Billie Nowak

Compound GW506U78 in refractory hematologic malignancies: relationship between cellular pharmacokinetics and clinical response.

PURPOSE In vitro investigations with arabinosylguanine (ara-G) demonstrated potent cytotoxicity to T-lymphoblastoid cell lines. The goals of the present study were to evaluate GW506U78, a prodrug of ara-G, against human hematologic malignancies and to determine its pharmacokinetics in plasma and cells. PATIENTS AND METHODS During a phase I multicenter trial of GW506U78, 26 patients were treated at M.D. Anderson Cancer Center (MDACC). Daily doses between 20 and 60 mg/kg were administered for 5 days. Parallel plasma and cellular pharmacokinetic studies were conducted. RESULTS Complete (n=5) or partial remission (n=5) was achieved in T-cell acute lymphoblastic leukemia (T-ALL), T-lymphoid blast crisis, T-lymphoma, and B-cell chronic lymphocytic leukemia (B-CLL) (n=13). In contrast, patients with B-ALL, B-lymphoma, acute myelogenous leukemia (AMI), or T-CLL did not respond. Peak plasma concentrations of GW506U78 and ara-G were dose-dependent. The elimination of GW506U78 (half-life [t1/2]=17 minutes) was faster than the elimination of ara-G (t1/2=3.7 hours). Median peak concentrations of ara-GTP were 23, 42, 85, and 93 micromol/L at 20, 30, 40, and 60 mg/kg, respectively. T-lymphoblasts accumulated significantly (P=.0008) higher peak arabinsylguanosine triphosphate (ara-GTP) (median, 140 micromol/L; n=7) compared with other diagnoses (median, 50 micromol/L; n=9) and normal mononuclear cells (n=3). The ara-GTP elimination was slow in all diagnoses (median, > 24 hours). Responders accumulated significantly (P=.0005) higher levels of ara-GTP (median, 157 micromol/L) compared with patients who failed to respond (median, 44 micromol/L). CONCLUSION GW506U78 is an effective prodrug and a potent agent for hematologic malignancies with major efficacy in T-cell diseases. The pharmacokinetics of ara-GTP in leukemia cells are strongly correlated with clinical responses to GW506U78.

High-performance liquid chromatography method for the determination and quantitation of arabinosylguanine triphosphate and fludarabine triphosphate in human cells

A gradient anion-exchange high-performance liquid chromatographic assay was developed for the simultaneous determination and quantitation of the cytotoxic triphosphates of arabinosylguanine (ara-GTP) and fludarabine (F-ara-ATP). The method was validated with respect to selectivity, recovery, linearity, precision, and accuracy using authentic standards. To test this assay in a more complex biological matrix, perchloric acid extracts of circulating human leukemia cells spiked with known concentrations of ara-GTP and F-ara-ATP were examined. Finally, to assess the clinical utility of our method, perchloric acid extracts of circulating human leukemia cells isolated from patients treated with fludarabine and nelarabine were analyzed. The range of quantitation was 0.0125-10 nmol for the ara- and native NTPs in cellular extracts. This assay should be helpful in establishing the mechanistic rationales for drug scheduling and combinations of nelarabine and fludarabine, and for correlating the therapeutic efficacy and levels of the cytotoxic triphosphates in target cells.

Clinical and Pharmacokinetic Study of Clofarabine in Chronic Lymphocytic Leukemia: Strategy for Treatment

Purpose: Based on its mechanistic similarity to fludarabine and cladribine and the success of these analogues for treatment of chronic lymphocytic leukemia (CLL), we hypothesized that clofarabine would be effective for indolent leukemias. The present study was conducted to determine the efficacy and cellular pharmacology during clinical trials of single-agent clofarabine in CLL. Experimental Design: Previously treated patients with relapsed/refractory CLL were eligible for this study. Clofarabine was infused over 1 hour daily for 5 days. Most patients received 3 or 4 mg/m2/d × 5 days, whereas the other two were treated with 15 mg/m2/d × 5 days. Clinical outcome and associated pharmacologic end points were assessed. Results: Myelosuppression limited the maximum tolerated dose of clofarabine to 3 mg/m2/d on this schedule. Cellular pharmacokinetic studies showed a median clofarabine triphosphate concentration in CLL lymphocytes of 1.5 μmol/L (range, 0.2-2.3 μmol/L; n = 9). In the majority of cases, >50% of the analogue triphosphate was present 24 hours after infusion, indicating prolonged retention of the triphosphate in CLL cells. Although cytoreduction was observed, no patients achieved a response. In vitro clofarabine incubation of leukemic lymphocytes from 29 CLL patients showed that clofarabine monophosphate accumulated to a higher concentration compared with the triphosphate. Nonetheless, the triphosphate increased in a dose-dependent fashion and upon successive clofarabine infusions, suggesting benefit from greater doses given at less frequent intervals. Conclusion: Levels of clofarabine triphosphate at higher doses and prolonged maintenance of clofarabine triphosphate in leukemic lymphocytes provide a rationale to treat CLL in a weekly clofarabine schedule.

Phase I clinical, pharmacokinetic, and pharmacodynamic study of the Akt-inhibitor triciribine phosphate monohydrate in patients with advanced hematologic malignancies

Akt, a serine/threonine protein kinase, is constitutively phosphorylated and hyperactivated in multiple cancers, including acute myeloid leukemia. High levels are linked to poor survival and inferior responses to chemotherapy, making Akt inhibition an attractive therapeutic target. In this phase I/II study of TCN-PM, a small-molecule Akt inhibitor, TCN-PM therapy was well tolerated in patients with advanced hematological malignancies, and reduced levels of phosphorylation of Akt and its substrate Bad were shown, consistent with inhibition of this survival pathway and induction of cell death. Further investigation of TCN-PM alone or in combination in patients with high Akt levels is warranted.

Sapacitabine, the prodrug of CNDAC, is a nucleoside analog with a unique action mechanism of inducing DNA strand breaks.

Sapacitabine is an orally bioavailable prodrug of the nucleoside analog 2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosylcytosine (CNDAC). Both the prodrug and active metabolite are in clinical trials for hematologic malignancies and/or solid tumors. CNDAC has a unique mechanism of action: after incorporation into DNA, it induces single-strand breaks (SSBs) that are converted into double-strand breaks (DSBs) when cells go through a second S phase. In our previous studies, we demonstrated that CNDAC-induced SSBs can be repaired by the transcription-coupled nucleotide excision repair pathway, whereas lethal DSBs are mainly repaired through homologous recombination. In the current work, we used clonogenic assays to compare the DNA damage repair mechanism of CNDAC with two other deoxycytidine analogs: cytarabine, which is used in hematologic malignacies, and gemcitabine, which shows activity in solid tumors. Deficiency in two Rad51 paralogs, Rad51D and XRCC3, greatly sensitized cells to CNDAC, but not to cytarabine or gemcitabine, indicating that homologous recombination is not a major mechanism for repairing damage caused by the latter two analogs. This study further suggests clinical activity and application of sapacitabine that is distinct from that of cytarabine or gemcitabine.

Inhibition of ATP synthase by chlorinated adenosine analogue

8-Chloroadenosine (8-Cl-Ado) is a ribonucleoside analogue that is currently in clinical trial for chronic lymphocytic leukemia. Based on the decline in cellular ATP pool following 8-Cl-Ado treatment, we hypothesized that 8-Cl-ADP and 8-Cl-ATP may interfere with ATP synthase, a key enzyme in ATP production. Mitochondrial ATP synthase is composed of two major parts; F(O) intermembrane base and F1 domain, containing alpha and beta subunits. Crystal structures of both alpha and beta subunits that bind to the substrate, ADP, are known in tight binding (alpha(dp)beta(dp)) and loose binding (alpha(tp)beta(tp)) states. Molecular docking demonstrated that 8-Cl-ADP/8-Cl-ATP occupied similar binding modes as ADP/ATP in the tight and loose binding sites of ATP synthase, respectively, suggesting that the chlorinated nucleotide metabolites may be functional substrates and inhibitors of the enzyme. The computational predictions were consistent with our whole cell biochemical results. Oligomycin, an established pharmacological inhibitor of ATP synthase, decreased both ATP and 8-Cl-ATP formation from exogenous substrates, however, did not affect pyrimidine nucleoside analogue triphosphate accumulation. Synthesis of ATP from ADP was inhibited in cells loaded with 8-Cl-ATP. These biochemical studies are in consent with the computational modeling; in the alpha(tp)beta(tp) state 8-Cl-ATP occupies similar binding as ANP, a non-hydrolyzable ATP mimic that is a known inhibitor. Similarly, in the substrate binding site (alpha(dp)beta(dp)) 8-Cl-ATP occupies a similar position as ATP mimic ADP-BeF(3)(-). Collectively, our current work suggests that 8-Cl-ADP may serve as a substrate and the 8-Cl-ATP may be an inhibitor of ATP synthase.

Synthesis, Cytotoxicity and Metabolism of the 2′,2′-Difluoro-Analogs of Deoxyadenosine (dFdA)and Deoxyguanosine (dFdG)

Abstract 2′,2′-Difluoro analogs of deoxyadenosine (dFdA) and deoxyguanosine (dFdG) were synthesized. The in vitro toxicity and metabolism of dFdA and dFdG was studied in human leukemia cell lines.

Multifaceted actions of 8-amino-adenosine kill BCR-ABL positive cells.

Abstract Survival of chronic myelogenous leukemia (CML) cells is dependent on BCR–ABL kinase, the activity of which is contingent on the level of BCR–ABL protein and the availability of adenosine triphosphate (ATP). We hypothesized that 8-amino-adenosine (8-amino-Ado)-mediated reduction in cellular ATP level and inhibition of mRNA synthesis leading to a decrease in protein level would result in a multifaceted targeting of BCR–ABL. Using K562 cells, we demonstrated that there was a dose- and time-dependent increase in 8-amino-ATP accompanied by a > 95% decline in the endogenous ATP pool. In parallel, 8-amino-Ado inhibited RNA synthesis and resulted in a depletion of BCR–ABL transcript. Consistent with this, BCR–ABL and ABL protein levels were also decreased. These effects were associated with the initiation of cell death as visualized by poly(ADP-ribose) polymerase (PARP) cleavage, decreased clonogenicity and greater than additive interaction with imatinib. In imatinib-sensitive and -resistant KBM5 cells, 8-amino-Ado treatment augmented the imatinib effect on growth inhibition.

Mechanism-Based Drug Combinations with the DNA Strand–Breaking Nucleoside Analog CNDAC

CNDAC (2′-C-cyano-2′-deoxy-1-β-d-arabino-pentofuranosyl-cytosine, DFP10917) and its orally bioavailable prodrug, sapacitabine, are undergoing clinical trials for hematologic malignancies and solid tumors. The unique action mechanism of inducing DNA strand breaks distinguishes CNDAC from other deoxycytidine analogs. To optimize the clinical potentials of CNDAC, we explored multiple strategies combining CNDAC with chemotherapeutic agents targeting distinct DNA damage repair pathways that are currently in clinical use. The ability of each agent to decrease proliferative potential, determined by clonogenic assays, was determined in paired cell lines proficient and deficient in certain DNA repair proteins. Subsequently, each agent was used in combination with CNDAC at fixed concentration ratios. The clonogenicity was quantitated by median effect analysis, and a combination index was calculated. The c-Abl kinase inhibitor imatinib had synergy with CNDAC in HCT116 cells, regardless of p53 status. Inhibitors of PARP1 that interfere with homologous recombination (HR) repair or base excision repair (BER) and agents such as temozolomide that cause DNA damage repaired by the BER pathway were also synergistic with CNDAC. The toxicity of the nitrogen mustards bendamustine and cytoxan, or of platinum compounds, which generate DNA adducts repaired by nucleotide excision repair and HR, was additive with CNDAC. An additive cell killing was also achieved by the combination of CNDAC with taxane mitotic inhibitors (paclitaxel and docetaxel). At concentrations that allow survival of the majority of wild-type cells, the synergistic or additive combination effects were selective in HR-deficient cells. This study provides mechanistic rationales for combining CNDAC with other active drugs. Mol Cancer Ther; 15(10); 2302–13. ©2016 AACR.

Abstract 5667: Mechanism-based combinations of agents impacting the homologous recombination and nucleotide excision repair pathways

Sapacitabine, a prodrug of the nucleoside analog CNDAC [2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosylcytosine], has the advantage of oral bioavailability. CNDAC has a unique action mechanism of inducing single strand breaks following its incorporation into DNA through a β-elimination process. Such nicks caused by sapacitabine/CNDAC are partially repaired through the transcription-coupled nucleotide excision repair (NER) pathway [Cancer Res. 2008, 68:3881-89]. However, DNA replication across the unrepaired nicks generates double strand breaks which are mainly repaired by the ATM-dependent homologous recombination (HR) mechanism [Blood, 2010, 116:1737-46]. Sapacitabine is undergoing clinical trials with encouraging outcomes in AML or MDS patients [J. Clin.Oncol., 2010, 28:285-91]. A trial of sapacitabine in combination with cyclophosphamide and rituximab (SCR regimen) in chronic lymphocytic leukemia (CLL) relapsed from first line therapies has been initiated in populations lacking ATM function. Other candidates for clinical combinations with sapacitabine include bendamustine, an alkylating agent, and oxaliplatin, which are currently used for treatment of CLL. Primary adducts caused by each of these compounds are repaired by NER. To explore mechanistic interactions between sapacitabine/CNDAC and agents impacting the NER pathway, we used median effect analysis to compare cellular clonogenic survival of drug combinations with survival after single agents alone. First, cells deficient in HR components (Rad51D, Xrcc3 and Brca2) were all sensitized to 4-hydroperoxycyclophosphamide (4-HC, readily converted to the active metabolite of cyclophosphamide, 4-hydroxycyclophosphamide), bendamustine, ciplatin and oxaliplatin by 10- to 50-fold, whereas cells lacking the key NER endonuclease XPF were more sensitive to these agents. This indicates that HR is responsible for residual lesions not repaired by NER. Second, CNDAC in combination with 4-HC presented additive to mildly synergistic effect (combination index CI ≤ 1) in cells defective in Xrcc3, Rad51D, Brca2 or XPF. Third, combination of bendamustine with CNDAC showed additive loss of clonogenicity (CI ∼ 1) in cells lacking Rad51D or Xrcc3. Fourth, combination of cisplatin or oxaliplatin with CNDAC also exerted additive effects (CI ∼ 1) in HR- as well as NER-deficient cells. Finally, cisplatin/oxaliplatin combined with CNDAC, both at relatively high concentration ranges lethal to HR-defective cells, had no synergy in wild type cells either. Together these results suggest that agents causing DNA adducts, which are mainly repaired by NER process, are additive in their interaction with sapacitabine/CNDAC. Thus, this study provides rationales for sapacitabine-based combinational and targeted therapeutic strategies for translation into the design of clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5667. doi:1538-7445.AM2012-5667