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.

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.

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.

In vivo toxicity and pharmacokinetic features of B43(Anti-CD19)-Genistein immunoconjugate.

B43(anti-CD19)-Genistein immunoconjugate targets genistein, a naturally occurring protein tyrosine kinase inhibitory isoflavone to the membrane-associated anti-apoptotic CD19-LYN complexes and triggers apoptotic cell death. In this preclinical study, the toxicity profiles of B43-Genistein as well as unconjugated genistein were evaluated in mice. B43-Genistein and genistein were administered either as single bolus injections or daily injections for 10 consecutive days via the intraperitoneal route to mice. Genistein was not toxic to mice at the highest dose of 40 mg/kg and no test article-related histopathological lesions were found in any of the 64 genistein-treated mice. B43-Genistein had a significantly longer elimination half-life and slower plasma and tissue clearance than unconjugated genistein. B43-Genistein was not toxic to mice at the highest single dose of 40 mg/kg or highest cumulative dose of 100 mg/kg and no test article-related histopathological lesions were found in any of the 108 mice treated with B43-genistein. To our knowledge, this is the first preclinical toxicity and pharmacokinetic study of a tyrosine kinase inhibitor-containing immunoconjugate.

Treatment of human B-cell precursor leukemia in SCID mice by using a combination of the anti-CD19 immunotoxin B43-PAP with the standard chemotherapeutic drugs vincristine, methylprednisolone, and L-asparaginase.

We have compared the antileukemic activity of the investigational biotherapeutic agent B43-PAP to the antileukemic activities of the standard chemotherapeutic drugs vincristine (VCR), methylprednisolone (PDN), L-asparaginase (L-ASP) as single agents as well as in a 3-drug combination regimen ("VPL") using a SCID mouse model of human B-cell precursor (BCP) acute lymphoblastic leukemia (ALL). When mice (N = 95) were challenged with 1 x 10(6) NALM-6 leukemia cells, all of them died of disseminated leukemia with a median event-free survival (EFS) of 47 +/- 6 days. B43-PAP was more active than VCR, PDN, or L-ASP and the two-drug combinations VCR + B43-PAP, PDN + B43-PAP, or L-ASP + B43-PAP were not significantly more active than B43-PAP. The 120 days EFS outcome results were 46 +/- 13% for B43-PAP (Median EFS = 92 +/- 22 days), 0 +/- 0% for VCR (Median EFS = 49 +/- 1 days), 40 +/- 22% for PDN (Median EFS = 100 +/- 20 days), 0 +/- 0% for L-ASP (Median EFS = 41 +/- 1 days), 60 +/- 22% for VCR + B43-PAP (Median EFS = >120 days), 60 +/- 22% for PDN + B43-PAP (Median EFS = >120 days), and 50 +/- 25% for L-ASP + B43-PAP (Median EFS = 93 +/- 27 days), When mice (N = 61) were challenged with 5 x 10(6) NALM-6 cells, all of them rapidly died of disseminated leukemia with a median EFS of 37 +/- 3 days. The 3-drug combination "VPL" (Median EFS = 75 +/- 23 days) was slightly less active than B43-PAP (Median EFS = 84 +/- 19 days) (P = 0.09). Notably, the combination of "VPL" with B43-PAP (i.e., VPLB) resulted in 100% survival. By comparison, the combination of "VPL" with daunorubicin (i.e., VPLD) (Median EFS = 69 +/- 31 days) was not more active than VPL. To our knowledge, this preclinical study is the first to demonstrate the feasibility and superb antileukemic activity of immunochemotherapy using anti-CD19 immunotoxin in combination with the standard 3-drug combination "VPL" against BCP ALL.

Prognostic significance of B-lineage leukemic cell growth in SCID mice: a Children's Cancer Group Study.

Primary leukemic cells isolated from children (N = 681 ) with newly diagnosed B-lineage ALL enrolled on risk-adjusted treatment protocols of the Children's Cancer Group (CCG) were injected via the tail vein into 7-10 week old SCID mice. Leukemic cells from 104 of 681 patients (15.3%) were able to engraft and proliferate in one or more SCID mouse organs. These SCID+ patients were somewhat more likely than SCID patients to be older than 10 years of age (p = 0.03) and have WBC counts >20,000/microL (p = 0.04), but the groups were similar with respect to all other presenting features. Event-free survival (EFS) outcome at 3 years of follow-up was similar for SCID+ patients compared with SCID- patients (79.2%, SD = 5. 1% vs. 84.8%, SD = 2.8%; p = 0.20). Overall survival also was similar between the two groups (p = 0.93). This result was maintained within the subgroups of lower risk (N = 448) and higher risk (N = 233) patients. However, there were trends for poorer outcome among patients whose cells caused overt leukemia in SCID mice and infiltrated either 6 or more organs (p = 0.03), skeletal muscle (p = 0.0003), kidney (p = 0.05), or spleen (p = 0.06). Thus, engraftment of primary leukemic cells in SCID mice was not a significant predictor of outcome for the aggregate population of B-lineage ALL patients, the majority of whom were low risk, treated according to contemporary intensive chemotherapy programs of the CCG. However, development of disseminated overt leukemia and infiltration of SCID mouse skeletal muscle by primary leukemic cells from adjacent bone marrow may reflect a biologically more aggressive disease and identify patients at higher risk for treatment failure.

Antitumor activity of TP3(anti-p80)-pokeweed antiviral protein immunotoxin in hamster cheek pouch and severe combined immunodeficient mouse xenograft models of human osteosarcoma.

TP3-pokeweed antiviral protein (PAP) immunotoxin is directed against the p80 antigen on osteosarcoma cells. Previous studies have demonstrated that TP3-PAP kills clonogenic human osteosarcoma cells in vitro and shows significant antitumor activity in a murine soft tissue sarcoma model (P. M. Anderson, et al, Cancer Res., 55: 1321-1327, 1995.) In this study, we demonstrate that TP3-PAP elicits potent in vivo antitumor activity in a hamster cheek pouch model of human osteosarcoma. Furthermore, treatment with TP3-PAP at nontoxic dose levels significantly delayed the emergence and progression of leg tumors and markedly improved tumor-free survival in severe combined immunodeficient mice challenged with OHS human osteosarcoma cells. Thus, TP3-PAP may be useful in the treatment of poor risk osteosarcoma.

In vivo toxicity, pharmacokinetics, and anticancer activity of Genistein linked to recombinant human epidermal growth factor.

Epidermal growth factor receptor (EGFR)-associated protein tyrosine kinase (PTK) complexes have vital anti-apoptotic functions in human breast cancer cells. We have shown previously that targeting the naturally occurring PTK inhibitor genistein to the EGFR-associated PTK complexes using the EGF-Genistein (Gen) conjugate triggers rapid apoptotic cell death in human breast cancer cells and abrogates their in vitro clonogenic growth. In the present study, we examined the in vivo toxicity profile, pharmacokinetics, and anticancer activity of EGF-Gen. No toxicities were observed in mice treated with EGF-Gen at dose levels as high as 40 mg/kg administered i.p. as a single dose or 140 mg/kg administered i.p. over 28 consecutive days. EGF-Gen significantly improved tumor-free survival in a severe combined immune deficiency (SCID) mouse xenograft model of human breast cancer, when it was administered 24 h after inoculation of tumor cells. At 100 microg/kg/day x 10 days (1 mg/kg total dose), which is >100-fold less than the highest tested and nontoxic cumulative dose (ie., 140 mg/kg) in mice, EGF-Gen was more effective than cyclophosphamide (50 mg/kg/day x 2 days), Adriamycin (2.5 mg/kg x 1 day), or methotrexate (0.5 mg/kg x 1 day), the most widely used standard chemotherapeutic drugs for breast cancer, and resulted in 60% long-term tumor-free survival. Furthermore, treating SCID mice with established s.c. human breast cancer xenografts of 0.5-cm diameter with EGF-Gen at this dose level resulted in disappearance of the tumors in two of five mice and >50% shrinkage in three of five mice within 10 days, whereas all of the control tumors in five PBS-treated mice as well as five mice treated with unconjugated Gen (1 mg/kg/day x 10 days) showed >200% increase in diameter during the same observation period. EGF-Gen treatment reduced the growth rate of breast cancer xenografts of 1.0-cm diameter, but unlike with tumors of 0.5-cm diameter, it failed to cause shrinkage or disappearance of these larger tumors. The level of EGF-Gen systemic exposure that was effective in SCID mice was achieved in cynomolgus monkeys without any significant side effects detectable by clinical observation, laboratory studies, or histopathological examination of multiple organs. EGF-Gen might be useful in the treatment of breast cancer as well as other EGFR-positive malignancies.

Combined therapeutic efficacy of the thymidylate synthase inhibitor ZD1694 (Tomudex) and the immunotoxin B43(anti-CD19)-PAP in a SCID mouse model of human B-lineage acute lymphoblastic leukemia.

The quinazoline antifolate N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2-thenoyl)-L-glutamic acid (ZD1694; Tomudex) is a potent inhibitor of thymidylate synthase and causes cell death through disruption of DNA synthesis and repair by blocking the obligatory thymidine nucleotide synthesis. B43(anti-CD19)-PAP immunotoxin is a potent inhibitor of protein synthesis in CD19+ B-lineage acute lymphoblastic leukemia (ALL) cells and causes apoptosis. In this model, 100% of SCID mice challenged with 1 x 10(6) human NALM-6 B-lineage ALL cells develop overt and invariably fatal leukemia. All of the 22 control SCID mice treated with phosphate-buffered saline died of disseminated human leukemia between 31 and 61 days with a median survival of 41.2 days. Treatment with ZD 1694 resulted in improved leukemia-free survival with a median survival of 69.2 days (P < 0.001, log-rank test). B43-PAP treatment was more effective than ZD1694 (P=0.026) and resulted in 51.0% long-term leukemia-free survival with a median survival of 187.5 days (P < 0.0001. log-rank test). The combination of ZD1694 and B43-PAP was more effective than either agent alone and resulted in 100% long-term leukemia-free survival. To our knowledge, this preclinical study is the first to demonstrate the feasibility and therapeutic advantage of combining an anti-leukemia immunotoxin with a thymidylate synthase inhibitor.

In vivo toxicity and pharmacokinetic features of B43 (anti-CD19)-genistein immunoconjugate in nonhuman primates.

B43 (anti-CD19)-genistein immunoconjugate targets genistein, a naturally occurring protein tyrosine kinase-inhibitory isoflavone to the membrane-associated antiapoptotic CD19-LYN complexes and triggers apoptotic cell death. In this preclinical study, the toxicity profiles of B43-genistein as well as unconjugated genistein were evaluated in cynomolgus monkeys. B43-genistein and genistein were administered either as single bolus injections or daily injections for 5-10 consecutive days via the i.v. route to monkeys. Neither genistein nor B43-genistein was toxic to cynomolgus monkeys, and no test article-related histopathological lesions were found in any of the two genistein-treated or five B43-genistein-treated cynomolgus monkeys. B43-genistein showed a favorable pharmacokinetics in monkeys, with a plasma half-life of 10-23 h. Plasma samples from B43-genistein-treated monkeys elicited potent and CD19 antigenspecific antileukemic activity against human CD19+ leukemia cells in vitro. To our knowledge, this is the first preclinical toxicity and pharmacokinetic study of a tyrosine kinase inhibitor-containing immunoconjugate in nonhuman primates.

In vivo toxicity, pharmacokinetics, and antileukemic activity of TXU (anti-CD7)-pokeweed antiviral protein immunotoxin.

We evaluated the TXU (anti-CD7)-pokeweed antiviral protein (PAP) immunotoxin in both murine and nonhuman primate models. TXU-PAP caused dose-limiting cardiac toxicity in BALB/c mice. In a SCID mouse model of invariably fatal human T-lineage acute lymphoblastic leukemia (ALL), TXU-PAP therapy resulted in a marked improvement of leukemia-free survival without any side effects. Whereas 100% of control mice treated with PBS, unconjugated TXU antibody, or B43-PAP (an immunotoxin that does not react with T-lineage ALL cells) died of disseminated human leukemia within 80 days (median survival, 37 days), 80 +/- 13% of SCID mice treated with 15 microgram of TXU-PAP (median survival, >120 days) and 100% of mice treated with 30 microgram of TXU-PAP (median survival, > 120 days) remained alive and free of leukemia for >120 days. In cynomolgus monkeys, TXU-PAP showed favorable pharmacokinetics with an elimination half-life of 8.1-8.7 h. The monkeys treated with TXU-PAP at dose levels of 0.05 mg/kg/day x 5 days and 0.10 mg/kg/day x 5 days tolerated the therapy very well, without any significant clinical compromise or side effects, and at necropsy, no gross or microscopic lesions were found. This study provides a basis for further evaluation of TXU-PAP as an investigational biotherapeutic agent in the treatment of T-lineage ALL.

Cellular expression of antiapoptotic BCL-2 oncoprotein in newly diagnosed childhood acute lymphoblastic leukemia: a Children's Cancer Group Study.

We found a marked variation in BCL-2 oncoprotein expression levels of primary leukemic cells from 338 children with newly diagnosed acute lymphoblastic leukemia (ALL). None of the high-risk features predictive of poor treatment outcome in childhood ALL, such as older age, high white blood cell (WBC) count, organomegaly, T-lineage immunophenotype, ability of leukemic cells to cause overt leukemia in severe combined immunodeficient (SCID) mice, presence of MLL-AF4, and BCR-ABL fusion transcripts were associated with high levels of BCL-2 expression. Overall, high BCL-2 levels were not associated with slow early response, failure to achieve complete remission, or poor event-free survival. High BCL-2 levels in primary leukemic cells predicted slow early response only in T-lineage ALL patients, which comprised approximately 15% of the total patient population. Even for this small subset of patients, the level of BCL-2 expression did not have a significant impact on the short-term event-free survival.

Pharmacokinetic features, immunogenicity, and toxicity of B43(anti-CD19)-pokeweed antiviral protein immunotoxin in cynomolgus monkeys.

We studied the pharmacokinetic features, immunogenicity, and toxicity of B43-pokeweed antiviral protein (PAP) immunotoxin in 13 cynomolgus monkeys. The disposition of B43-PAP in two monkeys, when administered as a single i.v. bolus dose, was characterized by a slow clearance (1-2 ml/h/kg) with a very discrete peripheral distribution. B43-PAP was retained and distributed largely in the blood as the sole compartment with no significant equilibration with the extravascular compartment. The circulating B43-PAP immunotoxin detected in monkey plasma samples by ELISA and protein immunoblotting was both immunoreactive with, and active against, human leukemic cells in vitro. In systemic immunogenicity and toxicity studies, which involved 11 cynomolgus monkeys, each monkey received a total of seven i.v. doses of B43-PAP at a specific dose level of the dose escalation schedule. B43-PAP-treated monkeys mounted a dose-dependent humoral immune response against both the mouse IgG and PAP moieties of the immunotoxin. When administered i.v. either on an every-day or every-other-day schedule, B43-PAP was very well tolerated, with no significant clinical or laboratory signs of toxicity at total dose levels ranging from 0.007 to 0.7 mg/kg. A transient episode of a mild capillary leak with a grade 2 hypoalbuminemia and 2+ proteinuria was observed at total dose levels equal to or higher than 0.35 mg/kg. At total dose levels of 3.5 and 7.0 mg/kg, B43-PAP caused dose-limiting renal toxicity due to severe renal tubular necrosis. The present study completes the preclinical evaluation of B43-PAP and provides the basis for its clinical evaluation in children with therapy-refractory B-lineage acute lymphoblastic leukemia.

In vivo biotherapy of HL-60 myeloid leukemia with a genetically engineered recombinant fusion toxin directed against the human granulocyte macrophage colony-stimulating factor receptor.

Acute myeloid leukemia (AML) is the most common form of acute leukemia. Contemporary chemotherapy regimens fail to cure most patients with AML. We have genetically engineered a recombinant diphtheria toxin human granulocyte macrophage colony-stimulating factor (GMCSF) chimeric fusion protein (DTctGMCSF) that specifically targets the GMCSF receptor on fresh human AML cells and myeloid leukemia cell lines. At a nontoxic dose level, DTctGMCSF therapy was superior to the standard chemotherapeutic agents 1-beta-D-arabinofuranosylcytosine and Adriamycin, resulting in 60% long-term event-free survival of severe combined immunodeficient mice challenged with an otherwise invariably fatal cell dose of the human HL-60 myeloid leukemia. Notably, systemic exposure levels of DTctGMCSF, which were found to be therapeutic in the severe combined immunodeficient mouse xenograft model of human HL-60 myeloid leukemia, could be achieved in cynomolgus monkeys without any significant nonhematological toxicities. The recombinant DTctGMCSF fusion toxin might be useful in the treatment of AML patients whose leukemias have recurred and developed resistance to contemporary chemotherapy programs.

Treatment of human B-cell precursor leukemia in SCID mice using a combination of the investigational biotherapeutic agent B43-PAP with cytosine arabinoside.

Combined immunochemotherapy regimens using the investigational biotherapeutic agent B43(anti-CD19)-poke-weed antiviral protein (PAP) immunotoxin may offer an effective treatment for refractory B-cell precursor leukemias. The purpose of the present study was to explore and identify effective combinations of B43-PAP with standard chemotherapeutic drugs, including the anthracyclin doxorubicin, the epipodophyllotoxin etoposide, the nitrosurea carmustine, and the antimetabolite cytosine arabinoside. Here, we report that the B43-PAP plus cytosine arabinoside combination has potent antileukemic activity against human B-cell precursor leukemia in SCID mice and leads to 100% long-term event-free survival from an otherwise invariably fatal leukemia. Surprisingly, none of the other treatment protocols tested, including combinations of B43-PAP with carmustine, doxorubicin, or etoposide, proved more effective than B43-PAP alone.