Teratogenicity is more likely a function of primary and secondary pharmacology than caused by chemically reactive metabolites: a critical evaluation of 40 years of scientific research.

The number of therapeutic drugs known to be human teratogens is actually relatively small. This may reflect the rigorous animal testing and well defined labelling. Some of these drugs were identified to have reactive metabolites and this has been postulated, historically, to be their teratogenic mechanism. These drugs include thalidomide, various anticonvulsants and retinoic acid derivatives.Many of these experiments were conducted in a period where chemically reactive metabolites were being intensely investigated and associated with all forms of toxicity. The legacy of this is that these examples are routinely cited as well established mechanisms.Examination of mechanism leads to the conclusion that the teratogenicity in humans of these compounds is likely due to the primary and secondary pharmacology of the parent drug and stable circulating metabolites and that association of reactive metabolites to this toxicity is unwarranted.

Through a computer monitor darkly: artificial intelligence in absorption, distribution, metabolism and excretion science.

Artificial Intelligence (AI) is poised or has already begun to influence highly absorption, distribution, metabolism and excretion (ADME) science. It is not in the area expected, that of superior modelling of ADME data to increase its predictive power. It is influencing traditional exhaustive and careful literature research by providing almost perfect summaries of existing information. This will highly influence how people study, graduate and progress in the ADME sciences. The literature contains many flaws, protein binding influence on unbound drug concentration, is one of the examples cited, and without direction AI may help to popularise them.ADME science has a relatively small number of key assays and values but these are produced under widely varying conditions so large data sets, the best substrate for artificial intelligence, are not readily available to produce new more predictive systems. The use of AI to enrich the data bases may be a near term goal.AI is already contributing in other areas such as technical skill assimilation, maintenance of complex instruments (combined with virtual reality) and the processing of pharmacovigilance.

Ring-fused dimethoxybenzimidazole-benzimidazolequinone (DMBBQ): tunable halogenation and quinone formation using NaX/Oxone.

Ring-fused benzimidazolequinones are well-known anti-tumour agents, but dimeric ring-fused adducts are new. The alicyclic [1,2-a] ring-fused dimethoxybenzimidazole-benzimidazolequinone (DMBBQ) intermediate allows late-stage functionalization of bis-p-benzimidazolequinones. DMBBQs are chlorinated and brominated at the p-dimethoxybenzene site using nontoxic sodium halide and Oxone in HFIP/water. X-ray crystallography is used to rationalize site preference in terms of the discontinuity in conjugation in the DMBBQ system. Quinone formation occurs by increasing in situ halogen generation and water. Conversely, radical trifluoromethylation occurs at the quinone of the DMBBQ.

An in vitro toolbox to accelerate anti-malarial drug discovery and development.

BACKGROUND: Modelling and simulation are being increasingly utilized to support the discovery and development of new anti-malarial drugs. These approaches require reliable in vitro data for physicochemical properties, permeability, binding, intrinsic clearance and cytochrome P450 inhibition. This work was conducted to generate an in vitro data toolbox using standardized methods for a set of 45 anti-malarial drugs and to assess changes in physicochemical properties in relation to changing target product and candidate profiles. METHODS: Ionization constants were determined by potentiometric titration and partition coefficients were measured using a shake-flask method. Solubility was assessed in biorelevant media and permeability coefficients and efflux ratios were determined using Caco-2 cell monolayers. Binding to plasma and media proteins was measured using either ultracentrifugation or rapid equilibrium dialysis. Metabolic stability and cytochrome P450 inhibition were assessed using human liver microsomes. Sample analysis was conducted by LC-MS/MS. RESULTS: Both solubility and fraction unbound decreased, and permeability and unbound intrinsic clearance increased, with increasing Log D7.4. In general, development compounds were somewhat more lipophilic than legacy drugs. For many compounds, permeability and protein binding were challenging to assess and both required the use of experimental conditions that minimized the impact of non-specific binding. Intrinsic clearance in human liver microsomes was varied across the data set and several compounds exhibited no measurable substrate loss under the conditions used. Inhibition of cytochrome P450 enzymes was minimal for most compounds. CONCLUSIONS: This is the first data set to describe in vitro properties for 45 legacy and development anti-malarial drugs. The studies identified several practical methodological issues common to many of the more lipophilic compounds and highlighted areas which require more work to customize experimental conditions for compounds being designed to meet the new target product profiles. The dataset will be a valuable tool for malaria researchers aiming to develop PBPK models for the prediction of human PK properties and/or drug-drug interactions. Furthermore, generation of this comprehensive data set within a single laboratory allows direct comparison of properties across a large dataset and evaluation of changing property trends that have occurred over time with changing target product and candidate profiles.

Intracellular and Intraorgan Concentrations of Small Molecule Drugs: Theory, Uncertainties in Infectious Diseases and Oncology, and Promise.

The distribution of a drug within the body should be considered as involving movement of unbound drug between the various aqueous spaces of the body. At true steady state, even for a compound of restricted lipoidal permeability, unbound concentrations in all aqueous compartments (blood, extracellular, and intracellular) are considered identical, unless a compartment has a clearance/transport process. In contrast, total drug concentrations may differ greatly, reflecting binding or partitioning into constituents of each compartment. For most highly lipid permeable drugs, this uniform unbound concentration is expected to apply. However, many compounds have restricted lipoidal permeability and are subjected to transport/clearance processes causing a gradient between intracellular and extracellular unbound concentrations even at steady state. Additional concerns arise where the drug target resides in a site of limited vascularity. Many misleading assumptions about drug concentrations and access to drug targets are based on total drug. Correction, if made, is usually by measuring tissue binding, but this is limited by the lack of homogenicity of the organ or compartment. Rather than looking for technology to measure the unbound concentration it may be better to focus on designing high lipoidal permeable molecules with a high chance of achieving a uniform unbound drug concentration. It is hoped this paper will stimulate greater understanding of the path from circulation to cell interior, and thereby in part avoid or minimize the need to provide the experimentally very determining, and sometimes still questionable, answer to this problem.

UCT943, a Next-Generation Plasmodium falciparum PI4K Inhibitor Preclinical Candidate for the Treatment of Malaria.

The 2-aminopyridine MMV048 was the first drug candidate inhibiting Plasmodium phosphatidylinositol 4-kinase (PI4K), a novel drug target for malaria, to enter clinical development. In an effort to identify the next generation of PI4K inhibitors, the series was optimized to improve properties such as solubility and antiplasmodial potency across the parasite life cycle, leading to the 2-aminopyrazine UCT943. The compound displayed higher asexual blood stage, transmission-blocking, and liver stage activities than MMV048 and was more potent against resistant Plasmodium falciparum and Plasmodium vivax clinical isolates. Excellent in vitro antiplasmodial activity translated into high efficacy in Plasmodium berghei and humanized P. falciparum NOD-scid IL-2Rγ null mouse models. The high passive permeability and high aqueous solubility of UCT943, combined with low to moderate in vivo intrinsic clearance, resulted in sustained exposure and high bioavailability in preclinical species. In addition, the predicted human dose for a curative single administration using monkey and dog pharmacokinetics was low, ranging from 50 to 80 mg. As a next-generation Plasmodium PI4K inhibitor, UCT943, based on the combined preclinical data, has the potential to form part of a single-exposure radical cure and prophylaxis (SERCaP) to treat, prevent, and block the transmission of malaria.

Safety Assessment of Acyl Glucuronides-A Simplified Paradigm.

While simple O- (ether-linked) and N-glucuronide drug conjugates generally are unreactive and considered benign from a safety perspective, the acyl glucuronides that derive from metabolism of carboxylic acid-containing xenobiotics can exhibit a degree of chemical reactivity that is dependent upon their molecular structure. As a result, concerns have arisen over the safety of acyl glucuronides as a class, several members of which have been implicated in the toxicity of their respective parent drugs. However, direct evidence in support of these claims remains sparse, and due to frequently encountered species differences in the systemic exposure to acyl glucuronides (both of the parent drug and oxidized derivatives thereof), coupled with their instability in aqueous media and potential to undergo chemical rearrangement (acyl migration), qualification of these conjugates by traditional safety assessment methods can be very challenging. In this Commentary, we discuss alternative (non-acyl glucuronide) mechanisms by which carboxylic acids may cause serious adverse reactions, and propose a novel, practical approach to compare systemic exposure to acyl glucuronide metabolites in humans to that in animal species used in preclinical safety assessment based on relative estimates of the total body burden of these circulating conjugates.

Which metabolites circulate?

Characterization of the circulating metabolites for a new chemical entity in humans is essential for safety assessment, an understanding of their contributions to pharmacologic activities, and their potential involvement in drug-drug interactions. This review examines the abundance of metabolites relative to the total parent drug [metabolite-to-parent (M/P) ratio] from 125 drugs in relation to their structural and physicochemical characteristics, lipoidal permeability, protein binding, and fractional formation from parent (fm). Our analysis suggests that fm is the major determinant of total drug M/P ratio for amine, alcohol, N- and S-oxide, and carboxylic acid metabolites. Passage from the hepatocyte to systemic circulation does not appear to be limiting owing to the vast majority of metabolites formed being relatively lipid permeable. In some cases, active transport plays an important role in this process (e.g., carboxylic acid metabolites). Differences in total parent drug clearance and metabolite clearance are attenuated by the reduction in lipophilicity introduced by the metabolic step and resultant compensatory changes in unbound clearance and protein binding. A small subclass of these drugs (e.g., terfenadine) is unintentional prodrugs with very high parent drug clearance, resulting in very high M/P ratios. In contrast, arenol metabolites show a more complex relationship with fm due largely to the new metabolic routes (conjugation) available to the metabolite compared with the parent drug molecule. For these metabolites, a more thorough understanding of the elimination clearance of the metabolite is critical to discern the likelihood of whether the phenol will constitute a major circulating metabolite.

Evolution of ADME science: where else can modeling and simulation contribute?

The commentary describes progress in modeling and simulation in ADME science and focuses on lipoidal permeability as a central driver of drug molecule disposition. The tension between screening and in silico is outlined with practical suggestions on how to improve multiparameter models. The limitations on modeling drug metabolism and its enzymes are highlighted together with key features in molecules that lead to drug transport. Reservations about the quality of data and the imprecise classification of drug molecules are explained. Encouragement to move modeling and simulation to the forefront of project start-up is provided after examining the complexity of macromolecule-small molecule conjugate prodrugs.

Anti-infectives Developed as Racemic Drugs in the 21st Century: Norm or Exception?

This viewpoint outlines the case for developing new chemical entities (NCEs) as racemates in infectious diseases and where both enantiomers and racemate retain similar on- and off-target activities as well as similar PK profiles. There are not major regulatory impediments for the development of a racemic drug, and minimizing the manufacturing costs becomes a particularly important objective when bringing an anti-infective therapeutic to the marketplace in the endemic settings of infectious diseases.

Why do metabolites circulate?

The aim of most metabolism and excretion processes is to remove the drug and drug related material from the body; however, in most cases metabolites are present in abundance in circulation. To allow better in vitro/in vivo correlations a greater understanding of why metabolites formed in organs such as the liver are present in the circulation is necessary. Separating metabolites into highly lipid permeable and low lipid permeable allows the role of passive efflux from the liver and active transport to be dissected. Many drugs form glucuronide metabolites that circulate at high total concentrations and attention is drawn to low lipid permeability, efflux from the liver by MRP3, high plasma protein binding and restricted distribution as the explanation for this. The use of metabolite maps is suggested as a way of displaying complex processes in a simple form.

Are predictive biomarkers of toxicity worth having? An economic model.

In this paper we model the cost-benefit of excluding populations at risk through predictive toxicity biomarkers and diagnostics. False positives/ negatives inherent in predictive markers and the frequency and nature of adverse events determine whether biomarkers are beneficial and economically viable. We present a model that takes these and other factors into account using data largely in line with real world cases.

Radiolabelled mass-balance excretion and metabolism studies in laboratory animals: are they still necessary?

The conduct of excretion and metabolism studies using radiolabelled drugs in multiple laboratory animal species has been a mainstay of the suite of support activities provided by drug metabolism groups within pharmaceutical research and development organizations for decades. Drug metabolism scientists carry out exhaustive analyses of plasma and excretory matrices to comprehensively determine the profiles of metabolites in these species. While these analyses have taught us considerably regarding principles of drug metabolism and excretion, it is our contention that the routine conduct of such studies for every new drug development compound in every laboratory animal species used in toxicology studies is no longer necessary. The recently released regulatory guidance regarding metabolites and safety testing have better defined what we need to know regarding metabolite profiles in humans relative to animals. In this commentary, we propose a strategy wherein a radiolabel metabolism study is conducted only in humans, and that these data are utilized as a springboard to direct the exploration of steady-state human versus animal metabolite exposures. Such a strategy better serves the purpose of what is needed to support our understanding of the safety of a new drug candidate. Valuable expertise in drug metabolism and biotransformation can be redeployed to meet the burgeoning needs in drug design efforts to optimize structures with regard to metabolic clearance properties, understanding pharmacologically active metabolites, and reducing generation of chemically reactive metabolites.

Multiple factors govern the association between pharmacology and toxicity in a class of drugs: toward a unification of class effect terminology.

The term class effect has gained in use to describe a side effect including toxicity common to a series of drugs. There is no definition of what constitutes a class effect, and it is not applied against a rigid set of criteria.Thus, the finding of toxicity in one of a series of drugs can raise the concern of a class effect, especially if one or more of the others shows findings even slightly related or at very much lower incidence. This is particularly problematic when the term is used loosely or speculatively on initial events that are themselves of low incidence and serious. This speculation exaggerates and distorts the scientific process in establishing the true benefit risk of the individual drugs and can lead to lengthy development times, or highly restrictive labeling, to the detriment of patient welfare. To provide better definition and application of the term, we suggest that the term class effect toxicity is only used when a clear mechanistic link has been established between a safety concern and drug class based on (I) where the primary pharmacology delivers a clear rationale for the observed findings and toxicities; and (II) where the secondary pharmacology is obligate to the class of the molecule and not subject to variation of structure, and the selectivity cannot be impacted significantly by variations in potency introduced by structural manipulation. With these categorizations, we believe class effect toxicity will be mainly confined to I with examples such as the tetracycline class of antibacterials which inhibit protein synthesis both as a mechanism of antibacterial activity and to produce hepatic injury by mitochondrial injury in the liver.

Refining the in vitro and in vivo critical parameters for P-glycoprotein, [I]/IC50 and [I2]/IC50, that allow for the exclusion of drug candidates from clinical digoxin interaction studies.

The objective of this work was to further investigate the reasons for disconcordant clinical digoxin drug interactions (DDIs) particularly for false negative where in vitro data suggests no P-glycoprotein (P-gp) related DDI but a clinically relevant DDI is evident. Applying statistical analyses of binary classification and receiver operating characteristic (ROC), revised cutoff values for ratio of [I]/IC(50) < 0.1 and [I(2)]/IC(50) < 5 were identified to minimize the error rate, a reduction of false negative rate to 9% from 36% (based on individual ratios). The steady state total C(max) at highest dose of the inhibitor is defined as [I] and the ratio of the nominal maximal gastrointestinal concentration determined for highest dose per 250 mL volume defined [I(2)](.) We also investigated the reliability of the clinical data to see if recommendations can be made on values that would allow predictions of 25% change in digoxin exposure. The literature derived clinical digoxin interaction studies were statistically powered to detect relevant changes in exposure associated with digitalis toxicities. Our analysis identified that many co-meds administered with digoxin are cardiovascular (CV) agents. Moreover, our investigations also suggest that the presence of CV agents may alter cardiac output and/or kidney function that may act alone or are additional components to enhance digoxin exposure along with P-gp interaction. While we recommend digoxin as the probe substrate to define P-gp inhibitory potency for clinical assessment, we observed high concordance in P-gp inhibitory potency for calcein AM as a probe substrate.

Correction: Visible-light unmasking of heterocyclic quinone methide radicals from alkoxyamines.

Correction for 'Visible-light unmasking of heterocyclic quinone methide radicals from alkoxyamines' by Patrick Kielty et al., Chem. Commun., 2019, 55, 14665-14668, DOI: 10.1039/C9CC08261A.

The Critical Role of Passive Permeability in Designing Successful Drugs.

Passive permeability is a key property in drug disposition and delivery. It is critical for gastrointestinal absorption, brain penetration, renal reabsorption, defining clearance mechanisms and drug-drug interactions. Passive diffusion rate is translatable across tissues and animal species, while the extent of absorption is dependent on drug properties, as well as in vivo physiology/pathophysiology. Design principles have been developed to guide medicinal chemistry to enhance absorption, which combine the balance of aqueous solubility, permeability and the sometimes unfavorable compound characteristic demanded by the target. Permeability assays have been implemented that enable rapid development of structure-permeability relationships for absorption improvement. Future advances in assay development to reduce nonspecific binding and improve mass balance will enable more accurately measurement of passive permeability. Design principles that integrate potency, selectivity, passive permeability and other ADMET properties facilitate rapid advancement of successful drug candidates to patients.

Metabolites in safety testing (MIST): considerations of mechanisms of toxicity with dose, abundance, and duration of treatment.

In previous papers, we have offered a strategic framework regarding metabolites of drugs in humans and the need to assess these in laboratory animal species (also termed Metabolites in Safety Testing or MIST; Smith and Obach, Chem. Res. Toxicol. (2006) 19, 1570-1579). Three main tenets of this framework were founded in (i) comparisons of absolute exposures (as circulating concentrations or total body burden), (ii) the nature of the toxicity mechanism (i.e., reversible interaction at specific targets versus covalent binding to multiple macromolecules), and (iii) the biological matrix in which the metabolite was observed (circulatory vs excretory). In the present review, this framework is expanded to include a fourth tenet: considerations for the duration of exposure. Basic concepts of pharmacology are utilized to rationalize the relationship between exposure (to parent drug or metabolite) and various effects ranging from desired therapeutic effects through to severe toxicities. Practical considerations of human ADME (absorption-distribution-metabolism-excretion) data, to determine which metabolites should be further evaluated for safety, are discussed. An analysis of recently published human ADME studies shows that the number of drug metabolites considered to be important for MIST can be excessively high if a simple percentage-of-parent-drug criterion is used without consideration of the aforementioned four tenets. Concern over unique human metabolites has diminished over the years as experience has shown that metabolites of drugs in humans will almost always be observed in laboratory animals, although the proportions may vary. Even if a metabolite represents a high proportion of the dose in humans and a low proportion in animals, absolute abundances in animals frequently exceed that in humans because the doses used in animal toxicology studies are much greater than therapeutic doses in humans. The review also updates the enzymatic basis for the differences between species and how these relate to MIST considerations.

Assessment of three human in vitro systems in the generation of major human excretory and circulating metabolites.

An early understanding of key metabolites of drugs is crucial in drug discovery and development. As a result, several in vitro models typically derived from liver are frequently used to study drug metabolism. It is presumed that these in vitro systems provide an accurate view of the potential in vivo metabolites and metabolic pathways. However, no formal analysis has been conducted to validate their use. The goal of the present study was to conduct a comprehensive analysis to assess if the three commonly used in vitro systems, pooled human liver microsomes, liver S-9 fraction, and hepatocytes, adequately predict in vivo metabolic profiles for drugs. The second objective was to compare the overall capabilities of these three systems to generate in vivo metabolic profiles. Twenty-seven compounds in the Pfizer database and 21 additional commercially available compounds of diverse structure and routes of metabolism for which the human ADME data was available were analyzed in this study to assess the performance of the in vitro systems. The results suggested that all three systems reliably predicted human excretory and circulating metabolite profiles. Furthermore, the success in predicting primary metabolites and metabolic pathways was high (>70%), but the predictability of secondary metabolites was less reliable in the three systems. Thus, the analysis provides sufficient confidence in using in vitro systems to reliably produce primary in vivo human metabolites and supports their application in early discovery to identify metabolic spots for optimization of metabolic liabilities anticipated in humans in vivo. However, the in vitro systems cannot solely mitigate the risk of disproportionate circulating metabolites in humans and may need to be supplemented with metabolic profiling of plasma samples from first-in-human studies or early human radiolabeled studies.