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Background: The empirical dietary inflammation pattern score (EDIP), which measures the ability of the diet to regulate chronic inflammation, is associated with both higher adiposity and breast cancer (BC) risk. Mammographic density (MD) is an important risk factor for BC., Objective: We examined the associations between EDIP and mammographic features overall and stratified by menopausal status, and assessed the extent to which these associations are mediated by adiposity., Methods: We included 4145 participants without BC in the Nurses' Health Study (NHS) and NHSII. Cumulative average EDIP was assessed by food frequency questionnaires every 4-6 y. We assessed MD parameters (percent MD, dense area, and nondense area) and V (measure of grayscale variation). MD parameters were square-root transformed. Multivariable-adjusted linear regression models were used to analyze the associations between EDIP score and MD parameters. Baron and Kenny's regression method was used to assess the extent to which the associations of EDIP and mammographic traits were mediated by BMI., Results: In multivariable-adjusted models, EDIP was significantly inversely associated with percent MD [top compared with bottom quartile, β = -0.57; 95% confidence interval (CI): -0.78, -0.36]. Additional adjustment for BMI attenuated the association (β = -0.15; 95% CI: -0.34, 0.03), with 68% (β = 0.68, 20; 95% CI: 0.54, 0.86) mediation via BMI. In addition, EDIP was positively associated with nondense area after adjusting for BMI and other covariates. No associations were observed for dense area and V measure. Results were similar when stratified by menopausal status., Conclusions: EDIP score was inversely associated with percent MD and positively associated with nondense area, and these associations were largely mediated by BMI., Competing Interests: Conflict of interest PS received salary from Merck & Co., Inc. However, this had no role in the study design, data collection, analyses, and data interoperation, in writing the paper or in the decision to submit the paper for publication. All other authors report no conflicts of interest., (Copyright © 2024 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

Background: Mucosal melanoma, an aggressive type of malignancy different from the cutaneous melanomas commonly seen in the head and neck region, represents < 1% of all malignant melanomas. The pathogenesis of mucosal melanoma is unknown. Targetable mutations commonly seen in cutaneous melanoma, such as in the BRAF and NRAS genes, have a lower incidence in mucosal melanoma. Mucosal melanoma carries a distinct mutational pattern from cutaneous melanoma. Surgery with negative margins is the first-line treatment for mucosal melanoma, and systemic therapy is not well defined. Talimogene laherparepvec, an oncolytic viral immunotherapy, is United States Food and Drug Administration approved for the treatment of advanced malignant cutaneous melanoma, with local therapeutic benefits. Mucosal melanoma was initially excluded from talimogene laherparepvec's initial phase III clinical trial., Case Presentation: We present the case of a white female patient in her 40s with past medical history of systemic lupus erythematous, scleroderma, and estrogen-receptor-positive invasive ductal breast carcinoma. Following a bilateral mastectomy, the patient was found to have BRAF-negative mucosal melanoma of her hard palate with a soft palate skip lesion. Owing to the presence of a skip mucosal lesion as well as the anticipated defect and need for free-flap reconstructive surgery, nonsurgical management was considered. The patient was referred to medical oncology, where-based on the patient's complicated medical history and the risk of immunotherapy possibly worsening her prior autoimmune diseases-local talimogene laherparepvec injections were chosen as the primary therapy for her mucosal lesions. Though talimogene laherparepvec is approved for the treatment of cutaneous melanoma, there are limited data available on the use of talimogene laherparepvec in mucosal melanomas., Conclusion: The patient had a complete local tumor response at both the primary lesion as well as the skip lesion with the local injections. She had no side effects and maintained a high quality of life during treatment., (© 2024. The Author(s).)

Guideline Title: Management of Immune-Related Adverse Events in Patients Treated with Immune Checkpoint Inhibitor Therapy: ASCO Guideline Update RELEASE DATE: November 1, 2021 PRIOR VERSION(S): February 14, 2018 DEVELOPER: American Society of Clinical Oncology FUNDING SOURCE: American Society of Clinical Oncology TARGET POPULATION: Adult patients with cancer receiving treatment with immune checkpoint blockade inhibitors alone., (© 2023 The Authors. Journal of Hospital Medicine published by Wiley Periodicals LLC on behalf of Society of Hospital Medicine.)

Chimeric Antigen Receptor-T (CAR-T) cell immunotherapy has produced dramatic responses in hematologic malignancies. One of the challenges in the field is the lack of a simple assay for the detection of CARs on the surface of immune effector cells. In this study, we describe a novel luciferase-based assay, termed Topanga Assay, for the detection of CAR expression. The assay utilizes a recombinant fusion protein, called Topanga reagent, generated by joining the extra-cellular domain of a CAR-target in frame with one of the marine luciferases or their engineered derivatives. The assay involves incubation of CAR expressing cells with the Topanga reagent, a few washes and measurement of luminescence. The assay can detect CARs comprising either immunoglobulin- or non-immunoglobulin-based antigen binding domains. We further demonstrate that addition of epitope tags to the Topanga reagent not only allows its convenient one step purification but also extends its use for detection of CAR cells using flow cytometry. However, crude supernatant containing the secreted Topanga reagent can be directly used in both luminescence and flow-cytometry based assays without prior protein purification. Our results demonstrate that the Topanga assay is a highly sensitive, specific, convenient, economical and versatile assay for the detection of CARs.

A simple, accurate, sensitive and robust assay that can rapidly and specifically measure the death of target cells would have applications in many areas of biomedicine and particularly for the development of novel cellular- and immune-therapeutics. In this study, we describe a novel cytotoxicity assay, termed the Matador assay, which takes advantage of the extreme brightness, stability and glow-like characteristics of recently discovered novel marine luciferases and their engineered derivatives. The assay involves expression of a luciferase of interest in target cells in a manner so that it is preferentially retained within the healthy cells but is either released from dead and dying cells or whose activity can be preferentially measured in dead and dying cells. We demonstrate that this assay is highly sensitive, specific, rapid, and can be performed in a single-step manner without the need for any expensive equipment. We further validate this assay by demonstrating its ability to detect cytotoxicity induced by several cellular and immune-therapeutic agents including antibodies, natural killer cells, chimeric antigen receptor expressing T cells and a bispecific T cell engager.

Objective: To assess the bioequivalence of vildagliptin/metformin fixeddose combination (FDC) tablets (50/250 mg and 50/500 mg) to free combinations of vildagliptin and metformin and the effect of food on the pharmacokinetics (PK) of vildagliptin and metformin following administration of 50/500 mg FDC tablets., Methods: Two openlabel, randomized, single-center, singledose, 2-period crossover studies were conducted in Japanese healthy male volunteers. Participants were administered vildagliptin/ metformin FDC tablets (study I: 50/250 mg, study II: 50/500 mg) or their free combinations under fasted condition. Food effect (standard Japanese breakfast: fat, 20 - 30% with ~ 600 kcal in total) was assessed during an additional period in study II (50/500 mg). PK parameters (AUC, C(max), t(max), t(1/2)) were calculated for vildagliptin and metformin., Results: In both studies, vildagliptin/metformin FDC tablets were bioequivalent to their respective free combinations. Administration of FDC tablets after meals had no effect on vildagliptin PK parameters. The rate of absorption of metformin decreased when administered under fed condition, as reflected by a prolonged t(max) (3 hours in fasted state vs. 4 hours in fed state) and decrease in C(max) by 26%, however, the extent of absorption (AUC(last)) was similar to that in the fasted state., Conclusions: Vildagliptin/metformin FDC tablets were bioequivalent to their free combinations. Food decreased the C(max) of metformin by 26%, while AUC(last) was unchanged, consistent with previous reports. No food effect was observed on the C(max) or AUC(last) of vildagliptin. Thus, food had no clinically relevant effects on the PK of metformin or vildagliptin.

Metformin is the first-line pharmacotherapy choice for treating type-2 diabetes mellitus, alone or in combination with other antidiabetic drugs. During the development of immediate-release (IR) metformin containing novel fixed-dose combination (FDC) products, several health-authorities require sponsors to demonstrate bioequivalence between FDC products and the country-sourced metformin for market approval. Eight bioequivalence studies that compared metformin/vildagliptin FDC product (test) to metformin IR tablet sourced from various countries (reference) were conducted. A population pharmacokinetic (PPK) analysis of pooled metformin concentration-time data was performed to evaluate whether country-sourced metformin is a significant covariate. The PPK analysis demonstrated that there was no clinically relevant effect of metformin source or race/ethnicity on metformin pharmacokinetics. Also, noncompartmental analysis conducted showed that 90%CI of geometric mean ratios of test to reference metformin formulations, calculated for maximum-concentration and exposure parameters, were within the 80%-125% criteria, indicating comparable metformin exposure regardless of the country-sourced metformin IR formulation. These results are consistent with the biopharmaceutics properties of metformin and provide scientific evidence that after assessing in vitro dissolution of novel FDC formulation, additional bioequivalence studies with multiple countries' reference products may not be required once bioequivalence is established with 1 country-sourced IR metformin formulation., (© 2015, The American College of Clinical Pharmacology.)

Background and Objectives: Dipeptidyl peptidase-4 (DPP4) inhibition is a potential strategy to increase the engraftment rate of haematopoietic stem/progenitor cells. A recent clinical trial using sitagliptin, a DPP4 inhibitor approved for type 2 diabetes mellitus, has been shown to be a promising approach in adults with haematological malignancies after umbilical cord blood (UCB) haematopoietic cell transplantation (HCT). On the basis of data from this clinical trial, a semi-mechanistic model was developed to simultaneously describe DPP4 activity after multiple doses of sitagliptin in subjects with haematological malignancies after a single-unit UCB HCT., Methods: The clinical study included 24 patients who received myeloablative conditioning followed by oral sitagliptin with single-unit UCB HCT. Using a nonlinear mixed-effects approach, a semi-mechanistic pharmacokinetic-pharmacodynamic model was developed to describe DPP4 activity from these trial data, using NONMEM version 7.2 software. The model was used to drive Monte Carlo simulations to probe the various dosage schedules and the attendant DPP4 response., Results: The disposition of sitagliptin in plasma was best described by a two-compartment model. The relationship between sitagliptin concentrations and DPP4 activity was best described by an indirect response model with a negative feedback loop. Simulations showed that twice daily or three times daily dosage schedules were superior to a once daily schedule for maximal DPP4 inhibition at the lowest sitagliptin exposure., Conclusion: This study provides the first pharmacokinetic-pharmacodynamic model of sitagliptin in the context of HCT, and provides a valuable tool for exploration of optimal dosing regimens, which are critical for improving the time to engraftment in patients after UCB HCT.

Background and Objective: Tacrolimus is an immunosuppressive drug used for the prevention of the allograft rejection in kidney transplant recipients. It exhibits a narrow therapeutic index and large pharmacokinetic variability. Tacrolimus is mainly metabolized by cytochrome P450 (CYP) 3A4 and 3A5 and effluxed via ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp), encoded by ABCB1 gene. The influence of CYP3A5*3 on the pharmacokinetics of tacrolimus has been well characterized. On the other hand, the contribution of polymorphisms in other genes is controversial. In addition, the involvement of other efflux transporters than P-gp in tacrolimus disposition is uncertain. The present study was designed to investigate the effects of genetic polymorphisms of CYP3As and efflux transporters on the pharmacokinetics of tacrolimus., Subjects and Methods: A total of 500 blood concentrations of tacrolimus from 102 adult stable kidney transplant recipients were included in the analyses. Genetic polymorphisms in CYP3A4 and CYP3A5 genes were determined. In addition, the genes of efflux transporters including P-gp (ABCB1), multidrug resistance-associated protein (MRP2/ABCC2) and breast cancer resistance protein (BCRP/ABCG2) were genotyped. For ABCC2 gene, haplotypes were determined as follows: H1 (wild type), H2 (1249G>A), H9 (3972C>T) and H12 (-24C>T and 3972C>T). Population pharmacokinetic analysis was performed using nonlinear mixed effects modeling., Results: Analyses revealed that the CYP3A5 expressers (CYP3A5*1 carriers) and MRP2 high-activity group (ABCC2 H2/H2 and H1/H2) showed a decreased dose-normalized trough concentration of tacrolimus by 2.3-fold (p < 0.001) and 1.5-fold (p = 0.007), respectively. The pharmacokinetics of tacrolimus were best described using a two-compartment model with first order absorption and an absorption lag time. In the population pharmacokinetic analysis, CYP3A5 expressers and MRP2 high-activity groups were identified as the significant covariates for tacrolimus apparent clearance expressed as 20.7 × (age/50)(-0.78) × 2.03 (CYP3A5 expressers) × 1.40 (MRP2 high-activity group). No other CYP3A4, ABCB1 or ABCG2 polymorphisms were associated with the apparent clearance of tacrolimus., Conclusions: This is the first report showing that MRP2/ABCC2 has a crucial impact on the pharmacokinetics of tacrolimus in a haplotype-specific manner. Determination of the ABCC2 as well as CYP3A5 genotype may be useful for more accurate tacrolimus dosage adjustment.

1. Disposition of tacrolimus and its major metabolites, 13-O-desmethyl tacrolimus and 15-O-desmethyl tacrolimus, was evaluated in stable kidney transplant recipients in relation to diabetes mellitus and genetic polymorphism of cytochrome P450 (CYP) 3A. 2. Steady-state concentration-time profiles were obtained for 12-hour or 2-hour post-dose, in 20 (11 with diabetes) and 32 (24 with diabetes) patients, respectively. In addition, single nucleotide polymorphisms of the following genes: CYP3A4 (CYP3A4: CYP3A4*1B, -392A > G), 3A5 (CYP3A5: CYP3A5*3, 6986A > G) and P-glycoprotein (ABCB1: 3435C > T) were characterized. 3. Dose-normalized concentrations of tacrolimus or metabolites were higher in diabetic patients. CYP3A4*1B carriers and CYP3A5 expressers, independently or when assessed as a combined CYP3A4-3A5 genotype, had significantly lower dose-normalized pre-dose (C0/dose) and 2-hour post-dose (C2/dose) concentrations of tacrolimus and metabolites. Non-diabetic patients with at least one CYP3A4*1B and CYP3A5*1 allele had lower C0/dose as compared to the rest of the population. 4. Genetic polymorphism of CYP3A5 or CYP3A4 influence tacrolimus or metabolites dose-normalized concentrations but not metabolite to parent concentration ratios. The effect of diabetes on tacrolimus metabolism is subject to debate and requires a larger sample size of genetically stratified subjects.

Midazolam is an ultra short acting benzodiazepine derivative and a specific probe for phenotyping cytochrome P450 (P450) 3A4/5 activity. A rapid, sensitive, and selective LC-MS/MS method was developed for simultaneous quantitation of midazolam and its metabolites (1'-hydroxymidazolam and 4-hydroxymidazolam). Deuterated (D5) analog of midazolam was utilized as an internal standard. Sample preparation either from human plasma (100 microL) or liver microsomal incubations involved a simple protein precipitation using acetonitrile (900 microL) with an average recovery of >90% for all compounds. The chromatographic separation was achieved using Zorbax-SB Phenyl, Rapid Resolution HT (2.1 mm x 100 mm, 3.5 microm) and a gradient elution with 10 mM ammonium acetate in 10% methanol (A) and acetonitrile (B). The flow rate was 0.25 mL/min and total run time was 5.5 min. Calibration curves were linear over the concentration range of 0.100-250 ng/mL. The lower limit of quantitation (LLOQ) was 0.1 ng/mL for all three analytes. The accuracy and precision, estimated at LLOQ and three concentration levels of quality control samples in six replicates, were within 85-115%. In conclusion, a robust, simple and highly sensitive analytical method was developed and validated for the analysis of midazolam and its metabolites. This method is suitable for characterizing the P450 3A4/5 activity in vitro or in human pharmacokinetic studies allowing administration of smaller doses of midazolam., (Copyright 2010 Elsevier B.V. All rights reserved.)

Iodixanol is a widely used iso-osmolar contrast medium agent. Similar to iohexol, it can also be a good exogenous marker for the measurement of glomerular filtration rate (GFR). This article describes the development and validation of an HPLC-UV method for quantification of iodixanol in human plasma. Internal standard, iohexol (20 microl, 1 mg/ml), and perchloric acid (30 microl, 20%, v/v) were added to plasma samples (300 microl), followed by neutralization with 10 microl potassium carbonate (5M). Samples were centrifuged and 10 microl of the supernatant was injected onto a C(18) EPS analytical column (3 microm particle size, 150 mm x 4.6 mm). The extraction method yielded >95% recovery for both iodixanol and iohexol. The mobile phase consisted of 0.1% (w/v) sodium formate buffer and acetonitrile. Iohexol and iodixanol peaks were eluted at approximately 5 and 9 min, respectively using a fast gradient method. The assay lower limit of detection was 2.0 microg/ml and lower limit of quantification was 10 microg/ml. The calibration curves, assessed in six replicates, were linear over an iodixanol concentration range of 10-750 microg/ml. Intra- and inter-day accuracy was >95% and precision expressed as % coefficient of variation was <10%. This method is simple, accurate, precise and robust and can potentially be used for iodixanol quantification in large-scale clinical studies.