Determining Plasma Tacrolimus Concentrations Using High-Performance LC-MS/MS in Renal Transplant Recipients.

BACKGROUND: Whole-blood therapeutic drug monitoring of tacrolimus is conducted to maintain tacrolimus concentrations within a safe and effective range. Changes in hematocrit cause variability in blood concentrations of tacrolimus because it is highly bound to erythrocytes. Measuring plasma concentrations may eliminate this variability; however, current methods have limitations owing to the use of cross-reactive immunoassays, plasma separation at nonbiological temperatures, and lack of clinical validation. This study aimed to develop and validate a clinically applicable method to measure plasma tacrolimus concentrations in renal transplant recipients and to examine the concentration differences between genotypic CYP3A5 expressors and nonexpressors. METHODS: Plasma tacrolimus concentrations were measured in 9 stable renal transplant recipients who were genotypic CYP3A5 expressors or nonexpressors. Tacrolimus was extracted from plasma using solid-phase extraction, and liquid chromatography-tandem mass spectrometry was used for detection and quantitation. RESULTS: This assay was sensitive, selective, and linear between 100 and 5000 ng/L, with intraassay and interassay imprecision and inaccuracy <10% and <5% respectively. The extraction recovery of tacrolimus and ascomycin was 74%. Matrix ion suppression effects were 31.5% and 35% with overall recovery of 50.6% and 48.3% for tacrolimus and ascomycin, respectively. Whole-blood concentrations accounted for approximately 46% of the variation in plasma concentrations in CYP3A5 expressors and nonexpressors. No difference in dose-adjusted whole-blood and plasma concentrations was observed between CYP3A5 expressors and nonexpressors. CONCLUSIONS: This assay is clinically applicable with excellent performance and demonstrated that tacrolimus plasma concentrations highly correlated with whole-blood concentrations.

Site-specific contribution of Toll-like receptor 4 to intestinal homeostasis and inflammatory disease.

Toll-like receptor 4 (TLR4) is a highly conserved protein of innate immunity, responsible for the regulation and maintenance of homeostasis, as well as immune recognition of external and internal ligands. TLR4 is expressed on a variety of cell types throughout the gastrointestinal tract, including on epithelial and immune cell populations. In a healthy state, epithelial cell expression of TLR4 greatly assists in homeostasis by shaping the host microbiome, promoting immunoglobulin A production, and regulating follicle-associated epithelium permeability. In contrast, immune cell expression of TLR4 in healthy states is primarily centred on the maturation of dendritic cells in response to stimuli, as well as adequately priming the adaptive immune system to fight infection and promote immune memory. Hence, in a healthy state, there is a clear distinction in the site-specific roles of TLR4 expression. Similarly, recent research has indicated the importance of site-specific TLR4 expression in inflammation and disease, particularly the impact of epithelial-specific TLR4 on disease progression. However, the majority of evidence still remains ambiguous for cell-specific observations, with many studies failing to provide the distinction of epithelial versus immune cell expression of TLR4, preventing specific mechanistic insight and greatly impacting the translation of results. The following review provides a critical overview of the current understanding of site-specific TLR4 activity and its contribution to intestinal/immune homeostasis and inflammatory diseases.

Tacrolimus dose, blood concentrations and acute nephrotoxicity, but not CYP3A5/ABCB1 genetics, are associated with allograft tacrolimus concentrations in renal transplant recipients.

AIMS: Long-term use of the immunosuppressant tacrolimus is limited by nephrotoxicity. Following renal transplantation, the risk of nephrotoxicity may be determined more by allograft than by blood tacrolimus concentrations, and thus may be affected by donor CYP3A5 and ABCB1 genetics. Little is known regarding factors that determine tacrolimus intrarenal exposure. METHODS: This study investigated the relationship between trough blood (C0Blood ) and allograft (CGraft ) tacrolimus concentrations and tacrolimus dose, haematocrit, genetics, acute nephrotoxicity, rejection status, delayed graft function, and time post-transplant. C0Blood and CGraft were quantified in 132 renal transplant recipients together with recipient and donor CYP3A5 (rs776746) and ABCB1 3435 (rs1045642) genotypes. RESULTS: C0Blood ranged from 2.6 to 52.3 ng/mL and CGraft from 33 to 828 pg/mg tissue. Adjusting for dose, recipients who were CYP3A5 expressors had lower C0Blood compared to nonexpressors, whilst delayed graft function was associated with higher C0Blood . Linear regression showed that the significant predictors of CGraft were C0Blood (point-wise P = 7 × 10-10 ), dose (P = .004) acute nephrotoxicity (P = .002) and an interaction between C0Blood and acute tacrolimus nephrotoxicity (P = .0002), with an adjusted r2  = 0.35 and no contribution from donor or recipient CYP3A5 or ABCB1 genotype. The association between CGraft and acute nephrotoxicity depended on one very high CGraft (828 pg/mg tissue). CONCLUSIONS: Recipient and donor CYP3A5 and ABCB1 3435C>T genotypes are not determinants of allograft tacrolimus exposure in kidney transplant recipients. However, tacrolimus dose and C0Blood were significant predictors of CGraft , and the relationship between C0Blood and CGraft appeared to differ in the presence or absence of acute nephrotoxicity.

The bidirectional interaction of the gut microbiome and the innate immune system: Implications for chemotherapy-induced gastrointestinal toxicity.

Chemotherapy-induced gastrointestinal toxicity (CIGT) occurs in up to 80% of all patients undergoing cancer treatment, and leads to symptoms such as diarrhoea, abdominal bleeding and pain. There is currently limited understanding of how to predict an individual patient's risk of CIGT. It is believed the gut microbiome and its interactions with the host's innate immune system plays a key role in the development of this toxicity and potentially other toxicities, however comprehensive bioinformatics modelling has not been rigorously performed. The innate immune system is strongly influenced by the microbial environment and vice-versa. Ways this may occur include the immune system controlling composition and compartmentalisation of the microbiome, the microbiome affecting development of antigen-presenting cells, and finally, the NLRP6 inflammasome orchestrating the colonic host-microbiome interface. This evidence calls into question the role of pre-treatment risk factors in the development of gastrointestinal toxicity after chemotherapy. This review aims to examine evidence of a bidirectional interaction between the gut microbiome and innate immunity, and how these interactions occur in CIGT. In the future, knowledge of these interactions may lead to improved personalised cancer medicine, predictive risk stratification methods and the development of targeted interventions to reduce, or even prevent, CIGT severity.

Effect of tacrolimus dispositional genetics on acute rejection in the first 2 weeks and estimated glomerular filtration rate in the first 3 months following kidney transplantation.

BACKGROUND: CYP3A4/5 and P-glycoprotein (P-gp, ABCB1) affect tacrolimus (TAC) exposure in T cells and kidney cells. Genetic variability of these genes has been widely studied for effects on acute rejection and kidney function after transplantation, but findings remain contradictory. In addition, cytochrome P450 reductase (POR) is important for CYP3A4/5 activity, and the pregnane X receptor (NR1I2) regulates CYP3A4/5 and P-gp expression. However, the relationship between POR and NR1I2 genetics and acute rejection and kidney function has not been extensively investigated. OBJECTIVE: The aim of this study was to investigate the effect of ABCB1 (61A>G, 1199G>A, 1236C>T, 2677G>T, 3435C>T), CYP3A4*22, CYP3A5*3, NR1I2 (8055C>T, 63396C>T) and POR*28 genotypes/haplotypes on acute rejection and kidney function in the first 3 months after transplant. PARTICIPANTS AND METHODS: The study included 165 kidney transplant recipients, who received TAC, mycophenolate and prednisolone, and 129 donors. TAC dose was adjusted to target trough blood concentrations of 8-15 ng/ml by therapeutic drug monitoring. Recipient and donor genotype/haplotype differences in acute rejection incidence within the first 2 weeks after transplant were assessed by logistic regression, adjusting for induction therapy, human leucocyte antigen mismatches, kidney transplant number, peak panel-reactive antibodies and donor type. Recipient and donor genotype/haplotype differences in estimated glomerular filtration rate in the first 3 months after transplant were assessed by linear mixed effects analysis, adjusting for acute rejection, delayed graft function and donor type. RESULTS: No genetic factors significantly affected acute rejection or estimated glomerular filtration rate after correction for multiple comparisons (P>0.004). CONCLUSION: Recipient and donor dispositional genetics had no significant effect on short-term clinical outcomes in kidney transplant patients receiving TAC therapeutic drug monitoring.

The impact of liver transplant recipient and donor genetic variability on tacrolimus exposure and transplant outcome.

This study investigated the effect of recipient and donor genetic variability on dose-adjusted steady-state tacrolimus concentrations (Css ) and clinical outcomes 3 and 6 months after liver transplant. Twenty-nine recipients and matched donor blood samples were genotyped for 27 single nucleotide polymorphisms including CYP3A5*3 (rs776746), ABCB1 haplotype and immune genes. Associations between genetic variability and clinical parameters and Css and the occurrence of rejection and nephrotoxicity were analysed by multivariate and multinomial logistic regression modelling and Jonckheere-Terpstra tests examined the impact of combined donor/recipient CYP3A5 expression on Css . At 3 months post-transplant modelling revealed an association between tacrolimus Css and recipient CASP1 rs580523 genotype (P = 0.005), accounting for 52% Css variance. Jonckheere-Terpstra tests revealed that as combined donor/recipient CYP3A5 expression increased, Css decreased (P = 0.010 [3 months], 0.018 [6 months]). As this is the first report of CASP1 genetic variability influencing tacrolimus Css , further validation in larger cohorts is required.

Relationship between allograft cyclosporin concentrations and P-glycoprotein expression in the 1st month following renal transplantation.

The immunosuppressant cyclosporin is a P-glycoprotein (P-gp) substrate whose impaired function has been associated with an increased risk of cyclosporin-induced nephrotoxicity following renal transplantation. This study investigated the relationship between blood and allograft cyclosporin concentration, and the effect of P-gp expression. Fifty biopsy samples were obtained from 39 renal transplant recipients who received cyclosporin as part of maintenance immunosuppression. Blood cyclosporin concentrations (2 hours postdose) were obtained from clinical records, matching allograft cyclosporin concentrations were measured in frozen biopsy tissue by liquid chromatography-tandem mass spectrometry, and allograft P-gp expression was assessed by immunohistochemistry. Blood and allograft cyclosporin concentrations in the 1st month post-transplantation ranged from 505-2005 µg/L and 0.01-16.7 ng/mg tissue, respectively. Dose was the only significant predictor of allograft cyclosporin concentrations (adjusted R2  = .24, F-statistic = 11.52, P = .0019), with no effect of P-gp expression or blood cyclosporin concentrations. P-gp expression is not the major determinant of allograft cyclosporin concentrations.

Is There a Temporal Relationship Between Trough Whole Blood Tacrolimus Concentration and Acute Rejection in the First 14 Days After Kidney Transplantation?

BACKGROUND: There are inconsistent findings regarding the relationship between trough whole blood tacrolimus concentration (TAC C0) and acute kidney rejection in recipients undergoing TAC therapeutic drug monitoring (TDM). However, studies have not always assessed TAC C0 at the time of rejection or accounted for variability in hematocrit. Therefore, this study aimed to investigate the temporal relationship between TAC C0 and acute rejection, including when accounting for variation in hematocrit. METHODS: For 38 recipients who developed biopsy-proven acute rejection (BPAR) in the first 14 days after kidney transplantation, daily TAC C0 from TDM and hematocrit was collected from case notes. Differences in log10-transformed TAC C0 between the day of BPAR (log Cr), 1 day before BPAR (log Cr-1), and 2 days before BPAR (log Cr-2) and the combined median concentrations for the days preceding these (log Cprior) were examined by repeated-measures analysis of variance with Dunnett post hoc testing. Generalized linear mixed-effects regression (glmer) examined the ability of TAC C0 to predict acute rejection episodes with and without controlling for hematocrit. RESULTS: Log Cr-1 [mean difference (95% confidence interval) = -0.13 (-0.21 to -0.048), post hoc P = 0.002] and log Cr [-0.13 (-0.24 to -0.025), post hoc P = 0.013] were significantly lower than log Cprior. TAC C0 was a significant (P = 0.0078) predictor of rejection episodes (area under the receiver operating characteristic curve = 0.79) only in glmer models accounting for variability in hematocrit. CONCLUSIONS: In recipients who developed BPAR, there was a significant temporal relationship between TAC C0 and BPAR incidence under TAC TDM that may not be detected in cross-sectional studies, especially if variability in hematocrit is not addressed. This supports a TAC C0-rejection relationship, which differs between recipients, and may explain why some recipients do or do not experience rejection within or below the TDM range, respectively. However, studies with larger sample sizes are needed to confirm this finding.

Systematic review of agents for the management of cancer treatment-related gastrointestinal mucositis and clinical practice guidelines.

PURPOSE: The aim of this study was to update the clinical practice guidelines for the use of agents for the prevention and/or treatment of gastrointestinal mucositis (GIM). METHODS: A systematic review was conducted by the Mucositis Study Group of the Multinational Association of Supportive Care in Cancer/International Society for Oral Oncology (MASCC/ISOO). The body of evidence for each intervention, in each cancer treatment setting, was assigned an evidence level. Based on the evidence level, one of the following three guideline determinations was possible: Recommendation, Suggestion, and No Guideline Possible. RESULTS: A total of 78 papers across 13 interventions were examined of which 25 were included in the final review. No new guidelines were possible for any agent due to inadequate and/or conflicting evidence. Existing guidelines for probiotics and hyperbaric oxygen were unchanged. CONCLUSIONS: Of the agents studied for the prevention and treatment of GIM, the evidence continues to support use of probiotics containing Lactobacillus spp. for prevention of chemoradiotherapy and radiotherapy-induced diarrhea in patients with pelvic malignancy, and hyperbaric oxygen therapy to treat radiation-induced proctitis. Additional well-designed research is encouraged to enable a decision regarding palifermin, glutamine, sodium butyrate, and dietary interventions, for the prevention or treatment of GIM.

Mycophenolic acid concentrations in peripheral blood mononuclear cells are associated with the incidence of rejection in renal transplant recipients.

AIMS: Although therapeutic drug monitoring of plasma mycophenolic acid (MPA) concentrations has been recommended to individualize dosage in transplant recipients, little is known regarding lymphocyte concentrations of MPA, where MPA inhibits inosine monophosphate dehydrogenase (IMPDH). This study investigated the utility of measuring predose MPA concentrations in peripheral blood mononuclear cells (C0C ) and predose IMPDH activity, as predictors of graft rejection in renal transplant recipients. METHODS: Forty-eight patients commencing mycophenolate mofetil (1 g twice daily) in combination with tacrolimus and prednisolone were recruited. Blood was collected for determination of trough total (C0P ) and unbound (C0u ) plasma MPA concentrations. Peripheral blood mononuclear cells were isolated for determination of C0C and IMPDH activity. The incidence of rejection within 2 days of sample collection was determined histologically and classified according to the Banff 2007 criteria. RESULTS: There was no association between MPA C0C and C0P (rs  = 0.28, P = 0.06), however, MPA C0C were weakly correlated with MPA C0u (rs  = 0.42, P = 0.013). Multivariate analysis indicated that MPA C0C was the only covariate independently associated with rejection (FDR-adjusted P = 0.033). The receiver operating characteristic area under the curve (AUC) for the prediction of severe rejection using MPA C0C was 0.75 (P = 0.013), with 73% sensitivity and specificity at a C0C threshold of 0.5 ng 10-7 cells. However, predose IMPDH activity was not a predictor of rejection (P > 0.15). CONCLUSIONS: MPA C0C measurement within the early post-transplant period may be useful to facilitate early titration of MPA dosing to significantly reduce rejection.

Chemotherapy-induced gut toxicity and pain: involvement of TLRs.

PURPOSE: Chemotherapy-induced gut toxicity is associated with significant pain, and pain influences gut function. Toll-like receptors (TLRs) that regulate gut homeostasis are activated by tissue damage and microbes, and their altered expression following chemotherapy may change cellular responses. This study examined the interaction between chemotherapy-induced gut toxicity and pain and related these to gut TLR and glial fibrillary acidic protein (GFAP) expression. METHODS: Female tumor bearing Dark Agouti rats received irinotecan (175 mg/kg, n = 34) or vehicle (n = 5) and were assessed over 120 h for gut toxicity (diarrhea, weight loss), pain (facial), and GFAP, TLR2, 4, 5, and 9 gut expression. RESULTS: Irinotecan caused diarrhea (72 % of animals grade ≥ 1), weight loss (11.1 ± 6.6 %, P < 0.0001), and pain (5 (0-5), P < 0.0001) all peaking at 72 h. Higher pain scores were observed in rats with diarrhea versus those without: median (range) of 2.0 (0-5) versus 0 (0-5), P = 0.01. Irinotecan also caused a decrease in TLR4 and 5, and an increase in GFAP expression in jejuna crypt at 96 and 120 h (all P < 0.05); with lower TLR4 expression associated with lower pain (P = 0.012). CONCLUSIONS: The association between gut toxicity and pain suggests these toxicities are linked, possibly via TLR-mediated inflammatory pathways. Further, as TLR4 and 5 expression was absent during recovery in the jejuna and GFAP expression was increased in the jejuna, this implies expression of these may be critical in the healing phase following chemotherapy. Detailed studies of gut TLRs and GFAP are now warranted.

Alcohol-induced sedation and synergistic interactions between alcohol and morphine: a key mechanistic role for Toll-like receptors and MyD88-dependent signaling.

Increasing evidence demonstrates induction of proinflammatory Toll-like receptor (TLR) 2 and TLR4 signaling by morphine and, TLR4 signaling by alcohol; thus indicating a common site of drug action and a potential novel innate immune-dependent hypothesis for opioid and alcohol drug interactions. Hence, the current study aimed to assess the role of TLR2, TLR4, MyD88 (as a critical TLR-signaling participant), NF-κB, Interleukin-1ß (IL-1ß; as a downstream proinflammatory effector molecule) and the µ opioid receptor (MOR; as a classical site for morphine action) in acute alcohol-induced sedation (4.5g/kg) and alcohol (2.5g/kg) interaction with morphine (5mg/kg) by assessing the loss of righting reflex (LORR) as a measure of sedation. Wild-type male Balb/c mice and matched genetically-deficient TLR2, TLR4, and MyD88 strains were utilized, together with pharmacological manipulation of MOR, NF-κB, TLR4 and Interleukin-1ß. Alcohol induced significant LORR in wild-type mice; this was halved by MyD88 and TLR4 deficiency, and surprisingly nearly completely eliminated by TLR2 deficiency. In contrast, the interaction between morphine and alcohol was found to be MOR-, NF-κB-, TLR2- and MyD88-dependent, but did not involve TLR4 or Interleukin-1ß. Morphine-alcohol interactions caused acute elevations in microglial cell counts and NF-κB-p65 positive cells in the motor cortex in concordance with wild-type and TLR2 deficient mouse behavioral data, implicating neuroimmunopharmacological signaling as a pivotal mechanism in this clinically problematic drug-drug interaction.

CYP2B6*6 allele and age substantially reduce steady-state ketamine clearance in chronic pain patients: impact on adverse effects.

AIMS: Ketamine analgesia is limited by low intrinsic efficacy compounded by large interindividual variability in drug responses, possibly due to the heterogeneity in drug concentration. The CYP2B6*6 allele is associated with substantially reduced ketamine metabolism in vitro and, therefore, may affect ketamine clearance. Our aims were to examine the impact of the CYP2B6*6 allele on ketamine plasma clearance and on adverse effects in chronic pain patients. METHODS: CYP2B6 genotypes were identified in 49 chronic pain patients who received 24 h continuous subcutaneous infusions of ketamine. Steady-state plasma concentrations of ketamine (Css,k ) and norketamine (Css,nk ) were determined using HPLC. RESULTS: The median plasma clearance of ketamine after 100 mg 24 h(-1) dose was significantly lower in patients with the CYP2B6*6/*6 (21.6 l h(-1) ) and CYP2B6*1/*6 (40.6 l h(-1) ) genotypes compared with patients with the CYP2B6*1/*1 genotype (68.1 l h(-1) , P < 0.001). The ketamine : norketamine plasma metabolic ratio was significantly higher in patients with the CYP2B6*6/*6 genotype than in those with the CYP2B6*1/*6 and the CYP2B6*1/*1 genotypes (P < 0.001). Patients who experienced adverse effects had lower plasma clearance (45.6 l h(-1) ) than those who did not (52.6 l h(-1) , P = 0.04). The CYP2B6*6 genotype and age, and their combined impact explained 40%, 30% and 60% of the variation in Css,k , respectively. Similar results were observed after higher doses. CONCLUSIONS: The CYP2B6*6 allele is associated with a substantial decrease in steady-state ketamine plasma clearance in chronic pain patients. The decreased clearance and resultant higher plasma concentrations may be associated with a higher incidence of ketamine adverse effects.

Predictive model for risk of severe gastrointestinal toxicity following chemotherapy using patient immune genetics and type of cancer: a pilot study.

PURPOSE: Severe chemotherapy-induced gastrointestinal toxicity (CIGT) is common and results in treatment delays, dose reductions, and potential premature treatment discontinuation. Currently, there is no diagnostic marker to predict CIGT. Proinflammatory cytokines, produced via toll-like receptor signaling, are key mediators of this toxicity. Hence, this pilot study investigated the association between immune genetic variability and severe CIGT risk. METHODS: Genomic DNA from 34 patients (10 with severe CIGT) who had received 5-fluoruracil-based chemotherapy regimens was analyzed for variants of IL-1B, IL-2, IL-6, IL-6R, IL-10, TNF-a, TGF-b, TLR2, TLR4, MD2, MYD88, BDNF, CRP, ICE, and OPRM1. Multivariate logistic regression created a prediction model of severe CIGT risk. RESULTS: There were no significant differences between patients with and without severe CIGT with regards to age, sex, type of cancer, or chemotherapy treatment regimens. The prediction model of severe CIGT risk included TLR2 and TNF-a genetic variability and cancer type (colorectal and gastric). This prediction model was both specific and sensitive, with a receiver operator characteristic area under the curve of 87.3 %. CONCLUSIONS: This is the first report of immune genetic variability, together with cancer type, being predictive of severe CIGT risk. These outcomes are being validated in a larger patient population.

Dietary interventions and tumor response to chemotherapy in breast cancer: A comprehensive review of preclinical and clinical data.

BACKGROUND & AIMS: Optimizing treatment efficacy is still a critical part in advancing the treatment of breast cancer. Dietary interventions have drawn significant attention for their potential to increase tumor sensitivity, with a plethora of strategies evaluated both preclinically and clinically. The aim of this paper is to explore these strategies, ranging from entire dietary programs to specific supplements, for their potential to directly enhance tumor sensitivity and chemotherapy adherence. METHODS: PubMed, Scopus, Embase and Web of Science databases were searched up to September 2023. In this comprehensive review, preclinical and clinical research on dietary interventions used in conjunction with chemotherapy for breast cancer was examined and synthesized, to identify potential causal mechanisms. RESULTS: 42 studies in total were identified and synthesized, 32 pre-clinical and 8 clinical studies. CONCLUSION: Although a topic of intense interest, the heterogeneity in approaches has resulted in a large but minimally impactful evidence base, further complicated by a limited understanding of the mechanisms at play. This review highlights the areas for further research to increase opportunities for nutritional-based interventions as adjuvant to chemotherapy for breast cancer.

NK1 tachykinin receptor antagonist treatment reduces cerebral edema and intracranial pressure in an ovine model of ischemic stroke.

Following ischemic stroke, substance P (SP)-mediated neurogenic inflammation is associated with profound blood-brain barrier (BBB) dysfunction, cerebral edema, and elevated intracranial pressure (ICP). SP elicits its effects by binding the neurokinin 1 tachykinin receptor (NK1-R), with administration of an NK1-R antagonist shown to ameliorate BBB dysfunction and cerebral edema in rodent and permanent ovine stroke models. Given the importance of reperfusion in clinical stroke, this study examined the efficacy of NK1-R antagonist treatment in reducing cerebral edema and ICP in an ovine model of transient middle cerebral artery occlusion (tMCAo). Anesthetized sheep (n = 24) were subject to 2-hours tMCAo and randomized (n = 6/group) to receive early NK1-R treatment (days 1-3 post-stroke), delayed NK1-R treatment (day 5 post-stroke), or saline vehicle. At 6-days post-stroke animals were re-anaesthetized and ICP measured, followed by MRI to evaluate infarction, edema and BBB dysfunction. Following both early and delayed NK1-R antagonist administration, ICP was significantly reduced on day 6 compared to vehicle animals (p < 0.05), accompanied by a reduction in cerebral edema, midline shift and BBB dysfunction (p < 0.05). This study demonstrates that NK1-R antagonist treatment is an effective novel therapy for cerebral edema and elevated ICP following stroke in an ovine model, warranting future clinical evaluation.

The CYP2B6*6 allele significantly alters the N-demethylation of ketamine enantiomers in vitro.

Ketamine is primarily metabolized to norketamine by hepatic CYP2B6 and CYP3A4-mediated N-demethylation. However, the relative contribution from each enzyme remains controversial. The CYP2B6*6 allele is associated with reduced enzyme expression and activity that may lead to interindividual variability in ketamine metabolism. We examined the N-demethylation of individual ketamine enantiomers using human liver microsomes (HLMs) genotyped for the CYP2B6*6 allele, insect cell-expressed recombinant CYP2B6 and CYP3A4 enzymes, and COS-1 cell-expressed recombinant CYP2B6.1 and CYP2B6.6 protein variant. Effects of CYP-selective inhibitors on norketamine formation were also determined in HLMs. The two-enzyme Michaelis-Menten model best fitted the HLM kinetic data. The Michaelis-Menten constants (K(m)) for the high-affinity enzyme and the low-affinity enzyme were similar to those for the expressed CYP2B6 and CYP3A4, respectively. The intrinsic clearance for both ketamine enantiomers by the high-affinity enzyme in HLMs with CYP2B6*1/*1 genotype were at least 2-fold and 6-fold higher, respectively, than those for CYP2B6*1/*6 genotype and CYP2B6*6/*6 genotype. The V(max) and K(m) values for CYP2B6.1 were approximately 160 and 70% of those for CYP2B6.6, respectively. N,N'N'-triethylenethiophosphoramide (thioTEPA) (CYP2B6 inhibitor, 25 µM) and the monoclonal antibody against CYP2B6 but not troleandomycin (CYP3A4 inhibitor, 25 µM) or the monoclonal antibody against CYP3A4 inhibited ketamine N-demethylation at clinically relevant concentrations. The degree of inhibition was significantly reduced in HLMs with the CYP2B6*6 allele (gene-dose P < 0.05). These results indicate a major role of CYP2B6 in ketamine N-demethylation in vitro and a significant impact of the CYP2B6*6 allele on enzyme-ketamine binding and catalytic activity.

Validation of an LC-MS/MS method to measure tacrolimus in rat kidney and liver tissue and its application to human kidney biopsies.

BACKGROUND: Tacrolimus (TAC) has a narrow therapeutic index and high interindividual and intraindividual pharmacokinetic variability, necessitating therapeutic drug monitoring to individualize dosage. Recent evidence suggests that intragraft TAC concentrations may better predict transplant outcomes. This study aimed to develop a method for the quantification of TAC in small biopsy-sized samples of rat kidney and liver tissue, which could be applied to clinical biopsy samples from kidney transplant recipients. METHODS: Kidneys and livers were harvested from Mrp2-deficient TR- Wistar rats administered TAC (4 mg·kg·d for 14 days, n = 8) or vehicle (n = 10). Tissue samples (0.20-1.00 mg of dry weight) were solubilized enzymatically and underwent liquid-liquid extraction before analysis by liquid chromatography tandem mass spectrometry method. TAC-free tissue was used in the calibrator and quality control samples. Analyte detection was accomplished using positive electrospray ionization (TAC: m/z 821.5 → 768.6; internal standard ascomycin m/z 809.3 → 756.4). RESULTS: Calibration curves (0.04-2.6 µg/L) were linear (R > 0.99, n = 10), with interday and intraday calibrator coefficients of variation and bias <17% at the lower limit of quantification and <15% at all other concentrations (n = 6-10). Extraction efficiencies for TAC and ascomycin were approximately 70%, and matrix effects were minimal. Rat kidney TAC concentrations were higher (range 109-190 pg/mg tissue) than those in the liver (range 22-53 pg/mg of tissue), with median tissue/blood concentrations ratios of 72.0 and 17.6, respectively. In 2 transplant patients, kidney TAC concentrations ranged from 119 to 285 pg/mg of tissue and were approximately 20 times higher than whole blood trough TAC concentrations. CONCLUSIONS: The method displayed precision and accuracy suitable for application to TAC measurement in human kidney biopsy tissue.

The Association between the Gut Microbiome and Development and Progression of Cancer Treatment Adverse Effects.

Adverse effects are a common consequence of cytotoxic cancer treatments. Over the last two decades there have been significant advances in exploring the relationship between the gut microbiome and these adverse effects. Changes in the gut microbiome were shown in multiple clinical studies to be associated with the development of acute gastrointestinal adverse effects, including diarrhoea and mucositis. However, more recent studies showed that changes in the gut microbiome may also be associated with the long-term development of psychoneurological changes, cancer cachexia, and fatigue. Therefore, the aim of this review was to examine the literature to identify potential contributions and associations of the gut microbiome with the wide range of adverse effects from cytotoxic cancer treatments.

Contribution of TLR4 to colorectal tumor microenvironment, etiology and prognosis.

PURPOSE: Toll-like receptor 4 (TLR4) is increasingly recognized for its ability to govern the etiology and prognostic outcomes of colorectal cancer (CRC) due to its profound immunomodulatory capacity. Despite widespread interest in TLR4 and CRC, no clear analysis of current literature and data exists. Therefore, translational advances have failed to move beyond conceptual ideas and suggestions. METHODS: We aimed to determine the relationship between TLR4 and CRC through a systematic review and analysis of published literature and datasets. Data were extracted from nine studies that reported survival, CRC staging and tumor progression data in relation to TLR4 expression. Primary and metastatic tumor samples with associated clinical data were identified through the Cancer Genome Atlas (TCGA) database. RESULTS: Systematic review identified heterogeneous relationships between TLR4 and CRC traits, with no clear theme evident across studies. A total of 448 datasets were identified through the TCGA database. Analysis of TCGA datasets revealed TLR4 mRNA expression is decreased in advanced CRC stages (P < 0.05 for normal vs Stage II, Stage III and Stage IV). Stage-dependent impact of TLR4 expression on survival outcomes were also found, with high TLR4 expression associated with poorer prognosis (stage I vs III (HR = 4.2, P = 0.008) and stage I vs IV (HR = 11.3, P < 0.001)). CONCLUSION: While TLR4 mRNA expression aligned with CRC staging, it appeared to heterogeneously regulate survival outcomes depending on the stage of disease. This underscores the complex relationship between TLR4 and CRC, with unique impacts dependent on disease stage.