Permeability and protein binding

The absorption properties of a drug is due to the aqueous solubility, limited membrane permeability, and/or chemical or biological degradation, i.e., limited absorption. To measure the drug permeation across the intestinal membrane, Caco-2 and MDR1-MDCK cell lines are the two major cell lines being used to estimate the ability of potential drug compounds to cross the intestinal epithelium. Caco-2 cells express transporter proteins, efflux proteins, and Phase II conjugation enzymes to model a variety of transcellular pathways as well as metabolic transformation of test substances. MDR1-MDCK cell line is Madin-Darby Canine Kidney (MDCK) Cells with high expression of transfected P-gp. P-gp substrates, low permeability marker, high permeability markers, and P-gp inhibitor could be dosed to Caco-2 or MDR1-MDCK cell line as reference to investigate in vitro drug permeability. Our team can perform the permeability assays to evaluate such properties of the compounds.

Upon entering into plasma through permeability, most drugs bind rapidly to blood constituents, including blood cells, proteins, etc. Characterization of protein binding of a drug and the effects of various pathophysiological conditions, such as disease states and concomitant medications, on protein binding are important for the understanding of the pharmacokinetic behavior of a compound or drug candidate. Our Drug Metabolism Department can perform protein binding via rapid equilibrium device and ultracentrifugation to investigate protein binding with (1) plasma or whole blood from various species, (2) microsomal protein from various species, (3) human serum albumin (HSA), (4) human alpha-1 acid glycoprotein (AAG), (5) other tissue protein binding, etc. Positive controls (e.g., warfarin) are used as reference, along with recovery measurement, to ensure system reliability.

Metabolic stability, CYP inhibition, and metabolite profiling

Drug metabolic clearance is a measure of the ability of the body or an organ to eliminate a drug or compound from the blood circulation. Systemic clearance is a measure of the ability of the entire body to eliminate the drug, whereas organ clearance such as hepatic or renal clearance is a measure of the ability of a particular organ to eliminate the drug. Measurement of metabolic stability in vitro is used to predict in vivo clearance. The in vitro T1/2 by in vitro calculation can be a suitable approach to measure in vitro CLint, which can be scaled up to the in vivo situation and used in the prediction of human clearance. Currently, we can perform metabolic stability in different species of fresh and cryopreserved hepatocytes, liver microsomes, liver S9 fraction and liver cytosol. The study could also be performed in extrahepatic tissues, such as intestine, brain, kidney, lung, skin, etc.

Furthermore, metabolic stability studies serve to investigate the metabolic pathways and profiles of a drug or compound and to address potential metabolism-related issues such as toxic metabolites, metabolic interactions, and polymorphic metabolism. Our Drug Metabolism Department has standardized protocols for direct and mechanism-based inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 to determine IC50, mechanism-based inhibition and reaction phenotyping using LC-MS/MS in human liver microsomes (HLM), or in recombinant CYP enzymes. Inhibitive constant (Ki) and time-dependent inhibitive constant (Kinact) can be further identified. We can also study the non-CYP involved metabolism, such as FMO metabolism, MAO inhibition in HLM or recombinant FMO, MAO A or MAO B. Metabolite profiling, isolation, purification and identification could be further conducted to understand the drug metabolism by identifying which metabolites are formed during in vitro or in vivo studies. Metabolites can be investigated in a number of different matrices including in vitro microsomal incubations, hepatocyte incubations, expressed enzyme incubations as well as in vivo samples.

Drug-drug interaction and enzyme induction

Drug-drug interaction include drug-drug inhibition and drug-drug induction. Administration of certain xenobiotics results in a selective increase in the concentration of metabolizing enzymes in both Phase I and Phase II metabolism, and their activities. Enzyme induction becomes important especially when polypharmacy involves drugs with narrow therapeutic windows, since the induced drug metabolism could result in a significant decrease in its exposure and therapeutic effects. In addition, enzyme induction may cause toxicity, associated with increased production of toxic metabolites. Our Drug Metabolism Department can detect the induction levels from gene, protein to activity of phase I and II enzymes. Cell or tissue based samples will be analyzed through RNA extraction, cDNA transcription, real-time PCR, western blot evaluation, and activity analysis by LC-MS/MS. Standard protocols have been established, as well as optimized platform including.

Drug-drug interaction studies, Alliance Pharma has the capability to detect the induction levels from gene, protein to activity of Phase I and Phase II enzymes. Cell or tissue based samples will be analyzed through RNA extraction, cDNA transcription, real-time PCR, western blot evaluation, and activity analysis by LC-MS/MS. With the latest Hamilton automation robot, we can perform high-throughput ADME screening assays.

In vivo animal PK

Alliance Pharma provides rapid in vivo pharmacokinetic (PK) screening studies in rodent and non-rodent species. From standard screening to more complex studies, our team can assist in designing the best strategy and protocols customized to suit any drug discovery program. We can rapidly initiate a study from protocol approval, animal sourcing and dosing, sample collection, to LC-MS/MS sample bioanalysis, PK parameter calculation, and report submission. Our in vivo PK team utilizes a wide variety of dose regimens and administration routes in the study of mice and rats; while we collaborate with partners on studies using other animal species. We have standardized designs, and can draft unique protocols to fit client’s specific requirements. We also use validated WinNonlin PK software to perform PK data analysis and submit full analytical report.

Our PK team performs the following studies:

  • In-house dosing and sampling from mice and rats
  • In-life study collaboration with partners on other animal species
  • Sample bioanalysis under GLP (Tox study) or non-GLP (discovery PK)
  • PK parameter calculation
  • Documentation for IND filing


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