Michael Lobb

Colonic microbiota can promote rapid local improvement of murine colitis by thioguanine independently of T lymphocytes and host metabolism.

Objective Mercaptopurine (MP) and pro-drug azathioprine are ‘first-line’ oral therapies for maintaining remission in IBD. It is believed that their pharmacodynamic action is due to a slow cumulative decrease in activated lymphocytes homing to inflamed gut. We examined the role of host metabolism, lymphocytes and microbiome for the amelioration of colitis by the related thioguanine (TG). Design C57Bl/6 mice with or without specific genes altered to elucidate mechanisms responsible for TGs actions were treated daily with oral or intrarectal TG, MP or water. Disease activity was scored daily. At sacrifice, colonic histology, cytokine message, caecal luminal and mucosal microbiomes were analysed. Results Oral and intrarectal TG but not MP rapidly ameliorated spontaneous chronic colitis in Winnie mice (point mutation in Muc2 secretory mucin). TG ameliorated dextran sodium sulfate-induced chronic colitis in wild-type (WT) mice and in mice lacking T and B lymphocytes. Remarkably, colitis improved without immunosuppressive effects in the absence of host hypoxanthine (guanine) phosphoribosyltransferase (Hprt)-mediated conversion of TG to active drug, the thioguanine nucleotides (TGN). Colonic bacteria converted TG and less so MP to TGN, consistent with intestinal bacterial conversion of TG to so reduce inflammation in the mice lacking host Hprt. TG rapidly induced autophagic flux in epithelial, macrophage and WT but not Hprt−/− fibroblast cell lines and augmented epithelial intracellular bacterial killing. Conclusions Treatment by TG is not necessarily dependent on the adaptive immune system. TG is a more efficacious treatment than MP in Winnie spontaneous colitis. Rapid local bacterial conversion of TG correlated with decreased intestinal inflammation and immune activation.

Development, validation and application of an HPLC–MS/MS method for the determination of fentanyl and nor-fentanyl in human plasma and saliva

Monitoring fentanyl concentration in saliva and plasma may be useful in pharmacokinetic/pharmacodynamic studies. Salivettes(®) have been used widely for collecting saliva samples. However due to its lipophilicity, fentanyl adsorbs to the cotton dental bud (CDB) used in this device. Furthermore, due to dry mouth being a common adverse effect seen in patients treated with opioids, obtaining enough saliva for analysis is often a challenge. Hence, a simple simultaneous method to quantify fentanyl and its metabolite in both human plasma and saliva was developed and validated. A novel extraction method was also developed and validated to recover fentanyl in saliva directly from the CDB. This extraction method utilises acetonitrile to recover the fentanyl directly from the CDB rather than recovery by centrifugation, which is not always possible. Reverse phase chromatographic separation was performed on a Shimadzu LC 20A HPLC system using gradient elution. The electrospray ion source (ESI) was operated in positive ion mode using an Applied Biosystems API 3200 LC/MS/MS as detector. Deuterated fentanyl (D5) and nor-fentanyl (D5) were used as internal standards (IS). The retention times for fentanyl and nor-fentanyl were 3.70 min and 3.20 min respectively. The lower limit of quantitation (LLOQ) was determined to be 0.030 μg/L in plasma and 0.045 in saliva for fentanyl and nor-fentanyl. Acceptable linearity for fentanyl and nor-fentanyl in both plasma and saliva was demonstrated from 0.02 to 10 μg/L (R(2) 0.9988-0.9994). Accuracy for fentanyl and nor-fentanyl in both plasma and saliva samples was between 96% and 108%. Total imprecision expressed as the co-efficient of variation was between 1.0 and 15.5% for both analytes in both matrices. The validated method was applied successfully in 11 paired plasma and saliva samples obtained from patients with cancer pain receiving transdermal fentanyl (Duragesic(®)) at doses from 25 μg to 100 μg.

Saliva versus Plasma for Pharmacokinetic and Pharmacodynamic Studies of Fentanyl in Patients with Cancer

PURPOSE Fentanyl is widely used to relieve cancer pain. However there is great interpatient variation in the dose required to relieve pain and little knowledge about the pharmacokinetic and pharmacodynamic (PK/PD) relationship of fentanyl and pain control. Patients with cancer are fragile and there is reluctance on the part of health professionals to take multiple plasma samples for PK/PD studies. The relationship between plasma and saliva fentanyl concentrations was investigated to determine whether saliva could be a valid substitute for plasma in PK/PD studies. METHODS One hundred sixty-three paired plasma and saliva samples were collected from 56 patients prescribed transdermal fentanyl (Durogesic, Janssen-Cilag Pty Limited, NSW, Australia) at varying doses (12-200 µg/h). Pain scores were recorded at the time of sampling. Fentanyl and norfentanyl concentrations in plasma and saliva were quantified using HPLC-MS/MS. FINDINGS Saliva concentrations of fentanyl (mean = 4.84 μg/L) were much higher than paired plasma concentrations of fentanyl (mean = 0.877 μg/L). Both plasma and saliva mean concentrations of fentanyl were well correlated with dose with considerable interpatient variation at each dose. The relationship between fentanyl and norfentanyl concentrations was poor in both plasma and saliva. No correlation was observed between fentanyl concentration in plasma and saliva (r(2) = 0.3743) or free fentanyl in plasma and total saliva concentrations (r(2) = 0.1374). Pain scores and fentanyl concentration in either of the matrices were also not correlated. IMPLICATIONS No predictive correlation was observed between plasma and saliva fentanyl concentration. However the detection of higher fentanyl concentrations in saliva than plasma, with a good correlation to dose, may allow saliva to be used as an alternative to plasma in PK/PD studies of fentanyl in patients with cancer.

Quantitative determination of the enantiomers of methadone in human plasma and saliva by chiral column chromatography coupled with mass spectrometric detection.

Methadone is a potent lipophilic synthetic opioid that is effective in the treatment of cancer pain and perceived benefit in difficult pain control scenarios (especially in cases of neuropathic pain). The use of methadone in clinical practice is challenging however, due to the narrow therapeutic window and large inter- and intra-individual variability in therapeutic response. Quantitation of the enantiomers d- and l-methadone (d- and l-MTD) in plasma and saliva provides a basis for studying its pharmacokinetics in patients with cancer and for monitoring efficacy, toxicity and side-effects. This assay involves quantitation of the enantiomers of methadone using their respective deuterated internal standards, in plasma and saliva matrices with no impact of ion suppression in either matrix. The analytical recoveries of d- and l-MTD from the saliva collection devices (Salivette®) are optimised in this novel method with an accurate and simple extraction method employing dichloromethane. Optimal enantioselective separations were achieved using an α1-acid glycoprotein chiral stationary phase and triple quadrupole tandem mass spectrometer. Linearity was demonstrated over 0.05-1000µg/L for both enantiomers in plasma and in saliva with correlation coefficients greater than 0.998. The lower limit of quantitation (LLOQ) was determined to be 0.1µg/L in plasma and saliva for d- and l-MTD. Accuracy of the method ranges from 100% to 106% even at the LLOQ and total precision, expressed as the coefficient of variation, was between 0.2% and 4.4% for both analytes in both matrices. A simple one step extraction procedure resulted in recoveries greater than 95% for both analytes, at concentrations as low as 0.5µg/L, from the Salivette®. The validated method was applied successfully in 14 paired plasma and saliva samples obtained from adult patients with cancer pain receiving methadone.

Protein binding of fentanyl and its metabolite nor-fentanyl in human plasma, albumin and α-1 acid glycoprotein

Abstract 1. Fentanyl is a highly lipophilic opioid commonly used to treat cancer pain. Plasma protein binding (PPB) of fentanyl in human plasma is reported as 80–85%, however it is unclear whether fentanyl binds primarily to albumin (ALB) or α-1 acid glycoprotein (AAG) and no studies have been conducted on the metabolite, nor-fentanyl. Fentanyl is also known to bind to plasticware and ultrafiltration (UF) devices which impacts adversely on binding experiments. 2. PPB of fentanyl and nor-fentanyl to ALB and AAG in isotonic phosphate buffer solution and seeded human plasma was quantified. PPB was also performed in plasma samples obtained from cancer patients receiving transdermal fentanyl. The adsorption of fentanyl and nor-fentanyl to UF devices and plasticware commonly used in PPB studies was also assessed. 3. Fentanyl was shown to bind primarily to ALB as opposed to AAG, with nor-fentanyl exhibiting negligible binding to plasma proteins. Total PPB of fentanyl was 86–89% in seeded human plasma. PPB in 56 cancer patient samples was 95.1 ± 3.52% for fentanyl and 32.4 ± 21.9% for nor-fentanyl. 4. UF was shown to be a reliable and convenient method for PPB studies, thereby removing the need for complex testing for adsorption of the drug to plasticware during UF.

Blood collection technique for pharmacokinetic studies of doxorubicin in paediatric patients

Aims: Dihydropyrimidine dehydrogenase (DPD) is the initial enzyme in the catabolism of 5-fluorouracil (5FU) and DPD deficiency is an important pharmacogenetic syndrome. The main purpose of this study was to develop a limited sampling strategy to evaluate the pharmacokinetics of 5FU and to detect decreased 5FU elimination in patients with c.1905+1G>A (IVS14+1G>A) mutation related DPD deficiency. Methods: Thirty patients, heterozygous for the c.1905+1G>A (IVS14+1G>A) mutation in DPYD, and 16 control patients received an intravenous dose administered over 2 min of 300 mg 5FU/m2 and/or 450 mg 5FU/m2, followed by blood sampling over 2 hrs and subsequently pharmacokinetic analysis of the 5FU plasma levels. A population pharmacokinetic analysis was performed in order to develop a quantitative compartmental pharmacokinetic model suitable for limited sampling strategy. Clinical aspects of treating DPD patients with 5FUbased chemotherapy was assessed from the retrospectively collected clinical data. Results: A two compartment model with Michaelis-Menten elimination from the central compartment was used in the population pharmacokinetic analysis. The mean (€ SD) AUC (mg.h/L), T1/2 s (h), Km (mg/L) and Vmax (mg/h) of 5FU in DPD deficient patients vs. controls were 9.1 € 4.0 vs. 6.0 € 2.1 (AUC), 0.268 € 0.116 vs. 0.128 € 0.043 (T1/2 s), 4.81 € 0.39 vs. 4.39 € 0.72 (Km) and 942 € 310 vs. 1749 € 380 (Vmax) at a dose of 300 mg 5FU/m2. For a dose of 450 mg 5FU/m2, the PK values in DPD deficient patients vs. controls were 17.7 € 5.4 vs. 13.4 € 3.9 (AUC), 0.306 € 0.103 vs. 0.181 € 0.041 (T1/2 s), 5.02 € 0.32 vs. 4.58 € 0.25 (Km) and 900 € 194 vs. 1370 € 267 (Vmax). The differences in AUC, T1/2 s and Vmax between DPD deficient patients and controls were all highly significant (p <0.05). Using a single sample t = 30 min limited sampling strategy, the positive predictive value and negative predictive value for Vmax were 96% and 88 %, respectively. Conclusion: Profound differences in pharmacokinetics of 5FU existed between DPD deficient patients and control patients. Pharmacokinetic 5FU profiling using a limited sampling model may be useful for identification of DPD deficient patients in order to reduce severe toxicity.

Erratum to: Population pharmacokinetic modelling of doxorubicin and doxorubicinol in children with cancer: is there a relationship with cardiac troponin profiles? (Cancer Chemotherapy and Pharmacology, (2017), 80, 1, (15-25), 10.1007/s00280-017-3309-6)

The author would like to correct the errors in the Appendix in the original publication of the article. The correct Appendix is given in the following page. The original article was corrected. (Table Presented.).

Microbial metabolism of thiopurines: A method to measure thioguanine nucleotides.

Thiopurines are anti-inflammatory prodrugs. We hypothesised that bacteria may contribute to conversion to active drug. Escherichia coli strain DH5α was evaluated to determine whether it could metabolise the thiopurine drugs, thioguanine or mercaptopurine, to thioguanine nucleotides. A rapid and reliable high performance liquid chromatography (ultraviolet detection) method was developed to quantify indirectly thioguanine nucleotides, by measuring thioguanine nucleoside.