In vitro co-culture systems of hepatic and intestinal cells for cellular pharmacokinetic and pharmacodynamic studies of capecitabine against colorectal cancer.

BACKGROUND: As a prodrug of 5-fluorouracil (5-FU), orally administrated capecitabine (CAP) undergoes preliminary conversion into active metabolites in the liver and then releases 5-FU in the gut to exert the anti-tumor activity. Since metabolic changes of CAP play a key role in its activation, a single kind of intestinal or hepatic cell can never be used in vitro to evaluate the pharmacokinetics (PK) and pharmacodynamics (PD) nature. Hence, we aimed to establish a novel in vitro system to effectively assess the PK and PD of these kinds of prodrugs. METHODS: Co-culture cellular models were established by simultaneously using colorectal cancer (CRC) and hepatocarcinoma cell lines in one system. Cell Counting Kit-8 (CCK-8) and flow cytometric analysis were used to evaluate cell viability and apoptosis, respectively. Apoptosis-related protein expression levels were measured using western blot analysis. A selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for cellular PK in co-culture models. RESULTS: CAP had little anti-proliferative effect on the five monolayer CRC cell lines (SW480, LoVo, HCT-8, HCT-116 and SW620) or the hepatocarcinoma cell line (HepG2). However, CAP exerted marked anti-tumor activities on each of the CRC cell lines in the co-culture models containing both CRC and hepatocarcinoma cell lines, although its effect on the five CRC cell lines varied. Moreover, after pre-incubation of CAP with HepG2 cells, the culture media containing the active metabolites of CAP also showed an anti-tumor effect on the five CRC cell lines, indicating the crucial role of hepatic cells in the activation of CAP. CONCLUSION: The simple and cost­effective co-culture models with both CRC and hepatocarcinoma cells could mimic the in vivo process of a prodrug dependent on metabolic conversion to active metabolites in the liver, providing a valuable strategy for evaluating the PK and PD characteristics of CAP-like prodrugs in vitro at the early stage of drug development.

The IMBG Test for Evaluating the Pharmacodynamic Response to Immunosuppressive Therapy in Kidney Transplant Patients: Current Evidence and Future Applications.

Immunosuppressive drugs are widely used to prevent rejection after kidney transplantation. However, the pharmacological response to a given immunosuppressant can vary markedly between individuals, with some showing poor treatment responses and/or experiencing serious side effects. There is an unmet need for diagnostic tools that allow clinicians to individually tailor immunosuppressive therapy to a patient's immunological profile. The Immunobiogram (IMBG) is a novel blood-based in vitro diagnostic test that provides a pharmacodynamic readout of the immune response of individual patients to a range of immunosuppressants commonly used in kidney transplant recipients. Here, we discuss the current approaches used to measure the pharmacodynamic responses of individual patients to specific immunosuppressive drugs in vitro, which can then be correlated with patient's clinical outcomes. We also describe the procedure of the IMBG assay, and summarize the results obtained using the IMBG in different kidney transplant populations. Finally, we outline future directions and other novel applications of the IMBG, both in kidney transplant patients and other autoimmune diseases.

Chemotherapeutic dosing implicated by pharmacodynamic modeling of in vitro cytotoxic data: a case study of paclitaxel.

Conventional maximum tolerated doses (MTD) in chemotherapy are recently challenged by an alternative dosing method with low doses and high dosing frequency (LDHF). Still, it remains unclear which chemotherapies would potentially benefit from LDHF. The pharmacokinetic (PK) differences between MTD and LDHF are drug exposure magnitude (concentration) and exposure duration (time), two fundamental PK elements that are associated with the pharmacodynamics (PD) of chemotherapies. Here we hypothesized that quantitatively analyzing the contribution of each PK element to the overall cytotoxic effects would provide insights to the selection of the preferred chemotherapeutic dosing. Based on in vitro cytotoxic data, we developed a cellular PD model, which assumed that tumor cells were generally comprised of two subpopulations that were susceptible to either concentration- or time-dependent cytotoxicity. The developed PD model exhibited high flexibility to describe diverse patterns of cell survival curves. Integrated with a PK model, the cellular PD model was further extended to predict and compare the anti-tumor effect of paclitaxel in two dosing regimens: multiple MTD bolus and continuous constant infusion (an extreme LDHF). Our simulations of paclitaxel in treatment of three types of cancers were consistent with clinical observations that LDHF yielded higher patient efficacy than MTD. Our further analysis suggested that the ratio between drug steady-state concentrations and its cytotoxic sensitivity (C ss /KC 50 ) was a critical factor that largely determines favored dosing regimen. LDHF would produce higher efficacy when the ratio C ss /KC 50 is greater than 1. Otherwise MTD was favored. The developed PD model presented an approach simply based on in vitro cytotoxic data to predict the potentially favored chemotherapeutic dosing between MTD and LDHF.

The additive damage model: a mathematical model for cellular responses to drug combinations.

Mathematical models to describe dose-dependent cellular responses to drug combinations are an essential component of computational simulations for predicting therapeutic responses. Here, a new model, the additive damage model, is introduced and tested in cases where varying concentrations of two drugs are applied with a fixed exposure schedule. In the model, cell survival is determined by whether cellular damage, which depends on the concentrations of the drugs, exceeds a lethal threshold, which varies randomly in the cell population with a prescribed statistical distribution. Cellular damage is assumed to be additive, and is expressed as a sum of separate terms for each drug. Each term has a saturable dependence on drug concentration. The model has appropriate behavior over the entire range of drug concentrations, and is predictive, given single-agent dose-response data for each drug. The proposed model is compared with several other models, by testing their ability to fit 24 data sets for platinum-taxane combinations and 21 data sets for various other combinations. The Akaike Information Criterion is used to assess goodness of fit, taking into account the number of unknown parameters in each model. Overall, the additive damage model provides a better fit to the data sets than any previous model. The proposed model provides a basis for computational simulations of therapeutic responses. It predicts responses to drug combinations based on data for each drug acting as a single agent, and can be used as an improved null reference model for assessing synergy in the action of drug combinations.

The Immunobiogram, a novel in vitro diagnostic test to measure the pharmacodynamic response to immunosuppressive therapy in kidney transplant patients.

BACKGROUND: Diagnostic tools to measure the response to individual immunosuppressive drugs for transplant patients are currently lacking. We previously developed the blood-based Immunobiogram bioassay for in-vitro characterization of the pharmacodynamic response of patients' own immune cells to a range of immunosuppressants. We used Immunobiogram to examine the association between patients' sensitivity to their prescribed immunosuppressants and clinical outcome. METHODS: We conducted an international, multicenter, observational study in a kidney transplant population undergoing maintenance immunosuppressive therapy. Patients were selected by clinical course poor [PCC] N = 53 (with renal dysfunction, and rejection signs in biopsy or/and an increase in DSA strength in last 12 months) versus good [GCC] N = 50 (with stable renal function and treatment, no rejection and no DSA titers). Immunobiogram dose-response curve parameters were compared between both subgroups in patients treated with mycophenolate, tacrolimus, corticosteroids, cyclosporine A or everolimus. Parameters for which significant inter-group differences were observed were further analyzed by univariate and subsequent multivariate logistic regression. RESULTS: Clinical outcome was associated with following parameters: area over the curve (AOC) and 25% (ID25) and 50% (ID50) inhibitory response in mycophenolate, tacrolimus, and corticosteroid-treated subgroups, respectively. These statistically significant associations persisted in mycophenolate (OR 0.003, CI95% <0.001-0.258; p = 0.01) and tacrolimus (OR < 0.0001, CI95% <0.00001-0.202; p = 0.016) subgroups after adjusting for concomitant corticosteroid treatment, and in corticosteroid subgroup after adjusting for concomitant mycophenolate or tacrolimus treatment (OR 0.003; CI95% <0.0001-0.499; p = 0.026). CONCLUSIONS: Our results highlight the potential of Immunobiogram as a tool to test the pharmacodynamic response to individual immunosuppressive drugs.

The Immunobiogram, a Novel In Vitro Assay to Evaluate Treatment Resistance in Patients Receiving Immunosuppressive Therapy.

Immunosuppressive drugs are widely used to treat several autoimmune disorders and prevent rejection after organ transplantation. However, intra-individual variations in the pharmacological response to immunosuppressive therapy critically influence its efficacy, often resulting in poor treatment responses and serious side effects. Effective diagnostic tools that help clinicians to tailor immunosuppressive therapy to the needs and immunological profile of the individual patient thus constitute a major unmet clinical need. In vitro assays that measure immune cell responses to immunosuppressive drugs constitute a promising approach to individualized immunosuppressive therapy. Here, we present the Immunobiogram, a functional pharmacodynamic immune cell-based assay for simultaneous quantitative measurement of a patient's immune response to a battery of immunosuppressive drugs. Peripheral blood mononuclear cells collected from patients are immunologically stimulated to induce activation and proliferation and embedded in a hydrogel mixture in which they are exposed to a concentration gradient of the immunosuppressants of interest. Analysis of samples from kidney transplant patients using this procedure revealed an association between the sensitivity of individual patients to the immunosuppressive regimen and their immunological risk of transplant rejection. Incorporation of the Immunobiogram assay into clinical settings could greatly facilitate personalized optimization and monitoring of immunosuppressive therapy, and study of the mechanisms underlying resistance to immunosuppressants.