Disrupting B and T-cell collaboration in autoimmune disease: T-cell engagers versus CAR T-cell therapy?

B and T cells collaborate to drive autoimmune disease (AID). Historically, B- and T-cell (B-T cell) co-interaction was targeted through different pathways such as alemtuzumab, abatacept, and dapirolizumab with variable impact on B-cell depletion (BCD), whereas the majority of patients with AID including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and organ transplantation benefit from targeted BCD with anti-CD20 monoclonal antibodies such as rituximab, ocrelizumab, or ofatumumab. Refractory AID is a significant problem for patients with incomplete BCD with a greater frequency of IgD-CD27+ switched memory B cells, CD19+CD20- B cells, and plasma cells that are not directly targeted by anti-CD20 antibodies, whereas most lymphoid tissue plasma cells express CD19. Furthermore, B-T-cell collaboration is predominant in lymphoid tissues and at sites of inflammation such as the joint and kidney, where BCD may be inefficient, due to limited access to key effector cells. In the treatment of cancer, chimeric antigen receptor (CAR) T-cell therapy and T-cell engagers (TCE) that recruit T cells to induce B-cell cytotoxicity have delivered promising results for anti-CD19 CAR T-cell therapies, the CD19 TCE blinatumomab and CD20 TCE such as mosunetuzumab, glofitamab, or epcoritamab. Limited evidence suggests that anti-CD19 CAR T-cell therapy may be effective in managing refractory AID whereas we await evaluation of TCE for use in non-oncological indications. Therefore, here, we discuss the potential mechanistic advantages of novel therapies that rely on T cells as effector cells to disrupt B-T-cell collaboration toward overcoming rituximab-resistant AID.

Calculation of an Apical Efflux Ratio from P-Glycoprotein (P-gp) In Vitro Transport Experiments Shows an Improved Correlation with In Vivo Cerebrospinal Fluid Measurements in Rats: Impact on P-gp Screening and Compound Optimization.

P-glycoprotein (P-gp) is a major blood-brain barrier (BBB) efflux transporter. In vitro approaches, including bidirectional efflux ratio (ER), are used to measure P-gp-mediated transport, but findings can be inconsistent across models. We propose a novel, more physiologically relevant, in vitro model: unidirectional apical efflux ratio (AP-ER)-a ratio of permeability rates at the apical side of the BBB with and without P-gp inhibitor. To test our approach, ER and AP-ER were calculated for 3227 structurally diverse compounds in porcine kidney epithelial cells (LLC-PK1) overexpressing human or mouse P-gp and classified based on their passive transcellular P-gp permeability or charged properties. In vivo rat infusion studies were performed for selected compounds with high ER but low AP-ER. One-third of the 3227 compounds had bidirectional ER that was much higher than AP-ER; very few had AP-ER higher than ER. Compounds with a large difference between AP-ER and ER were typically basic compounds with low-to-medium passive permeability and high lipophilicity and/or amphiphilicity, leading to strong membrane binding. Outcomes in the human model were similar to those in mice, suggesting AP-ER/ER ratios may be conserved for at least two species. AP-ER predicted measured cerebrospinal fluid (CSF) concentration better than ER for the five compounds tested in our in vivo rat infusion studies. We report superior estimations of the CSF concentrations of the compounds when based on less resource-intensive AP-ER versus classic ER. Better understanding of the properties leading to high P-gp-mediated efflux in vivo could support more efficient brain-penetrant compound screening and optimization. SIGNIFICANCE STATEMENT: To address inconsistencies associated with the historical, bidirectional efflux ratio (ER) calculation of P-glycoprotein-mediated transport, we propose to use the novel, more physiologically relevant, unidirectional apical efflux ratio (AP-ER) model. In vitro experiments suggested that compounds with strong membrane binding showed the largest difference between AP-ER and ER, and in vivo infusion studies showed that AP-ER predicted cerebrospinal fluid concentrations of compounds better than ER; outcomes in the human model were similar to those in mice.

Differential Monocyte Actuation in a Three-Organ Functional Innate Immune System-on-a-Chip.

A functional, human, multiorgan, pumpless, immune system-on-a-chip featuring recirculating THP-1 immune cells with cardiomyocytes, skeletal muscle, and liver in separate compartments in a serum-free medium is developed. This in vitro platform can emulate both a targeted immune response to tissue-specific damage, and holistic proinflammatory immune response to proinflammatory compound exposure. The targeted response features fluorescently labeled THP-1 monocytes selectively infiltrating into an amiodarone-damaged cardiac module and changes in contractile force measurements without immune-activated damage to the other organ modules. In contrast to the targeted immune response, general proinflammatory treatment of immune human-on-a-chip systems with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) causes nonselective damage to cells in all three-organ compartments. Biomarker analysis indicates upregulation of the proinflammation cytokines TNF-α, IL-6, IL-10, MIP-1, MCP-1, and RANTES in response to LPS + IFN-γ treatment indicative of the M1 macrophage phenotype, whereas amiodarone treatment only leads to an increase in the restorative cytokine IL-6 which is a marker for the M2 phenotype. This system can be used as an alternative to humanized animal models to determine direct immunological effects of biological therapeutics including monoclonal antibodies, vaccines, and gene therapies, and the indirect effects caused by cytokine release from target tissues in response to a drug's pharmacokinetics (PK)/pharmacodynamics (PD) profile.

Are Biotransformation Studies of Therapeutic Proteins Needed? Scientific Considerations and Technical Challenges.

For therapeutic proteins, the currently established standard development path generally does not foresee biotransformation studies by default because it is well known that the clearance of therapeutic proteins proceeds via degradation to small peptides and individual amino acids. In contrast to small molecules, there is no general need to identify enzymes involved in biotransformation because this information is not relevant for drug-drug interaction assessment and for understanding the clearance of a therapeutic protein. Nevertheless, there are good reasons to embark on biotransformation studies, especially for complex therapeutic proteins. Typical triggers are unexpected rapid clearance, species differences in clearance not following the typical allometric relationship, a mismatch in the pharmacokinetics/pharmacodynamics (PK/PD) relationship, and the need to understand observed differences between the results of multiple bioanalytical methods (e.g., total vs. target-binding competent antibody concentrations). Early on during compound optimization, knowledge on protein biotransformation may help to design more stable drug candidates with favorable in vivo PK properties. Understanding the biotransformation of a therapeutic protein may also support designing and understanding the bioanalytical assay and ultimately the PK/PD assessment. Especially in cases where biotransformation products are pharmacologically active, quantification and assessment of their contribution to the overall pharmacological effect can be important for establishing a PK/PD relationship and extrapolation to humans. With the increasing number of complex therapeutic protein formats, the need for understanding the biotransformation of therapeutic proteins becomes more urgent. This article provides an overview on biotransformation processes, proteases involved, strategic considerations, regulatory guidelines, literature examples for in vitro and in vivo biotransformation, and technical approaches to study protein biotransformation. SIGNIFICANCE STATEMENT: Understanding the biotransformation of complex therapeutic proteins can be crucial for establishing a pharmacokinetic/pharmacodynamic relationship. This article will highlight scientific, strategic, regulatory, and technological features of protein biotransformation.

Multi-organ system for the evaluation of efficacy and off-target toxicity of anticancer therapeutics.

A pumpless, reconfigurable, multi-organ-on-a-chip system containing recirculating serum-free medium can be used to predict preclinical on-target efficacy, metabolic conversion, and measurement of off-target toxicity of drugs using functional biological microelectromechanical systems. In the first configuration of the system, primary human hepatocytes were cultured with two cancer-derived human bone marrow cell lines for antileukemia drug analysis in which diclofenac and imatinib demonstrated a cytostatic effect on bone marrow cancer proliferation. Liver viability was not affected by imatinib; however, diclofenac reduced liver viability by 30%. The second configuration housed a multidrug-resistant vulva cancer line, a non-multidrug-resistant breast cancer line, primary hepatocytes, and induced pluripotent stem cell-derived cardiomyocytes. Tamoxifen reduced viability of the breast cancer cells only after metabolite generation but did not affect the vulva cancer cells except when coadministered with verapamil, a permeability glycoprotein inhibitor. Both tamoxifen alone and coadministration with verapamil produced off-target cardiac effects as indicated by a reduction of contractile force, beat frequency, and conduction velocity but did not affect viability. These systems demonstrate the utility of a human cell-based in vitro culture system to evaluate both on-target efficacy and off-target toxicity for parent drugs and their metabolites; these systems can augment and reduce the use of animals and increase the efficiency of drug evaluations in preclinical studies.

Understanding Molecular Drivers of Melanin Binding To Support Rational Design of Small Molecule Ophthalmic Drugs.

Binding of drugs to ocular melanin is a prominent biological phenomenon that affects the local pharmacokinetics and pharmacodynamics in the eye. In this work, we report on the development of in vitro and in silico tools for an early assessment and prediction of melanin binding properties of small molecules. A robust high-throughput assay has been established to study the binding of large sets of compounds to melanin. The extremely randomized trees approach was used to develop an in silico model able to predict the extent of melanin binding from the molecular properties of the compounds. After the last iteration of the model, strong melanin binders could prospectively be identified with 91% accuracy. On the basis of in vitro data generated for approximately 3400 chemically diverse drug-like small molecules, pronounced correlations were observed between the extent of melanin binding and the basicity, lipophilicity, and aromaticity of the compounds.

A Sensitive In Vitro Approach to Assess the Hybridization-Dependent Toxic Potential of High Affinity Gapmer Oligonucleotides.

The successful development of high-affinity gapmer antisense oligonucleotide (ASO) therapeutics containing locked nucleic acid (LNA) or constrained ethyl (cEt) substitutions has been hampered by the risk of hepatotoxicity. Here, we present an in vitro approach using transfected mouse fibroblasts to predict the potential hepatic liabilities of LNA-modified ASOs (LNA-ASOs), validated by assessing 236 different LNA-ASOs with known hepatotoxic potential. This in vitro assay accurately reflects in vivo findings and relates hepatotoxicity to RNase H1 activity, off-target RNA downregulation, and LNA-ASO-binding affinity. We further demonstrate that the hybridization-dependent toxic potential of LNA-ASOs is also evident in different cell types from different species, which indicates probable translatability of the in vitro results to humans. Additionally, we show that the melting temperature (Tm) of LNA-ASOs maintained below a threshold level of about 55°C greatly diminished the hepatotoxic potential. In summary, we have established a sensitive in vitro screening approach for assessing the hybridization-dependent toxic potential of LNA-ASOs, enabling prioritization of candidate molecules in drug discovery and early development.

2 H-1,2,3-Triazole-Based Dipeptidyl Nitriles: Potent, Selective, and Trypanocidal Rhodesain Inhibitors by Structure-Based Design.

Macrocyclic inhibitors of rhodesain (RD), a parasitic cysteine protease and drug target for the treatment of human African trypanosomiasis, have shown low metabolic stability at the macrocyclic ether bridge. A series of acyclic dipeptidyl nitriles was developed using structure-based design (PDB ID: 6EX8 ). The selectivity against the closely related cysteine protease human cathepsin L (hCatL) was substantially improved, up to 507-fold. In the S2 pocket, 3,4-dichlorophenylalanine residues provided high trypanocidal activities. In the S3 pocket, aromatic residues provided enhanced selectivity against hCatL. RD inhibition ( Ki values) and in vitro cell-growth of Trypanosoma brucei rhodesiense (IC50 values) were measured in the nanomolar range. Triazole-based ligands, obtained by a safe, gram-scale flow production of ethyl 1 H-1,2,3-triazole-4-carboxylate, showed excellent metabolic stability in human liver microsomes and in vivo half-lives of up to 1.53 h in mice. When orally administered to infected mice, parasitaemia was reduced but without complete removal of the parasites.

Repurposing a Library of Human Cathepsin L Ligands: Identification of Macrocyclic Lactams as Potent Rhodesain and Trypanosoma brucei Inhibitors.

Rhodesain (RD) is a parasitic, human cathepsin L (hCatL) like cysteine protease produced by Trypanosoma brucei ( T. b.) species and a potential drug target for the treatment of human African trypanosomiasis (HAT). A library of hCatL inhibitors was screened, and macrocyclic lactams were identified as potent RD inhibitors ( Ki < 10 nM), preventing the cell-growth of Trypanosoma brucei rhodesiense (IC50 < 400 nM). SARs addressing the S2 and S3 pockets of RD were established. Three cocrystal structures with RD revealed a noncovalent binding mode of this ligand class due to oxidation of the catalytic Cys25 to a sulfenic acid (Cys-SOH) during crystallization. The P-glycoprotein efflux ratio was measured and the in vivo brain penetration in rats determined. When tested in vivo in acute HAT model, the compounds permitted up to 16.25 (vs 13.0 for untreated controls) mean days of survival.

Application of Imaging Techniques to Cases of Drug-Induced Crystal Nephropathy in Preclinical Studies.

A number of drugs can cause precipitates within renal tubules leading to crystal nephropathy. Crystal nephropathy is usually an exposure-related finding and is not uncommon in preclinical studies, where high doses are tested. An understanding of the nature of precipitates is important for human risk assessment and further development. Our aim was to investigate the ability of various imaging techniques to detect the presence of drugs or metabolites in renal crystals. We applied matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS) imaging, Raman and infrared microspectroscopy, scanning electron microscopy coupled with energy dispersive X-ray (SEM/EDX) spectroscopy and standard histopathology to cases of drug-induced crystal nephropathy, induced in rodents and primates by 4 compounds. MALDI-FTICR MS imaging enabled the identification of the drug-related crystal content in all 4 cases of nephropathy, without reference material and with high accuracy. Crystals were composed of unchanged parent drug and/or metabolites. Similar results were obtained using Raman and infrared microspectroscopy for 2 compounds. In the absence of reference standards of metabolites, Raman and infrared microspectroscopy showed that the crystals consisted of components similar, but not identical, to the administered drug for the other compounds, a limitation for these techniques. SEM/EDX showed which counter ions were colocalized with the identified drug-related material, complementing the MALDI-FTICR MS findings. Therefore, we recommend MALDI-FTICR MS as a first-line methodology to characterize crystal nephropathies. Raman and infrared microspectroscopy may be useful when MALDI-FTICR MS imaging cannot be applied. SEM/EDX could be considered as a complementary technology.

Electro-mechanical conditioning of human iPSC-derived cardiomyocytes for translational research.

RATIONALE: Impaired maturation of human iPSC-derived cardiomyocytes (hiPSC-CMs) currently limits their use in experimental research and further optimization is required to unlock their full potential. OBJECTIVE: To push hiPSC-CMs towards maturation, we recapitulated the intrinsic cardiac properties by electro-mechanical stimulation and explored how these mimetic biophysical cues interplay and influence the cell behaviour. METHODS AND RESULTS: We introduced a novel device capable of applying synchronized electrical and mechanical stimuli to hiPSC-CM monolayers cultured on a PDMS membrane and evaluated effects of conditioning on cardiomyocyte structure and function. Human iPSC-CMs retained their cardiac phenotype and displayed adaptive structural responses to electrical (E), mechanical (M) and combined electro-mechanical (EM) stimuli, including enhanced membrane N-cadherin signal, stress-fiber formation and sarcomeric length shortening, most prominent under the EM stimulation. On the functional level, EM conditioning significantly reduced the transmembrane calcium current, resulting in a shift towards triangulation of intracellular calcium transients. In contrast, E and M stimulation applied independently increased the proportion of cells with L-Type calcium currents. In addition, calcium transients measured in the M-conditioned samples advanced to a more rectangular shape. CONCLUSION: The new methodology is a simple and elegant technique to systematically investigate and manipulate cardiomyocyte remodelling for translational applications. In the present study, we adjusted critical parameters to optimize a regimen for hiPSC-CM transformation. In the future, this technology will open up new avenues for electro-mechanical stimulation by allowing temporal and spatial control of stimuli which can be easily scaled up in complexity for cardiac development and disease modelling.

Toward in vitro-to-in vivo translation of monoclonal antibody pharmacokinetics: Application of a neonatal Fc receptor-mediated transcytosis assay to understand the interplaying clearance mechanisms.

Monoclonal antibodies (mAbs) are a rapidly growing drug class for which great efforts have been made to optimize certain molecular features to achieve the desired pharmacokinetic (PK) properties. One approach is to engineer the interactions of the mAb with the neonatal Fc receptor (FcRn) by introducing specific amino acid sequence mutations, and to assess their effect on the PK profile with in vivo studies. Indeed, FcRn protects mAbs from intracellular degradation, thereby prolongs antibody circulation time in plasma and modulates its systemic clearance. To allow more efficient and focused mAb optimization, in vitro input that helps to identify and quantitatively predict the contribution of different processes driving non-target mediated mAb clearance in vivo and supporting translational PK modeling activities is essential. With this aim, we evaluated the applicability and in vivo-relevance of an in vitro cellular FcRn-mediated transcytosis assay to explain the PK behavior of 25 mAbs in rat or monkey. The assay was able to capture species-specific differences in IgG-FcRn interactions and overall correctly ranked Fc mutants according to their in vivo clearance. However, it could not explain the PK behavior of all tested IgGs, indicating that mAb disposition in vivo is a complex interplay of additional processes besides the FcRn interaction. Overall, the transcytosis assay was considered suitable to rank mAb candidates for their FcRn-mediated clearance component before extensive in vivo testing, and represents a first step toward a multi-factorial in vivo clearance prediction approach based on in vitro data.

Inhibition of EGF Uptake by Nephrotoxic Antisense Drugs In Vitro and Implications for Preclinical Safety Profiling.

Antisense oligonucleotide (AON) therapeutics offer new avenues to pursue clinically relevant targets inaccessible with other technologies. Advances in improving AON affinity and stability by incorporation of high affinity nucleotides, such as locked nucleic acids (LNA), have sometimes been stifled by safety liabilities related to their accumulation in the kidney tubule. In an attempt to predict and understand the mechanisms of LNA-AON-induced renal tubular toxicity, we established human cell models that recapitulate in vivo behavior of pre-clinically and clinically unfavorable LNA-AON drug candidates. We identified elevation of extracellular epidermal growth factor (EGF) as a robust and sensitive in vitro biomarker of LNA-AON-induced cytotoxicity in human kidney tubule epithelial cells. We report the time-dependent negative regulation of EGF uptake and EGF receptor (EGFR) signaling by toxic but not innocuous LNA-AONs and revealed the importance of EGFR signaling in LNA-AON-mediated decrease in cellular activity. The robust EGF-based in vitro safety profiling of LNA-AON drug candidates presented here, together with a better understanding of the underlying molecular mechanisms, constitutes a significant step toward developing safer antisense therapeutics.

From the Cover: The Minipig is a Suitable Non-Rodent Model in the Safety Assessment of Single Stranded Oligonucleotides.

Non-human primates (NHPs) are currently considered to be the non-rodent species of choice for the preclinical safety assessment of single-stranded oligonucleotide (SSO) drugs. We evaluated minipigs as a potential alternative to NHPs to test the safety of this class of compounds. Four different phosphorothioated locked nucleic acid-based SSOs (3 antisense and 1 anti-miR), all with known safety profiles, were administered to minipigs using similar study designs and read-outs as in earlier NHP studies with the same compounds. The studies included toxicokinetic investigations, in-life monitoring, clinical and anatomic pathology. In the minipig, we demonstrated target engagement by the SSOs where relevant, and a similar toxicokinetic behavior in plasma, kidney, and liver when compared with NHPs. Clinical tolerability was similar between minipig and NHPs. For the first time, we showed similar and dose-dependent effects on the coagulation and complement cascade after intravenous dosing similar to those observed in NHPs. Similar to NHPs, morphological changes were seen in proximal tubular epithelial cells of the kidney, Kupffer cells, hepatocytes, and lymph nodes. Minipigs appeared more sensitive to the high-dose kidney toxicity of most of the selected SSOs than NHPs. No new target organ or off-target toxicities were identified in the minipig. The minipig did not predict the clinical features of human injection site reactions better than the NHPs, but histopathological similarities were observed between minipigs and NHPs. We conclude that there is no impediment, as default, to the use of minipigs as the non-rodent species in SSO candidate non-clinical safety packages.

Metabolic Profiling of Human Long-Term Liver Models and Hepatic Clearance Predictions from In Vitro Data Using Nonlinear Mixed-Effects Modeling.

Early prediction of human clearance is often challenging, in particular for the growing number of low-clearance compounds. Long-term in vitro models have been developed which enable sophisticated hepatic drug disposition studies and improved clearance predictions. Here, the cell line HepG2, iPSC-derived hepatocytes (iCell®), the hepatic stem cell line HepaRG™, and human hepatocyte co-cultures (HµREL™ and HepatoPac®) were compared to primary hepatocyte suspension cultures with respect to their key metabolic activities. Similar metabolic activities were found for the long-term models HepaRG™, HµREL™, and HepatoPac® and the short-term suspension cultures when averaged across all 11 enzyme markers, although differences were seen in the activities of CYP2D6 and non-CYP enzymes. For iCell® and HepG2, the metabolic activity was more than tenfold lower. The micropatterned HepatoPac® model was further evaluated with respect to clearance prediction. To assess the in vitro parameters, pharmacokinetic modeling was applied. The determination of intrinsic clearance by nonlinear mixed-effects modeling in a long-term model significantly increased the confidence in the parameter estimation and extended the sensitive range towards 3% of liver blood flow, i.e., >10-fold lower as compared to suspension cultures. For in vitro to in vivo extrapolation, the well-stirred model was used. The micropatterned model gave rise to clearance prediction in man within a twofold error for the majority of low-clearance compounds. Further research is needed to understand whether transporter activity and drug metabolism by non-CYP enzymes, such as UGTs, SULTs, AO, and FMO, is comparable to the in vivo situation in these long-term culture models.

Minimizing the risk of chemically reactive metabolite formation of new drug candidates: implications for preclinical drug design.

Many pharmaceutical companies aim to reduce reactive metabolite formation by chemical modification at early stages of drug discovery. A practice often applied is the detection of stable trapping products of electrophilic intermediates with nucleophilic trapping reagents to guide rational structure-based drug design. This contribution delineates this strategy to minimize the potential for reactive metabolite formation of clinical candidates during preclinical drug optimization, exemplified by the experience at Roche over the past decade. For the majority of research programs it was possible to proceed with compounds optimized for reduced covalent binding potential. Such optimized candidates are expected to have a higher likelihood of succeeding throughout the development processes, resulting in safer drugs.

Extension of the dissolution-precipitation model for kinetic elucidation of solvent-mediated polymorphic transformations.

Thorough understanding and control of the different crystal forms of a drug product is key for fine chemistry and materials science; it ultimately determines the product's physicochemical properties and performance. In this work, we extend the application of a mechanistic dissolution-precipitation model to solvent-mediated solid form transformations. To address the relevance of the model, various kinetic solvent-mediated polymorphic transition studies were retrieved from the literature. Our model succeeds in accurately describing the experimental data, shedding light on the molecular steps driving the polymorphic conversion. Given its simplicity and mechanistic character, the model can be viewed as a useful tool to monitor, predict and optimize the solvent-mediated transformations of solid forms.

Establishment of a Predictive In Vitro Assay for Assessment of the Hepatotoxic Potential of Oligonucleotide Drugs.

Single stranded oligonucleotides (SSO) represent a novel therapeutic modality that opens new space to address previously undruggable targets. In spite of their proven efficacy, the development of promising SSO drug candidates has been limited by reported cases of SSO-associated hepatotoxicity. The mechanisms of SSO induced liver toxicity are poorly understood, and up to now no preclinical in vitro model has been established that allows prediction of the hepatotoxicity risk of a given SSO. Therefore, preclinical assessment of hepatic liability currently relies on rodent studies that require large cohorts of animals and lengthy protocols. Here, we describe the establishment and validation of an in vitro assay using primary hepatocytes that recapitulates the hepatotoxic profile of SSOs previously observed in rodents. In vitro cytotoxicity upon unassisted delivery was measured as an increase in extracellular lactate dehydrogenase (LDH) levels and concomitant reduction in intracellular glutathione and ATP levels after 3 days of treatment. Furthermore, toxic, but not safe, SSOs led to an increase in miR-122 in cell culture supernatants after 2 days of exposure, revealing the potential use of miR122 as a selective translational biomarker for detection of SSO-induced hepatotoxicity. Overall, we have developed and validated for the first time a robust in vitro screening assay for SSO liver safety profiling which allows rapid prioritization of candidate molecules early on in development.

Recent developments in using mechanistic cardiac modelling for drug safety evaluation.

On the tenth anniversary of two key International Conference on Harmonisation (ICH) guidelines relating to cardiac proarrhythmic safety, an initiative aims to consider the implementation of a new paradigm that combines in vitro and in silico technologies to improve risk assessment. The Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative (co-sponsored by the Cardiac Safety Research Consortium, Health and Environmental Sciences Institute, Safety Pharmacology Society and FDA) is a bold and welcome step in using computational tools for regulatory decision making. This review compares and contrasts the state-of-the-art tools from empirical to mechanistic models of cardiac electrophysiology, and how they can and should be used in combination with experimental tests for compound decision making.

Shedding light on minipig drug metabolism - elevated amide hydrolysis in vitro.

1. In recent years, the minipig is increasingly used as a test species in non-clinical assessment of drug candidates. While there is good scientific evidence available concerning cytochrome P450-mediated metabolism in minipig, the knowledge of other metabolic pathways is more limited. 2. The aim of this study was to provide an understanding of when, why, and how drug metabolism in minipig differs from other species commonly used in non-clinical studies. In-house cross-species metabolite profile comparisons in hepatocytes and microsomes of 38 Roche development compounds were retrospectively analyzed to compare the metabolism among minipig, human, rat, dog, monkey, rabbit and mouse. 3. A significant contributor to the elevated metabolism observed for certain compounds in minipig was identified as amide hydrolysis. The hepatic amide hydrolysis activity in minipig was further investigated in subcellular liver fractions and a structure-activity relationship was established. When structural motifs according to the established SAR are excluded, coverage of major human metabolic pathways was shown to be higher in minipig than in dog, and only slightly lower than in cynomolgus monkey. 4. A strategy is presented for early identification of drug compounds which might not be suited to further investigation in minipig due to excessive hydrolytic metabolism.