First-in-Human Study of AT13148, a Dual ROCK-AKT Inhibitor in Patients with Solid Tumors.

PURPOSE: AT13148 is an oral AGC kinase inhibitor, which potently inhibits ROCK and AKT kinases. In preclinical models, AT13148 has been shown to have antimetastatic and antiproliferative activity. PATIENTS AND METHODS: The trial followed a rolling six design during dose escalation. An intrapatient dose escalation arm to evaluate tolerability and a biopsy cohort to study pharmacodynamic effects were later added. AT13148 was administered orally three days a week (Mon-Wed-Fri) in 28-day cycles. Pharmacokinetic profiles were assessed using mass spectrometry and pharmacodynamic studies included quantifying p-GSK3ß levels in platelet-rich plasma (PRP) and p-cofilin and p-MLC2 levels in tumor biopsies. RESULTS: Fifty-one patients were treated on study. The safety of 5-300 mg of AT13148 was studied. Further, the doses of 120-180-240 mg were studied in an intrapatient dose escalation cohort. The dose-limiting toxicities included hypotension (300 mg), pneumonitis, and elevated liver enzymes (240 mg), and skin rash (180 mg). The most common side effects were fatigue, nausea, headaches, and hypotension. On the basis of tolerability, 180 mg was considered the maximally tolerated dose. At 180 mg, mean C max and AUC were 400 nmol/L and 13,000 nmol/L/hour, respectively. At 180 mg, ≥50% reduction of p-cofilin was observed in 3 of 8 posttreatment biopsies. CONCLUSIONS: AT13148 was the first dual potent ROCK-AKT inhibitor to be investigated for the treatment of solid tumors. The narrow therapeutic index and the pharmacokinetic profile led to recommend not developing this compound further. There are significant lessons learned in designing and testing agents that simultaneously inhibit multiple kinases including AGC kinases in cancer.

Metabolomic changes of the multi (-AGC-) kinase inhibitor AT13148 in cells, mice and patients are associated with NOS regulation.

INTRODUCTION: To generate biomarkers of target engagement or predictive response for multi-target drugs is challenging. One such compound is the multi-AGC kinase inhibitor AT13148. Metabolic signatures of selective signal transduction inhibitors identified in preclinical models have previously been confirmed in early clinical studies. This study explores whether metabolic signatures could be used as biomarkers for the multi-AGC kinase inhibitor AT13148. OBJECTIVES: To identify metabolomic changes of biomarkers of multi-AGC kinase inhibitor AT13148 in cells, xenograft / mouse models and in patients in a Phase I clinical study. METHODS: HILIC LC-MS/MS methods and Biocrates AbsoluteIDQ™ p180 kit were used for targeted metabolomics; followed by multivariate data analysis in SIMCA and statistical analysis in Graphpad. Metaboanalyst and String were used for network analysis. RESULTS: BT474 and PC3 cells treated with AT13148 affected metabolites which are in a gene protein metabolite network associated with Nitric oxide synthases (NOS). In mice bearing the human tumour xenografts BT474 and PC3, AT13148 treatment did not produce a common robust tumour specific metabolite change. However, AT13148 treatment of non-tumour bearing mice revealed 45 metabolites that were different from non-treated mice. These changes were also observed in patients at doses where biomarker modulation was observed. Further network analysis of these metabolites indicated enrichment for genes associated with the NOS pathway. The impact of AT13148 on the metabolite changes and the involvement of NOS-AT13148- Asymmetric dimethylarginine (ADMA) interaction were consistent with hypotension observed in patients in higher dose cohorts (160-300 mg). CONCLUSION: AT13148 affects metabolites associated with NOS in cells, mice and patients which is consistent with the clinical dose-limiting hypotension.

AT13148, a first-in-class multi-AGC kinase inhibitor, potently inhibits gastric cancer cells both in vitro and in vivo.

The AGC kinase family is important cell proliferation and survival. Dysregulation of this family contributes to gastric cancer progression. Here, we evaluated the potential activity of AT13148, a first-in-class multi-AGC kinase inhibitor, against gastric cancer cells. Our results showed that AT13148 exerted potent cytotoxic and anti-proliferative activities against a panel human gastric cancer cell lines (HGC-27, AGS, SNU-601, N87 and MKN-28), possibly via inducing cancer cell apoptotic death. Apoptosis inhibition by the Caspase blockers dramatically attenuated AT13148-caused cytotoxicity against gastric cancer cells. Intriguingly, same AT13148 treatment was not cytotoxic/pro-apoptotic to the non-cancerous human gastric epithelial GEC-1 cells. At the signaling level, AT13148 treatment in gastric cancer cells dramatically suppressed activation of multiple AGC kinases, including Akt (at p-Thr-308), p70S6 kinase (p70S6K), glycogen synthase kinase 3ß (GSK-3ß) and p90 ribosomal S6 kinase (RSK). Our in vivo studies demonstrated that daily oral gavage of AT13148 at well-tolerated doses significantly inhibited HGC27 xenograft tumor growth in nude mice. AGC activity was also dramatically decreased in AT13148-administrated HGC27 tumors. Therefore, targeting AGC kinases by AT13148 demonstrates superior anti-gastric cancer activity both in vitro and in vivo. The preclinical results of this study support the progression of this molecule into future evaluation as a valuable anti-gastric cancer candidate.

Rho kinase inhibitors block melanoma cell migration and inhibit metastasis.

There is an urgent need to identify new therapeutic opportunities for metastatic melanoma. Fragment-based screening has led to the discovery of orally available, ATP-competitive AKT kinase inhibitors, AT13148 and CCT129254. These compounds also inhibit the Rho-kinases ROCK 1 and ROCK 2 and we show they potently inhibit ROCK activity in melanoma cells in culture and in vivo. Treatment of melanoma cells with CCT129254 or AT13148 dramatically reduces cell invasion, impairing both "amoeboid-like" and mesenchymal-like modes of invasion in culture. Intravital imaging shows that CCT129254 or AT13148 treatment reduces the motility of melanoma cells in vivo. CCT129254 inhibits melanoma metastasis when administered 2 days after orthotopic intradermal injection of the cells, or when treatment starts after metastases have arisen. Mechanistically, our data suggest that inhibition of ROCK reduces the ability of melanoma cells to efficiently colonize the lungs. These results suggest that these novel inhibitors of ROCK may be beneficial in the treatment of metastasis.

Inhibition of human non-small cell lung cancers with a targeted cytotoxic somatostatin analog, AN-162.

Human non-small cell lung cancers (NSCLCs) express receptors for somatostatin. The cytotoxic analog of somatostatin AN-162 (AEZS-124), consisting of doxorubicin linked to a somatostatin analog RC-121 binds to receptors for somatostatin and is targeted to tumors expressing these receptors. The aim of this study was to investigate the effect of targeted cytotoxic somatostatin analog AN-162 on a panel of human NSCLC cell lines (A549, H460, H838, H1299) in vitro (at 0.5-100 microM concentrations) and in vivo on H460 and H1299 NSCLCs xenografted into nude mice (at the dose of 2.5 micromol/kg, i.v., once a week). The expression of mRNA for somatostatin receptor subtypes was investigated by RT-PCR in cell lines and tumor tissues. Somatostatin receptor proteins were also characterized by ligand competition assay and Western blotting. AN-162 significantly decreased cell proliferation in vitro and tumor growth (p<0.05 vs. all groups) of H460 and H1299 NSCLCs in vivo. Based on real-time PCR array data, AN-162 induced several apoptosis-related genes in vivo in both models. Our results suggest that cytotoxic somatostatin analog AN-162 (AEZS-124) should be considered for the further development of a therapy of patients with NSCLC.

The drug LY79771 affects fat regain by starved and refed BHE rats.

The effect of the drug LY79771 on the fat rebound response of BHE rats to starvation-refeeding was studied. Three experiments were conducted. Experiment 1 determined the effect of the drug on the composition of the regained weight following a period of starvation. The drug-treated rats had significantly less body fat after refeeding than did the control rats. Experiment 2 measured the liver and fat pad lipid levels and the activities of two NADP-linked enzymes after starvation-refeeding. The classic two- to threefold hepatic glucose-6-phosphate dehydrogenase and malic enzyme overshoot and increase in liver and fat pad lipid levels were seen in refed controls but not in refed LY79771-treated rats. Experiment 3 measured de novo fatty acid synthesis in LY79771-treated and control rats. Treatment with LY79771 resulted in lower hepatic fatty acid synthesis in starved and refed rats. These observations suggest that LY79771 can be effective in preventing fat regain following energy deprivation.

The thermogenic role of adipose tissue in the dog.

Brown adipose tissue was clearly present in neonatal dogs. In the adult the tissue was superseded by a tissue with the gross characteristics of white adipose. However despite their appearance adult adipose tissue depots may contribute to non-shivering thermogenesis. Regional blood flow measurements using injected radioactive microspheres indicated large increases in blood flow to adipose depots during infusion of noradrenaline. Coupled with blood flow estimations, measurement of arteriovenous differences in dissolved oxygen across the bladder fat depot demonstrated a quantitative increase in oxygen extraction by the depot during noradrenaline infusion. Acute activation of non-shivering thermogenesis in the dog was not associated with increased mitochondrial GDP-binding in adipose tissue. However chronic treatment with a beta-stimulant (LY79730) which increased capacity for non-shivering thermogenesis was associated with increased mitochondrial GDP-binding and cytochrome oxidase activity in peri-renal adipose tissue.

Clinical and urodynamic effects of norfenefrine in women with stress incontinence.

20 women with stress incontinence were treated with an alpha-adrenoceptor stimulating agent norfenefrine (Nevadral Retard) for 3 months. The mean dose administered was 60 mg given in slow-release tablets. Significant beneficial effect on the symptom and the 'sign' of stress incontinence was found as well as a significant increase in the maximum urethral closure pressure. 7 patients (35%) became subjectively and objectively continent. Only minor side effects were observed during treatment.

[Release of norfenefrine from sustained-release formulations by an in vitro dissolution model. Simulation of "drug levels" by calculation using pharmacokinetical constants and comparison with in vivo course of action (author's transl)]. / Freisetzung von Norfenefrin-HCl aus Depotpräparaten in einem In-vitro-Lösemodell. Rechnerische Simulation von Wirkstoffprofilen mit Hilfe pharmakokinetischer Parameter und Vergleich mit In-vivo-Wirkungsverläufen.

Drug release and dissolution behaviour of 1-(3'-hydroxyphenyl)-2-amino-ethan-1-ol hydrochloride (norfenefrine-HCl) from a sustained-release norfenefrine preparation (Esbuphon) were tested by an in vitro dissolution model in comparison with other norfenefrine formulations. By using pharmacokinetical constants, relative "drug levels" were calculated and compared with the activity curves from animal experiments. Conformities in time course were shown. Variations are discussed. The application of the model is justified if determination of serum concentration of norfenefrine cannot be done.