Programmed Cell Death
Cell death has become recognized as a physiological process important in normal development, hormonal regulation of various tissues, and in regulation of the receptor repertoires of both T and B lymphocytes. A major unresolved problem is the lack of defined molecular pathways for such programmed cell death (PCD). The finding that a pattern of morphological changes is common to many examples of PCD led to the suggestion of a common mechanism, and the term apoptosis was defined to include both the morphological features and the mechanism common to such cell death (Kerr et al., Br. J. Cancer 26:239 (1972)). This concept was extended by the finding that nuclear DNA fragmentation correlates well with apoptotic morphology (Arends et al., Am. J. Pathol. 136:593 (1990)), and the scientific literature contains many examples of PCD accompanied by these features. The relationship between the DNA fragmentation and cell death pathways has not been elucidated. Furthermore, there are clear examples of PCD in the absence of apoptotic morphology or DNA fragmentation (Clarke, Anat. Embry. 181:195 (1990), Martin et al, J. Cell Biol. 106:829 (1988), and Ishigami et al., J. Immunol. 148:360 (1992)). New approaches to defining the molecular pathways of PCD and agents for inhibiting this process are clearly needed.
Lymphocyte-mediated cytotoxicity has been considered to be an example of apoptotic death since the target cells often (but not always) show DNA fragmentation (Sellins et al., J. Immunol. 147:795 (1991)) and apoptotic morphology (Webb et al., Cell 63:1249 (1990)). However, since in most cases there is no requirement for RNA or protein synthesis, this type of cell death has been considered to be a different category of apoptotic death (Goldstein et al., Immunol. Rev. 121:29 (1991)).
Infection by the human immunodeficiency viruses, HIV-1 and HIV-2, typically results in an early asymptomatic phase. During this period there is a selective loss of the ability of helper T (T.sub.H) cells to proliferate in vitro in response to self major histocompatibility complex (MHC) class II restricted recall antigens while retaining the capacity to respond to mitogens such as phytohemagglutinin (PHA), and to alloreactive cells. Thus, some antigen-specific memory T.sub.H cells are present and, while failing to proliferate, they show at least partial activation after stimulation in vitro.
HIV infection affects CD4.sup.+ T.sub.H cells in two different ways: qualitative CD4.sup.+ T.sub.H cell functional defects can be detected very early, when only 1 in 10,000 to 1 in 1,000 T.sub.H cells are infected, followed several months or years later by quantitative decrease in the T.sub.H cell population that will lead to acquired immune deficiency syndrome (AIDS). It has been proposed that an inappropriate activation-induced T cell death, PCD, can account for both functional and numerical abnormalities of T.sub.H cells from HIV-infected patients, which leads to near complete collapse of a patient's immune system. Ameisen and Capron, Immunol. Today 12: 102-104 (1991); Ameisen, Immunol. Today 13: 388-392 (1992). According to this theory, HIV infection leads to an early priming of T.sub.H cells for a suicide process upon stimulation by antigen. In vivo, T.sub.H cell death after activation will progressively lead to a detectable decrease in the T.sub.H cell population. Thus, T.sub.H cells do not proliferate, but rather, the antigen induces T.sub.H cell death.
According to the programmed cell death model for HIV pathogenesis, the decline in immune reactivity and number of CD4+ lymphocytes associated with HIV infection is due to T cell receptor-induced programmed cell death (PCD) in CD4+ lymphocytes after contact with antigen. This abnormal response to antigen has been shown to be triggered by antigen in vitro after prior cross-linking of CD4 with either anti-CD4 antibody or with viral gp120 and anti-gp120. Calpain
Calpain is a calcium-activated neutral protease located in the cytoplasm of many cell types. It is a member of the cysteine protease family, in common with several lysosomal cathepsins. There are two isoenzymes known as calpain I and II, which differ in their in vitro calcium requirements of activation, and in the amino acid sequence of the larger of the two peptide chains comprising the enzyme. Both calpain heavy chains and the common light chain have been sequenced after cDNA cloning (Suzuki, in Intracellular Ca-Dependent Proteolysis, Mellgren and Murachi, eds., CRC Press, Boca Raton, Fla., pp. 26-35 (1990)). All contain a calmodulin domain. Calpain activity regulation in situ is complex, being affected by the endogenous inhibitory protein calpastatin, proteolytic processing, interaction with membranes, and cytoplasmic calcium levels. Croall and Demartino, Physiol. Rev. 71: 813 (1991). Lymphocyte activation triggers a rapid rise in internal calcium, and lymphocytes have also been found to have the highest levels of calpains I and II of any cells measured (Murachi, Biochem. Int. 18:263 (1989)).
In spite of considerable study, the physiological role of calpain is not established (Croall and Demartino, supra). In platelets, calpain has been implicated in activation-induced vesiculation, apparently by cleavage of platelet cytoskeletal proteins (Fox et al., J. Biol. Chem. 266: 13289 (1991)).
A number of calpain inhibitors have been developed (Wang, Trends Pharmacol. Sci. 11: 139 (1990)). Some of these, e.g., E-64 and leupeptin, are effective on cysteine proteases generally, while others such as ALLnM have a selective effect on calpain. Although ALLnM is quite selective for calpain, it does react slowly with papain, another member of the cysteine protease family.
To date, calpain inhibitors have been of therapeutic interest principally in two clinical situations. One is in stroke, where there is evidence that increases in cytoplasmic calcium are responsible for nerve cell damage and death associated with ischemia. Since a specific calpain inhibitor blocked both neuronal death and brain spectrin proteolysis induced by ischemia, it has been suggested that calpain inhibitors may be useful as a treatment for stroke (Lee et al., Proc. Natl. Acad. Sci. 88: 7233 (1991)).
The second disease in which calpain inhibitors have been considered for use in therapy is muscular dystrophy. The most severe form of this disease, Duchenne muscular dystrophy, is a fatal genetic disease caused by a lack of expression of the cytoskeletal protein dystrophin. Muscle degeneration in this condition is accompanied by increases in intracellular calcium and Z-band loss, suggesting calpain activation. It was hypothesized that blocking calpain would prevent the subsequent steps in muscle cell damage and death, and calpain inhibitors were thus sought for therapy (Satoyoshi, Intern. Med. 31: 841 (1992)).
The cysteine protease inhibitor E-64 is a natural product, secreted by the fungus Aspergillis japonica. It is remarkably non-toxic to cells in vitro, and a number of experiments have been described in which it was given in vivo (Amamoto et al., Biochem. Biophys. Res. Comm. 118: 117 (1984); Hanada et al., in Proteinase Inhibitors: Medical and Biological Aspects, Katunuma, ed., Springer-Verlag, Tokyo, pp. 25-36(1983)). Since it inhibits lysosomal cathepsins B, H, and L, as well as calpain, it was considered as a potential agent which could block the muscle damage associated with muscular dystrophy. A series of E-64 derivatives has been prepared and their properties studied with respect to potential therapeutic use (Hanada et al., supra). Modification of E-64 to the compound E-64c, gave approximately equivalent potency to E-64. Both E-64 and E-64c are active when injected intraperitoneally but inactive when delivered orally. The ethyl ester of E-64c, termed EST (also known as E-64d, Loxistatin, or Ep-453) is readily absorbed from the gut and produce .mu.molar levels of E-64c in plasma several hours after administration (Hanada et al., supra).
What is needed in the art is a means to treat or prevent the decline in immune function that is typically associated with immunodeficiency diseases, particularly infection by HIV, that results in a collapse of the immune system and eventual death of the patient from secondary infections. Desirably, the treatment would be effective in the early asymptomatic phases of disease, when then progressive decline in the ability of T.sub.H cells to proliferate in response to antigens is initiated. Moreover, it would be extremely helpful to identify agents that are capable of restoring an immune system that has been devastated to near total loss by PCD. Quite surprisingly, the present invention fulfills these and other related needs.