UC San Diego Drug Development Pipeline

The assessment of in vitro metabolic stability provides a means to measure the susceptibility of a test compound to biotransformation by the liver. Following incubations in either microsomes or hepatocytes, an in vitro intrinsic clearance (CL_int) and half-life are reported. These parameters may be used to rank-order investigational compounds to facilitate lead selection. Additionally, CL_int can be scaled to predict in vivo hepatic clearance in humans and animal models. Advantages of enhanced metabolic stability include (i) increased oral bioavailability, (ii) longer in vivo half life with less frequent dosing requirements, (iii) reduced interpatient pharmacokinetic variability, and (iv) reduced likelihood of off-target toxicities due to metabolites. Liver microsomes primarily assess cytochrome P450 system-mediated metabolism (phase I metabolism) while hepatocytes represent a complete, undisrupted metabolic system, including cofactors, and thus assess both phase I and phase II metabolism. Microsomes and hepatocytes from different species can be tested to characterize interspecies differences in metabolism.

The assessment of in vitro metabolic stability provides a means to measure the susceptibility of a test compound to biotransformation by the liver. Following incubations in either microsomes or hepatocytes, an in vitro intrinsic clearance (CL_int) and half-life are reported. These parameters may be used to rank-order investigational compounds to facilitate lead selection. Additionally, CL_int can be scaled to predict in vivo hepatic clearance in humans and animal models. Advantages of enhanced metabolic stability include (i) increased oral bioavailability, (ii) longer in vivo half life with less frequent dosing requirements, (iii) reduced interpatient pharmacokinetic variability, and (iv) reduced likelihood of off-target toxicities due to metabolites. Liver microsomes primarily assess cytochrome P450 system-mediated metabolism (phase I metabolism) while hepatocytes represent a complete, undisrupted metabolic system, including cofactors, and thus assess both phase I and phase II metabolism. Microsomes and hepatocytes from different species can be tested to characterize interspecies differences in metabolism.

Inhibition of cytochrome P450 (CYP450) enzyme activity is a major mechanism underlying drug-drug interactions. In vitro inhibition studies may be conducted to predict the existence and magnitude of in vivo metabolism-based drug-drug interactions. To determine whether a test compound inhibits the activity of an individual CYP enzyme, changes in the metabolism of a specific probe substrate are determined in human liver microsomes or hepatocytes in the presence of varying concentrations of the test compound. Screening is available for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. Potency of the inhibition for a particular enzyme is reported as IC₅₀ or K_i values.

Induction of cytochrome P450 (CYP450) enzymes involves an increase in the expression and activity of drug metabolizing CYPs, which in turn leads to increased metabolism of co-administered drug substrates. CYP induction is assessed in vitro using hepatocytes, whereby CYP3A4, CYP1A2, and CYP2B6 metabolic activity and mRNA levels are characterized following exposure to a range of concentrations of the test compound.

An evaluation of in vivo pharmacokinetics can be combined with in vitro ADME screening in order to identify compound liabilities and guide necessary structural modifications or other development strategies to mitigate problems. Multiple rodent and non-rodent species are available. In addition, pharmacokinetic studies may be conducted as either conventional (full) PK studies with dense sampling in more animals, or as snapshot PK with fewer samples in a small number of animals, which is generally appropriate for hit-to-lead screening. Additional services include bioavailability, multiple dose PK, tissue distribution, and dose proportionality.

Metabolite identification may be added on to both in vitro metabolism studies and in vivo pharmacokinetic studies. Using the MS/MS spectra of parent compound and observed metabolites, a structure and molecular formula of formed metabolites may be proposed. Metabolites with high in vivo exposure may be flagged for further evaluation of pharmacological activity or toxicity.

The efficacy of a drug is affected by the degree to which it binds to proteins within blood plasma, as highly bound drugs are unable to cross cell membranes and interact with target receptors to elicit effects. Protein binding also affects PK parameters. Therefore, an understanding of unbound drug concentrations is important to guide further development and to generate PK-PD models. Plasma protein binding is assessed by equilibrium dialysis or ultrafiltration with subsequent LC-MS/MS quantitation.

Membrane permeability may be evaluated to predict the oral absorption of a test compound. In vitro model systems can assess both passive membrane permeability (parallel artificial membrane permeability assay; PAMPA) and intestinal permeability including active drug efflux (Caco-2 polarized epithelial monolayers).

Physicochemical properties can impact oral absorption, bioavailability, formulation, and drug delivery. Profiling of of solubility, lipophilicity, pKa, and stability is available.

Quantitative bioanalytical chemistry is available for all experimental steps of the UC San Diego Drug Development Pipeline. The Laboratory is also available to provide sensitive and specific measurements of drug concentrations in a variety of biological fluids and tissues to support clinical pharmacokinetic studies. The laboratory is staffed with experts in bioanalytical chemistry and operates as a GLP facility with standard GLP policies, procedures, and SOPs. Assays are conducted with either tandem triple-quadrupole mass spectrometry (LC-MS/MS) or HPLC. The Laboratory has acted as the FDA Reference Laboratory for INDs/NDAs for drugs brought forward by pharmaceutical companies or within NIH supported networks.