Targeting the Epidermal Growth Factor Receptor with Molecular Degraders: State-of-the-Art and Future Opportunities.

Epidermal growth factor receptor (EGFR) is an oncogenic drug target and plays a critical role in several cellular functions including cancer cell growth, survival, proliferation, differentiation, and motility. Several small-molecule tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs) have been approved for targeting intracellular and extracellular domains of EGFR, respectively. However, cancer heterogeneity, mutations in the catalytic domain of EGFR, and persistent drug resistance limited their use. Different novel modalities are gaining a position in the limelight of anti-EGFR therapeutics to overcome such limitations. The current perspective reflects upon newer modalities, importantly the molecular degraders such as PROTACs, LYTACs, AUTECs, and ATTECs, etc., beginning with a snapshot of traditional and existing anti-EGFR therapies including small molecule inhibitors, mAbs, and antibody drug conjugates (ADCs). Further, a special emphasis has been made on the design, synthesis, successful applications, state-of-the-art, and emerging future opportunities of each discussed modality.

Improving Drug Delivery While Tailoring Prodrug Activation to Modulate Cmax and Cmin by Optimization of (Carbonyl)oxyalkyl Linker-Based Prodrugs of Atazanavir.

Structure-property relationships associated with a series of (carbonyl)oxyalkyl amino acid ester prodrugs of the marketed HIV-1 protease inhibitor atazanavir (1), designed to enhance the systemic drug delivery, were examined. Compared to previously reported prodrugs, optimized candidates delivered significantly enhanced plasma exposure and trough concentration (Cmin at 24 h) of 1 in rats while revealing differentiated PK paradigms based on the kinetics of prodrug activation and drug release. Prodrugs incorporating primary amine-containing amino acid promoieties offered the benefit of rapid bioactivation that translated into low circulating levels of the prodrug while delivering a high Cmax value of 1. Interestingly, the kinetic profile of prodrug cleavage could be tailored for slower activation by structural modification of the amino terminus to either a tertiary amine or a dipeptide motif, which conferred a circulating depot of the prodrug that orchestrated a sustained release of 1 along with substantially reduced Cmax and a further enhanced Cmin.

U.S. FDA Approved Drugs from 2015-June 2020: A Perspective.

In the present work, we report compilation and analysis of 245 drugs, including small and macromolecules approved by the U.S. FDA from 2015 until June 2020. Nearly 29% of the drugs were approved for the treatment of various types of cancers. Other major therapeutic areas of focus were infectious diseases (14%); neurological conditions (12%); and genetic, metabolic, and cardiovascular disorders (7-8% each). Itemization of the approved drugs according to the year of approval, sponsor, target, chemical class, major drug-metabolizing enzyme(s), route of administration/elimination, and drug-drug interaction liability (perpetrator or/and victim) is presented and discussed. An effort has been made to analyze the pharmacophores to identify the structural (e.g., aromatic, heterocycle, and aliphatic), elemental (e.g., boron, sulfur, fluorine, phosphorus, and deuterium), and functional group (e.g., nitro drugs) diversity among the approved drugs. Further, descriptor-based chemical space analysis of FDA approved drugs and several strategies utilized for optimizing metabolism leading to their discoveries have been emphasized. Finally, an analysis of drug-likeness for the approved drugs is presented.

(Carbonyl)oxyalkyl linker-based amino acid prodrugs of the HIV-1 protease inhibitor atazanavir that enhance oral bioavailability and plasma trough concentration.

We describe the design, synthesis and pharmacokinetic (PK) evaluation of a series of amino acid-based prodrugs of the HIV-1 protease inhibitor atazanavir (1) derivatized on the pharmacophoric secondary alcohol using a (carbonyl)oxyalkyl linker. Prodrugs of 1 incorporating simple (carbonyl)oxyalkyl-based linkers and a primary amine in the promoiety were found to exhibit low chemical stability. However, chemical stability was improved by modifying the primary amine moiety to a tertiary amine, resulting in a 2-fold enhancement of exposure in rats following oral dosing compared to dosing of the parent drug 1. Further refinement of the linker resulted in the discovery of 22 as a prodrug that delivered the parent 1 to rat plasma with a 5-fold higher AUC and 67-fold higher C24 when compared to oral administration of the parent drug. The PK profile of 22 indicated that plasma levels of this prodrug were higher than that of the parent, providing a more sustained release of 1 in vivo.

Qualified method for the estimation of di-18:1 bis(monoacylglycero)phosphate in urine, a noninvasive biomarker to monitor drug-induced phospholipidosis.

Aim: Our objective was to develop and qualify a bioanalytical method for the estimation of di-18:1-bis(monoacylglycero)phosphate (di-18:1 BMP) as a urinary biomarker for the assessment of drug-induced phospholipidosis and demonstrate its application in a preclinical study. Methodology/results: di-18:1 BMP was extracted by liquid-liquid extraction using n-butanol and analyzed by LC-MS/MS. The qualified method was selective, precise, robust and accurate across the linearity range (0.2-250 ng/ml). Qualified method was then used to assess chloroquine-induced phospholipidosis in rats dosed at 120 mg/kg for 5 days. A fivefold increase in di-18:1 BMP was observed on Day 5 compared with predose. Conclusion: Di-18:1 BMP can be used as a noninvasive biomarker to assess/screen compounds that could cause drug-induced phospholipidosis in rats.

Impact of quercetin on pharmacokinetics of quetiapine: insights from in-vivo studies in wistar rats.

Quercetin (QCN) is commonly used in high doses as a dietary supplement for weight loss. Psychotic patients are at greater risk of developing obesity than the general population. The present study was designed to understand the impact of QCN on the exposure of quetiapine (QTE), an anti-psychotic drug with narrow therapeutic index and brain penetrating capability. The content of QTE in rat plasma was analyzed through liquid chromatography-tandem mass spectrometry. The results showed a significant (p < 0.05) increase in exposure of QTE (peroral dosed) in the animals pre-treated with QCN as compared to the control group. All the animals pre-treated with QCN, succumbed to death within 3-5 min of intravenous dosing of QTE (1 mg/kg). The studies in rat liver S9 fraction indicated that QCN could increase the metabolic stability of QTE by inhibiting the activity of CYP enzymes. The brain to plasma ratio of QTE increased upon QCN pre-treatment (2.6 vs 7.7), which could be attributed to P-glycoprotein inhibition at the blood-brain barrier by QCN. The current set of studies indicated that serious herb-drug interaction between QCN and QTE might occur when they are co-administered. Caution is advised for concomitant use of QCN rich dietary supplements with QTE.

Rapid and cost-effective LC-MS/MS method for determination of hydroxycitric acid in plasma: Application in the determination of pharmacokinetics in commercial Garcinia preparations.

Garcinia cambogia is one of the most commonly used anti-obesity dietary supplements, and hydroxycitric acid (HCA) is a major constituent in the commercial preparations of Garcinia. High doses of HCA are often consumed without much awareness of its pharmacokinetic and toxicokinetic parameters, and therefore, a complete understanding of its effects is lacking. The first step in understanding these parameters is the availability of a reliable bioanalytical method. Here, we present the first report on a UPLC-MS/MS method for analysis of HCA in rat plasma after a simplified and cost-effective protein precipitation. Chromatographic separation of the analytes in the supernatant was achieved using hydrophilic interaction liquid chromatography, where mass parameters were optimized and a rapid 5-min quantitative assay was developed. The method was highly sensitive, accurate, precise and linear in the concentration range of 10.5-5000 ng/mL (validated according to the United States Food and Drug Administration guidelines). Further, the method was successfully used to describe the pharmacokinetic profile of HCA in rat plasma after the administration of pure HCA and commercial Garcinia preparations.

Design, Synthesis, and Pharmacokinetic Evaluation of Phosphate and Amino Acid Ester Prodrugs for Improving the Oral Bioavailability of the HIV-1 Protease Inhibitor Atazanavir.

Phosphate and amino acid prodrugs of the HIV-1 protease inhibitor (PI) atazanavir (1) were prepared and evaluated to address solubility and absorption limitations. While the phosphate prodrug failed to release 1 in rats, the introduction of a methylene spacer facilitated prodrug activation, but parent exposure was lower than that following direct administration of 1. Val amino acid and Val-Val dipeptides imparted low plasma exposure of the parent, although the exposure of the prodrugs was high, reflecting good absorption. Screening of additional amino acids resulted in the identification of an l-Phe ester that offered an improved exposure of 1 and reduced levels of the circulating prodrug. Further molecular editing focusing on the linker design culminated in the discovery of the self-immolative l-Phe-Sar dipeptide derivative 74 that gave four-fold improved AUC and eight-fold higher Ctrough values of 1 compared with oral administration of the drug itself, demonstrating a successful prodrug approach to the oral delivery of 1.

Identification of novel glutathione conjugates of terbinafine in liver microsomes and hepatocytes across species.

1. Terbinafine (TBF), a common antifungal agent, has been associated with rare incidences of hepatotoxicity. It is hypothesized that bioactivation of TBF to reactive intermediates and subsequent binding to critical cellular proteins may contribute to this toxicity. In the present study, we have characterized the bioactivation pathways of TBF extensively in human, mouse, monkey, dog and rat liver microsomes and hepatocytes. 2. A total of twenty glutathione conjugates of TBF were identified in hepatocytes; thirteen of these conjugates were also detected in liver microsomes. To the best of our knowledge, only two of these conjugates have been reported previously. The conjugates were categorized into three groups based on their mechanism of formation: (a) alkene/alkyne oxidation followed by glutathione conjugation, with or without N-demethylation, (b) arene oxidation followed by glutathione conjugation, with or without N-demethylation, and (c) N-dealkylation followed by glutathione conjugation of the allylic aldehyde, alcohol and acid intermediates. 3. Differences were observed across species in the contributions of these pathways toward overall metabolic turnover. We conclude that, in addition to the glutathione conjugates known to form by Michael addition to the allylic aldehyde, there are other pathways involving the formation of arene oxides and alkene/alkyne epoxides that may be relevant to the discussion of TBF-mediated idiosyncratic drug reactions.

Coupling of an Acyl Migration Prodrug Strategy with Bio-activation To Improve Oral Delivery of the HIV-1 Protease Inhibitor Atazanavir.

HIV-1 protease inhibitors (PIs), which include atazanavir (ATV, 1), remain important medicines to treat HIV-1 infection. However, they are characterized by poor oral bioavailability and a need for boosting with a pharmacokinetic enhancer, which results in additional drug-drug interactions that are sometimes difficult to manage. We investigated a chemo-activated, acyl migration-based prodrug design approach to improve the pharmacokinetic profile of 1 but failed to obtain improved oral bioavailability over dosing the parent drug in rats. This strategy was refined by conjugating the amine with a promoiety designed to undergo bio-activation, as a means of modulating the subsequent chemo-activation. This culminated in a lead prodrug that (1) yielded substantially better oral drug delivery of 1 when compared to the parent itself, the simple acyl migration-based prodrug, and the corresponding simple l-Val prodrug, (2) acted as a depot which resulted in a sustained release of the parent drug in vivo, and (3) offered the benefit of mitigating the pH-dependent absorption associated with 1, thereby potentially reducing the risk of decreased bioavailability with concurrent use of stomach-acid-reducing drugs.

Offline derivatization LC-MS/MS method for simultaneous estimation of vanillin and vanillic acid in guinea pig plasma.

AIM: Vanillin used as a positive control substrate of aldehyde oxidase activity gets metabolized to vanillic acid. Low MW and low sensitivity in negative ion mode are challenges with these analytes. Our objective was to develop a simple offline derivatization LC-MS/MS method to address these challenges. METHODOLOGY/RESULTS: A simple dansyl chloride derivatization of the phenolic groups on vanillin and vanillic acid was adopted to enable easy ionization in commonly used acidic mobile phases. Calibration curves were linear over the concentrations of 4.88-1250 nM with an LLOQ of 0.64 fmoles on column for both analytes. CONCLUSION: The qualified method was successfully applied to simultaneously measure vanillin and vanillic acid in plasma and urine from a guinea pig pharmacokinetic study.

Unanticipated Cleavage of 2-Nitrophenyl-Substituted N-Formyl Pyrazolines under Bechamp Conditions: Unveiling the Synthesis of 2-Aryl Quinolines and Their Mechanistic Exploration via DFT Studies.

We herein report for the first time an unusual decomposition of 2-nitrophenyl-substituted N-formyl pyrazolines under Bechamp reduction condition employed to yield 2-aryl quinolines exclusively instead of pyrazolo[1,5-c]quinazolines. The reaction investigation suggests acid-mediated cleavage of 1 followed by a retro-Michael addition, and a subsequent in situ intramolecular reductive cyclization through a modified Friedlander mechanism afforded 2-aryl quinolines (2) in good yields. The proposed mechanistic pathways were supported via experimental evidence and density functional theory studies. B3LYP/6-31+G(d) analysis indicated the involvement of trans-2-hydroxyaminochalcone as a key intermediate and its isomerization and cyclization, leading to unusual product formation.

Comprehensive evaluation of liver microsomal cytochrome P450 3A (CYP3A) inhibition: comparison of cynomolgus monkey and human.

1. Members of the cytochrome P450 3A (CYP3A) subfamily metabolize numerous compounds and serve as the loci of drug-drug interactions (DDIs). Because of high amino acid sequence identity with human CYP3A, the cynomolgus monkey has been proposed as a model species to support DDI risk assessment. 2. Therefore, the objective of this study was to evaluate 35 known inhibitors of human CYP3A using human (HLM) and cynomolgus monkey (CLM) liver microsomes. Midazolam was employed as substrate to generate IC50 values (concentration of inhibitor rendering 50% inhibition) in the absence and presence of a preincubation (30 mins) with NADPH. 3. In the absence of preincubation, the IC50 values generated with CLM were similar to those obtained with HLM (86% within 2-fold; 100% within 3-fold difference). However, significant differences (up to 48-fold) in preincubation IC50 were observed with 17% of the compounds (raloxifene, bergamottin, nicardipine, mibefradil, ritonavir, and diltiazem). 4. Our results indicate that in most cases the cynomolgus monkey can be a viable DDI model. However, significant species differences in time-dependent CYP3A inhibition can be observed for some compounds. In the case of raloxifene, such a difference can be ascribed to a specific CYP3A4 amino acid residue.

Absolute quantification of imipramine and its metabolites in vivo utilizing calibrators from radiolabeled in vitro incubations.

BACKGROUND: We have demonstrated the use of a single-point calibration approach, derived from in vitro metabolite identification studies utilizing radiolabeled imipramine, that allows for the quantitation of metabolites from in vivo studies in the absence of metabolite synthetic standards. RESULTS: From the in vivo study of imipramine in rats, the concentration of parent and metabolites were determined using the single-point calibration approach. Sixty seven percent of the dosed imipramine was recovered within 24 h, with 95 and 5% of drug-related material detected in feces and urine, respectively. CONCLUSION: Using a novel single-point calibration approach from radiolabeled in vitro studies, we quantified metabolites in vivo and determined various disposition pathways.

Absorption and cleavage of enalapril, a carboxyl ester prodrug, in the rat intestine: in vitro, in situ intestinal perfusion and portal vein cannulation models.

In recent years prodrug strategy has been used extensively to improve the pharmacokinetic properties of compounds exhibiting poor bioavailability. Mechanistic understanding of the absorption and the role of intestine and liver in the activation of oral prodrugs is crucial. Enalapril, a carboxyl ester prodrug, is reported to be metabolized by human carboxylesterase-1 (CES1) but not by carboxylesterase-2 (CES2) to its active metabolite enalaprilat. Further, it has been reported that the small intestines of both rat and human contain mainly CES2. The objective of this work was to understand whether enalapril remains unchanged as it is absorbed through the intestine into the portal circulation. This was evaluated using different intestinal preparations, an in situ intestinal perfusion experiment and a portal vein cannulated rat model. No turnover of enalapril was seen with commercial rat intestinal S9 and microsomes, but reasonable turnover was observed with freshly prepared rat intestinal and mucosal homogenate and S9. In the intestinal perfusion study, both enalapril and enalaprilat were observed in the mesenteric plasma with the data suggesting 32% hydrolysis of enalapril in the intestine. In the portal vein cannulated rat, about 51% of enalapril absorbed into intestine was converted to enalaprilat. Overall, it was demonstrated that even though enalapril has been shown to be a specific substrate for CES1, it is converted to enalaprilat to a significant extent in the intestine. Such experimental techniques can be applied by other scientific groups who are working on prodrugs to determine the region and extent of activation. Copyright © 2015 John Wiley & Sons, Ltd.

Role of hepatic blood flow and metabolism in the pharmacokinetics of ten drugs in lean, aged and obese rats.

1. The effect of age and obesity on the pharmacokinetics (PK), hepatic blood flow (HBF) and liver metabolism of 10 compounds was determined in rats. The animals fed a high-fat diet were defined as the diet-induced obese (DIO) group, while the animals that were aged similar to the DIO rats but not fed with high-fat diet were called the age-matched (AM) group. 2. The clearance (CL) values of high CL compounds (CL > 50 mL/min/kg, namely propranolol, diazepam, phenytoin, ethinylestradiol, lorcaserin and fenfluramine) decreased significantly (1.5- to 6-fold) in DIO and AM rats as compared to lean rats, while there was no clear trend for change in CL for the low-to-moderate CL compounds (CL < 50 mL/min/kg, namely atenolol, chlorzoxazone, vancomycin and sibutramine). Hepatocytes incubations revealed a change in half life (t1/2) only for phenytoin. The body weight normalized liver weights and HBF of AM and DIO rats were found to be 2- to 3-fold lower than in lean rats. 3. Our findings suggest that age, and diet to a lesser extent, can reduce HBF and body normalized liver weights and, hence, also reduce CL values for high CL compounds in rats.

Expression and characterization of cynomolgus monkey cytochrome CYP3A4 in a novel human embryonic kidney cell-based mammalian system.

Cynomolgus monkeys are a commonly used species in preclinical drug discovery, and have high genetic similarity to humans, especially for the drug-metabolizing cytochrome P450s. However, species differences are frequently observed in the metabolism of drugs between cynomolgus monkeys and humans, and delineating these differences requires expressed CYPs. Toward this end, cynomolgus monkey CYP3A4 (c3A4) was cloned and expressed in a novel human embryonic kidney 293-6E cell suspension system. Following the preparation of microsomes, the kinetic profiles of five known human CYP3A4 (h3A4) substrates (midazolam, testosterone, terfenadine, nifedipine, and triazolam) were determined. All five substrates were found to be good substrates of c3A4, although some differences were observed in the Km values. Overall, the data suggest a strong substrate similarity between c3A4 and h3A4. Additionally, c3A4 exhibited no activity against non-h3A4 probe substrates, except for a known human CYP2D6 substrate (bufuralol), which suggests potential metabolism of human cytochrome CYP2D6-substrates by c3A4. Ketoconazole and troleandomycin showed similar inhibitory potencies toward c3A4 and h3A4, whereas non-h3A4 inhibitors did not inhibit c3A4 activity. The availability of a c3A4 preparation, in conjunction with commercially available monkey liver microsomes, will support further characterization of the cynomolgus monkey as a model to assess CYP3A-dependent clearance and drug-drug interactions.