Drug Metabolite Concentrations Research Articles

Tandem Mass Spectrometry (LC-MS-MS): a Predominant Role in Bioassays for Pharmacokinetic Studies

Plasma concentrations of drugs and drug metabolites in bioavailability studies are routinely bioassayed with high performance liquid chromatography (HPLC), gas chromatography (GC), and liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) methods. In the 1980s and 1990s, HPLC had achieved a relevant role compared to GC and other techniques in pharmacokinetic bioassays. However, in the last few years the LC-MS-MS technique, known as tandem mass spectrometry, has attained a predominant role over all other techniques. This is because it requires a less amount of matrix, possesses better specificity and sensitivity, and requires a shorter operating time. In the experience of the authors, LC-MS-MS in fact requires on average 3-4 fold shorter time to complete a bioequivalence study when performed by one operator than does HPLC. The higher cost of the apparatus and technical assistance of LC-MS-MS are fully compensated by the shorter operating time. Part or most of the HPLC apparatuses have been or are being replaced by LC-MS-MS systems in laboratories operating in clinical and pre-clinical pharmacokinetics and, to a large extent, in those involved in assessing permeability in screening procedures of new chemical entities. This paper analyses the growing development of the LC-MS-MS technique and compares four couples of methods validated with HPLC or GC versus LC-MS-MS, giving analytical details of the two approaches.

Joint Population Pharmacokinetic Analysis of Zidovudine, Lamivudine, and Their Active Intracellular Metabolites in HIV Patients

The population pharmacokinetic parameters of zidovudine (AZT), lamivudine (3TC), and their active intracellular metabolites in 75 naïve HIV-infected patients receiving an oral combination of AZT and 3TC twice daily as part of their multitherapy treatment in the COPHAR2-ANRS 111 trial are described. Four blood samples per patient were taken after 2 weeks of treatment to measure drug concentrations at steady state. Plasma AZT and 3TC concentrations were measured in 73 patients, and among those, 62 patients had measurable intracellular AZT-TP and 3TC-TP concentrations. For each drug, a joint population pharmacokinetic model was developed and we investigated the influence of different covariates. We then studied correlations between the mean plasma and intracellular concentrations of each drug. A one-compartment model with first-order absorption and elimination best described the plasma AZT concentration, with an additional compartment for intracellular AZT-TP. A similar model but with zero-order absorption was found to adequately described concentrations of 3TC and its metabolite 3TC-TP. The half-lives of AZT and 3TC were 0.81 h (94.8%) and 2.97 h (39.2%), respectively, whereas the intracellular half-lives of AZT-TP and 3TC-TP were 10.73 h (69%) and 21.16 h (44%), respectively. We found particularly a gender effect on the apparent bioavailability of AZT, as well as on the mean plasma and intracellular concentrations of AZT, which were significantly higher in females than in males. Relationships between mean plasma drug and intracellular metabolite concentrations were also highlighted both for AZT and for 3TC. Simulation with the model of plasma and intracellular concentrations for once- versus twice-daily regimens suggested that a daily dosing regimen with double doses could be appropriate.

Accelerator Mass Spectrometry Measurement of Intracellular Concentrations of Active Drug Metabolites in Human Target Cells In Vivo

Accelerator mass spectrometry (AMS) is an ultrasensitive technique to detect radiolabeled compounds. We administered a microdose (100 µg) of (14)C-labeled zidovudine (ZDV) with or without a standard unlabeled dose (300 mg) to healthy volunteers. Intracellular ZDV-triphosphate (ZDV-TP) concentration was measured using AMS and liquid chromatography-tandem mass spectrometry (LC/MS/MS). AMS analysis yielded excellent concordance with LC/MS/MS and was 30,000-fold more sensitive. The kinetics of intracellular ZDV-TP formation changed several-fold over the dose range studied (100 µg-300 mg). AMS holds promise as a tool for quantifying intracellular drug metabolites and other biomediators in vivo.

Usefulness of the measurement of azathioprine metabolites in the assessment of non-adherence

Azathioprine is a thiopurine immunosuppressive antimetabolite used to chronically treat inflammatory bowel disease and autoimmune hepatitis. Azathioprine treatment is a long-term therapy and therefore it is at risk for non-adherence, which is considered an important determinant of treatment inefficacy. Measurement of 6-thioguanine and 6-methylmercaptopurine nucleotides has been recently suggested as a screener for non-adherence detection.We describe four young patients in which non-adherence to azathioprine therapy was detected only through the measurement of drug metabolite concentrations, and the criterion for non-adherence was undetectable metabolite levels. After the identification of non-adherence, patients and their families were approached and the importance of a correct drug administration was thoroughly enlightened and discussed; this allowed obtaining a full remission in all subjects.Our observations support the use of undetectable metabolite levels as indicators of non-adherence to therapy in azathioprine treated patients. The additional level of medical supervision given by this assay allows getting a better adherence to medical treatment, which results in an improvement in the response to therapy; these benefits may justify the costs associated with the assay.

Using adaptive model predictive control to customize maintenance therapy chemotherapeutic dosing for childhood acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is a common childhood cancer in which nearly one-quarter of patients experience a disease relapse. However, it has been shown that individualizing therapy for childhood ALL patients by adjusting doses based on the blood concentration of active drug metabolite could significantly improve treatment outcome. An adaptive model predictive control (MPC) strategy is presented in which maintenance therapy for childhood ALL is personalized using routine patient measurements of red blood cell mean corpuscular volume as a surrogate for the active drug metabolite concentration. A clinically relevant mathematical model is developed and used to describe the patient response to the chemotherapeutic drug 6-mercaptopurine, with some model parameters being patient-specific. During the course of treatment, the patient-specific parameters are adaptively identified using recurrent complete blood count measurements, which sufficiently constrain the patient parameter uncertainty to support customized adjustments of the drug dose. While this work represents only a first step toward a quantitative tool for clinical use, the simulated treatment results indicate that the proposed mathematical model and adaptive MPC approach could serve as valuable resources to the oncologist toward creating a personalized treatment strategy that is both safe and effective.

The development of an objective methodology to measure medication adherence to oral thiopurines in paediatric patients with acute lymphoblastic leukaemia—an exploratory study

To develop a method that prospectively assesses adherence rates in paediatric patients with acute lymphoblastic leukaemia (ALL) who are receiving the oral thiopurine treatment 6-mercaptopurine (6-MP). A total of 19 paediatric patients with ALL who were receiving 6-MP therapy were enrolled in this study. A new objective tool (hierarchical cluster analysis of drug metabolite concentrations) was explored as a novel approach to assess non-adherence to oral thiopurines, in combination with other objective measures (the pattern of variability in 6-thioguanine nucleotide erythrocyte concentrations and 6-thiouric acid plasma levels) and the subjective measure of self-reported adherence questionnaire. Parents of five ALL patients (26.3%) reported at least one aspect of non-adherence, with the majority (80%) citing "carelessness at times about taking medication" as the primary reason for non-adherence followed by "forgetting to take the medication" (60%). Of these patients, three (15.8%) were considered non-adherent to medication according to the self-reported adherence questionnaire (scored > or = 2). Four ALL patients (21.1%) had metabolite profiles indicative of non-adherence (persistently low levels of metabolites and/or metabolite levels clustered variably with time). Out of these four patients, two (50%) admitted non-adherence to therapy. Overall, when both methods were combined, five patients (26.3%) were considered non-adherent to medication, with higher age representing a risk factor for non-adherence (P < 0.05). The present study explored various ways to assess adherence rates to thiopurine medication in ALL patients and highlighted the importance of combining both objective and subjective measures as a better way to assess adherence to oral thiopurines.

Reversal of Diabetes by Pancreatic Islet Transplantation into a Subcutaneous, Neovascularized Device

Transplantation of pancreatic islets for the treatment of type 1 diabetes allows for physiologic glycemic control and insulin-independence when sufficient islets are implanted via the portal vein into the liver. Intrahepatic islet implantation requires specific infrastructure and expertise, and risks inherent to the procedure include bleeding, thrombosis, and elevation of portal pressure. Additionally, the relatively higher drug metabolite concentrations in the liver may contribute to the delayed loss of graft function of recent clinical trials. Identification of alternative implantation sites using biocompatible devices may be of assistance improving graft outcome. A desirable bioartificial pancreas should be easy to implant, biopsy, and retrieve, while allowing for sustained graft function. The subcutaneous (SC) site may require a minimally invasive procedure performed under local anesthesia, but its use has been hampered so far by lack of early vascularization, induction of local inflammation, and mechanical stress on the graft. Chemically diabetic rats received syngeneic islets into the liver or SC into a novel biocompatible device consisting of a cylindrical stainless-steel mesh. The device was implanted 40 days prior to islet transplantation to allow embedding by connective tissue and neovascularization. Reversal of diabetes and glycemic control was monitored after islet transplantation. Syngeneic islets transplanted into a SC, neovascularized device restored euglycemia and sustained function long-term. Removal of graft-bearing devices resulted in hyperglycemia. Explanted grafts showed preserved islets and intense vascular networks. Ease of implantation, biocompatibility, and ability to maintain long-term graft function support the potential of our implantable device for cellular-based reparative therapies.

Relationship between Temperature, Dopaminergic Neurotoxicity, and Plasma Drug Concentrations in Methamphetamine-Treated Squirrel Monkeys

To examine the relationship between temperature (ambient and core), dopaminergic neurotoxicity, and plasma drug [methamphetamine (METH)] and metabolite [amphetamine (AMPH)] concentrations, two separate groups of squirrel monkeys (n = 4-5 per group) were treated with METH (1.25 mg/kg, given twice, 4 h apart) or vehicle (same schedule) at two different ambient temperatures (26 and 33 degrees C). Core temperatures and plasma drug concentrations were measured during the period of drug exposure; striatal monoaminergic neuronal markers in the same monkeys were determined 1 week later. At the temperature range examined, the higher ambient temperature did not significantly enhance METH-induced hyperthermia or METH-induced dopaminergic neurotoxicity, although there were trends toward increases. Acute METH-induced increases in core temperature correlated highly and directly with subsequent decreases in striatal dopaminergic markers. Squirrel monkeys with the greatest increases in core temperature (and largest dopaminergic deficits) had the highest plasma drug metabolite (AMPH) concentrations. There was substantial interanimal variability, both with regard to elevations in core temperature and plasma drug concentrations. Pharmacokinetic studies in six additional squirrel monkeys revealed comparable individual differences in METH metabolism. These results, which provide the first available data on the within-subject relationship between temperature (ambient and core), plasma concentrations of METH (and AMPH), and subsequent dopaminergic neurotoxic changes, suggest that, as in rodents, core temperature can influence METH neurotoxicity in primates. In addition, they suggest that interanimal differences presently observed in thermal and neurotoxic responses to METH may be related to individual differences in drug metabolism.

Structural identification of bitespiramycin metabolites in rat: A single oral dose study

Bitespiramycin is a macrolide antibiotic consisting of a mixture of some nine spiramycin ester derivatives. It has a similar spectrum of antibiotic activity to that of spiramycin but has superior pharmacokinetic properties. In this study, a rapid and facile LC/ESI-MSn method was applied to study the metabolism of bitespiramycin in rat following a single oral dose (80 mg kg−1). Concentrations of parent drug constituents and metabolites were determined in plasma, urine, feces and bile. Concentrations of parent drug constituents and metabolites in plasma were very low. In urine, feces and bile, parent drug constituents and 38 metabolites were identified on the basis of their chromatographic and mass spectrometric properties. The identity of 17 metabolites was confirmed by comparison with reference substances. The principal metabolites were the corresponding spiramycins formed by hydrolysis of the 4′′-(3-methylbutanoate) groups. Other important metabolic pathways were: hydrolytic loss of the forosamine and mycarose sugars; aldehyde reduction; cysteine conjugation of the aldehyde group; and hydrolysis of the lactone ring. Products formed by lactone ring opening were found only in urine, and those formed by aldehyde reduction were found only in feces. Aldehyde reduction and hydrolytic loss of forosamine represent novel biotransformation pathways for spiramycin derivatives.

Maternal sertraline treatment and serotonin transport in breast-feeding mother-infant pairs.

Pharmacological treatment of postpartum depression is frequently complicated by the mother's desire to breast-feed. Although breast milk levels of several selective serotonin reuptake inhibitors (SSRIs) have been reported to be relatively low, a critical question is whether SSRI exposure during nursing results in clinically significant blockade of serotonin (5-HT) reuptake in infants. This study determined the degree of transporter blockade in infants exposed to sertraline through maternal breast milk. The extent of maternal and infant transporter blockade was assessed by measurement of platelet levels of 5-HT in 14 breast-feeding mother-infant pairs before and after 6-16 weeks of maternal treatment with sertraline for major depression with postpartum onset. Plasma sertraline and desmethylsertraline levels were obtained in 13 of these mothers and 11 of their infants. Marked declines in platelet 5-HT levels of 70%-96% were observed in mothers after sertraline treatment, 25-200 mg/day. In contrast, infants showed little or no change in platelet 5-HT levels after exposure through breast-feeding. Mean levels of maternal plasma sertraline and its major metabolite, desmethylsertraline, were 30.7 ng/ml and 45.3 ng/ml, respectively. Drug and drug metabolite concentrations in the infants were at or below the lower limit of quantitation. The data indicate that while mothers receiving clinical doses of sertraline experience substantial blockade of the platelet 5-HT transporter, platelet 5-HT uptake in nursing infants of treated mothers is unaltered. The observations suggest that mothers taking sertraline can breast-feed without appreciably affecting peripheral or central 5-HT transport in their infants.

Accuracy of five on-site immunoassay drugs-of-abuse testing devices.

Many current "on-site" urine drug-testing products claim performance equivalent to laboratory testing. Five commercially available products (PharmScreen, Roche TestCup, Accusign DOA 2, Status DS, and American Bio Medica-Rapid Drug Screen) were challenged with quality-control specimens of known drug metabolite concentrations, 25% above and 25% below the SAMHSA cutoffs, and with known positive and negative donor specimens previously analyzed by immunoassay and gas chromatography-mass spectrometry. The results indicate discrepancies between claims and performance for all products, particularly with amphetamines. The implications for employer-based drug testing are discussed.

CEDIA dau Benzodiazepine screening assay: a reformulation.

The CEDIA dau Benzodiazepine assay has been reformulated to include online hydrolysis of urinary benzodiazepine glucuronide conjugates. The new antibody possesses enhanced cross-reactivities toward the low-dose benzodiazepines, which are excreted at low urinary drug-metabolite concentrations. The screening method was evaluated using lorazepam as the probe benzodiazepine. Four subjects each consumed a 1-mg lorazepam tablet. Sequential urine voids over the same time intervals were collected for the next 48 h. Twelve postdose urine samples were collected from each subject. Positive results were obtained from 5-24 h to 2-35 h using a 200-ng/mL nitrazepam calibration cutoff. There was no practical difference between hydrolyzing online with the supplied E. coli beta-glucuronidase or offline with Helix pomatia beta-glucuronidase purchased separately. Without hydrolysis, all urine samples tested negative. The cross-reactivities of lorazepam in terms of nitrazepam calibration equivalents, varied from 108 to 178% for lorazepam concentrations between 50 and 2500 ng/mL. Lorazepam glucuronide gave cross-reactivities (expressed as lorazepam base) between 72 and 136% using the online hydrolysis procedure with E. coli beta-glucuronidase. Offline hydrolysis with Helix pomatia gave cross-reactivities between 84 and 134%. Without hydrolysis, lorazepam glucuronide gave less than 4% cross-reactivity in the assay.

Impact of nutrition on pharmacokinetics of anti-neoplastic agents.

It has been estimated that approximately 80% of the world's pediatric population lives in countries with limited resources, and that 43% of these children are malnourished. In children with cancer, malnutrition may antedate the diagnosis or be a result of aggressive chemotherapeutic regimens. Studies have shown that children with cancer and malnutrition have a less favorable prognosis, a higher risk of early relapse, and tolerate chemotherapy poorly when compared with children with normal nutritional status. Improvements in nutritional status may improve tolerance to chemotherapy. An understanding of the mechanisms responsible for the effects of malnutrition on drug disposition and pharmacodynamic response is important, especially for anti-neoplastic agents, which have a narrow therapeutic index and may be associated with potentially severe or life-threatening side-effects. Several factors related to malnutrition have been suggested to alter drug disposition. Diminished protein "status" in malnourished children results in lower amounts of plasma proteins, increasing the concentration of free drug available to exert its cytotoxic effect. Severely malnourished individuals also exhibit decreased oxidative metabolism and reduced glomerular filtration rate (GFR), potentially increasing concentrations of parent drug or active metabolites. Malnourished children receiving chemotherapy for the treatment of an underlying malignancy may need specifically "tailored" protocols to achieve therapeutic response while minimizing adverse acute and long-term side effects. The role of specific interventions, such as correction of nutritional status or pharmacokinetic drug monitoring, should be evaluated in this context.

Pharmacokinetics of mercaptopurine: plasma drug and red cell metabolite concentrations after an oral dose.

Measurement of red cell 6-mercaptopurine (MP) derived 6-thioguanine nucleotide (TGN) and methylmercaptopurine metabolites (MeMPs) can be used to monitor therapy in children who are administered MP for childhood lymphoblastic leukemia. Red cell TGNs are not influenced by the time of blood sampling in relation to the last MP dose. The purpose of this study was to find out whether the same is true for the MeMPs. Plasma MP and red cell MP metabolite pharmacokinetics were studied in seven children immediately before and for 4 hours after a protocol standardized dose of MP. Duplicate blood samples were taken, one was processed immediately whereas one was left at an ambient temperature for 24 hours. The variation in TGN and MeMP metabolites over the 0- to 4-hour period (10 time points per child) was within the error of the assays used. The coefficients of variation for the TGNs ranged from 2.7% to 7% and for the MeMPs, 4% to 10.7%. There was no difference in the TGN and MeMP concentrations measured when the blood samples were left for 24 hours. If a child takes a MP tablet immediately before a clinic appointment, it has no major influence on MeMP measurements.

Profile of non-compliance in lymphoblastic leukaemia

A nationwide study of intracellular drug metabolite concentrations in children prescribed 6-mercaptopurine for the treatment of lymphoblastic leukaemia was carried out to assess interpatient variability at a standardised dose. Nine...

Mercaptopurine metabolism and risk of relapse in childhood lymphoblastic leukaemia

Many months' treatment with daily oral mercaptopurine is an important part of therapy for nearly all children with lymphoblastic leukaemia (ALL). Even when prescribed the same dose based on body surface area, patients have widely different intracellular concentrations of drug metabolites. Whether this variation matters in terms of disease control is not yet clear. To find out, we followed up a large group of children with ALL in whom mercaptopurine-derived thioguanine nucleotides in the red cells were measured during treatment with an identical dose of mercaptopurine early in first remission. 172 unselected children (100 boys, 72 girls) were recruited between 1980 and 1992. At median follow-up of 5 years from diagnosis 42 (24%) had relapsed; 30 had erythrocyte thioguanine nucleotide concentrations below the group median (284 pmol per 8 × 108 erythrocytes) and 12 had values above the median. The actuarial relapse-free survival at 5 years was 63% in the below-median group and 84% in the above-median group (difference 21% [95% Cl 3-39%], p=0·0018). Multivariate analysis showed that erythrocyte thioguanine nucleotide concentration was independent of other prognostic variables including age, leukaemia immunophenotype, white-blood-cell count at diagnosis, trial protocol, and sex. Whatever the cause, in childhood ALL variable formation of intracellular mercaptopurine metabolites seems to be clinically important. Therapeutic schedules that include long-term daily oral mercaptopurine might be more effective if such metabolites are monitored.

Drug screening of meconium in infants of drug-dependent mothers: An alternative to urine testing

Meconium specimens (first 3 days' stool) obtained from 20 infants of drug-dependent mothers and five control infants were analyzed by radioimmunoassay for the metabolites of three commonly abused drugs, heroin, cocaine, and cannabinoids. Control stools contained no drug. Meconium from the infants of drug-dependent mothers showed the presence of at least one drug metabolite: 80% of the infants of drug-dependent mothers showed cocaine (range 0.14 to 19.91 micrograms/gm stool), 55% showed morphine (range 0.41 to 14.97 micrograms/gm stool), and 60% showed cannabinoid (range 0.05 to 0.67 micrograms/gm stool). The concentrations of metabolites were highest during the first 2 days; some stools tested positive up to the third day. In contrast, only 37% of the infants had positive results on a urine screen (fluorescent polarization immunoassay method). Paired urine and meconium specimens, both analyzed by radioimmunoassay, showed a higher concentration of drug metabolites in the latter; eight urine samples had no detectable drugs despite a corresponding positive stool test result. We conclude that meconium is useful for drug screening in the neonate.

Kinetics of drug action: An overview

The intensity and time course of action of directly and reversibly acting drugs are related to and determined largely by the time course of drug and active drug metabolite concentrations in the body. When the pharmacokinetics and the concentration-effect relationship of a drug are known, it is often possible to predict the temporal pattern of its pharmacologic effect(s), including the maximum intensity and duration of action. Sites of action may not be immediately accessible to a drug even if it is injected intravenously; this may be reflected by a gradual increase in the intensity of effect despite decreasing drug concentrations in plasma, with maximum effects occurring later than maximum drug concentrations in plasma. Pharmacologic effects may persist well beyond the time when drug concentrations in plasma are no longer determinable; this is often caused by localization of the drug in an extravascular compartment. Drug distribution kinetics, pharmacologically active metabolites, and development of functional (as opposed to metabolic) tolerance may be responsible for time-dependent changes in drug concentration—pharmacologic effect relationships. Interindividual differences in patients' response to drug therapy may have a pharmacokinetic basis, a pharmacodynamic basis, or both. In clinical assessments of pharmacologic response, it is important to measure the therapeutically relevant effect, to determine interindividual and intraindividual variability, and to explore the possible influence of underlying diseases and other physiologic variables on drug concentration-effect relationships.

Mechanism of action of thioureylene antithyroid drugs: factors affecting intrathyroidal metabolism of propylthiouracil and methimazole in rats.

Experiments were performed with rats to test the physiological significance of a previously proposed mechanism of action of thioureylene antithyroid drugs, which had been derived from results obtained with a model system containing purified thyroid peroxidase. Two features of the previously proposed scheme were tested:1) the effects of drug dosage and 2) the effects of iodine deficiency. In the dosage experiments, rats were injected with graded doses of [35S]PTU (0.18-59 mumol or [35S]mmi (0.16-18 mumol). Thyroid glands were removed 1 and 6--8 h later and 35S distribution in the homogenates was determined by paper chromatography. Serum samples were also analyzed by the same procedure. From the measured 35S activity in the various components and from the known specific activity of the injected drugs, it was possible to calculate thyroidal concentrations of unchanged drug and drug metabolites. At low doses, thyroidal concentrations of unchanged 6-propyl-2-thiouracil (PTU) and 1-methyl-2-mercaptoimidazole (MM) significantly exceeded their concentrations in serum, as reported by previous investigators. A major new finding in the present investigation was the observation that intrathyroidal metabolism of PTU and MMI is greatly affected by dosage. Marked inhibition of intrathyroidal drug metabolism was observed at 6-8 when the dosage was increased from 5.9 to 18 mumol for [35S]PTU and from 0.88 to 2.2 mumol for [35S]MMI (per 200 g rat). These findings demonstrate that with increasing dosage, PTU and MMI inhibit their own intrathyroidal metabolism. These dosage effects are similar to results previously reported for in vitro oxidation of PTU and MMI by the thyroid peroxidase system, and they offer support for the physiological significance of the previously proposed scheme. Further evidence for the physiological validity of this scheme was obtained in the experiments with iodine-deficient rats. As predicted from the in vitro findings, intrathyroidal metabolism of [35S]PTU and [35S]MMI was markedly reduced in rats on low iodine diet.

Use of Stable Isotopes in Measuring Low Concentrations of Drugs and Drug Metabolites by GC—MS—COM Procedures

Abstract Certain drugs in urine, plasma, breast milk, and amniotic fluid have been quantitatively analyzed in the picogram to nanogram range by selective ion detection with two gas chromatograph—mass spectrometer—computer systems: an LKB 9000/PDP 12 operated in the electron impact mode and a Finnigan 1015/PDP 8/I operated in the chemical ionization mode. With the latter system, it is possible to monitor several drugs and drug metabolites and an internal standard in a single temperature-programmed analysis. Internal standards labeled with stable isotopes are preferred for selective ion detection. Diphenylhydantoin-2,4,5-13C has been used to quantify diphenylhydantoin, and pentobarbital-2,4(6) ,5-13C has been used to quantify amobarbital, secobarbital, caffeine, meperidine ("Demerol"), and phenobarbital in 50-200 µl samples of plasma and breast milk.