Structure-based design of novel anticancer agents

Recently identified agents that interact with cytoskeletal elements such as tubulin include synthetic spiroketal pyrans (SPIKET) and monotetrahydrofuran compounds (COBRA compounds). SPIKET compounds target the spongistatin binding site of beta-tubulin and COBRA compounds target a unique binding cavity on alpha-tubulin. At nanomolar concentrations, the SPIKET compound SPIKET-P causes tubulin depolymerization and exhibits potent cytotoxic activity against cancer cells. COBRA-1 inhibits GTP-induced tubulin polymerization. Treatment of human breast cancer and brain tumor cells with COBRA-1 caused destruction of microtubule organization and apoptosis. Other studies have identified some promising protein tyrosine kinase inhibitors as anti-cancer agents. These include EGFR inhibitors such as the quinazoline derivative WHI-P97 and the leflunomide metabolite analog LFM-A12. Both LFM-A12 and WHI-P97 inhibit the in vitro invasiveness of EGFR positive human breast cancer cells at micromolar concentrations and induce apoptotic cell death. Dimethoxyquinazoline compounds WHI-P131 and WHI-P154 inhibit tyrosine kinase JAK3 in leukemia cells. Of particular interest is WHI-P131, which inhibits JAK3 but not JAK1, JAK2, SYK, BTK, LYN, or IRK at concentrations as high as 350 microM. Studies of BTK inhibitors showed that the leflunomide metabolite analog LFM-A13 inhibited BTK in leukemia and lymphoma cells. Consistent with the anti-apoptotic function of BTK, treatment of leukemic cells with LFM-A13 enhanced their sensitivity to chemotherapy-induced apoptosis.     

Lack of adverse effects on fertility of female CD-1 mice exposed to repetitive intravaginal gel-microemulsion formulation of a dual-function anti-HIV agent: aryl phosphate derivative of bromo-methoxy-zidovudine (compound WHI-07)

5-bromo-6-methoxy-5,6-dihydro-3(')-azidothymidine-5(')-(p-bromophenyl) methoxyalaninyl phosphate (WHI-07), a novel bromo-methoxy-substituted aryl phosphate derivative of zidovudine (ZDV), is a potent dual-function contraceptive agent with anti-HIV activity. Its potential for reproductive toxicity was assessed in a series of experiments using CD-1 mice under the conditions of its intended use as an intravaginal microbicide. Female CD-1 mice were exposed intravaginally to a gel-microemulsion formulation containing 0%, 0.5%, 1.0% or 2.0% WHI-07 for up to 13 weeks. On a molar basis, these concentrations represent 1400-5700 times its in vitro spermicidal IC(50) and 1.4-5.7(x10(6)) times its in vitro anti-HIV IC(50). We examined the effects of intravaginally administered WHI-07 on: ovulation efficiency; in vivo fertilization and early embryonic, fetal development; and reproductive outcome, including neonatal survival and pup development. Compound WHI-07 was administered intravaginally during superovulation, organogenesis and prior to mating for 5 and 10 consecutive days and for 13 weeks, respectively. Mice were evaluated for ovulation efficiency and fertilization rate and cleavage 14 and 40 h after human chorionic gonadotropin (hCG) injection, respectively. Pregnant mice were administered 2% WHI-07 intravaginally during gestation days (GD) 6-15 and measures of teratogenicity were evaluated on GD 17. For short-term toxicity study, mice were given intravaginal treatment of gel-microemulsion containing 0%, 0.5%, 1.0% and 2.0% WHI-07 for 13 weeks and then mated with untreated males to evaluate potential reproductive and developmental effects. Repeated intravaginal exposure of mice to 2% WHI-07 had no adverse effects on ovulation response, mean number of eggs recovered or the percentage of eggs fertilized or cleaved. No evidence of reproductive toxicity, fetal toxicity or teratogenicity was found following repetitive intravaginal application of 2% WHI-07 during the period of organogenesis. Furthermore, repeated intravaginal exposure of mice to 0.5-2.0% WHI-07 for 13 weeks had no adverse effect on the subsequent reproductive capability, perinatal outcome or growth and development of the offspring. Compound WHI-07 shows unique clinical potential as a safe, dual-function vaginal contraceptive for curbing mucosal and perinatal HIV transmission.     

Apoptosis inducing novel anti-leukemic agent, bis(4,7-dimethyl-1,10 phenanthroline) sulfatooxovanadium(IV) [VO(SO4)(Me2-Phen)2] depolarizes mitochondrial membranes

Bis(4,7-dimethyl-1,10 phenanthroline) sulfatooxovanadium(IV) [VO(SO(4) )(Me(2)-Phen)(2)] induces apoptosis in human NALM-6 leukemia cells. In the present report, we demonstrate that VO(SO(4) )(Me(2)-Phen)(2)-induced apoptosis is mediated through the generation of reactive oxygen species (ROS), depletion of glutathione and depolarization of mitochondrial membrane potential (DeltaPsim). Using multilaser flow cytometry methods, we further mapped out the death sequence that occurs in VO(SO(4))(Me(2)-Phen)(2)-treated leukemic cells. Triple labeling method to measure ROS, DeltaPsim and glutathione coupled with multilaser excitation flow cytometry showed that induction of ROS took place before the loss of mitochondrial permeability transition and depletion of glutathione. Correlated two parameter plots of glutathione content versus DeltaPsim showed that loss of DeltaPsim and depletion of glutathione closely follows each other. Translocation of phosphatidylserine to the outer leaflet of the cell membrane was the final step in the process before the cells became apoptotic. These results demonstrate that the mitochondrial permeability transition takes place during VO(SO(4))(Me(2)-Phen)(2)-induced apoptosis and is mediated through induction of ROS and depletion of glutathione.     

Expression of TEL-AML1 fusion transcripts and response to induction therapy in standard risk acute lymphoblastic leukemia.

We prospectively examined the frequency of the t(12;21)TEL-AML1 fusion in 504 children with newly diagnosed standard risk ALL using RT-PCR assays. Cells from 95 patients (18.8%) were TEL-AML1+. There was a significantly higher frequency of pseudodiploidy among the TEL-AML1+ cases (39.4% versus 14.1%, P = 0.001), primarily because structural abnormalities involving 12p and del(6q) occurred more frequently in the TEL-AML1+ group. TEL-AML1+ ALL was more sensitive to the induction chemotherapy than TEL-AML1- ALL. The percentage of "rapid early responders", i.e., patients who achieved an M1 (< 5% blasts) or M2 (5-25% blasts) marrow status on day 7 of induction chemotherapy, was significantly higher among TEL-AML1+ cases. The quality of remission of RT-PCR positive cases was excellent, as evidenced by the very low to absent MRD burden of their end-of-induction bone marrow specimens. TEL-AML1+ patients also had an excellent early EFS outcome. The probability of EFS at 30 months from study entry were 98.9 +/- 1.0% for the TEL-AML1+ group and 92.1 +/- 1.5% for the TEL-AML1- group (P = 0.0001). This prospective study significantly expands the knowledge gained from previous studies regarding the prognostic significance of t(12;21)TEL-AML1 fusion in pediatric ALL.     

Hypodiploidy with less than 45 chromosomes confers adverse risk in childhood acute lymphoblastic leukemia: a report from the children's cancer group

We have determined the prognostic significance of hypodiploidy (<46 chromosomes) in a large cohort of children with acute lymphoblastic leukemia (ALL) treated by the Children's Cancer Group. Among 1,880 patients, 110 (5.8%) had hypodiploid karyotypes: 87 had 45 chromosomes, 15 had 33 to 44 chromosomes, none had 29 to 32 chromosomes, and 8 had 24 to 28 chromosomes (near-haploidy). Six-year event-free survival (EFS) estimates for patients with 45 chromosomes, 33 to 44 chromosomes, or 24 to 28 chromosomes were 65% (standard deviation [SD], 8%), 40% (SD, 18%), and 25% (SD, 22%), respectively (log rank, P =.002; test for trend, P =.0009). The combined hypodiploid group had worse outcome than nonhypodiploid patients, with 6-year EFS of 58% (SD, 7%) and 76% (SD, 2%), respectively (P <.0001). EFS for the subgroup with 45 chromosomes was similar to that of patients with pseudodiploidy (P =.43) or 47 to 50 chromosomes (P =.76). None of the patients with 24 to 28 chromosomes had a t(4;11), a t(9;22), or a t(1;19), and most received highly intensive therapy. The adverse risk associated with 33 to 44 and 24 to 28 chromosomes remained significant in multivariate analyses adjusted for important risk factors including age, white blood cell count, and Philadelphia chromosome status. Thus, hypodiploidy with less than 45 chromosomes, particularly 24 to 28 chromosomes, is a significant adverse risk factor despite treatment with contemporary intensive therapies.     

A critical risk-benefit assessment argues against the use of anthracyclines in induction regimens for newly diagnosed childhood acute lymphoblastic leukemia.

Although anthracyclines are associated with significant cardiac toxicity and their benefit remains unclear, they are included in nearly all current protocols for the treatment of childhood acute lymphoblastic leukemia (ALL). Currently open trials from most major groups use anthracyclines in the induction phase for all high-risk patients and in the delayed intensification phase for all patients regardless of risk classification. Our review of published randomized studies reveals no benefit for the addition of anthracyclines to induction phase of childhood ALL regimens consisting of vincristine, prednisone, and L-asparaginase (VPL), with or without a delayed intensification phase. No randomized studies have evaluated the use of anthracyclines in the delayed intensification phase of therapy. Furthermore, studies of relapsed patients indicated no benefit for the addition anthracyclines to maintenance regimens. Recent evidence from preclinical studies suggests that a combination of VPL with an anti-CD19 immunotoxin is more effective than VPL plus anthracyclines combination. Accumulated evidence exists that anthracyclines are associated with late-onset cardiac morbidity in about 25% of childhood ALL and other cancer survivors, and about 5% develop overt heart failure, with some requiring cardiac transplantation. Anthracycline-induced cardiotoxicity in children has no safe dose threshold and all doses are likely to cause significant myocardial damage. New data suggests that a unique cardiac mitochondrial exogenous NADH dehydrogenase is responsible for the anthracycline-induced oxygen radicals damage to the heart, and that chelators currently evaluated may not prevent late-onset cardiotoxicity in children. In view of these findings we urge extreme caution in using anthracyclines as part of multimodality ALL treatment programs, and strongly recommend reevaluation of what should be considered the best induction regimen for high-risk childhood ALL.     

In vivo toxicity and pharmacokinetic features of the janus kinase 3 inhibitor WHI-P131 [4-(4'hydroxyphenyl)-amino-6,7- dimethoxyquinazoline.

4-(4'Hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P131) is a potent and selective inhibitor of the Janus kinase 3, which triggers apoptosis in human acute lymphoblastic leukemia (ALL) cells. In this preclinical study, we evaluated the pharmacokinetics and toxicity of WHI-P131 in rats, mice, and cynomolgus monkeys. Following i.v. administration, the terminal elimination half-life of WHI-P131 was 73.2 min in rats, 103.4 min in mice, and 45.0 min in monkeys. The i.v. administered WHI-P131 showed a very wide tissue distribution in mice. Following i.p. administration, WHI-P131 was rapidly absorbed in both rats and mice, and the time to reach the maximum plasma concentration (tmax) was 24.8 min in rats and 10.0 min in mice. Subsequently, WHI-P131 was eliminated with a terminal elimination half-life of 51.8 min in rats and 123.6 min in mice. The estimated i.p. bioavailability was 95% for rats, as well as for mice. WHI-P131 was quickly absorbed after oral administration in mice with a tmax of 5.8 min, but its oral bioavailability was relatively low (29.6%). The elimination half-life of WHI-P131 after oral administration was 297.6 min. WHI-P131 was not acutely toxic to mice at single i.p. bolus doses ranging from 0.5-250 mg/kg. Two cynomolgus monkeys treated with 20 mg/kg WHI-P131 and one cynomolgus monkey treated with 100 mg/kg WHI-P131 experienced no side effects. Plasma samples from WHI-P131-treated monkeys exhibited potent antileukemic activity against human ALL cells in vitro. To our knowledge, this is the first preclinical toxicity and pharmacokinetic study of a Janus kinase 3 inhibitor. Further development of WHI-P131 may provide the basis for new and effective treatment programs for relapsed ALL in clinical settings.