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Clinical Cancer Research Vol. 8, 1224-1233, May 2002
© 2002 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

In Vivo Pharmacokinetic Features, Toxicity Profile, and Chemosensitizing Activity of {alpha}-Cyano-ß-hydroxy-ß- methyl-N-(2,5-dibromophenyl)propenamide (LFM-A13), a Novel Antileukemic Agent Targeting Bruton’s Tyrosine Kinase

Fatih M. Uckun1, Yaguo Zheng, Marina Cetkovic-Cvrlje, Alexei Vassilev, Elizabeth Lisowski, Barbara Waurzyniak, Hao Chen, Randy Carpenter and Chun-Lin Chen

Parker Hughes Cancer Center [F. M. U., M. C-C., A. V., C-L. C.], Departments of Pharmaceutical Sciences [H. C., C-L. C.], Chemistry [Y. Z.], Experimental Oncology [M.C-C., A. V., E. L.], and Pathology [B. W., R. C.], and the Drug Discovery Program [F. M. U.], Parker Hughes Institute, St. Paul, Minnesota 55113


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The purpose of the present study was to examine the in vivo pharmacokinetics and activity of {alpha}-cyano-ß-hydroxy-ß-methyl-N-(2,5-dibromophenyl)propenamide (LFM-A13), a novel antileukemic agent targeting Bruton’s tyrosine kinase (BTK). We have applied an analytical high-performance liquid chromatography method for the quantitative detection of LFM-A13 in plasma samples. Our findings indicate that LFM-A13 is quickly absorbed, with the time required to reach the maximum plasma drug concentration (tmax) being 10–18 min after i.p. administration with nearly complete bioavailability. LFM-A13 had an elimination half-life of 17–32 min after i.p. administration at dose levels of 10–50 mg/kg. LFM-A13 exhibited a dose-dependent and significant increase in the values of normalized area under the curve and maximum concentration (Cmax) as well as a dose-dependent and significant decrease in clearance values, suggesting a saturable clearance mechanism. LFM-A13 was not toxic to mice when administered systemically at dose levels ranging from 10 to 80 mg/kg. Highly effective BTK-inhibitory and apoptosis-promoting plasma concentrations of LFM-A13 could be achieved in mice without toxicity. LFM-A13 exhibited a favorable pharmacokinetic behavior that was not adversely affected by the standard chemotherapy drugs vincristine, methylprednisolone, or L-asparaginase (when used as combination treatment, VPL) and significantly improved the chemotherapy response and survival outcome of mice challenged with BCL-1 leukemia cells. Whereas only 14% of mice treated with the standard triple-drug combination VPL became long-term survivors, 41% of mice treated with this combination plus LFM-A13 survived long-term. LFM-A13 prolonged the median survival time of VPL-treated mice from 37 to 58 days. Our results confirm and extend previous studies regarding the role of BTK chemotherapy resistance of B-lineage leukemic cells (S. Mahajan et al., J. Biol. Chem., 274: 9587–9599, 1999). BTK inhibitors such as LFM-A13 may be useful as a new class of chemosensitizing and apoptosis-promoting antileukemic agents for treatment of patients with chemotherapy-resistant B-lineage leukemias or lymphomas.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis is a common mode of eukaryotic cell death that is triggered by an inducible cascade of biochemical events leading to activation of endonucleases that cleave the nuclear DNA into oligonucleosome-length fragments (1, 2, 3, 4) . Several of the biochemical events that contribute to apoptotic cell death as well as both positive and negative regulators of apoptosis have been identified recently (1, 2, 3, 4) . Apoptosis plays a pivotal role in the development and maintenance of a functional immune system by ensuring the timely self-destruction of autoreactive immature and mature lymphocytes as well as any emerging target neoplastic cells by cytotoxic T cells (1, 2, 3, 4, 5, 6, 7) . Inappropriate apoptosis may contribute to the development as well as chemotherapy resistance of human leukemias and lymphomas (5, 6, 7) .

BTK,2 a member of the BTK/Tec family of protein tyrosine kinases that includes TecI, TecII, Itk, Bmx/Etk, and DSrc28C (found in Drosophila; Ref. 8 ), is a cytoplasmic protein tyrosine kinase involved in signal transduction pathways regulating growth, differentiation, and survival of B-lineage lymphoid cells (9, 10, 11) . Recent observations suggest the involvement of BTK in signal transduction pathways affecting gene transcription (12) . Recent studies have further revealed a nucleocytoplasmic shuttling system for BTK that has implications regarding potential targets inside the nucleus, which may be critical in gene regulation during B-cell development and differentiation as well as apoptosis (13) . The recognition of an antigen by the BCR triggers a signal transduction cascade that culminates in activation of multiple genes controlling activation, proliferation, differentiation, and survival of B cells. BCR stimulation leads to the activation of transcription factor nuclear factor-{kappa}B, which in turn regulates genes controlling B-cell growth. BTK is essential for the BCR-mediated activation of the nuclear factor-{kappa}B/Rel family of transcription factors (14 , 15) . BTK has also been shown to regulate the nuclear localization and transcriptional activity of the multifunctional transcription factor BAP-135/TFII-I (16) . BAP-135/TFII-I is a ubiquitously expressed multifunctional transcription initiation factor capable of binding to several promoter elements, including initiator elements (Refs. 17, 18, 19 ; e.g., VpreB, TdT, and possibly RAG, CD5, Bcl-2, and Bcl-xL), that is tyrosine phosphorylated in B cells after engagement of the BCR (20) . Most recently, we discovered a functional interaction between the transcription factor STAT5A and BTK (21) . BTK is the first cytoplasmic non-Janus kinase to be identified as a positive regulator of STAT5A in B cells. Our findings point to a novel pathway through which B-cell-specific signals mediated by BTK might communicate with target genes via STAT5A.

In murine B cells, BTK has been shown to act as an antiapoptotic protein upstream of bcl-xL in the BCR (but not the CD40 receptor) activation pathway (22) . Our recent studies provided biochemical and genetic evidence that BTK is an inhibitor of the Fas/APO-1 death-inducing signaling complex in B-lineage lymphoid cells (23) . Furthermore, we found that BTK also prevents ceramide- and VCR-induced apoptosis (24) . The fate of leukemia/lymphoma cells may reside in the balance between the opposing proapoptotic effects of caspases activated by the death-inducing signaling complex and an upstream antiapoptotic regulatory mechanism involving BTK and/or its substrates (23) .

In a systematic effort to design potent inhibitors of BTK as antileukemic agents with apoptosis-promoting properties, we have constructed a novel three-dimensional homology model of the BTK kinase domain (24) . Advanced docking procedures were used for the rational design of LFM analogues with a high likelihood to bind favorably to the catalytic site within the kinase domain of BTK. We reported recently the identification of the LFM analogue LFM-A13 as a potent and specific inhibitor of BTK (24) . LFM-A13 is the first BTK-specific tyrosine kinase inhibitor and the first antileukemic agent targeting BTK (25 , 26) . LFM-A13 inhibited purified recombinant BTK in vitro with an IC50 of 7.5 µM. Besides its remarkable inhibitory activity in cell-free and cellular BTK kinase assays, LFM-A13 was also discovered to be a highly specific inhibitor of BTK. Even at concentrations as high as 300 µM, this novel inhibitor did not affect the enzymatic activity of other protein tyrosine kinases, including JAK1, JAK3, HCK, epidermal growth factor receptor kinase, and insulin receptor kinase. Notably, treatment of leukemic cells with LFM-A13 resulted in abrogation of BTK activity at concentrations >=10 µM. LFM-A13 disrupted BTK-Fas association and rendered resistant leukemic cells sensitive to Fas-mediated apoptosis (23) . In accordance with the antiapoptotic function of BTK, treatment of BTK+ B-lineage leukemia cells with LFM-A13 enhanced their sensitivity to VCR- and ceramide-induced apoptosis (24) . Therefore, LFM-A13 shows potential as an antileukemic agent with apoptosis-promoting and chemosensitizing properties. The purpose of the present study was to evaluate the pharmacodynamic features of this promising antileukemic agent in BALB/c mice challenged with chemotherapy-resistant B-lineage leukemia cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Deionized water was prepared through a Milli-Q purification system (Millipore Corp., Medford, MA). Methanol, acetonitrile, and ammonium phosphate were obtained from Fisher Chemicals (Fair Lawn, NJ). All other chemicals were either HPLC grade or analytical grade.

Synthesis and Characterization of LFM-A13.
LFM-A13 was synthesized and characterized as described previously (24, 25, 26, 27) . Stock solutions of LFM-A13 were prepared in methanol and stored at -20°C. These stock solutions were diluted further in 90% methanol to yield appropriate working solutions for the preparation of standards to calibrate.

Analysis of BTK Expression Levels in BCL-1 Leukemia Cells.
BCL-1 cells, BALB/c splenocytes, and NALM-6 human B-lineage ALL cells treated for 18 h at 37°C with vehicle or 100 µM LFM-A13 were lysed in 1% TX 100 lysis buffer (150 mM NaCl, 15 mM EGTA, 1% Triton X-100, 3% sodium deoxycholate, 0.3% SDS, 3 mM phenylmethylsulfonyl fluoride, 100 µM Na3VO4, 60 µg/ml leupeptin, 60 µg/ml aprotinin, and 50 mM Tris-HCl, pH 7.4) for 20 min at 4°C. Whole cell lysates were examined for BTK expression levels by immunoblotting and enhanced chemiluminescence (Amersham, Arlington Heights, IL) on polyvinylidene difluoride membranes (Millipore, Bedford, MA), as reported previously (21 , 23 , 24) . In parallel, BTK was immunoprecipitated from whole-cell lysates, and immune complex kinase assays using 10 µCi of [32P]ATP were performed to examine its enzymatic activity as measured by autophosphorylation and expressed in phosphorimager units as well as activity index, as described previously (21 , 23 , 24) . The phosphorimaging of kinase gels dried onto Whatman 3M filter papers was performed on a Molecular Imager (Bio-Rad, Hercules, CA), as reported previously (24) .

HPLC Determination of Plasma LFM-A13 Levels.
Plasma LFM-A13 levels were determined by an analytical HPLC method. In brief, for extraction and determination of LFM-A13 levels in plasma, 75 µl of methanol were added to a 25-µl plasma sample, and the mixture was vortexed thoroughly for 1 min. After centrifugation (300 x g for 5 min), the supernatant was used for HPLC analysis using a system composed of a Hewlett Packard series 1100 instrument equipped with an automated electronic degasser, a quaternary pump, an autosampler, an automatic thermostatic column compartment, diode array detector, and a computer with a Chemostation software program for data analysis (28, 29, 30) . Acetonitrile:ammonium phosphate buffer (10 mM, pH 3.7; 35:65, v/v) was used as the mobile phase for separating LFM-A13. The analytical column (Lichrospher RP-18) was equilibrated and eluted under isocratic conditions using a flow rate of 1.0 ml/min at ambient temperature. The wavelength of detection was set at 294 nm. All extraction procedures were carried out at room temperature.

Animals.
Female BALB/c mice from Taconic (Germantown, NY) and CD-1 mice from Charles River Laboratories (Hartford, CT) were housed in a controlled SPF environment (12-h light/12-h dark photoperiod, 22 ± 1°C, 60 ± 10% relative humidity), which is fully accredited by the United States Department of Agriculture. All mice were housed in microisolator cages (Lab Products, Inc., Maywood, NY) containing autoclaved bedding. All male Lewis rats used in the toxicity studies were obtained from the SPF breeding facilities of Harlan Sprague Dawley (Indianapolis, IN) at 14 weeks of age. All husbandry and experimental contact made with the rats maintained SPF conditions. The rats were kept in microisolater cages (Allentown Caging Equipment Co., Inc., Allentown, NJ) containing autoclaved food, water, and bedding. Animal studies were approved by Parker Hughes Institute Animal Care and Use Committee, and all animal care procedures conformed to the Guide for the Care and Use of Laboratory Animals (31) .

Murine Leukemia Model.
In this study, we used a highly aggressive subclone of murine BCL-1 B-lineage leukemia, reported previously (32) . BCL-1 leukemia was first described by Slavin and Strober (33) . The parent BCL-1 cell line was generously provided by Drs. Jonathan Uhr and Ellen Vitetta at the University of Texas Health Sciences Center (Dallas, TX) and maintained in vivo by i.p. passage in BALB/c mice (32) . Two weeks after i.p. inoculation of 1 x 106 BCL-1 cells in 0.2 ml of PBS, mice were killed, and their splenocytes were used as a source of BCL-1 leukemia cells for further passage. Spleens of leukemic mice with massive hepatosplenomegaly were removed using sterile techniques. A tissue grinder was used to gently prepare cell suspensions from small spleen pieces. After the clumps and debris were allowed to settle, the cell suspensions were passed through a fine-pore nylon cell strainer (100 µm; Becton Dickinson) and Pasteur pipettes to prepare single-cell suspensions. Erythrocytes were lysed using a 45-s wash with distilled water, their membranes were removed by centrifugation, and erythrocyte-depleted BCL-1-enriched splenocyte suspensions were suspended in PBS and used in the experiments described. The standard inoculum consisted of 1 x 106 cells in 0.5 ml of RPMI 1640 with L-glutamine (Cellgro). When BALB/c recipients reached the end stages of their progressive leukemia and became moribund, they were sacrificed by CO2 inhalation. The survival outcome of mice in the different treatment groups was subjected to comparative statistical analysis using standard life table methods (34) .

Drug Treatment.
After i.v. injection of BCL-1 cells, BALB/c mice were given i.p. injections of a single drug or a combination of drugs in 0.2 ml of PBS. VCR (Vincasar PFS, 1 mg/ml; Pharmacia, Inc., Kalamazoo, MI) was given at 0.05 mg/kg once/week. L-ASP (Elspar, 2000 IU/ml after resuspension in 5 ml of distilled H2O; Merck & Co., Inc., West Point, PA) was given at 200 IU/kg/day, three times/week. MP (Depo-Medrol, 40 mg/ml; Pharmacia & Upjohn, Inc., Kalamazoo, MI) was given at 2 mg/kg/day, once daily. LFM-A13 was resuspended in DMSO in the concentration of 25 mg/ml and given to mice in a dose of 50 mg/kg/day, twice daily. Control mice were treated with 10% DMSO in PBS. Treatments lasted until mice developed a terminal stage of leukemia.

Toxicity Studies of LFM-A13 in Mice and Rats.
The toxicity profile of LFM-A13 in CD-1 mice and rats was examined, as reported previously for the JAK3 inhibitor WHI-P131 (35) . In brief, groups of 40 mice were treated with vehicle alone (15% DMSO/PBS, 0.2 ml) or LFM-A13 at 10-, 20-, 40-, or 80-mg/kg dose levels. Mice were allowed free access to autoclaved standard pellet food and tap water throughout the experiments and were monitored daily for morbidity and mortality. Groups of 20 mice were electively sacrificed on day 7 or day 30 to determine the acute and subacute toxicity of LFM-A13 by examining their blood chemistry profiles and blood counts and by evaluating multiple organs for the presence of toxic lesions. No sedation or anesthesia was used throughout the treatment period. To study the toxicity of LFM-A13 in rats, groups of 10 rats were treated with vehicle alone (15% DMSO/PBS, 0.8 ml) or LFM-A13 at 20-, 40-, or 80-mg/kg dose levels. Rats were monitored daily for morbidity and mortality. On day 7, rats were electively sacrificed to determine the acute toxicity of LFM-A13 by examining their blood chemistry profiles and blood counts and by evaluating multiple organs for the presence of toxic lesions. At the time of necropsy, several tissues (bone, bone marrow, brain, cecum, heart, kidney, large intestine, liver, lung, lymph node, ovary, pancreas, skeletal muscle, skin, small intestine, spleen, stomach, thymus, thyroid gland, urinary bladder, and uterus, as available) were collected immediately from mice and rats for histopathological examination. For histopathological studies, tissues were fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin by routine methods. Glass slides with affixed 6-µm-thick tissue sections were prepared and stained with H&E.

Pharmacokinetic Studies of LFM-A13 in BALB/c Mice.
We studied the pharmacokinetics of LFM-A13 in BALB/c mice. In pharmacokinetic studies, mice were given i.p injections of 10-, 20-, 25-, 40-, or 50-mg/kg LFM-A13 or i.v. injections with 25-mg/kg LFM-A13 in vehicle. The mice were anesthetized with methoxyflurane, and 200-µl blood samples were obtained from the ocular plexus by retro-orbital venipuncture at 0, 3, 5, 10, 15, 30, and 45 min and at 1, 1.5, 2, 4, and 6 h after the i.p. administration of LFM-A13. All collected blood samples were heparinized and centrifuged at 7000 x g for 5 min in a microcentrifuge to obtain plasma. The plasma samples were stored at -20°C until analysis. Aliquots of plasma were used for extraction and HPLC determination of plasma LFM-A13 levels.

To study the effect of L-ASP, VCR, and MP on the pharmacokinetics of LFM-A13, BALB/c mice were first treated with i.p. injections of L-ASP (200 IU/kg/day), VCR (0.05 mg/kg/day), MP (2 mg/kg/day), or a standard triple-drug combination of VPL for 7 days. On day 7, mice received a 25-mg/kg i.p. bolus dose of LFM-A13 in 50 µl of DMSO as a vehicle, and blood samples were obtained from the ocular venous plexus by retro-orbital venipuncture at 0, 3, 5, 10, 15, 30, and 45 min and 1, 1.5, 2, 4, and 6 h after the administration of LFM-A13 to determine the pharmacokinetics of LFM-A13 as described above.

Blood was obtained from each of 25 mice once or twice only during a pharmacokinetics study. Of the 25 mice, 4 were used for 5-min and 6-h samples, 4 were used for 10-min and 4-h samples, 4 were used for 15-min and 1-h samples, 4 were used for 30-min and 2-h samples, 4 were used for 45-min and 1.5-h samples, 4 were used for 3-min samples, and 1 was used for the zero time point (blank control).

Pharmacokinetic Modeling Studies and Statistical Analysis.
Pharmacokinetic modeling and pharmacokinetic parameter estimations were carried out using the pharmacokinetics software, WinNonlin Program, Professional version 3.0 (Pharsight, Inc., Mountain View, CA; Refs. 29 , 30 , 35 ). An appropriate pharmacokinetic model was chosen on the basis of lowest sum of weighted squared residuals, lowest Schwartz Criterion, lowest Akaike’s Information Criterion value, lowest SE of the fitted parameters, and dispersion of the residuals. The elimination half-life was estimated by linear regression analysis of the terminal phase of the plasma concentration-time profile. The systemic clearance (CL) was determined by dividing the dose by the AUC.

Statistical analysis was performed using the Instat Program V.3.0 (GraphPad Software, San Diego, CA). Statistical differences between pharmacokinetic parameter values were analyzed using a two-tailed t test; P < 0.05 was considered significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Toxicity Profile of LFM-A13 in Mice and Rats.
LFM-A13, when administered as a single i.p. bolus injection, was not toxic to mice (n = 160) at dose levels ranging from 10 to 80 mg/kg. None of the 40 mice treated with 80-mg/kg LFM-A13, 40 mice treated with 40-mg/kg LFM-A13, 40 mice treated with 20-mg/kg LFM-A13, or 40 mice treated with 10-mg/kg LFM-A13 showed any signs of morbidity (Tables 1Citation and 2Citation ). Groups of 20 mice were sacrificed on day 7 (day 7 group) and day 30 (day 30 group), respectively.


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  		Table 1 Effects of LFM-A13 on health, blood chemistry profile, and peripheral blood counts in mice at day 7

 

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  		Table 2 Effects of LFM-A13 on health, blood chemistry profile, and peripheral blood counts in mice at day 30

 
The majority of the vehicle-treated as well as LFM-A13-treated mice in the day 7 group gained weight during the 7-day observation period. The average weight gain was 0.455 ± 0.196 g (2.1 ± 0.8%) for vehicle-treated control mice and 0.625 ± 0.163 g (2.8 ± 0.7%) for mice treated with the highest dose of LFM-A13 (Table 1)Citation . As shown in Table 1Citation , the blood chemistry profiles and cell counts of these mice were virtually identical to those of vehicle-treated control mice. In particular, LFM-A13-treated mice showed: (a) no evidence of kidney toxicity, as documented by their normal serum BUN and creatinine levels; and (b) no evidence of hematological toxicity, as documented by their normal WBC, ANC, ALC, and RBC counts (Table 1)Citation . However, there was evidence of mild hepatotoxicity as reflected by elevated AST and bilirubin levels. Histopathological examination of multiple tissues from LFM-A13-treated mice in the day 7 group (n = 80) did not reveal any test article-related toxic lesions except for a mild, subacute focal serositis in the liver. The lesion in the liver was observed only in 6 of 20 mice (30%) treated with the highest dose of LFM-A13. However, there was no statistically significant relationship between elevated AST levels and focal serositis in the liver. The mean (±SE) AST levels were 199 ± 83 IU/liter for the 6 mice with serositis and 146 ± 12 IU/liter for the 14 mice without serositis (P = 0.3). The lesion was characterized by thickening of the capsule by fibroblasts with slight infiltration of the thickened capsule by inflammatory cells (lymphocytes and neutrophils), with or without focal aggregation of neutrophils under the thickened capsule, and mesothelial cell hypertrophy. No lesions were found in the myocardium, kidney, pancreas, lungs, large/small intestine, or brain.

Virtually all of the vehicle-treated as well as LFM-A13-treated mice in the day 30 group gained weight during the 30-day observation period. The average weight gain was 4.030 ± 0.511 g (17.4 ± 2.2%) for vehicle-treated control mice and 4.517 ± 0.445 g (19.1 ± 2.0%) for mice treated with the highest dose of LFM-A13 (Table 2)Citation . As shown in Table 2Citation , the blood chemistry profiles and cell counts of these mice were virtually identical to those of vehicle-treated control mice. In particular, LFM-A13-treated mice showed: (a) no evidence of kidney toxicity, as documented by their normal serum BUN and creatinine levels; and (b) no evidence of hematological toxicity, as documented by their normal WBC, ANC, ALC, and RBC counts (Table 2)Citation . Unlike the results from the day 7 group, the AST and bilirubin levels of the day 30 group were within normal limits, suggesting that mice recover from the mild, subacute hepatotoxicity they experience within the first 7 days after exposure to LFM-A13. Histopathological examination of multiple tissues from LFM-A13-treated mice in the day 30 group (n = 80) did not reveal any test article-related toxic lesions. In particular, no lesions were found in the myocardium, kidney, liver, pancreas, lungs, large/small intestine, or brain. Thus, LFM-A13 was overall very well tolerated by mice at dose levels up to 80 mg/kg.

We next examined the toxicity of i.v.-administered LFM-A13 in rats. None of the 30 rats treated with 20 mg/kg (n = 10), 40 mg/kg (n = 10), or 80 mg/kg (n = 10) LFM-A13 showed any signs of toxicity. One hundred % of vehicle-treated as well as LFM-A13-treated rats gained weight throughout the 7-day observation period. The average weight gain was 20.4 ± 1.3 g (6.7 ± 0.4%) for vehicle-treated control rats and 21.1 ± 2.1 g (6.9 ± 0.7%) for rats treated with the highest dose of LFM-A13 (Table 3)Citation . All rats were healthy when sacrificed on day 7. As shown in Table 3Citation , the blood chemistry profiles and cell counts of these rats were virtually identical to those of vehicle-treated control rats. LFM-A13-treated rats showed: (a) no evidence of kidney toxicity, as documented by their normal serum BUN and creatinine levels; (b) no evidence of liver toxicity, as documented by their normal AST, bilirubin, lactate dehydrogenase, and ammonia levels; (c) no evidence of hematological toxicity, as documented by their normal WBC, ANC, ALC, and RBC counts (Table 3)Citation . Histopathological examination of multiple tissues from LFM-A13-treated rats (n = 30) did not reveal any test article-related toxic lesions. In particular, no lesions were found in the myocardium, liver, kidney, pancreas, lungs, large/small intestine, or brain. Thus, LFM-A13 was not toxic to rats at dose levels up to 80 mg/kg.


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  		Table 3 Effects of LFM-A13 on blood chemistry profile and peripheral blood counts in rats

 
Pharmacokinetics of LFM-A13 in Mice.
We next examined the pharmacokinetics of LFM-A13 in BALB/c mice (Fig. 1)Citation . LFM-A13 was administered either i.p. at five different nontoxic dose levels ranging from 10 to 50 mg/kg or i.v. at a 25-mg/kg dose level. A one-compartment, first-order pharmacokinetic model was fit to the pharmacokinetic data. The computer-fitted pharmacokinetic parameter values are shown in Table 4Citation . The estimated values for AUC and Cmax after i.p. administration showed a linear relationship to the applied dose level. The average AUC/Cmax values were 63 ± 3 µM·h/94 ± 8 µM at 10 mg/kg, 183 ± 14 µM·h/243 ± 16 µM at 20 mg/kg, 653 ± 47 µM·h/640 ± 44 µM at 40 mg/kg, and 1023 ± 31 µM·h/940 ± 16 µM at 50 mg/kg (Table 4ACitation ; Fig. 1ACitation ). The dose-adjusted "normalized" AUC and Cmax values were not superimposed. The observed dose-dependent decrease in systemic clearance indicates a saturable clearance mechanism.



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  		Fig. 1. Pharmacokinetics of LFM-A13. A, plasma concentration versus time profiles of LFM-A13 in BALB/c mice after i.p. bolus injection at doses of 10 (black), 20 (blue), 40 (green), and 50 (red) mg/kg (n = 4 mice/time point). B, plasma concentration versus time profiles of LFM-A13 in BALB/c mice after i.v. (red) or i.p. (black) bolus injection at doses of 25 mg/kg (n = 4 mice/time point).

 

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  		Table 4 Pharmacokinetic profile of LFM-A13 in BALB/c micea

 
The plasma concentration-time curves of LFM-A13 in vehicle-treated BALB/c mice after the i.v. injection of a 25-mg/kg bolus dose are depicted in Fig. 1BCitation . A two-compartment pharmacokinetic model was best fit to the plasma concentration time curves. The pharmacokinetic parameter values are shown in Table 4ACitation . LFM-A13 showed a moderate to fast elimination (t1/2, 44 min) and systemic clearance (CL, 184 ml/h/kg) after i.v. administration. The values for AUC and Cmax were 384 ± 30 µM·h and 731 ± 22 µM, respectively. The relatively large volume of distribution at the central compartment [95 ml/kg; ~2-fold greater than the plasma volume (50 ml/kg) or less than the total body of water (725 ml/kg); Ref. 36 ] suggests that LFM-A13 is poorly distributed into extravascular compartments. After administration of a single i.v. bolus dose of 25 mg/kg or i.p. bolus dose of 50 mg/kg in mice, LFM-A13 plasma concentrations of >10 µM, which exhibit potent chemosensitizing activity against leukemic cells in vitro (23 , 24) , were achieved within 3 min and maintained for >2 h (Fig. 1)Citation .

Effects of Standard Antileukemic Drugs on the Pharmacokinetics of LFM-A13 in Mice.
We next sought to determine whether the standard antileukemic drugs VCR, MP, or L-ASP alter the pharmacokinetics of LFM-A13. Mice were treated with these drugs or a VPL triple-drug combination for 7 days before the administration of LFM-A13. As shown in Table 4BCitation , VCR and L-ASP did not affect the pharmacokinetics of LFM-A13. Intriguingly, MP treatment resulted in slower clearance (156 ± 9 versus 234 ± 29 ml/h/kg; P < 0.05) and smaller Vc (75 ± 7 versus 144 ± 18 ml/kg; P < 0.05) with higher Cmax (526 ± 21 versus 383 ± 35 µM; P < 0.05) and systemic exposure levels (AUC; 449 ± 26 versus 308 ± 31 µM·h; P < 0.05) of LFM-A13 (Table 4B)Citation . Similarly, treatment of mice with VPL resulted in greater systemic exposure levels as reflected by the higher AUC values (444 ± 4 versus 308 ± 31 µM·h; P < 0.05) and higher Cmax (500 ± 17 versus 383 ± 35 µM; P < 0.05; Table 4BCitation ).

In Vivo Antileukemic Activity of LFM-A13, VPL, and LFM-A13 + VPL against BCL-1 Leukemia.
We reported recently the ability of LFM-A13 to chemosensitize human B-lineage ALL cell line NALM-6 by inhibiting the BTK kinase (24) . As shown in Fig. 2ACitation , the BTK expression level in BCL-1 leukemia cells was similar to the BTK expression levels of NALM-6 leukemia cells and normal BALB/c mouse splenocytes. LFM-13 treatment did not cause a significant change in BTK protein expression levels in BCL-1 cells, NALM-6 cells, or normal BALB/c splenocytes (Fig. 2A)Citation . The BTK activity index of BCL-1 leukemia cells was virtually identical to that of human NALM-6 leukemia cells and ~5-fold less than the BTK activity index of BALB/c splenocytes (Fig. 2B)Citation . Treatment with LFM-13 abolished the enzymatic activity of BTK in BCL-1 cells without affecting the BTK protein expression levels as it did in NALM-6 cells and normal BALB/c splenocytes (Fig. 2B)Citation .



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  		Fig. 2. A, BTK expression levels. The expression levels of BTK in whole-cell lysates of LFM-A13- or vehicle-treated BCL-1 cells, NALM-6 cells, or BALB/c splenocytes were measured by Western blot analysis as reported previously (21 , 23 , 24) . B, inhibition of BTK activity in the cells pretreated by LFM-A13. BTK was immunoprecipitated from lysates of LFM-A13-treated or vehicle-treated cells and subjected to immune complex kinase assays. The autophosphorylation activity was expressed in phosphorimager units (PIU). The amount of BTK protein in each lane was measured by Western blot analysis and expressed in densitometric scan units (DSU).

 
We next examined the chemosensitizing activity of LFM-A13 (25-mg/kg/dose, twice/day) in BALB/c mice challenged with 1 x 106 BCL-1 leukemia cells (Fig. 3)Citation . The probability of survival of vehicle-treated control mice (n = 43) after inoculation of BCL-1 cells was 47 ± 8% at 2 weeks and 2 ± 2% at 3 weeks with a median survival of 13.5 days. The probability of survival of LFM-A13-treated mice (n = 39) after inoculation of BCL-1 cells was 54 ± 8% at 2 weeks and 15 ± 6% at 3 weeks with a median survival of 15 days, which was only slightly better than that of vehicle-treated control mice (P = 0.02). All 82 vehicle-treated (n = 43) or LFM-A13-treated (n = 39) mice died of leukemia with massive hepatosplenomegaly within 4 weeks (Fig. 3)Citation . The standard VPL treatment, which consisted of VCR (0.05 mg/kg/day, once/week), MP (2 mg/kg/day, once/day), and L-ASP (200 IU/kg/day, three times/week), exhibited potent antileukemic activity and improved the probability of survival (Fig. 3)Citation . One hundred % of mice treated with VPL were alive at 3 weeks, and the probability of long-term survival (>12 weeks) was 14 ± 9% with a median survival of 37 days (P [VPL versus vehicle] < 0.0001; P [VPL versus LFM-A13] < 0.0001). All 12 treatment failures in the VPL group were attributable to progressive leukemia as reflected by massive hepatosplenomegaly at the time of death, and the remaining 2 mice that were electively sacrificed in moribund condition at 12 weeks also had massive hepatosplenomegaly. Notably, addition of LFM-A13 to this treatment regimen further improved the survival outcome, as reflected by higher cumulative proportions of mice surviving at 4, 8, or >=12 weeks (Fig. 3B)Citation . The probability of long-term survival (>12 weeks) was 41 ± 14% with a median survival of 58 days (P [LFM-A13 + VPL versus VPL] = 0.049). Of the 6 long-term survivors treated with LFM-A13 + VPL that were electively sacrificed in healthy condition at 12 weeks, 4 had evidence of residual leukemia, as reflected by a moderately enlarged spleen, whereas 2 had no hepatosplenomegaly.



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  		Fig. 3. In vivo antileukemic activity of LFM-A13. BALB/c mice were challenged with 1 x 106 BCL-1 leukemia cells and then were treated with vehicle alone, LFM-A13, VPL, or LFM-A13 + VPL. A, cumulative proportions of mice surviving event free are shown according to the time after the inoculation of BCL-1 cells. B, statistical comparisons using the log-rank test were performed, as reported previously (34) . 2W, 3W, 4W, 8W, and 12W, 2, 3, 4, 8, and 12 weeks, respectively.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have applied a highly sensitive analytical HPLC method for the quantitative detection of LFM-A13 in plasma samples. Our findings indicate that LFM-A13 is absorbed quickly, with tmax values of 10–18 min after intraperitoneal administration. The i.p. bioavailability was estimated to be ~100%. LFM-A13 had an elimination half-life of 17–32 min after i.p. administration at dose levels of 10–50 mg/kg. Pretreatment of mice with VCR or L-ASP had no significant effects on the pharmacokinetic features of LFM-A13. However, pretreatment with MP resulted in higher LFM-A13 Cmax and AUC values. These changes may be caused by a significant decrease in volume of distribution and a slight decrease in clearance. The mechanism of this previously unknown interaction between LFM-A13 and methylprednisone remains to be deciphered.

LFM-A13 is a rationally designed derivative of LFM (also known as A77 1726, the metabolite of LFM; Ref. 37 ). The anti-inflammatory agent LFM (HWA486) is a water-insoluble prodrug that is rapidly converted to its primary metabolite, A77 1726, in vivo. In rabbits, 95% of A77 1726 is associated with the lipoprotein-free fraction of plasma. A77 1726 has excellent bioavailability, a small volume of distribution, and a short elimination half-life (38) . The small volume of distribution and short elimination half-life of LFM-A13 are reminiscent of those for A77 1726. LFM-A13 exhibited a dose-dependent and significant increase in the values of normalized AUC and Cmax as well as a dose-dependent and significant decrease in clearance values. Because the i.p. bioavailability is almost complete for LFM-A13, a dose-dependent and significant increase in the values of normalized AUC and Cmax is attributed to the dose-dependent and significant decrease in clearances, suggesting that there may be a saturable clearance mechanism for LFM-A13. The metabolism and elimination of LFM-A13 will be the subject of a separate study pending the identification of its key metabolites.

ALL is the most common childhood malignancy. Dramatic improvements in the multiagent chemotherapy of children with ALL resulted in cure rates of 70–75% (39) . Despite recent improvements in therapy, perhaps one of five patients will eventually suffer a leukemic relapse. Currently, the major challenge in the treatment of childhood ALL is to cure patients who have relapsed despite intensive multiagent chemotherapy (40 , 41) . For patients who have relapsed while receiving therapy or shortly after elective cessation of therapy (duration of initial remission, <18 months), the overall survival is very poor. The poor outcome of this population makes a significant contribution to the overall childhood cancer mortality rate, despite the excellent outcome for the substantial majority of children with ALL (40 , 41) . Treatment of these children has generally been either intensive chemotherapy to achieve a second remission and subsequent use of either nonablative chemotherapy or ablative radiochemotherapy followed by bone marrow transplant. Recurrence of leukemia is the major obstacle to the success of either approach. Intensification of cytotoxic therapy using conventional drugs will likely cause overlapping toxicities and may result in delays that may erode the intensity of therapy. Consequently, the development of new potent anti-ALL drugs and the design of combinative treatment protocols using these new agents have emerged as exceptional focal points for research in modern therapy of relapsed ALL (39, 40, 41) .

Recent studies indicate that antiapoptotic tyrosine kinases, such as BTK, play a pivotal role in survival of human ALL cells and therefore may be used as targets in rational drug design against ALL (21 , 24) . In a recent study, we found that the LFM analogue LFM-A13 is a potent and specific inhibitor of BTK (24) . In vitro treatment of ALL cells with LFM-A13 enhanced their sensitivity to chemotherapy drugs (24) . In the present study, we investigated whether LFM-A13 can enhance the in vivo chemosensitivity of leukemic cells. To this end, we examined the pharmacodynamic features of this promising antileukemic agent in BALB/c mice challenged with the chemotherapy-resistant BCL-1 B-lineage leukemia. In this model, 100% of mice die within 3 weeks after i.v. inoculation of 1 x 106 BCL-1 cells with a median survival of 13.5 days. LFM-A13 was not toxic to mice when administered systemically at dose levels ranging from 1 to 100 mg/kg. Highly effective BTK-inhibitory and apoptosis-promoting plasma concentrations (>=10 µM; Ref. 24 ) of LFM-A13 could be achieved in mice without toxicity. LFM-A13 exhibited a favorable pharmacokinetic profile that was not adversely affected by the standard chemotherapy drugs VCR, MP, or L-ASP and significantly improved the chemotherapy response and survival outcome of mice challenged with BCL-1 leukemia cells. Whereas only 14% of mice treated with the standard triple-drug combination VPL became long-term survivors, 41% of mice treated with this combination plus LFM-A13 survived long term. LFM-A13 prolonged the median survival time of VPL-treated mice from 37 to 58 days. Our results confirm and extend previous studies regarding the role of BTK chemotherapy resistance of B-lineage leukemic cells (24) . BTK inhibitors such as LFM-A13 may be useful as a new class of chemosensitizing and apoptosis-promoting antileukemic agents for treatment of patients with relapsed ALL. Finally, the HPLC-based accurate and precise analytical detection method and pilot pharmacokinetic studies described herein provide the basis for advanced preclinical pharmacodynamic studies of LFM-A13.


   ACKNOWLEDGMENTS
 
We thank Thao Tran, Bert Roers, Christina Tague, Greg Micheltree, Heidi Bergstrom, and Linda Eddy for skillful assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Parker Hughes Institute, 2699 Patton Road, St. Paul, MN 55113. Phone: (651) 796-5450; Fax: (651) 796-5493; E-mail: Fatih_Uckun{at}mercury.ih.org Back

2 The abbreviations used are: BTK, Bruton’s tyrosine kinase; BCR, B-cell antigen receptor; LFM, leflunomide; LFM-A13, {alpha}-cyano-ß-hydroxy-ß-methyl-N-(2,5-dibromophenyl)propenamide; HPLC, high-performance liquid chromatography; ALL, acute lymphoblastic leukemia; SPF, specific pathogen-free; VCR, vincristine; MP, methylprednisolone; L-ASP, L-asparaginase; VPL, vincristine + MP + L-ASP; AUC, area under the curve; BUN, blood urea nitrogen; ANC, absolute neutrophil count; ALC, absolute lymphocyte count; AST, aspartate aminotransferase. Back

Received 8/30/01; revised 1/22/02; accepted 2/ 1/02.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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