Phase 1 dose-escalation trial of clofarabine followed by escalating dose of fractionated cyclophosphamide in adults with relapsed or refractory acute leukaemias.

The prognosis of patients with relapsed and refractory acute leukaemia (RRAL) is very poor. Forty patients with RRAL were enroled [28 acute myeloid leukaemia (AML), 12 acute lymphoblastic leukaemia (ALL)] in this Phase 1 dose-escalation trial of daily-infused clofarabine (CLO) followed by cyclophosphamide (CY) for four consecutive days (CLO-CYx4). The median age was 48·5 years. The median number of prior regimens was 2 (range 1-5), and 6/40 patients (15%) had prior allogeneic haematopoietic stem cell transplant. 28/40 patients (70%) had adverse genetic features. 6/40 patients (15%) died within 60 d of induction (two infections, four progressive disease). The average time to neutrophil recovery (absolute neutrophil count ≥0·5 × 10(9) /l was 34 d, (range, 17-78). The overall response rate (ORR) was 33% (13/40), with seven complete remissions (18%), four complete remissions with incomplete recovery of blood counts (10%), and two partial remissions (5%). ORR was 25% (7/28), and 50% (6/12), for AML and ALL respectively. Notably, the clinical responses were independent of dose level. 7/17 patients (41%) exhibited CLO-mediated enhancement of CY-induced DNA, which was associated with, but not necessary for, improved clinical outcomes. In summary, the CLO-CYx4 regimen was well tolerated and had activity in patients with RRAL, especially relapsed ALL. Therefore, CLO-CYx4 can be considered a salvage therapy for adults with RRALs, and warrants further investigations.

Phase I dose-escalation trial of clofarabine followed by escalating doses of fractionated cyclophosphamide in children with relapsed or refractory acute leukemias.

BACKGROUND: By inhibiting DNA repair, clofarabine (CLO) may augment cyclophosphamide (CY)-induced DNA damage and apoptosis. We performed a Phase I study for refractory and/or relapsed (R/R) leukemia in children to determine maximum-tolerated dose (MTD) of time-sequential CLO followed by CY. PROCEDURE: Thirteen patients with (R/R) ALL (n = 8) and AML (N = 5), median age 9 years (range: 2-12 years), were treated with escalating doses of CLO on days 1, 2, 3 and 8, 9, 10 and CY 200 mg/m(2) /day on days 0 and 1 then 400 mg/m(2) /day on days 2, 3, 8, 9, and 10. Ten patients were treated at dose level 1 (DL1) (CLO 20 mg/m(2) /day) and three patients at DL2 (CLO 30 mg/m(2) /day). The average number of prior chemotherapies was 8.9. DNA damage testing was performed before treatment on day 0, and 2 hours after CY on day 0, before sequential CLO, CY treatment on day 1, and 2 hours after CLO followed by CY on day 1. RESULTS: Two patients at DL2 had dose-limiting toxicities (DLTs) that included hypotension with cardio-respiratory failure (1) and hepato-renal failure (1). Complete remission (CR) was achieved in 2/11 (18.2%) and partial remission (PR) in 2/11 (18.2%) for an overall response (OR) of 36.4%. The use of CLO before CY augmented CY-induced DNA damage in leukemic blasts compared to CY alone. CONCLUSION: In pediatric patients with R/R leukemia, 20 mg/m(2) /day is the MTD for CLO in timed sequential combination with CY. Increased DNA damage with the use of this combination suggests a mechanism for the sequential timing of these two chemotherapeutic agents.

Active oral regimen for elderly adults with newly diagnosed acute myelogenous leukemia: a preclinical and phase 1 trial of the farnesyltransferase inhibitor tipifarnib (R115777, Zarnestra) combined with etoposide.

The farnesyltransferase inhibitor tipifarnib exhibits modest activity against acute myelogenous leukemia. To build on these results, we examined the effect of combining tipifarnib with other agents. Tipifarnib inhibited signaling downstream of the farnesylated small G protein Rheb and synergistically enhanced etoposide-induced antiproliferative effects in lymphohematopoietic cell lines and acute myelogenous leukemia isolates. We subsequently conducted a phase 1 trial of tipifarnib plus etoposide in adults over 70 years of age who were not candidates for conventional therapy. A total of 84 patients (median age, 77 years) received 224 cycles of oral tipifarnib (300-600 mg twice daily for 14 or 21 days) plus oral etoposide (100-200 mg daily on days 1-3 and 8-10). Dose-limiting toxicities occurred with 21-day tipifarnib. Complete remissions were achieved in 16 of 54 (30%) receiving 14-day tipifarnib versus 5 of 30 (17%) receiving 21-day tipifarnib. Complete remissions occurred in 50% of two 14-day tipifarnib cohorts: 3A (tipifarnib 600, etoposide 100) and 8A (tipifarnib 400, etoposide 200). In vivo, tipifarnib plus etoposide decreased ribosomal S6 protein phosphorylation and increased histone H2AX phosphorylation and apoptosis. Tipifarnib plus etoposide is a promising orally bioavailable regimen that warrants further evaluation in elderly adults who are not candidates for conventional induction chemotherapy. These clinical studies are registered at www.clinicaltrials.gov as #NCT00112853.

Screening a combinatorial peptide library to develop a human glandular kallikrein 2-activated prodrug as targeted therapy for prostate cancer.

OBJECTIVE: Prostate cancer cells secrete the unique protease human glandular kallikrein 2 (hK2) that represents a target for proteolytic activation of cytotoxic prodrugs. The objective of this study was to identify hK2-selective peptide substrates that could be coupled to a cytotoxic analogue of thapsigargin, a potent inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase pump that induces cell proliferation-independent apoptosis through dysregulation of intracellular calcium levels. METHODS: To identify peptide sequence requirements for hK2, a combination of membrane-bound peptides (SPOT analysis) and combinatorial chemistry using fluorescence-quenched peptide substrates was used. Peptide substrates were then coupled to 8-O-(12[L-leucinoylamino]dodecanoyl)-8-O-debutanoylthapsigargin (L12ADT), a potent analogue of thapsigargin, to produce a prodrug that was then characterized for hK2 hydrolysis, plasma stability, and in vitro cytotoxicity. RESULTS: Both techniques indicated that a peptide with two arginines NH2-terminal of the scissile bond produced the highest rates of hydrolysis. A lead peptide substrate with the sequence Gly-Lys-Ala-Phe-Arg-Arg (GKAFRR) was hydrolyzed by hK2 with a Km of 26.5 micromol/L, kcat of 1.09 s(-1), and a kcat/Km ratio of 41,132 s(-1) mol/L(-1). The GKAFRR-L12ADT prodrug was rapidly hydrolyzed by hK2 and was stable in plasma, whereas the GKAFRR-L peptide substrate was unstable in human plasma. The hK2-activated thapsigargin prodrug was not activated by cathepsin B, cathepsin D, and urokinase but was an excellent substrate for plasmin. The GKAFRR-L12ADT was selectively cytotoxic in vitro to cancer cells in the presence of enzymatically active hK2. CONCLUSION: The hK2-activated thapsigargin prodrug represents potential novel targeted therapy for prostate cancer.

A rapid and sensitive method for determination of veliparib (ABT-888), in human plasma, bone marrow cells and supernatant by using LC/MS/MS.

A rapid and sensitive method was developed and validated using a liquid chromatographic method with tandem mass spectrometry detection (LC/MS/MS) for determination of veliparib (ABT-888) in plasma, bone marrow supernatant, and bone marrow cells. Sample preparation involved a single protein precipitation step by the addition of the sample with acetonitrile. Separation of veliparib and the internal standard, A620223.69, was achieved on a Atlantis dC(18) column (100mmx2.1mm, 3microm) column using a mobile phase consisting of acetonitrile-ammonium acetate (2mM) containing formic acid (0.1%, v/v) using isocratic flow at 0.2mL/min for 3min. The analyte and internal standard were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 5-1000nM. The values for both within day and between day precision and accuracy were well within the generally accepted criteria for analytical methods. This method was subsequently used to measure concentrations of veliparib in cancer patients receiving an oral daily dose of 10mg with demonstration of drug accumulation in the marrow compartment and in the target leukemia bone marrow cells.

A phase 1 clinical-laboratory study of clofarabine followed by cyclophosphamide for adults with refractory acute leukemias.

Clofarabine has shown impressive response rates in patients with acute leukemias. In vitro investigations with clofarabine in combination with cyclophosphamide in primary cells have demonstrated synergistic cytotoxicity and inhibition of DNA repair. Based on these clinical and laboratory observations, we designed a mechanism-based combination protocol with clofarabine and cyclophosphamide for patients with relapsed acute leukemias. Eighteen patients were treated with cyclophosphamide (200 mg/m(2)) alone on day 0 and with clofarabine plus cyclophosphamide on day 1. Clinical responses, toxicity, DNA damage measured as H2AX phosphorylation, and accumulation of clofarabine triphosphate (TP) were analyzed. At dose level 1 (20 mg/m(2) clofarabine + cyclophosphamide, 6 patients) and dose level 0 (10 mg/m(2) clofarabine + cyclophosphamide, 12 patients) overall response rates were 50% and 30%, respectively, with responses in 4 (67%) of 6 patients with refractory acute lymphoblastic leukemia. Dose-limiting toxicity occurred at dose level 1 with prolonged marrow aplasia. Four (22%) patients died from prolonged aplasia (1), fungal pneumonia (1), or multiorgan failure (2). In 12 of 13 patient samples, increased DNA damage (gammaH2AX) was observed with clofarabine and cyclophosphamide compared with cyclophosphamide alone. In conclusion, pharmacodynamic end points along with clinical results suggest usefulness of this combination strategy, whereas toxicity data suggest reduction in chemotherapeutic intensity. This clinical trial is registered with the National Cancer Institute's PDQ at www.clinicaltrials.gov as no. JHOC-J0561.

Comparative study of PSMA expression in the prostate of mouse, dog, monkey, and human.

BACKGROUND: Intraprostatic PSMA targeted prodrugs/protoxins are under development in our laboratory. Future toxicologic studies of these therapies require identification of animal models that express PSMA within the prostate. METHOD: PSMA enzymatic activity and protein expression was determined. PSMA expression in the prostates of mouse, dog, and monkey were compared to humans by real-time PCR analysis. RESULTS: No substrate hydrolysis was observed in dog or monkey prostate homogenates. Monkey prostate was negative for PSMA protein expression. No significant PSMA mRNA levels were detected by real time PCR in mouse, dog, or monkey prostate tissue compared to PSMA negative tissues. CONCLUSIONS: PSMA is not expressed in any significant amount in the prostates of mouse, beagle dog, or macaque monkeys in this study but is expressed in high levels by human prostate. These non-human species, therefore, are not suitable toxicologic models to assess prostate damage from PSMA-activated intraprostatic prodrug/protoxin therapies.

Pharmacokinetics, biodistribution, and antitumor efficacy of a human glandular kallikrein 2 (hK2)-activated thapsigargin prodrug.

BACKGROUND: Prostate cancer cells secrete unique proteases such as prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) that represent targets for the activation of prodrugs as systemic treatment of metastatic prostate cancer. Previously, a combinatorial peptide library was screened to identify a highly active peptide substrate for hK2. The peptide was coupled to an analog of the potent cytotoxin thapsigargin, L12ADT, to generate an hK2-activated prodrug that was efficiently hydrolyzed by purified hK2, stable to hydrolysis in human and mouse plasma in vitro and selectively toxic to hK2 producing prostate cancer cells in vitro. METHODS: In the current study, toxicology, pharmacokinetics, prodrug biodistribution, and antitumor efficacy studies were performed to evaluate the hK2-activated prodrug in vivo. RESULTS: The single intravenous maximally tolerated dose of prodrug was 6 mg/kg (i.e., 3.67 micromole/kg) which produced peak serum concentration of approximately 36 microM and had a half-life of approximately 40 min. In addition, over a 24 hr period <0.5% of free L12ADT analog was observed in plasma. The prodrug demonstrated significant antitumor effect in vivo while it was being administered, but prolonged intravenous administration was not possible due to local toxicity to tail veins. Subcutaneous administration of equimolar doses produced lower plasma AUC compared to intravenous dosing but equivalent intratumoral levels of prodrug following multiple doses. CONCLUSIONS: The hK2-activated prodrug was stable in vivo. The prodrug, however, was rapidly cleared and difficult to administer over prolonged dosing interval. Additional studies are underway to assess antitumor efficacy with prolonged administration of higher subcutaneous doses of prodrug. Second-generation hK2-activated thapsigargin prodrugs with increased half-lives and improved formulations are also under development.

Dissociation between androgen responsiveness for malignant growth vs. expression of prostate specific differentiation markers PSA, hK2, and PSMA in human prostate cancer models.

BACKGROUND: A detailed understanding is evolving as to how androgen receptor (AR) functions as a transcriptional regulator via its binding to androgen response elements (ARE) within promoter and enhancer regions of prostate-specific differentiation markers such as PSA, hK2, and PSMA. It has been assumed that an understanding of regulation of expression of these marker proteins would also provide an understanding of the mechanisms whereby AR interactions regulate proliferation and survival of malignant prostate cells. In order to validate this hypothesis, we used a series of human prostate cancer models [i.e., LAPC-4, CWR22Rv1, MDA PCA-2b, LNCaP, and C4-2B (derived from LNCaP)] to test whether there is a consistent concordance between androgen responsive regulation for malignant growth vs. regulation of expression of prostate differentiation specific markers PSA, hK2, and PSMA. METHODS: In order to define androgen growth responsiveness in vivo, human prostate cancer cell lines were inoculated as xenografts into intact vs. surgically castrated adult male nude mice and the subsequent tumor growth response monitored. To assess androgen regulation of PSA and hK2 expression in these cell lines, the concentration of PSA and hK2 in the conditioned standard media and charcoal stripped media +/- androgen from each cell line was determined using an immunoassay system. PSMA enzymatic activity was determined using the PSMA substrate (3)H N-acetylaspartylglutamate ((3)H NAAG). RESULTS: Wild-type AR expressing LAPC-4 cells are androgen responsive for their in vivo growth. This cell line is also androgen sensitive for the expression of both PSA and hK2 in vitro and express PSMA. CWR22Rv1 cells have a mutated AR and are androgen responsive for growth in vivo and androgen sensitive for hk2 but not PSA expression. CWR22Rv1 produce approximately 1.4-fold more PSA, approximately 18-fold more hK2, and have 21-fold higher PSMA activity than LAPC-4 cells. MDA PCA-2b cells are androgen responsive for growth in vivo and androgen sensitive for PSA expression. MDA PCA-2b cells produce approximately 250-fold more PSA but almost equivalent amounts of hK2 compared to LAPC-4 and have approximately 19-fold higher PSMA activity. Both late passage LNCaP and C4-2B are androgen independent for growth in vivo but remain androgen sensitive for both PSA and hK2 expression. LNCaP cells produce approximately 50-fold more PSA, approximately 35-fold more hK2, and have 28-fold higher PSMA activity compared to LAPC-4. C4-2B cells produce approximately 80-fold higher levels of PSA, approximately 250-fold higher levels of hK2. C4-2B also the highest PSMA activity of the cell lines with 105-fold higher PSMA activity than LAPC-4 and approximately 4-fold higher activity than late passage LNCaP cells. CONCLUSIONS: Androgen can coordinately regulate both the tumor growth and expression of prostate specific marker genes as observed for the LAPC-4 human prostate cancer cells. Such coordinated regulation, however, is not universal. In all of the other cell lines, there is a dissociation between androgen responsive regulation of malignant growth vs. regulation of expression of prostate specific markers PSA and hK2. In addition, PSMA activity in these cell lines increases as cells become more androgen independent for growth in vivo. These results emphasize that tumor growth and the expression of the specific secretory genes are independently regulated molecular events even if they share a requirement for androgen and/or AR function. Additional independent mechanisms occur in prostate cancer cells for regulation of expression for even the highly related PSA and hK2 genes. Further studies are needed to clarify the mechanisms for androgen ligand-independent, AR-dependent regulation of the genes that directly effect the growth of androgen (i.e., ligand) independent prostate cancer cells. Unfortunately, the data in this present report do not validate the use of the PSA or hK2 gene as surrogates for a model system for such critically important mechanistic studies. Prostate prostate cancer cells. Unfortunately, the data in this present report do not validate the use of the PSA or hK2 gene as surrogates for a model system for such critically important mechanistic studies.