Toxicity Profile of the Investigational New Biotherapeutic Agent, B43 (Anti-CD 19)-Pokeweed Antiviral Protein Immunotoxin

The investigational biotherapeutic agent, B43(anti-CD19)-pokeweed antiviral protein (PAP) immunotoxin, has shown substantial anti-leukemic activity in SCID mouse models of human B-lineage leukemia and lymphoma. In this report, we describe the results of a comprehensive preclinical toxicity study which determined the toxicity profile of B43-PAP in BALB/c mice. Administration of un-conjugated B43 monoclonal antibody was not associated with any toxicity, whereas B43-PAP caused dose-limiting and cardiac and renal toxicities which were fatal. In addition, B43-PAP also caused multifocal skeletal myofiber necrosis, which was associated with abnormal gait and lethargy. Notably, parenteral administrations of methylprednisolone, pentoxyphylline, or dopamine were able to markedly reduce B43-PAP related toxicity. This study provides a basis for further evaluation of the toxicity of B43-PAP in monkeys and humans.     

Effects of recombinant growth factors on radiation survival of human bone marrow progenitor cells

The purpose of this study was to evaluate the individual radioprotective effects of 4 distinct purified recombinant human hematopoietic growth factors, namely recombinant human granulocyte-macrophage colony stimulating factor (rGM-CSF), recombinant human granulocyte colony stimulating factor (rG-CSF), recombinant human interleukin 1 (rIL-1), and recombinant human interleukin 2 (rIL-2) on human myeloid (CFU-GM) and erythroid (BFU-E) bone marrow progenitor cells. We demonstrate that (a) preconditioning with rGM-CSF, rG-CSF, or rIL-1 enables CFU-GM to repair sublethal radiation damage and renders CFU-GM less radiosensitive, (b) preconditioning with rGM-CSF or rIL-1 enables BFU-E to repair sublethal radiation damage, and (c) preconditioning with rIL-2 does not increase the radiation survival of CFU-GM or BFU-E. The effects of recombinant growth factors, in particular rGM-CSF, on the radiation damage repair, radiosensitivity, and proliferative activity of bone marrow progenitor cells resulted in a substantial increase in the mean numbers of progenitor cell-derived hematopoietic colonies in irradiated marrow samples. The effects of rGM-CSF on the radiation response of CFU-GM and BFU-E, and the effects of rG-CSF as well as rIL-1 on the radiation response of CFU-GM did not appear to require the presence of T-cells/T-cell precursors, NK-cells, B-cells/B-cell precursors, monocytes, macrophages, MY8 antigen positive non-CFU-GM myeloblasts, promyelocytes, myelocytes, metamyelocytes, granulocytes, or glycophorin A positive erythroid cells since virtually identical results were obtained with unsorted marrow samples or highly purified fluorescence activated cell sorter (FACS) isolated progenitor cell suspensions. To our knowledge, this report represents the first study on recombinant human growth factor-induced modulation of the radiation responses of normal human bone marrow progenitor cells.     

Structure-based design of potent inhibitors of EGF-receptor tyrosine kinase as anti-cancer agents

In a systematic effort to design inhibitors of the epidermal growth factor receptor (EGFR) family protein tyrosine kinases (PTK) as anti-cancer agents, we have constructed a three-dimensional homology model of the EGFR kinase domain and used molecular modeling methods for the structure-based design of analogs of the active metabolite of leflunomide (LFM) with potent and specific inhibitory activity against EGFR. These docking studies identified alpha-cyano-beta-hydroxy-beta-methyl-N-[4-(trifluoromethoxy)phenyl]-p ropenamide (LFM-A12) as our lead compound, which was predicted to bind to the EGFR catalytic site in a planar conformation. LFM-A12 inhibited the proliferation (IC50 = 26.3 microM) and in vitro invasiveness (IC50 = 28.4 microM) of EGFR positive human breast cancer cells in a concentration-dependent fashion. Similarly, the model of the EGFR binding pocket was used in combination with docking procedures to predict the favorable placement of chemical groups with defined sizes at multiple modification sites on another class of EGFR inhibitors, the 4-anilinoquinazoline. This approach has led to the successful design of a dibromo quinazoline derivative, WHI-P97, which had an estimated Ki value of 0.09 microM from modeling studies and a measured IC50 value of 2.5 microM in EGFR kinase inhibition assays. WHI-P97 effectively inhibited the in vitro invasiveness of EGFR-positive human cancer cells in a concentration-dependent manner. However, unlike LFM-A12, the quinazoline compounds are not specific for EGFR.     

AZT-5'-(p-bromophenyl methoxyalaninyl phosphate) as a potent and non-toxic anti-human immunodeficiency virus agent

The potency and selectivity index of the AZT-phenyl phosphate derivatives in thymidine kinase (TK)-deficient T cells were substantially enhanced by introducing a single para-bromo substitutent in the phenyl moiety. AZT-5'-(p-bromophenyl methoxyalaninyl phosphate) was 43-fold more potent than AZT-5'-(phenyl methoxyalaninyl phosphate) and was fivefold more potent than AZT in inhibiting human immunodeficiency virus (HIV) replication in TK-deficient CEM cells.     

Prognostic significance of T-lineage leukemic cell growth in SCID mice: a Children's Cancer Group study

Contemporary intensive therapies are effective for the majority of pediatric T-lineage acute lymphoblastic leukemia (ALL) patients, thus current challenge is to identify patients who may benefit from alternative treatment modalities. Previously, we demonstrated that human leukemic cell growth in the severe combined immunodeficiency (SCID) mouse was a significant prognostic factor for very high risk B-lineage ALL patients. In the current report we show that primary leukemic cells from 24 of 88 (27%) T-lineage ALL patients (SCID+) caused histopathologically detectable leukemia in SCID mice. These SCID+ patients were similar to SCID- (n = 64) patients with respect to virtually all presenting features, including age, sex, race, and leukocyte count. Growth of primary leukemic cells in SCID mice was not a significant predictor of outcome for the aggregate population of T-lineage ALL patients. Two-year event-free survival (EFS) outcomes for SCID+ patient and SCID- patients were 76.2% (SD = 5.6%) and a 64.0% (SD = 10.4%; p = 0.20). Overall survival also was similar between the two groups (p = 0.36). Among the subset of patients with M1 or M2 marrow status by day 7 of induction chemotherapy (rapid early responders), those who were SCID+ had poorer outcomes than those who were SCID-, with a 2-year EFS of 68.4% (SD = 11.9%) vs. 85.7% (SD = 6.0%) and relative hazard rate of 3.06 (p = 0.06). These data suggest that leukemic cell growth in SCID mice may identify a subset of T-lineage ALL patients who are at higher risk for relapse despite achieving a rapid early response to induction chemotherapy.     

Evaluation of temozolomide in a SCID mouse model of human B-cell precursor leukemia

We used a SCID mouse model of human B-lineage acute lymphoblastic leukemia to examine the antileukemic activity of temozolomide in comparison to as well as in combination with B43-PAP anti-CD19 immunotoxin. One hundred percent of the 20 PBS-treated control mice died of disseminated human B-lineage ALL at 32 to 64 days after the inoculation of 1x10(6) NALM-6 cells, with a median event free survival time of 43 +/- 1 days. Temozolomide, when administered i.p. for 5 consecutive days at a dose level of 411 mg/m2 or as a single 750 mg/m2 bolus dose, elicited significant antileukemic activity and improved survival in this SCID mouse model of human B-lineage ALL. The median survival times were 43 +/- 1 days for PBS-treated mice, 56 +/- 16 days for mice injected with the 5-day temozolomide program, and 64 +/- 15 days for mice treated with a single bolus dose of temozolomide. However, temozolomide was not as effective as B43-PAP. Whereas only 40 +/- 21% of mice treated with temozolomide survived beyond 120 days, B43-PAP treatment resulted in 74 +/- 7% survival in the same model system. The combination of temozolomide with B43-PAP was well tolerated by mice but it was not significantly more effective than B43-PAP alone. Temozolomide may have very limited potential as an antileukemic agent for treatment of B-lineage ALL.     

Antileukemic activity and cellular metabolism of the aryl phosphate derivative of bromo-methoxy zidovudine (compound WHI-07)

The novel aryl phosphate derivative of bromo-methoxy zidovudine (ZDV/AZT) (compound WHI-07, CAS 213982-96-8) was found to be a potent antileukemic agent against human leukemia, lymphoma, and multiple myeloma cell lines in MTT and clonogenic assays with low micromolar IC50 values. In addition, WHI-07 was antimitotic, leading to cell fusion and developmental arrest in the Zebrafish model of rapid cell proliferation. WHI-07 was cytotoxic to drug-sensitive (NALM-6, MOLT-3, HL-60, P388) and multi-drug resistant (MDR) leukemia cell lines (HL-60/VCR, HL-60/ADR, P388/ ADR). Treatment of leukemia cells with WHI-07 showed rapid and dramatic depletion of all cellular nucleoside diphosphate and triphosphate (NDP/NTP) pools, which would contribute to the overall reduction of nucleic acid synthesis and cell death. WHI-07 was rapidly metabolized to alaninyl ZDV monophosphate (Ala-ZDV-MP), the levels of which inversely correlated with cytotoxic IC50 values of WHI-07. Glutathione was found to mediate the in vitro and in vivo detoxification pathway of WHI-07 to 3'-azidothymidine-5'-p-bromophenylmethoxyalaninyl phosphate and Ala-ZDV-MP, respectively. The proposed intracellular metabolic pathway for WHI-07 involves a thiol-mediated dehalogenation step followed by the paraoxon-sensitive carboxylesterase-mediated reaction leading to the formation of Ala-ZDV-MP as the major intracellular metabolite.     

Protease-mediated enzymatic hydrolysis and activation of aryl phosphoramidate derivatives of stavudine

Several proteases are capable of hydrolyzing the aryl substituted phosphoramidate derivatives of stavudine resulting in the formation of the active metabolite, alaninyl d4T monophosphate. Subtilisin Protease A, Subtilisin Griseus, Subtilisin Carlsberg, Papaya, Bacillus were amongst the most effective proteases in hydrolyzing stavudine derivatives and specificity of their activity was confirmed using several protease inhibitors to block the hydrolysis of these phosphoramidate derivatives. We found that these proteases exhibit chiral selectivity at the phosphorus center of stavudine derivatives. Our results indicate that cellular proteases may be responsible for the activation of these phosphoramidate derivatives. In addition, we show that the enzymatic hydrolysis takes place at the carboxymethyl ester side chain of these pro-drugs and the direct attack on the phosphorus center by these enzymes does not occur. Finally, we describe a novel activation pathway hitherto unknown for the activation and viral inhibitory characteristic shown by these phosphoramidate derivatives of stavudine.     

Targeting JAK3 and BTK tyrosine kinases with rationally-designed inhibitors

Multifunctional rational drug design of protein tyrosine kinases inhibitors allows a potent drug to be utilized to treat more than one disease for greater patient benefits. Many protein tyrosine kinases (PTK), including Janus kinase 3 (JAK3) and Bruton's tyrosine kinase (BTK), have been identified as potential drug targets to treat diverse diseases including cancer and disorders of the immune system. Here we review advances in JAK3 and BTK inhibitors and describe the therapeutic potential of these potent agents in the clinical setting.     

Effect of targeted disruption of signal transducer and activator of transcription (Stat)4 and Stat6 genes on the autoimmune diabetes development induced by multiple low doses of streptozotocin

The MLDS (multiple low doses of streptozotocin) model of diabetes was induced in Stat4(-/-), Stat6(-/-), and double-deficient Stat4(-/-)/6(-/-) mice to examine the role of STAT4/STAT6 deficiency in development of autoimmune diabetes. Cytokine production of T-cells from Stat4(-/-) mice confirmed a predominantly Th2-type immune response. Stat4(-/-) mice exhibited delayed onset and reduced severity of disease compared to wild-type (WT) mice. In contrast, STAT6 deficiency, with a predominant Th1 response, did not influence the kinetics or severity of MLDS-induced autoimmune diabetes. Interestingly, Stat4(-/-)/6(-/-) mice, with a prominent Th1-type response, experienced an accelerated and aggravated course of diabetes after MLDS, implicating a STAT4-independent Th1 response in the immunopathogenesis of MLDS-induced autoimmune diabetes. The sensitivity of islet cells from Stat4(-/-) or Stat4(-/-)/6(-/-) mice to cytokines and STZ was not different from that of islet cells of WT mice. Hence, the observed effects of STAT4 and STAT4/6 deficiency on MLDS-induced autoimmune diabetes are likely due to their effects on T-cell responses.