ISSX 2011, Poster 145

Published:  14 September 2011

Esomeprazole, omeprazole sulfone, 5-O-desmethyl omeprazole and 5-hydroxylansoprazole are in vitro metabolism-dependent inhibitors of CYP2C19

The intensively researched interaction between clopidogrel and proton pump inhibitors (PPIs) and the impact of CYP2C19 poor metabolizer phenotype on clopidogrel efficacy have prompted warnings from the FDA and EMA in recent years (2010). The FDA specifically warns against coadministration of clopidogrel and omeprazole and further specifically suggests that pantoprazole may be a safer alternative (2010).

ISSX 2011, Poster 209

Published:  14 September 2011

Evaluating the potential for lysosomal trapping in immortalized human hepatocytes (Fa2N-4 cells)

Sequestration of high concentrations of lipophilic amines (a.k.a cationic amphiphilic drugs or CADs) in the acidic (pH 4-5) environment of lysosomes contributes to their presystemic clearance (by lysosomal trapping in liver and lung), their large volume of distribution (Vd) and their propensity to cause phospholipidosis (Hanumegowda et al, 2010; Daniel and Wojcikowski, 1999). In a related study with Lysotracker Red, a fluorescent probe that, by virtue of being a lipophilic amine, accumulates in lysosomes, we demonstrated that Fa2N-4 cells (immortalized human hepatocytes) contain functional lysosomes, similar to those observed in non-transformed human hepatocytes (Kazmi et al, 2011).

ISSX 2011, Poster 140

Published:  14 September 2011

Can Ki values for direct inhibition of CYP enzymes be reliably estimated from IC50 values?

Regulatory agencies recommend that the potential for a drug candidate to cause clinically relevant, direct inhibition of cytochrome P450 (CYP) enzymes be estimated based on the ratio of [I]/Ki (or 1+[I]/Ki) where [I] is the in vivo concentration of drug candidate and Ki is the dissociation constant for the enzyme-inhibitor complex for direct inhibition (US FDA, 2006). Typically, inhibition of CYP enzymes by a drug candidate is first evaluated in vitro by determining the concentration of drug candidate that causes 50% inhibition of a specific CYP enzyme activity (IC50) using a marker substrate concentration approximately equal to Km for the marker substrate reaction. Determining the mechanism of direct inhibition (competitive, uncompetitive, noncompetitive and mixed) and measuring the Ki value requires an in vitro evaluation of the effects of multiple concentrations of the drug candidate versus multiple concentrations of CYP probe substrate (the former spanning Ki and later spannign Km).

ISSX 2011, Poster 112

Published:  14 September 2011

An in vitro test system to evaluate drug-drug interactions with biologics

Inflammation, infection, vaccination, and some marketed therapeutic proteins (biologics) are associated with cytokine-mediated suppression (down-regulation) of drug-metabolizing enzymes (DME). Biologics, such as monoclonal antibodies, can trigger the release of pro-inflammatory cytokines (e.g. IL-1ß. IL-6, INFy and TNFα); extreme cases of which are known as a cytokine storm. Protein therapeutics may change the clearance of small molecule drugs (SMDs) by affecting DME expression and thereby precipitate drug-drug interactions (DDIs). Potential of biologics to cause DDI is a safety concern recognized by the FDA (Lee et al). In the present study, we developed an in vitro method to evaluate the potential of biologics to elicit DDIs with SMDs via alteration of DME expression. This method involves treating fresh human blood with a biologic to stimulate the release of pro-inflammatory cytokines from peripheral blood mononuclear cells (PBMCs), after which plasma is prepared and added to primary human hepatocytes co-cultured with Kupffer cells to evaluate effects of biologics on cytochrome P450 (CYP) enzyme expression. E. coli lipopolysaccharide (LPS) and murine anti-CD28 monoclonal antibody were evaluated for their potential to perpetrate DDI with SMDs.

ISSX 2011, Poster 147

Published:  14 September 2011

Evaluation of dilution, dialysis and ultracentrifugation methods to assess the reversibility of metabolism-dependent inhibitors (MDIs) of cytochrome P450 (CYP) enzymes

Metabolism-dependent inhibition (MDI) of P450 enzymes is a well-recognized cause of clinically significant drug-drug interactions (DDI). For this reason, the US Food & Drug Administration (FDA) and the European Medicines Agency (EMA) have both published draft guidance documents on DDI that require an in vitro assesment of the ability of drug candidates to cause MDI of the major drug-metabolizing P450 enzymes (2006, 2010). The most recent PhRMA publication and EMA draft guidance discuss in vitro experiments, to not only identify the potential for a drug candidate to cause MDI. (e.g. IC50 shift experiments), but also to evaluate whether MDI involves reversible or irreversible inhibition (Grimm et al., 2009).

ISSX 2011, Poster 203

Published:  14 September 2011

A robust method to identify compounds that undergo intracellular lysosomal sequestration

Lysosomes are acidic organelles (pH 4-5) that play a key role in various metabolic processes such as turnover of phospholipids, the breakdown of waste products (including bacteria and viruses) and apoptosis. Lipophilic and amphiphilic drugs (a.k.a. cationic amphiphilic amines or CADs) with ionizable amines (pKa>6) can accumulate in lysosomes (a process known as lysosomal trapping), which contributes to presystemic clearance in lysosome-rich organs (such as liver and lung) and, together with the binding of lipophilic amines to phospholipids, is associated with a large volume of distribution of numerous cardiovascular and CNS drugs (MacIntyre and Cutler, 1988; Daniel and Wojcikowski, 1999; Houston and Halifax, 2007). The prolonged accumulation of lipophilic amines in lysosomes and their binding to phospholipids, both of which disrupt lysosomal function, have been implicated as the major cause of phospholipidosis, where an excessive accumulation of phospholipids occurs in various tissues (Hanumegowda et al., 2010). Furthermore, elevated levels of CADs in lysosomes can lead to high organ to blood ratios of drugs that can be mistaken for active drug transport.

ISSX 2011, Poster 170

Published:  14 September 2011

Temporal changes in CYP3A4 mRNA and activity following treatment of cultured human hepatocytes with interleukin-6 (IL-6): Implications for study design and endpoint selection

In recent years the focus of pharmaceutical drug development (once dominated by small molecule (NCE) therapies) has shifted and is now shared with a significant number of new therapies emerging from biological (New Biological Entities or NBE's) development. Since the approval of the first biological treatment in the United States (recombinant insulin, 1982), more than 250 biologics have reached the market, representing roughly one-quarter of all new drugs approved by U.S. and European Union authorities (Trusheim et al, 2010). Biologics include a broad range of therapies including (but not limited to) vaccines, cell or gene therapies, therapeutic protein hormones, cytokines and tissue growth factors, and monoclonal antibodies.