Pharmacokinetics,Toxicity,and Functional Studies of the Selective Kv1.3 Channel Blocker 5-(4-Phenoxybutoxy)Psoralen in Rhesus Macaques

Abstract

The small molecule 5-(4-phenoxybutoxy)psoralen (PAP-1) is a selective blocker of the voltage-gated potassium channel Kv1.3 that is highly expressed in cell membranes of activated effector memory T cells (TEMs). The blockade of Kv1.3 results in membrane depolarization and inhibition of TEM proliferation and function. In this study, the in vitro effects of PAP-1 on T cells and the in vivo toxicity and pharmacokinetics (PK) were examined in rhesus macaques (RM) with the ultimate aim of utilizing PAP-1 to define the role of TEMs in RM infected with simian immunodeficiency virus (SIV). Electrophysiologic studies on T cells in RM revealed a Kv1.3 expression pattern similar to that in human T cells. Thus, PAP-1 effectively suppressed TEM proliferation in RM. When administered intravenously, PAP-1 showed a half-life of 6.4 hrs; the volume of distribution suggested extensive distribution into extravascular compartments. When orally administered, PAP-1 was efficiently absorbed. Plasma concentrations in RM undergoing a 30-day, chronic dosing study indicated that PAP-1 levels suppressive to TEMs in vitro can be achieved and maintained in vivo at a non-toxic dose. PAP-1 selectively inhibited the TEM function in vivo, as indicated by a modest reactivation of cytomegalovirus (CMV) replication. Immunization of these chronically treated RM with the live influenza A/PR8 (flu) virus suggested that the development of an in vivo, flu-specific, central memory response was unaffected by PAP-1. These RM remained disease-free during the entire course of the PAP-1 study. Collectively, these data provide a rational basis for future studies with PAP-1 in SIV-infected RM.

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References

Chandy KG, Wulff H, Beeton C, Pennington M, Gutman GA, Cahalan MD. K+ channels as targets for specific immunomodulation. Trends Pharmacol Sci 25:280–289, 2004.

Freedman BD, Price MA, Deutsch CJ. Evidence for voltage modulation of IL-2 production in mitogen-stimulated human peripheral blood lymphocytes J Immunol 149:3784–3794, 1992.

Leonard RJ, Garcia ML, Slaughter RS, Reuben JP. Selective blockers of voltage-gated K+ channel depolarize human T lymphocytes: mechanism of the antiproliferative effect of charybdotoxin. Proc Natl Acad Sci U S A 89:10094–10098, 1992.

Lin CS, Boltz RC, Blake JT, Nguyen M, Talento A, Fischer PA, Springer MS, Sigal NH, Slaughter RS, Garcia ML, Kaczorowski GJ, Koo GC. Voltage-gated potassium channels regulate calcium-dependent pathways involved in human T lymphocyte activation. J Exp Med 177:637–645, 1993.

Wulff H, Calabresi PA, Allie R, Yun S, Pennington M, Beeton C, Chandy KG. The voltage-gated Kv1.3 K+ channel in effector memory T cells as a new target for MS. J Clin Invest 111:1703–1713, 2003.

Wulff H, Knaus H-G, Pennington M, Chandy KG. K+ channel expression during B cell differentiation: implications for immunomodulation and autoimmunity. J Immunol 173:776–786, 2004.

Schmitz A, Sankaranarayanan A, Azam P, Schmidt-Lassen K, Homerick D, Hansel W, Wulff H. Design of PAP-1, a selective small molecule Kv1.3 blocker, for the suppression of effector memory T cells in autoimmune diseases. Mol Pharmacol 68:1254–1270, 2005.

Wulff H, Miller MJ, Hansel W, Grissmer S, Cahalan MD, Chandy KG. Design of a potent and selective inhibitor of the intermediate-conductance Ca2+-activate K+ channel, IKCa1: a potential immunosuppressant. Proc Natl Acad Sci U S A 97:8151–8156, 2000.

Beeton C, Pennington MW, Wulff H, Singh S, Nugent D, Crossley G, Khaytin I, Calabresi PA, Chen CY, Gutman GA, Chandy KG. Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases. Mol Pharmacol 67:1369–1381, 2005.

10.

Beeton C, Wulff H, Barbaria J, Clot-Faybesse O, Pennington M, Bernard D, Cahalan MD, Chandy KG, Beraud E. Selective blockade of T lymphocyte K+ channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. Proc Natl Acad Sci U S A 98:13942–13947, 2001.

11.

Beeton C, Wulff H, Standifer NE, Azam P, Mullen KM, Pennington MW, Kolski-Andreaco A, Wei E, Grino A, Counts DR, Wang PH, LeeHealey CJ, Andrews BS, Sankaranarayanan A, Homerick D, Roeck WW, Tehranzadeh J, Stanhope KL, Zimin P, Havel PJ, Griffey S, Knaus H-G, Nepom GT, Gutman GA, Calabresi PA, Chandy KG. Kv1.3 channels are a therapeutic target for T cell-mediated autoimmune diseases. Proc Natl Acad Sci U S A 103:17414–17419, 2006.

12.

Firpo PP, Axberg I, Scheibel M, Clark EA. Macaque CD4+ T-cell subsets: influence of activation on infection by simian immunodeficiency viruses (SIV). AIDS Res Hum Retroviruses 8:357–366, 1992.

13.

Kaur A, Grant RM, Means RE, McClure H, Feinberg M, Johnson RP. Diverse host responses and outcomes following simian immunodeficiency virus SIVmac239 infection in sooty mangabeys and rhesus macaques. J Virol 72:9597–9611, 1998.

14.

Veazey RS, Tham IC, Mansfield KG, DeMaria M, Forand AE, Shvetz DE, Chalifoux LV, Sehgal PK, Lackner AA. Identifying the target cell in primary simian immunodeficiency virus (SIV) infection: highly activated memory CD4(+) T cells are rapidly eliminated in early SIV infection in vivo. J Virol 74:57–64, 2000.

15.

Finzi D, Hermankova M, Pierson T, Carruth LM, Buck C, Chaisson RE, Quinn TC, Chadwick K, Margolick J, Brookmeyer R, Gallant J, Markowitz M, Ho DD, Richman DD, Siliciano RF. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 278:1295–1300, 1997.

16.

Finzi D, Siliciano RF. Viral dynamics in HIV-1 infection. Cell 93: 6314–6319, 1998.

17.

Hazenberg MD, Otto SA, Benthem BHV, Roos MT, Coutinho RA, Lange JM, Hamann D, Prins M, Miedema F. Persistent immune activation in HIV-1 infection is associated with progression to AIDS. AIDS 17:1881–1888, 2003.

18.

Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector memory T cell subsets: Function, Generation, and Maintenance. Ann Rev Immunol 22:745–763, 2004.

19.

Song K, Rabin RL, Hill BJ, Rosa SCD, Perfetto SP, Zhang HH, Foley JF, Reiner JS, Liu J, Mattapallil JJ, Douek DC, Roederer M, Farber JM. Characterization of subsets of Cd4+ memory T cells reveals early branched pathways of T cell differentiation in humans. Proc Natl Acad Sci U S A 102:7916–7921, 2005.

20.

Kuroda MJ, Schmitz JE, Charini WA, Nickerson CE, Lifton MA, Lord CI, Forman MA, Letvin NL. Emergence of CTL coincides with clearance of virus during primary simian immunodeficiency virus infection in rhesus monkeys. J Immunol 162:5127–5133, 1999.

21.

National Research Council. Guide for the care and use of laboratory animals. Washington DC: National Academic Press, 1996.

22.

Ansari AA, Bostik P, Mayne AE, Villinger F. Failure to expand influenza and tetanus toxoid memory T cells in vitro correlates with disease course in SIV infected rhesus macaques. Cell Immunol 210: 125–142, 2001.

23.

Pitcher CJ, Hagen SI, Walker JM, Lum R, Mitchell BL, Maino VC, Axthelm MK, Picker LJ. Development and homeostasis of T cell memory in rhesus macaque. J Immunol 168:29–43, 2002.

24.

Knuchel M, Bednarik DP, Chikkala N, Ansari AA. Biphasic in vitro regulation of retroviral replication by CD8+ cells from nonhuman primates J Acquir Immune Defic Syndr 7:438–446, 1994.

25.

Knuchel M, Bednarik DP, Chikkala N, Villinger F, Folks TM, Ansari AA. Development of a novel quantitative assay for the measurement of chloramphenicol acetyl transferase (CAT) mRNA. J Virol Methods 48: 325–338, 1994.

26.

Kaur A, Hale CL, Noren B, Kassis N, Simon MA, Johnson RP. Decreased frequency of cytomegalovirus (CMV)-specific CD4+ T lymphocytes in simian immunodeficiency virus-infected rhesus macaques: inverse relationship with CMV viremia. J Virol 76:3646–3658, 2002.

27.

Grissmer S, Dethlefs B, Wasmuth JJ, Goldin AL, Gutman GA, Cahalan MD, Chandy KG. Expression and chromosomal localization of a lymphocyte K+ channel gene. Proc Natl Acad Sci U S A 87:9411–9415, 1990.

28.

Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708–712, 1999.

29.

Bennett JS, Gossett KA, McCarthy MP, Simpson ED. Effects of ketamine hydrochloride on serum biological and hematological variables in rhesus monkeys (Macaca mulatta). Vet Clin Pathol 21: 15–18, 1992.

30.

Kim C-Y, Lee H-S, Han S-C, Heo J-D, Kwon M-S, Ha C-S, Han S-S. Hematological and serum biochemical values in cynomologus monkeys anesthesized with ketamine hydrochloride. J Med Primatol 34:96–100, 2005.

31.

Kaur A, Daniel MD, Hempel D, Martin D, Hirsch S, Johnson RP. Cytotoxic T-Lymphocyte Responses to Cytomegalovirus Normal and Simian Immunodeficiency Virus-Rhesus Macaques. J Virol 70:7725–7733, 1996.

32.

Villinger F, Brice GT, Mayne AE, Bostik P, Mori K, June CH, Ansari AA. Adoptive transfer of simian immunodeficiency virus (SIV) naive autologous CD4+ cells to macaques chronically infected with SIV is sufficient to induce long-term nonprogressor status. Blood 99:590–599, 2002.

33.

Azam P, Sankaranarayanan A, Homerick D, Griffey S, Wulff H. Targeting effector memory T cells with the small molecule Kv1.3 blocker PAP-1 suppresses allergic contact dermatitis. J Invest Dermatol 127:1419–1429, 2007.

34.

Freedman BD, Fleischmann BK, Punt J, Hashimoto Y, Gaulton G, Kotlikoff MI. Identification of Kv1.1 expression by murine CD4-CD8-thymocytes: A role for voltage-dependent K + channels in murine thymocyte development. J Biol Chem 270:22406–22411, 1995.

35.

Lewis RS, Cahalan MD. Subset-specific expression of potassium channels in developing murine T lymphocytes. Science 239:771–775, 1988.

36.

Beeton C, Chandy KG. Potassium channels, memory T cells, and multiple sclerosis. Neuroscientist 11:550–562, 2005.

37.

Koo GC, Blake JT, Talento A, Nguyen M, Lin S, Sirotina A, Shah K, Mulvany K, Jr DH, Cunningham P, Wunderler DL, McManus OB, Slaughter R, Bugianesi R, Felix J, Garcia M, Williamson J, Kaczorowski G, Sigal NH, Springer MS, Feeney W. Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo. J Immunol 158:5120–5128, 1997.

38.

Asher DM, Jr CJG, Lang DJ, Gajdusek DC. Persistent shedding of cytomegalovirus in the urine of healthy monkeys (37897). Proc Soc Exp Biol Med 145:794–801, 1974.

39.

Baskin GB. Disseminated cytomegalovirus infection in immunodeficient rhesus monkeys. Am J Pathol 129:345–352, 1987.

40.

Osborn KG, Prahalada S, Lowenstine LJ, Gardner MB, Maul DH, Henrickson RV. The pathology of an epizootic of acquired immunodeficiency in in rhesus macaques. Am J Pathol 114:94–103, 1984.

41.

Vogel P, Weigler BJ, Kerr H, Hendrickx AG, Barry PA. Seroepidemiologic studies of cytomegalovirus infection in a breeding population of rhesus macaques. Lab Anim Sci 44:25–30, 1994.

42.

Kaur A, Kassis N, Hale CL, Simon M, Elliott M, Gomez-Yafal A, Lifson JD, Desrosiers RC, Wang F, Barry P, Mach M, Johnson RP. Direct relationship between suppression of virus-specific immunity and emergence of cytomelgalovirus disease in simian AIDS. J Virol 77: 5749–5758, 2003.

43.

Kean LS, Adams AB, Strobert E, Hendrix R, Gangappa S, Jones TR, Shirasugi N, Rigby MR, Hamby K, Jiang J, Bello H, Anderson D, Cardona K, Durham MM, Pearson TC, Larsen CP. Induction of chimerism in rhesus macaques through stem cell transplant and costimulation blockade-based suppression. Am J Transplant 7:320–335, 2007.

44.

Shah K, Blake JT, Huang C, Fischer P, Goo GC. Immunosuppressive effects of a Kv1.3 inhibitor. Cell Immunol 221:100–106, 2003.

45.

Silvestri G, Fedanov A, Germon S, Kozyr N, Kaiser WJ, Garber DA, McClure H, Feinberg MB, Staprans SI. Divergent host responses during primary simian immunodeficiency virus SIVsm infection of natural sooty mangabey and nonnatural rhesus macaque hosts. J Virol 79:4043–4054, 2005.

46.

Silvestri G, Sodora DL, Koup RA, Paiardini M, O’Neil SP, McClure HM, Staprans SI, Feinberg MB. Nonpathogenic SIV infection of sooty mangabeys is characterized by limited bystander immunopathology despite chronic high-level viremia. Immunity 18:441–452, 2003.