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1.  The Role of Bystander Effects in the Antitumor Activity of the Hypoxia-Activated Prodrug PR-104 
Frontiers in Oncology  2013;3:263.
Activation of prodrugs in tumors (e.g., by bioreduction in hypoxic zones) has the potential to generate active metabolites that can diffuse within the tumor microenvironment. Such “bystander effects” may offset spatial heterogeneity in prodrug activation but the relative importance of this effect is not understood. Here, we quantify the contribution of bystander effects to antitumor activity for the first time, by developing a spatially resolved pharmacokinetic/pharmacodynamic (SR-PK/PD) model for PR-104, a phosphate ester pre-prodrug that is converted systemically to the hypoxia-activated prodrug PR-104A. Using Green’s function methods we calculated concentrations of oxygen, PR-104A and its active metabolites, and resultant cell killing, at each point of a mapped three-dimensional tumor microregion. Model parameters were determined in vitro, using single cell suspensions to determine relationships between PR-104A metabolism and clonogenic cell killing, and multicellular layer (MCL) cultures to measure tissue diffusion coefficients. LC-MS/MS detection of active metabolites in the extracellular medium following exposure of anoxic single cell suspensions and MCLs to PR-104A confirmed that metabolites can diffuse out of cells and through a tissue-like environment. The SR-PK/PD model estimated that bystander effects contribute 30 and 50% of PR-104 activity in SiHa and HCT116 tumors, respectively. Testing the model by modulating PR-104A-activating reductases and hypoxia in tumor xenografts showed overall clonogenic killing broadly consistent with model predictions. Overall, our data suggest that bystander effects are important in PR-104 antitumor activity, although their reach may be limited by macroregional heterogeneity in hypoxia and reductase expression in tumors. The reported computational and experimental techniques are broadly applicable to all targeted anticancer prodrugs and could be used to identify strategies for rational prodrug optimization.
doi:10.3389/fonc.2013.00263
PMCID: PMC3791487  PMID: 24109591
anticancer prodrugs; hypoxia-activated prodrugs; PR-104; bystander effect; extravascular drug transport; multicellular layers; pharmacokinetic/pharmacodynamic modeling; NADPH:cytochrome P450 oxidoreductase
2.  Design of Optimized Hypoxia-Activated Prodrugs Using Pharmacokinetic/Pharmacodynamic Modeling 
Frontiers in Oncology  2013;3:314.
Hypoxia contributes to resistance of tumors to some cytotoxic drugs and to radiotherapy, but can in principle be exploited with hypoxia-activated prodrugs (HAP). HAP in clinical development fall into two broad groups. Class I HAP (like the benzotriazine N-oxides tirapazamine and SN30000), are activated under relatively mild hypoxia. In contrast, Class II HAP (such as the nitro compounds PR-104A or TH-302) are maximally activated only under extreme hypoxia, but their active metabolites (effectors) diffuse to cells at intermediate O2 and thus also eliminate moderately hypoxic cells. Here, we use a spatially resolved pharmacokinetic/pharmacodynamic (SR-PK/PD) model to compare these two strategies and to identify the features required in an optimal Class II HAP. The model uses a Green’s function approach to calculate spatial and longitudinal gradients of O2, prodrug, and effector concentrations, and resulting killing in a digitized 3D tumor microregion to estimate activity as monotherapy and in combination with radiotherapy. An analogous model for a normal tissue with mild hypoxia and short intervessel distances (based on a cremaster muscle microvessel network) was used to estimate tumor selectivity of cell killing. This showed that Class II HAP offer advantages over Class I including higher tumor selectivity and greater freedom to vary prodrug diffusibility and rate of metabolic activation. The model suggests that the largest gains in class II HAP antitumor activity could be realized by optimizing effector stability and prodrug activation rates. We also use the model to show that diffusion of effector into blood vessels is unlikely to materially increase systemic exposure for realistic tumor burdens and effector clearances. However, we show that the tumor selectivity achievable by hypoxia-dependent prodrug activation alone is limited if dose-limiting normal tissues are even mildly hypoxic.
doi:10.3389/fonc.2013.00314
PMCID: PMC3873531  PMID: 24409417
tumor hypoxia; hypoxia-activated prodrugs; bystander effect; extravascular drug transport; pharmacokinetic/pharmacodynamic modeling; rational drug design; tirapazamine; PR-104
3.  PR-104 a bioreductive pre-prodrug combined with gemcitabine or docetaxel in a phase Ib study of patients with advanced solid tumours 
BMC Cancer  2012;12:496.
Background
The purpose of this phase Ib clinical trial was to determine the maximum tolerated dose (MTD) of PR-104 a bioreductive pre-prodrug given in combination with gemcitabine or docetaxel in patients with advanced solid tumours.
Methods
PR-104 was administered as a one-hour intravenous infusion combined with docetaxel 60 to 75 mg/m2 on day one given with or without granulocyte colony stimulating factor (G-CSF) on day two or administrated with gemcitabine 800 mg/m2 on days one and eight, of a 21-day treatment cycle. Patients were assigned to one of ten PR-104 dose-levels ranging from 140 to 1100 mg/m2 and to one of four combination groups. Pharmacokinetic studies were scheduled for cycle one day one and 18F fluoromisonidazole (FMISO) positron emission tomography hypoxia imaging at baseline and after two treatment cycles.
Results
Forty two patients (23 females and 19 males) were enrolled with ages ranging from 27 to 85 years and a wide range of advanced solid tumours. The MTD of PR-104 was 140 mg/m2 when combined with gemcitabine, 200 mg/m2 when combined with docetaxel 60 mg/m2, 770 mg/m2 when combined with docetaxel 60 mg/m2 plus G-CSF and ≥770 mg/m2 when combined with docetaxel 75 mg/m2 plus G-CSF. Dose-limiting toxicity (DLT) across all four combination settings included thrombocytopenia, neutropenic fever and fatigue. Other common grade three or four toxicities included neutropenia, anaemia and leukopenia. Four patients had partial tumour response. Eleven of 17 patients undergoing FMISO scans showed tumour hypoxia at baseline. Plasma pharmacokinetics of PR-104, its metabolites (alcohol PR-104A, glucuronide PR-104G, hydroxylamine PR-104H, amine PR-104M and semi-mustard PR-104S1), docetaxel and gemcitabine were similar to that of their single agents.
Conclusions
Combination of PR-104 with docetaxel or gemcitabine caused dose-limiting and severe myelotoxicity, but prophylactic G-CSF allowed PR-104 dose escalation with docetaxel. Dose-limiting thrombocytopenia prohibited further evaluation of the PR104-gemcitabine combination. A recommended dose was identified for phase II trials of PR-104 of 770 mg/m2 combined with docetaxel 60 to 75 mg/m2 both given on day one of a 21-day treatment cycle supported by prophylactic G-CSF (NCT00459836).
doi:10.1186/1471-2407-12-496
PMCID: PMC3495895  PMID: 23098625
4.  Pharmacokinetic/pharmacodynamic modeling identifies SN30000 and SN29751 as tirapazamine analogs with improved tissue penetration and hypoxic cell killing in tumors 
Clinical Cancer Research  2010;16(20):4946-4957.
Purpose
Tirapazamine (TPZ) has attractive features for targeting hypoxic cells in tumors but limited clinical activity, in part because of poor extravascular penetration. Here we identify improved TPZ analogs by using a spatially resolved pharmacokinetic/pharmacodynamic (SR-PKPD) model that considers tissue penetration explicitly during lead optimization.
Experimental design
The SR-PKPD model was used to guide progression of 281 TPZ analogs through a hierarchical screen. For compounds exceeding hypoxic selectivity thresholds in single cell cultures, SR-PKPD model parameters (kinetics of bioreductive metabolism, clonogenic cell killing potency, diffusion coefficients in multicellular layers, plasma pharmacokinetics at well tolerated doses in mice) were measured to prioritize testing in xenograft models in combination with radiation.
Results
SR-PKPD-guided lead optimization identified SN29751 and SN30000 as the most promising hypoxic cytotoxins from two different structural subseries. Both were reduced to the corresponding 1-oxide selectively under hypoxia by HT29 cells, with an oxygen dependence quantitatively similar to that of TPZ. SN30000, in particular, showed higher hypoxic potency and selectivity than TPZ in tumor cell cultures and faster diffusion through HT29 and SiHa multicellular layers. Both compounds also provided superior plasma PK in mice and rats at equivalent toxicity. In agreement with SR-PKPD predictions, both were more active than TPZ with single dose or fractionated radiation against multiple human tumor xenografts.
Conclusions
SN30000 and SN29751 are improved TPZ analogs with potential for targeting tumor hypoxia in humans, and illustrate the utility of novel SR-PKPD modeling approaches for lead optimization during anticancer drug development.
doi:10.1158/1078-0432.CCR-10-1439
PMCID: PMC3390971  PMID: 20732963
Hypoxia; benzotriazine N-oxides; tirapazamine; SN29751; SN30000; extravascular transport; PKPD models
5.  A phase I trial of PR-104, a pre-prodrug of the bioreductive prodrug PR-104A, given weekly to solid tumour patients 
BMC Cancer  2011;11:432.
Background
The phosphate ester PR-104 is rapidly converted in vivo to the alcohol PR-104A, a nitrogen mustard prodrug that is metabolised to hydroxylamine (PR-104H) and amine (PR-104M) DNA crosslinking agents by one-electron reductases in hypoxic cells and by aldo-keto reductase 1C3 independently of oxygen. In a previous phase I study using a q 3 week schedule of PR-104, the maximum tolerated dose (MTD) was 1100 mg/m2 and fatigue, neutropenic fever and infection were dose-limiting. The primary objective of the current study was to determine the dose-limiting toxicity (DLT) and MTD of weekly PR-104.
Methods
Patients with advanced solid tumours received PR-104 as a 1-hour intravenous infusion on days 1, 8 and 15 every 28 days with assessment of pharmacokinetics on cycle 1 day 1. Twenty-six patients (pts) were enrolled (16 male/10 female; median age 58 yrs, range 30 to 70 yrs) who had received a median of two prior chemotherapy regimens (range, 0 to 3) for melanoma (8 pts), colorectal or anal cancer (3 pts), NSCLC (3 pts), sarcoma (3 pts), glioblastoma (2 pts), salivary gland tumours (2 pts) or other solid tumours (5 pts). PR-104 was administered at 135 mg/m2 (3 pts), 270 mg/m2 (6 pts), 540 mg/m2 (6 pts), 675 mg/m2 (7 pts) and 900 mg/m2 (4 pts) for a median of two treatment cycles (range, 1 to 7 cycles) and five infusions (range, 1 to 18) per patient.
Results
Dose-limiting toxicities (DLTs) during cycle one included grade four thrombocytopenia at 540 mg/m2 (1 of 6 pts) and grade four thrombocytopenia and neutropenia at 900 mg/m2 (2 of 4 pts). At an intermediate dose of 675 mg/m2, there were no DLTs among a total of seven patients given 12 treatment cycles but all experienced moderate to severe (grade 2 to 4) haematological toxicity. Thrombocytopenia was delayed in its onset and nadir, and its recovery was protracted and incomplete in many patients. There were no complete or partial tumour responses. PR-104-induced thrombocytopenia and neutropenia correlated with plasma AUC of PR-104, PR-104A and an oxidative semi-mustard metabolite (PR-104S1), but no more strongly than with PR-104 dose-level. There was no significant correlation between plasma AUC for the reduced metabolites and myelotoxicity.
Conclusions
Thrombocytopenia, and to a lesser extent neutropenia, was the DLT of weekly PR-104. The MTD was 675 mg/m2/week. PR-104 given weekly may be a suitable protocol for further clinical evaluation as a short course of treatment with fractionated radiotherapy or haematopoietic stem cell support, as its duration of dosing is restricted by delayed-onset and protracted thrombocytopenia.
doi:10.1186/1471-2407-11-432
PMCID: PMC3205073  PMID: 21982454
6.  Effectiveness of Combined Modality Radiotherapy of Orthotopic Human Squamous Cell Carcinomas in Nu/Nu Mice Using Cetuximab, Tirapazamine and MnSOD-Plasmid Liposome Gene Therapy 
In vivo (Athens, Greece)  2010;24(1):1-8.
Hypoxic regions limit the radiocontrollability of head and neck carcinomas. Whether or not combinations of plasmid/liposome mediated overexpression of normal tissue protective manganese superoxide dismutase (MnSOD), cetuximab (C225), and the hypoxic cytotoxin tirapazamine (TPZ) enhanced radiotherapeutic effects was tested in a CAL-33 orthotopic mouse cheek tumor model. The tumor volume continued to increase in the control (untreated) mice, with a ninefold increase by 10 days when the tumors exceeded 2 cm3. The mice receiving 14 Gy only showed reduced tumor growth to 3.1±0.1 fold at day 10. The mice receiving MnSOD-PL, C225, TPZ plus 14 Gy had the best outcome with 0.7±0.1 fold increase in tumor volume by 10 days (p=0.015) compared to irradiation only. The addition of MnSOD-PL, TPZ, and C225 to irradiation optimized the therapeutic ratio for the local control of hypoxic region-containing CAL-33 orthotopic tumors.
PMCID: PMC2899489  PMID: 20133969
Orthotopic tumors; MnSOD-PL; cetuximab; tirapazamine
7.  Tricyclic [1,2,4]Triazine 1,4-Dioxides As Hypoxia Selective Cytotoxins 
Journal of medicinal chemistry  2008;51(21):6853-6865.
A series of novel tricyclic triazine-di-N-oxides (TTOs) related to tirapazamine have been designed and prepared. A wide range of structural arrangements with cycloalkyl, oxygen- and nitrogen-containing saturated rings fused to the triazine core, coupled with various side chains linked to either hemisphere, resulted in TTO analogues that displayed hypoxia-selective cytotoxicity in vitro. Optimal rates of hypoxic metabolism and tissue diffusion coefficients were achieved with fused cycloalkyl rings in combination with both the 3-aminoalkyl or 3-alkyl substituents linked to weakly basic soluble amines. The selection was further refined using pharmacokinetic/pharmacodynamic model predictions of the in vivo hypoxic potency (AUCreq) and selectivity (HCD) with 12 TTO analogues predicted to be active in vivo, subject to the achievement of adequate plasma pharmacokinetics.
doi:10.1021/jm800967h
PMCID: PMC2690574  PMID: 18847185
8.  Rapid Development of Bilateral Internal Carotid Artery Aneurysm from Sphenoid Sinus Aspergillosis 
Skull base surgery  1998;8(4):211-214.
The purpose of this case report is to familiarize the sinus surgeon with the possibility of the rapid development of internal carotid artery aneuryams from fungal infections of the sphenoid sinuses. A renal dialysis patient with progressive loss of vision was treated with high doses of steroids for the presumed diagnosis of temporal arteritis. Subsequent work-up included a magnetic resonance arteriogram (MRA) and computed tomography (CT) with contrast that failed to demonstrate aneurysmal changes of the carotid arteries but suggested the presence of a mycotic infection of the sphenoid sinuses. During a sphenoidotomy two days later, in addition to the anticipated aspergillus infection of the sinuses, an aneurysm extending from the left internal carotid artery into the sphenoid sinus was encountered. An emergency arteriogram immediately following the surgery revealed a second newly developed large mycotic aneurysm of the right internal carotid artery filling the right sphenoid sinus as well. This case report documents the rapidity with which mycotic aneurysms can develop from a sphenoid sinus infection secondary to aspergillosis in an immunocompromised host.
Images
PMCID: PMC1656709  PMID: 17171068
9.  Zinc Finger Nuclease Mediated Knockout of ADP-Dependent Glucokinase in Cancer Cell Lines: Effects on Cell Survival and Mitochondrial Oxidative Metabolism 
PLoS ONE  2013;8(6):e65267.
Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines.
doi:10.1371/journal.pone.0065267
PMCID: PMC3683018  PMID: 23799003
10.  Effects of triiodothyronine-induced hypermetabolism on factor VIII and fibrinogen in man 
Journal of Clinical Investigation  1969;48(4):768-774.
Triiodothyronine (liothyronine sodium) (400-500 μg/day for 14 days) was given to six normal subjects. Factor VIII (antihemophilic globulin) activity increased from 109 to 167% (P < 0.05); fibrinogen increased from 344 to 581 mg/100 ml (P < 0.01). To test whether the increases in factor VIII activity and fibrinogen were mediated by beta adrenergic receptors, propranolol (20 mg every 6 hr) was given orally to four other normal subjects in addition to triiodothyronine for 14 days. Factor VIII increased from 100 to 161%; fibrinogen increased from 374 to 564% (P < 0.01). Factor VIII activity did not change in a severe classical hemophiliac made hypermetabolic with triiodothyronine, but it increased from 39 to 82% in a patient with von Willebrand's disease. Triiodothyronine-induced hypermetabolism increased the incorporation of selenomethionine-75Se into plasma fibrinogen. These results suggest that the increases in clotting factor activity during triiodothyronine-induced hypermetabolism reflect an effect of increased protein synthesis rather than enhanced stimulation of beta adrenergic receptors.
Images
PMCID: PMC322281  PMID: 5304832
14.  Pronounced Hypoxia in Models of Murine and Human Leukemia: High Efficacy of Hypoxia-Activated Prodrug PR-104 
PLoS ONE  2011;6(8):e23108.
Recent studies indicate that interactions between leukemia cells and the bone marrow (BM) microenvironment promote leukemia cell survival and confer resistance to anti-leukemic drugs. There is evidence that BM microenvironment contains hypoxic areas that confer survival advantage to hematopoietic cells. In the present study we investigated whether hypoxia in leukemic BM contributes to the protective role of the BM microenvironment. We observed a marked expansion of hypoxic BM areas in immunodeficient mice engrafted with acute lymphoblastic leukemia (ALL) cells. Consistent with this finding, we found that hypoxia promotes chemoresistance in various ALL derived cell lines. These findings suggest to employ hypoxia-activated prodrugs to eliminate leukemia cells within hypoxic niches. Using several xenograft models, we demonstrated that administration of the hypoxia-activated dinitrobenzamide mustard, PR-104 prolonged survival and decreased leukemia burden of immune-deficient mice injected with primary acute lymphoblastic leukemia cells. Together, these findings strongly suggest that targeting hypoxia in leukemic BM is feasible and may significantly improve leukemia therapy.
doi:10.1371/journal.pone.0023108
PMCID: PMC3154919  PMID: 21853076

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