The present invention relates to pharmaceutical preparations and methods for treating individuals infected with the human immunodeficiency virus (HIV). The pharmaceutical preparations comprise an immunomodulating agent and a anti-retroviral compound. The pharmaceutical preparations are used to treat HIV infected patients, particularly for gastrointestinal complications arising from viral infection. In addition, the pharmaceutical preparations of the present invention have the effect of raising the levels of CD4.sup.+ single positive and CD4.sup.+ and CD8.sup.+ double positive T cells, thus promoting restoration and normalization of the immune system following HIV infection.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical preparations and methods for treating HIV and AIDS, and in particular, to novel combinations of immunomodulatory peptides and anti-retroviral agents.

BACKGROUND OF THE INVENTION

Human immunodeficiency virus type 1 (HIV-1) infection is characterized as a systemic immunosuppressive disorder caused by the viral-mediated depletion of CD4.sup.+ T cells, which develops into the profound immunodeficiency that underlies AIDS. The targeting of CD4.sup.+ lymphocytes by HIV-1 is thought to result from expression of cell surface CD4 (Dalgleish et al., Nature 312:763 767 (1984)), the chemokine receptor CXCR4 (Feng et al., Science 272:872 877 (1996)) and (upon activation) CCR5 (Alkhatib et al., Science 272:1955 58 (1996)), which act as receptors for the attachment and entry of HIV-1 (Imlach et al., J. Virol. 75(23):11555 11564 (2001)). HIV infection may also result in a particularly massive reduction in the double-positive CD4.sup.+ CD8.sup.+ T cell population, possibly due to reduced expression of Bcl-2 and concomitant sensitivity to apoptosis (Guillemard et al., Blood 98(7):2166 2174 (2001)). CD4 and CCR5 are also thought to be responsible for HIV infection of macrophages and macrophage-derived cell types in vivo, although the effect this has on the immune system is unresolved (Guillemard et al., supra). The severe immunodeficiency caused by HIV infection is due not only to the low CD4.sup.+ T cell numbers but also to the qualitative dysfunction of the lymphocytes (Bogner et al., Infection 29:32 36 (2001)).

The progression of HIV-1 infection is clearly associated with an increase in the viral load in plasma as well as the progressive depletion of CD4.sup.+ T cells. Treatment of HIV-1-infected individuals with potent combinations of anti-retroviral drugs can result in a dramatic decline of the viral load to undetectable HIV-1 RNA levels in the majority of patients (Pakker et al., Nat. Med. 4(2):208 14 (1998)). Apart from controlling viral replication, however, the major goal of these antiviral therapies is to achieve a degree of immune reconstitution. Although increases in CD4.sup.+ T cell numbers have been observed, the mechanisms underlying T cell repopulation and restoration of function are still unclear, and complete quantitative or qualitative reconstitution of the immune system may not be achieved or may take a long time to be achieved (Pakker et al., supra). The renewal proceeds slowly, suggesting, in some cases, a severe impairment of T-cell progenitors, depending on the stage of the disease and the age of the patient (Chene et al., J. Virol. 73:7533 7542 (1999)). In many cases, recovery of immune functions to almost normal levels has not been achieved (Plana et al., AIDS 14(13):1921 1933 (2000); Hejdeman et al., AIDS Res. Hum. Retro. 17:277 286 (2001)).

The clinical abnormalities correlated with the presence of HIV infection include immune suppression as well as morphologic and histopathologic changes in intestinal mucosa. Pathologic changes in small intestinal tissues from HIV-infected patients include crypt hyperplasia, villus atrophy, and inflammation. Functional changes have included decreased digestive enzyme activities (Heise et al., Gastroenterology 100(6):1521 1527 (1991); Ullrich et al. Ann Intern Med 111 (1):15 21 (1989)) and intestinal permeability (Keating, J. et al., Gut 37(5):623 629 (1995)), indicative of abnormalities in absorptive epithelial cells. Aberrant mucosal antibody responses and compromised epithelial barrier function may contribute to intestinal disease in HIV infection (Janoff et al., J. Infect. Dis. 170(2):299 307 (1994)).

Gastrointestinal complications commonly seen in HIV-infected patients are nutrient malabsorption, malnutrition, diarrhea and weight loss. These symptoms are associated with a rapid clinical course (Ehrenpreis et al., J. Acquir. Immune Defic. Syndr. 5(10):1047 1050 (1992); Ehrenpreis et al., Am J Clin. Pathol. 97(1):21 28 (1992). With the onset of immunodeficiency, opportunistic enteric pathogens contribute to the severity of intestinal disease (Smith et al., Gastroenterol Clin. North Am. 17(3):587 598 (1988); Kotler, D. P. et al., Ann. Intern. Med. 113(6):444 449 Greenson et al., Ann. Intern. Med. 114(5):366 72 (1991)). However, in many instances, intestinal abnormalities are often detected prior to advanced stages of immunodeficiency and in the absence of detectable enteric pathogens (Gillin et al., Ann. Intern. Med. 102(5):619 622 (1985); Heise et al., supra; Ullrich et al., supra; Kotler et al., supra; Greenson et al., supra; and Miller, A. R. et al., Q J Med. 69(260):1009 1019 (1988). Thus the onset of the intestinal mucosal immune system dysregulation may occur early in infection.

The role of tumor necrosis factor (TNF) in gastrointestinal inflammation in HIV infected individuals is unclear. In SIV-infected rhesus monkeys, expression of TNF is known to be variable throughout the disease course; significant levels were present in intestinal mucosa of 5 of 7 asymptomatic animals, and 6 of 8 terminal animals. It was undetectable in the majority of animals in the acute stage of infection, regardless of viral inoculum. A reciprocal relationship was observed between TNF and IL-10. This suggests that the presence of IL-10 in the intestinal mucosa inhibits TNF production by resident macrophages, as has been described previously in other systems (Fiorentino, et al., J. Immunol. 147(11):3815 3822 (1991); de Waal Malefyt et al., J. Exp. Med. 174(5):1209 1220 (1991)).

TNF is also known to increase HIV replication in various monocyte and T cell model systems (Chene et al., J. Virol. 73(9):7533 7542 (1999); Marshall et al., J. Immunol. 162(10):6016(1999); Heguy et al., Antivir. Chem. Chemother. 9(2):149 155 (1998); Munoz-Fernandez et al., J. Allergy Clin. Immunol. 100:838 845 (1997)). Addition of neutralizing anti-TNF antibodies to primary cultures of HIV-infected human T lymphocytes drastically reduces p24 antigen release and prevents CD4.sup.+ cell depletion associated with infection (Munoz-Fernandez et al., supra). Anti-TNF also prevents nuclear factor-kappa B activation, which is involved in the activation of HIV replication. On the other hand recent reports suggest that TNF suppresses HIV replication in freshly infected peripheral blood monocytes and alveolar macrophages (Herbein et al., J. Virol. 70(11):7388 7397 (1996)). Additional studies with a large number of patients will be necessary to evaluate the effect of anti-TNF antibody therapy on disease progression.

As indicated above, significant challenges still remain in the scientific and clinical battle against HIV and AIDS. What is needed are improved compositions and methods capable of accelerating and enhancing the immune reconstitution of infected individuals, and effectively treating gastrointestinal complications resulting from HIV infection.

Relevant Literature

Buelow et al., Transplantation 59:649 654 (1995) and references cited therein. Manolios et al., Nature Medicine 3:84 88 (1997) describes oligopeptides derived by rational design which modulate T cell activity. WO 95/13288 by Clayberger et al. which describes peptides capable of modulating T cell activity. References describing methods for designing compounds by computer using structure activity relationships include Grassy et al., J. of Molecular Graphics 13:356 367 (1995); Haiech et al., J. of Molecular Graphics 13:46 48 (1995); Yasri et al., Protein Engineering 11: 959 976 (1996); Ashton et al., Drug Discovery Today 1:71 78 (1996); and Iyer et al., Curr. Pharm. Des. 8:2217 2229 (2002)

SUMMARY OF THE INVENTION

The present invention relates to pharmaceutical preparations and methods for treating HIV infection and the resultant Acquired Immune Deficiency Disorder, or AIDS. In particular, novel combinations of immunomodulatory peptides and anti-retroviral agents are provided to accelerate and enhance immune reconstitution and normalization in gut-associated lymphoid tissue (GALT), and to alleviate the gastrointestinal abnormalities and dysfunction resulting from HIV infection. As described herein, the synergistic combination of the subject immunomodulatory peptides with anti-retroviral therapies accelerates and enhances CD4+ T cell repopulation in GALT well beyond that obtained using anti-retroviral therapies alone, and further results in a dramatic increase in double-positive CD4+CD8+ T cells in GALT. Thus, methods are provided utilizing the combination therapies described herein to increase the level of single-positive CD4+ T cells and/or double-positive CD4+ CD8+ T cells in GALT of individuals infected with HIV, along with kits for carrying out the subject methods.

Suitable immunomodulatory peptides for use in the subject compositions and methods are capable of modulating the activity of various immune system cells, particularly T cells, and/or inhibiting the production of inflammatory cytokines. In a preferred embodiment, the subject peptides comprise one or more of the cytomodulating peptides disclosed in co-pending U.S. patent applications U.S. Ser. No. 09/028,083 & U.S. Ser. No. 08/838,918 as well as corresponding International Publication WO 98/46633, the disclosures of which are expressly incorporated herein by reference. In a particularly preferred embodiment, the immunomodulating peptide comprises the sequence Arg-nL-nL-nL-Arg-nL-nL-nL-Gly-Tyr (SEQ ID NO:1), where nL is norleucine and all amino acids are the D-stereoisomer.

In one aspect, the peptides have amino acid extensions at the N- or C-terminus to provide additional functionality, such as targeting the peptide to the affected tissue, increasing half-life, or for attachment of various compounds. In another aspect, the cytomodulating peptides are oligomers, particularly dimers of the active sequence, or are in the form of cyclic peptides. The peptides may comprise naturally-occurring amino acids or, more preferably, one or more D-stereoisomers.

Suitable anti-retroviral agents for use in the therapeutic compositions and methods described herein include nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, co-receptor antagonists, retroviral integrase inhibitors, viral adsorption inhibitors, viral specific transcription inhibitors, and cyclin dependent kinase inhibitors.

In one embodiment, a pharmaceutical preparation is provided comprising a novel combination of an immunomodulating peptide and an anti-retroviral agent for simultaneous or sequential administration to a patient infected with HIV-1. In a preferred embodiment, the anti-retroviral agent is a nucleoside reverse transcriptase inhibitor selected from the group consisting of Azidothymidine, Lamivudine, Didanosine, Zalcitabine, Stavudine, Abacavir, and Tenofovir. In another preferred embodiment, the anti-retroviral agent is a non-nucleoside reverse transcriptase inhibitor selected from the group consisting of Nevirapine, Dlavirdine, and Efavirenz. In yet another preferred embodiment, the anti-retroviral agent is a protease inhibitor selected from the group consisting of Indinavir, Saquinavir, Ritonavir, Nelfinavir, Amprenavir, and Lopinavir.

In another preferred embodiment, combinations of a plurality of antiviral agents may be used, thus increasing the efficacy of the therapy and lessening the occurrence of resistance to the anti-viral drugs. Various combinations may be made and used by those skilled in the art. In one aspect, the immunomodulatory peptides, particularly a peptide comprising sequence Arg-nL-nL-nL-Arg-nL-nL-nL-Gly-Tyr (SEQ ID NO:1), wherein all the amino acids are D-stereoisomers is used with a combination of retroviral agents comprising a reverse transcriptase inhibitor and a protease inhibitor. In another aspect, the combination of antiviral agents comprises different protease inhibitors or different reverse transcriptase inhibitors.

In the present invention, a method for treating a patient infected with HIV-1 is provided, comprising the administration of a therapeutically effective amount of an immunomodulatory peptide, either alone or in combination with one or more anti-retroviral agent(s). When used in combination with an anti-retroviral agent, the immunomodulatory peptide may be administered simultaneously or sequentially with the anti-retroviral agent. In a preferred embodiment, the dose is effective to increase the number of single-positive CD4.sup.+ T cells in GALT of said patient. In another preferred embodiment, the dose is effective to increase the number of double-positive CD4.sup.+ CD8.sup.+ T cells in GALT of said patient. In a further and preferred embodiment, said dose is also effective to alleviate gastrointestinal abnormalities associated with HIV infection.

Generally, the pharmaceutical preparations and methods disclosed herein accelerate the process of immune reconstitution in GALT of HIV-infected individuals and enhance the level of immune reconstitution achieved, resulting in a more normalized T cell population in GALT. Thus, the present invention further contemplates general methods for enhancing or improving immune reconstitution in individuals suffering from T cell depletion such as, e.g., the CD4.sup.+ T cell depletion caused by HIV infection, as well as general methods for normalizing T cell populations in tissues affected by such depletion.
 

Claim 1 of 19 Claims

1. A method for treating an individual infected with HIV, comprising administering to an HIV infected individual a pharmaceutically effective amount of a) an immunomodulatory peptide; and b) at least one anti-retroviral agent; wherein said immunomodulatory peptide is an oligopeptide is selected from the group consisting of: (a) Arg-Leu-Leu-Leu-Arg-Leu-Leu-Leu-Gly-Tyr (SEQ ID NO:10); (b) Arg-Val-Leu-Leu-Arg-Leu-Leu-Leu-Gly-Tyr (SEQ ID NO:11); (c) Arg-Ile-Leu-Leu-Arg-Leu-Leu-Leu-Gly-Tyr (SEQ ID NO:12); (d) Arg-Leu-Val-Leu-Arg-Leu-Leu-Leu-Gly-Tyr (SEQ ID NO:13); (e) Arg-Leu-Ile-Leu-Arg-Leu-Leu-Leu-Gly-Tyr (SEQ ID NO:14); (f) Arg-Leu-Leu-Val-Arg-Leu-Leu-Leu-Gly-Tyr (SEQ ID NO:15); (g) Arg-Leu-Leu-Ile-Arg-Leu-Leu-Leu-Gly-Tyr (SEQ ID NO:16); (h) Arg-Leu-Leu-Leu-Arg-Val-Leu-Leu-Gly-Tyr (SEQ ID NO:17); (i) Arg-Leu-Leu-Leu-Arg-Ile-Leu-Leu-Gly-Tyr (SEQ ID NO:18); (j) Arg-Leu-Leu-Leu-Arg-Leu-Val-Leu-Gly-Tyr (SEQ ID NO:19); (k) Arg-Leu-Leu-Leu-Arg-Leu-Ile-Leu-Gly-Tyr (SEQ ID NO:20); (l) Arg-Leu-Leu-Leu-Arg-Leu-Leu-Val-Gly-Tyr (SEQ ID NO:21); (m) Arg-Leu-Leu-Leu-Arg-Leu-Leu-Ile-Gly-Tyr (SEQ ID NO:22); (n) Arg-Trp-Leu-Leu-Arg-Leu-Leu-Leu-Gly-Tyr (SEQ ID NO:23); (o) Arg-Leu-Trp-Leu-Arg-Leu-Leu-Leu-Gly-Tyr (SEQ ID NO:24); (p) Arg-Leu-Leu-Trp-Arg-Leu-Leu-Leu-Gly-Tyr (SEQ ID NO:25); (q) Arg-Leu-Leu-Leu-Arg-Trp-Leu-Leu-Gly-Tyr (SEQ ID NO:26); (r) Arg-Leu-Leu-Leu-Arg-Leu-Trp-Leu-Gly-Tyr (SEQ ID NO:27); (s) Arg-Leu-Leu-Leu-Arg-Leu-Leu-Trp-Gly-Tyr (SEQ ID NO:28); (t) Arg-Tyr-Leu-Leu-Arg-Leu-Leu-Leu-Gly-Tyr (SEQ ID NO:29); (u) Arg-Leu-Tyr-Leu-Arg-Leu-Leu-Leu-Gly-Tyr (SEQ ID NO:30); (v) Arg-Leu-Leu-Tyr-Arg-Leu-Leu-Leu-Gly-Tyr (SEQ ID NO:31); (w) Arg-Leu-Leu-Leu-Arg-Tyr-Leu-Leu-Gly-Tyr (SEQ ID NO:32); (x) Arg-Leu-Leu-Leu-Arg-Leu-Tyr-Leu-Gly-Tyr (SEQ ID NO:33); (y) Arg-Leu-Leu-Leu-Arg-Leu-Leu-Tyr-Gly-Tyr (SEQ ID NO:34); and (z) Arg-nL-nL-nL-Arg-nL-nL-nL-Gly-Tyr (SEQ ID NO: 1).
 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.