Birgitta Pettersson

Differences between children and adults in thiopurine methyltransferase activity and metabolite formation during thiopurine therapy: Possible role of concomitant methotrexate

This study examined the role of thiopurine methyltransferase (TPMT) polymorphism in the metabolism and clinical effects of azathioprine and 6-mercaptopurine in the treatment of inflammatory bowel disease and childhood leukemia. The current hypothesis is that the cytotoxic effects of thiopurines are caused by the incorporation of thioguanine nucleotides into DNA. In this context, S-methylation catalyzed by TPMT can be regarded as a competing metabolic pathway. The authors assayed the TPMT activity in red blood cells from 122 patients treated with azathioprine or 6-mercaptopurine (83 adults with inflammatory bowel disease and 39 children with acute lymphoblastic leukemia) and in 290 untreated controls (219 adult blood donors and 71 children). The concentrations of thioguanine nucleotides and methylthioinosine monophosphate were also assayed in red blood cells from the patients. The TPMT activity and the concentrations of methylthioinosine monophosphate and thioguanine nucleotides were higher in children than in adults. All children but no adult patient received concomitant methotrexate. Interaction between methotrexate and 6-mercaptopurine has been described, and may explain the results. Low TPMT activity in adult patients with inflammatory bowel disease correlated to an increased incidence of adverse drug reactions. However, there was no correlation between TPMT activity and the red blood cell concentrations of methylthioinosine monophosphate or thioguanine nucleotides, or between the concentrations of these metabolites and the occurrence of adverse effects. The results show that the role of thiopurine metabolism for drug effects is complex.

Thionations Using a P4S10−Pyridine Complex in Solvents Such as Acetonitrile and Dimethyl Sulfone

Tetraphosphorus decasulfide (P(4)S(10)) in pyridine has been used as a thionating agent for a long period of time. The moisture-sensitive reagent has now been isolated in crystalline form, and the detailed structure has been determined by X-ray crystallography. The thionating power of this storable reagent has been studied and transferred to solvents such as acetonitrile in which it has proven to be synthetically useful and exceptionally selective. Its properties have been compared with the so-called Lawesson reagent (LR). Particularly interesting are the results from thionations at relatively high temperatures (∼165 °C) in dimethyl sulfone as solvent. Under these conditions, for instance, acridone and 3-acetylindole could quickly be transformed to the corresponding thionated derivatives. Glycylglycine similarly gave piperazinedithione. At these temperatures, LR is inefficient due to rapid decomposition. The thionated products are generally cleaner and more easy to obtain because in the crystalline reagent, impurities which invariably are present in the conventional reagents, P(4)S(10) in pyridine or LR, have been removed.

One-Pot Eschenmoser Episulfide Contractions in DMSO: Applications to the Synthesis of Fuligocandins A and B and a Number of Vinylogous Amides

Practical total syntheses of the natural products fuligocandin A (2a) and fuligocandin B (3) have been achieved through a convergent strategy depending on the Eschenmoser episulfide contraction as a key step. Conducting the reaction in DMSO proved to be an efficient and general method for the synthesis of a variety of vinylogous amides, such as azepan-2-ylidenepropan-2-one.

Simultaneous quantitation of methotrexate and its two main metabolites in biological fluids by a novel solid-phase extraction procedure using high-performance liquid chromatography

We have developed an assay for the simultaneous determination of methotrexate (MTX) and its main metabolites, 7-hydroxymethotrexate (7-OHMTX) and 2,4-diamino-N10-methylpteroic acid (DAMPA) in plasma, urine and saliva meeting the requirement of rapidity for routine use in high-dose MTX therapy and the requirement of sensitivity for its potential use in therapeutic drug monitoring in low-dose MTX therapy. Sample preparation is based on solid-phase extraction using C8 Isolute cartridges. Chromatographic separation was achieved with a reversed-phase column (C18), and quantitation by subsequent exposure to UV light of 254 nm, which converted MTX and its two metabolites by photolytic oxidation to fluorescent products. The recoveries of MTX, 7-OHMTX and DAMPA from plasma at 100 nmol/l were 85.8, 91.1 and 102.3%, respectively. The limits of detection for MTX, 7-OHMTX and DAMPA in plasma and saliva were 0.1 nmol/l. In urine the limit of detection was 10 nmol/l for all compounds. The limits of quantitation in plasma and saliva were 0.5 nmol/l for all compounds.

High-performance liquid chromatography with fluorometric detection for monitoring of etoposide and its cis-isomer in plasma and leukaemic cells

The podophyllotoxin derivative etoposide, extensively used in anticancer therapy, is highly protein-bound (95%) in plasma. It is a chiral drug and only the trans-isomer is pharmacologically active. Isomerisation to the inactive cis-lactone occurs in plasma. The cis-lactone is often present in ultrafiltrates of plasma from patients treated with etoposide, therefore it is important to separate the isomers when free etoposide concentrations are assayed. There is reason to believe that free and cellular concentrations are more important for the effect of etoposide therapy than total plasma concentrations. A high-performance liquid chromatographic (HPLC) method for quantification of etoposide and its cis-isomer in plasma, total and non-protein-bound concentrations, and in leukaemic cells is described. After addition of teniposide as internal standard the drugs were extracted with chloroform. Etoposide, its cis-isomer, teniposide and endogenous substances were separated isocratically on a Spherisorb phenyl reversed-phase column. Detection was performed fluorometrically, lambda ex/em = 230/330 nm. Non-protein-bound concentrations were determined after ultrafiltration. The detection limit for etoposide was 10 ng/ml plasma, 25 ng/ml ultrafiltrate and 10 ng/50 x 10(6) cells. The sensitivity of the assay for the cis-lactone was twice as high due to higher fluorescence. The protein binding of the cis-lactone in plasma from ten healthy blood donors was 54.5 +/- 4.8% (mean +/- S.D.). Thus, the free fraction was about ten-fold higher than that of the mother compound. The assay is convenient and sensitive enough for the determination of free and cellular fractions of etoposide.

In Vivo Accumulation of Etoposide in Peripheral Leukemic Cells in Patients Treated for Acute Myeloblastic Leukemia; Relation to Plasma Concentrations and Protein Binding

Since etoposide interacts with the nuclear enzyme topoisomerase II, the drug concentrations in the malignant cells during chemotherapy may have clinical correlates. Plasma protein binding of etoposide is extensive (94%) and alterations of the non-proteinbound fraction affect pharmacokinetic behavior of the drug. The pharmacokinetics of etoposide was therefore studied in plasma, total and non-proteinbound concentrations, and in leukemic cells isolated from peripheral blood samples from 22 patients after the first dose of the induction treatment for acute myelocytic leukemia. Fourteen patients received 100 mg/m2 and eight patients 200 mg/m2 as a 1 h infusion. The mean area under the concentration versus time curve AUC(0-infinity) in plasma was at the lower dose level 78.4 +/- 29.1 (mean +/- S.D.) micrograms/ml x h and 201.0 +/- 56.5 micrograms/ml x h at the higher dose level. The fraction of non-proteinbound etoposide in plasma was 5.2 +/- 3.4 and 5.4 +/- 2.1% in the two treatment groups. AUC(0-16h) in leukemic cells was 8.4 +/- 8.7 and 22.4 +/- 12.1 micrograms/ml x h at the two dose levels, respectively. The cellular etoposide concentration was 12.1 +/- 7.9 and 14.7 +/- 5.1% of the plasma concentration at the end of the infusion. The interpatient variability in cellular drug levels was considerable and exceeded the variability in plasma concentrations. Cellular accumulation of etoposide could be important for treatment outcome.

Ultrafiltration and subsequent high performance liquid chromatography for in vivo determinations of the protein binding of etoposide

Etoposide is extensively (approximately 94%) bound to plasma proteins and the free non-protein-bound levels have been shown to correlate more closely to toxicity than total drug concentrations. A rapid and easily performed method, compared to the time consuming equilibrium dialysis, to obtain the free fraction is needed. The aim of this study was to evaluate ultrafiltration and subsequent high performance liquid chromatography (HPLC) for the determination of protein binding of etoposide. Spiked plasma from healthy, drug-free volunteers was used to compare ultrafiltration, using Amicon Centrifree filters, with equilibrium dialysis at 37 degrees C. The variability (CV) of the ultrafiltration method was 6.1 and 13.5% (n = 6) at 37 degrees C and room temperature (RT), respectively. The relative size of the free fraction obtained by ultrafiltration at 37 degrees C and RT was 1.22 (P = 0.0005) and 0.37 (P = 0.0001), respectively, compared with equilibrium dialysis at 37 degrees C. The chromatographic separation of metabolites from the mother compound when free etoposide is analyzed is crucial. It is shown that a hydroxy-acid metabolite of etoposide is quite dominant in a protein-free plasma fraction. The free concentrations were determined throughout a dose interval of 24 h in a patient receiving etoposide 100 mg/m2 daily. Ultrafiltration and subsequent HPLC is considered convenient and suitable for in vivo pharmacokinetic investigations.

Determination of plasma azathioprine and 6-mercaptopurine in patients with rheumatoid arthritis treated with oral azathioprine.

Two specific high-performance liquid chromatography methods for determining plasma concentrations of azathioprine and 6 mercaptopurine after oral administration of azathioprine are presented. It was shown that azathioprine is unstable in the blood samples unless immediately cooled in ice water. The 2-amino analog, guaneran, was used as internal standard for azathioprine, which was extracted from plasma with ethylacetate. A Nucleosil C18 column was used for the separation. The detection limit was 6 nM. For quantification of 6-mercaptopurine, 6-thioguanine was used as internal standard. Plasma was deproteinized with HC104 and the sample was purified on mercurial cellulose. A Beckman ODS column was used and the detection limit was 5 nM. Phar-macokinetic data from two patients are presented. Unchanged azathioprine was seen until 6 h after an oral dose of 32 mg/m2.

On the interaction between cytosine arabinoside and etoposide in vivo and in vitro.

Abstract: Cytosine arabinoside (ara‐C) and etoposide are often used in combination in the treatment of acute myelocytic leukemia (AML). The intracellular phosphorylation of ara‐C to its 5′‐triphosphate (ara‐CTP) is a prerequisite for its cytotoxic effects. It has been shown in vitro that etoposide can impair the formation of ara‐CTP in leukemia cells. The present study was undertaken in order to elucidate whether this interaction may be of clinical importance. Leukemia cells were isolated from 3 patients with acute myelocytic leukemia and incubated in medium (RPMI‐1640) with or without 10% fetal calf serum or in human plasma. When the cells were incubated in RPMI‐1640 with ara‐C (10 μmol/l) and etoposide during 2 h, the formation of ara‐CTP was decreased to 71 ± 18 (mean ± S.D.) and 30 ± 15% of control at 1 and 10 μg/ml etoposide, respectively. When the cells were incubated in human plasma, the formation of ara‐CTP was not influenced by the presence of etoposide (101 ± 6 and 103 ± 20% at 1 and 10 μg/ml etoposide). When incubated in RPMI supplemented with 10% fetal calf serum, the corresponding figures were 81 ± 8 and 70 ± 20%. Six patients with AML were therefore treated with ara‐C 0.5 or 1.0 g/m2 as a 2‐h infusion every 12 h and, during 1 h before the second ara‐C infusion, 100 or 200 mg/m2 etoposide was administered. The median change in the AUC of cellular ara‐CTP between the first and second ara‐C dose was 0% (‐37 to +21%). The corresponding median change in rate of accumulation of ara‐CTP in leukemia cells was 12% (‐26 to +110%). The concentration of etoposide in plasma during the ara‐C infusion was 18.7 ± 5.1 μg/ml while the non‐protein bound etoposide was 0.73 ± 0.34 μg/ml. Thus, despite exposure to higher etoposide concentrations in vivo than in vitro, no impairment of ara‐CTP formation was seen in the patients. This corresponds to the results obtained when leukemic cells were incubated in plasma. It is concluded that the inhibition of ara‐CTP formation by etoposide seen in vitro is offset by the high protein binding of etoposide in plasma (96%) and that etoposide does not impair the formation of ara‐CTP in leukemia cells in vivo during treatment with standard‐dose etoposide.

Analysis of Azathioprine and 6-Mercaptopurine in Plasma in Renal Transplant Recipients After Administration with Oral Azathioprine

Abstract Studies on the pharmacokinetics of azathioprine (AZA) and its main metabolite, 6-mercaptopurine (6-MP) in patients treated with oral AZA, such as renal transplant recipients, require an analytical method with a high sensitivity. Since the substances differ substantially in their physicochemical properties, it is hardly feasible to develop an extraction procedure and select chromatographic conditions for the simultaneous determination of both substances. Therefore, we have developed two specific chromatographic assays for determination of AZA and 6-MP in plasma. A solid-phase extraction (C8 Isolute) procedure was used for AZA with guaneran as internal standard (IS), while 6-MP was purified on mercurial cellulose and 6-mercaptopurine arabinoside was used as IS. Chromatographic analyses were achieved using C8 and C18 reversed phase columns for AZA and 6-MP, respectively. The methods were reproducible with intra- and inter-assay coefficients of variations below 6 %. The average recoveries of AZA, 6-M...