Leveraging Modeling and Simulation to Enhance the Efficiency of Bioequivalence Approaches for Generic Drugs: Highlights from the 2023 Generic Drug Science and Research Initiatives Public Workshop.

The 2023 Generic Drug Science and Research Initiative Public Workshop organized by the U.S. Food and Drug Administration (FDA) discussed the research needs to improve and enhance bioequivalence (BE) approaches for generic drug development. FDA takes such research needs and panel discussions into account to develop its Generic Drug User Fee Amendments III (GDUFA III) Science and Research Initiatives specific to generics. During the five workshop sessions, presentations and panel discussions focused on identifying and addressing scientific gaps and research needs related to nitrosamine impurity issues, BE assessment for oral products, innovative BE approaches for long-acting injectable products, alternative BE approaches for orally inhaled products, and advanced BE methods for topical products. Specifically, this report highlights the discussions on how to improve BE assessment for developing generic drug products based on research priorities for leveraging quantitative methods and modeling, as well as artificial intelligence/machine learning (AI/ML).

Development of Mechanistic In Vitro-In Vivo Extrapolation to Support Bioequivalence Assessment of Long-Acting Injectables.

Long-acting injectable (LAI) formulations provide sustained drug release over an extended period ranging from weeks to several months to improve efficacy, safety, and compliance. Nevertheless, many challenges arise in the development and regulatory assessment of LAI drug products due to a limited understanding of the tissue response to injected particles (e.g., inflammation) impacting in vivo performance. Mechanism-based in silico methods may support the understanding of LAI-physiology interactions. The objectives of this study were as follows: (1) to use a mechanistic modeling approach to delineate the in vivo performance of DepoSubQ Provera® and formulation variants in preclinical species; (2) to predict human exposure based on the knowledge gained from the animal model. The PBPK model evaluated different elements involved in LAI administration and showed that (1) the effective in vivo particle size is potentially larger than the measured in vitro particle size, which could be due to particle aggregation at the injection site, and (2) local inflammation is a key process at the injection site that results in a transient increase in depot volume. This work highlights how a mechanistic modeling approach can identify critical physiological events and product attributes that may affect the in vivo performance of LAIs.

Colletotrichum siamense leaf blight/spot: Characterization of a newly identified disease on Cinnamomum tamala.

Cinnamomum tamala (bay leaf) is widely used for culinary and medicinal purposes in South Asia. A leaf blight/spot disease was first discovered on nearly 90% of C. tamala plants with a mean severity of 48% to 74.4% in Gazipur and Bogura, Bangladesh, in 2019. The present study identified and characterized the causal organism and formulated the optimum growth conditions and effective fungicides for the chemical control of the pathogen. The characteristic symptoms on the infected leaves appeared circular to oval reddish-brown spots with raised margins and often developed in tear-stain patterns. Severe infection of C. tamala sapling resulted in dieback symptoms with leaf defoliation. A fungus with floccose, dense, white colonies with well-differentiated acervuli was isolated from the infected leaves. Combined cultural, morphological, and molecular characteristics identified the pathogen as Colletotrichum siamense. Inculcating healthy leaves and 1-year-old saplings of C. tamala with a conidial suspension of the fungus reproduced the same symptoms observed in the bay leaf orchard. The highest mycelial growth was recorded on V-8 Juice Agar media, while the maximum radial mycelial growth and level of sporulation of the fungus were significantly higher in incubation temperature 30°C. Fungicide trials showed that carbendazim 50 WP, azoxystrobin, mancozeb, and trifloxystrobin, either singly or in combination, successfully reduced fungal mycelial growth in vitro. Therefore, disease management strategies should be opted to halt the further spread of this issue. To our knowledge, this is the first study to document the incidence of Colletotrichum leaf blight on C. tamala in Bangladesh and even in the world.

Mechanistic modeling of ophthalmic, nasal, injectable, and implant generic drug products: A workshop summary report.

For approval, a proposed generic drug product must demonstrate it is bioequivalent (BE) to the reference listed drug product. For locally acting drug products, conventional BE approaches may not be feasible because measurements in local tissues at the sites of action are often impractical, unethical, or cost-prohibitive. Mechanistic modeling approaches, such as physiologically-based pharmacokinetic (PBPK) modeling, may integrate information from drug product properties and human physiology to predict drug concentrations in these local tissues. This may allow clinical relevance determination of critical drug product attributes for BE assessment during the development of generic drug products. In this regard, the Office of Generic Drugs of the US Food and Drug Administration has recently established scientific research programs to accelerate the development and assessment of generic products by utilizing model-integrated alternative BE approaches. This report summarizes the presentations and panel discussion from a public workshop that provided research updates and information on the current state of the use of PBPK modeling approaches to support generic product development for ophthalmic, injectable, nasal, and implant drug products.

Mechanistic modeling of drug products applied to the skin: A workshop summary report.

The development of a generic drug product involves demonstrating that there is no significant difference in the rate and extent to which the active ingredient becomes available at the site of action, relative to the reference listed drug product. This remains challenging for many locally acting topical dermatological products because measuring the concentration of the active ingredient at the site of action in the skin may not be straightforward, and, in most instances, there are no established relationships between skin and plasma pharmacokinetic profiles. In recent years, the Office of Generic Drugs of the US Food and Drug Administration (FDA) established scientific research programs with the goal of enhancing patient access to high quality, affordable topical dermatological generics. A key strategy of these research programs was to leverage modeling and simulation methodologies that accelerate the development of these generics by facilitating alternative bioequivalence approaches for dermatological drug products. This report summarizes relevant insights and discussions from a 2021 FDA public workshop titled "Regulatory Utility of Mechanistic Modeling to Support Alternative Bioequivalence Approaches," which illustrated how mechanistic modeling and simulation approaches can be utilized (and have been used) to inform generic drug product development and regulatory decisions during the assessment of generic drug applications submitted to the FDA.

Assessing Trans-Inhibition of OATP1B1 and OATP1B3 by Calcineurin and/or PPIase Inhibitors and Global Identification of OATP1B1/3-Associated Proteins.

Organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 are key determinants of drug-drug interactions (DDIs). Various drugs including the calcineurin inhibitor (CNI) cyclosporine A (CsA) exert preincubation-induced trans-inhibitory effects upon OATP1B1 and/or OATP1B3 (abbreviated as OATP1B1/3) by unknown mechanism(s). OATP1B1/3 are phosphoproteins; calcineurin, which dephosphorylates and regulates numerous phosphoproteins, has not previously been investigated in the context of preincubation-induced trans-inhibition of OATP1B1/3. Herein, we compare the trans-inhibitory effects exerted on OATP1B1 and OATP1B3 by CsA, the non-analogous CNI tacrolimus, and the non-CNI CsA analogue SCY-635 in transporter-overexpressing human embryonic kidney (HEK) 293 stable cell lines. Preincubation (10-60 min) with tacrolimus (1-10 µM) rapidly and significantly reduces OATP1B1- and OATP1B3-mediated transport up to 0.18 ± 0.03- and 0.20 ± 0.02-fold compared to the control, respectively. Both CsA and SCY-635 can trans-inhibit OATP1B1, with the inhibitory effects progressively increasing over a 60 min preincubation time. At each equivalent preincubation time, CsA has greater trans-inhibitory effects toward OATP1B1 than SCY-635. Preincubation with SCY-635 for 60 min yielded IC50 of 2.2 ± 1.4 µM against OATP1B1, which is ~18 fold greater than that of CsA (0.12 ± 0.04 µM). Furthermore, a proteomics-based screening for protein interactors was used to examine possible proteins and processes contributing to OATP1B1/3 regulation and preincubation-induced inhibition by CNIs and other drugs. A total of 861 and 357 proteins were identified as specifically associated with OATP1B1 and OATP1B3, respectively, including various protein kinases, ubiquitin-related enzymes, the tacrolimus (FK506)-binding proteins FKBP5 and FKBP8, and several known regulatory targets of calcineurin. The current study reports several novel findings that expand our understanding of impaired OATP1B1/3 function; these include preincubation-induced trans-inhibition of OATP1B1/3 by the CNI tacrolimus, greater preincubation-induced inhibition by CsA compared to its non-CNI analogue SCY-635, and association of OATP1B1/3 with various proteins relevant to established and candidate OATP1B1/3 regulatory processes.

Physicochemical surrogates for in vitro toxicity assessment of liposomal amphotericin B.

Pharmaceutical toxicity evaluations often use in vitro systems involving primary cells, cell lines or red blood cells (RBCs). Cell-based analyses ('bioassays') can be cumbersome and typically rely on hard-to-standardize biological materials. Amphotericin B (AmB) toxicity evaluations are primarily based on potassium release from RBCs and share these limitations. This study evaluates the potential substitution of two physicochemical AmB toxicity approaches for the bioassay: Ultraviolet-visible spectroscopy (UV-vis) and in vitro drug release kinetics. UV-vis spectral analyses indicated that liposomal AmB's (L-AmB) main peak position (λmax) and peak ratio (OD346/OD322) are potential toxicity surrogates. Similarly, two first-order release parameters derived from USP-4 in vitro drug release analyses also provided linear relationships with toxicity. These were the initial, overall drug release rate and the ratio of loose to tight AmB pools. Positive slopes and high correlation coefficients (R2 > 0.9) characterized all interrelations between physicochemical parameters and toxicity. These tests converted the manufacturing variables' nonlinear (i.e., curvilinear) relationships with in vitro toxicity to linear responses. Three different toxicity attenuation approaches (2 manufacturing, 1 formulation), covering formulation composition and process aspects, support this approach's universality. These data suggest that one or more spectral and kinetic physicochemical tests can be surrogates for L-AmB in vitro toxicity testing.

Amphotericin B release rate is the link between drug status in the liposomal bilayer and toxicity.

Amphotericin B (AmB) is an amphiphilic drug commonly formulated in liposomes and administered intravenously to treat systemic fungal infections. Recent studies on the liposomal drug product have shed light on the AmB aggregation status in the bilayer, which heat treatment (curing) modifies. Although toxicity was found related to aggregation status - loose aggregates significantly more toxic than tight aggregates - the precise mechanism linking aggregation and toxicity was not well understood. This study directly measured drug release rate from various AmB liposomal preparations made with modified curing protocols to evaluate correlations among drug aggregation state, drug release, and in vitro toxicity. UV-Vis spectroscopy of these products detected unique curing-induced changes in the UV spectral features: a ∼25 nm blue-shift of the main absorption peak (λmax) in aqueous buffer and a decrease in the OD346/OD322 ratio upon thermal curing, reflecting tighter aggregation. In vitro release testing (IVRT) data showed, by applying and fitting first-order release kinetic models for one or two pools, that curing impacts two significant changes: a 3-5-fold drop in the overall drug release rate and a ten-fold decrease in the ratio between the loosely aggregated and the tightly aggregated, more thermodynamically stable drug pool. The kinetic data thus corroborated the trend independently deduced from the UV-Vis spectral data. The in vitro toxicity assay indicated a decreased toxicity with curing, as shown by the significantly increased concentration, causing half-maximal potassium release (TC50). The data suggest that the release of AmB requires dissociation of the tight complexes within the bilayer and that the reduced toxicity relates to this slower rate of dissociation. This study demonstrates the relationship between AmB aggregation status within the lipid bilayer and drug release (directly measured rate constants), providing a mechanistic link between aggregation status and in vitro toxicity in the liposomal formulations.

Mechanistic Modeling of In Vitro Skin Permeation and Extrapolation to In Vivo for Topically Applied Metronidazole Drug Products Using a Physiologically Based Pharmacokinetic Model.

Physiologically based pharmacokinetic (PBPK) modeling has increasingly been employed in dermal drug development and regulatory assessment, providing a framework to integrate relevant information including drug and drug product attributes, skin physiology parameters, and population variability. The current study aimed to develop a stepwise modeling workflow with knowledge gained from modeling in vitro skin permeation testing (IVPT) to describe in vivo exposure of metronidazole locally in the stratum corneum following topical application of complex semisolid drug products. The initial PBPK model of metronidazole in vitro skin permeation was developed using infinite and finite dose aqueous metronidazole solution. Parameters such as stratum corneum lipid-water partition coefficient (Ksclip/water) and stratum corneum lipid diffusion coefficient (Dsclip) of metronidazole were optimized using IVPT data from simple aqueous solutions (infinite) and MetroGel (10 mg/cm2 dose application), respectively. The optimized model, when parameterized with physical and structural characteristics of the drug products, was able to accurately predict the mean cumulative amount permeated (cm2/h) and flux (µg/cm2/h) profiles of metronidazole following application of different doses of MetroGel and MetroCream. Thus, the model was able to capture the impact of differences in drug product microstructure and metamorphosis of the dosage form on in vitro metronidazole permeation. The PBPK model informed by IVPT study data was able to predict the metronidazole amount in the stratum corneum as reported in clinical studies. In summary, the proposed model provides an enhanced understanding of the potential impact of drug product attributes in influencing in vitro skin permeation of metronidazole. Key kinetic parameters derived from modeling the metronidazole IVPT data improved the predictions of the developed PBPK model of in vivo local metronidazole concentrations in the stratum corneum. Overall, this work improves our confidence in the proposed workflow that accounts for drug product attributes and utilizes IVPT data toward improving predictions from advanced modeling and simulation tools.

Multi-phase multi-layer mechanistic dermal absorption (MPML MechDermA) model to predict local and systemic exposure of drug products applied on skin.

Physiologically-based pharmacokinetic models combine knowledge about physiology, drug product properties, such as physicochemical parameters, absorption, distribution, metabolism, excretion characteristics, formulation attributes, and trial design or dosing regimen to mechanistically simulate drug pharmacokinetics (PK). The current work describes the development of a multiphase, multilayer mechanistic dermal absorption (MPML MechDermA) model within the Simcyp Simulator capable of simulating uptake and permeation of drugs through human skin following application of drug products to the skin. The model was designed to account for formulation characteristics as well as body site- and sex- population variability to predict local and systemic bioavailability. The present report outlines the structure and assumptions of the MPML MechDermA model and includes results from simulations comparing absorption at multiple body sites for two compounds, caffeine and benzoic acid, formulated as solutions. Finally, a model of the Feldene (piroxicam) topical gel, 0.5% was developed and assessed for its ability to predict both plasma and local skin concentrations when compared to in vivo PK data.

Physiologically-Based Pharmacokinetic Modeling to Support Determination of Bioequivalence for Dermatological Drug Products: Scientific and Regulatory Considerations.

Physiologically-based pharmacokinetic (PBPK) modeling and simulation provides mechanism-based predictions of the pharmacokinetics of an active ingredient following its administration in humans. Dermal PBPK models describe the skin permeation and disposition of the active ingredient following the application of a dermatological product on the skin of virtual healthy and diseased human subjects. These models take into account information on product quality attributes, physicochemical properties of the active ingredient and skin (patho)physiology, and their interplay with each other. Regulatory and product development decision makers can leverage these quantitative tools to identify factors impacting local and systemic exposure. In the realm of generic drug products, the number of US Food and Drug Administratioin (FDA) interactions that use dermal PBPK modeling to support alternative bioequivalence (BE) approaches is increasing. In this report, we share scientific considerations on the development, verification and validation (V&V), and application of PBPK models within the context of a virtual BE assessment for dermatological drug products. We discuss the challenges associated with model V&V for these drug products stemming from the fact that target-site active ingredient concentrations are typically not measurable. Additionally, there are no established relationships between local and systemic PK profiles, when the latter are quantifiable. To that end, we detail a multilevel model V&V approach involving validation for the model of the drug product of interest coupled with the overall assessment of the modeling platform in use while leveraging in vitro and in vivo data related to local and systemic bioavailability.

Knowledge, attitude and prevention practices of garment factory workers regarding the largest Dengue outbreak on record in Bangladesh.

The aim of this cross-sectional study was to assess the knowledge, attitudes and prevention practices (KAP) among the garment factory worker population in Bangladesh regarding a historical dengue outbreak in 2019. A total of 400 participants were selected by simple random sampling, and questionnaire-based interviews were conducted. The average score of knowledge, attitude and prevention practice was 8.33 ± 2.35, 6.32 ± 1.20 and 6.31 ± 1.50, respectively. Only 76 out of 400 participants (19%) scored above 10 (all university-educated). Participant workers reported both negative and positive attitudes regarding dengue fever (DF). Negative attitudes included an expectation of increased mortality and strained family finances from DF attacks. A significantly high number of participants (92%) believed that death from DF was inevitable. Positive attitudes included optimism about DF eradication potentials and eagerness to help and donate blood to sick relatives. Participants primarily learned about the DF prevention from mass media (244/400; 61.0%) and social media (97/400; 24.25%). The most popular prevention measures adopted were mosquito repellent incense (344/400; 86.0%) and mosquito nets (389/400; 97.25%). While most participants (358/400; 89.5%) cleaned areas where mosquitos lay eggs, only 169 out of 400 (42.25%) regularly treated with chemical sprays. Only 182 out of 400 (45.5%) reported receiving DF prevention training in the factory. Correlation between DF knowledge and education was statistically significant (r = .38, p < .01, n = 398). Correlation between DF knowledge and work experience was insignificant (r = .01, p > .01, n = 398). Age and DF knowledge were not correlated (r = 0.07, p > .01, n = 398). In conclusion, gaps in KAP for dengue could be addressed by government-sponsored educational programmes that utilize the power of mass/social media for dengue prevention and control. More KAP surveillance studies are needed for other sectors of the society.

Assessing OATP1B1- and OATP1B3-Mediated Drug-Drug Interaction Potential of Vemurafenib Using R-Value and Physiologically-Based Pharmacokinetic Models.

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 (IC50,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 (fu,plasma) and after addition into the uptake assay plate (fu,plasma,inc) were determined by rapid equilibrium dialysis. There was no statistically significant difference between fu,plasma and fu,plasma,inc. Vemurafenib IC50,total values against OATP1B1 and OATP1B3 are 175 ± 82 and 231 ± 26 µM, respectively. The R-values [R = 1 + fu,plasma × Iin,max/(fu,plasma,inc × IC50,total)] were then simplified as R = 1+Iin,max/IC50,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.

Characterization of Plasma Membrane Localization and Phosphorylation Status of Organic Anion Transporting Polypeptide (OATP) 1B1 c.521 T>C Nonsynonymous Single-Nucleotide Polymorphism.

PURPOSE: Membrane transport protein organic anion transporting polypeptide (OATP) 1B1 mediates hepatic uptake of many drugs (e.g. statins). The OATP1B1 c.521 T > 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.521 T > 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 32P-orthophosphate labeling and [3H]estradiol-17ß-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.

Preincubation With Everolimus and Sirolimus Reduces Organic Anion-Transporting Polypeptide (OATP)1B1- and 1B3-Mediated Transport Independently of mTOR Kinase Inhibition: Implication in Assessing OATP1B1- and OATP1B3-Mediated Drug-Drug Interactions.

Organic anion transporting polypeptides (OATP)1B1 and OATP1B3 mediate hepatic uptake of many drugs including lipid-lowering statins. Current studies determined the OATP1B1/1B3-mediated drug-drug interaction (DDI) potential of mammalian target of rapamycin (mTOR) inhibitors, everolimus and sirolimus, using R-value and physiologically based pharmacokinetic models. Preincubation with everolimus and sirolimus significantly decreased OATP1B1/1B3-mediated transport even after washing and decreased inhibition constant values up to 8.3- and 2.9-fold for OATP1B1 and both 2.7-fold for OATP1B3, respectively. R-values of everolimus, but not sirolimus, were greater than the FDA-recommended cutoff value of 1.1. Physiologically based pharmacokinetic models predict that everolimus and sirolimus have low OATP1B1/1B3-mediated DDI potential against pravastatin. OATP1B1/1B3-mediated transport was not affected by preincubation with INK-128 (10 µM, 1 h), which does however abolish mTOR kinase activity. The preincubation effects of everolimus and sirolimus on OATP1B1/1B3-mediated transport were similar in cells before preincubation with vehicle control or INK-128, suggesting that inhibition of mTOR activity is not a prerequisite for the preincubation effects observed for everolimus and sirolimus. Nine potential phosphorylation sites of OATP1B1 were identified by phosphoproteomics; none of these are the predicted mTOR phosphorylation sites. We report the everolimus/sirolimus-preincubation-induced inhibitory effects on OATP1B1/1B3 and relatively low OATP1B1/1B3-mediated DDI potential of everolimus and sirolimus.

Regulation of Organic Anion Transporting Polypeptides (OATP) 1B1- and OATP1B3-Mediated Transport: An Updated Review in the Context of OATP-Mediated Drug-Drug Interactions.

Organic anion transporting polypeptides (OATP) 1B1 and OATP1B3 are important hepatic transporters that mediate the uptake of many clinically important drugs, including statins from the blood into the liver. Reduced transport function of OATP1B1 and OATP1B3 can lead to clinically relevant drug-drug interactions (DDIs). Considering the importance of OATP1B1 and OATP1B3 in hepatic drug disposition, substantial efforts have been given on evaluating OATP1B1/1B3-mediated DDIs in order to avoid unwanted adverse effects of drugs that are OATP substrates due to their altered pharmacokinetics. Growing evidences suggest that the transport function of OATP1B1 and OATP1B3 can be regulated at various levels such as genetic variation, transcriptional and post-translational regulation. The present review summarizes the up to date information on the regulation of OATP1B1 and OATP1B3 transport function at different levels with a focus on potential impact on OATP-mediated DDIs.

Characterization of Liver- and Cancer-type-Organic Anion Transporting Polypeptide (OATP) 1B3 Messenger RNA Expression in Normal and Cancerous Human Tissues.

BACKGROUND: Membrane transport protein organic anion transporting polypeptide (OATP) 1B3 mediates the cellular uptake of many clinically important drugs including anti-cancer drugs (e.g., paclitaxel). In addition to the well-recognized hepatic expression and function of OATP1B3 [herein named liver-type (Lt) OATP1B3], OATP1B3 also expresses in cancers and has been postulated to play a role in cancer therapy, presumably by facilitating the influx of anti-cancer drugs. Recently, a cancer type (Ct)-OATP1B3 mRNA variant was identified in colon and lung cancer tissues, which encodes truncated Ct-OATP1B3 with negligible transport activity. Other than in colon and lung cancers, reports on mRNA expression of OATP1B3 in other cancers cannot distinguish between the Ltand Ct-OATP1B3. OBJECTIVE: The current studies were designed to characterize the expression of Lt- and Ct-OATP1B3 mRNA in ovarian, prostate, bladder, breast, and lung tissues. METHODS: Lt- and Ct-OATP1B3 isoform-specific PCR primers were utilized to determine the mRNA levels of Lt- and Ct-OATP1B3, respectively. An expression vector expressing green fluorescent protein (GFP)-tagged Lt-OATP1B3 was transiently transfected into the ovarian cancer cell line SKOV3. Confocal live-cell microscopy was utilized to determine the localization of GFP-Lt-OATP1B3 in SKOV3 cells. RESULTS: For the first time, Lt-OATP1B3 mRNA was detected in ovarian, prostate, bladder and breast cancers. The localization of GFP-Lt-OATP1B3 on the plasma membrane of SKOV3 cells after transient transfection was readily detected by confocal microscopy. CONCLUSION: Our findings are supportive of the potential role of Lt-OATP1B3 in cancer cells.

Autonomic Reflex Screen Test Abnormalities in Cold-Induced Sweating Syndrome Type 1.

Cold-induced sweating syndrome (CISS) is a rare autosomal recessive disease due to mutation in the Cytokine receptor-like factor 1 (CRLF1). The characteristic symptom of CISS is the tendency to sweat profusely especially in the upper body and hands when the patient is exposed to cold temperature. We sought to first report the findings of autonomic reflex screen in a case of CISS type 1 with Cytokine receptor-like factor 1 mutation. Valsalva morphology, Valsalva ratio, and heart rate response to deep breathing were normal for the patient's age. Quantitative sudomotor axon reflex test showed nonlength dependent decrease in the sweat volume. Tilt table revealed evidence of reflex (vasovagal) "syncope," however, the patient was asymptomatic without loss of consciousness.

Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner.

OATP1B1 and OATP1B3 mediate hepatic uptake of many drugs (e.g., statins) and can mediate transporter-mediated drug-drug-interactions (DDIs). Bortezomib is the first-in-class proteasome inhibitor drug approved by the U. S. Food and Drug Administration for the treatment of multiple myeloma. The potential of bortezomib to cause OATP-mediated DDIs has not been assessed. The current study investigated the involvement of the ubiquitin-proteasome system (UPS) in OATP1B1 and OATP1B3 degradation and determined the effects of proteasome inhibitors on OATP1B1- and OATP1B3-mediated transport. Co-immunoprecipitation of FLAG-OATP1B1/1B3 and HA-ubiquitin was observed in human embryonic kidney (HEK) 293 cells co-transfected with FLAG-tagged OATP1B1/OATP1B3 and hemagglutinin (HA)-tagged ubiquitin, suggesting that OATP1B1 and OATP1B3 can be ubiquitin-modified. Although blocking proteasome activity by bortezomib treatment (50 nM, 7 h) increased the endogenous ubiquitin-conjugated FLAG-OATP1B1 and FLAG-OATP1B3 in HEK293-FLAG-OATP1B1 and-OATP1B3 cells, such treatment did not affect the total protein levels of OATP1B1 and OATP1B3, suggesting that the UPS plays a minor role in degradation of OATP1B1 and OATP1B3 under current constitutive conditions. Pretreatment with bortezomib (50-250 nM, 2-7 h) significantly decreased transport of [3H]CCK-8, a specific OATP1B3 substrate, in HEK293-OATP1B3 and human sandwich-cultured hepatocytes (SCH). However, bortezomib pretreatment had negligible effects on the transport of [3H]E217ßG and [3H]pitavastatin, dual substrates of OATP1B1 and OATP1B3, in HEK293-OATP1B1/1B3 cells and/or human SCH. Compared with vehicle control treatment, bortezomib pretreatment significantly decreased the maximal transport velocity (Vmax) of OATP1B3-mediated transport of CCK-8 (92.25 ± 14.2 vs. 133.95 ± 15.5 pmol/mg protein/min) without affecting the affinity constant (Km) values. Treatment with other proteasome inhibitors MG132, epoxomicin, and carfilzomib also significantly decreased OATP1B3-mediated [3H]CCK-8 transport. In summary, the current studies for the first time report ubiquitination of OATP1B1 and OATP1B3 and the apparent substrate-dependent inhibitory effect of bortezomib on OATP1B3-mediated transport. The data suggest that bortezomib has a low risk of causing OATP-mediated DDIs.

MicroRNA 375 regulates proliferation and migration of colon cancer cells by suppressing the CTGF-EGFR signaling pathway.

MicroRNA 375 (MIR375) is significantly down regulated in human colorectal cancer (CRC) tissues; we have previously identified MIR375 as a colon cancer associated microRNA (miRNA). We identified putative MIR375 target genes by comparing the mRNA microarray analysis data of MIR375-overexpressing cells with the candidate MIR375 target genes predicted by public bioinformatic tools. We investigated that the connective tissue growth factor (CTGF) is a direct target gene of MIR375. Expression of CTGF, a ligand of epidermal growth factor receptor (EGFR), was markedly enhanced in human CRC tissues in comparison with the corresponding normal colon tissues. We demonstrated that the expression levels of molecules in EGFR signaling pathways were regulated by MIR375 in colorectal cells. Using immunohistochemistry and the xenograft of MIR375-overexpressing colorectal cells in mice, we showed that MIR375 regulates cell growth and proliferation, angiogenesis, cell migration, cell cycle arrest, apoptosis, and necrosis in colon cells. Furthermore, results of MIR375 overexpression and cetuximab treatment indicated that the apoptosis and necrosis in colon cells were synergistically enhanced. Our results suggest that the down-regulation of MIR375 modulates EGFR signaling pathways in human colorectal cells and tissues by increasing CTGF expression; therefore, MIR375 may have a therapeutic value in relation to human CRC.