Lee Roy Morgan

Tumor Physiology and Charge Dynamics of Anticancer Drugs: Implications for Camptothecin-based Drug Development

Charge is an important characteristic of drug molecules, since ionization sites determine the pKa at a particular pH. The pKa in turn can affect many parameters, including solubility, dissolution rate, reaction kinetics, formulation, cell permeability, tissue distribution, renal elimination, metabolism, protein binding and receptor interactions. The impact of charge dynamics is amplified in human solid tumors that exhibit the glycolytic phenotype and associated acidic extracellular microenvironment. This phenotype is driven by hypoxia and creates a pH gradient in tumors that favors uptake of weak acids and exclusion of weak bases. Established anticancer drugs exhibit a range of pKas and thus variable ability to exploit the tumor pH gradient. The camptothecins are a prime example as they represent a diverse class of approved anticancer drugs and drug candidates whose charge distribution varies with pH. An in silico method was used to predict charge distribution of camptothecins at physiological versus acidic pH in both the lactone and carboxylate forms. A significant amount of uncharged carboxylate was predicted at acidic pH that could enter tumor cells and accumulate in mitochondria to inhibit mitochondrial topoisomerase I. A model is presented to describe the charge dynamics of a new camptothecin analog and the impact on nuclear and mitochondrial mechanism(s) of action. This example illustrates the importance of integrating tumor physiology and charge dynamics into anticancer drug development.

Pentamethylpyrromethene boron difluoride complexes in human ovarian cancer photodynamic therapy

Quasiaromatic heterocycles (QAM) such as substituted 1 , 3 , 5 , 7 , 8-pentamethylpyrromethene boron difluorides (PMP-BF2) and - (dimethoxyphosphinylmethyl, methyl) bimane have been evaluated for their abilities to produce cellular toxicities when used in photodynamic therapy (PDT) for ovarian cancer. The most active QAH tested to date has been the disodiuxn salt of PMP-2,6-disulfonate--BF2 (PMPDS-BF2). Human ovarian cancer cells from fifteen different patients have been grown in culture. Cells were obtained from biopsy material and grown in RPMI medium with 10% FBA plus penicillin and streptomycin. Cells were harvested and as single cell suspensions exposed to PMP-BF2 complexes or bimanes in concentrations of 0.004-0.4 ug/106 cells/ml of medium. Initially the cells were exposed to the chemicals for 30 minutes in a 5% CO2 incubator (37°C) with gentle shaking. The cells were washed with plain RPMI medium, then resuspended in the enriched RPMI medium and exposed to a sunlamp for 10-20 minutes. Cells were then allowed to grow in an soft agar culture media at 37°C (5% C02) for 14 days. When compared to controls (only light or only chemicals) there was 100% inhibition of all cellular growth for PMPDSBF2 at the 0.4 ug/mi concentrations. There was variations in concentrations of the chemical needed to produce 100% inhibition when the 15 different ovarian cancer cell specimens were compared at all concentrations. PMP-BF2 complexes are characterized by extremely high extinction coefficients, superior laser activity and little if any triplet-triplet absorption. The biamanes share these properties however are less active in ovarian cancer cell The lasing properties of PMP-BF2, and bimanes will be compared to their PDT effectiveness.

Acyl derivatives of demethylpenclomedine, an antitumor-active, non-neurotoxic metabolite of penclomedine

Abstract. Purpose: The purpose of this investigation was to compare the antitumor activities of a series of acyl derivatives of 4-demethylpenclomedine (DM-PEN), the major plasma metabolite of penclomedine (PEN) observed to be an active antitumor agent in vivo and non-neurotoxic in a rat model with that of DM-PEN. Methods: Acyl derivatives were prepared from DM-PEN and evaluated in vivo against human MX-1 breast tumor xenografts implanted subcutaneously (s.c.) or intracerebrally (i.c.). Several derivatives were also evaluated against other human tumor xenografts and murine P388 leukemia cell lines. Results: Several of the acyl derivatives were found to be superior to DM-PEN against MX-1, human ZR-75-1 breast tumor, human U251 CNS tumor and the P388 leukemia parent cell line and lines resistant to cyclophosphamide and carmustine. 4-Demethyl-4-methoxyacetylpenclomedine showed inferior activity to current clinical brain tumor drugs against a glioma cell line, superior activity to temozolomide and procarbazine against the derived mismatch repair-deficient cell line, and superior activity to cyclophosphamide and carmustine but inferior activity to temozolomide against two ependymoma cell lines, all of which were implanted s.c. Conclusion: Proposed mechanisms of activation and action of DM-PEN and the acyl derivatives support the potential clinical superiority of the acyl derivatives.

Abstract 4479: Preclinical toxicology and pharmacology for BACPTDP, a camptothecin that targets hypoxic/acidic tumors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC BACPTDP is a dipeptide prodrug of the 7-butyl-10-amino analog of camptothecin (BACPT) that is nearing clinical development. BACPTDP was selected based on its ability to exploit the tumor pH gradient that exists in tumors growing in hypoxic and acidic extracellular microenvironments. The drug has two known molecular targets: nuclear topoisomerase I and the transcription factor HIF-1α. BACPTDP has broad spectrum preclinical antitumor activity, but was particularly active against Panc-1 murine xenografts, a model for advanced pancreatic cancer (Adams, D.J., et al. Cancer Chemother. Pharmacol. 67, 855-865, 2011). The goals of the current work were to evaluate toxicology in vivo and to identify a sensitive biomarker of drug response in vitro. Results: Acute toxicity of a single intravenous bolus dose was assessed in mice and rats, where the LD10/LD50 was 15/23 mg/kg and 7.5/13 mg/kg, respectively. The cause of death in both rodent models was cardiovascular/respiratory arrest. Liver function abnormalities (AST/ALT) and bone marrow suppression were noted in rats at 5 mg/kg; neither renal nor CNS toxicity (seizures) was observed in any studies. Dog studies are pending. The phosphorylated histone γH2AX was found to be a sensitive biomarker for BACPT response in both human PBMCs and in Panc-1 cells. γH2AX could be detected in 20% of Panc-1 cells treated for 2 h at 10 nM by flow cytometry assay. Of note, when cells were cultured at 10% oxygen, the level seen by circulating tumor cells (CTCs), the effective drug concentration decreased by 25-fold. The biomarker could also be detected in 30% of HT29 colon carcinoma cells treated for 2 h with 100 nM BACPT, spiked into human blood samples and analyzed by the Veridex CellSearch technology for CTCs. This response was equivalent to that observed with a ten-fold higher concentration of topotecan. Interaction of BACPT with gemcitabine was evaluated in Panc-1, MiaPaCa-2 and BxPC-3 pancreatic cancer cell lines under atmospheric versus 10% oxygen conditions. Antiproliferative activity of BACPT alone was not significantly affected by oxygen concentration (cell line mean IC50 = 5 nM versus 2 nM, respectively). Likewise, drug interaction assessed by median effect analysis indicated that lower oxygen tension did not dramatically affect drug interaction, with additive to synergistic interactions that were independent of sequence of administration. Conclusions: these preclinical data support the development of a Phase I clinical trial to evaluate BACPTDP in pancreatic cancer as a single agent and in Phase II studies in combination with gemcitabine, the standard-of-care chemotherapy. In addition, γH2AX can serve as a sensitive biomarker of response to BACPTDP in both PBMCs and in circulating tumor cells, consistent with previously published studies with topotecan. Citation Format: David J. Adams, Lee Roy Morgan, Andrew H. Rodgers, Branko S. Jursic, Govindarajan Manikumar, Mansukh C. Wani. Preclinical toxicology and pharmacology for BACPTDP, a camptothecin that targets hypoxic/acidic tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4479. doi:10.1158/1538-7445.AM2013-4479