Pharmacological Assessment Research Articles

Fabrication and Evaluation of Controlled Release Transdermal Drug Delivery System of Carvedilol using Design Expert Software for the Management of Hypertension

Published on:August 2022Journal of Young Pharmacists, 2022; 14(3):295-301Original Article | doi:10.5530/jyp.2022.14.58 Authors: Saurabh Maan1, Roop Krishen Khar2, Rupa Mazumder3,*, Nishant Yadav1, Urooj Ahmed Khan4,* 1Department of Pharmaceutics, B.S. Anangpuria Institute of Pharmacy, Faridabad, Haryana, INDIA. 2Department of Pharmaceutics, B.S. Anangpuria Educational Institute, Faridabad, Haryana, INDIA. 3Department of Pharmacy, Noida Institute of Engineering and Technology, Greater Noida, Uttar Pradesh, INDIA. 4Department of Pharmaceutics, School of Medical and Allied Sciences, K.R Mangalam University, Sohna Road, Gurugram, Haryana, INDIA. Abstract: Objectives: Design, development and characterization of controlled release transdermal drug delivery system of carvedilol using Design Expert® software for managing hypertension. Materials and Methods: CV undergoes extensive first-pass metabolism due to low oral bioavailability approx ~24% and biological half-life ~6 hr. Available CV preparations have drawback of poor patient compliance due to increase in dosing frequency and making the therapy less effective. Phospholipid E80, glycerol and cholesterol were used for preparing CVGs via film hydration technique. Optimization was done using Central Composite Design under Design Expert software. While PVP and EC in a ratio of 4:1 in chloroform (5 mL) with plasticizer dibutyl phthalate (30%) were used for preparing Matrix type transdermal patch containing CVGs. Results: Optimized CVGs showed 115.7 nm particle size, and -16.4 mV zeta potential. TEM analysis also showed similar vesicle size and reveals globular structure of CVGs. Nearly 0.31 mm thickness, 0.14 % g weight variation, 99.13% flatness and 98.72% drug content was found in Matrix type transdermal patch having CVGs Transdermal patch with Franz diffusion cell showed approx. 90% release upto 48 hr during in-vitro permeation studies. In-vivo pharmacological assessment was done for efficacy estimation of GVs transdermal patch by N-nitro-L-arginine methyl ester which produced significant hypertension in rats. The application of CVGs transdermal patch resulted in a gradual decrease in BP, with the maximum effect from the patch observed at 10 hr (p < 0.001), the effect continued for 48 hr clearly indicating the gradual release of drug by transdermal patch for a long period. Conclusion: Developed novel transdermal patch having CV loaded glycerosomes may be considered as a promising approach for controlled release of drug with effective hypertension management. Keywords: Transdermal, Carvedilol, Design Expert, Glycerosomes, Hypertension, CCD.

Review of five years of comprehensive NPS analytics and insights into the substance identification workflow of the ADEBAR project

The ADEBAR project aims to comprehensively characterize emerging substances relevant for forensic toxicological casework providing collaborative structure elucidation and instrumental analysis services for forensic science institutes in and around Germany. The ADEBAR project addresses the need of the forensic science community for structural elucidation of novel new psychoactive substances (NPS) and other substances appearing on the illicit market and the digital provision of the analytical datasets. High-quality reference substances as well as court-proof analytical data are required to keep screening methods and databases up to date in a timely and cost-efficient manner. The ADEBAR project represents the competence network of the Federal Criminal Police Office, seven State Bureaus of Criminal Investigation, and the German Customs, co-funded by the EU's Internal Security Fund since July 2017. Additionally, the Universities of Mainz and Freiburg have been involved in the anticipatory synthesis and pharmacological assessment of NPS variants since 2019. Forensic institutes, mostly German police and customs laboratories, submit samples to the ADEBAR consortium. New substances emerging on the drug market (seized or purchased) are elucidated and comprehensively characterized using chromatographic, spectroscopic, and spectrometric techniques [GC-MS, (N)-IR, GC-sIR, NMR, LC-(HR)MS, and Raman spectroscopy]. The workflow chart illustrates the sequence of analytical processes within the ADEBAR project. The results of the analytical characterization are documented in a publishable comprehensive report. The report and the corresponding analytical datasets are published digitally through national and international channels (forum of the GTFCh, EDND of the EMCDDA, and NPS Data Hub) and formal reports are submitted to the early warning system (EWS) of the EMCDDA. Over the period of four and a half years, a total of 549 substances have been analytically characterized, leading to close to 400 reports published. Seized materials, test purchases in online shops, and designer drug derivatives synthesized within the ADEBAR project were analyzed. The minimum requirement of the ADEBAR project for an unequivocal identification is applying two analytical techniques of category A (referring to the SWGDRUG classification scheme) to differentiate structurally related isomers reliably. Synthetic cannabinoids were the largest and (designer) pharmaceuticals the second largest group of substances analyzed so far. Identified opioids shifted from fentanyl analogs like cyclopentanoylfentanyl, 3-methylcrotonylfentanyl, and “U-type” compounds (mainly detected in 2017 to 2018) to buccinazine (AP-237, 2-Me-AP-237, and AP-238) and benzimidazole type opioids like isotonitazene, etonitazene, and etonitazepipne from 2019 onwards. In addition, 106 fully characterized and precisely quantified NPS reference substances have been issued to all German forensic institutes together with a certificate of analysis as a continuous service of the ADEBAR project. The ADEBAR project has been contributing to the availability of analytical data and reference material relevant to the daily work of police and customs laboratories. A total of 3619 mass spectra have been published, together with NMR, IR, GC-sIR, and NIR spectra. The datasets were published worldwide and free of charge and facilitate the identification of these new compounds by other forensic laboratories.

Structural, spectral inspection, electronic properties in different solvents, Fukui functions, 6-acetyl-2H-1,4-benzoxazin-3(4H)-one – Multiple sclerosis and auto immune disorders therapeutics

• Spectroscopic (FT-IR, FT-Raman) analysis has been carried out. • Electronic properties (NLO, HOMO-LUMO, UV and MEP) have been analyzed. • NBO analysis and Fukui functions have been examined. • Using Multiwavefunction, properties ELF and LOL have been studied. • Pharmacological assessment has been done by docking and drug likeness. Using DFT method along with the basis function 6-311++G(d,p), the compound 6-acetyl-2H-1,4-benzoxazin-3(4H)-one was analyzed with the help of vibrational spectroscopy. The vibrational wavenumbers have been computed and scaled down. These were compared with the experimental values. The geometry was optimized. The values of the parameters bond length and bond angle have been generated. Using gas and solvents water, DMSO, ethanol and benzene, the properties of NLO, analysis of HOMO-LUMO and MEP studies have been carried out. UV spectra of the title compound were stimulated by TD-DFT method and IEFPCM model in the solvents mentioned above. The investigation of inter and intra molecular reactions of the molecule has been done by NBO analysis. The region of reactive sites were found by Fukui functions. Using Multiwave function, the parameters ELF and LOL have been investigated in gas. The drug like parameters were found and the biological nature of the compound was observed by molecular docking studies.

The anticholinergic burden: from research to practice.

SUMMARYDrugs with anticholinergic effects are known to cause adverse effects such as dry mouth, constipation and urinary retention. In older people drugs with anticholinergic effects may contribute to cognitive decline and a loss of functional capacity.Many drugs that are not in the anticholinergic drug class also have anticholinergic effects. They include antidepressants, antipsychotics and antihistamines.Taking multiple drugs with anticholinergic effects creates an anticholinergic burden. It is important that clinicians identify which patients are at risk. There are several tools to assess the anticholinergic burden.Clinicians can use these tools to make a pharmacological risk assessment when reviewing a patient’s medicines. This can assist decisions about continuing or stopping drugs with anticholinergic effects.Deprescribing drugs with anticholinergic effects has several potential benefits in older people. In addition to reversing adverse effects, deprescribing may prevent problems such as falls.

Validation of wound healing property of Siddha herbal extractive preparation Ellukai Thol Uppu (salt extraction from pericarp of Sesamum indicum)

Background: The belief of general public and globalization on Siddha medicine led to the stable growth of Indian traditional medicine, thereby necessitating the standardization of Siddha formulation and pharmacopeia standards for their quality control. Ellukai Thol Uppu (ETU) is a Siddha formulation extracted from Sesamum indicum by Siddha traditional method used for excision wound. The present study aimed to study the standardization parameters and screen the antimicrobial and wound-healing properties. Objective: The present study aimed to study the standardization parameters and screen the antimicrobial and wound-healing properties. Materials and Methods: ETU was prepared from Sesamum indicum as per the literature, and subjected to study the standardization parameters such as physicochemical analysis and heavy metal analysis were done by using AYUSH testing protocol, and screening of acute toxicity was done by organisation for economic co-operation and development method, and wound healing property was done by using excision wound method. Result: The study results revealed that the drug ETU is free from any microbial contaminations and has no toxicity effect. The drug ETU possesses antimicrobial and wound-healing properties. Conclusion: The derived results could serve as a ready reference for quality control and pharmacological property assessment of the drug.

Abstract 3594: Exportin 1 inhibition as a therapeutic strategy for small cell lung cancer

Abstract Small cell lung cancer (SCLC) is an aggressive disease characterized by early metastasis and exceptional lethality, comprising 13% of all lung cancer cases. With few treatment options, typically resulting in only transient responses, SCLC is responsible for approximately 250,000 deaths globally per year. The backbone of SCLC treatment over the past several decades has been platinum-based doublet chemotherapy, with the recent addition of immunotherapy to first-line chemotherapy showing limited benefit in a small subset of patients. Major hurdles to improving SCLC treatment include development of rapid chemoresistance and ineffective second line therapies. The identification of more durably effective therapeutic strategies is a major unmet clinical need. Here, we performed an in vitro CRISPR screen in SCLC cell lines from all major SCLC subtypes, including short-term cultured cells from patient-derived xenografts (PDXs), to identify potential therapeutic targets to enhance sensitivity to chemotherapy. Candidate hits were validated genetically and pharmacologically with in vitro synergy assays, in vivo clonal competition assays and pharmacologic assessments in PDX models. Signaling pathways were studied by RNA sequencing and western blot, and toxicity studies were performed in vivo to assess the safety of the agents at pharmacologically effective doses. We performed immunohistochemistry (IHC) to assess expression of candidate targets in tissue microarrays (TMAs). Our CRISPR screen revealed the nuclear exporter exportin 1 (encoded by the XPO1 gene) as a promising target sensitizing to chemotherapy, independently of the SCLC subtype. We found that XPO1 mRNA expression was higher in SCLC than in any other solid tumor or hematological malignancy, and demonstrated consistently high protein expression by IHC in clinical TMAs. A potent and selective exportin 1 inhibitor, selinexor, is approved for use in hematological malignancies. Combination of selinexor with cisplatin or irinotecan demonstrated synergy in vitro and efficacy in vivo in an array of chemonäive and chemoresistant SCLC PDXs, including all major SCLC subtypes. The combinations were well tolerated in mice. The chemo-sensitizing effects of selinexor were associated with suppression of chemotherapy-induced AKT activation. In conclusion, exportin 1 inhibition strongly enhances sensitivity of SCLC tumors to cisplatin and irinotecan, used in first line and second line treatment of SCLC tumors, respectively, and these effects are independent of the SCLC subtype. These results provide preclinical rationale for the combination of selinexor with cisplatin or irinotecan in naïve and relapsed SCLC. The clinical availability of selinexor will allow rapid clinical translation of these results in a disease setting with extremely limited therapeutic options. Citation Format: Alvaro Quintanal-Villalonga, Hirokazu Taniguchi, Yuan Hao, Andrew Chow, Yingqian A. Zhan, Fathema Uddin, Viola Allaj, Parvathy Manoj, Nisargbhai S. Shah, Umesh K. Bhanot, Juan Qiu, Elisa de Stanchina, Richard P. Koche, Triparna Sen, John T. Poirier, Charles M. Rudin. Exportin 1 inhibition as a therapeutic strategy for small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3594.

Abstract LB567: Dramatic in vivo efficacy of the EZH2-inhibitor tazemetostat in PBRM1-mutated human chordoma xenograft

Abstract Background: Chordomas are considered as rare bone neoplasms characterized by a high level of recurrence and a poor long-term prognosis. Considering their chemo-radio-resistance, alternative treatment strategies are therefore strongly requested, but their development is limited by the paucity of relevant preclinical models. Mutations affecting genes of the SWI/SNF complex including PBMR1 are frequently found in chordomas, suggesting a potential therapeutic effect of epigenetic regulators in this indication. Methods: Thirty-eight chordoma specimens derived from primary patients’ tumors were grafted into nude mice. Relationship between grafted tumors and clinical, biological and therapeutic features of corresponding patients were investigated. We subjected the established PDX models to histopathological and genetic examination; finally, a therapeutic program focused on the EZH2-inhibition was performed in one PDX model. Results: Twelve PDX models were established and characterized on histological and biomolecular features. Patients whose tumors were able to grow into mice had a statistically significant lower progression-free survival than those whose tumors did not grow after in vivo transplantation (p=0.007). All PDXs maintained the same histopathological features as patients’ tumors. Homozygous deletions of CDKN2A/2B (58.3%) and PBRM1 (25%) variants were the most common genomic alterations found. In the tazemetostat treated PDX model harboring a PBRM1 variant, an overall survival of 100% was observed. Conclusion: Our panel of chordoma PDXs represents a useful preclinical tool for both pharmacologic and biological assessments. The first demonstration of a high antitumor activity of tazemetostat in a PDX model harboring a PBRM1variant supports further evaluation of EZH2-inhibitors in this subgroup of chordomas. Citation Format: Thibault Passeri, Ahmed Dahmani, Julien Masliah-Planchon, Adnan Naguez, Marine Michou, Rania El-Botty, Sophie Vacher, Rachida Bouarich, André Nicolas, Marc Polivka, Coralie Franck, Anne Schnitzler, Fariba Némati, Sergio Roman-Roman, Franck Bourdeaut, Homa Adle-Biassette, Hamid Mammar, Sébastien Froelich, Ivan Bièche, Didier Decaudin. Dramatic in vivo efficacy of the EZH2-inhibitor tazemetostat in PBRM1-mutated human chordoma xenograft [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB567.

Abstract 6013: TST005, a bifunctional fusion protein of PD-L1/TGF-βRII, demonstrates potent anti-tumor activities with good safety profiles

Abstract Antibodies targeting PD1/PD-L1 are emerging as effective cancer immunotherapies, however, not all patients with PD-L1 expression respond to the treatment. A growing body of data demonstrates the pivotal roles of the master regulators from the tumor microenvironment (TME), such as transforming growth factor β (TGF-β), in the development of resistance to Immune checkpoint inhibitors. Therefore, co-inhibition of TGF-β signaling is expected to enhance the antitumor responses of PD1/PD-L1-based immunotherapies. TST005 is a bifunctional fusion protein composed of the truncated extracellular domain of the TGF-βRII receptor (a TGF-β trap) fused to a humanized anti-PD-L1 IgG1 antibody with ablated Fc immune effector function. Previously we showed the potent antitumor activity of TST005 in MC38/hPD-L1 and EMT6/hPD-L1 xenograft models. Here we report TST005 anti-tumor activities in MC38 colorectal cancer and EMT-6 breast cancer models compared to M7824 (Merck’s PD-L1/TGF-βRII) analog and its safety profiles following single or repeated doses in rats and non-human primates (NHP). TGF-β1depletion from the plasma of MC38 tumor-bearing mice was observed even at the lowest dose (1mg/kg). At higher dose (10mg/kg), TGF-β1 depletion persisted longer. The same trend was also found in MC38 tumor tissue. In EMT-6 tumor model upon TST005 treatment (30mg/kg), TST005 showed better tumor inhibition compared to M7824 analog at high dose (30mg/kg), which correlates well with the extent of TGF-β1depletion. For TST005, TGF-β1depletion was persistent throughout all time points tested (up to Day 14) in plasma and tumor tissue compared to M7824 analog at the same dose/dosing frequency. TST005 treatment (3mg/kg,10mg/kg) significantly lowered phosphorylated SMAD2 (pSMAD2) in MC38 tumors. TST005 was well tolerated in both rats and monkeys, no significant safety issues were observed following single or repeated doses in general toxicity studies and safety pharmacology assessment, the exposure of TST005 in both species was comparable between two genders and increased in an approximately dose-proportional manner. No cytokine release was observed in monkeys. Based on the exposure of TST005 in monkeys and predicted exposure at First in Human (FIH) dose, the safety margin of TST005 will be higher than 200 and 500 folds calculated on Cmax and AUC respectively. In conclusion, we have demonstrated the antitumor activity of TST005 in PD/PD-L1 sensitive and resistant tumor models as well as the safety profile in NHP, TST005 has been granted for phase 1 clinical trials in USA (NCT04958434). Citation Format: Huanhuan Guo, Xinlai Yao, Chunming Wang, Linlin Mao, Hongjun Li, Shuang Lu, Di Sun, Chengwei Jiang, Shenjie Zhang, Lisa Zheng, Fan Zhang, Fei Teng, Steven Yu, Yi Gu, Xueming Qian. TST005, a bifunctional fusion protein of PD-L1/TGF-βRII, demonstrates potent anti-tumor activities with good safety profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6013.

COMPUTATIONAL PREDICTION OF BIOACTIVE COMPOUNDS AS POTENTIAL INHIBITORS OF COVID-19 MAIN PROTEASE, SPIKE GLYCOPROTEIN RECEPTOR-BINDING DOMAIN, AND RNA-DEPENDENT RNA POLYMERASE

Objectives: It has been known for ages that natural products have potent antiviral activity and hence show inhibitory effects on SARS-CoV-2 infections. In this study, some promising bioactive compounds from natural sources for drug development against SARS-CoV-2 were studied. Methods: The study was based on a computational approach using different phytochemicals for evaluating their potential against non-structural (main protease [MPro], RNA-dependent RNA polymerase [RdRp], and structural [spike [S] glycoprotein receptor-binding domain]) viral proteins. Molecular docking was conducted systematically using PyRx and AutoDock 4.2 to determine the binding affinities between bioactive compounds and Mpro, spike RBD, and RdRp. Twenty-two ligands were selected in this study from different sources including three known inhibitors of the virus remdesivir, favipiravir, and nelfinavir. The pharmacological assessment of the ligands was achieved using ADMET filters. Results: The docking results revealed that β-carotene, piperine, and cianidanol were the best antagonists for Mpro, isovitexin, quercitin, β-carotene, piperine, and cianidanol were the best antagonists for RdRp, and in case of the spike RBD, capsaicin, cianidanol, curcumin, gingerol, isovitexin, piperine, quercitin, rhapontin, and riboflavin were found to be best. Conclusion: All of these bioactive compounds could be considered potential drug candidates for COVID-19 inhibition due to their promising binding affinities with the viral structural and non-structural proteins.

Characterization of JNJ-2482272 [4-(4-Methyl-2-(4-(Trifluoromethyl)Phenyl)Thiazole-5-yl) Pyrimidine-2-Amine] As a Strong Aryl Hydrocarbon Receptor Activator in Rat and Human.

[4-(4-Methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-yl)pyrimidine-2-amine] (JNJ-2482272), under investigation as an anti-inflammatory agent, was orally administered to rats once daily at 60 mg/kg for 6 consecutive days. Despite high plasma exposure after single administration (Cmax of 7.1 μM), JNJ-2482272 had plasma concentrations beneath the lower limit of quantification (3 ng/ml) after 6 consecutive days of dosing. To determine if JNJ-2482272 is an autoinducer in rats, plated rat hepatocytes were treated with JNJ-2482272 for 2 days. The major hydroxylated metabolites of JNJ-2482272 were isolated and characterized by mass spectrometry and NMR analyses. Compared with the vehicle-treated cells, a concentration-dependent increase was observed in the formation of phase I- and II-mediated metabolites coinciding with greater expression of cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) in rat hepatocytes. CYP1A1, CYP1A2, CYP1B1, and UGT1A6 transcripts were predominantly induced, suggesting that JNJ-2482272 is an activator of the aryl hydrocarbon receptor (AhR). In a human AhR reporter assay, JNJ-2482272 demonstrated potent AhR activation with an EC50 value of 0.768 nM, a potency more comparable to the strong AhR activator and toxin 2,3,7,8-tetrachloro-dibenzodioxin than to weaker AhR activators 3-methylcholanthrene, β-naphthoflavone, and omeprazole. In plated human hepatocytes, JNJ-2482272 induced CYP1A1 gene expression with an EC50 of 20.4 nM and increased CYP1A activity >50-fold from basal levels. In human recombinant P450s, JNJ-2482272 was exclusively metabolized by the CYP1 family of enzymes and most rapidly by CYP1A1. The summation of these in vitro findings bridges the in vivo conclusion that JNJ-2482272 is a strong autoinducer in rats and potentially in humans through potent AhR activation. SIGNIFICANCE STATEMENT: Drugs that induce their own metabolism (autoinducers) can lack sustained exposures for pharmacology and safety assessment hindering their development. JNJ-2482272 is demonstrated herein as a strong aryl hydrocarbon receptor (AhR) activator and CYP1A autoinducer, explaining its near complete loss of exposure after repeat administration in rat, which is likely translatable to human (if progressed further) considering its nanomolar potency comparable to "classical" AhR ligands like 2,3,7,8-tetrachloro-dibenzo-dioxin despite bearing a "nonclassical" drug structure.

Comprehensive Nonclinical Safety Assessment of Nirmatrelvir Supporting Timely Development of the SARS-COV-2 Antiviral Therapeutic, Paxlovid™.

COVID-19 is a potentially fatal infection caused by the SARS-CoV-2 virus. The SARS-CoV-2 3CL protease (Mpro) is a viral enzyme essential for replication and is the target for nirmatrelvir. Paxlovid (nirmatrelvir co-administered with the pharmacokinetic enhancer ritonavir) showed efficacy in COVID-19 patients at high risk of progressing to hospitalization and/or death. Nonclinical safety studies with nirmatrelvir are essential in informing benefit-risk of Paxlovid and were conducted to support clinical development. In vivo safety pharmacology assessments included a nervous system/pulmonary study in rats and a cardiovascular study in telemetered monkeys. Potential toxicities were assessed in repeat dose studies of up to 1 month in rats and monkeys. Nirmatrelvir administration (1,000 mg/kg, p.o.) to male rats produced transient increases in locomotor activity and respiratory rate but did not affect behavioral endpoints in the functional observational battery. Cardiovascular effects in monkeys were limited to transient increases in blood pressure and decreases in heart rate, observed only at the highest dose tested (75 mg/kg per dose b.i.d; p.o.). Nirmatrelvir did not prolong QTc-interval or induce arrhythmias. There were no adverse findings in repeat dose toxicity studies up to 1 month in rats (up to 1,000 mg/kg daily, p.o.) or monkeys (up to 600 mg/kg daily, p.o.). Nonadverse, reversible clinical pathology findings without clinical or microscopic correlates included prolonged coagulation times at ≥60 mg/kg in rats and increases in transaminases at 600 mg/kg in monkeys. The safety pharmacology and nonclinical toxicity profiles of nirmatrelvir support clinical development and use of Paxlovid for treatment of COVID-19.

Single-cell RNA sequencing unravels heterogeneity of skeletal progenitors and cell-cell interactions underlying the bone repair process.

IntroductionActivation of skeletal progenitors upon tissue injury and the subsequent cell fate specification are tightly coordinated in the bone repair process. Although known osteoimmunological signaling networks play important roles in the microenvironment of the bone defect sites, the molecular mechanism underlying the bone repair process has not been fully understood.MethodsTo better understand the behavior of the skeletal progenitors and the heterogeneity of the cells during bone repair at the microenvironmental level, we performed a combinatorial analysis consisting of lineage tracing for skeletal progenitors using the Sox9-CreERT2;R26RtdTomato mouse line followed by single-cell RNA sequencing (scRNA-seq) analysis using a mouse model of calvarial bone repair. To identify a therapeutic target for bone regeneration, further computational analysis was performed focusing on the identification of the cell–cell interactions, followed by pharmacological assessments with a critical-size calvarial bone defect mouse model.ResultsLineage tracing analysis showed that skeletal progenitors marked by Sox9 were activated upon bone injury and contributed to bone repair by differentiating into osteoblasts. The scRNA-seq analysis characterized heterogeneous cell populations at the bone defect sites; the computational analysis predicted a bifurcated lineage from skeletal progenitors toward osteogenic and adipogenic lineages. Chemokine C–C motif ligand 9 (Ccl9) was identified as a signaling molecule that regulates bone regeneration in the mouse model, possibly through the regulation of adipogenic differentiation at the bone defect site.ConclusionMultipotential skeletal progenitors and the direction of the cell differentiation were characterized at single cell resolution in a mouse bone repair model. The Ccl9 signaling pathway may be a key factor directing osteogenesis from the progenitors in the model and may be a therapeutic target for bone regeneration.

A database-guided integrated strategy for comprehensive chemical profiling of traditional Chinese medicine

A comprehensive chemical profiling of traditional Chinese medicine is the basic issue for further pharmacological research and quality assessment. To facilitate chemical identification and potential components discovery, the present study proposed an integrated identification strategy guided by a self-built component database constructed from literatures to carry out the global profiling of complex matrixes. Lanqin Oral Liquid was applied as example to validate the feasibility of this strategy. Based on LQL Component Database containing 710 compounds, modified MDF windows was established to extract the interested analogues, isoquinoline alkaloids, flavonoids and iridoid glycosides, according to their regular integral masses and mass defect. For compounds with characteristic substructures, such as quinic acids, crocins and some glycoside derivatives, the associated neutral losses and diagnostic fragment ions were collected to assist in profiling. Directly matching the m/z or formulas in database was proposed to components with limited regularity of accurate masses and substructures, like indole alkaloids, sesquiterpenes and some nucleosides. Eventually, 170 ions of 1038 precursor ions were identified or temporarily deduced, including 59 alkaloids, 36 flavonoids, 48 terpenoids, 24 organic acids and their derivatives, 2 oligosaccharides, and 1 lignans. Among them, 52 putative compounds were confirmed by chemical standards. The results indicated that the database-oriented identification strategy could locate potential components quickly and eliminate interfering ions, which have the potential for in-depth analysis of compounds.

Applications of Model-Based Target Pharmacology Assessment in Defining Drug Design and DMPK Strategies: GSK Experiences.

Many critical decisions faced in early discovery require a thorough understanding of the dynamic behavior of pharmacological pathways following target engagement. From fundamental decisions on the optimal target to pursue and the ultimate drug product profile (combination of modality, potency, and compound properties) expected to elicit the desired clinical outcome to tactical program decisions such as what chemical series to pursue, what chemical properties require optimization, and what compounds to synthesize and progress, all demand detailed consideration of pharmacodynamics. Model-based target pharmacology assessment (mTPA) is a computational approach centered around large-scale virtual exploration of pharmacokinetic and pharmacodynamic models built early in discovery to guide these decisions. The present work summarizes several examples (use cases) from programs at GlaxoSmithKline that demonstrate the utility of mTPA throughout the drug discovery lifecycle.

Addressing Race, Ethnicity, and Structural Inequality in Pharmacology Education and Assessment

OBJECTIVESocial determinants of health, including race, ethnicity, and gender, represent important factors in health care delivery and patient outcomes, and contribute to health disparities. Pharmacology, as an integrated discipline, lies at the nexus between foundational science and clinical practice, providing a platform for educators to discuss the impact of social determinants on patient outcomes. Our goal is to create a framework that provides guidance for topics in pharmacotherapeutics for educators, learners, and providers to use in promoting antiracism in medical curricula.METHODSThrough iterative discussions, we developed a conceptual framework that includes topics taught across medical schools, such as drug metabolism, pharmacogenomics, adverse drug reactions, prescribing guidelines, factors affecting medication adherence, pain management, and therapeutic outcomes. Under each topic, we describe how social constructs such as race, ethnicity, and gender can impact pharmacology and therapeutic outcomes.RESULTSLiterature reports show that some topics in pharmacology education have already established cultural competency and should be adopted widely by all pharmacology educators, e.g., using more appropriate terminology for drug reactions such as vancomycin‐infusion reaction. Other topics are less established, so a more nuanced discussion with students is required. Society guidelines for managing diseases such as hypertension include prescribing recommendations based on race, with which learners are expected to be familiar as they enter clinical training but are actively being debated. Similarly, inter‐ethnic differences in drug metabolism are often reported as a function of genetic polymorphisms without considering non‐genetic factors.CONCLUSIONSWe have developed a conceptual framework for promoting antiracism in pharmacology education by categorizing topics based on those where cultural competencies are clearly established and those where a more nuanced discussion is required with learners, as well as identifying and reducing bias in assessment. These are generalizable across health professions in which pharmacology education is a discipline in the curriculum.

Flavonoid components, chemotypes, and candidate chemical markers of Teucrium (Lamiaceae) species using HPLC-MQ-API-MS/MS

Belonging to Lamiaceae family, the genusTeucriumL. is a rich source of secondary metabolites used in pharmacology and industrial medicine. This study aimed to investigate the flavonoid contents of methanol extract and the chemical markers ofTeucriumat specific and infraspecific levels and identify the chemotypes of its accessions. Nineteen accessions, eight species, and five subspecies were collected from three sections (Teucris, Polium, andScordium) belonging to the natural habitats in the west, southwest, centre, and south of Iran. Total flavonoid extraction was attained by the air-dried leaf ofTeucriumspecies and 90% methanol. HPLC coupled with triple quadrupole mass analyser was applied to screen and identify the chemical constituents. Multivariate analyses were implemented by cluster and principal component analysis. Chemical investigation specified 148 chemical compounds, of which 135 flavonoids were tentatively identified. The most abundant derivatives belonged to flavones (41 derivatives), flavonols (37 derivatives), and isoflavonoids (22 derivatives). Based on the cluster analysis, 13 groups of chemical markers were formed in sectionsTeucrisandScordiumand five groups in sectionPoliumas chemotaxonomic indicators. The specified chemotypes were represented by ten in the sectionsTeucrisandScordiumand by six in the sectionPolium. A relationship was suggested between type and flavonoid quantity (number), altitude, and geographical location. The present study provided valuable information for pharmacological research, bioactivity assessment, and chemotaxonomic purposes. This is the first report on the presence of chemotypes, chemical markers, and quality assessments for the genusTeucrium.

Experimental Models for Testing the Efficacy of Pharmacological Treatments for Neonatal Hypoxic-Ischemic Encephalopathy.

Representing an important cause of long–term disability, term neonatal hypoxic-ischemic encephalopathy (HIE) urgently needs further research aimed at repurposing existing drug as well as developing new therapeutics. Since various experimental in vitro and in vivo models of HIE have been developed with distinct characteristics, it becomes important to select the appropriate preclinical screening cascade for testing the efficacy of novel pharmacological treatments. As therapeutic hypothermia is already a routine therapy for neonatal encephalopathy, it is essential that hypothermia be administered to the experimental model selected to allow translational testing of novel or repurposed drugs on top of the standard of care. Moreover, a translational approach requires that therapeutic interventions must be initiated after the induction of the insult, and the time window for intervention should be evaluated to translate to real world clinical practice. Hippocampal organotypic slice cultures, in particular, are an invaluable intermediate between simpler cell lines and in vivo models, as they largely maintain structural complexity of the original tissue and can be subjected to transient oxygen–glucose deprivation (OGD) and subsequent reoxygenation to simulate ischemic neuronal injury and reperfusion. Progressing to in vivo models, generally, rodent (mouse and rat) models could offer more flexibility and be more cost-effective for testing the efficacy of pharmacological agents with a dose–response approach. Large animal models, including piglets, sheep, and non-human primates, may be utilized as a third step for more focused and accurate translational studies, including also pharmacokinetic and safety pharmacology assessments. Thus, a preclinical proof of concept of efficacy of an emerging pharmacological treatment should be obtained firstly in vitro, including organotypic models, and, subsequently, in at least two different animal models, also in combination with hypothermia, before initiating clinical trials.

Pharmacological assessment of Ru(II) complex with GidA protein- A novel topoisomerase II inhibitor towards cancer therapeutics

The interactions of ruthenium(II) complex with Glucose inhibited division protein A (GidA protein) was studied through various spectroscopic techniques with the ultimate goal of preparing adducts with good selectivity for cancer cells. In all the cases, formation of a tight metal-protein conjugate was observed. The influence of pH, reducing agents and chelators on the formation of adduct was analysed by UV- visible spectroscopy. While there was no effect on the addition of sodium ascorbate, some alterations on some selected bands were seen on the UV-visible spectra on the addition of EDTA. The adduct was stable in the pH range of 5-8. Addition of ruthenium(II) complex effectively quenched the intrinsic fluorescence of GidA and it occurred through static quenching. The effect of ruthenium(II) complex on the conformation of GidA has been examined by analyzing CD spectrum. Though, there was some conformational changes observed in the presence of ruthenium(II) complex, α- helix in the secondary structure of GidA retained its identity. Molecular docking of ruthenium(II) complex with GidA also indicated that GidA docks through hydrophobic interaction. The stable semisynthetic complex (ruthenium(II) complex with GidA) was checked for topoisomerase II inhibition. Relaxation and decatenation assay proved topoisomerase II inhibition of semisynthetic complex. Communicated by Ramaswamy H. Sarma

Dramatic In Vivo Efficacy of the EZH2-Inhibitor Tazemetostat in PBRM1-Mutated Human Chordoma Xenograft.

Simple SummaryChordomas are rare bone tumors characterized by a high recurrence rate. Presently, no medical treatment is available for advanced diseases due to the lack of molecular data and preclinical models. The current study showed the establishment and characterization of the largest panel chordoma xenografts, allowing pharmacological studies. In one PBRM1-mutated model, we demonstrated a strong therapeutic efficacy of the EZH2-inhibitor tazemetostat, encouraging further research on EZH2-inhibitors in chordomas.Chordomas are rare neoplasms characterized by a high recurrence rate and a poor long-term prognosis. Considering their chemo-/radio-resistance, alternative treatment strategies are strongly required, but their development is limited by the paucity of relevant preclinical models. Mutations affecting genes of the SWI/SNF complexes are frequently found in chordomas, suggesting a potential therapeutic effect of epigenetic regulators in this pathology. Twelve PDX models were established and characterized on histological and biomolecular features. Patients whose tumors were able to grow into mice had a statistically significant lower progression-free survival than those whose tumors did not grow after in vivo transplantation (p = 0.007). All PDXs maintained the same histopathological features as patients’ tumors. Homozygous deletions of CDKN2A/2B (58.3%) and PBRM1 (25%) variants were the most common genomic alterations found. In the tazemetostat treated PDX model harboring a PBRM1 variant, an overall survival of 100% was observed. Our panel of chordoma PDXs represents a useful preclinical tool for both pharmacologic and biological assessments. The first demonstration of a high antitumor activity of tazemetostat in a PDX model harboring a PBRM1 variant supports further evaluation for EZH2-inhibitors in this subgroup of chordomas.

GlucoMedix®, an extract of Stevia rebaudiana and Uncaria tomentosa, reduces hyperglycemia, hyperlipidemia, and hypertension in rat models without toxicity: a treatment for metabolic syndrome

BackgroundThe objective of this in vivo study is to evaluate in five rat models the pharmacologic effects and toxicity of a commercial hydro-alcoholic extract, GlucoMedix®, derived from Stevia rebaudiana and the pentacyclic chemotype of Uncaria Tomentosa (Willd.) DC, for use as a treatment for metabolic syndrome. The extract contains phytochemicals of Stevia (e.g., steviol glycosides) and Uncaria (e.g., pentacyclic oxindole alkaloids, but lacks tetracyclic oxindole alkaloids).MethodsThe pharmacologic assessments in three rat models include reductions in chemically induced hyperglycemia, hyperlipidemia (cholesterol and triglycerides), and hypertension, all of which are comorbidities of metabolic syndrome. Acute toxicity and 28-day subacute toxicity were assessed in rat models at doses higher than those used in the efficacy models.ResultsThe acute oral toxicity was evaluated in Holtzman rats and the extract did not produce acute toxic effects or lethality, with the LD50 > 5000 mg/kg (extract wet weight). Furthermore, subacute oral toxicity was evaluated in rats for 28 days at daily doses as high as 2000 mg/kg without toxicity or abnormal clinical chemistry or hematological effects. Daily oral doses of 250 - 1000 mg/kg were used to evaluate the treatment effects in hyperglycemic (alloxan-induced and glibenclamide-controlled), hyperlipidemic (cholesterol-induced and atorvastatin-controlled), and hypertensive (L-NAME-induced and enalapril-controlled) rat models. Alloxan-induced hyperglycemia was reduced in a dose-dependent manner within 28 days or less. Cholesterol-induced hyperlipidemic rats exhibited dose-dependent reductions in cholesterol and triglycerides at 21 days. Furthermore, GlucoMedix® produced a dose-dependent decrease in systolic and diastolic arterial blood pressure in L-NAME-induced hypertensive rats at 28 days.ConclusionsThe five in vivo rat models revealed that the all-natural phytotherapy GlucoMedix® is a safe and effective treatment for hyperglycemia, hyperlipidemia, and hypertension. This extract is expected to affect multiple comorbidities of metabolic syndrome, without any acute or subacute oral toxicity in humans. Although multiple prescription drugs are well known for the treatment of individual comorbidities of metabolic syndrome, no drug monotherapy concurrently treats all three comorbidities.