Toxicokinetic link to Bioanalysis/Toxciokinetics and pharmacokinetic studies are required prior to exposing human subjects to a new investigational drug in the first in human (FiH) clinical trial.
We offer to determine the in vitro metabolism to either support or confirm the selection of appropriate toxicity species as well as the plasma protein binding of the drug candidate in human and animals in regulatory compliant assays to aid the non-clinical safety evaluation.
If needed and depending on the availability of data at this stage, the latter can also be supported by relevant in vitro and/or in vivo PK studies, e.g., intrinsic clearance, PK in rodent/non-rodent species , enabling the prediction of human pharmacokinetics to justify starting dose and potential dose escalation steps in the FiH trial.
In vitro investigations of drug-drug interactions (DDI) at least related to cytochrome P450 (CYP) enzymes are strongly recommended for FiH trials in patients to guide selection of participants, concomitant medications, and to define in- and exclusion criteria.
The intrinsic hepatic clearance reflects the inherent ability of hepatocytes or other subcellular systems (e.g., microsomes, S9 fractions) to eliminate unbound drug and once corrected for non-specific binding in the incubation is governed solely by the activity of metabolizing enzymes.
The use of in vitro metabolic clearance data from hepatocytes is a key approach to predict pharmacokinetics by in vitro-in vivo extrapolation (IVIVE) both in humans and animal species.
The intrinsic clearance determination of a drug candidate can be performed in absence or presence of serum (in order to avoid unspecific adsorption). The unbound intrinsic clearance considering the hepatocyte and plasma/serum protein binding in the assay medium is calculated and can be used to predict the human hepatic blood clearance applying the well-stirred liver model.
Our assay is generally conducted with unlabeled test compounds using cryopreserved human, minipig, dog, monkey, rabbit, rat, and mouse hepatocytes in suspensions (other species or test systems from special patient populations upon request).
Metabolism studies are integral parts of the development process which aid safety and efficacy assessments of drug candidates. Differences in metabolic profiles between humans and animal species used for toxicological testing should be investigated as early as possible to identify unique and/or disproportionate human metabolites.
The incubation of drug candidates with primary hepatocyte suspensions from human and animal species (e.g., minipig, dog, monkey, rabbit, rat, and mouse) aims to identify and quantify hepatic metabolites in order to enable or confirm the selection of species for toxicological studies. Occurrence of high abundant metabolites may also trigger further profiling for pharmacodynamic effects or drug-drug interaction (DDI) potential. Structure elucidation of identified metabolites is supported by orthogonal parameters e.g., accurate mass and MSn experiments.
Our cross-species metabolism assay is generally conducted with radiolabeled test compounds in which parent drug and metabolites are quantified by radioactive measurement applying off-line scintillation counting. The use of unlabeled test compounds is feasible, but allows only semi-quantitative assessments based on mass spectrometric responses.
The assay can be supplemented by the determination of covalent binding of radiolabeled test compounds to hepatocyte or microsomal proteins to obtain indications for the formation of reactive metabolites and to assess potential risks for idiosyncratic toxicity caused by the drug candidate.
In vivo metabolite profiling and structure elucidation can be offered in virtually all matrices collected from preclinical species in pharmacokinetic and toxicokinetic studies as well as from humans in clinical trials, including human mass balance (hADME).
We highly recommend investing in the identification of circulating human metabolites in individual or volume- and time-weighted Hamilton plasma pool samples even within the first in human (FiH, SAD) trial. Quantitation of metabolites can be accomplished by either using reference standards (if available) or radiolabeled calibration samples generated with relevant in vitro test systems (e.g., hepatocytes) and applying high resolution mass spectrometry (HR-MS) combined with off-line radio detection.
The evaluation of whether human metabolites are formed at sufficient levels by the animal toxicity species can be performed by analyzing matrix matched plasma samples, without requiring reference standards. The resulting relative metabolite exposures (human vs. preclinical species) will support the safety assessment of your drug candidate.
The assay can be supplemented by the determination of covalent binding of radiolabeled test compounds to hepatocyte or microsomal proteins to obtain indications for the formation of reactive metabolites and to assess potential risks for idiosyncratic toxicity caused by the drug candidate.
In vivo metabolite profiling and structure elucidation can be offered in virtually all matrices collected from preclinical species in pharmacokinetic and toxicokinetic studies as well as from humans in clinical trials, including human mass balance (hADME).
We highly recommend to already investing in the identification of circulating human metabolites in individual or volume- and time-weighted Hamilton plasma pool samples even within the first in human (FiH, SAD) trial. Quantitation of metabolites can be accomplished by either using reference standards (if available) or radiolabeled calibration samples generated with relevant in vitro test systems (e.g., hepatocytes) and applying high resolution mass spectrometry (HR-MS) combined with off-line radio detection.
The evaluation whether or not human metabolites are formed at sufficient levels by the animal toxicity species can be performed by analyzing matrix matched plasma samples, not even requiring reference standards. Resulting relative metabolite exposures (human vs. preclinical species) will support the safety assessment of your drug candidate.
Evaluation of possible drug-drug interactions (DDI) between two or more co-administered drugs to a patient is required to progress towards registration of new drugs.
Our assay package comprises different aspects of DDI such as drug metabolizing enzyme (DME) phenotyping, inhibition, and induction as well as drug transporter phenotyping and inhibition. It will, thus, address the potential of a drug candidate to act as DDI victim or perpetrator.
We can provide these IND/IMPD-enabling assays, results, and entire documentation in full compliance with international regulatory requirements to support your drug development.
We guide our pharmaceutical clients throughout all phases of the R&D value chain starting from early drug discovery, progressing into clinical development and marketing authorization.
Our service portfolio comprises PK consultancy, gap analyses and DMPK data assessments (e.g., for due diligences), project management including DMPK representation in project teams, support of candidate selection, generation of tailor-made NCE development plans, and scientific writing (study reports, DMPK sections of IND, IMPD, NCO, IB, or briefing books).
Aiming to inform the design of clinical trials regarding appropriate starting doses and concomitant medications, our DMPK experts can predict the human pharmacokinetics of your drug candidate based on allometric scaling approaches and other suitable methodologies and perform evaluations of in vitro drug-drug interactions (DDI) and Transporter Phenotyping and Inhibition using basic and mechanistic static modeling.
Assessing the permeability of drug candidates across biological membranes and investigating the involvement of drug transporters is important for oral bioavailability, distribution into tissues (e.g., ability to cross the blood-brain-barrier), and prediction of possible drug-drug interactions (DDI).
We can support the development of your drug candidate by different assays employing Caco-2 cells (human colon carcinoma cell line). Our permeability assay can be conducted in presence or absence of specific drug transporter inhibitors (allowing identification of active transport processes) and with unlabeled or radiolabeled test compounds.
RELATED TOPICS Transporter Phenotyping and Inhibition
It is generally assumed that only unbound drug can be transported across membranes and becomes subject to absorption, distribution, metabolism, and excretion (ADME) processes. Hence, plasma protein binding is an important parameter to understand the pharmacokinetic behavior of a drug as well as its pharmacological/toxicological effects. The binding of drug candidates in plasma of humans, animal species, and/or to isolated proteins such as human serum albumin or a-acid-glycoprotein is determined by equilibrium dialysis and expressed as unbound fraction. Our in vitro assay may include an investigation of concentration dependency in binding in all matrices.
The concentration ratio of drugs in whole blood and plasma provides an indication of drug binding to erythrocytes. It becomes important when clearance estimates are compared directly with organ blood flow rates e.g., in the liver, to obtain the extraction ratio of the organ clearance.
Our whole blood distribution assay is designed to determine the blood-to-plasma ratio of drugs candidates in early development. Data can be used e.g., to scale from in vitro to in vivo clearances and will, thus, allow an estimation of the hepatic first-pass extraction from a plasma clearance and to decide whether the bioanalysis for pharmacokinetic evaluation should be done in whole blood or in plasma.
Both in vitro distribution studies can be conducted with either radiolabeled or unlabeled test compounds applying liquid scintillation counting or tandem mass spectrometry for quantification, respectively.
Permeability-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are drug efflux transporter proteins situated in apical membranes of several organs including liver, kidney, testes, placenta, brain, and gastrointestinal tract. Both transporters can impact drug kinetics as well as safety and/or efficacy since they restrict the distribution of their substrates into these tissues and may also contribute to eliminating substrates via renal and biliary excretion as well as intestinal secretion.
Our in vitro drug transporter assays are designed to assess the P-gp and BCRP substrate properties of a (radiolabeled) drug candidate. The concentration and time-dependent bi-directional permeability and derived efflux ratio of the test compound are determined in Caco-2 monolayer cells. If polarized transport is observed indicating that it is a transporter substrate, the percentage inhibition of its efflux ratio is investigated under linear transport rate conditions in absence and presence of prototypical P-gp or BCRP inhibitors.
In order to evaluate the potential of a drug candidate to inhibit P-gp and BCRP, the bi-directional permeability and resulting efflux ratio of prototypical P‑gp and BCRP substrates are determined in Caco-2 monolayer cells in absence and presence of the test compound at different concentrations. If applicable, its concentration required for 50% inhibition (IC50) of the maximum transport mediated by P-gp or BCRP is calculated.
Evaluation of possible interactions between the drug candidate and other efflux and uptake transporters as required by regulatory agencies are available upon request.
RELATED TOPICS DDI package
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