Impaired transport as a mechanism of resistance to thiopurines in human T-lymphoblastic leukemia cells.

In order to better understand the mechanisms of resistance to thiopurines, we studied two sublines of the MOLT4 T-lymphoblastic leukemia cell line, resistant to 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). We found that the underlying mechanism of resistance in both resistant cell lines was a markedly reduction in initial transport of 6-MP (3- and 5-fold, respectively, in 6-MP- and 6-TG-resistant cells). No significant alteration of activities of hypoxanthine-guanine phosphoribosyl transferase, thiopurine methyltransferase or inosine monophosphate dehydrogenase, the key enzymes involved in the metabolism of thiopurines was detected. We conclude that defected initial transport of thiopurines by cells may very well explain their resistance to these drugs.

Molecular and biochemical mechanisms of fludarabine and cladribine resistance in a human promyelocytic cell line.

2F-Adenine arabinoside (fludarabine, Fara-A) and 2-chloro-2'-deoxyadenosine (cladribine, CdA) are nucleoside analogues with antineoplastic activity in vitro and in vivo. Lack of clinical resistance between CdA and Fara-A has been demonstrated in patients with chronic lymphocytic leukemia (G. Juliusson et al., N. Engl. J. Med., 327: 1056-1061, 1992). To clarify the differences in mechanism of resistance to CdA and Fara-A in vitro, we developed two stable, resistant cell lines, HL60/CdA and HL60/ Fara-A, by exposure to increasing concentrations of analogues over a period of 8 months. Resistant cells tolerated >8,000 and 5-fold higher concentrations of CdA and Fara-A, respectively. The specific activity of the nucleoside phosphorylating enzyme (using deoxycytidine as substrate) in cell extracts from HL60/CdA and HL60/Fara-A mutants was about 10 and 60%, respectively, compared with the parental cell line. Western blot analysis using a polyclonal antibody showed no detectable deoxycytidine kinase (dCK) protein in CdA-resistant cells, whereas in Fara-A-resistant cells, it was at the same level as in the parental cells. The mitochondrial enzyme deoxyguanosine kinase was not altered in resistant cell lines. The HL60/CdA cells showed cross-resistance to 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, Fara-A, arabinofuranosyl cytosine, difluorodeoxyguanosine, and difluorodeoxycytidine toxicity, most likely because of the decreased phosphorylation of these analogues by dCK. Using real-time quantitative PCR, the mRNA levels of dCK and cytosolic 5'-nucleotidase (5'-NT), a major nucleoside dephosphorylating enzyme, were measured. It was shown that the dCK mRNA levels in both CdA- and Fara-A resistant cells were decreased in parallel with the activity. The expression of 5'-NT mRNA was not significantly elevated in CdA- and Fara-A resistant cells, as compared with the parental cells. Ribonucleotide reductase maintains a balanced supply of deoxynucleotide triphosphate pools in the cell and may also be a major cellular target for CdA and Fara-A nucleotides. Except for the deoxycytidine triphosphate level, the intracellular deoxynucleotide triphosphate pools were significantly higher in Fara-A-resistant cells compared with the parental cell line. This might be a consequence of mutation or altered regulation of ribonucleotide reductase activity and may explain the 2-5-fold cross-resistance to several nucleoside analogues observed with HL60/Fara-A cells. It is likely that the resistance for CdA was mainly attributable to a dCK deficiency, and Fara-A-resistant cells might have another contributing factor to the resistance beyond the dCK deficiency.

On the bioavailability of oral and subcutaneous 2-chloro-2'-deoxyadenosine in humans: alternative routes of administration.

PURPOSE: The antimetabolite 2-chloro-2'-deoxyadenosine (CdA) is a promising alternative to alkylating agents for the treatment of lymphoproliferative disorders. Its use, however, is hampered by the need for intravenous (IV) administration. The aim of the present study was to determine the bioavailability of subcutaneously (SC) and orally administered CdA, and to establish an oral dose of CdA that could supersede IV administration. PATIENTS AND METHODS: A previously developed high-performance liquid chromatography method was used for the determination of plasma CdA concentrations in 13 patients. Ten patients were treated on alternate days with 0.14 mg/kg/d CdA as a 2-hour IV infusion or by a SC injection. Three of these patients were also given 0.14 mg/kg CdA orally in enteric-coated capsules. Ten patients were administered CdA orally that was dissolved in phosphate-buffered saline (PBS) after treatment with 20 mg omeprazole 1 and 6 hours before the administration of CdA. RESULTS: The bioavailability of SC CdA was 102% +/- 28% (mean +/- SD), and the bioavailability of CdA administered in enteric-coated capsules was 19%, 24%, and 60%. In the three patients who were given 0.14 mg/kg orally dissolved in PBS, the bioavailability was 48% +/- 8%, whereas in the seven patients who received 0.28 mg/kg, the bioavailability was 55% +/- 17%. In the 10 patients who were treated with the CdA solution orally, the coefficients of variation of the areas under the curve (AUCs) after oral and IV administration were similar. Thus, oral administration did not add to the interindividual variability. CONCLUSIONS: We conclude that orally administered CdA can supersede IV infusion if the dose is doubled. SC administration gives a high peak concentration of short duration with an AUC identical to that of IV infusion. Thus, SC injection can also be used as an alternative to IV infusion.

Subcutaneous injections of 2-chlorodeoxyadenosine for symptomatic hairy cell leukemia.

PURPOSE: To evaluate the clinical efficacy and safety of 2-chlorodeoxyadenosine (CdA) when administered by subcutaneous injection to patients with symptomatic hairy cell leukemia (HCL), and to evaluate predictive factors for response. PATIENTS AND METHODS: Seventy-three patients were given CdA as a subcutaneous injection once daily for 7 days. Complete remission (CR) required normalized blood counts and the absence of B-ly 7-positive bone marrow cells by flow cytometry. CdA concentrations in plasma following the first injection were analyzed by high-pressure liquid chromatography. RESULTS: Fifty-nine patients (81%) achieved a durable CR after one (n = 55) or two courses, and 10 had a partial remission (PR). With a median follow-up duration of 20 months, no patient had a clinical relapse. Neutropenic fever that required intravenous antibiotics occurred in 28 patients (38%). No toxicity at injection sites was observed. Incomplete response was predicted by an elevated lymphocyte count and serum beta 2-microglobulin level, and by a high percentage of hairy cells in the bone marrow. Plasma CdA levels were similar to those achieved from intravenous administration. CONCLUSION: Subcutaneous injection of CdA is safe and as effective as continuous infusion without problems associated with the mode of administration. Our schedule simplifies CdA treatment and can be generally recommended.

A late exclusion of bacteriophage T4 can be suppressed by Escherichia coli GroEL or Rho.

A litCon mutation in Escherichia coli TU6 results in exclusion of bacteriophage T4 during the late, morphogenetic stage of its development at low temperatures. DNA was synthesized continuously in the infected cells, but less than 10% of the DNA made by 90 min after infection was packaged into DNAase-resistant particles, few viable phage were formed, and the cells lysed poorly. The exclusion could be relieved by conditions leading to elevated levels, determined immunologically, of the E. coli Rho protein (believed to be involved in regulation of T4 transcription), or chromosomally encoded E. coli GroEL (a chaperone known to be involved in phage assembly), or by supplying GroEL in trans from a plasmid. The two suppressing proteins appeared to act independently of each other. GroEL-suppression restored packaging to normal levels, perhaps by preventing GP23 from activating the host Lit protein; in addition DNA synthesis was delayed and reduced and cell lysis enhanced, demonstrating involvement of GroEL in both these processes. Rho suppression was less efficient. Since both transcription-termination-proficient and transcription-termination-deficient Rho suppressed, the results raise the possibility that Rho has a role during T4 development not directly involving transcription regulation.

Real-time quantitative PCR assays for deoxycytidine kinase, deoxyguanosine kinase and 5'-nucleotidase mRNA measurement in cell lines and in patients with leukemia.

The relative levels of the deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK), and the 5'-nucleotidase (5'-NT) are of importance for the effect of many nucleoside analogues used in the treatment of hematological malignancies. To elucidate dCK, dGK and 5'-NT gene expressions in cell lines and in samples from patients with leukemia, we have established a real-time quantitative PCR (RQ-PCR) method. From the available dCK, dGK and 5'-NT cDNA sequences we designed specific primers and fluorogenic probes for the respective genes. The mRNA of dCK, dGK and 5'-NT was also measured by semi-quantitative RT-PCR, the enzyme activities by a radioactive substrate-based technique and Western blot was used to measure the amount of dCK and dGK protein. A MOLT-4 wild-type and its 9-beta-D-arabinofuranosylguanine (Ara-G)-resistant subline was used for the methods comparisons and the RQ-PCR assay was used in 35 samples from pediatric patients with ALL and AML. The results from RQ-PCR for the cell lines were in agreement with the semi-quantitative RT-PCR. The mRNA expression for dCK, dGK and 5'-NT (expressed as the ratio of the respective gene and the reference gene) in pediatric ALL and AML patients showed a large interindividual variability from 0.06 to 2.34, non-detectable to 0.06 and 0.04 to 0.30, respectively. These results show that the quantitative evaluation by RQ-PCR is a valuable tool in the determination of dCK, dGK and 5'-NT mRNA levels in cell lines and in clinical samples which were expressed at various levels. This rapid, convenient and specific method is suitable for further studies of these genes in clinical samples.

Biochemical pharmacology and resistance to 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, a novel analogue of cladribine in human leukemic cells.

The objective of the present study was to investigate the biochemical pharmacology of 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA)--a fluorinated analogue of cladribine [2-chloro-2'-deoxyadenosine, Leustatin (CdA)] with improved acid and metabolic stability--in human leukemic cell lines and in mononuclear cells isolated from patients with chronic lymphocytic leukemia (CLL) and acute myelocytic leukemia (AML). We have also made and characterized two cell lines that are not sensitive to the growth inhibitory and cytotoxic effects of CAFdA. Incubation of cells isolated from the blood of CLL and AML patients with various concentrations of CdA or of CAFdA accumulated CdA and CAFdA nucleotides in a dose-dependent manner. A significantly higher rate of phosphorylation to monophosphates was observed for CAFdA than for CdA in cells from CLL patients (n = 14; P = 0.04). The differences in the phosphorylation were even more pronounced for the respective triphosphates in both CLL (n = 14; P = 0.001) and AML (n = 4; P = 0.04) cells. Retention of CAFdA 5'-triphosphate (CAFdATP) was also longer than that for CdA 5'-triphosphate (CdATP) in cells from leukemic patients. The relative efficacy of CAFdA as a substrate for purified recombinant deoxycytidine kinase (dCK), the key enzyme in the activation of nucleoside analogues, was very high and exceeded that of CdA as well as the natural substrate, deoxycytidine, by a factor of 2 and 8, respectively. The Km for CAFdA with dCK was also lower than that for CdA, as measured in crude extracts from the human acute lymphoblastic leukemia cell line CCRF-CEM and the promyelocytic leukemia cell line HL60. Acquired resistance to CAFdA in HL60 and in CCRF-CEM cell lines was directly correlated to the decreased activity of the nucleoside phosphorylating enzyme, dCK. Resistant cells also showed a considerable degree of cross-resistance to analogues that were activated by dCK. These observations demonstrated that dCK phosphorylates CAFdA more efficiently than CdA. Furthermore, CAFdATP is apparently more stable than CdATP and the mechanisms of resistance to CAFdA are similar to those leading to CdA resistance. These results encourage studies on the clinical effect of CAFdA in lymphoproliferative diseases.

Pharmacokinetics of cladribine in plasma and its 5'-monophosphate and 5'-triphosphate in leukemic cells of patients with chronic lymphocytic leukemia.

The pharmacokinetic parameters of cladribine (CdA) in patient plasma and its intracellular nucleotides CdA 5'-monophosphate (CdAMP) and CdA 5'-triphosphate (CdATP) were delineated in circulating leukemia cells in 17 patients with chronic lymphocytic leukemia, after the last dose intake and up to 72 h thereafter. Patients were treated with 10 mg/m2 CdA p.o. on 3 consecutive days. A novel and specific ion-pair liquid chromatographic method, which separates the intracellular CdA nucleotides, was used. The area under the concentration versus time curve (AUC) of CdAMP in leukemia cells was generally higher (median, 47 micromol/liter x h) than the AUC of CdATP (median, 22 micromol/liter x h); however, in some patients (3 of 17), the reverse relationship was seen. The median ratio between the AUC values for CdATP and CdAMP was 0.60 (95% confidence interval, 0.4-1.0). The median half-life (t(1/2)) of CdAMP was 15 h, and that of CdATP was 10 h. The median terminal t(1/2) of CdA in plasma was 21 h. A significant correlation was found between the maximum plasma CdA and cellular CdAMP concentrations (r = 0.56, P = 0.02). There was no correlation between the AUC values of cellular CdAMP and CdATP (r = 0.224, P = 0.55). No correlation was found between deoxycytidine kinase activity and intracellular pharmacokinetic parameters of CdAMP or CdATP. The response to treatment was not significantly related to intracellular concentration of CdAMP or active metabolite CdATP. There is great heterogeneity among patients in terms of AUC and t(1/2) of CdAMP and CdATP. Furthermore, the results emphasize the differences between the pharmacokinetics of plasma CdA and those of the metabolites in circulating leukemic cells.

Treatment of normal and malignant cells with nucleoside analogues and etoposide enhances deoxycytidine kinase activity.

Deoxycytidine kinase (dCK), one of the rate-limiting enzymes in the intracellular metabolism of many antileukaemic drugs, was shown to be stimulated after treatment of human tonsillar lymphocytes by 2-chloro-2'-deoxyadenosine (cladribine, CdA) (Sasvári-Székely, et al., Biochem Pharmacol 1998, 56, 1175-1179). Here we present a comparative study of different normal and malignant cells in respect to the activation of dCK by CdA. G-phase lymphocytes showed a higher sensitivity for dCK stimulation than S-phase cells. Normal and leukaemic peripheral blood mononuclear cells, as well as the promyelocytic cell line HL60 responded to CdA treatment by a 2-5-fold increase in activity of dCK. However, no significant stimulation was detected either in CCRF-CEM T-lymphoblastoid cells, or in K562 myeloid cells. Thymidine kinase (TK) activity was not stimulated in any cases. Treatment of these cells with several other analogues beside CdA, such as 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA), 2-fluoro-1-beta-D-arabinosyladenine (Fludarabine, FaraA) and 1-beta-D-arabinosylcytosine (cytarabine, araC) gave similar results to CdA treatment. Enhancement of dCK activity could also be achieved with the topoisomerase II inhibitor, etoposide. In contrast, 2-chloro-riboadenosine (CrA) had no effect on the dCK at concentrations of 10 microM or less, while dCyd and 5-aza-dCyd caused slight inhibition. These results indicate that treatment of cells with several inhibitors of DNA synthesis potentiates the dCK activity. The drugs widely differ in their stimulatory effect on dCK, and there are also 'responsive' and 'non-responsive' cells with respect to dCK activation. Thus, enhancement of the dCK activity by specific drugs in 'responsive' cells might give a rationale for combination chemotherapy.

Deoxynucleoside anabolic enzyme levels in acute myelocytic leukemia and chronic lymphocytic leukemia cells.

The deoxynucleoside kinase reaction is often rate-limiting in the anabolism of pharmacologically active anti-cancer nucleosides. The levels of thymidine kinase (TK), deoxycytidine kinase, deoxyguanosine kinase (dGK), and thymidylate kinase were determined in leukocyte extracts from patients with chronic lymphocytic leukemia (CLL) and acute myelocytic leukemia (AML). The extracts from AML patients showed significantly higher TK activity than the ones from CLL patients. There were no differences in the levels of the other three kinases. In the case of dGK, the determinations were carried out with both an immunoblotting assay and selective enzyme activity measurements.

Identification and characterization of human deoxyguanosine kinase cDNA fragments.

Mitochondria require deoxyribonucleoside triphosphates for the synthesis of their DNA and one of the enzymes responsible for the initial phosphorylation of purine deoxyribonucleoside is deoxyguanosine kinase (dGK; EC 2.7.1.113). Recent studies have suggested that dGK in addition to deoxycytidine kinase phosphorylates several anti-cancer agents, such as 9-beta-D-arabinofuranosylguanine (Ara-G), cladribine (CdA), and fludarabine. There appear to coexist several mRNA fragments of dGK. In the present study we found 10 fragments, the longest fragment had 834bp, and represented the entire open reading frame of dGK (780bp). The nine additional fragments detected ranged from 807 to 269bp. All the fragments were found to contain the specific mitochondria translocation signal sequence. Expression of these fragments in Escherichia coli demonstrated that only the full-length dGK resulted in a protein that could phosphorylate CdA and Ara-G. Given the difficulty to measure the full-length dGK, these data are of value for studying the mRNA gene expression of dGK in cell lines and in leukemic cells from patients.

Analysis of DNA methylation of the 5' region of the deoxycytidine kinase gene in CCRF-CEM-sensitive and cladribine (CdA)- and 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA)-resistant cells.

DNA methylation of the CpG-rich 5' region of the deoxycytidine kinase (dCK) gene is potentially involved in the suppression of the gene and the resistance of tumour cells to arabinosylcytosine (ara-C). 2-Chlorodeoxyadenosine (cladribine, CdA) and 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA) are purine nucleoside analogues which are also phosphorylated by dCK. We observed a reduction in dCK activity in a number of CCRF-CEM-derived cell lines that are resistant to these drugs and hypothesized that this reduction is due to DNA methylation of the 5' region of the dCK gene. The DNA methylation state was analyzed at the DNA sequence level after bisulfite modification of genomic DNA. The investigated region included 0.3 kb of DNA upstream to the start site of transcription, exon 1 and part of intron 1. Sensitive cells (CCRF-CEM/0) and three resistant cell lines (CCRF-CEM/CdA4000, CCRF-CEM/CAFdA100 and CCRF-CEM/CAFdA4000) were investigated. The region that was analyzed contained no methylated cytosine residues in the parental cell line CCRF-CEM/0 or in the resistant cell lines. Therefore, it is highly unlikely that DNA methylation plays a role in the suppression of dCK gene expression in these cell lines.

Methotrexate plasma pharmacokinetics: importance of assay method.

Intravenous methotrexate (MTX) therapy is widely used for treatment of various neoplastic diseases in children. The optimization of the MTX dose and/or the subsequent leucovorin rescue is based on pharmacokinetic data calculated from plasma concentrations collected after cessation of the MTX administration. The influence of the MTX assay method on the subsequent pharmacokinetic evaluation was studied in 13 children with acute lymphoblastic leukemia. Plasma samples were collected after administration of MTX (5-8 g/m2) as 24 h infusions. All samples were analyzed by five different analytical procedures, viz. liquid chromatography (LC), enzyme inhibition assay (EIA), two fluorescence polarization immunoassays (FPIA1 and FPIA2) and enzyme multiplied immunoassay (EMIT). Using measurements from the four non-chromatographic procedures, only about 50% of determined pharmacokinetic parameters (area under the plasma concentration time curve, calculated by the trapezoidal rule and from pharmacokinetic modelling, and the terminal half life time) were within the range 75-125% of the values obtained from LC data. We conclude that the clinical outcome of MTX therapy using estimated MTX pharmacokinetics as guidelines for proper dosing of MTX and/or leucovorin rescue might be affected by the lack of accuracy of non-chromatographic procedures for MTX analysis. There is still a need for improving the accuracy of the procedures aimed at therapeutic drug monitoring of MTX.

In vitro topo II--DNA complex accumulation and cytotoxicity of etoposide in leukaemic cells from patients with acute myelogenous and chronic lymphocytic leukaemia.

Topoisomerase II (topo II) is the target enzyme of etoposide, and DNA--topo II complex accumulation is considered crucial for the cytotoxic effect. We used a SDS--KCl precipitation assay to determine the complex accumulation induced by etoposide in leukaemic cells isolated from 58 patients, 31 with acute myelogenous leukaemia (AML), and 27 with chronic lymphocytic leukaemia (CLL). To investigate whether the sensitivity towards etoposide was dependent on the complex accumulation in the cells, we investigated the drug-induced DNA damage using a DNA unwinding assay and the in vitro cytotoxicity of etoposide using the MTT assay. AML cells had higher complex accumulation (P=0.006) and more DNA damage (P=0.029) compared with CLL cells. The data support a relationship between etoposide-induced complex accumulation and DNA damage in leukaemic cells from AML and CLL patients. However, the induced DNA damage did not translate to in vitro cytotoxicity, suggesting that other factors, such as DNA repair and apoptosis functions, also play important roles to determine the etoposide sensitivity.

A limited sampling strategy for estimation of the cladribine plasma area under the concentration versus time curve after intermittent i.v. infusion, s.c. injection, and oral administration.

Cladribine is a newly developed antimetabolite with promising activity in lymphoproliferative disorders. Recent pharmacokinetics investigations have suggested that there is a relationship between its plasma area under the concentration versus time curve (AUC) and the degree of neutropenia posttreatment as well as the therapeutic outcome in hairy-cell leukemia. To enable a simple estimation of the plasma AUC, a limited sampling strategy was developed. Stepwise linear regression was used to determine which were the most important data points for estimation of the plasma AUC after 2-h i.v. infusion, s.c. injection (5 mg/m2), and oral administration (10 mg/m2) in 27 patients. The most important data points after i.v. infusion in 12 patients were 1, 4, and 24 h, in order of importance. The AUC could be estimated as 2.9081 x C1h + 5.1851 x C4h + 20.3265 x C24h. The accuracy and precision (mean value +/- SD for the determined/estimated AUC was 0.99 +/- 0.053) of the model could not be increased by the addition of more data points. A somewhat lower accuracy and precision (0.96 +/- 0.089) was seen with the 2-, 4-, and 24-h data points. These were used to test the regression technique prospectively for the estimation of the AUC after i.v. administration in another set of 10 patients. The accuracy and precision of the estimation of the AUC was similar in this group (1.01 +/- 0.109). In all, 11 patients were treated orally (10 mg/m2) and 10 patients were treated by s.c. injection (5 mg/m2). The most important data points for estimation of the AUC were 2.5, 24, and 0.5 h after oral administration (AUC = 0.8630 x C0.5h + 4.2337 x C2.5h + 45.4364 x C24h) and 9, 1, and 16 h after s.c. injection (AUC = 1.8821 x C1h + 16.4256 x C9h + 25.4518 x C16h). The accuracy and precision were 1.01 +/- 0.064 after oral dosing and 0.99 +/- 0.11 after s.c. injection. The derived mathematical models are reliable for estimation of the plasma AUC of cladribine after 2-h i.v. infusion, oral administration, and s.c. injection.

Multidrug resistance (Mdr1) gene expression in peripheral blasts from patients with acute leukemia only rarely increases during disease progression after combination chemotherapy.

Multidrug resistance gene (mdr1) RNA levels were determined in 55, and P-glycoprotein expression in 37 samples of peripheral leukemic cells from 17 patients with acute myeloblastic leukemia (AML) and 7 patients with acute lymphocytic leukemia (ALL). Between sample collections, patients were treated with various chemotherapy regimens. Mdr1 RNA levels were quantified by a RNA-RNA solution hybridization assay. P-glycoprotein was determined by Western blot analysis. Samples from 14 patients (9 AML, 5 ALL) had undetectable mdr1 RNA levels at initial analysis. Only two of these had detectable levels after chemotherapy. Ten patients (8 AML, 2 ALL) had detectable mdr1 RNA levels at initial analysis (median 1.0 transcript per cell, range 0.2-1.4). Increase of mdr1 RNA levels after chemotherapy were observed in cells from 3 patients, one patient had a lower level after chemotherapy and the 6 remaining patients had essentially unchanged mdr1 RNA levels in their leukemic cells. Samples from 13 patients were sequentially analysed for P-glycoprotein expression. In one patient, no P-glycoprotein was detectable at initial analysis but was weakly positive after chemotherapy. In the remaining 12 patients, P-glycoprotein levels stayed stable during disease progression. In conclusion, combination chemotherapy seems only rarely to be associated with an increase of mdr1 gene expression in residual leukemic cells. The addition of resistance modifiers to chemotherapy in order to overcome P-glycoprotein mediated resistance might therefore be more effective in chemotherapy naive patients since it is possible that during later disease progression additional mechanisms of resistance may be more operative.

Multidrug resistance gene (mdr1) RNA levels in relation to P-glycoprotein content of leukemic cells from patients with acute leukemia.

The clinical relevance of multidrug resistance gene (mdr1) expression in tumor cells remains largely unclear. Conflicting results regarding mdr1 gene expression and clinical outcome have been obtained. Little is known about regulation of mdr1 gene expression, and the conflicting results might be explained by the fact that mdr1 RNA levels do not reflect expression at the protein level. The aim of the present study was to investigate the relationship between mdr1 RNA levels and P-glycoprotein (Pgp) content of leukemic cells from patients with acute myelogenous or lymphocytic leukemia. Mdr1 RNA levels were determined by a quantitative RNA-RNA solution hybridization method, and Pgp by Western blot technique with enhanced chemiluminescence for immunodetection. Pgp was detected in 14/14 leukemic cell samples while mdr1 RNA was detectable (> 0.15 copies/cell) in cells from only six out of the 14 patients. Mdr1 RNA levels did not correlate with the Pgp content of leukemic cells (r = 0.284, p = 0.306). Relapsed leukemias had significantly (p = 0.016) higher levels of Pgp than de novo untreated leukemias (the mean and SD optical density units were 0.56 +/- 0.18 and 0.25 +/- 0.17 respectively) while no difference was found in RNA levels. The findings support post-transcriptional level regulation of mdr1 gene expression and stress the importance of accurate determinations of the Pgp content of tumor cells in studies of the relationship between mdr1 gene expression and clinical outcome.

Distribution of 2-chloro-2'-deoxyadenosine, 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, fludarabine and cytarabine in mice: a whole-body autoradiography study.

The distribution characteristics of tritiated nucleoside analogs, 2-chloro-2'-deoxyadeonosine (CdA), 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA), 2-fluoroarabinosyladenine (F-ara-A) and cytosine arabinoside (ara-C) were compared in mice using whole-body autoradiography. CdA, CAFdA and F-ara-A have quite similar molecular structures, but they differ substantially in clinical activity as well as the side effects. Eight mice were injected intravenously in couples. One mouse from each pair was killed 20 min postinjection and the other mouse from each pair 4 h after the injection. The distribution of the label was then analyzed by whole-body autoradiography. The distribution of the nucleoside analogs was rapid and uniform. High concentrations were found in highly perfused organs. After 4 h the overall concentration had decreased but relatively high activities were found in the skin for CdA and CAFdA, in the thymus for ara-C and the bone marrow for CdA. Both CdA and CAFdA were found in the brain, but the concentration was surprisingly lower after 4 h for CAFdA, a lipophilic and more stable analog as compared to CdA. There was an uptake of CdA, F-ara-A and CAFdA in the skin. There were signs of retention of ara-C in parts of the thymus. The present investigations indicate that the nucleoside analog transport to the brain in mice is not primarily dependent upon passive diffusion over a lipophilic barrier, but suggestive of a specific transport mechanism.

Bioavailability and bacterial degradation of rectally administered 2-chloro-2'-deoxyadenosine.

2-Chloro-2'-deoxyadenosine (CdA) is a new drug for the treatment of hairy cell leukemia and other lymphoproliferative diseases. It is generally administered as a continuous intravenous infusion during 5-7 days. The oral bioavailability is only 50%. The bioavailability after rectal administration was investigated in two patients with chronic lymphocytic leukemia. Five milligrams per square metre was given i.v. as a 2-h infusion and 24 h later the same dose was administered rectally in a gel formulation. The mean bioavailability was only 21% due to deglycosylation of CdA to 2-chloroadenine (CAde). To further elucidate the factors which are important for the rectal availability of CdA, the in vitro stability of CdA in bacterial cultures was tested. Clostridium perfringens and Escherichia coli as well as whole feces rapidly deglycosylated CdA to CAde while Bacteroides fragilis, Enterococcus faecalis as well as saliva only degraded CdA slowly or not at all. It is concluded that, due to bacterial degradation, rectal administration of CdA has no advantage over oral administration.

Relationship between cladribine (CdA) plasma, intracellular CdA-5'-triphosphate (CdATP) concentration, deoxycytidine kinase (dCK), and chemotherapeutic activity in chronic lymphocytic leukemia (CLL).

Seventeen patients with CLL were treated with oral 2-chloro-2'-deoxyadenosine (cladribine, CdA, 10 mg/m2) on 3 consecutive days and the pharmacokinetic parameters of CdA in patient plasma and its intracellular nucleotides (CdAMP, CdATP) in circulating leukemic cells were studied after the last dose intake and up to 72 h thereafter. The median terminal half life (t1/2) of CdA in plasma was 21.1 h and the area under the curve (AUC) was median 1.2 microMh. The median t1/2 was 14.6 h for CdAMP and 9.7 h for CdATP. The AUC of CdATP in leukemic cells is lower than the AUC of CdAMP (median ratio 0.60). There was no correlation between cellular CdATP and plasma CdA concentrations or dCK activity. The clinical response was related to higher Cmax values for plasma CdA (p = 0.05) and higher products of dCK activity and CdA Cmax of plasma (p = 0.02). The activity of dCK alone was not related to the clinical outcome in this patient group. The results suggest that further steps in the mechanism of action of CdA beyond its bioactivation may be more important, e.g. the extent of DNA fragmentation or the ability of the leukemic cell to go into apoptosis, than the concentration of CdA nucleotides alone.