Chlorambucil Pharmacokinetics and DNA Binding in Chronic Lymphocytic Leukemia Lymphocytes1

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[CANCER RESEARCH 49, 554-559, February 1, 1989] Chlorambucil Pharmacokinetics and DNA Binding in Chronic Lymphocytic Leukemia Lymphocytes1

Bruce B. Bank2, Denise Kanganis, Leonard F. Liebes, and Robert Silber3

Department of Medicine, New York University Medical Center, New York, New York 10016

ABSTRACT damaging these macromolecules (10). Hill, using tritiated CLB, demonstrated the presence of radioactivity in DNA from Yosh- Chlorambucil (CLB) uptake by chronic lymphocytic leukemia lympho ida ascites sarcoma cells exposed to the labeled drug (9). Data cytes was studied using a radiometrie and a newly developed high- performance liquid chromatography assay. CLB labeled with 14Cin either supporting this concept are also available for cisplatinum, mel phalan, the nitrosoureas, mitomycin C, and busulfan (11-15). the chloroethyl group or phenyl ring was used with identical results. Drug accumulation by the cells was found to peak at 30 s, was independent We used CLB labeled either in the phenyl ring or chloroethyl of temperature, and was proportional to medium CLB concentration over groups to investigate drug-DNA binding in CLL lymphocytes. a wide range. Efflux from cells loaded with CLB and resuspended in Gel filtration and HPLC analysis of DNA modified by enzyme drug-free medium was nearly complete at 30 s. The metabolic inhibitors digestion or neutral thermal hydrolysis was carried out to 2-deoxyglucose and NaN3, the nitrogen mustard transport inhibitor hem- investigate the nature of drug-DNA binding. icholinium-3, and another alkylating agent, melphalan, had no effect on drug uptake. We conclude that CLB enters and exits chronic lymphocytic leukemia lymphocytes by simple diffusion. Cells from 17 patients with MATERIALS AND METHODS all stages of chronic lymphocytic leukemia were studied including three Lymphocyte Isolation with CLB-resistant disease, and no heterogeneity was found in the peak cell-associated CLB content or in metabolite pattern on high-performance Blood from 20 patients who gave informed consent was collected liquid chromatography. These findings make it unlikely that transport or into heparinized syringes. All but three of these patients were untreated cellular drug metabolism are factors in drug resistance. for at least 1 year or had never received therapy. The three treated Drug-DNA binding was found to be temperature-sensitive and in patients were included because they had clinically resistant disease. creased with time of incubation. Gel filtration of DNA before and after Lymphocytes were isolated using Ficoll-Hypaque (Pharmacia, Pisca- enzymatic digestion indicated the presence of drug-DNA adducts. High- taway, NJ) centrifugation (16). Cell counts and sizing were done on a performance liquid chromatography analysis of digested DNA and DNA Coulter ZBI Counter and Channelyzer (Coulter Electronics, Hialeah, treated by neutral thermal hydrolysis suggested the presence of multiple FL). All patients had monoclonal B-cells as determined by a panel of adducts. Most of the radioactivity was found as purine adducts. Studies markers including B-l, Leu-1, and T-l 1 antisera. with CLB labeled at two different sites revealed the presence of the Cell Incubation. Cells (lOVml) were incubated in RPMI1640 medium phenyl group and ethyl chains in the adducts. A survey of patients showed (GIBCO, Grand Island, NY) at pH 7.4 containing human serum increased drug-DNA binding in cells from patients with clinical CLB albumin (Pentex, Miles Laboratories, Kankakee, IL) at a concentration resistance. of 0.4 mg/ml. Cells were counted and their viability tested by erythrosin B exclusion before incubation and at each time point. Cell viability exceeded 90% in all experiments. INTRODUCTION Drug Uptake and Efflux. CLB, melphalan, sodium azide, and hemi- CLB4 is an alkylating agent widely used in the treatment of cholinium-3 were purchased from Sigma, St. Louis, MO. [14C]CLB, patients with CLL. The drug has been shown to cause a reduc labeled in the phenyl ring, or in the chloroethyl group, was obtained from Amersham, Arlington Heights, IL, and from the National Cancer tion in the circulating lymphocyte count and total mass of Institute. The drug was purified before use by HPLC with an isocratic lymphoid tissue, while ameliorating symptoms in a majority of elution using solvent B (see below). Radiochemical purity after this previously untreated patients (1-4). Although CLB has been in procedure was found to be 90-95%. CLB was dissolved in dimethyl use for over 30 years, factors underlying drug action and resist sulfoxide, and was added to the cell suspension avoiding an organic ance are incompletely understood. solvent concentration of more than 0.1%. Uptake was usually studied Previous reports by Begleiter and Goldenberg using LSI78Y at a 330 fiM drug concentration (100 Mg/m')- Most experiments were (5) and by Hill, Harrap, and coworkers who investigated Yosh- performed at this high concentration to optimize the HPLC signal, but ida ascites sarcoma cell lines and CLL lymphocytes (6-9) experiments performed with 33 pM CLB showed similar results. So indicated that CLB uptake occurs by simple passive diffusion. dium azide was used at 10 HIM,and 2-deoxyglucose at a 5 HIMconcen tration. Melphalan stock was made as described (17). Hemicholinium- The present study combines a radiometrie with a newly devel 3 was dissolved directly in the medium to a final concentration of 100 oped HPLC technique, to further investigate this process in MM.Cells were preincubated with inhibitors for 15 min at 37Â°C. CLL lymphocytes. To measure drug efflux, cells were "loaded" by incubating with CLB Many authors attribute the chemotherapeutic action of al for 30 s, centrifugea at 1,100 x g for 1 min and then transferred to kylating agents to their ability to bind to DNA in target cells, drug-free medium. Quantitation of Cell-associated |14C|CLB. A modification of the Received 8/8/88; revised 10/17/88; accepted 10/27/88. The costs of publication of this article were defrayed in part by the payment method described by /.welling et al. (18) was used. Aliquots (0.3 ml) of of page charges. This article must therefore be hereby marked advertisement in cell suspension were layered on 0.4 ml of Versilube F-50 silicone oil accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (General Electric, Waterford, NY) in 1.5 ml conical plastic Eppendorf ' This work was supported by a grant from the NIH (CAI 1655) and by a grant tubes; after centrifugation at 4,400 x g for 1 min, the supernatant was from the Harry Winston Research Foundation. 2An American Cancer Society Fellow. Current address: Department of Med decanted and the inside of the tube blotted dry. The tip was then severed icine, UMDNJâ€”New Jersey Medical School, 185 South Orange Avenue, New and placed overnight in a counting vial containing 2 ml of 0.3 N KOH. ark, NJ 07103. Radioactivity was measured in a Beckman LS 7000 scintillation counter 3To whom requests for reprints should be addressed, at Department of following the addition of 15 ml of acidified Aquassure (New England Medicine, New York University Medical Center, 560 First Avenue, New York, Nuclear, Boston, MA). We determined medium trapping with [3H]- NY 10016. 4 The abbreviations used are: CLB, Chlorambucil; CLL, chronic lymphocytic inulin to be 0.6 Ml/sample; this volume was corrected for in all calcu leukemia; HPLC, high-performance liquid chromatography. lations. 554

Extraction of CLB for HPLC. The extraction procedure is a modifi patient with clinical CLB resistance was similar to that obtained cation of the one described for plasma by Ahmed et al. (19). The cell with cells from untreated patients. suspension (0.3 ml) was layered on 0.4-ml Versilube F-50 in 3-ml Pyrex As shown in Table 1, 2-deoxyglucose, sodium azide, hemi- conical tubes and centrifuged at 4,000 x g for 1 min. The supernatant cholinium-3, and melphalan, had no effect on the maximal was decanted, the inside of the tube blotted dry, and 1 ml of water amount of cell-associated drug. These data suggest that CLB added. The cells were dispersed by agitation on a Vortex Genie (Sci entific Industries, Bohemia, NY) and allowed to stand for IS min at influx occurs via passive diffusion. This conclusion is further 4Â°C;1.5 ml of acetonitrile containing 0.2% acetic acid was then added, supported by the linear and unsaturable drug accumulation over the mixture agitated again and frozen on dry ice. The tubes were then centrifuged at 4,000 x g for 5 min. One ml of the organic phase was transferred to glass test tubes and taken to dryness in a Speed-Vac concentrator (Savant Instruments, Hicksville, NY). Each sample was redissolved in 80 n\ of solvent B (see below), of which 50 ^1was injected into the HPLC apparatus. Control experiments showed that recovery of CLB standards was between 95 and 105%. Medium trapping using [3H]inulin was quantified at approximately 2 ^I/sample, accounting for 10-20% of the peak cell-associated CLB. The technique described above yielded organic, aqueous and solid phases. In some experiments, this method was applied to cells incubated with radioactive drug, and a sample of each phase removed for scintil 0.5 30 lation counting, to assess the partitioning of label in the cells. Time (minutes) In selected experiments following centrifugation of the cell suspen Fig. 1. Effect of temperature on CLB concentration in medium. Cells were sion through oil, 200 Â¡Aofmedium supernatant were extracted with incubated in RPMI 1640 with 0.4 mg/ml human serum albumin. The CLB 400 p\ of acetonitrile/0.2% acetic acid, using the same technique as for concentration was measured by HPLC and expressed as a percentage of the initial value, at 37'C (â€¢)and at 4Â°C(O). Results shown are an average of three cell lysates, to quantify CLB concentration. Quantitation of CLB by HPLC. All HPLC measurements were per experiments. formed on a Spectra Physics SP8000B apparatus equipped with a 5- ium end-capped Nucleosil 4.5 x 250 mm column. Solvent A was acetonitrile:water:acetic acid, 125:375:1, v/v/v. Solvent B was acetoni- 30 trile:water:acetic acid, 375:125:1, v/v/v. The gradient was developed as follows: 100% A x 3 min; linear 100% A to 73% B x 2 min; linear 73% B to 100% B x 8 min; 100% B x 2 min; linear 100% B to 100% S15 A x 2 min; 100% A x 3 min. Absorbance at 254 nm was measured. CLB had a retention time of 16 to 17 min under these conditions. A standard curve was linear over the range of 10 ng to 5 Mg- Resistant CLL Drug-DNA Interaction. DNA from CLL lymphocytes incubated with 0.5 30 60 [14C]CLBwas extracted using the phenol/chloroform method (20). The Time (minutes) DNA content was determined spectrophotometrically by the Ajeo-The Fig. 2. Effect of temperature on [I4C]CLB accumulation. Cell-associated bound CLB was measured in a liquid scintillation counter. The DNA [MC]CLB over time at 37'C (â€¢)and 4'C (O) with cells from untreated patients was applied to a 1 cm x 25 cm column of Sephadex (.Â¡-25and eluted (average of two experiments) compared to uptake by lymphocytes from a patient with clinically resistant CLL (A â€”¿A).Results corrected for average cell volume with phosphate buffered saline. and initial medium drug concentration. DNA was digested sequentially with DNase I, snake venom phosphodiesterase and alkaline phosphatase, according to Tomasz et al. Table 1 Effect of inhibitors on CLB uptake (21). The digest was Tillered through G-25 as described above. HPLC Cells were incubated as described in "Methods" for 30 s at 37'C. Uptake in analysis was carried out on a 1 cm x 25 cm semipreparative 5 um C-18 the presence of each inhibitor is expressed as a percentage of uptake after control column with a 1 cm x 5 cm guard column packed with the same 5-f/m incubation. material. To release purine-drug adducts, DNA was heated in a boiling CLB uptake bath as described (22), passed through a 0.2-^m Nalgene syringe filter Inhibitor (% of control) and injected into the HPLC apparatus. As a control, an HPLC elution 81.2Â±4.3%(A'= 2) profile was obtained on [I4C]CLB in aqueous medium and heated in 2-Deoxyglucose (5 mM) NaNj(lOmM) 85.3 Â±14.0% (N =5) the same way as the DNA. Hemicholinium-3 (100 nM) 107.3 Â±3.5% (N =2) Solvent C was acetonitrile:0.02 M potassium phosphate, pH 5.0, Melphalan (1:1 molar ratio) 95.6 Â±4.0% (N = 3) 90.1 Â±7.1%(/V=2) 8:92, v/v; Solvent D was acetonitrile:0.02 M potassium phosphate, pH Melphalan (2:1 molar ratio) 5.0, 50:50, v/v. The mobile phase was: 100% C x 25 min, linear 100% C to 100% D x 60 min, 100% D x 5 min, linear 100% D to 100% C x 5 min. 51,000 All experiments were performed at least twice.

RESULTS

Drug Influx and Efflux. CLB was found to be stable in RPMI 1640 containing 0.4 mg/ml HSA at 4"C, but not at 37Â°C,where the half-life was observed to be approximately 35 min (Fig. 1). Maximal cell-associated drug was observed within 30 s of incubation and was independent of temperature (Fig. 2). A 1.5 10 50 250 rapid decrease in cellular drug occurred with time at 37Â°C;at CLB (//g/ml) Fig. 3. Effect of concentration on cellular drug accumulation. Measurements 4"C, a small decline also took place, consistent with nonenzy- were obtained at 30 s of incubation at 37'C. Results of two experiments are matic catalysis. The pattern of drug uptake by cells from a shown. 555

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1989 American Association for Cancer Research. CLB UPTAKE AND DNA BINDING IN CLL LYMPHOCYTES a wide concentration range (Fig. 3). There was nearly immedi CLB resistance is not explained by differences in intracellular ate, temperature- and energy-independent efflux of drug from drug metabolism. Furthermore, as shown in Table 2, water- cells transferred to drug-free medium after loading with CLB soluble metabolites and presumably drug-macromolecule com (Fig. 4). These results indicated that influx and efflux of CLB plexes are generated, as indicated by the partitioning of radio occur via passive diffusion without receptor mediation. activity into the aqueous and solid phases after a 60 minute The peak cell-associated CLB was determined for the lym incubation at 37Â°C,but not at 4Â°C. phocytes from 17 patients (four with stage 0, four with stage 1, Drug-DNA Interaction. Since cellular DNA is commonly seven with stage 2, one with stage 3, one with stage 4). There believed to be the target of alkylating drugs, the binding of CLB was no significant heterogeneity, including three patients with and/or a metabolite to DNA was investigated. Radioactivity clinical drug resistance. Peak cell-associated drug in lympho bound to DNA increased over time in cells incubated for 2 h cytes from previously untreated patients was 19.9 Â±6.2 pmol/ with [14C]CLB at 37Â°Cbut not at 4Â°C(Fig. 7). This pattern was IO7cells by HPLC (JV= 11, mean Â±SD), 15.0 Â±2.6 pmol/107 in contrast to the time-dependent decrease in total cell-associ by radioassay (N = 16), and 16.5 Â±3.0 pmol/107 cells (N = 3) ated CLB seen in Fig. 2. Drug-DNA interaction at 60 min of by radioassay for lymphocytes from subjects with drug-resistant incubation at 37Â°Cwas found to increase with CLB concentra CLL. The above values are normalized for average cell volume tion (Fig. 8). No saturation was seen over a wide range, sug and corrected for initial medium CLB concentration. gesting a large excess of sites susceptible to alkylation. Drug Metabolism. A newly developed HPLC assay was uti Further characterization of this interaction revealed that the lized to investigate the intracellular fate of CLB. When cell- radioactivity was closely associated with macromolecular DNA associated drug was measured simultaneously by both methods when eluted on a column of Sephadex G-25; however, after (Fig. 5) similar values were obtained at 30 s but the amount of digestion of the DNA with DNase I, snake venom phosphodi- drug measured by radioassay exceeded that determined by esterase, and alkaline phosphatase, a different elution pattern HPLC at the later time points. This consistent discrepancy can was seen (Fig. 9). All of the radioactivity was eluted in two be explained by the inability of the radioassay to discriminate broad peaks in the internal volume of the column; two peaks of between the parent compound and its metabolites and adducts. material with a high absorbance at 260 nm, corresponding to Analysis of the organic phase by simultaneous measurement purines and pyrimidines, as determined by standards, were of A254and radioactivity during HPLC (Fig. 6) showed the noted. Identical results were obtained using [I4C]CLB labeled concurrent formation of two radioactive derivatives in medium in the phenyl ring or the chloroethyl groups. The above data and cells. No other metabolites were detected at 5 min and 30 suggest that covalent binding occurs between DNA and CLB min (data not shown). The material in the peak designated A and/or its metabolites. This interaction is temperature-sensitive was analyzed by mass spectrometry and found to contain no chloride moieties (data not shown); it was therefore considered to be a nonreactive product of spontaneous hydrolysis of the

Medium ot Medium ot chloroethyl groups. This characteristic pattern of metabolite 0.5mm fiOfnin formation was observed for all CLL cells studied, regardless of clinical resistance to CLB. These results suggest that metabo lism occurs by simple chemical breakdown of the drug and that

:= 400- II

Cells 01 60mm

10 20 30 Time (minutes) Fig. 4. Efflux of CLB. Cells were exposed to ['"CJCLB for 30 s, then centrifuged and resuspended in drug-free medium. Cellular radioactivity in cells resuspended at 37Â°C(â€¢ â€¢¿)and4'C (O O) is compared to control cells whose 10 20 1 10 20 incubation was continued in the presence of [UC]CLB at 37Â°C(â€¢ â€¢¿)and4Â°C Time (minutes) (O O). Results shown are representative of two experiments. Fig. 6. HPLC pattern of radioactivity and absorbance at 254 nm in medium and cell extracts. Top left, medium at 30 s; bottom left, cells at 30 s; top right, medium at 60 min; bottom right, cells at 60 min. Incubations were performed at 37Â°C.The mobile phase used is described in "Methods." Absorbance (full scale)

JO = 0.040. The peak of absorbance at the extreme left was found in cells incubated without drug, and therefore was of no relevance to CLB metabolism.

Table 2 Partitioning of label in cell extracts m Â£15 After incubation with [14C]CLB as described in "Methods," cells were lysed o i and extracted with acetonitrile. Radioactivity in each phase is expressed as a percentage of the total. 37'C 4-C

30 60 PhaseOrganicAqueousSolid0.5 Time (r min97%1.5%1.5%60min61%min 0.5 min97%1%2% Fig. 5. Cell-associated CLB as a function of time. Cells were incubated at 98%18% 37Â°C,and CLB as measured by HPLC (â€¢)and radioassay (O). A representative 1%21% experiment is shown. 1%60 556

0100%Solvent 400 400300 -^^ \* Enzymes ^ ^^T ^^'^

200 200100^ ' 100 1*â€ž /*J -Enzymes , . .-â€ž'."., ~!&^Â¿^^~~^S -- ^Solrtnl Cinni

0.5 60 120 15 10 15 20 25 30 35 45 50 55 Time of Incubotion (min) Fraction Number Fig. 7. Effect of temperature on drug-DNA binding. The amount of radioac tivity found in DNA from cells incubated with ['"CJCLB at 37Â°C(â€¢)and4Â°C(O). No significant drug-DNA binding occurred at 30 s and 37'C. as found in another expe riment (O ).

85 30 35 40 50 55 60 Fraction Number Fig. 10. Separation of drug-DNA adducts. DNA was treated as in Fig. 9, and analyzed by HPLC. Top, the patterns of digested (â€¢)and undigested (O) DNA are shown. The mobile phase is represented by the daiAerf line, solvents C and D 1,000 are described in "Methods." Bottom, DNA subjected to a boiling bath for 10 min CLB in neutral buffer (â€¢).The pattern obtained with ['"CjCLB in aqueous medium Fig. 8. Effect of CLB concentration on DNA binding. [I4C]CLB associated treated in the same way is shown (O). with DNA from cells incubated for 60 min at 37Â°Cwithincreasing concentrations of drug. Results of two experiments are shown. Table 3 binding DNA)"32.6(ng CLB/mg - 200 Patient1234567891011121314151617*18*19"20*Stage12111022104000104442Drug-DNA 2)30.9Â±5.9 (N = 2)57.7Â±3.3 (N = (N =1)34.5 2)68.4Â±4.2 (N = Â±37.4Â±24.2 (N = 3, mean (/V=1)47.0 2)36.8Â±19.9 (/V= 2)61.3Â±3.7 (/V= meanÂ±59.3Â±37.1 (/V=3, 5 10 15 20 25 30 35 40 Â±13.4(yV=2)51.4 Tube Number 2)36.7Â±2.1 (A'= (N=1)42.2 (/V=1)29.7 1.0 200 (N =1)59.0 2)77.4Â±2.6 (N = (/V=1)110.0Â± 2)75.4 11.6(JV= '0.5 100o~ Â±84.7Â±18.1 (N= 6, mean (JV=1)91.9 (N = 1)SD)SD)SD) " The amount of drug-DNA binding was measured in lymphocytes incubated 60 min at 37Â°C. 10 15 20 25 30 35 40 * Patient with clinically CLB-resistant CLL. Tube Number Fig. 9. Characterization of drug-DNA binding. DNA was extracted from CLL adducts from alkylated DNA (22). Again, more than one peak lymphocytes incubated with [I4C]CLB and eluted on Sephadex G-25 with PBS. was observed, and nearly all the radioactivity was eluted in the Top, undigested DNA; bottom, the same DNA following digestion as described in "Methods." two peaks seen. To determine that these peaks represented drug-DNA adducts, and not simply CLB metabolites, [14C]CLB was incubated in RPMI 1640, heated at 90Â°Cand analyzed by and both the ethyl groups and phenyl ring of the drug molecule are present in the resulting adduci. HPLC in the same manner as the DNA. A clearly different DNA exposed to DNase I, snake venom phosphodiesterase, elution profile was obtained, as shown in Fig. 10, bottom. The and alkaline phosphatase is degraded to nucleosides (21). If above data demonstrate the presence of multiple adducts and alkylated DNA is degraded, complexes between nucleosides indirectly indicate that most of these are purine-drug com and the alkylator moiety are released. HPLC analysis of the plexes. lymphocyte DNA after digestion showed two major and two Table 3 summarizes the quantitation of drug-DNA interac minor peaks of radioactivity, most likely representing drug- tion in cells from 20 patients. There was heterogeneity among nucleoside adducts (Fig. 10, top). The same pattern was ob these subjects; cells from patients with clinical resistance (Nos. tained regardless of the location of label in the [14C]CLB. HPLC 17, 18, 19, 20) had more radioactivity bound to the DNA than analysis was also performed on undigested DNA subjected to from untreated patients. In lymphocytes from untreated pa heating at 90Â°Cfor 10 min (Fig. 10, bottom). This process has tients, no correlation was found between drug-DNA binding been shown to cause the release of guanosyl and adenosyl and clinical stage. 557

DISCUSSION forming cross-links. This type of mechanism has been demon strated for several other alkylating agents (12, 14-15). The results presented above characterize two aspects of the By utilizing the neutral thermal hydrolysis technique de pharmacokinetics of CLB in CLL lymphocytes: drug uptake scribed by Beranek, et al. (22), we found that most of the and interaction with DNA. radioactivity in the DNA appeared to be present in purine Several lines of evidence support the concept that CLB enters adducts. This is analogous to the case of busulfan and mito- CLL lymphocytes by passive diffusion. Drug accumulation was mycin C (14, 15), two other alkylating agents. immediate, unsaturable over a wide concentration range and In the survey of CLL patients, we found a higher amount of temperature-independent. Neither sodium azide nor 2-deoxy- drug-DNA binding in cells from patients with clinically resis glucose had any effect on CLB uptake. Cellular uptake of CLB tant disease compared to untreated patients. This finding is in is through a different mechanism than that of nitrogen mustard contrast with results in Yoshida ascites sarcoma cells selected and melphalan, two other alkylating agents. It was not affected for CLB resistance (8). The cause for this unexpected result is by hemicholinium-3, an inhibitor of the choline transport sys unknown. If, as our data suggest, metabolic activation is nec tem by which nitrogen mustard is transported into LSI78Y essary for adduct formation, the above result could be explained cells (23), nor by melphalan, a drug transported via the L amino if the lymphocytes from patients with more advanced disease acid system (17). These results are in keeping with previous contained a higher percentage of metabolically active cells. reports about CLB uptake in Yoshida ascites sarcoma (6), CLL Also, cytotoxicity has been linked to cell cycle for several (7), and murine LSI78Y cells (5). As could be anticipated from alkylating agents (24-26). It is conceivable that populations of a passive diffusion process no differences in drug uptake were cells isolated from subjects with CLB-resistant CLL vary from observed between cells from patients with resistant CLL and the others in the proportion of lymphocytes in each phase of untreated patients. the cell cycle, accounting for the differences in drug-DNA In the present studies, isotope measurements were performed binding. in conjunction with a newly developed HPLC assay which discriminates between parent drug and other metabolites. This method thus provides more information than that obtainable ACKNOWLEDGMENTS by the older spectrophotometric assays (6, 7). Aqueous hydrol We are grateful to Dr. John Essigmann for helpful suggestions. ysis was found to be the only pathway of drug metabolism. Similar elution patterns were observed with cell extracts from untreated subjects as well as from patients with CLB-resistant REFERENCES disease. This suggests that differences in cellular metabolism of 1. Boggs, D. R., Soffermali, S. A., Wintrobe, M. M., and Cartwright, G. E. CLB are not the basis for clinical drug resistance and contrasts Factors influencing the duration of survival of patients with chronic lympho cytic leukemia. Am. J. Med., 40: 243-254, 1966. with an earlier report of a correlation between more rapid 2. Ezdinli, E. /.., and Stutsman, L. Chlorambucil therapy for lymphomas and intracellular breakdown of the phenyl ring and CLB resistance chronic lymphocytic leukemia. J. Am. Med. Assoc., 191(6): 100-106, 1965. in CLL lymphocytes (7). Another study of CLB uptake in 3. Gallon, D. A. G., Wiltshaw, E., Szur, L., and Dacie, J. V. The use of Chlorambucil and steroids in the treatment of chronic lymphocytic leukemia. 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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1989 American Association for Cancer Research. Chlorambucil Pharmacokinetics and DNA Binding in Chronic Lymphocytic Leukemia Lymphocytes

Bruce B. Bank, Denise Kanganis, Leonard F. Liebes, et al.

Cancer Res 1989;49:554-559.

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