13 is selected from the group consisting of L., I, A, S and T. V, K and R; 0297 Y4 is selected from the group consisting of T. S., I, 0322 Y14 is selected from the group consisting of L. S. K., N, H. R. Q., E and D; M. Y. N, Q, E, D, K, and R; and 0298 Y5 is selected from the group consisting of I. V. T. K, L, N, Q, D, E, R and H: 0323) each X independently is any amino acid. 0324. In another embodiment of the invention, there is 0299 Y6 is selected from the group consisting of any provided a peptide consisting of up to 51 amino acids amino acid except P. G and C: comprising the sequence 0300 Y7 is selected from the group consisting of I, L. V. N, Q, K, R, H, E and D: 0301 Y8 is selected from the group consisting of Q. H. R, N, E, D, K and P: 0326 Y4 is selected from the group consisting of T. S., I, 0302 Y9 is selected from the group consisting of Q. H. K., N, H. R. Q., E and D; N, E, D, K, R, L, and P: 0327 Y5 is selected from the group consisting of I. V. T. 0303 Y10 is selected from the group consisting of Q, H, K, L, N, Q, D, E, R and H: N, E, D, K and R: 0328 Y6 is selected from the group consisting of any 0304 Y11 is selected from the group consisting of N. S. amino acid except P. G and C: T, V, A and D; 0329 Y7 is selected from the group consisting of I. L. V. 0305 Y12 is selected from the group consisting of E. V. N, Q, K, R, H, E and D: K, G, R, Q, D., N, H., T and S: 0330 Y8 is selected from the group consisting of Q. H. 0306 Y13 is selected from the group consisting of L., I, R, N, E, D, K and P: V, K and R; 0331 Y9 is selected from the group consisting of Q. H. 0307 Y14 is selected from the group consisting of L, S. N, E, D, K, R, L, and P: M. Y. N, Q, E, D, K, and R; and 0332 Y10 is selected from the group consisting of Q, H, 0308) each X independently is any amino acid. N, E, D, K and R; 0333 Y11 is selected from the group consisting of N. S. 0309. In another embodiment of the invention, there is provided a peptide of up to 51 amino acids comprising the T, V, A and D; Sequence 0334 Y12 is selected from the group consisting of E. V. K, G, R, Q, D., N, H., T and S: 0335 Y13 is selected from the group consisting of L., I, V, K and R; 0311 Y3 is selected from the group consisting of I. V. L. 0336 Y14 is selected from the group consisting of L. S. A, S and T. M. Y. N, Q, E, D, K, and R; and 0312 Y4 is selected from the group consisting of T. S., I, 0337 each X independently is any amino acid. K., N, H. R. Q., E and D; 0338. In another embodiment of the invention, there is 0313 Y5 is selected from the group consisting of I. V. T. provided a peptide consisting of up to 51 amino acids K, L, N, Q, D, E, R and H: comprising the sequence 0314 Y6 is selected from the group consisting of any amino acid except P. G and C: 0315 Y7 is selected from the group consisting of I, L. V. 0340 Y5 is selected from the group consisting of I. V. T. N, Q, K, R, H, E and D: K, L, N, Q, D, E, R and H: 0316 Y8 is selected from the group consisting of Q. H. 0341 Y6 is selected from the group consisting of any R, N, E, D, K and P: amino acid except P. G and C: US 2007/0207952 A1 Sep. 6, 2007

0342 Y7 is selected from the group consisting of I, L. V. N, Q, K, R, H, E and D: 0343 Y8 is selected from the group consisting of Q. H. R, N, E, D, K and P: 0368 X1 is selected from the group consisting of M. L. 0344 Y9 is selected from the group consisting of Q. H. I, Q, T. R and K: N, E, D, K, R, L, and P: 0369 X2 is either E, D, Q and K: 0345 Y10 is selected from the group consisting of Q, H, 0370 X3 is selected from the group consisting of E, D N, E, D, K and R; and K. 0346 Y11 is selected from the group consisting of N. S. 0371 X4 is selected from the group consisting of K. R. T, V, A and D; E, Q, N and T: 0347 Y12 is selected from the group consisting of E. V. 0372 X5 is selected from the group consisting of E. L. R. K, G, R, Q, D., N, H., T is and S; K and Q; 0348 Y13 is selected from the group consisting of L., I, 0373 X6 is selected from the group consisting of N, D. V, K and R; S, E, Q. K, R, H, T, I and G: 0349 Y14 is selected from the group consisting of L, S. 0374 X7 is selected from the group consisting of N, Q, M. Y. N, Q, E, D, K, and R; and D, E, K, S, T and Y: 0350 each X independently is any amino acid. 0375 X8 is selected from the group consisting of Y. F. H. 0351. In another embodiment of the invention, there is I, V and S: provided a peptide consisting of up to 51 amino acids 0376 X9 is selected from the group consisting of G, K, comprising the sequence R, H, D, E, S, T, N and Q; 0377 X10 is selected from the group consisting of K, H, E, Q, T, V, I, L. M. A. Y. F, and P: 0353 Y6 is selected from the group consisting of any 0378 X11 is selected from the group consisting of H. K. amino acid except P. G and C: E, Y and F: 0354 Y7 is selected from the group consisting of I, L. V. 0379 X12 is selected from the group consisting of T. S. N, Q, K, R, H, E and D: Q, N, E, D, R, K, H. W. G, A, and M: 0355 Y8 is selected from the group consisting of Q. H. 0380 X13 is selected from the group consisting of D, E, R, N, E, D, K and P: Q, T, K, R, A, V and G: 0356. Y9 is selected from the group consisting of Q. H. 0381 X14 is selected from the group consisting of D, E, N, E, D, K, R, L, and P: K, H, Q. N, S, I. L. V. A and G: 0357 Y10 is selected from the group consisting of Q, H, 0382 X15 is selected from the group consisting of S. A N, E, D, K and R; and (P): 0358 Y11 is selected from the group consisting of N. S. 0383 X16 is selected from the group consisting of N, K, T, V, A and D; S, T, D, E, Y, I and V: 0359 Y12 is selected from the group consisting of E. V. 0384 X17 is selected from the group consisting of E, D, K, G, R, Q, D., N, H., T and S: N, K, G, and V: 0360 Y13 is selected from the group consisting of L., I, 0385 X18 is selected from the group consisting of K. R. V, K and R; H, D, E, N, Q, T. M., I, and Y: 0361 Y14 is selected from the group consisting of L, S. 0386 X19 is selected from the group consisting of E. V. M. Y. N, Q, E, D, K, and R; and Q. M, L. J. and G: 0362 each X independently is any amino acid. 0387 X20 is selected from the group consisting of Q, N. 0363. In another embodiment of the invention, there is E, K, R, H, L, and F: provided a peptide consisting of up to 51 amino acids 0388 X21 is selected from the group consisting of E, D, comprising the sequence N, S K, A, and G: 0389 X22 is selected from the group consisting of L., I, and Y, and 0390 X23 is selected from the group consisting of I, L. 0365) each X independently is any amino acid. M, Q, S, and Y. 0366. In another embodiment of the invention, there is 0391) In one variation of the above embodiment, the provided a peptide consisting of up to 51 amino acids peptide comprises a sequence selected from the group comprising the sequence consisting of the sequences shown in FIG. 1. US 2007/0207952 A1 Sep. 6, 2007 20

0392. In another embodiment of the invention, there is provided an isolated complex of the Formula I or Formula II:

(AV)-L1--Pr II AV-i-L2-(Pr).

0393 wherein: 0394 m is an integer from 1-5: 0395 n is an integer from 1-100; 04.05 peptide DP178 (T20); and 0396 o is an integer from 1-5; 04.06 peptide T-1249; 0397) p is an integer from 1-100; wherein: 0398 AV is an antiviral compound; 04.07 X1 is selected from the group consisting of M. L. 0399 L1 and L2 are polyvalent linkers covalently linking I, Q, T. R and K: AV to Pr, or where L1 and L2 are absent; 0400 Pr is a protein; and 0408) X2 is either E, D, Q and K: 04.01 wherein the complex possesses antiviral activity in 04.09 X3 is selected from the group consisting of E, D vivo. and K. 0402. In one variation of the above embodiment, the 0410 X4 is selected from the group consisting of K. R. antiviral compound is a peptide. In another variation, the E, Q, N and T: peptide has a mass of less than about 100 kDA. 0411 X5 is selected from the group consisting of E. L. R. In another variation, the peptide has a mass of less than K and Q; about 30 kDA. In yet another variation, the peptide has a 0412 X6 is selected from the group consisting of N, D. mass of less than about 10 kDA. S, E, Q. K, R, H, T, I and G: 0403. In one particular variation, the peptide is a pepti 0413 X7 is selected from the group consisting of N, Q, domimetic. D, E, K, S, T and Y: 0404 In another embodiment of the invention, the pep 0414 X8 is selected from the group consisting of Y. F. H. tide consists of up to 51 amino acids comprising a sequence I, V and S: selected from the group consisting of: 0415 X9 is selected from the group consisting of G, K, R, H, D, E, S, T, N and Q; 0416 X10 is selected from the group consisting of K, H, E, Q, T, V, I, L. M. A. Y. F, and P: 0417 X11 is selected from the group consisting of H. K. E, Y and F: 0418 X12 is selected from the group consisting of T. S. Q, N, E, D, R, K, H. W. G, A, and M: 0419 X13 is selected from the group consisting of D, E, Q, T, K, R, A, V and G: 0420 X14 is selected from the group consisting of D, E, K, H, Q. N, S, I. L. V. A and G: 0421 X15 is selected from the group consisting of S. A and (P): 0422 X16 is selected from the group consisting of N, K, S, T, D, E, Y, I and V: 0423 X17 is selected from the group consisting of E, D, N, K, G, and V: 0424 X18 is selected from the group consisting of K. R. H, D, E, N, Q, T. M., I, and Y: US 2007/0207952 A1 Sep. 6, 2007

0425 X19 is selected from the group consisting of E. V. not simply a prodrug of the peptides that are generated or Q. M. L. J. and G: released upon hydrolysis in vivo. 0426 X20 is selected from the group consisting of Q, N. 0437 WO 00/76550 (F. Kratz) discloses pharmaceuticals E, K, R, H, L, and F: and/or diagnostic active Substances attached to a spacer molecule that is attached to a thiol binding group, Such as 0427 X21 is selected from the group consisting of E, D, native or recombinant albumin. However, the disclosure N, S K, A, and G: teaches that the release of the pharmaceutical compounds or 0428 X22 is selected from the group consisting of L., I, the diagnostic active Substances is prefered since the low and Y, and molecular weight active Substance must interact with the target molecule to that it is pharmacologically active. In 0429 X23 is selected from the group consisting of I, L. addition, Kratz teaches that the spacer molecule are selected M, Q, S, and Y. from compounds that are hydrolytically and/or pH-depen 0430. The peptide sequence disclosed herein comprising dent and/or are eZymatically scissile. Preferably, these the fragment of the peptide that consists of up to 51 amino spacer molecules are acid sensitive or acid-unstable spacers. acids may be a fragment of the 51 amino acid peptide that is located at the N-terminus, the C-terminus or anywhere in 0438. The linker L1 or L2 can be a hydrophobic linker, a the interior of the 51 amino acid peptide. In one variation, hydrophilic linker, or combinations thereof when more than the peptides are the C-termininus amides ( CONH2) and one linker is present. The variety of different linker L1 or L2 their protected derivatives. In another variation, the peptides can be selected to provide different solubility characteristics are the C-terminus esters (i.e. —COOR, where R is substi and cell penetrability chracteristics. tuted or unsubstituted (Cs)alkyls). 0439 Where the antiviral compound of the present inven 0431 Optionally, the peptide sequence disclosed herein tion is a peptide that is attached to one or more linkers, the comprising the fragment may be further protected by stan linker L1 or L2 may be attached to the peptide at the dard protecting groups known in the art. Protected deriva N-terminus, the C-teminus, at a reactive side chain on an tives of these peptides are useful in the preparation of the internal amino acid(s) such as, for example, with a lysine, antiviral compounds or are useful in themselves as active aspartic acid, glutamic acid, or cysteine, or combinations antiviral compounds in their partially or fully protected thereof. forms. That is, the derivatized or protected or partially 0440. In one variation of the invention, the linker L1 or protected peptide fragments in the complex may still retain L2 comprises at least two functional groups covalently the ability to bind the target and manifest therapeutic bio linking AV to Pr. In another variation, the linker L1 or L2 is logical activities. For example, representative protecting hydrolytically stable in human serum for an extended period groups for amino groups of the peptide fragments include of time. acetyl, tert-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable and representative protecting groups can be 0441. It was determined that the complexes of the present found in T. W. Greene, Protecting Grouips in Organic invention are compounds that are themselves more stable Synthesis, 3rd edition, John Wiley & Sons, Inc. 1999. toward hydrolytic cleavage or degradation than the non complexed compounds. In one variation, the complex of the 0432. In one embodiment, the protecting group for the present invention are stable toward hydrolytic cleavage or peptide fragments comprise C-terminal amides and/or N-ter degradation, having half lives in human serum for a period minal acetyl groups and their derivatives. The peptides may of 4 hours to 120 days. In a particular variation, the complex have any functional groups of the amino acids, including of the present invention are stable toward hydrolytic cleav - NH, -SH, -OH, -COOH, and the like, that may be age or degradation for a period of about 8 hours to about 30 attached to the linker. days. 0433. In one variation of the invention, there is provided a complex of the above embodiments and variations wherein 0442. In one particular variation, there is provided the the protein is a blood component. In another variation, the complexes of the invention wherein the linker L1 or L2 is blood component is selected from the group consisting of stable in human serum for half lives of 8 hours to 30 days. red blood cells, immunoglobulins, IgM, IhC, serum albu 0443) In another particular variation of the above varia min, transferrin, P90 and P38, ferritin, a steroid binding tions and embodiments, the linker L1 or L2 is a derivative protein, thyroxin binding protein, and C-2-macroglobulin. In of a compound selected from the group consisting of acy yet another variation, the blood component is human serum loxymethylketones, aziridines, diazomethyl ketones, albumin and the linker is a peptide linker. epoxides, iodo-, bromo- or chloroacetamides, C.-haloesters, C.-haloketones, sulfoniums, chloroethylsulfides, O-alkyli 0434 In one particular variation, the blood component is Soureas, alkyl halides, vinylsulfones, acrylamides, acrylates, human serum albumin and the linker is a non-peptide linker. vinylpyridines, organometallic compounds, aryldisulfides, 0435. In one particular embodiment of the invention, the thiosulfonates, aldehydes, nitriles, C.-diketones, C.-ketoam complex is a fusion protein. ides, C.-ketoesters, diaminoketones, semicarbazones, and dihydrazides. 0436. In one variation of the invention, the linler L1 or L2 is a non-labile linker that is stable toward hydrolytic cleav 0444. In another particular variation of the above varia age in vivo. Therefore, the complexes of the present inven tions and embodiments, the linker L1 or L2 is a derivative tion provides compounds that are stable toward hydrolytic of a compound selected from the group consisting of azi cleavage in vivo. In addition, the complexes of the present dobenzoyl hydrazide, N-4-(p-azidosalicylamino)butyl-3- invention are also active compounds in themselves and are (2-pyridyldithio)propionamide, bis-sulfosuccinimidyl sub US 2007/0207952 A1 Sep. 6, 2007 22 erate, dimethyl adipimidate, disuccinimidyl tartrate, N-y- of the complex because the administration of the complex maleimidobutyryloxysuccinimide ester, N-hydroxy results in less irritation at the injection site, avoids the Sulfo Succinimidyl-4-azidobenzoate, N-succinimidyl-4-azi non-specific reaction with other proteins, and achieves an dophenyl-1,3'-dithiopropionate, N-succinimidyl 4-io improved therapeutic blood levels of the complex than the in doacetylaminobenzoate, glutaraldehyde, succinimidyl Vivo approach. 4-N-maleimidomethylcyclohexane-1-carboxylate, N-hy droxysulfoSuccinimide, maleimide-benzoyl-Succinimide, 0452. In another embodiment, the invention provides an Y-maleimido-butyryloxy Succinimide ester, maleimidopro anti-viral composition comprising a non-peptidic anti-viral pionic acid, N-hydroxySuccinimide, isocyanate, thioester, compound covalently linked to a blood component. thionocarboxylic acid ester, imino ester, carbodiimide, anhy 0453 According to each of the above embodiments and dride and carbonate ester. variations, there is provided according to the above embodi ments and variations a composition comprising the complex 0445. In one particular embodiment, there is provided a and a physiologically acceptable carrier. In one variation, the complex of the invention wherein the protein is albumin. In composition is formulated with saline or formulated without one variation of the above embodiment, the albuminis HSA saline. In another variation, the composition is formulated or recombinant HSA that is at least 10% pure on a dry matter for parenteral administration. basis. 0454 Administration of the composition of the present 0446. In another variation, the linkage is to a Cys-34 of invention may include parenteral administration, including human albumin. In yet another variation, the linkage is to a by injection through other route Such as Subcutaneous, lysine of human albumin. intramuscular, intraorbital, intracapsular, intraspinal, 0447. In one particular variation of the invention, there is intrasternal, intracerebral ventricular (ICV), intravenous, provided the above disclosed complex wherein m is 1, n is and the like. 1, and the protein is HSA or recombinant HSA. In another 0455. In one variation of the above, the composition is variation, n is 1, the protein is HSA or recombinant HSA, selected from the group consisting of solutions, dry products and wherein the complex is further purified to a purity of at for combining with a solvent prior to use, Suspensions, least 30%. In yet another particular variation, m is 1, n is 2, emulsions, and liquid concentrates. and the protein is HSA or recombinant HSA. 0456. In another embodiment of the invention, there is 0448. In one embodiment, the complex is prepared by provided a method for inhibiting the activity of HIV gp41 combining a stoichiometric ratio of (AV)-L1 with Pr or a and HIV in vivo, the method comprising: stoichiometric ratio of AV with L2-(Pr). Thus, the ratio of 0457 administering to the bloodstream of a mammalian (AV)-L1 to Pr, or the ratio of AV to L2-(Pr) are 1:1. In host an isolated conjugate complex of the above embodi another particular variation, the complex is prepared by ments and variations, wherein the complex is formed by combining a mixture of Pr to (AV)-L1 in a ratio of at least attaching an antiviral compound to a linker having at least about 1.3:1. one reactive functional group which reacts with the protein 0449 In one variation of the above embodiment, L1 and to form stable covalent bonds; and L2 are absent, and wherein the complex is prepared by 0458 wherein the isolated conjugate complex is admin forming an activated intermediate of AV followed by the istered in an amount to maintain an effective therapeutic condensation of the activated AV intermediate with Pr. In a effect in the bloodstream for an extended period of time as variation of the above, the activated intermediate of AV is compared to a non-conjugated antiviral compound. prepared from a mixed anhydride or N,N'-carbonyldiimida Zole reagent. 0459. In one variation of the above method, the method may be applicable to the complexes disclosed in the above 0450 According to the above variations, the complex is embodiments and variations. further purified to a purity of at least about 30%. Unexpect edly, it was determined that the formulation of the conjugate 0460. In another variation, the method employs a protein compound ex vivo produces unanticipated advantages over wherein the protein is HSA or recombinant HSA. forming of the conjugated compound in vivo. For example, 0461 In a particular variation of the above method, the in the case of the relatively insoluble antiviral agents, linker comprising a reactive functional group is a compound conjugation ex vivo forms a more soluble agent or complex. selected from the group consisting of acyloxymethylke In addition to improved the stability of the compounds, the tones, aziridines, diazomethyl ketones, epoxides, iodo-, formation of the complex of the present invention result in bromo- or chloroacetamides, C.-haloesters, C-haloketones, a more soluble complex for formulation, which is a signifi sulfoniums, chloroethylsulfides, O-alkylisoureas, alkyl cant advantage for the administration (via injection) over the halides, vinylsulfones, acrylamides, acrylates, vinylpy administration of the insoluble unconjugated drug. Because ridines, organometallic compounds, aryldisulfides, thiosul of the ex vivo conjugation of the antiviral agent forms a fonates, aldehydes, nitriles, C-diketones, C.-ketoamides, soluble drug formulation, the present method allows the C.-ketoesters, diaminoketones, semicarbazones, and dihy preparation of stable, physiological Solutions. The ability to drazides. prepare stable, soluble Solution compositions containing the 0462. In one embodiment, there is provided a method for complex of the present invention allows the preparation of eliciting antiviral activity in Vivo, said method comprising: physiological saline solution of the complex for ease of oral 0463 administering into the bloodstream of a mamma or parenteral administration. lian host the complex of the above embodiments and varia 0451. In addition, the formation of the complex ex vivo tions in an amount Sufficient to provide an effective amount has been found to be preferable over the in vivo formation for antiviral activity; US 2007/0207952 A1 Sep. 6, 2007

0464 whereby said complex is maintained in the blood 0478. In one variation, the invention provides a method stream over an extended period of time as compared to the of treating a subject Suffering from a viral infection, com lifetime of unbound antiviral compound. prising administering to said subject an effective amount of a composition of the above embodiments and variations. 0465. In another embodiment of the invention, there is According to the above variations, the Subject is suffering provided a method for eliciting antiviral activity in a host, from HIV infection. said method comprising: 0479. In another embodiment, the invention provides a 0466 a) preparing a compound AV-L1 or AV-L2 wherein method of prophylaxis in a patient Suspected of being AV is a peptide antiviral compound with a mass of less than exposed to a viral infection, comprising administering to 60 kD and L1 or L2 is a linker covalently bound to AV: said Subject an effective amount of a composition of the 0467 b) treating the compound AV-L1 or AV-L2 with above variations. isolated protein ex vivo for a time sufficient for the com 0480 Methods of Treatment pound AV-L1 or AV-L2 to covalently bond to the protein to 0481. The present invention takes advantage of the prop form the protein complex of the above embodiments and erties of existing anti-viral agents. The viruses that may be variations, and inhibited by the compounds of the present invention include, 0468 c) administering the treated protein complex to the but are not limited to all strains of viruses listed, e.g., in U.S. host. Pat. No. 6,013,263 and U.S. Pat. No. 6,017,536 at Tables V-VII and IX-XIV therein. These viruses include, e.g., 0469. In one variation of the above embodiment, the human retroviruses, including HIV-1, HIV-2, and human protein is albumin. In another variation of the above, the T-lymphocyte viruses (HTLV-I and HTLV-II), and non albumin is HSA or recombinant HSA. human retroviruses, including bovine leukosis virus, feline 0470 According to one variation, the albumin is obtained sarcoma virus, feline leukemia virus, simian immunodefi from blood, purified and isolated from blood prior to treating ciency virus (SIV), simian sarcoma virus, simian leukemia, the albumin with the compound AV-L1 or AV-L2. In another and sheep progress pneumonia virus. Non-retroviral viruses variation of the above methods, the albumin is purified to a may also be inhibited by the compounds of the invention, for purity level of at least 10% on a dry matter basis. In yet example human respiratory syncytial virus (RSV), canine another variation, the albumin is purified to a purity level of distemper virus, Newcastle Disease virus, human parainflu more than 95%. enza virus (HPIV), influenza viruses, measles viruses (MeV), Epstein-Barr viruses, hepatitis B viruses, and simian 0471. In another embodiment, the invention provides a Mason-Pfizer viruses. Non-enveloped viruses may also be method for eliciting antiviral activity in a host, said method inhibited, and include, but are not limited to, picomaviruses comprising: Such as polio viruses, hepatitis. A virus, enteroviruses, echo viruses, coxsackie viruses, papovaviruses such as papilloma 0472 a) preparing a compound AV-L1 or AV-L2 wherein virus, parvoviruses, adenoviruses, and reoviruses. AV is an antiviral compound peptide with a mass of less than 60 kD and L1 or L2 is a linker covalently bound to AV: 0482. The compounds of the present invention may be administered to patients according to the methods described 0473 b) treating the compound AV-L1 or AV-L2 with below and other methods known in the art. Effective thera isolated one or more protein Prex vivo for a time sufficient peutic dosages of the compounds derivatives may be deter for the compound AV-L1 or AV-L2 to covalently bond to one mined through procedures well known by those in the art. or more of the isolated proteins to form one or more modified protein complex of the above embodiments and 0483 The compounds also can be administered prophy lactically to previously uninfected individuals. This can be variations; and advantageous in cases where an individual has been exposed 0474 c) administering the modified protein or proteins to to a virus, as can occur when individual has been in contact the host. with an infected individual where there is a high risk of viral transmission. This can be especially advantageous where 0475. In one variation of the embodiment, the protein is there is no known cure for the virus, such as the HIV virus. albumin. In another variation, the albumin is obtained from As an example, prophylactic administration of a compound blood, purified and isolated from blood prior to treating with of the invention would be advantageous in a situation where the compound AV-L1 or AV-L2. In yet another variation, the a health care worker has been exposed to blood from an albumin is HSA or recombinant HSA. HIV-infected individual, or in other situations where an 0476. In one embodiment, the invention provides a phar individual engaged in high-risk activities that potentially maceutical composition comprising a therapeutically effec expose that individual to the HIV virus. tive amount of a complex of the above embodiments and 0484 The preferred route of administration of the com variations, or a physiologically acceptable salt thereof, and pounds of the invention is via intravenous administration, a pharmaceutically acceptable carrier, excipient, or diluent. which allows the compounds to circulate in the bloodstream and reach their desired target. However, the methods of the 0477. In one embodiment, the invention provides a pro present invention comprehend any method of administration cess for inhibiting the action of the HIV virus which process that permits circulation of the compounds in the body of the comprises administering to a host in recognized need of Such patient. treatment an effective amount of a complex of the above embodiments and variations, or a pharmaceutically accept 0485 The compounds and pharmaceutical compositions able salt thereof. of the present invention may be used alone or in combination US 2007/0207952 A1 Sep. 6, 2007 24 with other anti-viral compounds. For example, compounds 0492 Synthesis Protocol on Resin: and pharmaceutical compositions of the present invention may be used in a variety of drug cocktails, or combinations 0493 1. Deprotection-25% piperidine/DMF (5+25 min) of three or more antiretroviral agents, that can potently 0494 2. Washing DMF (6x1 min) Suppress viral replication and prevent or delay the onset of 0495 3. Coupling 3 eq. Fmoc-amino acid--3 eq. AIDS. TCTU+6 eq. DEA to negative Kaiser test, (approx. 1 h); 3 0486 The invention having been fully described can be h for Asn' and Lys further appreciated and understood with reference to the following non-limiting examples. 0496 4. Washing DMF (5x1 min) 0487 Compounds according to the present invention 0497 5. Terminal acetylation Acetic anhydride/DEEA may optionally be synthesized according to the following 0498 Cleavage From Resin: general reaction schemes: 0499 TFA m-cresol thioanisol triisopropylsilane Preparation of Complex of Formula I: (85:5:5:5). 2 h, RT evaporation in vacuum 0500 precipitation by ether (crude yield ~80%) Formula I 0501) Purification: mAV + L1 - (AV--L1 0502 HPLC using a Biosphere C-18 column n(AV)-L1 + Pr - > (AV)-L-Pr 0503) mobile phase-A: water/0.1% TFA B: acetonitrile/ O.1% TFA Preparation of Complex of Formula II: 0504 gradient—10-20% B/10 min, 20-40% B/90 min 0505) detection. UV 220 mm Formula II 0506 Fractions over 95% were collected and lyophilized 0488. As shown in the Scheme above for the preparation of the complex of Formula I, the antiviral is first attached to 0507 Analysis: the linker L1 to form the antiviral-linker AV-L1, which is followed by the reaction with protein Pr to form the complex 0508 of Formula I. However, it is also feasible to first attach the 0509 HPLC: column Luna C 18 linker L1 to the protein Pr to form a linker-protein com pound, L1 -Pr, which is then linked with the antiviral agent 0510 mobile phase A: water/0.04% HPO, B: aceto to form the complex of formula I. Similarly, the present nitrile/0.04% HPO. invention also teaches that the reverse sequence as noted 0511 gradient-5-65% B/30 min above may also be applicable for the preparation of the complex of Formula II. 0512 detection. UV 220 mm EXAMPLE 1. 0513) MSIMH." Design and Preparation of HIV Fusion Inhibitor 0514 TCTU=O-(1H-6-Chlorobenzotriazole-1-yl)-1,1,3, Peptides 3-tetramethyluronium tetrafluoroborate 0489 Sequences of putative HIV fusion inhibitor pep 0515 DIEA=diisopropylethylamine tides were modeled using the crystal structure of the gp41 trimeric helical fisogenic complex (reviewed in Jiang et al. EXAMPLE 2 2002). Peptide sequences were modeled to form helical segments that can fit into the grooves formed by the N-ter Evaluation of Antiviral Activity of Peptides minal triple helical core of the fusogenic complex. Evalua tion of the inner binding and outer exposed surfaces of the 0516 Antiviral potency of the peptides was analyzed modeled helical peptides were used to determine the against HIV-1 HXB2 or NLA-3 strains using a cytotoxicity sequence and composition of amino acids in the model assay with MT4 cells as previously described (ref 1-3) with peptides. Amino acids that have exposed side chains after minor modifications. MT-4 cells (1.5x10"/ml) were exposed complex formation are varied to improve solubility and to 200 50% tissue culture infective doses (TCID50) of other physical-chemical characteristics of the model peptide. viruses in the presence of various concentrations of test Amino acids that bind to the N-terminal triple helical core compound in 96 well microtiter plates and incubated at 37° are determinative of binding affinity and antiviral activity. C. for 5 days. Cytotoxicity of HIV was measured by the addition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazo 0490 Most of the peptides in Tables 1 and 2 reflect lium bromide (MMI) solution to each well to a final con changes in Surface residues and were predicted to be equally centration of 0.75 mg/ml, and incubation for 1 hour a 37°C. potent antiviral compounds against the HIV HXB2 strain. After incubation, cells were dissolved in isopropanoyrri All of the peptides in Table 1 have an acetyl group at the ton-X 100/HCl (1000:50:25) solution. Absorbance was N-terminus and a C-terminal amide. monitored in a microplate reader (Spectramax, Molecular 0491. The peptides were prepared using standard solid Devices) at 540 rm and 690 nm. MT-4 cells were obtained phase techniques on Tentagel-S-RAM resin (Rapp Poly from the AIDS Research and Reference Reagent Program mer), 0.25 mmol/g. All gave HPLC purities>90% and cor (ARRRP, Division of AIDS, NIAID, NIH: MT-4 from Dr. D. rect mass spec. Richman). Cells were propagated in RPMI 1640 growth US 2007/0207952 A1 Sep. 6, 2007

medium supplemented with 10% fetal bovine serum, 50U of penicillin and 50 ug of streptomycin per ml (Ivitrogen, TABLE 1-continued Carlsbad Calif.). ICso values for all compounds tested are listed in Table 1. Antiviral activity of unmodified peptides 0517 References IC nM 0518 1. Kodama, E. S. Shigeta, T. Sizuzki, E. De Clerq. HIW-1 HIW 1996. Application of a gastric cancer cell line (MKN-28) for IIIB NL 4-3 anti-adenovirus screening using the MTT method. Antiviral Res. 31:159-164. T1249 2.0 1.5 0519 2. Pauwels, R., J. Balzarini, M. Baba, R.Snoeck, D. c34 1 .. 4 1.7 Schols, P. Herdeweijn, J. Desmyter, E. De Clerq. 1988. Rapid and automated tetrazolium-based colorimetric assay SIW C34 3.8 72 for the detection of anti-HIV compounds. J. Virol. Methods. * average of 2 or 3 experiments 20:309-321. AC = acetyl 0520 3. Yoshimura, K. R. Kato, M. F. Kavlick, A. Nguyen, V. Maroun, K. Maeda, K. A. Hussain, A. K. Ghosh, EXAMPLE 3a S. V. Gulnik, J. W. Erickson, H. Mitsuya. 2002. A potent Preparation of Chemically-reactive Modified HIV HIV-1 protease inhibitor, UIC-94.003(TMC-126), and selec Fusion Inhibitor Peptides tion of novel (A28S) mutation in the protease active site. 0521. Analogues of peptides 2 and 7 demonstrate the J.Virol. 76:1349-1358. general applicability of the procedure for enhancing the pharmacokinetic activity of peptides with diverse sequences. TABLE 1. SPI-30014 and SPI-70038 (see below, Table 2) were pre Antiviral activity of unmodified peptides pared using Solid phase synthesis techniques as described above. Instead of acetylating the N-terminus it is reacted IC* nM with Fmoc-8-amino-3,6-dioxaoctanoic acid, TCTU, DIEA HIW-1 HIW for 3 h, washed as above and then reacted with 3-maleimi IIIB NL 4-3 dopropionic acid, TCTU, DIEA for 3 h. Cleavage and purification was as described above. 0522) SPI-30014 MH'4545 0523 SPI-7003.8 MH'4673 EXAMPLE 3b Preparation of Quenched, Modified HIV Fusion Inhibitor Peptides 0524) To generate unreactive controls maleirnide con taining peptides were quenched using an excess of 3-mer captoethanol. The resulting quenched peptides (SPI-30014O and SPI-70038Q) are unable to form covalent conjugates with HSA. The quenched derivatives were prepared as follows: 0525] 1 mg of SPI-30014 was dissolved in 22.0 ul DMSO. 5 ul of this solution was added to 45 ul of 53 mM aqueous 9-mercaptoethanol (final concentration 47.7 mM) and incubated for 5 hours at 37° C. The molar ratio of peptide: 3-mercaptoethanol was 1:51 and the final concen tration of peptide was 0.9 mM. 0526 Similarly 1 mg of SPI-70038 was dissolved in 21.4 ul DMSO. 5ul of this solution was added to 45ul of 53 mM aqueous B-mercaptoethanol (final concentration 47.7 mM) and incubated for 5 hours at 37° C. The molar ratio of peptide: B-mercaptoethanol was 1:58 and the final concen tration of peptide was 0.8 mM. EXAMPLE 4 Preparation of Long-acting HIV Fusion Inhibitor HSA-peptide Conjugates 0527 1 mg of SPI-30014 was dissolved in 22.0 ul DMSO. 10 ul of this solution was added to 90 ul 25% HSA US 2007/0207952 A1 Sep. 6, 2007 26

(Seracare) and incubated for 5 hours at 37° C. The molar EXAMPLE 6 ratio of peptide: HSA in the final reaction mixture is approxi mately 1:4 and final peptide concentration is 0.9 mM. Evaluation of Pharmacokinetic Properties of Long-acting HIV Fusion Inhibitors 0528) Similarly 1 mg of SPI-70038 was dissolved in 21.4 ul DMSO. 10 ul of this solution was added to 90 ul 25% 0531) Peptide-HSA conjugates (SPI-30014HSA and SPI HSA (Seracare) and incubated for 5 hours at 37° C. The 70038HSA) and quenched, unconjugated peptides (SPI molar ratio of peptide: HSA in the final reaction mixture is 30014Q and SPI-70038Q) were administered intravenously approximately 1:4 and final peptide concentration is 0.8 to Sprague-Dawley rats weighing between 400-500 g. Test mM. compounds were formulated in DMSO (peptides) or phos phate-buffered saline (HSA-peptide conjugates) and were 0529. The resulting HSA-peptide conjugates, SPI administered intravenously in a single dose of 0.5-0.6 umol/ 30014HSA and SPI-70038HSA, were tested for antiviral kg; rate of infusion was 2.0 ml/min: total infusion time was and pharmacokinetic profile compared to the reactive inter about 20 sec. Serum samples were collected at 5 min, 30 mediates (SPI-30014 and SPI-70038) and the quenched, min, 1, 2, 8, 24, 48, and 72 hours post dose. The concen unconjugated peptides (SPI-30014Q and SPI-70038Q). trations of antiviral peptides and conjugates in the rat plasma were analyzed using a cell-based antiviral bioassay. For each EXAMPLE 5 time point the serum sample was initially diluted 1:10 with growth medium and Subsequently used for multiple serial Evaluation of Antiviral Activity of Modified and dilutions analyzed in the standard antiviral assay using MT4 Conjugated HIV Fusion Inhibitor Peptides cells and HIV-1 HXB2 virus described above. The ICso 0530 Antiviral potency of maleimide containing pep value was determined and expressed as the percent of tides; quenched, unconjugated peptides; and, peptide-HSA sample serum necessary to inhibit 50% of the cytotoxic conjugates was analyzed against HBX2 HIV-1 using a activity of HIV-1 HXB2. A reference sample was prepared cytotoxicity assay with MT4 cells as described in Example that contains predose serum of the corresponding animal 2. ICs values for these compounds are listed in Table 2. The diluted 10-fold by medium. To this diluted sample a defined activities of the reactive and quenched peptides are similar. concentration of peptide or peptide-HSA conjugate in aque The antiviral activity of each HSA-peptide conjugates is ous DMSO was added. This sample was then analyzed in the reduced by about 34 fold. antiviral assay in the same way as test time point samples.

TABLE 2 Antiviral activity of modified peptides and conjugates

*average of 2-3 experiments ~Linker - refers to maleimidopropionylaminoethoxyethoxyacetyl O O

O --- Q-Linker refers to maleimidopropionylaminoethoxyethoxyacetyl derivative quenched using 3-mercaptoethanol rty---O O O X — Norleucine (unnatural amino acid) US 2007/0207952 A1 Sep. 6, 2007 27

This ICso reference value was determined and expressed as EXAMPLE 10 a concentration of peptide or peptide-HSA conjugate. The concentration of peptide or peptide-HSA conjugate in each Conjugation Reaction to Form the Complex of the Inven test time point serum sample was then calculated based on tion: the ICs values obtained with the reference sample in the 0537. A 10 mM solution of maleimidopropionylamino serum from the corresponding animal: ethoxyethoxyacetyl derivatized antiviral peptide in DMSO is added to a 25% water solution of HSA. The final peptide Concentration (nM)=LICso ref(nM), ICso(% of sample concentration in the reaction mixture is 1 mM and the molar serum)x100% ratio in reaction mixture of peptide: HSA is 1:1. The solution 0532. The concentration vs time profiles of test com pounds in rat plasma are shown in FIGS. 2 and 3 (average is incubated 5 hours at 37°C. Once incubation is complete, of 2-3 rats). The unconjugated (control) peptides display a the conjugate is stored at 4°C. rapid clearance profile: by 8 hours 80% of peptide is lost. In EXAMPLE 11 contrast, the terminal half-life of the two HSA-peptide conjugates ranged from 12 to 14 hours. The half-life of the Conjugation Reaction to Form the Complex of the Inven HSA-peptide conjugates is similar to the reported half-life of tion: HSA alone in rodents (15.8 hours) (1). I. M. Gaizutis, Pesce 0538 A 10 mM solution of maleimidopropionylamino A. J., Pollak V. E. 1975. Renal clearance of human and rat ethoxyethoxyacetyl derivatized antiviral peptide in DMSO albumins in the rat. Proc. Soc. Exp. Biol. Med. 148(4):947 is added to a 25% water solution of HSA. The final peptide 952. concentration in the reaction mixture is 1 mM and the molar 0533. These results indicate that the half-life, distribu ratio in reaction mixture of peptide: HSA is 10:1. The tion, and elimination of the antiviral compounds was deter solution is incubated 5 hours at 37° C. Once incubation is mined by the cloaking protein (HSA), while the antiviral complete, the conjugate is stored at 4°C. activity was determined by the warhead peptide. The pro longation of plasma activity in the animal is unexpected in ASSAY EXAMPLES light of the retention of potent biological activity of the conjugate. The latter finding would suggest that the warhead EXAMPLE 12 portion of the molecule is clearly exposed and therefore would be expected to be subject to metabolism, degradation, Binding Assay for Peptidyl Inhibitors of IV Fusion elimination and clearance processes in the body, but our 0539. The binding affinity of conjugated inhibitors of results suggest that it is protected from these processes. HIV fuision is measured using a chimeric peptide (IQN17) that contains a segment of GCN4 at the N-terminal and 17 EXAMPLE 7 residues from the first heptad repeat region of HIV-1 GP41 Conjugation Reaction to Form the Complex of the Inven at the C-terminal (Cell 99, 103-115). A 28-residue peptide tion: from the second heptad repeat region of GP41 (C28) is labeled with a fluorescent molecule Alexa-430 (Molecular 0534. A 10 mM solution of maleimidopropionylamino Probes) at its carboxyl terminal. The binding is measured by ethoxyethoxyacetyl derivatized antiviral peptide in DMSO titration of labeled C28 with IQN17. The concentrations of was added to a 25% water solution of HSA. The final peptide bound and unbound C28 were measured by capillary Zone concentration in the reaction mixture was 1 mM and the electrophoresis. At 3 uMC28 and 8 um IQN17, about 80% molar ratio in reaction mixture of peptide: HSA was 1:4. The C28 is bound to IQN17. For unlabeled peptides, the amount solution was incubated 5 hours at 37° C. Once incubation that competes 50% C28 off IQN17 gives its IC50 value. was complete, the conjugate was stored at 4°C. Renin Inhibitor Compounds and Complexes EXAMPLE 8 0540. The present invention relates to biologically active Conjugation Reaction to Form the Complex of the Inven compounds that may be used to react with proteins to form tion: covalently linked complexes wherein the resulting com plexes are found to exhibit renin inhibition activities in vivo. 0535 A 10 mM solution of trans-4-(maleimidylmethyl More specifically, the complexes are isolated complexes )cyclohexane-1-carbonyl derivatized antiviral peptide in comprising a renin inhibitor and a linkig group, and the DMSO is added to a 25% water Solution of HSA. The final blood component is a protein Such as albumin. The present peptide concentration in the reaction mixture is 1 mM and invention also provides methods for inhibiting renin activity the molar ratio in reaction mixture of peptide: HSA is 1:4. in Vivo comprising administering to the bloodstream of a The solution is incubated 5 hours at 37° C. Once incubation mammalian host the novel isolated complexes of the present is complete, the conjugate is stored at 4°C. invention. EXAMPLE 9 0541. In one embodiment, a pharmaceutical composition is provided that comprises a purified renin inhibitor complex Conjugation Reaction to Form the Complex of the Inven according to the present invention as an active ingredient. tion: Pharmaceutical compositions according to the invention 0536 A 10 mM solution of N-(3-2-2-(3-amino-pro may optionally comprise 0.001%-100% of one or more poxy)-ethoxy-ethoxy-ethoxy)-propyl)-2-bromoacetamide renin inhibitors complexes of this invention. These pharma derivatized antiviral peptide in DMSO is added to a 25% ceutical compositions may be admininistered or coadminis water solution of HSA. The final peptide concentration in tered by various methods known in the art for administering the reaction mixture is 1 mM and the molar ratio in reaction biologically active agents to the bloodstream. In a preferred mixture of peptide: HSA is 1:4. The solution is incubated 5 aspect of the invention, the compositions may be adminis hours at 37°C. Once incubation was complete, the conjugate tered by injection. In another preferred aspect, the compo is stored at 4° C. sitions may be administered by infusion. US 2007/0207952 A1 Sep. 6, 2007 28

0542. In another embodiment, methods and compositions are provided for delivery of isolated conjugated complexes -continued comprising biologically active agents, particularly therapeu tic agents such as renin inhibitors, where the complexes comprising the agents have an extended half-life in the bloodstream as compared to non-conjugated agents. OH 0543. The invention comprises using a biologically active compound covalently attached or linked to a linking HN group, the linking group comprising at least one chemically OH reactive moiety which is capable of forming covalent bonds with functionalities present on the protein. By preparing the isolated complexes before administration of the complexes r into the blood of the host, particularly the bloodstream of the O host, a biologically active complex is generated that main- N tain an effective therapeutic effect in the bloodstream for an HN 1N1 extended period of time as compared to a non-conjugated biologically active agent. ÖH 1N 0544. In one embodiment, the invention provides an isolated complex of the Formula I or Formula II: (Ih)-L1-Pr I O Ih-IL2-(Pr). II 0545 wherein: 0546 m is an integer from 1-5: 0547 n is an integer from 1-100; 0548 o is an integer from 1-5; 0549 p is an integer from 1-100; 0550 Ih is a renin inhibitor; 0551 L1 and L2 are polyvalent linkers covalently linking Th to Pr, or where L1 and L2areabsent; 0552) Pr is a protein; and 0553 wherein the complex possesses renin inhibitory acuV1lytivitv in1n vivo.V1Vo HN O1N 0554. In another embodiment, the renin inhibitor Ih is a peptide. In another embodiment, the peptide has a mass of less than about 60 kDA. In another embodiment, the peptide has a mass of less than about 10 kDA. In yet another embodiment, the peptide has a mass of less than about 1000 DA. 0555. In one particular embodiment, the peptide is a peptide mimetic. In another embodiment, the peptide is a transition state mimetic at the C-terminus. 0556. In one embodiment, the transition state mimetic at the C-terminus is selected from the group consisting of

11N HN n US 2007/0207952 A1 Sep. 6, 2007 29

-continued -continued

HN O n n-r ÖH O Nul / \ S. N N1R\ N / HN S N ÖH

O N YMe HN 1S-1N HN OH 1- H Nul OH O

O 21 O H N S. HN N 1N1N HN ; : NH N H O O

HN 1N1 S.

US 2007/0207952 A1 Sep. 6, 2007

Tyr-Lys, His-Pro-Phe-His-Leu-D-Leu-Val-Tyr-OH, Pro onic acid, N-hydroxysuccinimide, isocyanate, thioester, His-Pro-Phe-His-Leu(CH2NH)Val-Ile-His-Lys (H-142), thionocarboxylic acid ester, imino ester, carbodiimide, anhy Boc-Phe-His-Cha-(CH2NH)Val-NH2(S)-Me(Bu), Pro-His dride and carbonate ester. Pro-Phe-His-Leu-Phe-Val-Tyr-OH, Boc-His-Pro-Phe-His 0562. In one particular embodiment of the invention, the LeucCH(OH)CH2)Val-Ile-His-OH (H-26 1), and PEC protein is selected from the group consisting of red blood Phe-His-ACHPA-ILeNHC(CH2OH)2CH3. cells, and immunoglobulins, such as IgM and IgG, serum 0558. In another embodiment, Ih is a renin inhibitor albumin, transferrin, p90 and p38. In another particular peptide selected from the group consisting of Iva-Val-Val variation, the protein is albumin. In another variation, the Sta-Ala-Sta, Boc-Phe-His-Sta-Ile-AMP, Boc-Phe-His-Sta albumin is HSA or recombinant HSA that is at least 10% Ala-Sta-OMe, Boc-Phe-His-Sta-Leu-NHCH2Ph, Boc-Phe pure on a dry matter basis. In a further variation, the linkage His-ACHPA-Leu-AMB, Boc-Phe-His-Sta-Leu-AMB, Boc is to a Cys-34 of human albumin. In yet another variation, Pro-Phe-His-Sta-Ile-AMP, Iva-Phe-Noe-Sta-Ala-Sta, Iva the linkage is to a lysine of human albumin. His-Pro-Phe-His-Sta-Ala-Sta, Iva-His-Pro-Phe-His-Sta 0563. In one embodiment, the invention provides a com Leu-Phe-NH2, Ac-His-Pro-Phe-Val-Sta-Leu-Phe-NH2, plex of Formula I or Formula II, wherein m is 1, n is 1 or 2, Ac-His-Pro-Phe-His-ACHPA-Leu-Phe-NH2, Ac-Trp-Pro Phe-His-Sta-Ile-NH2, Ac-(HCO-Trp)-Pro-Phe-His-Sta-Ile and the protein is HSA or recombinant HSA. In another NH2, Pro-His-Pro-Phe-His-Sta-Ile-His-D-Lys, Pro-His-Pro variation of the above embodiment, n is 1, the protein is Phe-His-Sta-Ile-Phe-NH2, Z-Arg-Arg-Pro-Phe-His-Sta-Ile HSA or recombinant HSA, and wherein the complex is His-Lys(Boc)-OMe, Pro-His-Pro-Phe-His-Phe-Phe-Val further purified to a purity of at least 30%. In yet another Tyr-Lys, His-Pro-Phe-His-Leu-D-Leu-Val-Tyr-OH, Pro variation, m is 1., n is 2, and the protein is HSA or His-Pro-Phe-His-Leu(CH2NH)Val-Ile-His-Lys (H-142), recombinant HSA. Boc-Phe-His-Cha-(CH2NH)Val-NH-2(S)-Me (Bu), Pro 0564) In one variation, the complex is prepared by com His-Pro-Phe-His-Leu-Phe-Val-Tyr-OH, Boc-His-Pro-Phe bining a stoichiometric ratio of (Ih)m-L1 with Pr or a His-Leu(CH(OH)CH2)Val-Ile-His-OH (H-26 1), and stoichiometric ratio of Ih with L2-(Pr)o. In another variation, PEC-Phe-His-ACHPA-ILeNHC(CH2OH)2CH3, and Pr is the complex is prepared by combining a mixture of Pr to albumin. (Ih)m-L1 in a ratio of at least about 1.3:1. 0559). In a particular embodiment, the linker L1 or L2 0565. In another embodiment, the invention provide the comprises at least two functional groups covalently linking complex of Formula I or Formula II wherein L1 and L2 are Ih to Pr. In another embodiment, the linker L1 or L2 is absent, and wherein the complex is prepared by forming an hydrolytically stable in human serum for an extended period activated intermediate of Ih followed by the condensation of of time. In particular, the linker is sufficiently hydrolytically the activated Ih intermediate with Pr. In another variation, stable that, when administered to a subject, the active the activated intermediate of Ih is prepared from a mixed conjugate produces a Sustained decrease in blood pressure anhydride or N,N'-carbonyldiimidazole reagent. Optionally, over an extended period of time. In particular embodiments, in the above variations, the complex may be further purified the linker is Sufficiently stable that the conjugate can produce to a purity of at least about 30%. a Sustained decrease in blood pressure for 1 day, 2 days, 3 0566 In one embodiment of the invention, the renin days, 4 days, 5 days, 6 days, 7 days or more, or 14 days or inhibitor is a peptidomimetic with a mass of less than about more. In yet another embodiment, the linker L1 or L2 is 1OOODA stable in human serum for half lives of between 8 hours and 0567. In another embodiment, there is provided a com 30 days. position comprising the complex of Formula I or Formula II 0560. In another embodiment of the invention, the linker and a physiologically acceptable carrier. In another embodi L1 or L2 is a derivative of a compound selected from the ment, the composition above is formulated for parenteral group consisting of acyloxymethylketones, aziridines, dia administration. In another embodiment, the composition Zomethyl ketones, epoxides, iodo-, bromo- or chloroaceta above is selected from the group consisting of Solutions, dry mides, C-haloesters, C.-haloketones, Sulfoniums, chloroeth products for combining with a solvent prior to use, Suspen ylsulfides, O-alkylisoureas, alkyl halides, vinylsulfones, sions, emulsions, and liquid concentrates. acrylamides, acrylates, vinylpyridines, organometallic com 0568. In another embodiment, the invention provides a pounds, aryldisulfides, thiosulfonates, aldehydes, nitriles, method for inhibiting renin activity in vivo, said method C-diketones, C.-ketoamides, C.-ketoesters, diaminoketones, comprising: semicarbazones, and dihydrazides. 0569 administering to the bloodstream of a mammalian 0561. In one embodiment, the linker L1 or L2 is a host an isolated conjugate complex of Formula I or Formula derivative of a compound selected from the group consisting II, wherein the complex is formed by attaching a renin of azidobenzoyl hydrazide, N-4-(p-azidosalicylamino)bu inhibitor to a linker having at least one reactive functional tyl-3'-2'-pyridyldithio)propionamide), bis-sulfosuccinim group which reacts with the protein to form stable covalent idyl Suberate, dimethyl adipimidate, disuccinimidyl tartrate, bonds; and N-y-maleimidobutyryloxysuccinimide ester, N-hydroxy sul foSuccinimidyl-4-azidobenzoate, N-succinimidyl 4-azi 0570 wherein the isolated conjugate complex is admin dophenyl-1,3'-dithiopropionate, N-succinimidylA-io istered in an amount to maintain an effective therapeutic doacetylaminobenzoate, glutaraldehyde, succinimidyl effect in the bloodstream for an extended period of time as 4-N-maleimidomethylcyclohexane-1-carboxylate, N-hy compared to a non-conjugated renin inhibitor. droxysulfoSuccinimide, maleimide-benzoyl-Succinimide, 0571. In one variation, the invention provides a method emaleimido-butyryloxy Succinimide ester, maleimidopropi wherein the complex is the complex according to any of the US 2007/0207952 A1 Sep. 6, 2007 32 above complexes. In another variation of the above methods, 0585. In one variation of the above method, the protein is the protein is HSA or recombinant HSA. albumin. In another variation, the albumin is obtained from blood, purified and isolated from blood prior to treating with 0572 In one variation of the above methods, the linker comprises a reactive functional group is a compound the compound rh-L1 or Ih-L2. In yet another variation of the selected from the group consisting of acyloxymethylke method, the albumin is HSA or recombinant HSA. tones, aziridines, diazomethyl ketones, epoxides, iodo-, 0586. In one embodiment, there is provided a pharma bromo- or chloroacetamides, C-haloesters, oLhaloketones, ceutical composition comprising a therapeutically effective sulfoniums, chloroethylsulfides, O-alkylisoureas, alkyl amount of a complex as describe above, or a physiologically halides, vinylsulfones, acrylamides, acrylates, vinylpy acceptable salt thereof, and a pharmaceutically acceptable ridines, organometallic compounds, aryldisulfides, thiosul carrier, excipient, or diluent. In another variation, there is fonates, aldehydes, nitriles, a diketones, C.-ketoamides, provided a method of reducing the blood pressure of a C.-ketoesters, diaminoketones, semicarbazones, and dihy Subject comprising administering to the Subject a therapeu drazides. tically effective amount of the above composition. In yet another variation, the invention provides the above method, 0573. In one variation, the invention provides a method wherein the patient suffers from hypertension. In yet another for inhibiting renin activity in vivo, said method comprises: variation of the above method, the patient suffers from mild, 0574 administering into the bloodstream of a mamma moderate or severe hypertension. lian host the complex of Formula I or Formula II in an 0587. In another embodiment of the invention, the tran amount sufficient to provide an effective amount for renin sition state mimetic is a compound of the formula: inhibition; 0575 whereby said complex is maintained in the blood stream over an extended period of time as compared to the lifetime of unbound renin inhibitor. y NH 0576. In yet another embodiment, there is provided a method for inhibiting renin activity in a host, said method comprising: 0577 a) preparing a compound Ih-L1 or Ih-L2 wherein Ih 0588 wherein: is a renin inhibitor peptide with a mass of less than 60 kD 0589 R is selected from the group consisting of (C- and L1 or L2 is a linker covalently bound to Ih; lo)alkyl, (C-)cycloalkyl, carbonyl(C-o)alyl, Sulfo 0578 b) treating the compound rh-L1 or Ih-L2 with nyl(C)allyl, Sulfinyl(C)alkyl, (C-2)alkenyl, (C- isolated protein ex vivo for a time sufficient for the com 12)alkynyl, aryl, aryl (C-o)alkyl, heteroaryl, heteroaryl (C- pound Ih-L1 or Ih-L2 to covalently bond to the protein to lo)alkyl, each Substituted or unsubstituted; and form the protein complex of Formula I or Formula II, and 0590) R' is selected from the group consisting of (C- 0579) c) administering the treated protein complex to the lo)alkyl, (C-2)cycloalkyl, carbonyl(C-o)alkyl, (C- host lo)alkoxycarbonyl, (Co)alkylaminocarbonyl, Sulfonyl(C. 3)alkyl, Sulfinyl (C1-)alkyl, (C-12)alkenyl, (C-12)alkynyl, 0580. In one variation of the above method, the protein is aryl, aryl(C-o)alkyl, heteroaryl, heteroaryl(C-o)alkyl, albumin. In another variation, the albumin is HSA or recom alkylsulfonyl(C-o)alkyl, arylsulfonyl(C-o)alkyl, het binant HSA. In yet another variation of the above method, eroarylsulfonyl(C-o)alkyl, (C-o)alkylphosphonate and the albumin is obtained from blood, purified and isolated (C-o)alkyl phosphonyl, each substituted or unsubstituted. from blood prior to treating the albumin with the compound Ih-L1 or Ih-L2. In another variation, the albumin is purified 0591. In another embodiment, the transition state to a purity level of at least 10% on a dry matter basis. In yet mimetic is a compound of the formula: another variation, the albumin is purified to a purity level of more than 95%. R O 0581. In another embodiment, the invention provides a method for inhibiting renin activity in a host, said method comprising: Alls.O 0582 a) preparing a compound Ih-L1 or Ih-L2 wherein Ih is a renin inhibitor peptide with a mass of less than 60 kD and L1 or L2 is a linker covalently bound to Ih; 0592 wherein: 0583 b) treating the compound Ih-L1 or Ih-L2 with 0593 R is selected from the group consisting of (C- isolated one or more protein Prex vivo for a time sufficient lo)alkyl, (C-2)cycloalkyl, carbonyl(C-o)alkyl, Sulfo for the compound Ih-L1 or Ih-L2 to covalently bond to one nyl(C)alkyl, Sulfinyl(C)alkyl, (C-2)alkenyl, (C- or more of the isolated proteins to form one or more 12)alkynyl, aryl, aryl (C-o)alkyl, heteroaryl, heteroaryl (C- modified protein complex of Formula I or Formula II; and lo)alkyl, each Substituted or unsubstituted; and 0584) c) administering the modified protein or proteins to 0594 R" is selected from the group consisting of (C- the host. 4)alkyl, (C-2)cycloalkyl, heterocycloalkyl, bicycloalkyl, US 2007/0207952 A1 Sep. 6, 2007 carbonyl (C-o)alkyl, thiocarbonyl (Cs)alkyl, Sulfonyl 0602. The present invention relates to compounds and (C)alkyl, Sulfinyl(C)alkyl, amino, imino(C)alkyl, compositions that may be used as renin inhibitors with (C-o)alkoxy, aryloxy, heteroaryloxy, (C-12)alkenyl, (C2 extended lifetime as compared to a non-conjugated renin 12)alkynyl, aryl, aryl (C-o)alkyl, heteroaryl, heteroaryl (C- inhibitor. lo)allyl, (C-2)bicycloaryl, hetero(Cs)bicycloaryl, ami nosulfonyl, alkylsulfonyl, alkylsulfonyl(C-o)alkyl, 0603 The invention comprises using a biologically arylsulfonyl, arylsulfonyl(C-o)alkyl, heteroarylsulfonyl, active compourid covalently attached or linked to a linking heteroarylsulfonyl(C-o)alkyl, phosphonate, (C-o)alky group, the linking group comprising at least one chemically lphosphonyl, Sulfonyl group and Sulfinyl group, each Sub reactive moiety which is capable of forming covalent bonds stituted or unsubstituted. with functionalities present on a protein or a blood protein. 0595. In yet another embodiment, the transition state In one embodiment, the protein is albumin. By preparing the mimetic is a compound of the formula: isolated complex ex vivo before the administration of the complex into the blood of the host, particularly the blood stream of the host, the biologically active complex maintains R F F an effective therapeutic effect in the bloodstream for an R" extended period of time as compared to a non-conjugated NH biologically active agent. O O 0604 The extended life-time at a useful dosage will usually be at least 2 days, more preferably at least 5 days, even more preferably, at least 10 days, and most preferably 0596) wherein: at least 15 days. The protein that may be conjugated to 0597 R is selected from the group consisting of (C- include red blood cells, immunoglobulins, such as IgM and lo)alkyl, (C-2)cycloalkyl, carbonyl(C-o)alkyl, Sulfo IgG, serum albumin, transferrin, p90 and p38. In a preferred nyl(C)alkyl, sulfinyl(C)alkyl, (C-)alkenyl, (C- embodiment, the protein is albumin. In another embodiment, 12)alkynyl, aryl, aryl(C-o)alkyl, heteroaryl, heteroaryl(C- the protein is recombinant albumin. lo)alkyl, each Substituted or unsubstituted; and 0605) A large number of biologically active agents or 0598 R" is selected from the group consisting of (C- 4)alkyl, (C)cycloalkyl, heterocycloalkyl, bicycloalkyl, therapeutic agents may be used as the conjugate with the carbonyl (C-o)alkyl, thiocarbonyl (Cs)alkyl, Sulfonyl protein. In a preferred embodiment, the biologically active (C)alkyl, Sulfinyl(C)alkyl, amino, imino(C)alkyl, or therapeutic agent Ih, is a renin inhibitor. The renin (C-o)alkoxy, aryloxy, heteroaryloxy, (C-12)alkenyl, (C2 inhibitor, which may be depicted as Ih, comprises an active 12)alkynyl, aryl, aryl(C-o)alkyl, heteroaryl, heteroaryl(C- functional group that may be reacted with a liniding group, lo)alkyl, (C-2)bicycloaryl, hetero(Cs)bicycloaryl, ami depicted as L1 or L2, to form an inhibitor-linking group nosulfonyl, alkylsulfonyl, alkylsulfonyl(C-o)alkcyl. compound, Ih-L1 or Ih-L2, which may react with one or arylsulfonyl, arylsulfonyl(C-o)alkyl, heteroarylsulfonyl, more protein Pr. In one embodiment, the protein has a heteroarylsulfonyl(C-o)alkyl, phosphonate, (C-o)alky number of different functional groups which may react with lphosphonyl, Sulfonyl group and Sulfinyl group, each Sub the inhibitor-inking group compound to form a complex of stituted or unsubstituted. Formula I: 0599. In another embodiment, the transition state mimetic is a compound of the formula: 0606. In another embodiment, the protein has a number of different functional groups which may react with the inhibitor-linking group compound to form a complex of R Formula II: R NH l 11 Ih-IL2-(Pr). II 0607 wherein Ih is a biologically active agent, L1 and L2 H are linking groups that link Ih to Pr, Pr is a blood component, m and o are integers from 1-5, and n and pare integers from 0600 R is selected from the group consisting of (C- 1-100. lo)alkyl, (C-2)cycloalkyl, carbonyl(C-o)alkyl, Sulfo 0608. A number of functional groups are available on the nyl(C)alkyl, Sulfinyl(C)alkyl, (C-2)alkenyl, (C- protein Such as albumin. Non-limiting functional groups 12)alkynyl, aryl, aryl (C-o)alkyl, heteroaryl, heteroaryl (C- include amino groups, carboxyl groups and thio groups. lo)alkyl, each Substituted or unsubstituted; and While any of these functional groups in the protein may be 0601 R" is selected from the group consisting of (C- employed to form a covalent bond with the linker group, 4)alkyl, (C-2)cycloalkyl, heterocycloalkyl, bicycloalkyl, depending on the nature of the functional group(s) on the carbonyl (C-o)alkyl, thiocarbonyl (Cl-)alkyl, Sulfonyl linking group and the linker, certain functional groups will (C)alkyl, Sulfinyl(C)alkyl, amino, imino(C)alkyl, be preferred over the others. For example, the reaction of (Co.)alkoxy, aryloxy, heteroaryloxy, (C-2)alkenyl, (C- amine groups may form conjugates having an amide group. 12)alkynyl, aryl, aryl(C-o)alkyl, heteroaryl, heteroaryl(C- carboxyl groups may form conjugates having an amide or lo)alkyl, (C-2)bicycloaryl, and hetero(Cs)bicycloaryl, ester groups, and thio groups may form thioethers or each substituted or unsubstituted. tioesters. US 2007/0207952 A1 Sep. 6, 2007 34

The Biologically Active Agents Ih: ably, the agent is a peptide or peptidomimetic renin inhibitor. A large variety of renin inhibitors may be used in the present 0609. The biologically active agent Ih may be any com- invention. Non-exclusive examples of peptide or peptido pound. Such as an enzyme inhibitor, that will elicit a desired mimetic renin inhibitors are shown in the Table. Preferably, biological response and induce minimal immune response the renin inhibitors are peptidomimetics with a mass of less when administered in a mammalian host. Preferably, the than about 60 kDA, more preferably less than about 10 kDA, biologically active agent is a renin inhibitor. More prefer- and most preferably less than about 1000 DA.

TABLE Peptide or Peptidomimetic Renin Inhibitors

IC50 (nM) Human Inhibitor plasma renin References

Iva-Wal-Wal-Sta-Ala-Sta 14 OOO JMC 1152 (86) (pepstatin)

Boc-Phe-His-Sta---Ile-AMP 6 JMC 1837 (87)

Boc-Phe-His-Sta---Ala-Sta-OMe 27 JMC 1152 (86)

Boc-Phe-His-Sta---Leu-NHCHPh 26 JMC 1853 (87)

Boc-Phe-His-ACHPA---Leu-AMB 1 JMC 1918 (88)

Boc-Phe-His-Sta---Leu-AMB 9 JMC 1918 (88)

Boc-Pro-Phe-His-Sta---Ile-AMP 4.1 JMC 671 (88)

Iva-Phe-Nile-Sta-Ala-Sta 28 JMC 1152 (86), JMC 2287 (87)

Iva-His-Pro-Phe-His-Sta- --Ala-Sta 1.9 (Ki)

Iva-His-Pro-Phe-His-Sta---Leu-Phe-NH2 3 JMC 1853 (87), JMC 2080 (90), Nature 81 (83)

Ac-His-Pro-Phe-Val-Sta---Leu-Phe-NH2. 32 JMC 1679 (88)

Ac-His-Pro-Phe-His-ACHPA---Leu-Phe-NH2. O 5 JMC 1679 (88)

Ac-Trp-Pro-Phe-His-Sta---Ile-NH2. 16 JMC 18 (88)

Ac- (HCO-Trp)-Pro-Phe-His-Sta---Ile-NH2. 0.1 JMC 18 (88)

Pro-His-Pro-Phe-His-Sta---Ile-His-D-Lys 26 JMC 1377 (88)

Pro-His-Pro-Phe-His-Sta---Ile-Phe-NH2. 3 JMC 1287 (87)

Z-Arg-Arg-Pro-Phe-His-Sta--- 1 JMC 18 (88), Ille-His-Lys (Boc)-OMe Hypertension 797 (85)

Pro-His-Pro-Phe-His-Phe-Phe- 52 OO JMC 1287 (87), PNAS. Val-Tyr-Lys (RIP) 5476 (80), Tetrahedron 661 (88) His-Pro-Phe-His-Leu-D-Leu-Val-Tyr-OH Biochemistry 3877 (73)

Pro-His-Pro-Phe-His- 10 JMC 671 (88), Biochem Leu (CH2NH) Wal-Ile-His-Lys (H-142) Soc Trans1029 (85); Szelke review

Boc-Phe-His-Cha- (CH-NH) Val- 86 BBRC 982 (86) NH-2 (S)-Me (Bu.) Pro-His-Pro-Phe-His-Leu-Phe---Val-Tyr-OH Biochemistry 3892 (75) US 2007/0207952 A1 Sep. 6, 2007 35

TABLE-continued Peptide or Peptidomimetic Renin Inhibitors IC50 (nM) Human Inhibitor plasma renin References Boc-His-Pro-Phe-His- Of Szelke review Leu (CH (OH) CH2) Wal-Ile-His-OH (H-261) PEC-Phe-His-ACHPA- <0.01 J. Hypertens. S23 (87)

AMP 2-aminomethylpyridine AMB 3-aminomethylbenzylamine

The Linkers L1 and L2: formed in these complexes remain stable unless the biologi cally active subunit is intended to be released at the active 0610 A variety of different linkers or linking groups L1 site. and L2 may be used to link the blood component with the renin inhibitor. The linking groups may be divalent or 0.614 The linkers may comprise of compounds having polyvalent. For example, in the complex of Formula I, L1 bifunctional or polyfunctional groups that are available for may be n-valent where it is attached to Pr, and m-valent linking a protein Such as albuminto multiple renin inhibitors where it attaches to Ih where m and n are integers as defined or for linking multiple albumins to a single renin inhibitor. above. Similarly, in the complex of Formula II, L2 may be 0615. In a particular preferred embodiment, the linker o-valent where it is attached to Prand p-valent where it is comprises polyfunctional groups that link a HSA to one or attached to Ih, where o and p are as defined above. Non more renin inhibitors. In one embodiment, lining com exclusive examples of functional groups that may be present pounds as used herein include any compounds that can link in a linking group include compounds that have a hydroxyl the renin inhibitor to the protein in a single step. In another groups, such as N-hydroxysucciimmde, N-hydroxysulfos embodiment, the linking compounds are linked to the renin luccinimide, and other compounds such as maleimide-ben inhibitor first to forma inhibitor-linker intermediate that can Zoyl-succinimide, emaleimido-butyryloxy Succinimide be further reacted with the protein. In another embodiment, ester, maleimidopropionic acid, N-hydroxysuccinimide, iso the linking compounds are reacted with the protein first to cyanate, thioester, thionocarboxylic acid ester, imino ester, form a protein-linker intermediate that can be further reacted carbodiimide, anhydride, or ester. with the renin inhibitor. In each of the above permutations, optionally, the linked compounds may be further purified 0611 In addition, certain linng groups having functional and/or isolated before submitting to further reactions to form groups such as carboxylate, acid halide, azido, diazo, car the complex of Formula I or Formula II. bodiimide, anhydride, hydrazine, aldehydes, thiols, or amino 0616 Non-exclusive examples of such polyfunctional group may be used to form amides, esters, imines, thioet compounds include compounds having at least one func hers, disulfides, substituted amines, or the like. Other spe tional group selected from the group consisting of azido cific examples of functional groups that may be employed benzoyl hydrazide, N-4-(p-azidosalicylamino)butyl-3'-2'- include acyloxymethylketones, aziridines, diazomethyl pyridyldithio)propionamide), bis-sulfosuccinimidyl ketones, epoxides, iodo-, bromo- or chloroacetamides, olha Suberate, dimethyl adipimidate, disuccinimidyl tartrate, loesters, oe haloketones, Sulfoniums, chloroethylsulfides, N-y-maleimidobutyryloxysuccinimide ester, N-hydroxy sul O-alkylisoureas, alkyl halides, vinylsulfones, acrylamides, foSuccinimidyl-4-azidobenzoate, N-succinimidylA-azi vinylpyridines, organometallic compounds, aryldisulfides, dophenyl-1,3'-dithiopropionate, N-succinimidyl 4-io thiosulfonates, aldehydes, nitrites, C.-diketones, C.-ketoam doacetylaminobenzoate, glutaraldehyde, and Succinimidyl ides, C.-ketoesters, diaminoketones, semicarbazones, and dihydrazides. 4-N-maleimidomethylcyclohexane-1-carboxylate. 0.617. Any linker or linking group that is convenient for 0612 The nature and type of compounds that may be use and Subject to standard chemical transformations, or selected as the linker depends on the type of reactions, the linkers that form compounds that are physiologically accept relative reactivities, selectivities, reversibility and stability able at the desired dosages, and are stable in the bloodstream characteristics that are desired among the renin inhibitors, for the desired period of time, may be employed. The linking the linker and the functional groups on albuminor the blood group may be aliphatic, alicyclic, aromatic, heterocyclic, or component. For example, certain reactions that form the combinations thereof. Examples of groups that may be conjugate complex arise from an alkylation reaction, a employed as a linking group include alkylenes, arylenes, Michael type reaction, an addition-elimination reaction, an aralkylenes, cycloalkylenes, polyethers and the like. In a addition to Sulfur, carbonyl, or cyano groups, or the forma particular embodiment, polyfunctional polyethylene glycol tion of a metal bond. (PEG) and their derivatives may also be employed as 0613 Typically, the covalent bond that is formed from linkers. these reactions are stable during the active lifetime of the 0618. The linking groups may have at least one atom in renin inhibitor. In one embodiment, the covalent bond that is the linking chain, more preferably between 1 and 200 atoms US 2007/0207952 A1 Sep. 6, 2007 36 in the chain, most preferably between 2 and 50 atoms in the Porath et al.; FEBS Letters, vol. 185, p.306, 1985; K. L. chain. The atoms in the chain can be linear or the chain can Knudsen et al. Analytical Biochemistry, Vol 201, p. 170, be part of one or more rings, each Substituted or unsubsti 1992), and various resin matrices (WO 96/00735; WO tuted, and the chain may include carbons or heteroatoms 96/09 116). Certain blood components of established purity selected from the group consisting of ON, P and S. The rings are commercially available. may be aliphatic, heterocylic, aromatic or heteroaromatic or Preparation of Linked Compounds Ih-L1 and Ih-L2: mixtures thereof, each substituted or unsubstituted. In some embodiments, amino acids or peptides or amino acids 0626. In one embodiment, the linked compounds Ih-L1 employed with mixtures of the above may be used as a or Ih-L2 of the present invention may be prepared and used linking group. in the conjugation with albumin without further purification and/or isolation. The purity of the linked compounds will 0619. In one embodiment, L1 is absent and Ih is attached depend on the nature of the linker, the nature of Ih, and the directly to Pr. In another embodiment, L2 is absent and Ih is type of reaction and reaction conditions employed to attach attached directly to Pr. Ih to the linker. In another particular embodiment, the 0620. In another embodiment for the complex of Formula unpurified linked compounds are prepared and obtained with I, L1 is a linking group that is capable of linking more than a purity of at least 90%, preferably at least 95%, more one Ih to one Pr, for example, where m is 2 or is more. In preferably at least 97%, and most preferably at least 98%. one embodiment, m is 1, 2 or 3 and n is 1-30. In one 0627. In a particular embodiment, the present invention preferred embodiment for the complex of Formula I, Pr is relates to methods for the preparation of the isolated linked albumin and n is 1. In another particular embodiment, Pr is compounds, that is, Ih-L1 or Ih-L2. In a preferred embodi albumin, Ih is a renin inhibitor, and n is 2-25. ment, the isolated linked compounds Ih-L1 and Ihl 2 are 0621. In another embodiment for the complex of Formula renin inlubitors that are attached to a linker. In one embodi II, L2 is a linking group that is capable of linking more than ment, the isolated linked compounds may be purified before one Pr to one Ih, for example, in the case where o is 2 or conjugating with Pr. In another particular embodiment, the more. In one embodiment, Pr is albumin, Ih is a renin linked compounds Ih-L1 or Ih-L2 are isolated and purified inhibitor, o is 1, 2 or 3 and p is 1-5. to a purity of at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% or 0622. In another embodiment, the linking group may be O. absent in cases where the inhibitor, such as a renin inlnbitor, can be reacted directly with a protein, optionally using a 0628. The linked compounds may be prepared using standard methods known in the art of chemical synthesis. catalyst or coupling agent. Such that the complex that is The compounds may be purified using standard methods formed comprises only of the renin inhibitor that is directly known in the art, Such as by column chromatography or attached to the protein. An example of Such a direct coupling HPLC to provide purified products suitable for in vivo reaction is a mixed anhydride activated coupling reaction of applications. The linked compounds may be frther conju a carboxylic acid followed by the coupling reaction of the gated with a protein, such as albumin to form the complex intermediate mixed anhydride. of Formulae I and II. The Protein Component Pr: Preparation of Linked Compounds Pr-L1 and Pr-L2: 0623 Various blood components may be used to prepare 0629 For certain applications of the present invention, the isolated complexes of the present invention. Naturally the compounds as represented by Pr may be albumin, may occurring blood components include blood proteins, which be used as obtained from commercial sources without fur include red blood cells, and immunoglobulins, such as IgM ther purification or isolation, to prepare the linked com and IgG, serum albumin, transferrin, p90 and p38. In a pounds Pr-L1 and Pr-L2. In a particular embodiment, Pr is preferred embodiment, the blood component or blood pro HSA. In another embodiment, the albumin may be further tein is albumin. More preferably, the albumin is a protein purified using various methods known in the art as disclosed human serum albumin (HSA). herein. 0624 The albumin used in the present invention may also 0630. In one embodiment, the linked compounds Pr-L1 be recombinant albumin. For example, the recombinant and Pr-L2 may be prepared by treating a linker L1 or L2. human albumin may be produced by transforming a micro which may be derivatized or activated, with Pr. in a solution organism with a nucleotide coding sequence encoding the and monitoring the reaction mixture until the reaction is amino acid sequence of human serum albumin. substantially complete. In a particular preferred embodi 0625 Generally, there exists a very broad range of dif ment, Pr is a protein. In another preferred embodiment, the ferent methods available for the isolation of compounds protein is HSA or recombinant HSA. from blood or blood plasma that provide a very broad range 0631. In another preferred embodiment, the linked com of final purities, and yields of the product. Albumin is the pounds Pr-L1 or Pr-L2 obtained are substantially pure; that main protein present in blood plasma, and may be extracted is, the linked compounds are obtained with a purity of at from blood, for examples as disclosed in JP 03/258 728, EP least 10%, preferably at least 30%, and more preferably at 428 758, EP 452 753, and U.S. Pat. No. 6,638,740 and least 50%. Where the Pr is HSA or recombinant HSA, references cited therein. Further examples of non-exclusive components that may be present with the linked compounds methods for the isolation of various compounds may be may comprise of unreacted HSA and various biological based on selective reversible precipitation, ion exchange components that are present in the HSA starting material. chromatography, protein affinity chromatography, hydro Preferably, the HSA or recombinant HSA is at least 10% phobic chromatography, thiophilic chromatography (J. pure on a dry matter basis. US 2007/0207952 A1 Sep. 6, 2007 37

0632 An excess of HSA or HSA related biologically prepared by combining an excess of Pr relative to (Ih)m-L1. materials present with the linked compounds will not sig Preferably, the ratio of Pr to (Ih)m-L1 is about 50 to 100. In nificantly interfere with the Subsequent conjugation step another particular embodiment, the ratio is from about 10 to with Ih. In addition, the related biological materials and the 30. In yet another particular embodiment, the ratio is from conjugated complexes will also be pharmacologically safe about 2 to 5. for use in vivo. 0639. In one embodiment, Pr is added to (Ih)m-L1 in a 0633 However, in certain embodiments, the purity of the ratio of at least about 1.1:1, more preferably at least about linked compounds Pr-L1 or Pr-L2 may be at least 10% on a 1.2:1, and most preferably at least about 1.4:1. In the case dry matter basis to enable the selective reaction of the where Pr is albumin, the preferred ratios are based on the compounds with Ih without a significant amount of inter assumption that there is 0.7 free thiol per albumin. Prefer ferences or without the formation of undesirable by-products ably, the resulting complex is formed as a 1:1 complex, since obtained from the conjugate reaction with other undesired a Pr component such as albumin has only about 70% free blood components. However, the desired purity of Pr, such thiol functionality for conjugation. An excess of Pr. Such as as HSA or recombinant HSA, for example, will depend on HSA or recombinant HSA is pharmacologically safe and the nature of the functional groups on Ih as well as the may not require fither purification. Where there is an excess functional groups employed on the linker. Typically, higher of Pr in the product mixture, optionally, the conjugated purities of HSA or recombinant HSA is required if the complex may be purified to a purity of at least 10%. In a functional groups on the linker are more reactive and may particular embodiment, the conjugated complex may be form undesired by-products than functional groups on the purified to at least about 20% or at least about 30%. linker that are less reactive. 0.640. In another embodiment, the complex of Formula I 0634. The albumin maybe obtained from plasma or blood may be prepared by combining an excess of (Ih)m-L1 albumin from a host, purified to a desired level of purity, and relative to Pr. Preferably, the ratio of (Ih)m-L1 to Pr is about linked with the linker. Purification of the albumin from 50 to 100. In another particular embodiment, the ratio is blood or blood plasma may be performed using well estab from about 10 to 30. In yet another particular embodiment, lished standard methods known in the art for the purification the ratio is from about 2 to 5. Where there is an excess of of albumin. Using purified blood albumin, the isolated (Ih)m-L1 in the product mixture, optionally, the conjugated complexes of the present will comprise of a relatively complex may be purified to a purity of at least 10%. In a homogeneous population of functionalized proteins. particular embodiment, the conjugated complex maybe puri Preparation of the Complexes of Formula I or Formula II: fied to at least about 20% or at least about 30%. 0641. In another embodiment, the complexes of Formula 0635. In one embodiment, the complexes of Formula I or I or Formula II maybe prepared from a stoichiometric ratio Formula II may be prepared by the conjugation of lh-L1 or of (Ih)m-L1 with Pr or a stoichiometric ratio of Ih with Ih-L2 with Pr, the conjugation of Pr-L1 or Pr-L2 with Ih, or L2-(Pr)o, that is, in a 1:1 ratio. Optionally, the resulting the conjugation of Ih with Pr to form a complex wherein the product from these preparations may be further purified to a linker is absent. purity of at least 10%. In a particular embodiment, the 0636. In one embodiment, a solution of Ih-L1 or Ih-L2 is conjugated complex may be purified to at least about 20% or combined with Prunder conditions such that the conjugation to a purity of at least about 30%. In yet another particular reaction is deemed to be complete. In a particular embodi embodiment, the 1:1 conjugated complex may be further ment, the linked compound is a renin inhibitor that is purified to a purity of greater than about 90%. attached to a linker, and the linked compound is added to an 0642. In another embodiment, the conjugated cysteine aqueous solution of HSA. The resulting solution is incubated present in albumin is reduced to the free cysteine prior to the until the reaction is Substantially complete. reaction. 0637. In one embodiment, the Ih-L1 or Ih-L2 is com bined with an excess of HSA to ensure that the conjugation 0.643. Optionally, the complex formed from the conjugate reaction proceeds selectively to a single site on the HSA. For reaction may be further purified prior to administration. example, the formation of Ih-L1 on a single site on HSA may 0644. In one embodiment, the complexes of Formula I or permit ease of identification of a single complex of Formula Formula II obtained from the conjugate reaction may be I, for example, where n is 1. In one particular embodiment, administered without further processing or purification since the conjugate reaction of Ih-L1 or Ih-L2 with HSA occurs on an excess of HSA or HSA related biologically materials a single cysteine of HSA. Without being bound by any present with the complexes are pharmacologically safe for particular theory, for some reactions, it is believed that the use in vivo. conjugate reaction may also occur initially with a cysteine —SH group to form a kinetic product that is then rearranged 0.645. In each of the above embodiments, Ih is a peptide to another amino acid functional group. Such as a lysine, to or peptidomimetic renin inhibitor and Pr is HSA or recom form the thermodynamic product. binant HSA. 0638. In another embodiment, the conjugate reaction may 0.646. In one embodiment, the isolated complex compris form the complex of Formula I, for example, wherein more ing a protected or unprotected renin inhibitor with a linker than one Ih is linked to a single HSA to form the complex and albumin may be optionally further purified and then of Formula I; that is, wherein n is greater than 1. Optionally, returned to the host. m may be greater than 1 if the linker L1 is a polyfunctional 0647. The complexes formed from the methods of the linker that is capable of attaching more than one Ih group. present invention may be tested in animal or human hosts In one embodiment, the complex of Formula I may be until the physiology, pharmacokinetics, and safety profiles US 2007/0207952 A1 Sep. 6, 2007

are well established over an extended period of time. Typi vivo from purified components (specifically HSA, linker and cally, the measured half-life of the complexes is about 5 to a renin inhibitor) produces a remarkably efficient tissue vivo 7 days, more typically at least about 7 to 10 days, and distribution of the conjugate compared to conjugates that are preferably 15 to 20 days or more. In general, the duration is prepared by in vivo preparation of the conjugate by injection species dependent. For example, with human albumin, the of an activated compound that binds in situ to endogenous half life is about 17-19 days. Depending on the nature of the albumin in the blood stream of the subject. Moreover, the renin inhibitor, the linking group and the purity of the present inventors have found that substantially all of the albumin, the effective therapeutic concentration of the com conjugate remains in circulation for hours or even days plexes may be at least 1 month or more. following administration, compared to the dramatic losses of 0648 Half lives may be determined by serial measure compound that are observed when the conjugate is prepared ments of whole blood, plasma or serum levels of the in vivo. This efficiency reduces the number of times that the complexes of Formula I or Formula A, the Ih-L compounds, patient must be subjected to injection of active Substance, the L-Pr compounds, or the Ih compounds following label and also reduces the amount of renin inhibitor that must be ing of the complex or compounds with an isotope (e.g., 131I. given in a single administration. 125I, Tc, Cr, 3H, etc . . . ) or fluorochrome and injection of 0653. In the context of the present invention, a therapeu a known quantity of labeled complex or compound intra tically effective amount of a composition is understood to vascularly. Included are red blood cells (half life ca. 60 mean an amount that, when administered to a Subject, days), platelets (half life ca. 4-7 days), endothelial cells produces a desired physiological effect to a degree that is lining the blood vasculature, and long lived blood serum effective for treatment of a disease, condition, or syndrome proteins, such as albumin, Steroid binding proteins, ferritin, in the patient, or that is effective in alleviating the symptoms C.2-macroglobulin, transferrin, thyroxin binding protein, disease, condition, or syndrome. In particular, a therapeuti immunoglobulins, especially IgG, etc. In addition to pre cally effective amount of an antihypertensive complex or ferred half lives, the subject components are preferably in composition is understood to mean an amount that, upon cell count or concentration sufficient to allow binding of administration to a hypertensive Subject, produces a desired therapeutically useful amounts of the compound of the reduction in Systolic and/or diastolic pressure. present invention. For cellular long lived blood is compo nents, cell counts of at least 2,000/ul and serum protein Administration of the Isolated Complexes of Formula I and concentrations of at least 1 g/ml, usually at least about 0.01 Formula II: mg/ml, more usually at least about 1 mg/ml, are preferred. 0654. In one embodiment, the administration of the iso 0649. However, where the nature of the complex is lated complex of the present invention may be accomplished designed Such that the biologically active agent Ih, Such as using a bolus, but may be introduced slowly over time by a renin inhibitor, is to be cleaved from the complex and transfusion using metered flow, or the like. released into the host, the desired half life for the effective 0655 The complex of the present invention may be therapeutic concentration of the complex and/or the biologi administered in a physiologically acceptable medium, e.g. cally active agent may vary from the measured half-life deionized water, phosphate buffered saline, Saline, mannitol, above. The rate of release of the biologically active agent aqueous glucose, alcohol, vegetable oil, or the like. A single depends in part, on the Valency or the functionality on the injection may be employed although more than one injection biological agent which is to be released, the nature of the may be used, if desired. The complex may be administered linking group, the purity and type of the protein, the com by any convenient means, including Syringe, trocar, catheter, position for administration, the manner of administration, or the like. The particular manner of administration, will and the like. Ihus, various linking groups and biological vary depending upon the amount to be administered, agents may be employed, where the environment of the whether a single bolus or continuous administration, or the blood, components of the blood, particularly enzymes, activ like. The administration may be intravascularly, where the ity in the liver, or other agent may result in the cleavage of site of introduction is not critical to this invention, preferably the linking group with release of the biological agent in the at a site where there is rapid blood flow, e.g. intravenously, host at a desired rate. peripheral or central vein. Other routes may find use where 0650. The isolated complexes of the present invention the administration is coupled with slow release techniques or provides biological active compounds that have improved a protective matrix. pharmacokinetics, solubility, bioavailability, distribution, 0656 Surprisingly, it is noted that the administration of and/or immunogenicity characteristics as compared to the the isolated complexes prepared by the methods of the non-conjugated compounds. present invention, for example, from isolated blood protein, 0651 Surprisingly, the complexes of Formula I and For Such as albumin, results in renin inhibitor conjugate com mula II, when prepared and used according to the methods plexes that maintain an effective therapeutic effect in the of the present invention, provides an effective therapeutic bloodstream for an extended period of time as compared to concentration for a significantly longer time than the Ih a non-conjugated renin inhibitor or as compared to com component by itself. In addition, the complexes of the plexes that are not prepared from isolated blood protein such present invention provide improved solubility, distribution, as albumin. pharmacokinetics, and result in decrease immunogenicity 0657. In one embodiment, the present invention provides when compared to the administration of the Ib component the compounds in the form of a pharmaceutically acceptable by itself. salt. 0652 The present inventors surprisingly have found that 0.658. In another embodiment, the present invention pro administration to a subject of a conjugate that is prepared ex vides the compounds present in a mixture of Stereoisomers. US 2007/0207952 A1 Sep. 6, 2007 39

In yet another embodiment, the present invention provides pound with respect to its therapeutic activity in the body. An the compounds as a single stereoisomer. example of a pharmacokinetic property that may be favor ably affected is the manner in which the compound is 0659. In yet another embodiment, the present invention transported across cell membranes, which in turn may provides pharmaceutical compositions comprising the com directly and positively affect the absorption, distribution, pound as an active ingredient. In yet another particular biotransformation and excretion of the compound. While the variation, the present invention provides pharmaceutical route of administration of the pharmaceutical composition is composition wherein the composition is a tablet or a solid important, and various anatomical, physiological and patho for administration as a depot. In another particular variation, logical factors can critically affect bioavailability, the solu the present invention provides the pharmaceutical compo bility of the compound is usually dependent upon the sition wherein the composition is a liquid formulation character of the particular salt form thereof, which is uti adapted for IV or subcutaneous administration. In yet lized. One of skill in the art will appreciate that an aqueous another particular variation, the present invention provides solution of the compound will provide the most rapid pharmaceutical composition wherein the composition is a absorption of the compound into the body of a subject being liquid formulation adapted for parenteral administration. treated, while lipid solutions and Suspensions, as well as 0660. It is noted in regard to all of the embodiments, and Solid dosage forms, will result in less rapid absorption of the any further embodiments, variations, or individual com compound. pounds described or claimed herein that all such embodi Indications for Use of Renin Inhibitors ments, variations, and/or individual compounds are intended 0.665. The complexes of Formula I and Formula II of the to encompass all pharmaceutically acceptable salt forms present invention may also be used as renin inhibitors. Renin whether in the form of a single stereoisomer or mixture of is an endopeptidase which plays an important role in the Stereoisomers unless it is specifically specified otherwise. control of blood pressure. The renin angiotension system is Similarly, when one or more potentially chiral centers are a multiregulated proteolytic cascade in which renin cleaves present in any of the embodiments, variations, and/or indi the protein Substrate angiotensinogen to give angiotensin I. vidual compounds specified or claimed herein, both possible Angiotensin converting enzyme (ACE) catalyses the chiral centers are intended to be encompassed unless it is removal of the terminal dipeptide from the decapeptide specifically specified otherwise. angiotensin I to form angiotensin II which exhibits potent 0661 Prodrug derivatives of compounds according to the pressor activity. Renin is an aspartyl protease with high present invention can be prepared by modifying Substituents substrate specificity and is the first proteolytic step in the of compounds of the present invention that are then con renin-angiotensin System which is involved in the control of verted in vivo to a different substituent. It is noted that in blood pressure. Renin inhibitors have been shown to lower many instances, the prodrugs themselves also fall within the blood pressure in primates, J. Hypertension, 1,399 (1983), Scope of the range of compounds according to the present J. Hypertension 1 (suppl 2), 189 (1983) and in man, Lancet invention. For example, prodrugs can be prepared by react II, 1486 (1983), Trans. Assoc. Am. Physicians, 96, 365 ing a compound with a carbamylating agent (e.g., 1,1- (1983), J. Hypertension, 3, 653 (1985 and thus are poten acyloxyalkylcarbonochloridate, para-nitrophenyl carbonate, tially useful in the control of hypertension. or the like) or an acylating agent. Further examples of Injectables methods of making prodrugs are described in Saulnier et 0.666 The present invention is also directed to composi al.(1994), Bioorganic and Medicinal Chemistry Letters, Vol. tions designed to administer the renin inhibitors of the 4, p. 1985. present invention by parenteral administration, generally 0662 Protected derivatives of compounds of the present characterized by injection, either Subcutaneously, intramus invention can also be made. Examples of techniques appli cularly or intravenously. Injectables may be prepared in any cable to the creation of protecting groups and their removal conventional form, for example as liquid Solutions or Sus can be found in T. W. Greene, Protecting Groups in Organic pensions, Solid forms suitable for solution or Suspension in Synthesis, 3rd edition, John Wiley & Sons, Inc. 1999. liquid prior to injection, or as emulsions. 0667 Examples of excipients that maybe used in con 0663 Compounds of the present invention may also be junction with injectables according to the present invention conveniently prepared, or formed during the process of the include, but are not limited to water, Saline, dextrose, invention, as Solvates (e.g. hydrates). Hydrates of com glycerol, ethanol, or DMSO. The injectable compositions pounds of the present invention may be conveniently pre may also is optionally comprise minor amounts of non-toxic pared by recrystallization from an aqueous/organic solvent auxiliary Substances such as wetting or emulsifying agents, mixture, using organic Solvents such as dioxane, tetrahydro pH buffering agents, stabilizers, solubility enhancers, and furan or methanol. other Such agents, such as for example, Sodium acetate, 0664) A “pharmaceutically acceptable salt”, as used Sorbitan monolaurate, triethanolamine oleate and cyclodex herein, is intended to encompass any compound according trins. Implantation of a slow-release or Sustained-release to the present invention that is utilized in the form of a salt system, Such that a constant level of dosage is maintained thereof, especially where the salt confers on the compound (see, e.g., U.S. Pat. No. 3,710.795) is also contemplated improved pharmacokinetic properties as compared to the herein. The percentage of active compound contained in free form of compound or a different salt form of the Such parenteral compositions is highly dependent on the compound. The pharmaceutically acceptable salt form may specific nature thereof, as well as the activity of the com also initially confer desirable pharmacokinetic properties on pound and the needs of the Subject. the compound that it did not previously possess, and may 0668 Parenteral administration of the formulations even positively affect the pharmacodynarnics of the com includes intravenous, Subcutaneous and intramuscular US 2007/0207952 A1 Sep. 6, 2007 40 administrations. Preparations for parenteral administration 0677 Unit-dose parenteral preparations may be packaged include sterile solutions ready for injection, sterile dry in an ampule, a vial or a syringe with a needle. All prepa soluble products, such as the lyophilized powders described rations for parenteral administration should be sterile, as is herein, ready to be combined with a solvent just prior to use, known and practiced in the art. including hypodermic tablets, sterile Suspensions ready for injection, Sterile dry insoluble products ready to be com 0678 Injectables may be designed for local and systemic bined with a vehicle just prior to use and sterile emulsions. administration. Typically a therapeutically effective dosage The Solutions may be either aqueous or nonaqueous. is formulated to contain a concentration of at least about 0.1% w/w up to about 90% w/w or more, preferably more 0669 When administered intravenously, examples of than 1% w/w of the renin inhibitor to the treated tissue(s). Suitable carriers include, but are not limited to physiological The renin inhibitor complexes may be administered at once, saline or phosphate buffered saline (PBS), and solutions or may be divided into a number of smaller doses to be containing thickening and solubilizing agents, such as glu administered at intervals of time. It is understood that the cose, polyethylene glycol, and polypropylene glycol and precise dosage and duration of treatment will be a function mixtures thereof. of the location of where the composition is parenterally administered, the carrier and other variables that may be 0670) Examples of pharmaceutically acceptable carriers determined empirically using known testing protocols or by that may optionally be used in parenteral preparations extrapolation from in vivo or in vitro test data. It is to be include, but are not limited to aqueous vehicles, nonaqueous noted that concentrations and dosage values may also vary vehicles, antimicrobial agents, isotonic agents, buffers, anti with the age of the individual treated. It is to be further oxidants, local anesthetics, Suspending and dispersing understood that for any particular Subject, specific dosage agents, emulsifying agents, sequestering or chelating agents regimens may need to be adjusted over time according to the and other pharmaceutically acceptable Substances. individual need and the professional judgment of the person 0671 Examples of aqueous vehicles that may optionally administering or Supervising the administration of the for be used include Sodium Chloride Injection, Ringers Injec mulations. Hence, the concentration ranges set forth herein tion, Isotonic Dextrose Injection, Sterile Water Injection, are intended to be exemplary and are not intended to limit Dextrose and Lactated Ringers Injection. the scope or practice of the claimed formulations. 0672 Examples of nonaqueous parenteral vehicles that 0679 The renin inhibitor complexes may optionally be may optionally be used include fixed oils of vegetable suspended in micronized or other suitable form or may be origin, cottonseed oil, corn oil, sesame oil and peanut is oil. derivatized to produce a more soluble active product or to produce a prodrug. The form of the resulting mixture 0673 Antimicrobial agents in bacteriostatic or fungistatic depends upon a number of factors, including the intended concentrations may be added to parenteral preparations, mode of administration and the solubility of the compound particularly when the preparations are packaged in multiple in the selected carrier or vehicle. The effective concentration dose containers and thus designed to be stored and multiple aliquots to be removed. Examples of antimicrobial agents is Sufficient for ameliorating the symptoms of the disease that may used include phenols or cresols, mercurials, benzyl state and may be empirically determined. alcohol, chlorobutanol, methyl and propyl p-hydroxyben 0680 Suitable formulations for each of these methods of Zoic acid esters, thinerosal, benzalkonium chloride and administration may be found in, for example, "Remington: benzethonium chloride. The Science and Practice of Pharmacy’. A. Gennaro, ed., 20th edition, (2000), Lippincott, Williams & Wilkins, Phila 0674) Examples of isotonic agents that may be used delphia, Pa. include sodium chloride and dextrose. Examples of buffers that may be used include phosphate and citrate. Examples of antioxidants that maybe used include sodium bisulfite. REFERENCES Examples of local anesthetics that may be used include 0681 Various methods for the alkylation of albumin have procaine hydrochloride. Examples of Suspending and dis been reported, for example: persing agents that may be used include Sodium carboxym ethylcellulose, hydroxypropyl methylcellulose and polyvi 0682. Self-quenched fluorogenic substrates for pro nylpyrrolidone. Examples of emulsifying agents that may be teolytic enzymes have been prepared by alkylation of thiol used include Polysorbate 80 (TWEEN 80). A sequestering or groups in reduced bovine serum albumin with iodoaceta chelating agent of metal ions include EDTA. midofluorescein or iodoacetamidoeosin. Anal. Biochem. 176:261-264. 0675 Pharmaceutical carriers may also optionally include ethyl alcohol, polyethylene glycol and propylene 0683 Fluorescent derivative with acrylodan. Biophysical glycol for water miscible vehicles and sodium hydroxide, Journal Volume 75 August 1998 1084-1096. hydrochloric acid, citric acid or lactic acid for pH adjust 0684. The alkylating reagents iodoacetamide, 4-vinylpy ment. ridine, and acrylamide are all successful in improving the 0676 The concentration of a renin inhibitor complex in sequence coverage for albumin. the parenteral formulation may be adjusted so that an 0685 Alkylation of Cysteines: injection administers a pharmaceutically effective amount sufficient to produce the desired pharmacological effect. The 0686 Benzyl chlorides: Saunders; BIJOAK; Biochem.J.; exact concentration of a renin inhibitor complex and/or 28; 1934; 1977; Kwon, Yeondae; Zhang, Ruoheng; Bem dosage to be used will ultimately depend on the age, weight querer, Marcelo P: Tominaga, Mineko; Hojo, Hironobu; and condition of the patient or animal as is known in the art. Aimoto, Saburo; Chem. Lett.; EN: 5; 1993; 881-884. US 2007/0207952 A1 Sep. 6, 2007

0687 Alkyl halide: Foti, Salvatore: Saletti, Rosaria; Mar ticular Stereochemistry is specified, recitation of a com letta, Donata; Org.Mass Spectrom.; EN: 26; 10; 1991; pound is intended to encompass all of the different possible 903-907; Jin, Lixia; Baillie, Thomas A.: Chem.Res. Toxicol., Stereoisomers. EN: 10: 3; 1997; 318-327; Franzen, Henry M.: Nagren, Kjell; Grehn, Leif Langstroem, Bengt. Ragnarsson, Ulf. 0703 Various methods for separating mixtures of differ ent stereoisomers are known in the art. For example, a J.Chem.Soc. Perkin Trans. 1; EN: 1988: 497-502. racemic mixture of a compound may be reacted with an 0688 Bromoacetamide; Ziegler.E. et al.: Z. Naturfor optically active resolving agent to form a pair of diastere sch.B. Anorg. Chem. Org. Chem. Biochem. Biophys. Biol.: oisomeric compounds. The diastereomers may then be sepa GE: 25; 1970; 1417-1420. rated in order to recover the optically pure enantiomers. Dissociable complexes may also be used to resolve enanti 0689 AZiridines: Hata, Yoshiteru; Watanabe, Masamichi: omers (e.g., crystalline diastereoisomeric salts). Diastere Tetrahedron; EN: 43; 17: 1987: 3881-3888. omers typically have sufficiently distinct physical properties 0690 Methacrylate: Kasai, Takanori; Nishitoba, Tsuy (e.g., melting points, boiling points, solubilities, reactivity, oshi; Shiroshita, Yoshinari; Sakamura, Sadao; Agric. Biol. etc.) that they can be readily separated by taking advantage Chem.; EN: 48; 9; 1984; 2271-2278. of these dissimilarities. For example, diastereomers can typically be separated by chromatography or by separation/ 0691 Vinyl sulfones: Homer, L.; Lindel, H.; Phosphorus resolution techniques based upon differences in Solubility. A Sulfur; GE; 15; 1983: 1-8. more detailed description of techniques that can be used to 0692 C.-Halo ketones: Silva, Claudius D'; Seddon, resolve Stereoisomers of compounds from their racemic Andrew P.; Douglas, Kenneth T.; J. Chem. Soc. Perkin mixture can be found in Jean Jacques Andre Collet, Samuel Trans. 1; EN: 1981: 3029-3033: Chari, Ravi V. J.: Kozarich, H. Wilen, Enantiomers, Racemates and Resolutions, John John W.; J. Amer. Chem. Soc.; EN: 105; 24; 1983; 7169 Wiley & Sons, Inc. (1981). 7171. 0704 Compounds according to the present invention can 0693 Haloacetate: Climie, Ian J. G.; Evans, David A.: also be prepared as a pharmaceutically acceptable acid Tetrahedron; EN: 38: 5; 1982; 697-711. addition salt by reacting the free base form of the compound 0694 Unsaturated ketones: Spanton, Stephen G.; with a pharmaceutically acceptable inorganic or organic Prestwich, Glenn D.; Tetrahedron; EN: 38: 13; 1982; 1921 acid. Alternatively, a pharmaceutically acceptable base addi 1930. Biophysical Journal Volume 75 August 1998 1084 tion salt of a compound can be prepared by reacting the free 1096. acid form of the compound with a pharmaceutically accept able inorganic or organic base. Inorganic and organic acids 0695) Acrylonitrile: Climie, Ian J. G.; Evans, David A.: and bases suitable for the preparation of the pharmaceuti Tetrahedron; EN: 38: 5; 1982; 697-711. cally acceptable salts of compounds are set forth in the 0696 Acrylamide: Harrison, M. E.; Baldwin, M. A.; Org. definitions section of this Application. Alternatively, the salt Mass Spectrom.; EN: 24; 1989: 689-693. forms of the compounds can be prepared using salts of the starting materials or intermediates. 0697 B-Chloroketones: Vince, R.; Daluge, S.; J. Med. Chem.; EN; 14: 1971; 35-37. 0705 The free acid or free base forms of the compounds can be prepared from the corresponding base addition salt or 0698 Epoxide: Jin, Lixia; Baillie, Ihomas A.: Chem. Res. acid addition salt form. For example, a compound in an acid Toxicol.: EN: 10:3; 1997; 318-327. addition salt form can be converted to the corresponding free 0699 Allyl halide: Jin, Lixia; Baillie, Ihomas A.: Chem. base by treating with a suitable base (e.g., ammonium Res. Toxicol.: EN: 10:3; 1997; 318-327. hydroxide solution, sodium hydroxide, and the like). A compound in a base addition salt form can be converted to 0700. The entire disclosure of all documents cited the corresponding free acid by treating with a Suitable acid throughout this application are incorporated herein by ref (e.g., hydrochloric acid, etc). CCC. 0706 Protected derivatives of the compounds can be made by methods known to those of ordinary skill in the art. RENIN INHIBITOR EXAMPLES A detailed description of the techniques applicable to the Preparation of Renin Inhibitors Conjugate Complexes creation of protecting groups and their removal can be found in T. W. Greene, Protecting Groups in Organic Synthesis, 0701 Various methods may be developed for synthesiz ing compounds according to the present invention. Repre 3rd edition, John Wiley & Sons, Inc. 1999. sentative methods for synthesizing these compounds are 0707 Compounds according to the present invention provided in the Examples. It is noted, however, that the may be conveniently prepared, or formed during the process compounds of the present invention may also be synthesized of the invention, as Solvates (e.g. hydrates). Hydrates of by other synthetic routes that others may devise. compounds of the present invention may be conveniently prepared by recrystallization from an aqueous/organic Sol 0702. It will be readily recognized that certain com pounds according to the present invention have atoms with vent mixture, using organic solvents such as dioxin, tetrahy linkages to other atoms that confer a particular stereochem drofuran or methanol. istry to the compound (e.g., chiral centers). It is recognized 0708 Compounds according to the present invention can that synthesis of compounds according to the present inven also be prepared as their individual stereoisomers by react tion may result in the formation of mixtures of different ing a racemic mixture of the compound with an optically Stereoisomers (enantiomers, diastereomers). Unless a par active resolving agent to form a pair of diastereoisomeric US 2007/0207952 A1 Sep. 6, 2007 42 compounds, separating the diastereomers and recovering the molar ratio in reaction mixture of peptide: HSA was 1:4. The optically pure enantiomer. While resolution of enantiomers solution was incubated 5 hours at 37° C. Once incubation can be carried out using covalent diasteromeric derivatives was complete, the conjugate was stored at 4° C. of compounds, dissociable complexes are preferred (e.g., crystalline diastereoisomeric salts). Diastereomers have dis EXAMPLE 1.4 tinct physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) and can be readily separated by Conjugation Reaction to Form the Complex taling advantage of these dissimilarities. The diastereomers can be separated by chromatography or, preferably, by 0716 A 10 mM solution of trans4-(maleimidyhmethyl separation/resolution techniques based upon differences in )cyclohexane-1-carbonyl derivatized peptide in DMSO is solubility. The optically pure enantiomer is then recovered, added to a 25% water solution of HSA. The final peptide along with the resolving agent, by any practical means that concentration in the reaction mixture is 1 mM and the molar would not result in racemization. ratio in reaction mixture of peptide: HSA is 1:4. The solution is incubated 5 hours at 37° C. Once incubation was com 0709 As used herein the symbols and conventions used plete, the conjugate is stored at 4°C. in these processes, schemes and examples are consistent with those used in the contemporary scientific literature, for EXAMPLE 1.5 example, the Journal of the American Chemical Society or the Journal of Biological Chemistry. Standard single-letter Conjugation Reaction to Form the Complex or thee-letter abbreviations are generally used to designate amino acid residues, which are assumed to be in the L-con 0717. A 10 mM solution of N-(3-2-2-(3-amino-pro figuration unless otherwise noted. Unless otherwise noted, poxy)-ethoxy-ethoxy-ethoxy)-propyl)-2-bromoacetamide all starting materials are obtained from commercial Suppliers derivatized peptide in DMSO is added to a 25% water and used without further purification. solution of HSA. The final peptide concentration in the reaction mixture is 1 mM and the molar ratio in reaction Synthetic Schemes for Renin Inhibitors of the Present Inven mixture of peptide: HSA is 1:4. The solution is incubated 5 tion hours at 37°C. Once incubation IS complete, the conjugate 0710 Renin inlubitors according to the present invention is stored at 4° C. may be synthesized according to a variety of reaction schemes. Some illustrative schemes are provided herein in EXAMPLE 16 the examples. Other reaction schemes could be readily devised by those skilled in the art. Conjugation Reaction to Form the Complex 0711. In the reactions described hereinafter it may be 0718. A 10 mM solution of maleimidopropionylamino necessary to protect reactive functional groups, for example ethoxyethoxyacetyl derivatized peptide in DMSO is added hydroxy, amino, imino, thio or carboxy groups, where these to a 25% water solution of HSA. The final peptide concen are desired in the final product, to avoid their unwanted tration in the reaction mixture is 1 mM and the molar ratio participation in the reactions. Conventional protecting in reaction mixture of peptide: HSA is 1:1. The solution is groups may be used in accordance with standard practice, incubated 5 hours at 37°C. Once incubation is complete, the for examples see T. W. Greene and P. G. M. Wuts in conjugate is stored at 4° C. “Protective Groups in Organic Chemistry' John Wiley and Sons, 1991. EXAMPLE 17 0712 Compounds according to the present invention Conjugation Reaction to Form the Complex may optionally be synthesized according to the following general reaction schemes: 0719. A 10 mM solution of maleimidopropionylamino ethoxyethoxyacetyl derivatized peptide in DMSO is added Preparation of Complex of Formula I: to a 25% water solution of HSA. The final peptide concen tration in the reaction mixture is 1 mM and the molar ratio 0713) in reaction mixture of peptide: HSA is 10:1. The solution is incubated 5 hours at 37°C. Once incubation is complete, the conjugate is stored at 4° C. Preparation of Complex of Formula II: 0720. It will be apparent to those skilled in the art that various modifications and variations can be made to the 0714) compounds, compositions, kits, and methods of the present invention without departing from the spirit or scope of the pL2-(Pr)+Ih-Ih-L2-(Pr). Formula II invention. Ihus, it is intended that the present invention cover the modifications and variations of this invention EXAMPLE 13 provided they come within the scope of the appended claims and their equivalents. Conjugation Reaction to Form the Complex Examples of In vitro Assays 0715. A 10 mM solution of maleimidopropionylamino ethoxyethoxyacetyl derivatized peptide in DMSO was 0721 Various assays to measure renin inhibition activity added to a 25% water solution of HSA. The final peptide are described in Cartledge, et al. Ann. Clin. Biochem. concentration in the reaction mixture was 1 mM and the 262-278 (2000). US 2007/0207952 A1 Sep. 6, 2007

EXAMPLE 1.8 0734) 3. Wang GT, Chung C C, Holzman T F, Kraft G A. A continuous fluorescence assay of renin activity. Anal Measurement of Renin Inhibitory Activity In Vitro Biochem. 1993, 210(2):351-9. 0722 Fluorescence Measurement of Renin Inhibitory EXAMPLE 1.8 Activity 0723. One method of measuring renin enzyme activity In Vivo Testing uses the cleavage of a synthetic peptide Substrate in a 0735. The conjugate is administered intravenously to fluorescence-based microplate reader. The peptide substrate rats. The inhibitor not conjugated with albumin is adminis for renin, is linked to a fluorophore (5-(aminoethyl)aminon tered in a control group. Serum samples are collected at 5 aphthalene sulfonate, EDANS) at one end and to a nonfluo min, 30 min, 1 h, 2 h, 8 h, 24 h, 48 h, and 72 h post dose. rescent chromophore (4'-dimethylaininoaZobenzene-4-car Renin inhibitory activity is measured by one of the methods boxylate, DABCYL) at the other. After cleavage by renin, described above. Serum concentrations of peptide or pep the product (peptide-EDANS) is brightly fluorescent. A 500 tide-HSA conjugates were calculated from the calibration uM stock solution of renin substrate can be prepared by curves. Based on results of these experiments the following adding 877 uL of dimethyl sulfoxide (DMSO) to 1 mg of conclusions maybe drawn: substrate. This stock solution is added into the assay buffer 0736. The control peptide displayed a clearance profile to a final concentration of 2 uM. A small amount (<3% of the final Volume) of renin-containing Solution is diluted in the with rapid elimination. assay buffer. The initial rate of cleavage of fluorogenic 0737. The terminal half-life of HSA conjugates range Substrate is measures by monitoring the increase in fluores from 12 to 14 hours, similar to that of HSA in this species. cence signal at 490 nm for 5-8 min at 37°C. Our conjugates 0738 Antihypertensive activity due to human renin inhi show activity from Subnanomolar to high micromolar in this bition can be measured in hypertensive rats doubly trans assay. genic for human angiotensinogen with endogenous pro 0724) 1. J Protein Chem 10, 553 (1991) moter and human renin with endogenous promoter. 0725 2. Anal Biochem 210, 351 (1993) 0739 Bohlender, et al. Hypertension 428-434 (1997) 0740 For cases where the inhibition of rat renin is 0726) 3. Science 247, 954 (1990) comparable to that of human renin antihypertensive activity 0727 4. J Protein Chem 9,663 (1990). can be measured in Sodium depleted rats. 0728. Measurement of Plasma Renin Inhibitors 0741. Allan, et al. JPET 283:661-665, (1997). 0729) Plasma renin activity is determined based on EXAMPLE 19 method originally described by Haber et al. (1). Briefly, plasma samples are divided on two aliquots. One aliquot is Bio Activity of Renin Inhibitor Derivatives incubated for 3-18 h at 37 C, while another aliquot is kept on ice. The angiotensin I concentration is determined using 0742 The information presented above clearly demon commercial kits in RIA or ELISA format according to strates that the biotin ring on the Ih-L-Pr complex is acces manufacturer protocol. The angiotensin I concentration in sible for binding to avidin. The next series of experiments is the aliquot kept at 0-4 C is subtracted from that in 37 C designed to address whether Ih-L-Pr complex, which has an IC50 of about 50 nM in its soluble free acid form, is still aliquot to give a measure of renin activity. bioactive after conjugation to target proteins. 0730 Activity of renin in plasma can also be measured 0743 Materials and Methods: The following procedures towards externally added renin peptide Substrate using are done under sterile conditions. Rabbit plasma is obtained HPLC separation of cleavage products. Cleavage products from freshly drawn heparinized blood. One 8 mL aliquot of may be detected by LC-MS analysis. Alternatively, peptide plasma is incubated with 5 micromoles of the Ih-L-Pr substrate can be modified by fluorophore or chromophore to complex for 60 minutes at room temperature. Another equal allow spectrophotometric detection. Plasma proteins can be aliquot is similarly incubated with 5 micromoles of the removed by precipitation prior the HPLC analysis. Ih-L-Pr complex. The reaction mixtures are stored at 4°C. 0731 Concentration of renin inhibitor and/or renin overnight. Aliquots of these samples are saved for analysis inhibitor-HSA conjugate in plasma is determined by of total renin inhibitor content by a standard renin radioim enzyme-based assay (2), which measures inhibitory poten munoassay (RIA). After warming to 37° C., the plasma tial of plasma sample towards externally added recombinant samples are injected into two autologous rabbits. The rabbits human renin using commercially available quenched fluo are then bled at defined intervals. The blood is centrifiged for rescent substrate (3) at pH 7.0-8.0. 5 minutes at 2500 rpm and then aliquots of the plasma are analyzed by RIA. 0732 1. Haber E. Koerner T. Page L B. Kliman B, Purnode A. Application of a radioimmunoassay for angio 0744) Results: Plasma proteins derivatized with the NHS tensin I to the physiologic measurements of plasma renin ester of the Ih-L-Pr complex did indeed maintain the inhibi activity in normal human subjects. J Clin Endocrinol Metab. tor in a conformation which remained bioavailable and 1969, 29(10): 1349-55 inhibitory after an extended period of circulation in the blood. Again, the amount of inhibitor detectable has been 0733 2. Gulnik S., Erickson, J. W., Yu, B. Protease assay normalized for the effect of dilution of the plasma by the for therapeutic drug monitoring. 2003, WO03040390 volume of blood in circulation. US 2007/0207952 A1 Sep. 6, 2007 44

0745) The data shows that the level of free acid of the 21. The complex of claim 19, wherein the peptide has a renin inhibitor Ih falls rapidly and is not detectable after one mass of less than about 30 kDA. hour. On the other hand, the modified plasma proteins as the 22. The complex of claim 19, wherein the peptide has a Ih-L-Pr complexes are inhibitory in the renin assay, indicat mass of less than about 10 kDA. ing that the conjugation did not destroy the bioactivity of the inhibitor Ih. Furthermore, the level of the inhibition 23. The complex of claim 19 wherein the peptide is a observed does not significantly decrease until day 10. Sev peptidomimetic. eral abundant plasma proteins (albumin and immunoglobu 24. The complex of claim 19 wherein the peptide consists lins) are long-lived and could account for this delivery of up to 51 amino acids comprising a sequence selected from profile. These results, therefore, clearly demonstrate that the group consisting of: covalent attachment of a derivatized renin inhibitor to plasma proteins. Such as albumin, does not destroy the bioactivity of the molecule and significantly increases the lifetime of the inhibitor Ih in the blood. 0746. It is evident from the above results that the subject invention provides for greatly improved treatment involving renin inhibition Ih by the use of the complexes of Formula I and Formula II. By use of the subject invention, the renin inhibitors Ih maintain for extended periods of time, so that repetitive dosages are not required, compliance by the patient is not required, and protection is ensured. The derivatized renin inhibitors of the present invention covalently attach to erythrocytes, plasma proteins and vari ous other vascular components to form the complexes of Formula I and Formula II, while retaining biological activity and are not immunogenic. 0747 While the invention has been described in connec tion with specific embodiments thereof, it will be understood that it is capable of further modifications, and this applica tion is intended to cover any variations, uses or adaptations of the invention following, in general, the principles of the invention, and including Such departures from the present description as come within known or customary practice within the art to which the invention pertains, and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims. 1-17. (canceled) 18. An isolated complex of the Formula I or Formula II:

(AV)-L1--Pr II AV-i-L2-(Pr).

wherein: peptide DP178 (T-20); and m is an integer from 1-5; peptide T-1249; n is an integer from 1-100: wherein: o is an integer from 1-5. X1 is selected from the group consisting of M. L., I, Q, T. p is an integer from 1-100: R and K; AV is an antiviral compound; X2 is either E. D. Q and K: L1 and L2 are polyvalent linkers covalently linking AV to X3 is selected from the group consisting of E, D and K: Pr, or where L1 and L2 are absent; X4 is selected from the group consisting of K, R, E, Q, N Pr is a protein; and and T. wherein the complex possesses antiviral activity in vivo. X5 is selected from the group consisting of E., I, R. K and 19. The complex of claim 18, wherein the antiviral compound is a peptide. Q: 20. The complex of claim 19 wherein the peptide has a X6 is selected from the group consisting of N, D, S, E, Q, mass of less than about 100 kDA. K, t, H, T, I and G: US 2007/0207952 A1 Sep. 6, 2007 45

X7 is selected from the group consisting of N, Q, D, E, K, 53. A method for inhibiting the activity of HIV gp41 and S, T and Y: HIV in vivo, the method comprising: X8 is selected from the group consisting of Y. F. H., I, V administering to the bloodstream of a mammmalian host and S; an isolated conjugate complex of claim 18, wherein the complex is formed by attaching an antiviral compound X9 is selected from the group consisting of G. K. R. H. D. to a linker having at least one reactive functional group E, S, T, N and Q; which reacts with the protein to form stable covalent X10 is selected from the group consisting of K. H. E. Q. bonds; and T, V, I, L, M, A, Y, F, and P: wherein the isolated conjugate complex is administered in an amount to maintain an effective therapeutic effect in X11 is selected from the group consisting of H. K. E. Y the bloodstream for an extended period of time as and F: compared to a non-conjugated antiviral compound. X12 is selected from the group consisting of T. S. Q. N. 54-80. (canceled) E, D, R, K, H. W. G, A, and M: 81. An isolated complex of the Formula I or Formula II: X13 is selected from the group consisting of D, E, Q, T. (Ih)-L1-Pr I K, R, A, V and G: Ih-IL2-(Pr). II X14 is selected from the group consisting of D, E, K, H, wherein m is an integer from 1-5; n is an integer from Q, N. S., I, L. V. A and G: 1-100; o is an integer from 1-5; p is an integer from 1-100; Ih is a renin inhibitor; L1 and L2 are polyvalent X15 is selected from the group consisting of S, A and (P): linkers covalently linking Ih to Pr, or where L1 and L2 are absent; Pr is a protein; and wherein the complex X16 is selected from the group consisting of N, K, S, T, possesses renin inhibitory activity in vivo. D, E, Y, I and V: 82-135. (canceled) X17 is selected from the group consisting of E, D, N, K, 136. An isolated compound comprising a pharmacologi G, and V: cally active moiety covalently conjugated to a macromo X18 is selected from the group consisting of K. R. H. D. lecular carrier, E., N, Q, T. M., I, and Y: wherein the carrier is pharmacologically inert, X19 is selected from the group consisting of E. V. Q. M. wherein the linkage between said pharmacologically L. J. and G: active moiety and said carrier is stable in Vivo, X20 is selected from the group consisting of Q, N. E. K wherein the intact compound Substantially retains the I, H, L, and F: pharmacological activity of said pharmacologically active moiety, X21 is selected from the group consisting of E. D. N. S. and wherein the active half-life of said compound when K, A, and G: administered to a mammal is at least about twice that of X22 is selected from the group consisting of L., I, and Y: said pharmacologically active moiety. and 137. The compound according to claim 136, wherein said macromolecular carrier is a protein. X23 is selected from the group consisting of I. L. M., Q. 138. The compound according to claim 136, wherein said S, and Y. macromolecular carrier is an albumin of homologous origin 25. The complex of claim 24 wherein the protein is a to said mammal. blood component. 139. The compound according to claim 138, wherein said 26. The complex of claim 25, wherein the blood compo albumin is a human serum albumin. nent is selected from the group consisting of red blood cells, 140. The compound according to claim 136, wherein said immunoglobulins, IgM, IhC, serum albumin, transferrin, pharmacologically active moiety is conjugated to said car P90 and P38, ferritin, a steroid binding protein, thyroxin rier via a linker moiety. binding protein, and C-2-macroglobulin. 141. The compound according to claim 136, wherein said 27. The complex of claim 25, wherein the blood compo pharmacologically active moiety is directly linked to said nent is human serum albumin and the linker is a peptide carrier. linker. 142. The compound according to claim 136, wherein at 28. The complex of claim 25, wherein the blood compo least two phannacologically active moiety molecules are nent is human serum albumin and the linker is a non-peptide conjugated to said carrier. linker. 143. The compound according to claim 137, wherein the 29. (canceled) linkage to said carrier is via a lysine side chain on said 30. The complex of claim 18, wherein the linler L1 or L2 carrier. is a non-labile linker that is stable toward hydrolytic cleav 144. The compound according to claim 137, wherein the age in vivo. linkage to said carrier is via a cysteine side chain on said 31-47. (canceled) carrier, 48. An anti-viral composition comprising a non-peptidic 145. The compound according to claim 136, wherein said anti-viral compound covalently linked to a blood compo carrier is HSA and the linkage is via C34 of the HSA. nent. 49-52. (canceled) k k k k k