Because of its requirement for signaling by multiple cytokines, Janus kinase 3 (JAK3) is an excellent target for clinical immunosuppression. We report the development of a specific, orally active inhibitor of JAK3, CP-690,550, that significantly prolonged survival in a murine model of heart transplantation and in cynomolgus monkeys receiving kidney transplants. CP-690,550 treatment was not associated with hypertension, hyperlipidemia, or lymphoproliferative disease. On the basis of these preclinical results, we believe JAK3 blockade by CP-690,550 has potential for therapeutically desirable immunosuppression in human organ transplantation and in other clinical settings.

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Purpose: The insulin-like growth factor (IGF) signaling pathway is implicated in cellular mitogenesis, angiogenesis, tumor cell survival, and tumorigenesis. Inhibition of this pathway results in decreased cell growth, inhibition of tumor formation in animal models, and increased apoptosis in cells treated with cytotoxic chemotherapy. We generated and characterized a human monoclonal antibody that targeted the IGF receptor.Experimental Design: By use of XenoMouse technology, we generated CP-751,871, a fully human IgG2 antibody with high affinity (K d = 1.5 nmol/L) for human IGF-1R and evaluated its biological, pharmacologic, and antitumor properties.Results: This antibody blocks binding of IGF-1 to its receptor (IC 50 1.8 nmol/L), IGF-1-induced receptor autophosphorylation (IC 50 0.42 nmol/L) and induced the downregulation of IGF-1R in vitro and in tumor xenografts. The extent of IGF-1R down-regulation in vivo was proportional to CP-751,871 concentrations in the serum of tumor-bearing mice. Pharmacokinetic profiles in cynomolgus monkeys indicated a close to linear increase of exposure following i.v. dosing of antibody in the range of 3 to 100 mg/kg. CP-751,871 showed significant antitumor activity both as a single agent and in combination with Adriamycin, 5-fluorouracil, or tamoxifen in multiple tumor models. A biomarker assay was developed to establish the relationship between circulating antibody concentrations and down-regulation of IGF-1R in peripheral blood cells. The concentration of CP-751,871 required to down-regulate 50% of IGF-1R on peripheral blood cells was 0.3 nmol/L. Conclusion:These data suggest that inhibition of the IGF cascade by use of this monoclonal antibody may be of clinical benefit in the treatment of human cancers.

The erbB-2 oncogene encodes a transmembrane protein tyrosine kinase which plays a pivotal role in signal transduction and has been implicated when overexpressed in breast, ovarian, and gastric cancers. Naturally occurring benzoquinoid ansamycin antibiotics herbimycin A, geldanamycin (GDM), and dihydrogeldanamycin were found to potently deplete p185, the erbB-2 oncoprotein, in human breast cancer SKBR-3 cells in culture. Chemistry efforts to modify selectively the quinoid moiety of GDM afforded derivatives with greater potency in vitro and in vivo. Analogs demonstrated inhibition of p185 phosphotyrosine in cell culture and in vivo after systemic drug administration to nu/nu nude mice bearing Fisher rat embryo cells transfected with human erbB-2 (FRE/erbB-2). Specifically, dosed intraperitoneally at 100 mg/kg, 17-(allylamino)-17-demethoxygeldanamycin and other 17-amino analogs were effective at reducing p185 phosphotyrosine in subcutaneous flank FRE/erbB-2 tumors. Modifications to the 17-19-positions of the quinone ring revealed a broad structure-activity relationship in vitro.

Heregulin is a ligand for the erbB3 and erbB4 receptors, with a region of high homology to epidermal growth factor (EGF). Despite this homology, these ligands bind to their corresponding receptors with great specificity. We report here the synthesis of heregulin beta 177-241 and show that a region consisting of amino acids 177-226 is sufficient both for binding and stimulation of receptor phosphorylation. Studies of chimeric EGF/heregulin peptides revealed that amino acids 177-181 of heregulin provide the specificity for binding to the heregulin receptor. The substitution of amino acids 177-181 of heregulin for the N terminus of EGF produced a unique bifunctional agonist that binds with high affinity to both the EGF receptor and the heregulin receptor.

Virulence of poxviruses, the causative agents of smallpox, depends on virus-encoded growth factors related to the mammalian epidermal growth factor (EGF). Here we report that the growth factors of Shope fibroma virus, Myxoma virus and vaccinia virus (SFGF, MGF and VGF) display unique patterns of specificity to ErbB receptor tyrosine kinases; whereas SFGF is a broad-specificity ligand, VGF binds primarily to ErbB-1 homodimers, and the exclusive receptor for MGF is a heterodimer comprised of ErbB-2 and ErbB-3. In spite of 10- to 1000-fold lower binding affinity to their respective receptors, the viral ligands are mitogenically equivalent or even more potent than their mammalian counterparts. This remarkable enhancement of cell growth is due to attenuation of receptor degradation and ubiquitination, which leads to sustained signal transduction. Our results imply that signal potentiation and precise targeting to specific receptor combinations contribute to cell transformation at sites of poxvirus infection, and they underscore the importance of the often ignored low-affinity ligand-receptor interactions.

Evidence is presented that the liver effects an essentially complete degradation of plasma uridine in a single pass and replaces it largely from hepatic pools of acid-soluble uridine nucleotides. The concentration of uridine in the hepatic vein of the rat was essentially the same as that in the arterial circulation and portal vein. However, the isolated perfused rat liver degraded more than 90 percent of infused [5-3H]uridine in a single passage. Similar results were found in vivo when tracer amounts of [3H]uridine and [14C]uridine were infused into the portal vein of an intact rat. Furthermore, less than 2 percent of the infused uridine entered the acid-soluble nucleotide pools of the liver after 30 minutes of infusion. Intraperitoneal injection of [3H]orotate allowed selective labeling of liver (and kidney) pyrimidines. After 3 hours, the specific activity of uridine in the hepatic vein was more than three times that in the arterial circulation. This unusual exchange, which is not saturated even at uridine concentrations as high as 50 microM, contributes to the rapid turnover of plasma uridine and explains its inefficient utilization in peripheral tissues.

A high-performance-liquid-chromatography (h.p.l.c.) separation was developed, which resolves isomers of inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) in a single run. In GH3 cells labelled with [3H]inositol, treated with Li+ and thyrotropin-releasing hormone (TRH), radiolabelled components identified as inositol 1-phosphate (I1P), inositol 2-phosphate (I2P), inositol 4-phosphate (I4P), inositol 1,4-bisphosphate [I(1,4)P2], inositol 1,3,4-trisphosphate [I(1,3,4)P3] and inositol 1,4,5-trisphosphate [I(1,4,5)P3] are present, as are multiple unidentified IP2 peaks. After TRH stimulation, both I1P and I4P increase, the increase in I4P preceding that of I1P; I(1,4)P2 and an unknown IP2 increase; and both I(1,3,4)P3 and I(1,4,5)P3 increase, the increase in I(1,4,5)P3 being rapid and transient, whereas the increase in I(1,3,4)P3 is slower and more sustained. The most rapidly appearing inositol phosphates produced after TRH stimulation are I(1,4)P2 and I(1,4,5)P3.