Discovery of CBPD-268 as an Exceptionally Potent and Orally Efficacious CBP/p300 PROTAC Degrader Capable of Achieving Tumor Regression.

CBP/p300 proteins are key epigenetic regulators and promising targets for the treatment of castration-resistant prostate cancer and other types of human cancers. Herein, we report the discovery and characterization of CBPD-268 as an exceptionally potent, effective, and orally efficacious PROTAC degrader of CBP/p300 proteins. CBPD-268 induces CBP/p300 degradation in three androgen receptor-positive prostate cancer cell lines, with DC50 ≤ 0.03 nM and Dmax > 95%, leading to potent cell growth inhibition. It has an excellent oral bioavailability in mice and rats. Oral administration of CBPD-268 at 0.3-3 mg/kg resulted in profound and persistent CBP/p300 depletion in tumor tissues and achieved strong antitumor activity in the VCaP and 22Rv1 xenograft tumor models in mice, including tumor regression in the VCaP tumor model. CBPD-268 was well tolerated in mice and rats and displayed a therapeutic index of >10. Taking these results together, CBPD-268 is a highly promising CBP/p300 degrader as a potential new cancer therapy.

Insights into Peptidoglycan-Targeting Radiotracers for Imaging Bacterial Infections: Updates, Challenges, and Future Perspectives.

The unique structural architecture of the peptidoglycan allows for the stratification of bacteria as either Gram-negative or Gram-positive, which makes bacterial cells distinguishable from mammalian cells. This classification has received attention as a potential target for diagnostic and therapeutic purposes. Bacteria's ability to metabolically integrate peptidoglycan precursors during cell wall biosynthesis and recycling offers an opportunity to target and image pathogens in their biological state. This Review explores the peptidoglycan biosynthesis for bacteria-specific targeting for infection imaging. Current and potential radiolabeled peptidoglycan precursors for bacterial infection imaging, their development status, and their performance in vitro and/or in vivo are highlighted. We conclude by providing our thoughts on how to shape this area of research for future clinical translation.

Bioanalysis of six antibiotics from volumetric microsamples: a new tool for precision dosing in critically ill children.

Background: Volumetric absorptive microsamples (VAMS) can support pharmacokinetic / pharmacodynamic studies. We present the bioanalytical method development for the simultaneous quantification of ampicillin, cefepime, ceftriaxone, meropenem, piperacillin, tazobactam, and vancomycin from VAMS. Methods & results: Optimal extraction, chromatographic, and mass spectrometry conditions were identified. Maximum extraction recoveries included 100 µl of water for rehydration and methanol for protein precipitation. Chromatographic separation used Phenomenex Kinetex™ Polar C18 column with a mobile phase comprising water/acetonitrile with formic acid and was fully validated. Hematocrit effects were only observed for vancomycin. Samples were stable for 90 days at -80°C except for meropenem, which was stable for 60 days. Conclusion: Multiple antibiotics can be assayed from a single VAMS sample to facilitate pharmacokinetic/pharmacodynamic studies.

Discovery of ARD-1676 as a Highly Potent and Orally Efficacious AR PROTAC Degrader with a Broad Activity against AR Mutants for the Treatment of AR + Human Prostate Cancer.

We report herein the discovery and extensive characterization of ARD-1676, a highly potent and orally efficacious PROTAC degrader of the androgen receptor (AR). ARD-1676 was designed using a new class of AR ligands and a novel cereblon ligand. It has DC50 values of 0.1 and 1.1 nM in AR+ VCaP and LNCaP cell lines, respectively, and IC50 values of 11.5 and 2.8 nM in VCaP and LNCaP cell lines, respectively. ARD-1676 effectively induces degradation of a broad panel of clinically relevant AR mutants. ARD-1676 has an oral bioavailability of 67, 44, 31, and 99% in mice, rats, dogs, and monkeys, respectively. Oral administration of ARD-1676 effectively reduces the level of AR protein in the VCaP tumor tissue in mice and inhibits tumor growth in the VCaP mouse xenograft tumor model without any sign of toxicity. ARD-1676 is a highly promising development candidate for the treatment of AR+ human prostate cancer.

Discovery of ARD-2051 as a Potent and Orally Efficacious Proteolysis Targeting Chimera (PROTAC) Degrader of Androgen Receptor for the Treatment of Advanced Prostate Cancer.

We report the discovery of ARD-2051 as a potent and orally efficacious androgen receptor (AR) proteolysis-targeting chimera degrader. ARD-2051 achieves DC50 values of 0.6 nM and Dmax >90% in inducing AR protein degradation in both the LNCaP and VCaP prostate cancer cell lines, potently and effectively suppresses AR-regulated genes, and inhibits cancer cell growth. ARD-2051 achieves a good oral bioavailability and pharmacokinetic profile in mouse, rat, and dog. A single oral dose of ARD-2051 strongly reduces AR protein and suppresses AR-regulated gene expression in the VCaP xenograft tumor tissue in mice. Oral administration of ARD-2051 effectively inhibits VCaP tumor growth and causes no signs of toxicity in mice. ARD-2051 is a promising AR degrader for advanced preclinical development for the treatment of AR+ human cancers.

Volumetric Absorptive Microsampling to Enhance the Therapeutic Drug Monitoring of Tacrolimus and Mycophenolic Acid: A Systematic Review and Critical Assessment.

BACKGROUND: Volumetric absorptive microsampling (VAMS) is an emerging technique that may support multisample collection to enhance therapeutic drug monitoring in solid organ transplantation. This review aimed to assess whether tacrolimus and mycophenolic acid can be reliably assayed using VAMS and to identify knowledge gaps by providing granularity to existing analytical methods and clinical applications. METHODS: A systematic literature search was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The PubMed, Embase, and Scopus databases were accessed for records from January 2014 to April 2022 to identify scientific reports on the clinical validation of VAMS for monitoring tacrolimus and mycophenolic acid concentrations. Data on the study population, sample sources, analytical methods, and comparison results were compiled. RESULTS: Data from 12 studies were collected, including 9 studies pertaining to tacrolimus and 3 studies on the concurrent analysis of tacrolimus and mycophenolic acid. An additional 14 studies that provided information relevant to the secondary objectives (analytical validation and clinical application) were also included. The results of the clinical validation studies generally met the method agreement requirements described by regulatory agencies, but in many cases, it was essential to apply correction factors. CONCLUSIONSS: Current evidence suggests that the existing analytical methods that use VAMS require additional optimization steps for the analysis of tacrolimus and mycophenolic acid. The recommendations put forth in this review can help guide future studies in achieving the goal of improving the care of transplant recipients by simplifying multisample collection for the dose optimization of these drugs.

Design and Synthesis of Orally Active Quinolyl Pyrazinamides as Sigma 2 Receptor Ligands for the Treatment of Pancreatic Cancer.

Sigma 2 receptor (σ2R) is overexpressed in select cancers and is regarded as a biomarker for tumor proliferation. σ2R ligands are emerging as promising theranostics for cancer and neurodegenerative diseases. Herein, we describe the design and synthesis of a series of novel quinolyl pyrazinamides as selective and potent σ2R ligands that show sub-micromolar potency in pancreatic cancer cell lines. Compounds 14 (JR1-157) and 17 (JR2-298) bind σ2R with Ki of 47 and 10 nM, respectively. Importantly, compound 14 has an oral bioavailability of 60% and shows significant in vivo efficacy without obvious toxicity in a syngeneic model of pancreatic cancer. The cytotoxicity of the quinolyl pyrazinamides significantly enhanced in the presence of copper and diminished in the presence of the copper-chelator tetrathiomolybdate. In conclusion, compound 14 is water-soluble, metabolically stable, orally active, and increases the expression of the autophagy marker LC3B and warrants further development for the treatment of pancreatic cancer.

Application of a new volumetric microsampling device for quantitative bioanalysis of immunosuppression.

Background: Volumetric absorptive microsampling may reduce the blood collection burden associated with therapeutic drug monitoring of immunosuppression to prevent organ transplant rejection. This work describes the development of a laboratory and analytical technique for quantifying tacrolimus and mycophenolic acid (MPA) from the Tasso-M20™ in human whole blood using bead-based impact-assisted extraction. Results: The sampled blood volume was accurate with estimated volumes within <2% of the expected 20 µl. Recovery using impact-assisted extraction was 73-87% for MPA and 100% for tacrolimus and was hematocrit-independent for both analytes. The LC-MS/MS assay is precise and accurate within the acceptance criteria of 15%. Conclusion: The sampling and extraction procedures allowed for accurate quantification of tacrolimus and MPA. Exploration of abuse scenarios identified important education points for patients conducting home-based sample collections in the future.

Development and validation of a LC-HRMS method for the quantification of cannabinoids and their metabolites in human plasma.

The resurgence of Cannabis therapeutic discoveries have led to the need for sensitive and selective analytical methods for the detection of cannabinoids and their metabolites in biological matrices. High resolution mass spectrometry (HRMS) enables good sensitivity and provides more selectivity due to its accurate mass measurement of the targeted compounds. The aim of this study was to develop and validate a sensitive liquid chromatography high resolution mass spectrometry (LC-HRMS) method for the quantitative analysis of cannabidiol (CBD), cannabinol (CBN), Δ9-tetrahydrocannabinol (Δ9-THC) and its major metabolites 11-Hydroxy-Δ9-THC and 11-Nor-9-carboxy-Δ9-THC in human plasma. The method utilized a simple liquid-liquid extraction of the cannabinoids from plasma samples followed by an isocratic chromatographic separation and detection by ESI-HRMS Q-Exactive plus platform. The lower limit of quantification (LLOQ) was 0.2 ng/ mL for the targeted cannabinoids and its metabolites with sample volume of 0.5 mL plasma. The method was linear from 0.2 to 100.0 ng/mL with an average correlation coefficient of >0.995 using weighted (1/x) linear least-squares regression. No significant carry-over was noticed for all analytes and the extraction recovery ranged from 60.4 % to 85.4 %. Dilution results indicated no influence on the accuracy of analysis. The method's intra-day and inter-day precision (CV %) ranged from 2.90 to 10.80 % and accuracy within -0.9 to 7.0 from nominal. Matrix effect ranged from 1.1 % to 49.8 %. The analytes were stable in the autosampler for 6 and 12 h, respectively. This method was sensitive and can be applicable to cannabinoids pharmacokinetics study.

Household representative sample strategy for COVID-19 large-scale population screening.

In the advent of COVID-19 pandemic, testing is highly essential to be able to identify, isolate, treat infected persons, and finally curb transmission of this infectious respiratory disease. Group testing has been used previously for various infectious diseases and recently reported for large-scale population testing of COVID-19. However, possible sample dilution as a result of large pool sizes has been reported, limiting testing methods' detection sensitivity. Moreover, the need to sample all individuals prior to pooling overburden the limited resources such as test kits. An alternative proposed strategy where test is performed on pooled samples from individuals representing different households is presented here. This strategy intends to improve group testing method through the reduction in the number of samples collected and pooled during large-scale population testing. Moreover, it introduces database system which enables continuous monitoring of the population's virus exposure for better decision making.

Development and validation of an LC-MS/MS method for the quantification of goserelin in a Pheroid® formulation, in simulated intestinal fluid.

The purpose of this reported study was to develop and validate an LC-MS/MS method for the quantification of goserelin in a Pheroid® formulation simulated intestinal fluid. Biopharmaceuticals are formulated in drug delivery systems to improve their gastrointestinal stability. Goserelin, a peptide drug was formulated in Pheroid® delivery system and its gastrointestinal stability assessed using simulated intestinal fluid, which required an assay to determine the varying amounts of goserelin remaining after a specific time. Several extraction methods and solvents investigated to extract goserelin from complex matrix led to either poor recovery, peak shape or high background interference. A rapid gradient reversed-phase method coupled to tandem mass spectrometry detection was optimized for the separation and quantification of the extracted peptide. A simple, reproducible and good recovery extraction procedure for goserelin quantification was achieved through simultaneous acetonitrile protein precipitation and water-saturated n-butanol liquid-liquid extraction with water dilution. The method was found to be rapid, specific, precise and accurate, and successfully applied to determine goserelin remaining content in a simulated intestinal fluid, with potential use in other lipid-based formulation evaluated in simulated intestinal fluids.

Genetic incorporation of 1,2-aminothiol functionality for site-specific protein modification via thiazolidine formation.

Here we report a new site-specific conjugation strategy to modify proteins via thiazolidine ligation. Proteins harbouring a 1,2-aminothiol moiety introduced by amber codon suppression technology could be modified chemoselectively with aldehyde-functionalized reagents, such as a biotin-labeled peptide or ubiquitin, under mild conditions to yield homogeneous biotinylated or ubiquitinated products.

Ambient ionization MS for bioanalysis: recent developments and challenges.

Ambient ionization MS has become very popular in analytical science and has now evolved as an effective analytical tool in metabolomics, biological tissue imaging, protein and small molecule drug analysis, where biological samples are probed in a rapid and direct fashion with minimal sample preparation at ambient conditions. However, certain inherent challenges continue to hinder the vibrant prospects of these methods for in situ analyses or to replace conventional methods in bioanalysis. This review provides an introduction to the field and its application in bioanalysis, with an emphasis on the most recent developments and applications. Furthermore, ongoing challenges or limitations related to quantitation, sensitivity, selectivity, instrumentation and mass range of these ambient methods will also be discussed.

Application of paper spray-MS in PK studies using sunitinib and benzethonium as model compounds.

AIM: Current bioanalytical methods applied in nonclinical PK studies for screening drug candidates demand significant amount of time and resources, hence, the need to develop alternative methods. RESULTS: A proof-of-concept paper spray-MS method for the detection and quantitation of small molecules in plasma has been developed and validated using sunitinib and benzethonium as model compounds. The method includes single oral or intravenous administration of sunitinib to mice and serial micro-volume (20 µl) blood collection at different time intervals. The method is rapid with overall analysis time of 1 min and a full PK profile of sunitinib was obtained from a single mouse. CONCLUSION: The paper spray-MS approach is simple, sensitive and can potentially enable significant reduction of animal use and cost.