Growth hormone secretagogue increases muscle strength during immobilization after canine hindlimb immobilization

Summary: Twenty-two beagles were divided into two equal groups, and the right hindlimb of each animal was immobilized at 105" of knee flexion by external fixation. After 10 weeks of fixation, the device was removed, allowing free mobility for the following 5 weeks. Each day throughout the 1. 5 weeks, one group received a growth hormone secretagogue (treatment) at a dose of 5 mg/kg, and the other received a lactose placebo (control). A t weeks 0, 10, and 15, strength as indicated by maximum isometric extension torque was measured in the right hindlimb, biopsies of the vastus lateralis muscle were taken, and the dogs were weighed. Weekly blood samples were analyzed for insulin-like growth factor-1, blood urea nitrogen, and creatine phosphokinase. Between weeks 0 and 10, tetanic torque declined by about 60% (p < 0.001) in both groups, with no significant difference between the groups (p > 0.7). Between weeks 10 and 15, tetanic torque in the treated group increased by 0.81 Nm; this was significantly greater than the increase of 0.25 Nm in the placebo group (p < 0.05). The diameters of slow (type-1) and fast (type-2) fibers measured from the vastus lateralis muscle followed the same trend. At all time points, fiber diameter correlated strongly with torque; this argues against nonmuscular causes such as nerve injury for strength loss. The mean levels of insulin-like growth factor-1 increased 100% by week 4 in the treated group and remained elevated by about 60% throughout the experiment. Levels of insulin-like growth factor-1 in the placebo group decreased 30% within week 1 and remained depressed throughout the experiment. Our interpretation of these data suggests that the growth hormone secretagogue elevated levels of serum insulin-like growth factor-1, which in turn increased the size and strength of the quadriceps muscle during remobilization. These data may ultimately have therapeutic application to humans during rehabilitation after prolonged inactivity. - The strengthening of skeletal muscle after surgery or prolonged disuse remains a primary goal of rehabilitation. Many approaches -including electrical stimulation (lo), voluntary exercise (15), continuous passive motion (7), and hormone therapy (32) -have been used to strengthen atrophied muscles. In some cases, treatment modalities are limited by a patient's access to rehabilitation professionals or rehabilitation devices. Medical treatment of muscle atrophy with hormone therapy offers the advantage of requiring relatively little effort on the part of the patient to achieve the desired therapeutic result. There is ample evidence of hypertrophy of skeletal muscle in response to anabolic steroids. strong correlations were demonstrated between muscle strength and serum testosterone concentration in elderly men in whom a marked loss of muscle function was correlated with low testosterone levels relative to stronger age-matched controls (1). In related studies, the administration of testosterone to elderly men (to increase serum testosterone to a range observed in younger men) increased lean body mass (32) and grip strength (25). The effect of this hormone may be mediated, at least in part, by serum insulin-like growth factor-1 (IGF-1) (4). The anabolic effects on humans of I G F -1 administration have been well documented. For example, recombinant IGF-1 attenuated the catabolic effects of glucocorticoids (23) and increased muscle protein synthesis in young men (9). Similar results were obtained in vitro with other muscle growth factors such as fibroblast growth factor (FGF). At the cellular level, skeletal muscle myotubes grown in culture in the presence of FGF demonstrated a marked increase in protein synthesis. This effect was enhanced 51

Structure-Guided Evolution of Potent and Selective CHK1 Inhibitors through Scaffold Morphing

Pyrazolopyridine inhibitors with low micromolar potency for CHK1 and good selectivity against CHK2 were previously identified by fragment-based screening. The optimization of the pyrazolopyridines to a series of potent and CHK1-selective isoquinolines demonstrates how fragment-growing and scaffold morphing strategies arising from a structure-based understanding of CHK1 inhibitor binding can be combined to successfully progress fragment-derived hit matter to compounds with activity in vivo. The challenges of improving CHK1 potency and selectivity, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space were tackled by multiple scaffold morphing steps, which progressed through tricyclic pyrimido[2,3-b]azaindoles to N-(pyrazin-2-yl)pyrimidin-4-amines and ultimately to imidazo[4,5-c]pyridines and isoquinolines. A potent and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was identified, which potentiated the efficacies of irinotecan and gemcitabine in SW620 human colon carcinoma xenografts in nude mice

Multiparameter Lead Optimization to Give an Oral Checkpoint Kinase 1 (CHK1) Inhibitor Clinical Candidate: (R)-5-((4-((Morpholin-2-ylmethyl)amino)-5-(trifluoromethyl)pyridin-2-yl)amino)pyrazine-2-carbonitrile (CCT245737)

Multiparameter optimization of a series of 5-((4-aminopyridin-2-yl)amino)pyrazine-2-carbonitriles resulted in the identification of a potent and selective oral CHK1 preclinical development candidate with in vivo efficacy as a potentiator of deoxyribonucleic acid (DNA) damaging chemotherapy and as a single agent. Cellular mechanism of action assays were used to give an integrated assessment of compound selectivity during optimization resulting in a highly CHK1 selective adenosine triphosphate (ATP) competitive inhibitor. A single substituent vector directed away from the CHK1 kinase active site was unexpectedly found to drive the selective cellular efficacy of the compounds. Both CHK1 potency and off-target human ether-a-go-go-related gene (hERG) ion channel inhibition were dependent on lipophilicity and basicity in this series. Optimization of CHK1 cellular potency and in vivo pharmacokinetic–pharmacodynamic (PK–PD) properties gave a compound with low predicted doses and exposures in humans which mitigated the residual weak in vitro hERG inhibition

Acquainting veterinary students with careers in the pharmaceutical industry

Careers in the pharmaceutical industry were revealed in modules facilitated by senior scientists from companies that sponsor the Cornell Leadership Program for Veterinary Students. One module was structured as a series of interviews for different positions in industry, the other as a competition between hypothetical companies created by students. The interview-based module stimulated wide-ranging discussion of the activities and responsibilities of veterinarians employed in a discovery-intensive pharmaceutical firm and of the characteristics such companies seek in prospective employees, from both professional and personal perspectives. The second module explored the drug discovery and development process from the perspective of animal-health companies that are competitors in the market for animal health care products. The exercise provided insights into the manner in which companies discover new chemical entities, screen candidate drugs, allocate resources, and pursue the development of products through testing, licensing, and distribution

The Preclinical Pharmacology and Therapeutic Activity of the Novel CHK1 Inhibitor SAR-020106

Genotoxic antitumor agents continue to be the mainstay of current cancer chemotherapy. These drugs cause DNA damage and activate numerous cell cycle checkpoints facilitating DNA repair and the maintenance of genomic integrity. Most human tumors lack functional p53 and consequently have compromised G1-S checkpoint control. This has led to the hypothesis that S and G2-M checkpoint abrogation may selectively enhance genotoxic cell killing in a p53-deficient background, as normal cells would be rescued at the G1-S checkpoint. CHK1 is a serine/threonine kinase associated with DNA damage–linked S and G2-M checkpoint control. SAR-020106 is an ATP-competitive, potent, and selective CHK1 inhibitor with an IC50 of 13.3 nmol/L on the isolated human enzyme. This compound abrogates an etoposide-induced G2 arrest with an IC50 of 55 nmol/L in HT29 cells, and significantly enhances the cell killing of gemcitabine and SN38 by 3.0- to 29-fold in several colon tumor lines in vitro and in a p53-dependent fashion. Biomarker studies have shown that SAR-020106 inhibits cytotoxic drug–induced autophosphorylation of CHK1 at S296 and blocks the phosphorylation of CDK1 at Y15 in a dose-dependent fashion both in vitro and in vivo. Cytotoxic drug combinations were associated with increased ?H2AX and poly ADP ribose polymerase cleavage consistent with the SAR-020106–enhanced DNA damage and tumor cell death. Irinotecan and gemcitabine antitumor activity was enhanced by SAR-020106 in vivo with minimal toxicity. SAR-020106 represents a novel class of CHK1 inhibitors that can enhance antitumor activity with selected anticancer drugs in vivo and may therefore have clinical utility

Preclinical Pharmacology, Antitumor Activity, and Development of Pharmacodynamic Markers for the Novel, Potent AKT Inhibitor CCT128930

AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT128930 is a novel ATP-competitive AKT inhibitor discovered using fragment- and structure-based approaches. It is a potent, advanced lead pyrrolopyrimidine compound exhibiting selectivity for AKT over PKA, achieved by targeting a single amino acid difference. CCT128930 exhibited marked antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G1 arrest in PTEN-null U87MG human glioblastoma cells, consistent with AKT pathway blockade. Pharmacokinetic studies established that potentially active concentrations of CCT128930 could be achieved in human tumor xenografts. Furthermore, CCT128930 also blocked the phosphorylation of several downstream AKT biomarkers in U87MG tumor xenografts, indicating AKT inhibition in vivo. Antitumor activity was observed with CCT128930 in U87MG and HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts, consistent with its pharmacokinetic and pharmacodynamic properties. A quantitative immunofluorescence assay to measure the phosphorylation and total protein expression of the AKT substrate PRAS40 in hair follicles is presented. Significant decreases in pThr246 PRAS40 occurred in CCT128930-treated mouse whisker follicles in vivo and human hair follicles treated ex vivo, with minimal changes in total PRAS40. In conclusion, CCT128930 is a novel, selective, and potent AKT inhibitor that blocks AKT activity in vitro and in vivo and induces marked antitumor responses. We have also developed a novel biomarker assay for the inhibition of AKT in human hair follicles, which is currently being used in clinical trials

CCT244747 Is a Novel Potent and Selective CHK1 Inhibitor with Oral Efficacy Alone and in Combination with Genotoxic Anticancer Drugs

Purpose: Many tumors exhibit defective cell-cycle checkpoint control and increased replicative stress. CHK1 is critically involved in the DNA damage response and maintenance of replication fork stability. We have therefore discovered a novel potent, highly selective, orally active ATP-competitive CHK1 inhibitor, CCT244747, and present its preclinical pharmacology and therapeutic activity. Experimental Design: Cellular CHK1 activity was assessed using an ELISA assay, and cytotoxicity a SRB assay. Biomarker modulation was measured using immunoblotting, and cell-cycle effects by flow cytometry analysis. Single-agent oral CCT244747 antitumor activity was evaluated in a MYCN-driven transgenic mouse model of neuroblastoma by MRI and in genotoxic combinations in human tumor xenografts by growth delay. Results: CCT244747 inhibited cellular CHK1 activity (IC50 29–170 nmol/L), significantly enhanced the cytotoxicity of several anticancer drugs, and abrogated drug-induced S and G2 arrest in multiple tumor cell lines. Biomarkers of CHK1 (pS296 CHK1) activity and cell-cycle inactivity (pY15 CDK1) were induced by genotoxics and inhibited by CCT244747 both in vitro and in vivo, producing enhanced DNA damage and apoptosis. Active tumor concentrations of CCT244747 were obtained following oral administration. The antitumor activity of both gemcitabine and irinotecan were significantly enhanced by CCT244747 in several human tumor xenografts, giving concomitant biomarker modulation indicative of CHK1 inhibition. CCT244747 also showed marked antitumor activity as a single agent in a MYCN-driven neuroblastoma. Conclusion: CCT244747 represents the first structural disclosure of a highly selective, orally active CHK1 inhibitor and warrants further evaluation alone or combined with genotoxic anticancer therapies