Mitapivat increases ATP and decreases oxidative stress and erythrocyte mitochondria retention in a SCD mouse model.

Polymerization of deoxygenated sickle hemoglobin (HbS) leads to erythrocyte sickling. Enhancing activity of the erythrocyte glycolytic pathway has anti-sickling potential as this reduces 2,3-diphosphoglycerate (2,3-DPG) and increases ATP, factors that decrease HbS polymerization and improve erythrocyte membrane integrity. These factors can be modulated by mitapivat, which activates erythrocyte pyruvate kinase (PKR) and improves sickling kinetics in SCD patients. We investigated mechanisms by which mitapivat may impact SCD by examining its effects in the Townes SCD mouse model. Control (HbAA) and sickle (HbSS) mice were treated with mitapivat or vehicle. Surprisingly, HbSS had higher PKR protein, higher ATP, and lower 2,3-DPG levels, compared to HbAA mice, in contrast with humans with SCD, in whom 2,3-DPG is elevated compared to healthy subjects. Despite our inability to investigate 2,3-DPG-mediated sickling and hemoglobin effects, mitapivat yielded potential benefits in HbSS mice. Mitapivat further increased ATP without significantly changing 2,3-DPG or hemoglobin levels, and decreased levels of leukocytosis, erythrocyte oxidative stress, and the percentage of erythrocytes that retained mitochondria in HbSS mice. These data suggest that, even though Townes HbSS mice have increased PKR activity, further activation of PKR with mitapivat yields potentially beneficial effects that are independent of changes in sickling or hemoglobin levels.

Quantitation of ivosidenib in human plasma via LC-MS/MS and its application in clinical trials.

Aim: Ivosidenib is a potent and selective small molecule inhibitor of mutant isocitrate dehydrogenase 1. Accurate measurement of ivosidenib is the key to ivosidenib pharmacokinetics in clinical trials. Materials & methods: Quantitation of ivosidenib was conducted by using a stable isotope labeled compound (ivosidenib-d4) as the internal standard. Results: This assay was validated and successfully applied to support multiple clinical trials. Selected clinical samples were also tested by a chiral LC-MS/MS method against four ivosidenib isomer standards to exclude the possibility of in vivo racemization of ivosidenib. Conclusion: A robust LC-MS/MS method was validated for ivosidenib in human plasma. This is the first time for ivosidenib bioanalytical method in any human matrix to be reported.

The pyruvate kinase activator mitapivat reduces hemolysis and improves anemia in a ß-thalassemia mouse model.

Anemia in ß-thalassemia is related to ineffective erythropoiesis and reduced red cell survival. Excess free heme and accumulation of unpaired α-globin chains impose substantial oxidative stress on ß-thalassemic erythroblasts and erythrocytes, impacting cell metabolism. We hypothesized that increased pyruvate kinase activity induced by mitapivat (AG-348) in the Hbbth3/+ mouse model for ß-thalassemia would reduce chronic hemolysis and ineffective erythropoiesis through stimulation of red cell glycolytic metabolism. Oral mitapivat administration ameliorated ineffective erythropoiesis and anemia in Hbbth3/+ mice. Increased ATP, reduced reactive oxygen species production, and reduced markers of mitochondrial dysfunction associated with improved mitochondrial clearance suggested enhanced metabolism following mitapivat administration in ß-thalassemia. The amelioration of responsiveness to erythropoietin resulted in reduced soluble erythroferrone, increased liver Hamp expression, and diminished liver iron overload. Mitapivat reduced duodenal Dmt1 expression potentially by activating the pyruvate kinase M2-HIF2α axis, representing a mechanism additional to Hamp in controlling iron absorption and preventing ß-thalassemia-related liver iron overload. In ex vivo studies on erythroid precursors from patients with ß-thalassemia, mitapivat enhanced erythropoiesis, promoted erythroid maturation, and decreased apoptosis. Overall, pyruvate kinase activation as a treatment modality for ß-thalassemia in preclinical model systems had multiple beneficial effects in the erythropoietic compartment and beyond, providing a strong scientific basis for further clinical trials.

Mesenchymal stem cell treatment for peripheral nerve injury: a narrative review.

Peripheral nerve injuries occur as the result of sudden trauma and lead to reduced quality of life. The peripheral nervous system has an inherent capability to regenerate axons. However, peripheral nerve regeneration following injury is generally slow and incomplete that results in poor functional outcomes such as muscle atrophy. Although conventional surgical procedures for peripheral nerve injuries present many benefits, there are still several limitations including scarring, difficult accessibility to donor nerve, neuroma formation and a need to sacrifice the autologous nerve. For many years, other therapeutic approaches for peripheral nerve injuries have been explored, the most notable being the replacement of Schwann cells, the glial cells responsible for clearing out debris from the site of injury. Introducing cultured Schwann cells to the injured sites showed great benefits in promoting axonal regeneration and functional recovery. However, there are limited sources of Schwann cells for extraction and difficulties in culturing Schwann cells in vitro. Therefore, novel therapeutic avenues that offer maximum benefits for the treatment of peripheral nerve injuries should be investigated. This review focused on strategies using mesenchymal stem cells to promote peripheral nerve regeneration including exosomes of mesenchymal stem cells, nerve engineering using the nerve guidance conduits containing mesenchymal stem cells, and genetically engineered mesenchymal stem cells. We present the current progress of mesenchymal stem cell treatment of peripheral nerve injuries.

Comparison of Serenoa repens With Tamsulosin in the Treatment of Benign Prostatic Hyperplasia: A Systematic Review and Meta-Analysis.

Studies reported that Serenoa repens was effective in relieving lower urinary tract symptoms (LUTS). This article carried out a systematic review and meta-analysis to compare Serenoa repens with tamsulosin in the treatment of benign prostatic hyperplasia (BPH) after at least 6-month treatment cycle. Four studies involving 1,080 patients (543 in the Serenoa repens group and 537 in the tamsulosin group) were included in the meta-analysis. The results were as follows: compared with tamsulosin, Serenoa repens had a same effect in treating BPH in terms of International Prostate Symptom Score (IPSS) (mean difference [MD] 0.63, 95% confidence interval [CI] [-0.33, 1.59], p = 0.20), quality of life (QoL) (MD 1.51, 95% CI [-1.51, 4.52], p = 0.33), maximum flow rate (Qmax) (MD 0.27, 95% CI [-0.15, 0.68], p = 0.21), postvoid residual volume (PVR) (MD -4.23, 95% CI [-22.97, 14.44], p = 0.65), prostate-specific antigen (PSA) (MD 0.46, 95% CI [-0.06, 0.97], p = 0.08) with the exception of prostate volume (PV) (MD -0.29, 95% CI [-0.41, -0.17], p < 0.00001). For side effects, Serenoa repens was well tolerated compared with tamsulosin especially in ejaculation disorders (odds ratio [OR] = 12.56, 95% CI [3.83, 41.18], p < 0.0001) and decreased libido (OR = 5.40; 95% CI [1.17, 24.87]; p = 0.03). This study indicated that Serenoa repens had the same effect in treating BPH compared with tamsulosin in terms of IPSS, QoL, and PVR after at least 6-month treatment cycle, however, the latter had a greater improvement in PV compared with the former. And Serenoa repens did not increase the risk of adverse events especially with respect to ejaculation disorders and libido decrease.

Severe Japanese encephalitis with multiple intracranial hemorrhages: A case report.

RATIONALE: Intracranial hemorrhage occurs infrequently in Japanese encephalitis (JE), and even less frequently with hemorrhage occurring twice. In this report, we describe the clinical features and outcomes of a patient with confirmed JE combined with hemorrhage twice. PATIENT CONCERNS: The patient, a 71-year-old Asian woman, was admitted to the hospital with symptoms of hemiplegia following fever and diarrhea. Soon her condition worsened and a decreased level of consciousness, respiratory failure, and paralysis of extremities occurred.The brain diffusion-weighted imaging sequence showed suspicious abnormal signals in bilateral thalami. Japanese encephalitis virus immunoglobulin M antibody was detected in her serum and cerebrospinal fluid samples, so the patient was diagnosed with JE. During treatment, her condition became aggravated and the brain computed tomography (CT) scan showed multiple lobar hemorrhages. One month later, the multiple lobar hemorrhages occurred again, as observed by a brain CT scan. DIAGNOSIS: JE with multiple intracranial hemorrhages. INTERVENTIONS: The patient was treated comprehensively, including surgery, lowering her intracranial pressure and ventilator-assisted breathing. OUTCOMES: One month later, the patient underwent another surgical procedure for intracranial hemorrhage and suffered a serious neurological disorder. LESSONS: Severe intracranial hemorrhage may occur in elderly patients with JE, especially in those with poor vascular condition. Therefore, when treating such patients, great caution, as well as early detection and prevention, should be taken in case of the occurrence of severe intracranial hemorrhage.

Preclinical absorption, distribution, metabolism, excretion and pharmacokinetics of a novel selective inhibitor of breast cancer resistance protein (BCRP).

1. Breast cancer resistance protein (BCRP) plays an important role in drug absorption, distribution and excretion. It is challenging to evaluate BCRP functions in preclinical models because commonly used BCRP inhibitors are nonspecific or unstable in animal plasma. 2. In this work, in vitro absorption, distribution, metabolism and elimination (ADME) assays and pharmacokinetic (PK) experiments in Bcrp knockout (KO) (Abcg2-/-) and wild-type (WT) FVB mice and Wistar rats were conducted to characterize the preclinical properties of a novel selective BCRP inhibitor (ML753286, a Ko143 analog). 3. ML753286 is a potent inhibitor for BCRP, but not for P-glycoprotein (P-gp), organic anion-transporting polypeptide (OATP) or major cytochrome P450s (CYPs). It has high permeability, but is not an efflux transporter substrate. ML753286 has low to medium clearance in rodent and human liver S9 fractions, and is stable in plasma cross species. Bcrp inhibition affects oral absorption and clearance of sulfasalazine in rodents. A single dose of ML753286 at 50-300 mg/kg orally, and at 20 mg/kg intravenously or 25 mg/kg orally inhibits Bcrp functions in mice and rats, respectively. 4. These findings confirm that ML753286 is a useful selective inhibitor to evaluate BCRP/Bcrp activity in vitro and in rodent model systems.

Fasiglifam (TAK-875) Alters Bile Acid Homeostasis in Rats and Dogs: A Potential Cause of Drug Induced Liver Injury.

Fasiglifam (TAK-875), a Free Fatty Acid Receptor 1 (FFAR1) agonist in development for the treatment of type 2 diabetes, was voluntarily terminated in phase 3 due to adverse liver effects. A mechanistic investigation described in this manuscript focused on the inhibition of bile acid (BA) transporters as a driver of the liver findings. TAK-875 was an in vitro inhibitor of multiple influx (NTCP and OATPs) and efflux (BSEP and MRPs) hepatobiliary BA transporters at micromolar concentrations. Repeat dose studies determined that TAK-875 caused a dose-dependent increase in serum total BA in rats and dogs. Additionally, there were dose-dependent increases in both unconjugated and conjugated individual BAs in both species. Rats had an increase in serum markers of liver injury without correlative microscopic signs of tissue damage. Two of 6 dogs that received the highest dose of TAK-875 developed liver injury with clinical pathology changes, and by microscopic analysis had portal granulomatous inflammation with neutrophils around a crystalline deposition. The BA composition of dog bile also significantly changed in a dose-dependent manner following TAK-875 administration. At the highest dose, levels of taurocholic acid were 50% greater than in controls with a corresponding 50% decrease in taurochenodeoxycholic acid. Transporter inhibition by TAK-875 may cause liver injury in dogs through altered bile BA composition characteristics, as evidenced by crystalline deposition, likely composed of test article, in the bile duct. In conclusion, a combination of in vitro and in vivo evidence suggests that BA transporter inhibition could contribute to TAK-875-mediated liver injury in dogs.

The use of a rapid MS-based method for the quantification of the CYP 3A4 protein directly from hepatocyte cell lysate for CYP induction studies.

BACKGROUND: Most P450 protein quantitation methods involved the time-consuming preparation of microsomes and therefore are not amenable for high-throughput analysis. We here report a new method to measure P450 CYP3A4 protein levels directly from cell lysates. RESULTS: A direct sample preparation method from hepatocyte cell lysate has been developed for the quantification of CYP3A4 protein levels by combining a modified semi-automated precipitation with a filter-aided sample preparation. This novel LC-MS/MS-based method provides simple, subfemtomole sensitivity and rapid quantitation of CYP3A4 protein levels directly from hepatocyte lysate without the need for microsome preparation. CONCLUSION: A rapid, accurate and sensitive method has been developed and implemented to quantify CYP3A4 protein in hepatocytes down to 0.05 million cells in CYP induction studies. The number of cells required for quantitation was well below the typical 0.25 million cells used in a CYP induction study.

A simplified approach for the simultaneous qualification of bioanalytical methods for multiple species at late discovery stage.

BACKGROUND: Although not required to be GLP compliant, the bioanalytical methods intended to be used in supporting pharmacokinetic and exploratory toxicokinetic characterization of compounds in late drug-discovery stage are highly desirable to be qualified with respect to accuracy, precision, matrix effects and selectivity, as well as various stability assessments. Since the method qualification for each species is typically conducted separately, it is often resource-intensive and time-consuming. RESULTS: In this work, we report a simplified approach to perform a non-GLP, multiple-species, bioanalytical-method qualification. In this approach, QC samples prepared from multiple species were quantified against standard curves from a single species. More importantly, multiple stability assessments were combined to produce combination stability QC samples under the assumption that if the combination stability QC samples met the acceptance criteria, so did each individual stability assessment. CONCLUSION: Using this simplified approach, a method qualification was typically completed in less than 2 weeks compared with the 5-8 weeks of a conventional method qualification. This approach was found to be useful for bioanalytical methods developed for both plasma and whole blood matrices.

Direct analysis in real time coupled with dried spot sampling for bioanalysis in a drug-discovery setting.

BACKGROUND: Conventional mouse or rat pharmacokinetic/toxicokinetic (PK/TK) studies frequently require sacrifice or use of multiple animals for a full time-course in order to obtain adequate blood volume. Currently accepted LC-MS/MS analyses require tedious sample preparation and large blood volume, therefore, a bioanalytical method with a simpler blood-sampling procedure using fewer animals, lower sample volume and no additional sample preparation is desirable. RESULTS: We have developed a method that combines the direct analysis in real time (DART) open-air ambient ionization source and MS/MS to directly analyze dried blood spots (DBS) on glass from low volume whole blood samples without additional sample preparation or manipulation of the spots. Single mouse serial bleeding was performed for sample collection for DART-MS/MS and the results were comparable to the conventional terminal bleeding method for LC-MS/MS. CONCLUSION: The DART-MS/MS method was applied to DBS sampling for PK/TK studies and also for in vitro screening of absorption, distribution, metabolism and excretion properties. The results from the DART-MS/MS approach correlated well with the LC-MS/MS analyses for comparison.

P-glycoprotein and breast cancer resistance protein affect disposition of tandutinib, a tyrosine kinase inhibitor.

Tandutinib is a tyrosine kinase inhibitor under investigation for the treatment of solid and hematological tumors. We evaluated efflux transporter substrate specificity of tandutinib in Caco-2 cells, and the role of efflux transporters in the disposition of tandutinib in rats and efflux transporter knock-out mice. These studies demonstrated that tandutinib is a substrate of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in Caco-2 cells. In rats, administration of GF120918, before treatment with tandutinib orally resulted in approximately a seven-fold increase in the mean plasma area under the concentration-versus-time curve (AUC) compared to the vehicle control group. In mice, after intravenous administration of tandutinib, the mean plasma AUC values in the Bcrp1(-/-) mice and Mdr1a/b(-/-) mice was 1.53- and 1.20-fold greater than that of the wild type (WT) mice, respectively. After oral administration, the drug exposure in Mdr1a/b(-/-), Bcrp1(-/-), and Mdr1a/b(-/-)/Bcrp1(-/-) mice was higher than in the WT mice. The brain to plasma exposure ratio (B/P) of tandutinib in Mdr1a/b(-/-) mice increased by 2- to 3-fold over that in the WT mice. There was a 13-fold increase in B/P in Mdr1a/b(-/-)/Bcrp1(-/-) mice. This finding illustrates that P-gp and Bcrp play a role in oral absorption, systemic clearance, and brain penetration of tandutinib in the rodents. P-gp affected oral absorption and brain penetration of tandutinib to a greater extent than Bcrp, but Bcrp contribution to systemic clearance of tandutinib was greater than P-gp. Thus, co-administration of efflux pump inhibitors may be a useful strategy to enhance tandutinib absorption and brain penetration clinically.

Discovery and optimization of pyrazoline compounds as B-Raf inhibitors.

The discovery of novel pyrazoline derivatives as B-Raf (V600E) inhibitors is described in this report. Chemical modification of the pyrazoline scaffold led to the development of SAR and identified potent and selective inhibitors of B-Raf (V600E). Determination of the pharmacokinetic properties of selected inhibitors is also reported.

Bioanalysis without sample cleanup or chromatography: the evaluation and initial implementation of direct analysis in real time ionization mass spectrometry for the quantification of drugs in biological matrixes.

Two key bottlenecks in pharmaceutical bioanalysis are sample cleanup and chromatographic separations. Although multiple approaches have been developed in the past decade to either shorten or multiplex these steps, they remain the rate limiting steps as ADME (Absorption, Distribution, Metabolism, and Excretion) property screening is being routinely incorporated into the drug discovery process. In this work, a novel system incorporating an automated Direct Analysis in Real Time (DART) ionization source coupled with a triple-quadrupole mass spectrometer has been developed and evaluated for quantitative bioanalysis. This system has the capability of directly analyzing samples from their biological matrixes and therefore potentially eliminating the need for sample cleanup and chromatographic separations. A LEAP Technologies autosampler was customized to perform the automated sample introduction into the DART beam with high precision, which significantly improved the reproducibility of the method. Additional pumping was applied to the atmospheric pressure inlet on the mass spectrometer to compensate for the increased vacuum load because of the use of high-flow helium by the DART. This resulted in an improvement of detection sensitivity by a factor of 10 to 100 times. Matrix effects for a diversified class of compounds were evaluated directly from untreated raw plasma and were found to range from approximately 0.05 to 0.7. Precision and accuracy were also tested for multiple test compounds over a dynamic range of four orders of magnitude. The system has been used to analyze biological samples from both in vivo pharmacokinetic studies and in vitro microsomal/S9 stability studies, and the results generated were similar to those obtained with conventional LC/MS/MS methods. Overall, this new automated DART-triple quadrupole mass spectrometer system has demonstrated significant potential for high-throughput bioanalysis.

A novel liquid chromatography/tandem mass spectrometry based depletion method for measuring red blood cell partitioning of pharmaceutical compounds in drug discovery.

A novel liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based depletion method for measuring compound partitioning between human plasma and red blood cells (RBC) in a drug discovery environment is presented. Conventionally, RBC partitioning is determined by separate measurements of drug concentrations in equilibrating plasma and whole blood or RBC using separate standards prepared in their respective matrices, i.e., in plasma and whole blood or RBC lysates. The process is very tedious, labor-intensive, and difficult to automate. In addition, interferences from the heme and other highly abundant cellular composites make the measurement of the drug concentration in whole blood or RBC inevitably variable even with a highly specific LC/MS/MS method. Therefore, there is an imminent need to develop a straightforward and fast method to assess the partitioning of drug-like compounds in RBC. This work describes an LC/MS/MS-based depletion assay that measures the compound concentration in plasma that has been equilibrating with RBC. Compounds were spiked into fresh human whole blood and plasma respectively to a final concentration of 500 nM. Both the spiked whole blood and plasma control were incubated at 37 degrees C for up to 60 min. During the time course, aliquots of plasma and whole blood from both incubation mixtures were sampled at 10 and 60 min. The whole blood samples were centrifuged to yield the plasma. The plasma samples from both incubations were extracted using a protein precipitation method, and analyzed using LC/MS/MS under the multiple-reaction monitoring (MRM) mode. The RBC partitioning ratio was calculated using the analyte peak area responses of the plasma samples through an equation deduced in this work. The method was first tested using two commercial compounds, phenoprobamate and acetazolamide, to determine the optimal incubation conditions and the concentration dependency of the assay. The assay reproducibility was also assessed by three inter-day assays for phenoprobamate. This method was further evaluated using 20 commercial compounds of different classes with a wide range of RBC partitioning coefficients and the results were compared with those reported in the literature. Excellent correlation (R2=0.9396) was found between the measured and literature values. In addition, several proprietary compounds were assayed using both the new and traditional methods and the measured partitioning ratios from the two methods are equivalent. The experiments in this work demonstrate that the LC/MS/MS-based depletion method can provide direct and accurate measurement of RBC partitioning for compounds in drug discovery.

Application of a high-throughput HPLC-MS/MS assay to Arabidopsis mutant screening; evidence that threonine aldolase plays a role in seed nutritional quality.

Beyond their essential function as the building blocks of proteins, amino acids contribute to many aspects of plant biochemistry and physiology. Despite this, there are relatively large gaps in our understanding of the biochemical pathways and regulation of amino acid synthesis in plants. A rapid (1.5 min versus 20-90 min for standard methods) HPLC-MS/MS assay for separating 19 amino acids was developed for quantifying levels of free amino acids in plant tissue. This assay was used to determine the free amino acid content in the seeds of 10,000 randomly mutagenized Arabidopsis lines, and 322 Arabidopsis lines with increased levels of one or more amino acids were identified. The heritability of the mutant phenotype was confirmed for 43 lines with increased seed levels of the aspartate-derived amino acids Ile, Lys, Thr, or Met. Genetic mapping and DNA sequencing identified a mutation in an Arabidopsis threonine aldolase (AT1G08630, EC 4.1.2.5) as the cause of increased seed Thr levels in one mutant. The assay that was developed for this project has broad applicability to Arabidopsis and other plant species.

Quararibea Metabolites. 4.(1) Total Synthesis and Conformational Studies of (+/-)-Funebrine and (+/-)-Funebral.

Syntheses of racemic forms of the main secondary metabolites of Quararibea funebris, (+/-)-funebrine, (+/-)-funebral, and their biogenetic precursor (+/-)-(2S,3S,4R)-gamma-hydroxyalloisoluecine lactone have been developed. In synthetic studies, a new variation of the Paal-Knorr condensation employing titanium isopropoxide was utilized to construct the pyrrole lactone moiety. Two efficient synthetic approaches to the key (+/-)-gamma-amino lactone have been developed, one based on Claisen chemistry and the other on addition reactions to the butenolide ring of beta-angelicalactone. The restricted rotation around the C(sp(3))-N(sp(2)) bond in the pyrrole lactone structures of (+/-)-funebrine, (+/-)-funebral, and related aldehydes has been probed by conformational dynamic studies, and the barriers for interconversion between conformations have been measured by full NMR line-shape analysis. Molecular mechanics (MMX) and a (1)H-(1)H NOE study indicate a distinct preferred conformation for (+/-)-funebrine.