Development of resistance to 1-beta-D-arabinofuranosylcytosine after high-dose treatment in childhood lymphoblastic leukemia: analysis of resistance mechanism in established cell lines.

Cell lines PER-163 and PER-164 are derived from a patient with acute lymphoblastic leukemia who developed resistance to 1-beta-D-arabinofuranosylcytosine (ara-C) after high-dose (HD) therapy. Both lines are highly resistant to ara-C and have maintained stable resistance for more than 18 mo. The resistance in PER-164 cells is the result of a selection process in vivo only, while PER-163 cells have in addition been exposed to ara-C in culture. Comparison with cell line PER-145, which is sensitive to ara-C and was established from the same patient before HDara-C therapy, revealed no differences with respect to surface markers, morphology, cytochemical stains, or requirements for growth in vitro. The leukemic origin of the three cell lines is indicated by the close similarities of all three cell lines to the patient's fresh cells. The analysis of the two resistant cell lines shows that resistance to ara-C is not due to lower ara-C transport capacity nor to cytokinetic reasons, since the percentage of cells in S-phase is similar in all three cell lines. In addition, the resistant cell lines do not show any increased cytidine deaminase activity. PER-164 cells show a markedly reduced deoxycytidine kinase activity, 4.8 nmol/h/mg of protein, compared to PER-145 cells with an enzyme activity of 21.48 nmol/h/mg of protein. In PER-163 cells, no deoxycytidine kinase activity could be detected. Furthermore, the two resistant cell lines show significantly different dCTP levels. The sensitive PER-145 cells generated 97.9 pmol of 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP)/10(7) cells during a 45-min incubation period in the presence of 10(-6) M ara-C. This contrasts with 0.16 and 12 pmol of ara-CTP/10(7) cells for PER-163 and PER-164 cells, respectively. These investigations suggest that cell phenotypes with distinct features can be generated after HDara-C treatment and that decreased deoxycytidine kinase activity appears to be one of the major mechanisms of resistance.

The quantitation by radioimmunoassay of 2'-deoxyuridine 5'-triphosphate in extracts of thymidylate synthase-inhibited cells.

A radioimmunoassay (RIA) for dUTP, with a sensitivity of 3.78 fmol, has been developed. The antibody cross-reacted with dTTP so that affinity purification of the immunoglobulin G fraction was required before its use in the RIA. Cross-reactivity with UTP and with mono- and diphosphodeoxyuridylates has necessitated respectively sodium periodate oxidation and anion exchange chromatography of cell extracts, prior to RIA quantitation of dUTP directly in fractions from the chromatography column. Mean recovery rate of a range of concentrations of extracted dUTP standard taken through the entire procedure is 63.7% (7.8-31.3 pmol dUTP) although at a lower concentration (3.11 pmol) the recovery was only 36.2%. Results are reproducible with CV values of between 3.1 and 9.5%. The assay has been used to assess the presence of dUTP in A549 human lung carcinoma cells exposed to the thymidylate synthase inhibitor CB3717. The high sensitivity of the quantitation step has made it possible to measure dUTP in relatively small numbers (10(6)) of cells.

Cytosine arabinoside in the management of recurrent leukaemia.

A Phase II study of high dose cytosine arabinoside (ara-C) with different schedules in patients with recurrent acute myelogenous leukaemia (AML) and myeloid blast crisis of chronic myeloid leukaemia has been conducted at St. Bartholomew's Hospital. Ara-C was infused continuously for seven days at a dose of 100-200 mgs/m2 daily from day 1 with 1-2 g/m2 (3 h infusions) twice daily from day 2 for six days. Nineteen patients with acute myelogenous leukaemia and four patients with myeloid blast crisis of chronic myeloid leukaemia (CML) were treated. Complete remission was achieved in 4/19 patients with AML and in a further four patients an antileukaemic effect was observed. There were eight early deaths and three patients failed to show any response to therapy. All four patients with myeloid blast crisis of CML failed to respond to the treatment. Toxicity was considerable with gastro-intestinal and hepatotoxicity being the most serious problems. Pharmacokinetic studies revealed that mean basal levels achieved with continuous infusion prior to high-dose ara-C were 10(2) ng/ml and peak levels were of the order of 10(4) ng/ml. The considerable toxicity of the regimen, without clinical advantage over less intensive programmes, resulted in its termination.

The quantitative determination of 2'-deoxycytidine-5'-triphosphate in cell extracts by radioimmunoassay.

A radioimmunoassay (RIA) capable of quantitating dCTP in femtomolar amounts in cell extracts has been developed, and applied to human fibroblast cell lines and L5178Y mouse lymphoma lines. Cross reactivity of the antibody with CTP, though low (2.7%) has necessitated pre-RIA removal of CTP by either boronate affinity gel chromatography or sodium periodate oxidation. Fractions from the boronate gel column or aliquots of NaIO4-treated cell extract are quantitated directly by the RIA. Recovery of extracted dCTP standard taken through the entire procedure is quantitative and results are reproducible. Due to the high sensitivity of the quantitation step, dCTP can be accurately measured in relatively small numbers of cells--about 10(4) cells.

The pharmacokinetics of subcutaneous bolus cytosine arabinoside in an arachis oil plus aluminium distearate suspension.

An attempt was made to create a delayed release preparation of cytosine arabinoside (araC) which could be administered subcutaneously, and would produce plasma levels similar to steady state infusion concentrations. A thixotropic suspension of araC in arachis oil and aluminium distearate was formulated. This preparation was similar to that previously used with bleomycin oil suspension and procaine penicillin. Two hundred mg/ml of araC in arachis oil containing varying amounts of aluminium distearate were administered firstly to New Zealand White rabbits and then to patients with acute myelogenous leukaemia. This preparation was well tolerated by both rabbits and patients but did not delay the release of araC from the subcutaneous tissues.

Effect of dose and schedule on pharmacokinetics of high-dose cytosine arabinoside in plasma and cerebrospinal fluid.

The pharmacokinetics of high-dose cytosine arabinoside (ara-C) were studied in 18 patients with acute leukemia and high-grade non-Hodgkin's lymphoma. The plasma concentrations of ara-C increased in proportion to the dose over a range of 1-3 g/m2. The initial and terminal half-lives were not influenced by the dose or schedule of administration and no accumulation of ara-C occurred with repeated dosage in the same patients. These data suggest that cytidine deaminase is not saturated within this dose range. The cerebrospinal fluid (CSF) concentrations of ara-C also rose linearly with the increase in dose and varied from 347 ng/mL (1 g/m2) to 1,070 ng/mL (3 g/m2). The mean CSF concentrations of ara-C following high-dose infusions over three hours were 6%-22% of simultaneous plasma concentrations. Three hours after completion of the intravenous infusion the CSF concentrations were greater than the corresponding plasma concentrations owing to the long half-life of ara-C in CSF compared to that in plasma. These data demonstrate that therapy with intravenous high-dose ara-C given twice daily provides continuous levels in the CSF at concentrations that are likely to be of value in the treatment of central nervous system leukemia.

Relationship between protein binding and extravascular drug concentrations of a water-soluble drug, cytosine arabinoside.

The degree of binding of a drug to plasma proteins has a marked effect on its distribution, elimination, and pharmacological effect. Since only the unbound fraction is available for distribution into extravascular space, the ratio of drug in cerebrospinal fluid (CSF) or saliva to that in plasma is often regarded as a physiological measure of the free fraction of a drug. CSF: plasma and saliva: plasma ratios of cytosine arabinoside (araC) have been measured in patients with acute leukaemia and found to be 0.1-0.28, implying a binding of 72-90%. The protein binding of araC was measured by equilibrium dialysis in the plasma of patients with acute leukaemia at presentation. The mean binding ratio was 2.3 +/- 6.8, implying that there was little or no protein binding. There was no correlation between alpha-1 acid glycoprotein (AAG) levels and protein binding. The low CSF and saliva: plasma araC ratios found, suggest that drugs such as araC which have low lipid solubility do not pass freely into extravascular space. Thus the CSF or saliva: plasma ratio cannot be considered a good physiological measure of protein binding for drugs with poor lipid solubility.

Subcutaneous infusion of cytosine arabinoside. A practical alternative to intravenous infusion.

The administration of cytosine arabinoside (araC) by continuous IV infusion requires the patient to be in hospital and have prolonged IV cannulation. In this study the pharmacokinetics of araC during continuous IV infusion were compared with those of continuous SC infusion in six patients with acute myelogenous leukaemia. Each patient acted as his own control. The mean plasma levels of araC reached a plateau within 2 h and the plasma concentrations and the area under the curve were similar for both methods of administration. The mean area under the curve (AUC) was 1147 +/- 230 ng/ml for the IV infusion and 1017 +/- 238 ng/ml for the SC infusion. The plasma araC concentrations showed wide interpatient variation, and there was also considerable variability in the plasma concentrations of araC within the individual patients after a plateau had apparently been reached. Subcutaneous infusion was well tolerated by the patients without any local discomfort or excoriation and SC infusion of araC is thus a feasible alternative to IV infusion. It allows the patients the benefits of being at home, while avoiding unnecessary thrombophlebitis.

Evaluation of a commercially available radioimmunoassay kit for measurement of doxorubicin in plasma.

We evaluated a commercially available (Diagnostic Biochemistry Inc.) doxorubicin 125I radioimmunoassay kit. This kit gave a high apparent doxorubicin concentration (greater than 12 micrograms/L), which was not linearly related to dilution, for two pools of normal human serum and plasma and also for samples collected from patients before they received the drug. In contrast, a doxorubicin 3H radioimmunoassay developed by us gave a low blank (2 micrograms/L), which was linearly related to dilution, for the same pools and patients' samples. Doxorubicin concentrations in the plasma of patients receiving the drug were compared by the two methods; the kit gave results five- to 10-fold those obtained with our assay. High nonspecific interference by serum and plasma as measured by the 125I radioimmunoassay must therefore be borne in mind by users of the kit, and we suggest that results should be corrected for these nonspecific effects.

Controlled-release morphine tablets. A double-blind trial in dental surgery patients.

We report a randomized double-blind comparison of controlled-release morphine tablets (MST-1; 2 x 10 mg) and oral morphine sulphate in solution (20 mg) in 28 patients (20 females) who had undergone removal of impacted lower third molars or a dental clearance under general anaesthetic. The response in both groups was very poor: eight of 15 patients in MST-1 group and six of 13 patients in the standard group required "rescue" analgesics and were withdrawn from the study within the first 2h. No threshold plasma concentration of morphine corresponding to a particular analgesic effect was apparent. MST-1 produced significantly greater plasma concentrations at 8h compared with the standard preparation. Controlled-release morphine, or any oral formulation of morphine, may not be suitable for the treatment of acute pain after operation.

The pharmacokinetics of subcutaneous cytosine arabinoside in patients with acute myelogenous leukaemia.

1 The pharmacokinetics of subcutaneous cytosine arabinoside were compared with bolus intravenous injection and intravenous infusion in five patients with acute myelogenous leukaemia. 2 Subcutaneous cytosine arabinoside was rapidly absorbed and then declined biexponentially with initial and terminal half-lives similar to intravenous bolus injection. 3 Cytosine arabinoside levels declined rapidly after intravenous bolus and subcutaneous bolus injection, and fell below steady state infusion levels after a mean time of 40 min (intravenous bolus) and 100 min (subcutaneous injection).

Serum morphine concentration after oral administration of diamorphine hydrochloride and morphine sulphate.

1 Venous blood was obtained from patients with far-advanced cancer receiving either diamorphine (diacetylmorphine, heroin) hydrochloride (65 samples) or morphine sulphate (24 samples) regularly by mouth in doses from 2.5 mg to 90 mg every 4 h. 2 Samples were obtained within 30 min of the 09.00 h drug round. 3 Serial samples were also obtained over a 4 h period from three patients receiving diamorphine hydrochloride. 4 Assay of serum 'morphine equivalents' was by radioimmunoassay using an antibody that cross reacts almost equally with diamorphine, 6-0 monoacetylmorphine and morphine. 5 The serum concentration of opiates expressed as 'morphine equivalents' ranged from 11 ng/ml to 1440 ng/ml. 6 A highly significant positive linear correlation exists between the dose administered and the serum concentration (P less than 0.001) with respect to both drugs. 7 There was no difference between the two drugs in relation to the serum concentration achieved per 10 mg of opiate administered. 8 Higher oral doses of both diamorphine and morphine are now being used when indicated rather than, as before, resorting to injections when an oral dose in excess of 40 mg is indicated.

A radioimmunoassay for cytosine arabinoside.

A radioimmunoassay (RIA) for cytosine arabinoside (AraC) has been developed using antiserum raised in a sheep to an AraC monophosphate-ovalbumin conjugate. The antibody shows only 0.008% cross-reactivity with uracil arabinoside (AraU) and low (0.023%) cross-reactivity with other commonly co-administered drugs such as cytotoxic and antibacterial agents, and also a number of naturally occurring nucleosides and nucleotides. It does however cross-react by 125% with AraC monophosphate and by 109% with AraC triphosphate. As little as 1 ng/ml of AraC can be detected in plasma, serum, urine and cerebrospinal fluid (CSF) with no need for prior extraction. This RIA has been used to follow the disappearance of AraC from the plasma of patients receiving the drug.

Radioimmunoassay of methotrexate: use of 75Se-labelled methotrexate.

75Se-labelled methotrexate has been used as the radiolabel in a radioimmunoassay for methotrexate and its use compared with that of 3H-methotrexate. The gamma-labelled drug has proved to be extremely reliable and easier to use than 3H-methotrexate. Results obtained with it compare well with those obtained with tritiated drug. There are practical advantages in using a gamma emitter, and it is suggested that the modified radioimmunoassay is invaluable for routine monitoring of methotrexate in patients undergoing chemotherapy with the drug.