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.

Randomized controlled trial of perhexiline on regression of left ventricular hypertrophy in patients with symptomatic hypertrophic cardiomyopathy (RESOLVE-HCM trial).

BACKGROUND: The presence and extent of left ventricular hypertrophy (LVH) is a major determinant of symptoms in patients with hypertrophic cardiomyopathy (HCM). There is increasing evidence to suggest that myocardial energetic impairment represents a central mechanism leading to LVH in HCM. There is currently a significant unmet need for disease-modifying therapy that regresses LVH in HCM patients. Perhexiline, a potent carnitine palmitoyl transferase-1 (CPT-1) inhibitor, improves myocardial energetics in HCM, and has the potential to reduce LVH in HCM. OBJECTIVE: The primary objective is to evaluate the effects of perhexiline treatment on the extent of LVH, in symptomatic HCM patients with at least moderate LVH. METHODS/DESIGN: RESOLVE-HCM is a prospective, multicenter double-blind placebo-controlled randomized trial enrolling symptomatic HCM patients with at least moderate LVH. Sixty patients will be randomized to receive either perhexiline or matching placebo. The primary endpoint is change in LVH, assessed utilizing cardiovascular magnetic resonance (CMR) imaging, after 12-months treatment with perhexiline. SUMMARY: RESOLVE-HCM will provide novel information on the utility of perhexiline in regression of LVH in symptomatic HCM patients. A positive result would lead to the design of a Phase 3 clinical trial addressing long-term effects of perhexiline on risk of heart failure and mortality in HCM patients.

Is there scope for better individualisation of anthracycline cancer chemotherapy?

Anthracyclines are used to treat solid and haematological cancers, particularly breast cancers, lymphomas and childhood cancers. Myelosuppression and cardiotoxicity are the primary toxicities that limit treatment duration and/or intensity. Cardiotoxicity, particularly heart failure, is a leading cause of morbidity and mortality in cancer survivors. Cumulative anthracycline dose is a significant predictor of cardiotoxicity risk, suggesting a role for anthracycline pharmacokinetic variability. Population pharmacokinetic modelling in children has shown that doxorubicin clearance in the very young is significantly lower than in older children, potentially contributing to their higher risk of cardiotoxicity. A model of doxorubicin clearance based on body surface area and age offers a patient-centred dose-adjustment strategy that may replace the current disparate initial-dose selection tools, providing a rational way to compensate for pharmacokinetic variability in children aged <7 years. Population pharmacokinetic models in adults have not adequately addressed older ages, obesity, hepatic and renal dysfunction, and potential drug-drug interactions to enable clinical application. Although candidate gene and genome-wide association studies have investigated relationships between genetic variability and anthracycline pharmacokinetics or clinical outcomes, there have been few clinically significant reproducible associations. Precision-dosing of anthracyclines is currently hindered by lack of clinically useful pharmacokinetic targets and models that predict cumulative anthracycline exposures. Combined with known risk factors for cardiotoxicity, the use of advanced echocardiography and biomarkers, future validated pharmacokinetic targets and predictive models could facilitate anthracycline precision dosing that truly maximises efficacy and provides individualised early intervention with cardioprotective therapies in patients at risk of cardiotoxicity.

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.

Effect of the proton-pump Inhibitor pantoprazole on MycoPhenolic ACid exposure in kidney and liver transplant recipienTs (IMPACT study): a randomized trial.

BACKGROUND: Mycophenolic acid (MPA) is widely utilized as an immunosuppressant in kidney and liver transplantation, with reports suggesting an independent relationship between MPA concentrations and adverse allograft outcome. Proton-pump inhibitors (PPIs) may have variable effects on the absorption of different MPA formulations leading to differences in MPA exposure. METHODS: A multicentre, randomized, prospective, double-blind placebo-controlled cross-over study was conducted to determine the effect of the PPI pantoprazole on the MPA and its metabolite MPA-glucuronide (MPA-G) area under the curve (AUC) >12 h (MPA-AUC12 h) in recipients maintained on mycophenolate mofetil (MMF) or enteric-coated mycophenolate sodium (EC-MPS). We planned a priori to examine separately recipients maintained on MMF and EC-MPS for each pharmacokinetic parameter. The trial (and protocol) was registered with the Australian New Zealand Clinical Trials Registry on 24 March 2011, with the registration number of ACTRN12611000316909 ('IMPACT' study). RESULTS: Of the 45 recipients screened, 40 (19 MMF and 21 EC-MPS) were randomized. The mean (standard deviation) recipient age was 58 (11) years with a median (interquartile range) time post-transplant of 43 (20-132) months. For recipients on MMF, there was a significant reduction in the MPA-AUC12 h [geometric mean (95% confidence interval) placebo: 53.9 (44.0-65.9) mg*h/L versus pantoprazole: 43.8 (35.6-53.4) mg*h/L; P = 0.004] when pantoprazole was co-administered compared with placebo. In contrast, co-administration with pantoprazole significantly increased MPA-AUC12 h [placebo: 36.1 (26.5-49.2) mg*h/L versus pantoprazole: 45.9 (35.5-59.3) mg*h/L; P = 0.023] in those receiving EC-MPS. Pantoprazole had no effect on the pharmacokinetic profiles of MPA-G for either group. CONCLUSIONS: The co-administration of pantoprazole substantially reduced the bioavailability of MPA in patients maintained on MMF and had the opposite effect in patients maintained on EC-MPS, and therefore, clinicians should be cognizant of this drug interaction when prescribing the different MPA formulations.

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.

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.

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.

Drugs that Affect Cardiac Metabolism: Focus on Perhexiline.

Approaches to the pharmacotherapy of angina pectoris have previously centred on the concept that a transient imbalance between myocardial oxygen "demand" and supply within the myocardium can best be addressed by reducing demand (for example, with ß-adrenoceptor antagonist) or by increasing availability of blood (via coronary vasomotor reactivity adjustment or coronary revascularization). However, this principle is potentially challenged by the emergence of cases of angina unsuitable for such therapies (for example because of concomitant severe systolic heart failure) and by the recognition that impaired myocardial energetics may precipitate angina in the absence of fixed or variable coronary obstruction (for example in hypertrophic cardiomyopathy). The past 20 years have seen the re-emergence of a class of anti-anginal agents which act primarily by improving efficiency of myocardial oxygen utilization, and thus can correct impaired energetics, simultaneously treating angina and heart failure symptoms. We review the principles underlying the safe use of such agents, beginning with the prototype drug perhexiline maleate, which despite complex pharmacokinetics and potential hepato- or neuro-toxicity has emerged as an attractive management option in many "complicated" cases of angina pectoris.

Validation of a High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Perhexiline and Cis-Hydroxy-Perhexiline Plasma Concentrations.

BACKGROUND: The polymorphic nature of cytochrome P450 2D6 has made therapeutic drug monitoring of the anti-anginal agent perhexiline a compulsory step in reducing adverse events associated with plasma concentrations above the therapeutic range (0.15-0.60 mg/L). The aim of this study was to develop a high-performance liquid chromatography-mass spectrometry/mass spectrometry method for the determination of plasma perhexiline concentrations and its major metabolite cis-hydroxy-perhexiline to reduce sample extraction procedures and improve sample turnaround times. METHODS: The method was validated by determining the precision and accuracy of calibrators and quality control material, comparing quality assurance program samples and patient samples measured by a previously reported liquid-liquid extraction fluorescence (FL) detection high-performance liquid chromatography method and performing matrix effects investigations. RESULTS: Replicates of calibrators at concentrations of 3.00 and 0.05 mg/L demonstrated imprecision of <10.8% and inaccuracy of <8.2% for perhexiline and <10.1% and <4.5% for cis-hydroxy-perhexiline, respectively. All samples measured by the 2 methods (n = 102) demonstrated Deming regression of perhexiline = 1.20 FL + 0.00 (Sy.x = 0.08, 1/slope = 0.67); cis-hydroxy-perhexiline = 1.48 FL - 0.20 (Sy.x = 0.40, 1/slope = 0.67). CONCLUSIONS: The assay performance was deemed acceptable and integrated into the routine therapeutic drug monitoring program of the department.

Stereoselective handling of perhexiline: implications regarding accumulation within the human myocardium.

PURPOSE: Perhexiline is a prophylactic anti-ischaemic agent with weak calcium antagonist effect which has been increasingly utilised in the management of refractory angina. The metabolic clearance of perhexiline is modulated by CYP2D6 metaboliser status and stereoselectivity. The current study sought to (1) determine whether the acute accumulation of perhexiline in the myocardium is stereoselective and (2) investigate the relationship between duration of short-term therapy and the potential stereoselective effects of perhexiline within myocardium. METHOD: Patients (n = 129) from the active arm of a randomised controlled trial of preoperative perhexiline in cardiac surgery were treated with oral perhexiline for a median of 9 days. Correlates of atrial and ventricular concentrations of enantiomers were sought via univariate followed by multivariate analyses. RESULTS: Myocardial uptake of both (+) and (-) perhexiline was greater in ventricles than in atria, and there was more rapid clearance of (-) than (+) perhexiline. The main determinants of atrial uptake of both (+) and (-) perhexiline were the plasma concentrations [(+) perhexiline: ß = -0.256, p = 0.015; (-) perhexiline: ß = -0.347, p = 0.001] and patients' age [(+) perhexiline: ß = 0.300, p = 0.004; (-) perhexiline: ß = 0.288, p = 0.005]. Atrial uptake of (+) enantiomer also varied directly with duration of therapy (ß = 0.228, p = 0.025), while atrial uptake of (-) perhexiline varied inversely with simultaneous heart rate (ß = -0.240, p = 0.015). CONCLUSION: (1) Uptake of both perhexiline enantiomers into atrium is greater with advanced age and displays evidence of both saturability and minor stereoselectivity. (2) Atrial uptake of (-) perhexiline may selectively modulate heart rate reduction.

Comparison of CYP2D metabolism and hepatotoxicity of the myocardial metabolic agent perhexiline in Sprague-Dawley and Dark Agouti rats.

1. Perhexiline, a chiral anti-anginal agent, may be useful to develop new cardiovascular therapies, despite its potential hepatotoxicity. 2. This study compared Dark Agouti (DA) and Sprague-Dawley (SD) rats, as models of perhexiline's metabolism and hepatotoxicity in humans. Rats (n = 4/group) received vehicle or 200 mg/kg/d of racemic perhexiline maleate for 8 weeks. Plasma and liver samples were collected to determine concentrations of perhexiline and its metabolites, hepatic function and histology. 3. Median (range) plasma and liver perhexiline concentrations in SD rats were 0.09 (0.04-0.13) mg/L and 5.42 (0.92-8.22) ng/mg, respectively. In comparison, DA rats showed higher (p < 0.05) plasma 0.50 (0.16-1.13) mg/L and liver 24.5 (9.40-54.7) ng/mg perhexiline concentrations, respectively, 2.5- and 3.7-fold higher cis-OH-perhexiline concentrations, respectively (p < 0.05), and lower plasma metabolic ratio (0.89 versus 1.55, p < 0.05). In both strains, the (+):(-) enantiomer ratio was 2:1. Perhexiline increased plasma LDH concentrations in DA rats (p < 0.05), but had no effect on plasma biochemistry in SD rats. Liver histology revealed lower glycogen content in perhexiline-treated SD rats (p < 0.05), but no effects on lipid content in either strain. 4. DA rats appeared more similar to humans with respect to plasma perhexiline concentrations, metabolic ratio, enantioselective disposition and biochemical changes suggestive of perhexiline-induced toxicity.

Relationship between plasma, atrial and ventricular perhexiline concentrations in humans: insights into factors affecting myocardial uptake.

AIM: Little is known regarding the steady-state uptake of drugs into the human myocardium. Perhexiline is a prophylactic anti-anginal drug which is increasingly also used in the treatment of heart failure and hypertrophic cardiomyopathy. We explored the relationship between plasma perhexiline concentrations and its uptake into the myocardium. METHODS: Blood, right atrium ± left ventricle biopsies were obtained from patients treated with perhexiline for a median of 8.5 days before undergoing coronary surgery in the perhexiline arm of a randomized controlled trial. Perhexiline concentrations in plasma and heart tissue were determined by HPLC. RESULTS: Atrial biopsies were obtained from 94 patients and ventricular biopsies from 28 patients. The median plasma perhexiline concentration was within the therapeutic range at 0.24 mg l⁻¹ (IQR 0.12-0.44), the median atrial concentration was 6.02 mg kg⁻¹ (IQR 2.70-9.06) and median ventricular concentration was 10.0 mg kg⁻¹ (IQR 5.76-13.1). Atrial (r² = 0.76) and ventricular (r² = 0.73) perhexiline concentrations were closely and directly correlated with plasma concentrations (both P < 0.001). The median atrial : plasma ratio was 21.5 (IQR 18.1-27.1), ventricular : plasma ratio was 34.9 (IQR 24.5-55.2) and ventricular : atrial ratio was 1.67 (IQR 1.39-2.22). Using multiple regression, the best model for predicting steady-state atrial concentration included plasma perhexiline, heart rate and age (r² = 0.83). Ventricular concentrations were directly correlated with plasma perhexiline concentration and length of therapy (r² = 0.84). CONCLUSIONS: This study demonstrates that plasma perhexiline concentrations are predictive of myocardial drug concentrations, a major determinant of drug effect. However, net myocardial perhexiline uptake is significantly modulated by patient age, potentially via alteration of myocardial:extracardiac drug uptake.

Interaction of terbinafine (anti-fungal agent) with perhexiline: a case report.

Perhexiline is a unique anti-anginal agent that is frequently used in the treatment of chronic refractory angina. Its utility has been limited because of its complex pharmacokinetics that were only appreciated following the development of a therapeutic perhexiline assay. Perhexiline is cleared primarily via formation of mono-hydroxy metabolites (OH-perhexiline) by cytochrome P450 2D6 (CYP2D6). Drugs that are inhibitors of CYP2D6 may therefore inhibit perhexiline metabolism, increase plasma perhexiline concentration and may consequently increase the risk of toxicity. We report a case of a rise in perhexiline plasma concentration to a toxic level following the introduction of terbinafine hydrochloride; a moderate CYP2D6 inhibiting drug.

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.

Proteomic characterisation of perhexiline treatment on THP-1 M1 macrophage differentiation.

Background: Dysregulated inflammation is important in the pathogenesis of many diseases including cancer, allergy, and autoimmunity. Macrophage activation and polarisation are commonly involved in the initiation, maintenance and resolution of inflammation. Perhexiline (PHX), an antianginal drug, has been suggested to modulate macrophage function, but the molecular effects of PHX on macrophages are unknown. In this study we investigated the effect of PHX treatment on macrophage activation and polarization and reveal the underlying proteomic changes induced. Methods: We used an established protocol to differentiate human THP-1 monocytes into M1 or M2 macrophages involving three distinct, sequential stages (priming, rest, and differentiation). We examined the effect of PHX treatment at each stage on the polarization into either M1 or M2 macrophages using flow cytometry, quantitative polymerase chain reaction (qPCR) and enzyme linked immunosorbent assay (ELISA). Quantitative changes in the proteome were investigated using data independent acquisition mass spectrometry (DIA MS). Results: PHX treatment promoted M1 macrophage polarization, including increased STAT1 and CCL2 expression and IL-1ß secretion. This effect occurred when PHX was added at the differentiation stage of the M1 cultures. Proteomic profiling of PHX treated M1 cultures identified changes in metabolic (fatty acid metabolism, cholesterol homeostasis and oxidative phosphorylation) and immune signalling (Receptor Tyrosine Kinase, Rho GTPase and interferon) pathways. Conclusion: This is the first study to report on the action of PHX on THP-1 macrophage polarization and the associated changes in the proteome of these cells.

Perhexiline Therapy in Patients with Type 2 Diabetes: Incremental Insulin Resistance despite Potentiation of Nitric Oxide Signaling.

Perhexiline (Px) inhibits carnitine palmitoyltransferase 1 (CPT1), which controls uptake of long chain fatty acids into mitochondria. However, occasional cases of hypoglycaemia have been reported in Px-treated patients, raising the possibility that Px may also increase sensitivity to insulin. Furthermore, Px increases anti-aggregatory responses to nitric oxide (NO), an effect which may theoretically parallel insulin sensitization. We therefore sought to examine these relationships in patients with stable Type 2 diabetes (T2D) and cardiovascular disease (n = 30). Px was initiated, and dosage was titrated, to reach the therapeutic range and thus prevent toxicity. Investigations were performed before and after 2 weeks, to examine changes in insulin sensitivity and, utilizing aggregometry in whole blood, platelet responsiveness to the anti-aggregatory effects of the NO donor sodium nitroprusside (SNP). Other parameters that affect may affect NO signalling were also evaluated. Px substantially potentiated inhibition of platelet aggregation by SNP (from 16.7 ± 3.0 to 27.3 ± 3.7%; p = 0.005). Px did not change fasting blood glucose concentrations but reduced insulin sensitivity (HOMA-IR score increased from median of 4.47 to 6.08; p = 0.028), and increased fasting plasma insulin concentrations (median 16.5 to 19.0 mU/L; p = 0.014). Increases in SNP responses tended (r = -0.30; p = 0.11) to be reciprocally related to increases in HOMA-IR, and increases in HOMA-IR were greater (p = 0.002) in patients without NO-sensitizing effects. No patient developed symptomatic hypoglycaemia, nor was there any other short-term toxicity of Px. Thus, in patients with stable T2D and cardiovascular disease, Px increases anti-aggregatory responsiveness to NO, but is not an insulin sensitizer, and does not induce hypoglycaemia. Absence of NO-sensitizing effect occurs in approximately 30% of Px-treated patients with T2D, and is associated with induction of insulin resistance in these patients.

The Antianginal Drug Perhexiline Displays Cytotoxicity against Colorectal Cancer Cells In Vitro: A Potential for Drug Repurposing.

Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. Perhexiline, a prophylactic anti-anginal drug, has been reported to have anti-tumour effects both in vitro and in vivo. Perhexiline as used clinically is a 50:50 racemic mixture ((R)-P) of (-) and (+) enantiomers. It is not known if the enantiomers differ in terms of their effects on cancer. In this study, we examined the cytotoxic capacity of perhexiline and its enantiomers ((-)-P and (+)-P) on CRC cell lines, grown as monolayers or spheroids, and patient-derived organoids. Treatment of CRC cell lines with (R)-P, (-)-P or (+)-P reduced cell viability, with IC50 values of ~4 µM. Treatment was associated with an increase in annexin V staining and caspase 3/7 activation, indicating apoptosis induction. Caspase 3/7 activation and loss of structural integrity were also observed in CRC cell lines grown as spheroids. Drug treatment at clinically relevant concentrations significantly reduced the viability of patient-derived CRC organoids. Given these in vitro findings, perhexiline, as a racemic mixture or its enantiomers, warrants further investigation as a repurposed drug for use in the management of CRC.

Effects of aging, renal dysfunction, left ventricular systolic impairment, and weight on steady state pharmacokinetics of perhexiline.

MATERIALS AND METHODS: Two hundred patients at steady-state on long-term perhexiline were identified retrospectively. The ratio of maintenance dose to steady-state plasma concentration (dose:[Px]) was correlated with the following putative determinants via simple and multiple linear regression analyses: age, weight, left ventricular ejection fraction (LVEF), and creatinine clearance (CrCl, Cockroft-Gault formula). A Mann-Whitney U test was performed to determine if severe left ventricular systolic impairment affected maintenance dose. RESULTS: Advanced age, left ventricular systolic impairment, and renal impairment were frequently encountered. Using simple linear regression, age was a negative correlate of dose:[P] (R = 0.23, P = 0.001), whereas weight (R = 0.27, P = 0.0001) and CrCl (R = 0.30, P < 0.0001) were positive correlates. Mann-Whitney U analysis showed no difference between dose: [Px] among patients with LVEF of less than 30% versus 30% or greater. Advancing age was strongly associated with decreasing weight (R = -0.45, P < 0.00001) and calculated CrCl varied directly with weight, as expected (R = 0.66, P < 0.0001). Stepwise multiple linear regression using age, LVEF, CrCl, and weight as potential predictors of dose:[P] yielded only weight as a significant determinant. DISCUSSION: Perhexiline has become a "last-line" agent for refractory angina as a result of complex pharmacokinetics and potential toxicity. Use has increased predictably in the aged and infirm who have exhausted standard medical and surgical therapeutic options. Beyond genotype, the effect of patient characteristics on maintenance dose has not been explored in detail. In this study, dose requirement declined with age in a frail and wasting population as a result of weight-related pharmacokinetic factors. LVEF had no apparent effect on maintenance dose and should not be considered a contraindication to use. CONCLUSION: A weight-adjusted starting dose may facilitate the safe and effective prescription of perhexiline and is calculated by 50 + 2 × weight (kg) mg/d, rounded to the closest 50 mg/day.