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In the wake of emerging antimicrobial resistance, antibacterial drug development has become more critical. At the same time, development of antibacterial drugs targeting specific pathogens or resistance phenotypes that may have low prevalence presents challenges because it is difficult to conduct large, randomized controlled trials for such drugs. Animal models have increasingly supported clinical development of antibacterials; however, more work is needed to optimize the design and application of these animal models to ensure clear and actionable translation to further human investigation. This review discusses recent case studies of animal infection models used to support antibacterial drug development in order to illuminate considerations for future development of novel antibacterial drugs.

Organic anion transporting polypeptides (OATP)1B1 and OATP1B3 are liver-specific transport proteins that express on the basolateral membrane of human hepatocytes and mediate hepatic uptake of many drugs, including statins. They are important determinants of transporter-mediated drug-drug interactions (DDIs). It has been reported that pre-incubation with some OATP1B1 and OATP1B3 inhibitors potentiates the inhibitory effects, yielding reduced IC50 values. The US FDA draft guidance has recently recommended to use the lower IC50 values after inhibitor-preincubation to assess OATP1B1 and OATP1B3-mediated DDIs. However, it remains unknown whether the potentiation effects of inhibitor-preincubation on IC50 values occur in a physiologically relevant cell model. The current study was designed to determine the IC50 values of OATP1B1 and OATP1B3 inhibitors everolimus (EVR), sirolimus (SIR), and dasatinib against OATP1B substrates in physiologically relevant primary human hepatocytes with or without inhibitor-preincubation and to compare the OATP-mediated DDI prediction using data from primary human hepatocytes and that reported previously in transporter-expressing cell lines. Primary human hepatocytes were cultured in a sandwich configuration. Accumulation of [3H]-CCK-8 (1 µM, 1.5 min), [3H]-rosuvastatin (0.5 µM, 2 min) and [3H]-pitavastatin (1 µM, 0.5 min) was determined in human sandwich-cultured hepatocytes (SCH) in the presence of vehicle control or an inhibitor with or without inhibitor-preincubation at designated concentrations, and was utilized to determine the IC50 values for these inhibitors. R-value models were used to predict OATP-mediated DDIs. Pre-incubation with EVR at a clinically relevant concentration of 0.2 µM significantly reduced accumulation of [3H]-CCK-8 and [3H]-rosuvastatin even after washing. Reduced IC50 values following inhibitor pre-incubation were observed for all three inhibitors using [3H]-CCK-8 and [3H]-rosuvastatin as substrates in human SCH. The IC50 values after inhibitor-preincubation were lower or comparable in transporter-expressing cell lines compared with that in human SCH. For dasatinib, R-values from both cell lines and human SCH were greater than the US FDA cut-off value of 1.1. For EVR, R values from cell lines were 1.23 and were lowered to near 1.1 (1.08–1.09) in human SCH. For SIR, R values from either cell type were less than the cut-off values of 1.1. In conclusion, the current study is the first to report that pre-incubation with OATP1B inhibitors potentiates inhibitory effects in physiologically relevant primary human hepatocytes, supporting the rationale of the current US FDA draft guidance of including an inhibitor-preincubation step when assessing OATP-mediated DDIs in vitro. IC50 values after inhibitor-preincubation in transporter-expressing cell lines may be used for DDI prediction for the purpose of mitigating false-negative OATP-mediated DDI prediction.

Organic anion transporting polypeptides (OATP) 1B1 and OATP1B3 are important determinants of transporter-mediated drug-drug interactions (DDIs). Current studies assessed the OATP1B1 and OATP1B3-mediated DDI potential of vemurafenib, a kinase inhibitor drug with high protein binding and low aqueous solubility, using R-value and physiologically based pharmacokinetic (PBPK) models. The total half-maximal inhibitory concentration (IC(50,total)) values of vemurafenib against OATP1B1 and OATP1B3 were determined in 100% human plasma in transporter-overexpressing human embryonic kidney 293 stable cell lines. The unbound fraction of vemurafenib in human plasma before (f(u,plasma)) and after addition into the uptake assay plate (f(u,plasma,inc)) were determined by rapid equilibrium dialysis. There was no statistically significant difference between f(u,plasma) and f(u,plasma,inc). Vemurafenib IC(50,total) values against OATP1B1 and OATP1B3 are 175 ± 82 and 231 ± 26 μM, respectively. The R-values [R = 1 + f(u,plasma) × I(in,max)/(f(u,plasma,inc) × IC(50,total))] were then simplified as R=1+I(in,max)/IC(50,total), and were 1.76 and 1.57 for OATP1B1 and OATP1B3, respectively. The simulated pravastatin AUC ratio was 1.28 when a single dose of pravastatin (40 mg) was co-administered with vemurafenib (960 mg, twice daily) at steady-state, compared to pravastatin alone. Both R-value and PBPK models predict that vemurafenib has the potential to cause OATP1B1- and OATP1B3-mediated DDIs.

Quantitative assessment of MT1-MMP cell surface-associated proteolytic activity remains undefined. Presently, MT1-MMP was stably expressed and a cell-based FRET assay developed to quantify activity towards synthetic collagen-model triple-helices. To estimate the importance of cell surface localization and specific structural domains on MT1-MMP proteolysis, activity measurements were performed using a series of membrane-anchored MT1-MMP mutants and compared directly with those of soluble MT1-MMP. MT1-MMP activity (k(cat)/K(M)) on the cell surface was 4.8-fold lower compared with soluble MT1-MMP, with the effect largely manifested in k(cat). Deletion of the MT1-MMP cytoplasmic tail enhanced cell surface activity, with both k(cat) and K(M) values affected, while deletion of the hemopexin-like domain negatively impacted K(M) and increased k(cat). Overall, cell surface localization of MT1-MMP restricts substrate binding and protein coupled motions (based on changes in both k(cat) and K(M)) for catalysis. Comparison of soluble and cell surface-bound MT2-MMP revealed 12.9-fold lower activity on the cell surface. The cell-based assay was utilized for small molecule and triple-helical transition state analog MMP inhibitors, which were found to function similarly in solution and at the cell surface. These studies provide the first quantitative assessments of MT1-MMP activity and inhibition in the native cellular environment of the enzyme.

Present studies determined the effects of pretreatment with rifampicin, an organic anion-transporting polypeptide (OATP) inhibitor, and the tyrosine kinase inhibitor dasatinib on OATP1B1- and OATP1B3-mediated transport, and evaluated the OATP-mediated drug-drug interaction potential of dasatinib using the static R-value and dynamic physiologically based pharmacokinetic models. Rifampicin and dasatinib pretreatment significantly decreased OATP1B1- and OATP1B3-mediated transport. Rifampicin pretreatment also significantly decreased [ 3 H]-pitavastatin and [ 3 H]-CCK-8 accumulation in human sandwich-cultured hepatocytes. Present studies revealed that estrone-3-sulfate is a less-sensitive OATP1B1 substrate than estradiol-17β-glucuronide in assessing rifampicin pretreatment effects. Pretreatment with rifampicin and dasatinib reduced the inhibition constant (K i ) values against OATP1B1 by 3 and 2.1 fold and against OATP1B3 by 2.4 and 2.1 fold, respectively. The in vitro rifampicin K i values after preincubation are comparable to the estimated in vivo K i reported previously. Models predict that dasatinib has a low potential to cause OATP1B1- and OATP1B3-mediated drug-drug interactions. Time-lapse confocal microscopy demonstrated that rifampicin and dasatinib pretreatment did not affect plasma membrane localization of green-fluorescent protein–tagged OATP1B1 (GFP-OATP1B1) and GFP–OATP1B3 in human embryonic kidney 293 stable cell lines. In summary, we report novel findings that pretreatment with rifampicin and dasatinib potentiates the inhibitory effects toward OATP1B1 and OATP1B3 without affecting plasma membrane levels of the transporters.

PURPOSE: Membrane transport protein organic anion transporting polypeptide (OATP) 1B1 mediates hepatic uptake of many drugs (e.g. statins). The OATP1B1 c.521T>C (p. V174A) polymorphism has reduced transport activity. Conflicting in vitro results exist regarding whether V174A-OATP1B1 has reduced plasma membrane localization; no such data has been reported in physiologically relevant human liver tissue. Other potential changes, such as phosphorylation, of the V174A-OATP1B1 protein have not been explored. Current studies characterized the plasma membrane localization of V174A-OATP1B1 in genotyped human liver tissue and cell culture and compared the phosphorylation status of V174A- and wild-type (WT)-OATP1B1. METHODS: Localization of V174A- and WT-OATP1B1 were determined in OATP1B1 c.521T>C genotyped human liver tissue (n=79) by immunohistochemistry and in transporter-overexpressing human embryonic kidney (HEK) 293 and HeLa cells by surface biotinylation and confocal microscopy. Phosphorylation and transport of OATP1B1 was determined using (32)P-orthophosphate labeling and [(3)H]estradiol-17β-glucuronide accumulation, respectively. RESULTS: All three methods demonstrated predominant plasma membrane localization of both V174A- and WT-OATP1B1 in human liver tissue and in cell culture. Compared to WT-OATP1B1, the V174A-OATP1B1 has significantly increased phosphorylation and reduced transport. CONCLUSIONS: We report novel findings of increased phosphorylation, but not impaired membrane localization, in association with the reduced transport function of the V174A-OATP1B1.

Organic anion transporting polypeptide (OATP) 1B1 mediates the hepatic uptake of many drugs including lipid-lowering statins. Decreased OATP1B1 transport activity is often associated with increased systemic exposure of statins and statin-induced myopathy. Antimalarial drug chloroquine (CQ) is also used for long-term treatment of rheumatoid arthritis and systemic lupus erythematosus. CQ is lysosomotropic and inhibits protein degradation in lysosomes. The current studies were designed to determine the effects of CQ on OATP1B1 protein degradation, OATP1B1-mediated transport in OATP1B1-overexpressing cell line, and statin uptake in human sandwich-cultured hepatocytes (SCH). Treatment with lysosome inhibitor CQ increased OATP1B1 total protein levels in HEK293-OATP1B1 cells and in human SCH as determined by OATP1B1 immunoblot. In HEK293-FLAG-tagged OATP1B1 stable cell line, co-immunofluorescence staining indicated that intracellular FLAG-OATP1B1 is colocalized with lysosomal associated membrane glycoprotein (LAMP)-2, a marker protein of late endosome/lysosome. Enlarged LAMP-2-positive vacuoles with FLAG-OATP1B1 protein retained inside were readily detected in CQ-treated cells, consistent with blocking lysosomal degradation of OATP1B1 by CQ. In HEK293-OATP1B1 cells, without pre-incubation, CQ concentrations up to 100 μM did not affect OATP1B1-mediated [(3)H]E217G accumulation. However, pre-incubation with CQ at clinically relevant concentration(s) significantly decreased [(3)H]E217G and [(3)H]pitavastatin accumulation in HEK293-OATP1B1 cells and [(3)H]pitavastatin accumulation in human SCH. CQ pretreatment (25 μM, 2 h) resulted in ∼1.9-fold decrease in Vmax without affecting Km of OATP1B1-mediated [(3)H]E217G transport in HEK293-OATP1B1 cells. Pretreatment with monensin and bafilomycin A1, which also have lysosome inhibition activity, significantly decreased OATP1B1-mediated transport in HEK293-OATP1B1 cells. Pharmacoepidemiologic studies using data from the U.S. Food and Drug Administration Adverse Event Reporting System indicated that CQ plus pitavastatin, rosuvastatin, and pravastatin, which are minimally metabolized by the cytochrome P450 enzymes, led to higher myopathy risk than these statins alone. In summary, the present studies report novel findings that lysosome is involved in degradation of OATP1B1 protein and that pre-incubation with lysosomotropic drug CQ downregulates OATP1B1 transport activity. Our in vitro data in combination with pharmacoepidemiologic studies support that CQ has potential to cause OATP-mediated drug-drug interactions.

Primary hits that arise from screening one bead one compound (OBOC) libraries against a target of interest rarely have high potency. However, there has been little work focused on the development of an efficient workflow for primary hit improvement. In this study, we show that by characterizing the binding constants for all of the hits that arise from a screen, structure-activity relationship (SAR) data can be obtained to inform the design of "derivative libraries" of a primary hit that can then be screened under more demanding conditions to obtain improved compounds. Here, we demonstrate the rapid improvement of a primary hit against matrix metalloproteinase-14 using this approach.

The organic anion-transporting polypeptide (OATP) 1B3 is a membrane transport protein that mediates hepatic uptake of many drugs and endogenous compounds. Currently, determination of OATP-mediated drug-drug interactions in vitro is focused primarily on direct substrate inhibition. Indirect inhibition of OATP1B3 activity is under-appreciated. OATP1B3 has putative protein kinase C (PKC) phosphorylation sites. Studies were designed to determine the effect of PKC activation on OATP1B3-mediated transport in human hepatocytes using cholecystokinin-8 (CCK-8), a specific OATP1B3 substrate, as the probe. A PKC activator, phorbol-12-myristate-13-acetate (PMA), did not directly inhibit [(3)H]CCK-8 accumulation in human sandwich-cultured hepatocytes (SCH). However, pretreatment with PMA for as little as 10 minutes rapidly decreased [(3)H]CCK-8 accumulation. Treatment with a PKC inhibitor bisindolylmaleimide (BIM) I prior to PMA treatment blocked the inhibitory effect of PMA, indicating PKC activation is essential for downregulating OATP1B3 activity. PMA pretreatment did not affect OATP1B3 mRNA or total protein levels. To determine the mechanism(s) underlying the indirect inhibition of OATP1B3 activity upon PKC activation, adenoviral vectors expressing FLAG-Myc-tagged OATP1B3 (Ad-OATP1B3) were transduced into human hepatocytes; surface expression and phosphorylation of OATP1B3 were determined by biotinylation and by an anti-phosphor-Ser/Thr/Tyr antibody, respectively. PMA pretreatment markedly increased OATP1B3 phosphorylation without affecting surface or total OATP1B3 protein levels. In conclusion, PKC activation rapidly decreases OATP1B3 transport activity by post-translational regulation of OATP1B3. These studies elucidate a novel indirect inhibitory mechanism affecting hepatic uptake mediated by OATP1B3, and provide new insights into predicting OATP-mediated drug interactions between OATP substrates and kinase modulator drugs/endogenous compounds.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a zinc-dependent type-I transmembrane metalloproteinase involved in pericellular proteolysis, migration and invasion. Numerous substrates and binding partners have been identified for MT1-MMP, and its role in collagenolysis appears crucial for tumor invasion. However, development of MT1-MMP inhibitors must consider the substantial functions of MT1-MMP in normal physiology and disease prevention. The present review examines the plethora of MT1-MMP activities, how these activities relate to cancer initiation and progression, and how they can be monitored in real time. Examination of MT1-MMP activities and cell surface behaviors can set the stage for the development of unique, selective MT1-MMP inhibitors.

Background Nonclinical (animal and in vitro) models are commonly used during the development of antibacterial drugs. Pharmacokinetic (PK) and pharmacodynamic (PD) data obtained from these nonclinical models are used to generate a PK-PD target, which can then be bridged to humans in a probability of PK-PD target attainment (PTA) analysis to support selection of the dose regimen for phase 3 trials and in vitro susceptibility testing criteria (breakpoints) to guide clinical usage. Methods Two recently approved tetracycline antibacterial drugs, eravacycline (ERV) and omadacycline (OMD), were evaluated. PK-PD data from nonclinical models and clinical microbiological response were collected from each of the respective clinical pharmacology reviews and assessments published by FDA and EMA, respectively. The highest MICs (minimum inhibitory concentrations) reflecting 80% success in the ability of the drug to inhibit growth in the target bacteria were identified from clinical and nonclinical data and termed the MIC cutoff. The nonclinical MIC cutoff was obtained from the PTA analysis using the PK-PD targets from animal studies. The clinical MIC cutoff was obtained from microbiological response (microbiological intent-to-treat population) data from clinical trial experience. The ratios of the clinical and nonclinical MIC cutoffs were calculated and used to evaluate potential discrepancies between the animal model prediction and clinical trial experience. Results The drug development programs for ERV and OMD included murine infection models and in vitro models to characterize PK-PD. The clinical to nonclinical MIC cutoff ratios ranged from 4 to 32. Higher values of the MIC cutoff signify that the drug can treat larger proportions of the bacterial population; therefore, high clinical to nonclinical MIC cutoff ratios signify that the drugs had more activity in reducing the bacterial population in clinical than in nonclinical studies. Conclusion Thus, the nonclinical models for ERV and OMD under-predicted microbiological response and breakpoints. While nonclinical models are generally useful, more characterization of translational factors may be needed to allow nonclinical models to be more predictive of clinical trial outcomes. Disclosures All authors: No reported disclosures.

Intestinal alkaline phosphatases (IALPs) are widely expressed in the brush border of epithelial cells of the intestinal mucosa. Although their physiologic role is unclear, they are very significant when it comes to the release of bioactive parent from orally dosed phosphate prodrugs. Such prodrugs can be resistant to cleavage by IALP, or alternatively undergo rapid cleavage leading to the release and precipitation of the less soluble parent. Because purified IALPs from preclinical species are not commercially available, and species differences have not been investigated to date, an effort was made to recombinantly express, purify, and characterize rat and cynomolgus monkey IALP (rIALP). Specifically, recombinant IALP (rIALP)-catalyzed cleavage of five prodrugs (fosphenytoin, clindamycin phosphate, dexamethasone phosphate, ritonavir phosphate, and ritonavir oxymethyl phosphate) was tested in vitro and parent exposure was assessed in vivo (rat only) following an oral dose of each prodrug. It was determined that the rate of phosphate cleavage in vitro varied widely; direct phosphates were more resistant to bioconversion, whereas faster conversion was observed with oxymethyl-linked prodrugs. Overall, the rat rIALP-derived data were qualitatively consistent with in vivo data; prodrugs that were readily cleaved in vitro rendered higher parent drug exposure in vivo. Of the five prodrugs tested, one (ritonavir phosphate) showed no conversion in vitro and no in vivo parent exposure. Finally, the apparent K(m) values obtained for fosphenytoin and clindamycin phosphate in vitro suggest that IALP is not likely to be saturated at therapeutic doses.

Histidinol dehydrogenase, the product of the HisD gene, mediates the final step in the histidine biosynthetic pathway. This enzyme has captured attention for drug discovery studies in past few years. Recently, our group cloned and expressed Geotrichum candidum histidinol dehydrogenase and successful screening of substrate analog inhibitors of histidinol dehydrogenase led to some antifungal compounds with IC(50) values in micromolar range. In this study, we have done docking analysis of these antifungal agents in G. candidum. Two new compounds were designed based on the docking results and these compounds turned out to be potent inhibitors of G. candidum histidinol dehydrogenase, showing IC(50) values as low as 3.17 microM.

Introduction: MT1-MMP plays critical roles during tumor malignancy and is one of the best-validated proteolytic enzyme targets on cancer cells. It is up-regulated in several tumor types, including breast, cervical, and ovarian cancer and is significantly associated with adverse outcome. In spite of the large proteolytic repertoire and of the robust proteolytic activity of engineered soluble forms of the enzyme, several studies indicate that soluble MT1-MMP mediated proteolysis is not sufficient per se for efficient penetration of ECM barriers. Thus, localization of the enzyme at the cell surface is essential to translate its proteolytic activity into a modification of cell function. The transmembrane nature of MT1-MMP allows it to influence extracellular remodeling of the matrix surrounding tumor cells as well as intracellular signaling events involved in cell invasion. Like virtually all cell surface proteases, quantitative assessment of MT1-MMP in its native environment has not been achieved. A mechanistic examination of MT1-MMP at the cell surface would unravel the influences of binding partners on activities, and set the stage for the development of unique, non-active site inhibitors. Methods: ΔTM MT1-MMP (a deletion mutant lacking the TM domain) was transfected transiently in COS-1 cells to generate soluble MT1-MMP. Estimation of the amount of active enzyme was done by TIMP-2 titration. WT-MT1-MMP (Wild Type) was stably transfected in MCF-7 cells which are deficient in MT1-MMP. A cell-based FRET assay was developed in a 384-well format using fluorogenic triple-helical peptide substrates (fTHPs) that are either suitable for most collagenolytic MMPs or selective for different collagenolytic MMPs to quantify protease activity. Kinetic parameters were calculated and activity comparisons were made for soluble MT1-MMP and several surface-bound MT1-MMP mutants to evaluate the importance of the cell surface and specific MT1-MMP domains on activity. Results and Conclusion: Soluble MT1-MMP was expressed, purified, and quantified by TIMP-2 titration. Kinetic parameters KM, Vmax, and kcat/KM were determined by calculation of rates of hydrolysis of fTHP-9 in a FRET assay. A high level of MT1-MMP expression in transfected cells relative to those mock transfected cells was verified by western blot. Kinetic parameters determined with WT-MT1-MMP transfected cells as compared to mock transfected cell suggested that MT1-MMP has affinity for fTHP-9. kcat/KM for the soluble enzyme was 32800 sec−1M−1, three times higher as compared to membrane tethered enzyme. Thus, the catalytic efficiency of the enzyme is different in the cell surface environment. In this study, we have developed cell based assays useful for the mechanistic and quantitative evaluation of substrates and inhibitors of MT1-MMP in its native environment as well as to rank order potential inhibitors. Citation Format: Sonia Pahwa, Gregg Fields. Quantitative analysis of MT1-MMP activity on cell surface: A FRET assay model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5059. doi:10.1158/1538-7445.AM2013-5059