We report that TG101348, a selective small-molecule inhibitor of JAK2 with an in vitro IC50 of approximately 3 nM, shows therapeutic efficacy in a murine model of myeloproliferative disease induced by the JAK2V617F mutation. In treated animals, there was a statistically significant reduction in hematocrit and leukocyte count, a dose-dependent reduction/elimination of extramedullary hematopoiesis, and, at least in some instances, evidence for attenuation of myelofibrosis. There were no apparent toxicities and no effect on T cell number. In vivo responses were correlated with surrogate endpoints, including reduction/elimination of JAK2V617F disease burden assessed by quantitative genomic PCR, suppression of endogenous erythroid colony formation, and in vivo inhibition of JAK-STAT signal transduction as assessed by flow cytometric measurement of phosphorylated Stat5.

JAK2V617F and MPLW515L/K represent recently identified mutations in myeloproliferative disorders (MPD) that cause dysregulated JAK-STAT signaling, which is implicated in MPD pathogenesis. We developed TG101209, an orally bioavailable small molecule that potently inhibits JAK2 (IC 50 ¼ 6 nM), FLT3 (IC 50 ¼ 25 nM) and RET (IC 50 ¼ 17 nM) kinases, with significantly less activity against other tyrosine kinases including JAK3 (IC 50 ¼ 169 nM). TG101209 inhibited growth of Ba/F3 cells expressing JAK2V617F or MPLW515L mutations with an IC 50 of B200 nM. In a human JAK2V617F-expressing acute myeloid leukemia cell line, TG101209-induced cell cycle arrest and apoptosis, and inhibited phosphorylation of JAK2V617F, STAT5 and STAT3. Therapeutic efficacy of TG101209 was demonstrated in a nude mouse model. Furthermore, TG101209 suppressed growth of hematopoietic colonies from primary progenitor cells harboring JAK2V617F or MPL515 mutations. Leukemia (2007) IntroductionAcquisition of somatic mutations such as JAK2V617F 1 results in constitutive activation of JAK-STAT signaling, which is thought to play a primary role in the pathogenesis of myeloproliferative disorders (MPD) including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). In normal hematopoiesis, ligand-induced activation of a spectrum of hematopoietic cytokine receptors, including receptors for erythropoietin, thrombopoietin (MPL) and granulocyte colony stimulating factor, converges upon Janus kinase 2 (JAK2). The importance of JAK2 in hematopoiesis has been demonstrated in mice that are genetically deficient in JAK2, and have severe defects in erythropoiesis. 2 Dysregulated JAK-STAT signaling may be important in JAK2V617F-negative MPD as well, in that other activating JAK2 alleles have been identified in these patients, including JAK2 exon 12 mutations, JAK2D620E, JAK2DIREED, in addition to activating mutations in MPL at position W515. [3][4][5][6] Expression of JAK2V617F in vivo in a murine bone marrow transplant assay results in a phenotype resembling PV; 7,8 in contrast, MPLW515L expression in a similar assay results in a PMF-like phenotype. 9 Inhibition of JAK2 with small molecule 'tool' compounds that lack potential for clinical development, and that are not selective among JAK family members, induce apoptotic cell death in hematopoietic cell lines transformed either with JAK2V617F 10 or with MPLW515L, 9 and, similarly, may decrease the hematocrit in mouse models of JAK2V617F-induced disease. 8 MPD are currently not captured in the surveillance, epidemiology and end results (SEER) database or other cancer registries, but incidence of PV, ET and PMF has been estimated in the 1-5/100 000 per year range. 11 Because of relatively long survival after diagnosis, it has been estimated that the prevalence of MPD is on the order of 80 000-100 000 cases in the United States, significantly higher than that of BCR-ABLpositive chronic myeloid leukemia (CML). Although the MPD are relatively indolent, most patients ultimately develop...

Purpose: Injection of pharmacotherapy into the suprachoroidal space, between the sclera and choroid, is an alternative delivery technique developed with the rationale of providing higher drug concentrations to posterior ocular structures compared with other intraocular and periocular injection procedures. This study was conducted to evaluate the safety and efficacy of suprachoroidally injected triamcinolone acetonide formulation (CLS-TA), a suspension of triamcinolone acetonide, in improving vision among patients with noninfectious uveitis complicated by macular edema (ME).Design: Phase 3 masked, randomized trial.Participants: One hundred sixty patients with ME secondary to noninfectious uveitis. Patients were required to have a best-corrected visual acuity (BCVA) of 5 or more Early Treatment Diabetic Retinopathy Study (ETDRS) letters (Snellen equivalent, 20/800) and 70 or fewer ETDRS letters read (Snellen equivalent, 20/40) in the study eye.Methods: Patients were randomized 3:2 to suprachoroidally injected CLS-TA or sham treatment, with administrations at day 0 and week 12.Main Outcome Measures: The primary end point was improvement from baseline of 15 or more ETDRS letters in BCVA at week 24. The secondary end point was reduction from baseline in central subfield thickness (CST) at week 24.Results: In the CLS-TA arm, 47% of patients gained 15 or more ETDRS letters in BCVA versus 16% in the control arm (P < 0.001), meeting the primary end point. Mean reductions in CST from baseline were 153 mm versus 18 mm (P < 0.001). No serious adverse events (AEs) related to treatment were reported. Corticosteroidassociated AEs of elevated intraocular pressure occurred in 11.5% and 15.6% of the CLS-TA and control groups, respectively. Cataract AE rates were comparable (7.3% and 6.3%, respectively). Conclusions: Patients in the CLS-TA study arm experienced clinically significant improvement in vision relative to the sham procedure, demonstrating the efficacy of suprachoroidal injection of CLS-TA for the treatment of ME in a vision-threatening disorder.

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Although phosphoinositide 3-kinases (PI3Ks) play beneficial pro-cell survival roles during tissue ischemia, some isoforms (␥ and ␦) paradoxically contribute to the inflammation that damages these same tissues upon reperfusion. We therefore considered the possibility that selectively inhibiting proinflammatory PI3K isoforms during the reperfusion phase could ultimately limit overall tissue damage seen in ischemia/reperfusion injuries such as myocardial infarction. Panreactive and isoform-restricted PI3K inhibitors were identified by screening a novel chemical family; molecular modeling studies attributed isoform specificity based on rotational freedom of substituent groups. One compound (TG100-115) identified as a selective PI3K ␥/␦ inhibitor potently inhibited edema and inflammation in response to multiple mediators known to participate in myocardial infarction, including vascular endothelial growth factor and platelet-activating factor; by contrast, endothelial cell mitogenesis, a repair process important to tissue survival after ischemic damage, was not disrupted. In rigorous animal MI models, TG100-115 provided potent cardioprotection, reducing infarct development and preserving myocardial function. Importantly, this was achieved when dosing well after myocardial reperfusion (up to 3 h after), the same time period when patients are most accessible for therapeutic intervention. In conclusion, by targeting pathologic events occurring relatively late in myocardial damage, we have identified a potential means of addressing an elusive clinical goal: meaningful cardioprotection in the postreperfusion time period.edema ͉ inflammation ͉ myocardial infarct ͉ VEGF

Retinal and choroidal vascular diseases, with their associated abnormalities in vascular permeability, account for the majority of patients with vision loss in industrialized nations. VEGF is upregulated in ischemic retinopathies such as diabetes and is known to dramatically alter vascular permeability in a number of nonocular tissues via Src kinase-regulated signaling pathways. VEGF antagonists are currently in clinical use for treating the new blood vessels and retinal edema associated with neovascular eye diseases, but such therapies require repeated intraocular injections. We have found that vascular leakage following intravitreal administration of VEGF in mice was abolished by systemic or topical delivery of what we believe is a novel VEGFR2/Src kinase inhibitor; this was confirmed in rabbits. The relevance of Src inhibition to VEGF-associated alterations in vascular permeability was further substantiated by genetic studies in which VEGF injection or laser-induced vascular permeability failed to augment retinal vascular permeability in Src -/-and Yes -/-mice (Src and Yes are ubiquitously expressed Src kinase family members; Src -/-and Yes -/-mice lacking expression of these kinases show no vascular leak in response to VEGF). These findings establish a role for Src kinase in VEGF-mediated retinal vascular permeability and establish a potentially safe and painless topically applied therapeutic option for treating vision loss due to neovascular-associated retinal edema.

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Retinal gene therapy using adeno-associated viruses (AAVs) is constrained by the mode of viral vector delivery. Intravitreal AAV injections are impeded by the internal limiting membrane barrier, while subretinal injections require invasive surgery and produce a limited region of therapeutic effect. In this study, we introduce a novel mode of ocular gene delivery in rhesus macaques using transscleral microneedles to inject AAV8 into the subretinal or suprachoroidal space, a potential space between the choroid and scleral wall of the eye. Using in vivo imaging, we found that suprachoroidal AAV8 produces diffuse, peripheral expression in retinal pigment epithelial (RPE) cells, but it elicited local infiltration of inflammatory cells. Transscleral subretinal injection of AAV8 using microneedles leads to focal gene expression with transduction of RPE and photoreceptors, and minimal intraocular inflammation. In comparison, intravitreal AAV8 shows minimal transduction of retinal cells, but elicits greater systemic humoral immune responses. Our study introduces a novel mode of transscleral viral delivery that can be performed without vitreoretinal surgery, with focal or diffuse transgene expression patterns suitable for different applications. The decoupling of local and systemic immune responses reveals important insights into the immunological consequences of AAV delivery to different ocular compartments surrounding the bloodretinal barrier.