Gregg Smith

CYT387, a selective JAK1/JAK2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and primary cells from polycythemia vera patients.

Somatic mutations in Janus kinase 2 (JAK2), including JAK2V617F, result in dysregulated JAK-signal transducer and activator transcription (STAT) signaling, which is implicated in myeloproliferative neoplasm (MPN) pathogenesis. CYT387 is an ATP-competitive small molecule that potently inhibits JAK1/JAK2 kinases (IC50=11 and 18 nM, respectively), with significantly less activity against other kinases, including JAK3 (IC50=155 nM). CYT387 inhibits growth of Ba/F3-JAK2V617F and human erythroleukemia (HEL) cells (IC50 ∼1500 nM) or Ba/F3-MPLW515L cells (IC50=200 nM), but has considerably less activity against BCR–ABL harboring K562 cells (IC=58 000 nM). Cell lines harboring mutated JAK2 alleles (CHRF-288-11 or Ba/F3-TEL-JAK2) were inhibited more potently than the corresponding pair harboring mutated JAK3 alleles (CMK or Ba/F3-TEL-JAK3), and STAT-5 phosphorylation was inhibited in HEL cells with an IC50=400 nM. Furthermore, CYT387 selectively suppressed the in vitro growth of erythroid colonies harboring JAK2V617F from polycythemia vera (PV) patients, an effect that was attenuated by exogenous erythropoietin. Overall, our data indicate that the JAK1/JAK2 selective inhibitor CYT387 has potential for efficacious treatment of MPN harboring mutated JAK2 and MPL alleles.

Morphine-3-glucuronide : evidence to support its putative role in the development of tolerance to the antinociceptive effects of morphine in the rat

&NA; Antinociceptive tolerance to morphine (MOR) was induced in groups of Sprague‐Dawley rats receiving continuous intravenous infusions of morphine sulphate administered by 3 different MOR dosing regimes. At appropriate intervals throughout each infusion period, antinociceptive testing was performed using the tail‐flick latency test and blood samples were collected. Groups of saline (SAL)‐infused control rats also underwent antinociceptive testing and blood sample collection. Complete antinociceptive tolerance developed during each MOR infusion period and was characterized by a marked decline in the degree of antinociception from values greater than 90% of the maximum possible effect (%MPE) to pre‐dosing baseline values. By contrast, %MPE values in SAL‐infused control animals and in sham‐operated rats were not significantly different from pre‐dosing values throughout the infusion period, indicating that the experimental procedures themselves did not contribute to the development of antinociceptive tolerance to MOR. In addition, the rate of MOR tolerance development was inversely proportional to the MOR infusion rate. A very significant inverse relationship was observed between the mean degree of antinociception (%MPE) and the mean plasma molar concentration ratio, [morphine‐3‐glucuronide]/[MOR], for each of the 3 MOR dosing regimes and for the cumulated data. This relationship showed that near‐maximum antinociception was attainable at ratio values less than approximately 0.50, whilst at ratio values above approximately 1.5, little or no antinociception was observed. Although %MPE was highly inversely correlated with the mean plasma morphine‐3‐glucuronide (M3G) concentrations for rats receiving regimes A and B, this was not the case for rats receiving regime C where antinociceptive tolerance was partially reversed by an increase in the morphine infusion rate part‐way through the infusion period. In addition, a poor relationship was observed between %MPE and the mean plasma MOR concentration, possibly due to the confounding presence of M3G in all samples. Thus, we may conclude from this study in Sprague‐Dawley rats that irrespective of the rate of antinociceptive tolerance development, the level of antinociception achievable appears to be highly inversely correlated with the mean [M3G]/[MOR] plasma molar concentration ratio and poorly correlated with the plasma MOR concentration, consistent with the notion that it is perhaps the balance between the excitatory effects of M3G and the inhibitory effects of MOR at the functional level which is the important determinant. Further research is required in carefully conducted studies in cancer patients to evaluate the possible contribution of the MOR metabolites. M3G and morphine‐6‐glucuronide (MbG), to increasing dosing requirements of MOR. Other factors such as disease progression and increasing psychological distress must also be borne in mind, as these will have a major influence on increasing dosing requirements of MOR in cancer patients (Portenoy 1994).

Momelotinib inhibits ACVR1/ALK2, decreases hepcidin production and ameliorates anemia of chronic disease in rodents

Patients with myelofibrosis (MF) often develop anemia and frequently become dependent on red blood cell transfusions. Results from a phase 2 study for the treatment of MF with the Janus kinase 1/2 (JAK1/2) inhibitor momelotinib (MMB) demonstrated that MMB treatment ameliorated anemia, which was unexpected for a JAK1/2 inhibitor, because erythropoietin-mediated JAK2 signaling is essential for erythropoiesis. Using a rat model of anemia of chronic disease, we demonstrated that MMB treatment can normalize hemoglobin and red blood cell numbers. We found that this positive effect is driven by direct inhibition of the bone morphogenic protein receptor kinase activin A receptor, type I (ACVR1), and the subsequent reduction of hepatocyte hepcidin production. Of note, ruxolitinib, a JAK1/2 inhibitor approved for the treatment of MF, had no inhibitory activity on this pathway. Further, we demonstrated the effect of MMB is not mediated by direct inhibition of JAK2-mediated ferroportin (FPN1) degradation, because neither MMB treatment nor myeloid-specific deletion of JAK2 affected FPN1 expression. Our data support the hypothesis that the improvement of inflammatory anemia by MMB results from inhibition of ACVR1-mediated hepcidin expression in the liver, which leads to increased mobilization of sequestered iron from cellular stores and subsequent stimulation of erythropoiesis.

Phase I trial of CYT997, a novel cytotoxic and vascular-disrupting agent.

Background:CYT997 is a novel microtubule inhibitor and vascular-disrupting agent with marked preclinical anti-tumour activity.Methods:This phase I dose-escalation study assessed the safety, tolerability, pharmacokinetics and pharmacodynamics of CYT997 administered by continuous intravenous infusion over 24 h every 3 weeks to patients with advanced solid tumours.Results:Thirty-one patients received CYT997 over 12 dose levels (7–358 mg m−2). Doses up to 202 mg m−2 were well tolerated. Dose-limiting toxicities were observed at 269 and 358 mg m−2, consisting of grade 3 prolonged corrected QT interval in two patients and grade 3 hypoxia and grade 4 dyspnea in one patient. All toxicities were reversible. The pharmacokinetics of CYT997 were linear over the entire dose range. Dynamic contrast-enhanced magnetic resonance imaging scans showed significant changes in tumour Ktrans values consistent with vascular disruption in 7 out of 11 evaluable patients treated at CYT997 doses of ⩾65 mg m−2. Moreover, plasma levels of von Willebrand factor and caspase-cleaved cytokeratin-18 increased post-treatment at higher dose levels. Among 22 patients evaluable for response, 18 achieved stable disease for >2 cycles.Conclusions:CYT997 was well tolerated at doses that were associated with pharmacodynamic evidence of vascular disruption in tumours.

The microtubule depolymerizing agent CYT997 causes extensive ablation of tumor vasculature in vivo.

The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2-yl)phenyl)urea (CYT997), reported previously by us (Bioorg Med Chem Lett 19:4639–4642, 2009; Mol Cancer Ther 8:3036–3045, 2009), is potently cytotoxic to a variety of cancer cell lines in vitro and shows antitumor activity in vivo. In addition to its cytotoxic activity, CYT997 possesses antivascular effects on tumor vasculature. To further characterize the vascular disrupting activity of CYT997 in terms of dose and temporal effects, we studied the activity of the compound on endothelial cells in vitro and on tumor blood flow in vivo by using a variety of techniques. In vitro, CYT997 is shown to potently inhibit the proliferation of vascular endothelial growth factor-stimulated human umbilical vein endothelial cells (IC50 3.7 ± 1.8 nM) and cause significant morphological changes at 100 nM, including membrane blebbing. Using the method of corrosion casting visualized with scanning electron microscopy, a single dose of CYT997 (7.5 mg/kg i.p.) in a metastatic cancer model was shown to cause destruction of tumor microvasculature in metastatic lesions. Furthermore, repeat dosing of CYT997 at 10 mg/kg and above (intraperitoneally, b.i.d.) was shown to effectively inhibit development of liver metastases. The time and dose dependence of the antivascular effects were studied in a DLD-1 colon adenocarcinoma xenograft model using the fluorescent dye Hoechst 33342. CYT997 demonstrated rapid and dose-dependent vascular shutdown, which persists for more than 24 h after a single oral dose. Together, the data demonstrate that CYT997 possesses potent antivascular activity and support continuing development of this promising compound.

Biochemical synthesis, purification and preliminary pharmacological evaluation of normorphine-3-glucuronide

Normorphine was synthesised from morphine by thermal decomposition of an N-alpha-chloroethylchloroformate adduct, and purified (> 98% purity) using semi-preparative HPLC with ultraviolet detection. Normorphine-3-glucuronide (NM3G) was biochemically synthesised using the substrate normorphine, uridine diphosphoglucuronic acid and Sprague-Dawley rat liver microsomes in a 75% yield (relative to normorphine base). The synthesised NM3G was purified by precipitation and washing with acetonitrile. Determinations of purity using HPLC with electrochemical and ultraviolet detection confirmed that the NM3G produced was of high (> 99%) purity. Mass spectrometry, fourier transform infrared spectrophotometry and nuclear magnetic resonance spectrometry confirmed the structure, especially placement of the glucuronide moiety at the 3-phenolic position and not at the 17-nitrogen. Administration of NM3G by the intracerebroventricular (icv) route to rats in doses of 2.5 and 7.5 microg resulted in the development of central nervous system (CNS) excitatory behavioural effects including myoclonus, chewing, wet-dog shakes, ataxia and explosive motor behaviour. At an icv dose of 7.5 microg, NM3G also induced short periods of tonic-clonic convulsive activity. Thus, NM3G elicits CNS excitation following supraspinal administration in a manner analogous to morphine-3-glucuronide (M3G), the major metabolite of morphine (1). Further studies are required to determine whether NM3G attenuates morphine-induced antinociception in a similar manner to M3G.

Abstract A16: Antitumor activity of CYT997: A phase II vascular disrupting agent administered orally in combination with cisplatin in a colon adenocarcinoma xenograft model

CYT997 is an orally bioavailable small molecule vascular‐disrupting agent (VDA) that is currently being investigated in Phase II clinical studies. In vitro efficacy studies have demonstrated that CYT997 exhibits potent antiproliferative activity against the DLD‐1 cell line (IC50 21 nM), with a similar potency to doxorubicin, paclitaxel and vincristine (IC50 8 – 53 nM). The antitumor and vascular targeting activity of CYT997 has been demonstrated in the current series of studies following alternate dosing regimens (single dose vs metronomic sub‐maximal daily dose) and in combination with a standard chemotherapeutic agent, cisplatin. Female Balb/C immunodeficient nude mice were implanted subcutaneously with DLD‐1 human colon adenocarcinoma fragments and efficacy was assessed via tumor caliper measurements, in addition to histological assessment of tumor vascular shutdown (Hoechst 33342 staining) and necrotic area (haematoxylin and eosin staining). Animals were administered CYT997 (dihydrochloride salt) by oral gavage and efficacy was expressed relative to vehicle control. Administration of CYT997 resulted in a time and concentration dependent shutdown of tumor vasculature. Following a single CYT997 administration (40 mg/kg), vascular shutdown was extensive and rapid (ca 95% ablation compared with vehicle control at 1h post‐dose; p Daily oral administration of CYT997 (10 mg/kg/day) resulted in sustained shutdown of tumor vasculature over 10 days of treatment (57% shutdown compared to vehicle control; p These findings demonstrate that CYT997 exhibits rapid and long lasting antivascular effects following a single oral dose or following sub‐maximal metronomic dosing. There is clear potential for increased anti‐tumor efficacy when CYT997 is dosed in combination with other common anticancer therapies. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A16.