Although at one point it was unclear whether protein kinases would be therapeutically useful targets, there are now eight PTK inhibitors (imatinib, gefitinib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, sorafenib) approved by the Food and Drug Administration (FDA) for the therapy of several malignancies, including myeloproliferative as well as lymphoproliferative diseases and solid cancers. The first FDA-approved kinase inhibitor, imatinib, an inhibitor of Abl kinase, is indicated for the treatment of CML (123
). However, it is now clear that imatinib also inhibits other unrelated tyrosine kinases. This function is advantageous, in that it is efficacious in the treatment of solid cancers such as gastrointestinal stromal tumors and breast cancer. Other PTK inhibitors already approved or used in current clinical trials target the EGFR, vascular endothelial growth factor receptor, platelet-derived growth factor receptor, FLT-3, and Src family kinases. Yet, others are known to inhibit activity of multiple PTK as well as serine/threonine kinases. Interestingly, even kinase inhibitors with less specificity and inhibitory activity on multiple protein kinases (e.g. dasatinib and sunitinib) show clinical efficacy with tolerable toxicity. PTK inhibitors are even being tested in combination (e.g. dasatinib and imatinib or dasatinib and erlotinib).
Conceptually, all Jak family members might be targets in different settings; however, given the very limited function of Jak3, as exemplified in Jak3-SCID patients, the first efforts to generate Jak-selective inhibitors for therapeutic use focused on this kinase. Several advantages favor Jak3 as reasonable target to generate new immunosuppressive agents. In contrast to the other members of the Jak family, Jak3 expression appears to be most highly expressed in hematopoietic cells. Not only is Jak3 expression limited but also Jak3 function seems to be quite restricted due to its selective association with cytokine receptors. Among the Jak family members, Jak3 has an unique characteristic in that it only associates with one receptor, the γc chain, a subunit of the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. As a result, Jak3 mutations in human or germline deletion of Jak3 in mice result in immunodeficiency but not other abnormalities. Consequently, deficits are only present within lymphoid cells but have not been described in other organs.
Ten years ago, the quinazoline derivatives WHI-P131 and WHI-P154 were initially noted to have activity against glioblastoma, presumably due to activity against the EGFR (124
). These inhibitors were also thought to have activity against Jak3 kinase activity (125
), but in retrospect the compounds were neither selective nor potent Jak3 inhibitors (126
). Other compounds like tyrphostin AG490 or PNU156804 have been reported to inhibit Jak3 (127
). However, their selectivity has not been firmly established.
Potent and reasonably selective Jak3 inhibitors have been developed. Furthest along in development and clinical testing is the compound CP-690,550. A recent independent study showed that CP-690,550 has high affinity for Jak3, with little binding to unrelated kinases (129
). The in vivo
effect of CP-690,550 was first investigated in animal models of organ graft rejection. Efficacy could be shown for preventing heart or kidney rejection after transplantation without observing metabolic abnormalities or severe side effects due to immunosuppression (130
). Marked reduction in lymphocyte subsets was observed in a dose-dependent manner but was transient since after dosing cessation lymphocyte numbers began to normalize (131
). In cynomolgus monkeys, oral dosing of Jak3 mainly reduced numbers of NK cells and effector memory CD8+
T cells (132
). Prevention of graft rejection and prolongation of kidney allograft survival in cynomolgus monkeys has been suggested to be related to the decrease of NK cells and T cells and lower production levels of IFN-γ in CP-690,550-treated animals (133
). Jak3 inhibition with this compound was also effective in a transplantation setup in rodents and prevented allograft vasculopathy of transplanted aortas in rats (135
). In addition to the effects of Jak3 inhibition in transplantation models, CP-690,550 was recently shown to be effective in inflammatory diseases in rodents. Jak3 inhibition also prevented cartilage damage in a model of collagen-induced arthritis in mice and adjuvant-induced arthritis in rats. The main immunologic effect observed in the animals with experimental rheumatoid arthritis was the reduction of IL-6 production when treated with CP-690,550 (136
). The exact mechanism by which Jak3 inhibition inhibits IL-6 production, a critical cytokine that drives inflammatory destruction in rheumatoid arthritis, remains to be determined.
Since Jak3 affects the γc cytokine IL-4, it was reasonable to test CP-690,550 in asthma. In a mouse model of Th2-mediated asthma, treatment with CP-690,550 could effectively inhibit pulmonary eosinophilia. IL-4 is the central cytokine in this model of pulmonary disease and is dependent on functional association of Jak3 to the γc chain-containing IL-4R. CP-690,550 abrogated IL-4-mediated signals and inhibited IL-13, eotaxin, and the eosinophilic influx into the lungs (137
CP-690,550 is under current investigation in phase II clinical trials for the therapy of rheumatoid arthritis, psoriasis, and the prevention of renal transplant rejection. Preliminary data from first trials are promising, showing efficacy with acceptable toxicity. In a phase II study for rheumatoid arthritis, 70% to 81% of patients responded with an ACR20 improvement compared to 29% in the placebo group. ACR70 was achieved by 13 to 28% in the CP-690,550 group, whereas only in 3% of the patients in the placebo cohort an ACR70 response was observed (138
). Notably, these responses were achieved in patients that failed with standard of care therapies including methotrexate or biologics like tumor necrosis factor antagonists. Similarly, in psoriasis, a significant and dose-dependent reduction of inflammation and scaling was observed as measured by a modified PASI score (139
). A first phase I dose escalation study of CP-690,550 has also been performed in the setting of kidney transplantation. Twenty-eight patients were included in the study, 22 received CP-690,550 divided in different groups of 5 mg BID, 15 mg BID, and 30 mg BID, with 6 patients were in the placebo group. No graft loss was reported. At present there are more than a dozen clinical trials underway testing CP 690,550 in rheumatoid arthritis, psoriasis, and renal transplantation.
Although Jak3 is crucial for lymphocyte survival and proliferation and Jak3 deficiency results in severe lymphopenia in mice and human, no changes of the major CD4+
T-lymphocyte subsets have been observed in clinical studies. Consistent with preclinical studies, a decrease of the absolute CD16+
NK cell numbers has been observed in patients receiving CP-690,550 therapy (140
An important adverse event in the study of renal transplantation was an increased incidence of infections, although in this setting, the patients also received other immunosuppressive drugs. Laboratory parameters also revealed a significant reduction of hemoglobin concentration in the low and high dose group compared to the patients receiving placebo (140
). In this study, no major changes were observed in total or relative numbers of neutrophils, lymphocytes, eosinophils, basohphils, monocytes, or platelets. In contrast, dose-dependent neutropenia was noted in the rheumatoid arthritis study. A likely explanation for the effects of CP-690,550 on hemoglobulin concentrations and neutrophil counts might be related to the effect of this drug on Jak2. As discussed above, Jak2 signaling is important for Epo, although the importance of this kinase for granulocyte colony-stimulating factor signaling is less clear (38
). In vitro
, CP-690,550 has been reported to have considerable affinity for Jak2 and to a lesser extent for Tyk2 (129
). The ability of CP-690,550 to inhibit these other Jaks will be an important issue to study in the future. Because of the attractiveness of Jak3 as a target, a variety of companies have generated Jak3 inhibitors, which are at different levels of development. These include Rigel (R-348, Phase I), Pharmacopeia/Wyeth (PS-608504, preclinical), Vertex (VX-509, preclinical), and Cytopia/Novartis (preclinical).
Targeting Jak2 in MPD
The dramatic discovery that the mutations of Jak2 underlie nearly all cases of PV and many cases of ET and MPD provided a rationale for the use of Jak2 inhibitors in this setting (15
). At present, there are at least 15 clinical trials underway using various PTK inhibitors in the setting of MPD. Several companies have generated putatively selective Jak2 inhibitors, which are being tested in these disorders including S*BIO (SB1518), Exelixis (XL019), Incyte (INCB018424), TargeGen (TG101348), and Cephalon (lestaurtinib) (141
). The extent to which these compounds are truly Jak2 selective needs to be independently assessed. With respect to this issue, it is notable that the Incyte compound has been reported to inhibit both Jak1 and Jak2. Despite the fact that germline deletion of Jak1 and Jak2 are embryonically lethal, INCB018424 is presently being studied in rheumatoid arthritis and psoriasis. Moreover, the drug is also being tested in prostate cancer, multiple myeloma, AML, and CML. From all these trials, we will learn much about the consequences of inhibiting Jaks in human and the necessity for achieving specificity.
Lestaurtinib (CEP-701) is FDA designated as orphan drug for AML, which was originally thought to target FLT-3 and TrkA. However, lestaurtinib has also been reported to inhibit Jak2 and is therefore being tested in patients with Jak2 mutations (143
). Furthemore, dasatinib, a PTK inhibitor approved for use in CML, is an inhibitor of Src family PTKs and BCR-Abl. Dasatinib was not efficacious in an animal model of Jak2V617F-induced PV, but in vitro d
asatinib was able to inhibit myeloid and erythroid colony growth of peripheral blood cells from PV patients (144
). A recent analysis showed that at least at high doses (1μM), dasatinib can inhibit Jak2 activity in vitro
). How dasatinib inhibits Jak2V617F-driven proliferation is unclear, but clinical trials are underway. Finally, although imatinib has little activity towards Jak2, efficacy of imatinib is currently tested in phase II clinical trials for PV. It is very clear that the importance of selectively inhibiting PTKs is a moving target.
CP-690,550 has been reported to preferentially inhibit the signaling pathways activated by mutated Jak2 (146
). Interestingly, the inhibition observed with CP-690,550 on mutated Jak2 cells was more pronounced than in cells expressing wildtype Jak2. In vitro
, CP-690,550 at concentrations of 1μM induced apoptosis in erythroid progenitor cells of patients with PV but not from healthy controls (146
). The nanomolar binding capacity of CP-690,550 to Jak2 is very similar to the activity against the main target Jak3, and clinical trials have to show the efficacy of this primary Jak3 inhibitor in PV (129