The adiponectin-derived peptide ALY688 protects against the development of metabolic dysfunction-associated steatohepatitis.

Metabolic dysfunction-associated steatohepatitis (MASH) is the severe form of non-alcoholic fatty liver disease which has a high potential to progress to cirrhosis and hepatocellular carcinoma, yet adequate effective therapies are lacking. Hypoadiponectinemia is causally involved in the pathogenesis of MASH. This study investigated the pharmacological effects of adiponectin replacement therapy with the adiponectin-derived peptide ALY688 (ALY688-SR) in a mouse model of MASH. Human induced pluripotent stem (iPS) cell-derived hepatocytes were used to test cytotoxicity and signaling of unmodified ALY688 in vitro. High-fat diet with low methionine and no added choline (CDAHF) was used to induce MASH and test the effects of ALY688-SR in vivo. Histological MASH activity score (NAS) and fibrosis score were determined to assess the effect of ALY688-SR. Transcriptional characterization of mice through RNA sequencing was performed to indicate potential molecular mechanisms involved. In cultured hepatocytes, ALY688 efficiently induced adiponectin-like signaling, including the AMP-activated protein kinase and p38 mitogen-activated protein kinase pathways, and did not elicit cytotoxicity. Administration of ALY688-SR in mice did not influence body weight but significantly ameliorated CDAHF-induced hepatic steatosis, inflammation, and fibrosis, therefore effectively preventing the development and progression of MASH. Mechanistically, ALY688-SR treatment markedly induced hepatic expression of genes involved in fatty acid oxidation, whereas it significantly suppressed the expression of pro-inflammatory and pro-fibrotic genes as demonstrated by transcriptomic analysis. ALY688-SR may represent an effective approach in MASH treatment. Its mode of action involves inhibition of hepatic steatosis, inflammation, and fibrosis, possibly via canonical adiponectin-mediated signaling.

A novel blood-based bioassay to monitor adiponectin signaling.

The diverse beneficial effects of adiponectin-receptor signaling, including its impact on the regulation of inflammatory processes in vivo, have resulted in development of adiponectin receptor agonists as a treatment for metabolic disorders. However, there are no established non-invasive bioassays for detection of adiponectin target engagement in humans or animal models. Here, we designed an assay using small amounts of blood to assess adiponectin action. Specifically, we tested effects of the small 10-amino acid peptide adiponectin receptor agonist, ALY688, in a sublethal LPS endotoxemia model in mice. LPS-induced pro-inflammatory cytokine levels in serum were significantly reduced in mice treated with ALY688, assessed via multiplex ELISA in flow cytometry. Furthermore, ALY688 alone significantly induced TGF-ß release in serum 1 h after treatment and was elevated for up to 24 h. Additionally, using a flow-cytometry panel for detection of changes in circulating immune cell phenotypes, we observed a significant increase in absolute T cell counts in mice after ALY688 treatment. To assess changes in intracellular signaling effectors downstream of adiponectin, phospho-flow cytometry was conducted. There was a significant increase in phosphorylation of AMPK and p38-MAPK in mice after ALY688 treatment. We then used human donor immune cells (PBMCs) treated with ALY688 ex vivo and observed elevation of AMPK and p38-MAPK phosphorylation from baseline in response to ALY688. Together, these results indicate we can detect adiponectin action on immune cells in vivo by assessing adiponectin signaling pathway for AMPK and p38-MAPK, as well as pro-inflammatory cytokine levels. This new approach provides a blood-based bioassay for screening adiponectin action.

Cardioprotection by the adiponectin receptor agonist ALY688 in a preclinical mouse model of heart failure with reduced ejection fraction (HFrEF).

AIMS: Adiponectin has been shown to mediate cardioprotective effects and levels are typically reduced in patients with cardiometabolic disease. Hence, there has been intense interest in developing adiponectin-based therapeutics. The aim of this translational research study was to examine the functional significance of targeting adiponectin signaling with the adiponectin receptor agonist ALY688 in a mouse model of heart failure with reduced ejection fraction (HFrEF), and the mechanisms of cardiac remodeling leading to cardioprotection. METHODS AND RESULTS: Wild-type mice were subjected to transverse aortic constriction (TAC) to induce left ventricular pressure overload (PO), or sham surgery, with or without daily subcutaneous ALY688-SR administration. Temporal analysis of cardiac function was conducted via weekly echocardiography for 5 weeks and we observed that ALY688 attenuated the PO-induced dysfunction. ALY688 also reduced cardiac hypertrophic remodeling, assessed via LV mass, heart weight to body weight ratio, cardiomyocyte cross sectional area, ANP and BNP levels. ALY688 also attenuated PO-induced changes in myosin light and heavy chain expression. Collagen content and myofibroblast profile indicated that fibrosis was attenuated by ALY688 with TIMP1 and scleraxis/periostin identified as potential mechanistic contributors. ALY688 reduced PO-induced elevation in circulating cytokines including IL-5, IL-13 and IL-17, and the chemoattractants MCP-1, MIP-1ß, MIP-1alpha and MIP-3α. Assessment of myocardial transcript levels indicated that ALY688 suppressed PO-induced elevations in IL-6, TLR-4 and IL-1ß, collectively indicating anti-inflammatory effects. Targeted metabolomic profiling indicated that ALY688 increased fatty acid mobilization and oxidation, increased betaine and putrescine plus decreased sphingomyelin and lysophospholipids, a profile indicative of improved insulin sensitivity. CONCLUSION: These results indicate that the adiponectin mimetic peptide ALY688 reduced PO-induced fibrosis, hypertrophy, inflammation and metabolic dysfunction and represents a promising therapeutic approach for treating HFrEF in a clinical setting.

The slow-release adiponectin analog ALY688-SR modifies early-stage disease development in the D2.mdx mouse model of Duchenne muscular dystrophy.

Fibrosis is associated with respiratory and limb muscle atrophy in Duchenne muscular dystrophy (DMD). Current standard of care partially delays the progression of this myopathy but there remains an unmet need to develop additional therapies. Adiponectin receptor agonism has emerged as a possible therapeutic target to lower inflammation and improve metabolism in mdx mouse models of DMD but the degree to which fibrosis and atrophy are prevented remain unknown. Here, we demonstrate that the recently developed slow-release peptidomimetic adiponectin analog, ALY688-SR, remodels the diaphragm of murine model of DMD on DBA background (D2.mdx) mice treated from days 7-28 of age during early stages of disease. ALY688-SR also lowered interleukin-6 (IL-6) mRNA but increased IL-6 and transforming growth factor-ß1 (TGF-ß1) protein contents in diaphragm, suggesting dynamic inflammatory remodeling. ALY688-SR alleviated mitochondrial redox stress by decreasing complex I-stimulated H2O2 emission. Treatment also attenuated fibrosis, fiber type-specific atrophy, and in vitro diaphragm force production in diaphragm suggesting a complex relationship between adiponectin receptor activity, muscle remodeling, and force-generating properties during the very early stages of disease progression in murine model of DMD on DBA background (D2.mdx) mice. In tibialis anterior, the modest fibrosis at this young age was not altered by treatment, and atrophy was not apparent at this young age. These results demonstrate that short-term treatment of ALY688-SR in young D2.mdx mice partially prevents fibrosis and fiber type-specific atrophy and lowers force production in the more disease-apparent diaphragm in relation to lower mitochondrial redox stress and heterogeneous responses in certain inflammatory markers. These diverse muscle responses to adiponectin receptor agonism in early stages of DMD serve as a foundation for further mechanistic investigations.NEW & NOTEWORTHY There are limited therapies for the treatment of Duchenne muscular dystrophy. As fibrosis involves an accumulation of collagen that replaces muscle fibers, antifibrotics may help preserve muscle function. We report that the novel adiponectin receptor agonist ALY688-SR prevents fibrosis in the diaphragm of D2.mdx mice with short-term treatment early in disease progression. These responses were related to altered inflammation and mitochondrial functions and serve as a foundation for the development of this class of therapy.

Phase â /â ¡, Double-Masked, Randomized, Vehicle-Controlled Study of H-1337 Ophthalmic Solution for Glaucoma and Ocular Hypertension.

PURPOSE: To perform a phase Ⅰ/Ⅱ evaluation of an H-1337 ophthalmic solution in subjects with primary open-angle glaucoma (POAG) or ocular hypertension (OHT). DESIGN: This was a phase I/II, randomized, double-masked, vehicle-controlled, dose-response study conducted at 6 private practice sites in the United States. The study was registered with clinicaltrials.gov as NCT03452033. PARTICIPANTS: Eighty-seven subjects with bilateral POAG or OHT were enrolled. METHODS: After washout of ocular hypotensive medications as required, the subjects were randomized to receive either the H-1337 ophthalmic solution at 0.06%, 0.2%, and 0.6% or its vehicle twice daily unilaterally in the study eye for the first 3 days and then twice daily in both eyes from day 4 to 28. MAIN OUTCOME MEASURES: The primary efficacy end point was the mean change in intraocular pressure from baseline (day 0) for each group on day 28 at hour 4 compared with the vehicle. RESULTS: In the primary efficacy end point, i.e., mean change from the baseline on day 28 at hour 4, the mean change from the baseline was - 4.45 ± 3.801, - 5.16 ± 3.114, - 4.93 ± 3.110, and - 0.39 ± 2.355 in the 0.06%, 0.2%, and 0.6% H-1337 and vehicle groups, respectively. The difference between each active group and the vehicle group was statistically significant (P < 0.0001). Treatment-emergent adverse events (TEAEs) occurred in 49% of subjects who received H-1337 (range, 41% [0.2% arm]-64% [0.6% arm] across the H-1337 arms) and 18% of subjects who received the vehicle. The majority of TEAEs were mild in severity; 3 subjects who received H-1337 had a TEAE of moderate intensity (instillation site erythema, blurred vision, and muscle strain). CONCLUSIONS: The H-1337 ophthalmic solution showed clinically and statistically significant ocular hypotensive activity and was well tolerated, with a relatively low incidence of hyperemia. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

CSF1 receptor inhibition of tenosynovial giant cell tumor using novel disease-specific MRI measures of tumor burden.

Aim: Monitoring treatment of tenosynovial giant cell tumor (TGCT) is complicated by the irregular shape and asymmetrical growth of the tumor. We compared responses to pexidartinib by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 with those by tumor volume score (TVS) and modified RECIST (m-RECIST). Materials & methods: MRIs acquired every two cycles were assessed centrally using RECIST 1.1, m-RECIST and TVS and tissue damage score (TDS). Results: Thirty-one evaluable TGCT patients were treated with pexidartinib. From baseline to last visit, 94% of patients (29/31) showed a decrease in tumor size (median change: -60% [RECIST], -66% [m-RECIST], -79% [TVS]). All methods showed 100% disease control rate. For TDS, improvements were seen in bone erosion (32%), bone marrow edema (58%) and knee effusion (46%). Conclusion: TVS and m-RECIST offer potentially superior alternatives to conventional RECIST for monitoring disease progression and treatment response in TGCT. TDS adds important information about joint damage associated with TGCT.

Results from Phase I Extension Study Assessing Pexidartinib Treatment in Six Cohorts with Solid Tumors including TGCT, and Abnormal CSF1 Transcripts in TGCT.

PURPOSE: To assess the response to pexidartinib treatment in six cohorts of adult patients with advanced, incurable solid tumors associated with colony-stimulating factor 1 receptor (CSF1R) and/or KIT proto-oncogene receptor tyrosine kinase activity. PATIENTS AND METHODS: From this two-part phase I, multicenter study, pexidartinib, a small-molecule tyrosine kinase inhibitor that targets CSF1R, KIT, and FMS-like tyrosine kinase 3 (FLT3), was evaluated in six adult patient cohorts (part 2, extension) with advanced solid tumors associated with dysregulated CSF1R. Adverse events, pharmacokinetics, and tumor responses were assessed for all patients; patients with tenosynovial giant cell tumor (TGCT) were also evaluated for tumor volume score (TVS) and patient-reported outcomes (PRO). CSF1 transcripts and gene expression were explored in TGCT biopsies. RESULTS: Ninety-one patients were treated: TGCT patients (n = 39) had a median treatment duration of 511 days, while other solid tumor patients (n = 52) had a median treatment duration of 56 days. TGCT patients had response rates of 62% (RECIST 1.1) and 56% (TVS) for the full analysis set. PRO assessments for pain showed improvement in patient symptoms, and 76% (19/25) of TGCT tissue biopsy specimens showed evidence of abnormal CSF1 transcripts. Pexidartinib treatment of TGCT resulted in tumor regression and symptomatic benefit in most patients. Pexidartinib toxicity was manageable over the entire study. CONCLUSIONS: These results offer insight into outcome patterns in cancers whose biology suggests use of a CSF1R inhibitor. Pexidartinib results in tumor regression in TGCT patients, providing prolonged control with an acceptable safety profile.

Recurrent Mutations in Cyclin D3 Confer Clinical Resistance to FLT3 Inhibitors in Acute Myeloid Leukemia.

PURPOSE: Biomarkers of response and resistance to FLT3 tyrosine kinase inhibitors (TKI) are still emerging, and optimal clinical combinations remain unclear. The purpose of this study is to identify co-occurring mutations that influence clinical response to the novel FLT3 inhibitor pexidartinib (PLX3397). EXPERIMENTAL DESIGN: We performed targeted sequencing of pretreatment blasts from 29 patients with FLT3 internal tandem duplication (ITD) mutations treated on the phase I/II trial of pexidartinib in relapsed/refractory FLT3-ITD+ acute myeloid leukemia (AML). We sequenced 37 samples from 29 patients with available material, including 8 responders and 21 non-responders treated at or above the recommended phase II dose of 3,000 mg. RESULTS: Consistent with other studies, we identified mutations in NRAS, TP53, IDH2, and a variety of epigenetic and transcriptional regulators only in non-responders. Among the most frequently mutated genes in non-responders was Cyclin D3 (CCND3). A total of 3 individual mutations in CCND3 (Q276*, S264R, and T283A) were identified in 2 of 21 non-responders (one patient had both Q276* and S264R). No CCND3 mutations were found in pexidartinib responders. Expression of the Q276* and T283A mutations in FLT3-ITD MV4;11 cells conferred resistance to apoptosis, decreased cell-cycle arrest, and increased proliferation in the presence of pexidartinib and other FLT3 inhibitors. Inhibition of CDK4/6 activity in CCND3 mutant MV4;11 cells restored pexidartinib-induced cell-cycle arrest but not apoptosis. CONCLUSIONS: Mutations in CCND3, a gene not commonly mutated in AML, are a novel cause of clinical primary resistance to FLT3 inhibitors in AML and may have sensitivity to CDK4/6 inhibition.

Long-term outcomes of pexidartinib in tenosynovial giant cell tumors.

BACKGROUND: The objective of this study was to report on the long-term effects of pexidartinib on tenosynovial giant cell tumor (TGCT). METHODS: This was a pooled analysis encompassing 3 pexidartinib-treated TGCT cohorts: 1) a phase 1 extension study (NCT01004861; 1000 mg/d; n = 39), 2) ENLIVEN patients randomized to pexidartinib (1000 mg/d for 2 weeks and then 800 mg/d; n = 61), and 3) ENLIVEN crossover patients (NCT02371369; 800 mg/d; n = 30). Eligible patients were 18 years old or older and had a histologically confirmed TGCT that was unresectable and symptomatic. Efficacy endpoints included the best overall response (complete or partial response) and the duration of response (DOR) by the Response Evaluation Criteria in Solid Tumors (RECIST) and the tumor volume score (TVS). The safety assessment included the frequency of treatment-emergent adverse events (TEAEs) and hepatic laboratory abnormalities (aminotransferase elevations and mixed/cholestatic hepatotoxicity). The data cutoff was May 31, 2019. RESULTS: One hundred thirty patients with TGCT received pexidartinib (median treatment duration, 19 months; range, 1 to 76+ months); 54 (42%) remained on treatment at the end of the analysis (26 months after initial data cut of March 2017). The RECIST overall response rate (ORR) was 60%; the TVS ORR was 65%. The median times to response were 3.4 (RECIST) and 2.8 months (TVS), with 48 of the responding patients (62%) achieving a RECIST partial response by 6 months and with 72 (92%) doing so by 18 months. The median DOR was reached for TVS (46.8 months). Reported TEAEs were mostly low-grade, with hair color changes being most frequent (75%). Most liver abnormalities (92%) were aminotransferase elevations; 4 patients (3%) experienced mixed/cholestatic hepatotoxicity (all within the first 2 months of treatment), which was reversible in all cases (recovery spanned 1-7 months). CONCLUSIONS: This study demonstrates the prolonged efficacy and tolerability of long-term pexidartinib treatment for TGCT.

A phase 1/2 study of the oral FLT3 inhibitor pexidartinib in relapsed/refractory FLT3-ITD-mutant acute myeloid leukemia.

FMS-like tyrosine kinase 3 (FLT3) tyrosine kinase inhibitors (TKIs) have activity in acute myeloid leukemia (AML) patients with FLT3 internal tandem duplication (ITD) mutations, but efficacy is limited by resistance-conferring kinase domain mutations. This phase 1/2 study evaluated the safety, tolerability, and efficacy of the oral FLT3 inhibitor PLX3397 (pexidartinib), which has activity against the FLT3 TKI-resistant F691L gatekeeper mutation in relapsed/refractory FLT3-ITD-mutant AML. Ninety patients were treated: 34 in dose escalation (part 1) and 56 in dose expansion (part 2). Doses of 800 to 5000 mg per day in divided doses were tested. No maximally tolerated dose was reached. Plasma inhibitory assay demonstrated that patients dosed with ≥3000 mg had sufficient levels of active drug in their trough plasma samples to achieve 95% inhibition of FLT3 phosphorylation in an FLT3-ITD AML cell line. Based on a plateau in drug exposure, the 3000-mg dose was chosen as the recommended phase 2 dose. The most frequently reported treatment-emergent adverse events were diarrhea (50%), fatigue (47%), and nausea (46%). Based on modified response criteria, the overall response rate to pexidartinib among all patients was 21%. Twenty-three percent of patients treated at ≥2000 mg responded. The overall composite complete response rate for the study was 11%. Six patients were successfully bridged to transplantation. Median overall survival (OS) of patients treated in dose expansion was 112 days (90% confidence interval [CI], 77-150 days), and median OS of responders with complete remission with or without recovery of blood counts was 265 days (90% CI, 170-422 days). This trial was registered at www.clinicaltrials.gov as #NCT01349049.

Therapeutic Effect of Topical Adiponectin-Derived Short Peptides Compared with Globular Adiponectin in Experimental Dry Eye and Alkali Burn.

Purpose: To evaluate the efficacy of adiponectin (APN)-derived short peptides (ADPs) 355 compared with globular APN in a mouse model of experimental dry eye (EDE) and corneal alkali burn. Methods: EDE and chemical burn were induced in C57BL/6 mice by desiccating stress and application of NaOH, respectively. Eye drops consisting of 0.01% globular APN, 0.01% ADPs, 0.1% ADPs, or balanced salt solution (BSS) were applied. Tear volume, tear film break-up time, and corneal staining scores were measured. Concentrations of interleukin (IL)-1ß, interferon (IFN)-γ, IL-6, CXCL-9, and CXCL-10 using multiplex immunobead assay were evaluated, and flow cytometry were performed. Corneal epithelial defects and haze degree were analyzed, and enzyme-linked immunosorbent assay for IL-1ß and transforming growth factor (TGF)-ß levels were observed. Results: All treatment groups showed an improvement in clinical parameters and CD4+CCR5+ T cell and CD11b+ cell infiltrations in the conjunctiva (all P < 0.05). Both ADPs groups had significantly decreased concentrations of IL-1ß, IFN-γ, IL-6, CXCL-9, and CXCL-10 in the conjunctiva than the EDE or BSS group. Significantly improved parameters of epithelial defect, degree of haze, and concentrations of IL-1ß and TGF-ß were observed in all treatment groups. However, no significant differences were noted in clinical or experimental parameters among treatment groups. Conclusion: Topical ADPs could effectively improve clinical signs and inflammation of ocular surface in the EDE or alkali burn, and its efficacy and potency were similar to those of globular APN.

Pexidartinib versus placebo for advanced tenosynovial giant cell tumour (ENLIVEN): a randomised phase 3 trial.

BACKGROUND: Tenosynovial giant cell tumour (TGCT), a rare, locally aggressive neoplasm, overexpresses colony-stimulating factor 1 (CSF1). Surgery is standard with no approved systemic therapy. We aimed to evaluate pexidartinib, a CSF1 receptor inhibitor, in patients with TGCT to provide them with a viable systemic treatment option, especially in cases that are not amenable to surgical resection. METHODS: This phase 3 randomised trial had two parts. Part one was a double-blind study in which patients with symptomatic, advanced TGCT for whom surgery was not recommended were randomly assigned via an integrated web response system (1:1) to the pexidartinib or placebo group. Individuals in the pexidartinib group received a loading dose of 1000 mg pexidartinib per day orally (400 mg morning; 600 mg evening) for the first 2 weeks, followed by 800 mg per day (400 mg twice a day) for 22 weeks. Part two was an open-label study of pexidartinib for all patients. The primary endpoint, assessed in all intention-to-treat patients, was overall response at week 25, and was centrally reviewed by RECIST, version 1.1. Safety was analysed in all patients who received at least one dose of the study drug. This study is registered with ClinicalTrials.gov, number NCT02371369. FINDINGS: Between May 11, 2015, and Sept 30, 2016, of 174 patients assessed for eligibility, 120 patients were randomly assigned to, and received, pexidartinib (n=61) or placebo (n=59). There were 11 dropouts in the placebo group and nine in the pexidartinib group. Emergence of mixed or cholestatic hepatotoxicity caused the data monitoring committee to stop enrolment six patients short of target. The proportion of patients who achieved overall response was higher for pexidartinib than placebo at week 25 by RECIST (24 [39%] of 61 vs none of 59; absolute difference 39% [95% CI 27-53]; p<0·0001). Serious adverse events occurred in eight (13%) of 61 patients in the pexidartinib group and one (2%) of 59 patients in the placebo group. Hair colour changes (67%), fatigue (54%), aspartate aminotransferase increase (39%), nausea (38%), alanine aminotransferase increase (28%), and dysgeusia (25%) were the most frequent pexidartinib-associated adverse events. Three patients given pexidartinib had aminotransferase elevations three or more times the upper limit of normal with total bilirubin and alkaline phosphatase two or more times the upper limit of normal indicative of mixed or cholestatic hepatotoxicity, one lasting 7 months and confirmed by biopsy. INTERPRETATION: Pexidartinib is the first systemic therapy to show a robust tumour response in TGCT with improved patient symptoms and functional outcomes; mixed or cholestatic hepatotoxicity is an identified risk. Pexidartinib could be considered as a potential treatment for TGCT associated with severe morbidity or functional limitations in cases not amenable to improvement with surgery. FUNDING: Daiichi Sankyo.

The measurement of physical functioning among patients with Tenosynovial Giant Cell Tumor (TGCT) using the Patient-Reported Outcomes Measurement Information System (PROMIS).

BACKGROUND: Tenosynovial giant cell tumor (TGCT), a rare, locally aggressive neoplasm of the synovium of joints and tendon sheaths, is associated with joint destruction, pain and swelling. Impacts on physical function (PF) vary depending on tumor size and location. The aim of this study was to identify relevant items, and demonstrate the content validity of custom measures of lower extremity PF from the Patient-Reported Outcomes Measurement Information System Physical Function Physical Function (PROMIS-PF) item bank among patients with TGCT. METHODS: Patients were recruited for qualitative research interviews to identify predominant TGCT symptoms and impacts. Patients completed a checklist to evaluate the relevance of each PROMIS-PF item. The publicly available PROMIS-PF item response theory (IRT) parameters were used to select items representing the range of the latent PF trait. RESULTS: Participants (n = 20) were 75% female, mean age 42.5 years. TGCTs were located in the knee (n = 15), hip (n = 3), and ankle (n = 2). Fifty-four PROMIS-PF items were identified as relevant by ≥20% of the participants. PF concepts discussed by participants during the qualitative interviews were also used to select relevant items. Selected items (n = 13) were used to create a physical function subscale specific to lower extremity tumors. CONCLUSIONS: We describe a novel method of combining qualitative research and IRT-based item information to select a relevant and content valid subset of PROMIS-PF items to assess heterogeneous impacts on PF in TGCT, a rare disease population.

Patient-reported Symptoms of Tenosynovial Giant Cell Tumors.

PURPOSE: Tenosynovial giant cell tumor (TGCT), a rare locally aggressive neoplasm of the synovium of joints and tendon sheaths, is associated with joint destruction, inflammation, pain, and swelling, in part due to colony-stimulating factor 1 receptor-bearing macrophages recruited to the tumor by genetic elevation of colony-stimulating factor 1 activity. The most common treatment is surgery, although promising pharmacologic treatments are in development. Patient-reported outcome (PRO) instruments are critical end points in demonstrating the clinical relevance of standard oncologic outcome measures and the overall impact of novel pharmacologic therapies in nonmalignant neoplastic conditions such as TGCT. The content validity of PROs relevant to patients with TGCT has not been formally investigated, and instruments to evaluate such outcomes do not exist for this condition. METHODS: PRO instruments of potential relevance were evaluated by using a literature review and by clinical and PRO experts. Patients with TGCT were recruited through clinical sites and the Internet for participation in qualitative research interviews to identify predominant symptoms and to test the relevance and content validity of several PRO measures. Select PRO measures were included in a Phase I clinical trial, and preliminary results of the PRO end points are reported descriptively. FINDINGS: Of the 22 subjects who participated in qualitative interviews, 73% were female, and their mean age was 42.5 years (range, 27-56 years). The TGCTs (19 diffuse and 3 localized) were located in the knee (n = 15), hip (n = 3), ankle (n = 2), elbow (n = 1), and forearm (n = 1). The most common symptoms cited were pain (82%), swelling (86%), stiffness (73%), reduced range of motion (64%), and joint instability (64%), which were consistent with clinical expert input and with the content of instruments chosen by PRO experts. The worst pain numeric rating scale, Patient Reported Outcomes Measurement Information System physical functioning items, and the Western Ontario and McMaster Universities Osteoarthritis Index, as well as a worst stiffness numeric rating scale developed for TGCT, were confirmed as meaningful measures of TGCT patient symptoms and were well understood in qualitative interviews. Results from the Phase I trial showed trends of improvement in both pain and stiffness over time. IMPLICATIONS: This study is the first to gather information directly from patients with TGCT regarding their symptom experiences. Pain, stiffness, and physical functioning are important treatment outcomes in patients with TGCT. We have identified content-valid PRO measures of these concepts, which are included in an ongoing Phase III TGCT clinical trial with pexidartinib (PLX3397) (NCT02371369).

Orally administered colony stimulating factor 1 receptor inhibitor PLX3397 in recurrent glioblastoma: an Ivy Foundation Early Phase Clinical Trials Consortium phase II study.

BACKGROUND: The colony stimulating factor 1 receptor (CSF1R) ligands, CSF1 and interleukin-34, and the KIT ligand, stem cell factor, are expressed in glioblastoma (GB). Microglia, macrophages, blood vessels, and tumor cells also express CSF1R, and depletion of the microglia reduces tumor burden and invasive capacity. PLX3397 is an oral, small molecule that selectively inhibits CSF1R and KIT, penetrates the blood-brain barrier in model systems, and represents a novel approach for clinical development. METHODS: We conducted a phase II study in patients with recurrent GB. The primary endpoint was 6-month progression-free survival (PFS6). Secondary endpoints included overall survival response rate, safety, and plasma/tumor tissue pharmacokinetics. Exploratory endpoints included pharmacodynamic measures of drug effect in blood and tumor tissue. RESULTS: A total of 37 patients were enrolled, with 13 treated prior to a planned surgical resection (Cohort 1) and 24 treated without surgery (Cohort 2). PLX3397 was given at an oral dose of 1000 mg daily and was well tolerated. The primary efficacy endpoint of PFS6 was only 8.6%, with no objective responses. Pharmacokinetic endpoints revealed a median maximal concentration (Cmax) of 8090 ng/mL, with a time to attain Cmax of 2 hour in plasma. Tumor tissue obtained after 7 days of drug exposure revealed a median drug level of 5500 ng/g. Pharmacodynamic changes included an increase in colony stimulating factor 1 and reduced CD14(dim)/CD16+ monocytes in plasma compared with pretreatment baseline values. CONCLUSION: PLX3397 was well tolerated and readily crossed the blood-tumor barrier but showed no efficacy. Additional studies are ongoing, testing combination strategies and potential biomarkers to identify patients with greater likelihood of response.

Structure-Guided Blockade of CSF1R Kinase in Tenosynovial Giant-Cell Tumor.

BACKGROUND: Expression of the colony-stimulating factor 1 (CSF1) gene is elevated in most tenosynovial giant-cell tumors. This observation has led to the discovery and clinical development of therapy targeting the CSF1 receptor (CSF1R). METHODS: Using x-ray co-crystallography to guide our drug-discovery research, we generated a potent, selective CSF1R inhibitor, PLX3397, that traps the kinase in the autoinhibited conformation. We then conducted a multicenter, phase 1 trial in two parts to analyze this compound. In the first part, we evaluated escalations in the dose of PLX3397 that was administered orally in patients with solid tumors (dose-escalation study). In the second part, we evaluated PLX3397 at the chosen phase 2 dose in an extension cohort of patients with tenosynovial giant-cell tumors (extension study). Pharmacokinetic and tumor responses in the enrolled patients were assessed, and CSF1 in situ hybridization was performed to confirm the mechanism of action of PLX3397 and that the pattern of CSF1 expression was consistent with the pathological features of tenosynovial giant-cell tumor. RESULTS: A total of 41 patients were enrolled in the dose-escalation study, and an additional 23 patients were enrolled in the extension study. The chosen phase 2 dose of PLX3397 was 1000 mg per day. In the extension study, 12 patients with tenosynovial giant-cell tumors had a partial response and 7 patients had stable disease. Responses usually occurred within the first 4 months of treatment, and the median duration of response exceeded 8 months. The most common adverse events included fatigue, change in hair color, nausea, dysgeusia, and periorbital edema; adverse events rarely led to discontinuation of treatment. CONCLUSIONS: Treatment of tenosynovial giant-cell tumors with PLX3397 resulted in a prolonged regression in tumor volume in most patients. (Funded by Plexxikon; ClinicalTrials.gov number, NCT01004861.).

Characterizing and Overriding the Structural Mechanism of the Quizartinib-Resistant FLT3 "Gatekeeper" F691L Mutation with PLX3397.

UNLABELLED: Tyrosine kinase domain mutations are a common cause of acquired clinical resistance to tyrosine kinase inhibitors (TKI) used to treat cancer, including the FLT3 inhibitor quizartinib. Mutation of kinase "gatekeeper" residues, which control access to an allosteric pocket adjacent to the ATP-binding site, has been frequently implicated in TKI resistance. The molecular underpinnings of gatekeeper mutation-mediated resistance are incompletely understood. We report the first cocrystal structure of FLT3 with the TKI quizartinib, which demonstrates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and F830 within the highly conserved Asp-Phe-Gly motif in the activation loop. This reliance makes quizartinib critically vulnerable to gatekeeper and activation loop substitutions while minimizing the impact of mutations elsewhere. Moreover, we identify PLX3397, a novel FLT3 inhibitor that retains activity against the F691L mutant due to a binding mode that depends less vitally on specific interactions with the gatekeeper position. SIGNIFICANCE: We report the first cocrystal structure of FLT3 with a kinase inhibitor, elucidating the structural mechanism of resistance due to the gatekeeper F691L mutation. PLX3397 is a novel FLT3 inhibitor with in vitro activity against this mutation but is vulnerable to kinase domain mutations in the FLT3 activation loop.

Topical mecamylamine for diabetic macular edema.

PURPOSE: Stimulation of nicotinic acetylcholine (nACh) receptors on vascular endothelial cells promotes angiogenesis and vascular permeability in animal models. The safety and bioactivity of topical mecamylamine, an antagonist of nACh receptors, was tested in patients with diabetic macular edema. DESIGN: A multicenter phase I/II clinical trial. METHODS: Twenty-three patients with chronic diabetic macular edema received 1% mecamylamine topically twice daily for 12 weeks, the primary end point. Patients underwent safety assessments, measurement of best-corrected visual acuity (BCVA), and measurement of foveal thickness using optical coherence tomography at baseline, 1, 4, 8, 12, and 16 weeks. RESULTS: Mecamylamine drops were well tolerated and there were no drug-related safety problems. Mean improvement in BCVA at 1, 4, 8, 12, and 16 weeks was 2.8, 1.9, 2.4, 0.8, and 3.1 letters, respectively. There was little change in mean excess foveal thickness. There was substantial heterogeneity in response, because 8 patients showed convincing improvement in BCVA, foveal thickness, or both, 9 patients showed equivocal or no substantial changes, and 4 patients showed worsening. Five patients showed a substantial improvement in BCVA, foveal thickness, or both between their last visit while receiving mecamylamine and 1 month after stopping mecamylamine. CONCLUSIONS: This study suggested that administration of topical mecamylamine, a nonspecific nACh receptor blocker, may have heterogeneous effects in patients with diabetic macular edema. Variable expression of nACh receptor subtypes on endothelial cells that have different effects on permeability would provide an explanation for these results and should be investigated, because more specific nACh receptor blockers may dissociate antipermeability and propermeability effects.

Randomized study of adding inhaled iloprost to existing bosentan in pulmonary arterial hypertension.

RATIONALE: Small, open-label studies suggest that combinations of existing therapies may be effective for pulmonary arterial hypertension (PAH). OBJECTIVE: To evaluate the safety and efficacy of adding inhaled iloprost, a prostacyclin analog, to the endothelin receptor antagonist bosentan in patients with PAH. METHODS: In a randomized, multicenter, double-blind trial, inhaled iloprost (5 mug) or placebo was added to stable monotherapy with bosentan for 12 wk. Efficacy endpoints included change from baseline in 6-min-walk distance (6-MWD), modified New York Heart Association (NYHA) functional class, hemodynamic parameters, and time to clinical worsening. MEASUREMENTS AND MAIN RESULTS: A total of 67 patients with PAH (55% idiopathic PAH, 45% associated PAH, 94% NYHA class III, and mean baseline 6-MWD of 335 m) were randomized. At Week 12, patients receiving iloprost had a mean increase in 6-MWD of 30 m (p = 0.001); placebo patients had a mean 6-MWD increase of 4 m (p = 0.69), with a placebo-adjusted difference of +26 m (p = 0.051). NYHA status improved by one class in 34% of iloprost versus 6% of placebo patients (p = 0.002). Iloprost delayed the time to clinical worsening (p = 0.0219). Improvements were noted in postinhalation placebo-adjusted change in mean pulmonary artery pressure (-8 mm Hg; p < 0.001) and pulmonary vascular resistance (-254 dyn x s x cm(-5); p < 0.001). Combination therapy was well tolerated. CONCLUSIONS: Within the limitations of a relatively small sample size, results of this study demonstrate that the addition of inhaled iloprost in patients with PAH with reduced exercise capacity on bosentan monotherapy is safe and efficacious.

Iloprost inhalation solution for the treatment of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a condition that is characterised by increased pulmonary arterial pressure and vascular resistance that can lead to right ventricular failure and death. A variety of disturbances in pulmonary vascular endothelial and smooth muscle function are present in PAH, including reduced production of vasodilator and antiproliferative substances, such as nitric oxide and prostacyclin, and an overproduction of mitogens, such as endothelin. As a result of these observations, therapies have been developed for PAH that specifically target these pathogenic processes, including prostacyclin analogues and endothelin receptor antagonists. This article reviews iloprost inhalation solution, the most recently approved form of prostacyclin therapy that is delivered directly to the lungs by inhalation.