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Therapeutic potential of muscle growth promoters in a stress urinary incontinence model
No full textWeakness of urinary sphincter and pelvic floor muscles can cause insufficient urethral closure and lead to stress urinary incontinence (SUI). Bimagrumab is a novel myostatin inhibitor which blocks activin type II receptors, inducing skeletal muscle hypertrophy and attenuating muscle weakness. β2-adrenergic agonists, such as 5-hydroxybenzothiazolone derivative (5-HOB) and clenbuterol can enhance muscle growth. We hypothesized that promoting muscle growth would increase urethral pressure by facilitating muscle recovery in a dual injury (DI) SUI model. Rats underwent pudendal nerve crush (PNC) followed by vaginal distension (VD). One week after injury, each rat began subcutaneous (0.3ml/rat) treatment daily in a blinded fashion with either bimagrumab (DI+Bim), clenbuterol (DI+Clen), 5-HOB (DI+5HOB) or phosphate-buffered saline (PBS, DI+Sham). Sham-injured rats underwent sham PNC+VD and received PBS (SI). After two weeks of treatment, the rats were anesthetized for leak point pressure (LPP) and external urethral sphincter (EUS) electromyography recordings. Hind limb skeletal muscles and pelvic floor muscles were dissected and stained. At the end of 2 weeks of treatment, all 3 treatment groups had a significant increase in body weight and individual muscle weight compared to both sham treated and sham-injured rats. LPP in the DI+Bim group was significantly higher than LPP of DI+sham and DI+Clen rats. There were more consistent urethral striated muscle fibers, elastin fibers in the urethra and pelvic muscle recovery in DI+Bim rats compared with DI+Sham rats. In conclusion, bimagrumab was the most effective for increasing urethral pressure and continence by promoting injured EUS and pelvic floor muscle recovery
Toxicologic Pathology Forum: Opinion on Obligatory Microscopic Examination of Intermediate-Dose Groups in Toxicity Studies With Biotherapeutics in Cynomolgus Monkeys
No full textIn nonrodent toxicity studies that are usually conducted in cynomolgus monkeys or beagle dogs, the added value of examining all tissues from all dose groups (current practice) versus all tissues in only control and high-dose groups and target tissues in intermediate-dose groups by default, is a subject of debate. A previous retrospective review of 325 nonrodent toxicity studies that included a limited number of biotherapeutics suggested that the evaluation of all tissues from all groups was not justified as a routine practice and recommended the examination of all tissues in control and high-dose groups and only target tissues in intermediate-dose groups. In contrast, the present retrospective review which examined 213 nonrodent studies (212 in cynomolgus monkeys and 1 in dog) from 4 multinational pharmaceutical companies (Bristol-Myers Squibb, Novartis, Pfizer Inc, and Roche) conducted only with biotherapeutics showed that restricting the microscopic examination in intermediate-dose groups to target tissues has the potential to miss findings in 6.6% of studies, possibly impacting the overall study interpretation and conclusion. In conclusion and in the opinion of the authors, all tissues from all dose groups should be examined in toxicity studies with biotherapeutics conducted in nonrodent species
N-Butylpyrrolidinone for Solid-Phase Peptide Synthesis is Environmentally Friendlier and Synthetically Better than DMF
No full textSolid-phase peptide synthesis (SPPS) is the method of choice for the preparation of peptides in both laboratory scale and large production. Although the methodology has been improved during the last decades allowing the achievement of long peptides and challenging sequences in good yields and purities, the process was not revised from an environmental point of view. One of the main problems in this regard is the large amount of solvents used, and therefore the tons of generated waste. Moreover, the solvent of choice for the SPPS is N,N-dimethylformamide (DMF), which is considered as reprotoxic; thus, there is an urgent necessity to replace it with safer solvents. The DMF substitution by a green solvent is not a trivial task, because it should solubilize all the reagents and byproducts involved in the process, and, in addition to facilitating the coupling of the different amino acids, it should not favor the formation of side-reactions compared with DMF. Herein, it was demonstrated that the use of the green solvent N-butylpyrrolidinone (NBP) as a replacement of DMF was beneficial in two well-documented side reactions in peptide synthesis, racemization and aspartimide formation. The use of NBP rendered a lower or equal level of racemization in the amino acids more prone to this side reaction than DMF, whilst the aspartimide formation was clearly lower when NBP was used as solvent. Our findings demonstrate that the use of a green solvent does not hamper the synthetic process and could even improve it, making it environmentally friendlier and synthetically better
Metagenomic characterization of parental and production CHO cell lines for detection of adventitious viruses
No full textViral contamination is a major concern for biological products. Therefore, virus testing of raw materials and cells is essential for the safety of the final product. We used high-throughput sequencing to detect viral-like sequences in selected CHO cell lines. Our aim was to test various approaches of sample preparation, to establish a pipeline for metagenomic analysis and to characterize standard viral metagenome of production and parental CHO cell lines. The comparison of the metagenomics composition of the differently prepared samples showed that among four tested approaches sequencing of ribosomal RNA depleted total RNA is the most promising approach. The metagenomics investigation of one production and three parental CHO cell lines of diverse origin did not indicate the presence of adventitious viral agents in the investigated samples. The study revealed an expected background of virus-like nucleic acids in the samples, which originate from remains of expression vectors, endogenized viral elements and residuals of bacteriophages
Designing scalable ultrafiltration/diafiltration process of monoclonal antibodies via mathematical modeling by coupling mass balances and Poisson–Boltzmann equation
No full textUltrafiltration/diafiltration (UF/DF) operations are employed for achieving the desired therapeutic monoclonal antibody (mAb) formulations. Due to electrostatic interactions between the charged proteins, solute ions, and uncharged excipients, the final pH and concentration values are not always equal to those in the DF buffer. At high protein concentrations, typical for industrial formulations, this effect becomes predominant. To account for challenges occurring in industrial environments, a robust mathematical framework enabling the prediction of pH and concentration profiles throughout the UF/DF process is provided. The proposed mechanistic model combines a macroscopic mass balance approach with a molecular approach based on a Poisson–Boltzmann equation dealing with electrostatic interactions and accounting for protein exclusion volume effect. The mathematical model was validated with experimental data of two commercially relevant mAbs obtained from an industrial UF/DF process using scalable laboratory equipment. The robustness and flexibility of the model were tested by using proteins with different isoelectric points and net charges. The latter was determined via a titration curve, enabling realistic protein charge-pH evaluation. In addition, the model was tested for different DF buffer types containing both monovalent and polyvalent ions, with various types of uncharged excipients. The model generality enables its implementation for the UF/DF processes of other protein varieties
Reduced Mitochondrial Apoptotic Priming Drives Resistance to BH3 Mimetics in Acute Myeloid Leukemia
No full textBhatt et al. demonstrate that resistance to BCL-2 and MCL-1 antagonists emerges via selection for reduced mitochondrial apoptotic priming. Rapid measurements of drug-induced apoptotic signaling measured by dynamic BH3 profiling identify targeted agents with in vivo efficacy. BCL-2 and MCL-1 antagonist combinations overcome resistance to either single agent.
Acquired resistance to BH3 mimetic antagonists of BCL-2 and MCL-1 is an important clinical problem. Using acute myelogenous leukemia (AML) patient-derived xenograft (PDX) models of acquired resistance to BCL-2 (venetoclax) and MCL-1 (S63845) antagonists, we identify common principles of resistance and persistent vulnerabilities to overcome resistance. BH3 mimetic resistance is characterized by decreased mitochondrial apoptotic priming as measured by BH3 profiling, both in PDX models and human clinical samples, due to alterations in BCL-2 family proteins that vary among cases, but not to acquired mutations in leukemia genes. BCL-2 inhibition drives sequestered pro-apoptotic proteins to MCL-1 and vice versa, explaining why in vivo combinations of BCL-2 and MCL-1 antagonists are more effective when concurrent rather than sequential. Finally, drug-induced mitochondrial priming measured by dynamic BH3 profiling (DBP) identifies drugs that are persistently active in BH3 mimetic-resistant myeloblasts, including FLT-3 inhibitors and SMAC mimetics
Continuous, Automated Breathing Rate and Body Motion Monitoring of Rats With Paraquat-Induced Progressive Lung Injury
No full textAssessments of respiratory response and animal activity are useful endpoints in drug pharmacology and safety research. We investigated whether continuous, direct monitoring of breathing rate and body motion in animals in the home cage using the Vum Digital Smart House can complement standard measurements in enabling more granular detection of the onset and severity of physiologic events related to lung injury in a well-established rodent model of paraquat (PQ) toxicity. In rats administered PQ, breathing rate was significantly elevated while body motion was significantly reduced following dosing and extending throughout the 14-day study duration for breathing rate and at least 5 days for both nighttime and daytime body motion. Time course differences in these endpoints in response to the potential ameliorative test article bardoxolone were also readily detected. More complete than standard in-life measurements, breathing rate and body motion tracked injury progression continuously over the full study time period and aligned with, and informed on interval changes in clinical pathology. In addition, breathing rates correlated with terminal pathology measurements, such as normalized lung weights and histologic alveolar damage and edema. This study is a preliminary evaluation of the technology; our results demonstrate that continuously measured breathing rate and body motion served as physiologically relevant readouts to assess lung injury progression and drug response in a respiratory injury animal model
A phase 1b study of the MET inhibitor capmatinib combined with cetuximab in patients with MET-positive colorectal cancer who had progressed following anti-EGFR monoclonal antibody treatment
No full textBackground Overcoming resistance to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) in patients with KRAS wildtype (WT) metastatic colorectal cancer (mCRC) could help meet the needs of patients with limited treatment options. Methods In this phase 1b study, patients with N/KRAS WT, MET-positive mCRC who had progressed following anti-EGFR mAb treatment received escalating oral doses of capmatinib (150, 300, and 400 mg) twice daily plus weekly intravenous cetuximab (at the approved dose). The primary objective was to establish a recommended dose for expansion (RDE) of capmatinib in combination with cetuximab. Safety, preliminary activity, pharmacokinetics, and pharmacodynamics were also explored. Results Thirteen patients were enrolled. No patients experienced a dose-limiting toxicity at investigated doses; the RDE was established as capmatinib 400 mg twice daily plus cetuximab. All patients experienced adverse events (AEs) suspected to be related to the study treatment. Five patients (38.5%) reported study-drug–related AEs of grade 3/4 in severity. No patients achieved a complete or partial response according to RECIST v1.1; however, tumor shrinkage of 29–44% was observed in 4 patients. Conclusions Capmatinib plus cetuximab was well tolerated. Preliminary signs of activity were observed. Further investigation is warranted to obtain efficacy data and refine predictive biomarkers of response. Clinical trial registration NCT02205398
PD-1 Blockade in Anaplastic Thyroid Carcinoma
No full textPURPOSE: Anaplastic thyroid carcinoma is an aggressive malignancy that is almost always fatal and lacks effective systemic treatment options for patients with BRAF-wild type disease. As part of a phase I/II study in patients with advanced/metastatic solid tumors, patients with anaplastic thyroid carcinoma were treated with spartalizumab, a humanized monoclonal antibody against the programmed death-1 (PD-1) receptor. METHODS: We enrolled patients with locally advanced and/or metastatic anaplastic thyroid carcinoma in a phase II cohort of the study. Patients received 400 mg spartalizumab intravenously, once every 4 weeks. The overall response rate was determined according to RECIST v1.1. RESULTS: Forty-two patients were enrolled. Adverse events were consistent with those previously observed with PD-1 blockade. Most common treatment-related adverse events were diarrhea (12%), pruritus (12%), fatigue (7%), and pyrexia (7%). The overall response rate was 19%, including three patients with a complete response and five with a partial response. Most patients had baseline tumor biopsies positive for PD-L1 expression (n = 28/40 evaluable), and response rates were higher in PD-L1-positive (8/28; 29%) versus PD-L1-negative (0/12; 0%) patients. The highest rate of response was observed in the subset of patients with PD-L1 ≥ 50% (6/17; 35%). Responses were seen in both BRAF-nonmutant and BRAF-mutant patients and were durable, with a 1-year survival of 52.1% in the PD-L1-positive population. CONCLUSION: To our knowledge, this is the first clinical trial to show responsiveness of anaplastic thyroid carcinoma to PD-1 blockade
Application of Cell Impedance as a Screening Tool to Discover Modulators of Intraocular Pressure
No full textPurpose: To identify new targets and compounds involved in mediating cellular contractility or relaxation in trabecular meshwork (TM) cells and test their efficacy in an ex vivo model measuring outflow facility. Methods: A low-molecular weight compound library composed of 3,957 compounds was screened for cytoskeletal changes using the Acea xCelligence impedance platform in immortalized human NTM5 TM cells. Hits were confirmed by 8-point concentration response and were subsequently evaluated for impedance changes in 2 primary human TM strains, as well as cross-reactivity in bovine primary cells. A recently described bovine whole eye perfusion system was used to evaluate effects of compounds on aqueous outflow facility. Results: The primary screen conducted was robust, with Z' values >0.5. Fifty-two compounds were identified in the primary screen and confirmed to have concentration-dependent effects on impedance in NTM5 cells. Of these, 9 compounds representing distinct drug classes were confirmed to modulate impedance in both human primary TM cells and bovine cells. One of these compounds, wortmannin, an inhibitor of phosphoinositide 3-kinase, increased outflow facility by 11%. Conclusions: A robust phenotypic assay was developed that enabled identification of contractility modulators in immortalized TM cells. The screening hits were translatable to primary TM cells and modulated outflow facility in an ex vivo perfusion assay