| Drug Metabolism and Pharmacokinetics |
PharmaCadence is uniquely positioned to help address DMPK issues often faced at the Discovery and Lead Optimization stages. With years of experience doing the job at Merck research labs, we can provide big pharma experience with the flexibility and agility only a small group can offer.
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Solubility Assessment
One of the most common confounding factors for in vitro ADME assays is compound solubility in the assay solvent system. How many IC50s are really half max solubility rather than anything related to enzyme interaction? Is your compound free from drug interactions, or does the solubility in the assay buffer artificially limit the compound availability to the enzyme? Our rapid solubility assessment simply answers the question, is the compound present at the expected concentration in the assay? We can quickly and inexpensively, help reduce these concerns in your in vitro assays.
Cytochrome P450 Inhibition
A key component of a successful ADME profile for a potential new drug is the compound’s low drug-drug interaction (DDI) potential. The DDI potential of selected compounds may be established by determination of the extent of cytochrome P450 inhibition. A full range of important P450 metabolizing enzymes are available as separate assays, or as discovery panels. Cyp 3A4, 2D6, and 2C9 isoforms account for a large fraction of all drug metabolism. Our P3 screen is designed to determine the IC50 values of your compounds for each of these enzymes. Our P5 panel includes 3A4, 2D6, 2C9, 1A2, and 2C19 isoforms with three substrates for 3A4 for a more complete assessment of the inhibition profile of your most important compounds.
In vitro results can be verified in follow up in vivo inhibition testing in preclinical species.
P450 Phenotyping
The risk of severe drug-drug interactions in large populations can be reduced by ensuring that drug candidates are cleared by several mechanisms. One of the simplest ways to reduce the risk of DDI is to ensure that compounds are metabolized by multiple P450 enzymes, rather than a single P450 isoform. Of particular concern are the more common polymorphic cyps 2C19 and 2D6. The measurement of metabolic rates for the major P450 enzymes can help protect against advancing compounds with high risk of DDI. We use microsomal preparations from over-expressing specific human P450s as the enzyme source for individual P450 enzymes.
Protein Binding
Fraction unbound is a key parameter in pharmacokinetic calculations often interpreted as the concentration of drug available to interact with the target. While traditional in vitro and in vivo assays have focused on total drug, a growing body of research suggests that the free fraction is a more predictive measure of activity. We use rapid equilibrium dialysis 96 well plates and specified compound concentrations to determine free fraction. Full quantitative analysis by LC-MS/MS is performed on each compound tested to generate accurate and reproducible values.
Blood to Plasma Ratio
A common assumption in discovery PK is that the plasma concentration represents the total drug concentration in blood. In many cases, this is a good approximation, but on occasion, drug will partition into red blood cells to a much larger degree than plasma. The low plasma levels normally interpreted as poor bioavailability may be due to simple partitioning within the blood compartment. We offer blood to plasma ratio determinations in preclinical species and human to address this potential issue.
Microsomal Stability
Microsomal stability is typically used to screen out compounds with a high degree of metabolic instability, which could result in poor pharmacokinetic (PK) profiles. The metabolic stability of your compounds can be tested in human and preclinical liver microsomes, using full time course profiles or selected time point determinations.
Hepatic Stability
In some cases, microsomal preparations do not adequately represent the in vivo metabolic stability of a compound. This is often the case for compounds that are substrates for phase II metabolizing enzymes such as the glucuronyl transferases, sulfo transferases, and GSH transferase. In these cases, metabolic stability may be more predictively assessed in hepatocyte preparations. We can test the metabolic stability of compounds in human and preclinical hepatocyte cultures.
Km/Vmax
A more detailed understanding of the interaction of a compound with a metabolizing enzyme can be gained from determination of the Km/Vmax ratio. We have a screening design that allows rapid estimation of Km/Vmax. The data from this design can be used to estimate intrinsic hepatic clearance. Used in combination with in vivo PK, the importance of hepatic clearance to the overall compound clearance can be delineated.
Metabolite ID
Metabolite identification is an integral part of drug development. Special emphasis is placed on metabolites in safety testing as it is vital to know that preclinical models cover the human metabolites. A program of preclinical species and human in vitro metabolite ID combined with in vivo metabolite ID from preclinical species can reduce surprises in early clinical development. In vitro metabolite ID done in combination with metabolic stability and/or pharmacokinetics at the discovery and lead optimization stages can give medicinal chemists clues about important metabolic soft spots that can be inactivated through structural changes. In vitro metabolite ID testing in liver microsomes, liver S9, and hepatocytes can be designed to address your specific needs.
CaCO2 Permeability
Passive permeability is frequently an important factor in the absorption of drugs. Impermeable compounds, unless they are substrates for specific uptake transporters, are unlikely to have useful bioavailability. One of the most effective early screens for passive permeability is simple CaCO2 membrane permeability. More detailed questions regarding active transport operating in the cells, can be addressed with a bi-directional version of the assay.
Bi-directional Transporter Assays
Transport proteins are recognized as a major factor in drug distribution and elimination. Breast cancer resistant protein (BCRP) and p-glycoprotein (Pgp) are two of the most studied efflux transporters responsible for maintenance of the blood-brain barrier and the resistance of certain tumors to drug therapies. Efflux transporters are an expanding area of study that is attracting the attention of drug researchers, regulators, and the broader scientific community as an important element in understanding ADME of potential new drugs. We offer a bi-directional cell based assay capable of assessing the propensity of new compounds to be substrates for active transport proteins using cloned stable cell lines expressing preclinical species and human transport proteins.
BCS classification
BCS classification is arguably the simplest way to identify and classify the “developability” of new chemical entities as drugs. The combination of passive permeability and solubility are excellent predictors of the ease of formulating a compound for oral absorption, and a valuable predictor of drug distribution. Drugs with poor solubility and poor permeability are difficult to develop into successful drugs no matter what their biological characteristics might be. We can perform the assays necessary to classify your compounds according to published BCS classification guidelines, and we have the expertise to help place the results in perspective relative to other ADME and biological data.
Formulation Screening
During pharmacokinetic screening, oftentimes the most potent compounds are poorly bioavailable. Poor bioavailability results in more PK studies conducted to search for a suitable formulation that will increase bioavailability. In order to reduce the need for additional PK studies, we offer formulation screening where the 5 most common formulations are used to identify an optimal formulation.
Pharmacokinetics
Pharmacokinetics are at the backbone of any drug discovery program. We provide high quality LC-MS/MS assays to determine the concentration of drug candidates and their metabolites in preclinical fluids or tissues originating from any number of pharmacokinetic studies: screening PK with singly or co-administered compounds to guide discovery screening, PK characterization of lead compounds, PK/PD, drug disposition, mouse serial bleeds, mechanistic studies, formulation studies and bioavailabilty.
Dose Limiting Toxicity
Many of the discovery compounds that advance to preclinical development drop out due to toxicity findings at the safety assessment stage. Dose limiting toxicity studies in rodents may be performed with a few potential drug candidates to screen out compounds that exhibit toxicity, and thus allow your team to make timely decisions on the best candidate to bring forward.
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