Molecular and cellular pharmacokinetics/toxicokinetics

 

Molecular and cellular pharmacokinetics/toxicokinetics

The distribution of compounds in the human body results from an interplay of different processes in organs mediated by many proteins. We offer a broad range of experimental methods to investigate compound absorption, distribution, metabolism and elimination as well as toxicity.

During the development of agrochemical, chemical and pharmaceutical agents, experimental in vitro assays are often required. Compound metabolism and distribution are affected by highly specialised proteins which are predominantly expressed within tissues and organs such as intestine, kidney and the liver. Collectively, these proteins regulate the bioavailability, distribution and clearance of compounds and interactions occur as a result of induction and/or inhibition of one or more of these proteins. All our assays can be adapted to specific customer preferences and can be performed using radioactive compounds or dedicated LC-MS/MS assays validated using international guidelines. Here our list of validated in vitro assays:

Formulation characteristics

  • Release rate

Release of drugs or other components from formulations can have a major impact on absorption through different routes of administration. We can provide the quantification of release rate of compounds in different buffers simulating biological fluids such as gastrointestinal content, subcutaneous and intramuscular environments.

  • Tablet dissolution

Tablet, capsules and other formulations dissolve releasing their content in biological fluids. We offer the possibility of quantifying the dissolution of formulations over time at different pHs to mimic sections of the gastro-intestinal tract.

  • Solubility

Only the drug in solution is available to be absorbed and to interact with molecular targets. Consequently knowledge of drug solubility at different pHs is a relevant factor to evaluate distribution processes and optimised formulations as we have described in Moss et al Antimicrob Agents Chemother. 2012 Jun;56(6):3020-6. We can provide customisable assays to evaluate the effect of multiple factors, such as pHs, food, metals or concomitant drugs/compounds on solubility.

 Absorption

  • Caco2 and MDCK apparent permeability

Caco-2 and MDCK models can be used to investigate the suitability of compounds for oral administration and to predict intestinal permeability. We use a standardised and validated approach to measure the apparent permeability through monolayers of Caco-2 and MDCK in order to provide a quantitative evaluation of intestinal absorption as described in  McDonald TO et al Adv Healthc Mater. 2014 Mar;3(3):400-11.. Transporters inhibitors (such as verapamil for P-gp) can be used to clarify the potential role of uptake or efflux transporters and therefore to have a better understand of potential drug-drug interactions mediated by transporters.

Distribution

  • Plasma protein binding

Drug binding to plasma protein is one the main factors influencing distribution and metabolism, because only the drug unbound is able to penetrate in tissues and interact with targets. The unbound fraction can be characterised using microdyalisis or ultracentrifugation methods and we can provide the quantification of drug binding to specific plasma proteins such as alpha-1-acid glycoprotein or albumin in order to provide a better description of the molecular mechanisms mediating plasma binding.

  • Blood to plasma ratio

Once compounds reach the systemic circulation, they can diffuse in blood cells (erythrocytes, lymphocytes and monocytes). The blood to plasma ratio is a key variable and can influence the drug diffusion in tissues and organs and its elimination through the liver or kidney.

  • Cellular penetration (PBMC, erythrocytes, intestinal, hepatic, renal cell lines)

Drug targets are often located intracellularly, therefore the drug ability to penetrate in specific cell populations is a key factor influencing therapy efficacy and potentially toxicity. We can provide an accurate quantification of drug cellular accumulation in various cell populations as described in Kwan et al. Br J Clin Pharmacol. 2009 Sep; 68(3): 375–380.

Metabolism and drug-drug interactions

  • Plasma stability

Numerous drugs or compounds can be degraded and metabolised by enzymes present in the plasma such esterase. Drug stability can be quantified in human or animal plasma, providing an estimate of compounds half-life in plasma.

  • Metabolism rates and pathways

Several metabolism enzymes expressed in different tissues and organs are responsible for the enzymatic degradation of drugs/compounds. We offer an integrated and comprehensive service for the characterisation of drug metabolism mediated by enzymes. Our assays include the quantification of key parameters such as Vmax and Km through recombinant phase I and phase II enzymes as well as the apparent intrinsic clearance in primary human hepatocytes or enterocytes.

  • Enzyme / transporter induction and inhibition in primary cells or cell lines (human, rodent and mouse)

Numerous environmental factors can influence gene expression or directly inhibiting protein activity. Conseguently drug/compounds absorption and metabolism change due to the activity of numerous factors such as concomitant drugs or other chemicals and food. Our validated assays can help clarifying these molecular processes, providing a quantitative evaluation of the inhibitory and induction potential of compounds on key metabolism enzymes and transporters using standardised experimental approaches in multiple cell lines or primary cells. This approach was a pivotal element for our recent studies on the influence of anti-TB drugs on gene expression: Williamson et al. Antimicrob Agents Chemother. 2013 Dec;57(12):6366-9. and the investigation of the molecular mechanisms mediating potential drug-drug interactions for antiretrovirals Siccardi et al. Pharmacogenet Genomics. 2010 Dec;20(12):759-65.; Moss et al. Antimicrob Agents Chemother. 2013 Nov;57(11):5612-8. and Moss et al. Antimicrob Agents Chemother. 2011 Feb;55(2):879-87.

In vitro/ex vivo toxicology and compatibility

We have developed a number of, validated, techniques to assess the in vitro and ex vivo toxicity of conventional and nanoformulated APIs.

  • Sterility and endotoxin

Endotoxin, and microbial contamination, can be common occurrences in a laboratory setting. It is possible that such contamination may affect further immunological assays and therefore we have validated methods to examine possible microbial and/or endotoxin contamination. Kinetic turbidimetric LAL assay, Kinetic chromogenic LAL, Gel-clot LAL and endpoint chromogenic endotoxin EIA. Bacterial contamination via standard microbiological culture and mycoplasma detection by PCR

  • Cytotoxicity

These protocols examine the cytotoxicity of tested compounds by measuring the reduction of MTT and lactate dehydrogenase release. MTT and LDH assays (multiple cell lines, primary cells and time points)

  • Cellular Health

These assays examine cellular compatibility beyond cytotoxicity by measuring the impact of APIs or nanoformulations on aspects of cellular health that may not result in gross cytotoxicitymitochondrial membrane polarisation, oxidative stress (reactive oxygen species and reduced glutathione), caspase activation (poly-caspase, caspase-1 and caspase-3 and -7), autophagy, apoptosis and pyroptosis.

  • Immunology

immunotoxicology assessment is an integral component of API and nanoformulation development. We utilise a number of validated in vitro and ex vivo assays that have good correlation with in vivo effectsLeukocyte proliferation, cytokine secretion, complement activation, macrophage function/phenotype, dendritic cell maturation, immunophenotyping, phagocytosis, leukocyte procoagulant activity.

  • Haematology 

In addition to impact on immunological function we can also assess blood contact properties of newly developed conventional and nanoformulated APIs. Haemolysis, platelet activation and leukocyte interaction (flow cytometry), Interaction with plasma proteins (1D, 2D PAGE, proteomic analysis and flow cytometric analysis), plasma coagulation times.