Isophosphoramide Mustard Research Articles

An evaluation of the combination effect of zoledronate and chemotherapeutic agents in canine osteosarcoma cells.

Osteosarcoma (OSA) is an aggressive form of bone cancer in both dogs and humans. The treatment options for metastatic (stage III) OSA are currently limited and the prognosis is poor. Zoledronate, a second generation amino-bisphosphonate, is commonly used for palliation of cancer induced bone pain. Zoledronate has also demonstrated anti-cancer properties and possibly enhances the cytotoxicity of doxorubicin in a canine histiocytosis cell line and human prostatic cancer cell line. The goal of this study was to evaluate the combination effect of zoledronate and various chemotherapeutic drugs in canine OSA cells. Canine OSA cell line (D17), cells from two canine primary OSAs, and MDCK, a canine kidney cell line, were used to evaluate the therapeutic potential of these drugs. Carboplatin, doxorubicin, vinorelbine, toceranib, and isophosphoramide mustard (active metabolite of ifosfamide) were used as chemotherapeutic agents. First, cells were treated with either zoledronate or chemotherapy drug alone for 72 hours. Cell viability was assessed using CellTiter Glo and IC5, IC10, IC20, and IC50 were calculated. Second, cells were treated with a combination of zoledronate and each chemotherapeutic agent at their IC5, IC10, IC20, and IC50 concentrations. After 72 hours, cell viability was assessed by CellTiter Glo. Zoledronate, carboplatin, doxorubicin, vinorelbine, and isophosphoramide mustard showed concentration dependent decrease in cell viability. Toceranib showed decreased cell viability only at higher concentrations. When zoledronate was used in combination with chemotherapy drugs, while it showed potential synergistic effects with toceranib, potential antagonistic effects with vinorelbine and isophosphoramide mustard were observed. However, the results differed by cell line and thus, further evaluation is warranted to understand the exact mechanism of action.

Oxazaphosphorines combined with immune checkpoint blockers: dose-dependent tuning between immune and cytotoxic effects

BackgroundOxazaphosphorines (cyclophosphamide (CPA), ifosfamide (IFO)) are major alkylating agents of polychemotherapy protocols but limiting their toxicity and increasing their efficacy could be of major interest. Oxazaphosphorines are prodrugs that require...

Design, synthesis and evaluation of targeted hypoxia-activated prodrugs applied to chondrosarcoma chemotherapy.

The tumor microenvironment in chondrosarcoma (CHS), a chemo- and radio-resistant cancer provides unique hallmarks for developing a chondrosarcoma targeted drug-delivery system. Tumor targeting could be achieved using a quaternary ammonium function (QA) as a ligand for aggrecan, the main high negative charged proteoglycan of the extracellular matrix of CHS, and a 2-nitroimidazole as trigger that enables hypoxia-responsive drug release. In a previous work, ICF05016 was identified as efficient proteoglycan-targeting hypoxia-activated prodrug in a human extraskeletal myxoid chondrosarcoma model in mice and a first study of the structure-activity relationship of the QA function and the alkyl linker length was conducted. Here, we report the second part of the study, namely the modification of the nitro-aromatic trigger and the position of the proteoglycan-targeting ligand at the aromatic ring as well as the nature of the alkylating mustard. Synthetic approaches have been established to functionalize the 2-nitroimidazole ring at the N-1 and C-4 positions with a terminal tertiary alkyl amine, and to perform the phosphorylation step namely through the use of an amine borane complex, leading to phosphoramide and isophosphoramide mustards and also to a phosphoramide mustard bearing four 2-chloroethyl chains. In a preliminary study using a reductive chemical activation, QA-conjugates, except the 4-nitrobenzyl one, were showed to undergo efficient cleavage with release of the corresponding mustard. However N,N,N-trimethylpropylaminium tethered to the N-1 or C-4 positions of the imidazole seemed to hamper the enzymatic reduction of the prodrugs and all tested compounds featured moderate selectivity toward hypoxic cells, likely not sufficient for application as hypoxia-activated prodrugs.

An Intratumor Pharmacokinetic/Pharmacodynamic Model for the Hypoxia-Activated Prodrug Evofosfamide (TH-302): Monotherapy Activity is Not Dependent on a Bystander Effect

Tumor hypoxia contributes to resistance to anticancer therapies. Hypoxia-activated prodrugs (HAPs) selectively target hypoxic cells and their activity can extend to well-oxygenated areas of tumors via diffusion of active metabolites. This type of bystander effect has been suggested to be responsible for the single agent activity of the clinical-stage HAP evofosfamide (TH-302) but direct evidence is lacking. To dissect the contribution of bystander effects to TH-302 activity, we implemented a Green's function pharmacokinetic (PK) model to simulate the spatial distribution of O2, TH-302 and its cytotoxic metabolites, bromo-isophosphoramide mustard (Br-IPM) and its dichloro derivative isophosphoramide mustard (IPM), in two digitized tumor microvascular networks. The model was parameterized from literature and experimentally, including measurement of diffusion coefficients of TH-302 and its metabolites in multicellular layer cultures. The latter studies demonstrate that Br-IPM and IPM cannot diffuse significantly from the cells in which they are generated, although evidence was obtained for diffusion of the hydroxylamine metabolite of TH-302. The spatially resolved PK model was linked to a pharmacodynamic (PD) model that describes cell killing probability at each point in the tumor microregion as a function of Br-IPM and IPM exposure. The resulting PK/PD model accurately predicted previously reported monotherapy activity of TH-302 in H460 tumors, without invoking a bystander effect, demonstrating that the notable single agent activity of TH-302 in tumors can be accounted for by significant bioreductive activation of TH-302 even in oxic regions, driven by the high plasma concentrations achievable with this well-tolerated prodrug.

Transformation kinetics of cyclophosphamide and ifosfamide by ozone and hydroxyl radicals using continuous oxidant addition reactors

The detection of pharmaceuticals in water and wastewater has triggered human and ecological health concerns. As highly toxic compounds, chemotherapy agents (CAs), such as the cyclophosphamide (CYP) and ifosfamide (IFO) structural isomers, represent a unique threat. This research elucidated the fate of CYP and IFO during ozonation and advanced oxidation by hydroxyl radicals (HO•). Novel semi-batch reactors were used to determine the second-order rate constants for CYP and IFO with O3 and HO•. These reactors provided independent control of the oxidant exposure through continuous and constant aqueous ozone and peroxone (O3-H2O2) addition. The rate constants for transformation of CYP and IFO by ozone were 2.58 ± 0.40 M-1s-1 and 6.95 ± 0.21 M-1s-1, respectively, indicating that ozone alone is not suitable for treating CAs. Transformation of CYP and IFO by hydroxyl radicals was fast, with rate constants of 2.69(±0.17)×109 M-1s-1 and 2.73(±0.16)×109 M-1s-1, respectively. The major transformation products formed by O3 and HO attack consisted of the 4-hydroxy-, 4-keto-, dechloroethyl-, and imino- derivatives of CYP and IFO. Low yields of the active metabolites of the CAs, namely phosphoramide mustard and isophosphoramide mustard, were detected. These findings suggest that treated water may retain the ability to alkylate DNA and confer toxicity.

Influence of the alkylating function of aldo-Ifosfamide on the anti-tumor activity.

The present work investigates the influence of different DNA damages caused by different isophosphoramide mustards on the 3-hydroxypropanal-assisted apoptotic antitumor activity of oxazaphosphorine cytostatics using I-aldophosphamide-perhydrothiazine (IAP) and mesyl-I-aldophosphamide-perhydrothiazine (SUM-IAP) for in-vitro and in-vivo experiments. IAP and SUM-IAP hydrolyze spontaneously to the corresponding I-aldophosphamide derivatives. They differ in the chemical structure of the alkylating moiety, whereas IAP has two chlorethyl groups in the SUM-IAP molecule, one chlorethyl group is substituted by a mesylethyl group. With both substances, cytotoxicity studies on P388 tumor cells in vitro and therapy experiments in mice bearing advanced growing P388 tumors were carried out. IAP was significantly more cytotoxic in-vitro than SUM-IAP, but the antitumor activity of SUM-IAP was by order of magnitude higher than the antitumor activity of IAP. The reason for these findings is discussed with respect to the enzymatic cleavage of the various I-aldophosphamide derivatives to the corresponding isophosphoramide mustards and 3-hydroxypropanal. Overall, the findings indicate that antitumor activity of ifosfamide and derivatives of ifosfamide can be improved considerably by altering the alkylating moiety of the molecule, but retaining the aldophosphamide structure.

The chemomodulatory effects of glufosfamide on docetaxel cytotoxicity in prostate cancer cells.

Background. Glufosfamide (GLU) is a glucose conjugate of ifosfamide in which isophosphoramide mustard is glycosidically linked to the β-D-glucose molecule. Based on GLU structure, it is considered a targeted chemotherapy with fewer side effects. The main objective of the current study is to assess the cytotoxic potential of GLU for the first time in prostate cancer (PC) cells representing different stages of the tumor. Furthermore, this study examined the potential synergistic activity of GLU in combination with docetaxel (DOC).Methods. Two different cell lines were used, LNCaP and PC-3. Concentration-response curves were assessed. The tested groups per cell line were, control, GLU, DOC and combination. Treatment duration was 72 h. Cytotoxicity was assessed using sulforhodamine B (SRB) assay and half maximal inhibitory concentration (IC50) was calculated. Synergy analyses were performed using Calcusyn®software. Subsequent mechanistic studies included β-glucosidase activity assay, glucose uptake and apoptosis studies, namely annexin V-FITC assay and the protein expression of mitochondrial pathway signals including Bcl-2, Bax, Caspase 9 and 3 were assessed. Data are presented as mean ± SD; comparisons were carried out using one way analysis of variance (ANOVA) followed by Tukey-Kramer’s test for post hoc analysis.Results. GLU induced cytotoxicity in both cell lines in a concentration-dependent manner. The IC50 in PC-3 cells was significantly lower by 19% when compared to that of LNCaP cells. The IC50 of combining both drugs showed comparable effect to DOC in PC-3 but was tremendously lowered by 49% compared to the same group in LNCaP cell line. β-glucosidase activity was higher in LNCaP by about 67% compared to that determined in PC-3 cells while the glucose uptake in PC-3 cells was almost 2 folds that found in LNCaP cells. These results were directly correlated to the efficacy of GLU in each cell line. Treatment of PC cells with GLU as single agent or in combination with DOC induced significantly higher apoptosis as evidenced by Annexin V-staining. Apoptosis was significantly increased in combination group by 4.9 folds and by 2.1 Folds when compared to control in LNCaP cells and PC-3 cells; respectively. Similarly, the expression of Bcl-2 was significantly decreased while Bax, caspase 9 and 3 were significantly increased in the combined treatment groups compared to the control.Conclusion. GLU has a synergistic effect in combination with DOC as it increases the cell kill which can be attributed at least partially to apoptosis in both the tested cell lines and it is suggested as a new combination regimen to be considered in the treatment of the prostate cancer. Further experiments and clinical investigations are needed for assessment of that regimen.

A phase II trial to assess the activity and safety of the hypoxia-activated prodrug evofosfamide (TH-302) in combination with sunitinib in patients with disseminated grade 1 and 2 pancreatic neuroendocrine tumors (pNET) as a first-line approach: The GETNE-1408 trial.

TPS479 Background: Pancreatic-NETs are highly vascular tumors. The anti-angiogenetic sunitinib was approved in advanced pNETs based on prolongation of progression-free survival. Upregulation of several proangiogenic factors that reflects intratumor hypoxia conditions might drive resistance to sunitinib in pNETs. Evofosfamide is a prodrug that under hypoxic conditions preferentially releases a brominated version of isophosphoramide mustard and has shown activity in cell lines from neural crest derived tumors such as melanoma and glioblastoma/astrocytoma. We hypothesize that evofosfamide may have activity in the pathologic hypoxic conditions present in the tumor environment of neuroendocrine tumors inducing responses that may be consolidated and prolonged with sunitinib in patients with advanced pNETs that are naïve for systemic treatment. Methods: This is a prospective, non randomized, open-label, phase II study that is being conducted in 10 university sites belonging to the Spanish Task Force Group for NETs (GETNE) in Spain. Patients with histologically proven diagnosis of progressive unresectable or metastatic pNET with Ki67 < 20% and grade 1 or 2 will receive sunitinib orally at 37.5 mg PO daily on days 1 to 28 of a 28-day cycle (4 weeks) plus evofosfamide administered at 340 mg/m2 by IV infusion over 30-60 minutes on Days 8, 15 and 22 of a 28-day cycle (4 weeks). Hypoxia-related tumor markers, single nucleotide polymorphisms related to activity and metabolism of antiangiogenic agents (VEGFR2, VEGFR3, PDGFR-α, VEGF-A, IL8, CYP3A4, CYP3A5, ABCB1-2) will be correlated with clinical outcome. In this study it is planned to include 43 patientsbased on a two-stage Simon’s phase II design (α = 0.05, β = 80%). If the trial shows 8 responses or more among 43 patients, the treatment will be considered for further investigation. EudraCT number: 2014-004072-30 Clinical trial information: NCT02402062.

Abstract 4537: Geranyloxy-ifosfamide: A pre-activated ifosfamide analogue showing an increased therapeutic index

Abstract Ifosfamide (IFO) is an alkylating agent used in routine clinical practices for treatment of cancer for many years. As a prodrug it requires cytochrome P450 bio-activation (Giraud et al. Expert Opin.Drug Metab. Toxicol. 2010, 6, 919-938) oxidising the position C-4 leading to the formation of the 4-HO-IFO (10%) which releases of the active compound (isophosphoramide mustard) responsible for the DNA cross links. However, during high dose protocols, this bio-activation leads to the release of two toxic metabolites: i) acrolein, an urotoxic compound, concomitantly generated with the active isophosphoramide mustard and, ii) chloroacetaldehyde, a neurotoxic and nephrotoxic compound, arising from the oxidation of the side chains (Kerbusch et al. Clin Pharmacokinet. 2001, 40, 41-62) responsible for limiting side-effects. To circumvent these toxic side-effects, we have designed several pre-activated compounds by covalent binding of several O- and S-alkyl moieties on the carbon C-4 to avoid cytochrome bio-activation favoring the release of the 4-HO-IFO, control this release by the physico-chemical properties of the engrafted moiety and limit the chloroacetaldehyde release. Among these designed compounds, some showed a self assembling property leading to nano-assemblies formulation. Moreover, the in vitro evaluation of these compounds in absence of cytochrome has revealed that one of them: the geranyloxy-ifosfamide showed a greater activity under nano-assemblies (600nM) compared to the bulk form (15μM) on rhabdomyosarcoma. This interesting finding has led us to deepen our investigation of this compound to understand and explain the meaning of this increased activity. We present here the latest results obtained for this compound from additional in vitro evaluation on a wide panel of cancer cell line, internalization studies within the cell. Moreover, transmission electron microscopy imaging of this formulation allows the determination of the shape of these nano-assemblies. Previous studies regarding the release kinetic profile of 4-HO-IFO of this compound in vitro in mice plasma at 37°C showed an interesting release profile which was increased when tested as nano-assemblies compared to the bulk form. To validate this finding, a pharmacokinetic study has also been investigated compared to IFO, showing a higher release of 4-HO-IFO compound suggesting that this compound might have an increased therapeutic index. Also, in vivo studies are being carried out at different dose to evaluate the efficacy of this compound (as bulk and nano-assemblies) on rhabdomyosarcoma xenografted mice compared to IFO. These results increase acknowledge of this compound which could become a promising candidate for both oral and IV administration in a wide panel of tumors. Citation Format: Charles Skarbek, Didier Desmaele, Alain Deroussent, Lea Lesueur, Estelle Daudigeos-Dubus, Ludivine Le Dret, Michael Rivard, Thierry Martens, Gilles Vassal, Patrick Couvreur, Angelo PACI. Geranyloxy-ifosfamide: A pre-activated ifosfamide analogue showing an increased therapeutic index. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4537. doi:10.1158/1538-7445.AM2015-4537

The Neurotoxicity and Pharmacokinetics of Oral Ifosfamide

Purpose:Ifosfamide can cause an unexplained encephalopathy. The incidence after intravenous infusion is 10%, but is much higher after oral administration. This study assesses the pharmacokinetics of oral ifosfamide in relation to neurotoxicity. Patients and Methods:Eleven patients received oral ifosfamide 500 mg twice daily for 14 days, with concurrent oral mesna. The concentrations of ifosfamide, isophosphoramide mustard, 2-dechloroethylifosfamide, 3-dechloroethylifosfamide, carboxyifosfamide, ketoifosfamide, chloroethylamine and 3-oxazolidine-2-one were measured using GC-MS. Patients were evaluated clinically, and also with the EEG, psychometric testing, the national adult reading test, and the mini-mental state examination. Results:A decrease in the electroencephalogram alpha frequency was observed, with the development of pathological slow wave activity. Psychometric performance was also impaired. Neurotoxicity was progressive during treatment, and the incidence of grade 3 neurotoxicity was 22%. The mean day 14 / day 1 Cmax ratios for 2-dechloroethylifosfamide and 3-dechloroethylifosfamide were 2.73 (± 2.11) and 2.04 (± 1.32) respectively. The metabolite with the lowest ratio was isophosphoramide mustard 1.07 (± 0.39). High chloroethylamine Cmax values were associated with lower alpha frequencies, and increased clinical neurotoxicity. Conclusion:Oral ifosfamide 500 mg twice daily for 14 days causes unacceptable neurotoxicity. It was not possible to identify one particular metabolite responsible for the neurotoxicity, although the dechloroethyl metabolites and chloroethylamine are implicated.

Preactivated oxazaphosphorines designed for isophosphoramide mustard delivery as bulk form or nanoassemblies: synthesis and proof of concept.

Oxazaphosphorines are alkylating agents used in routine clinical practices for treatment of cancer for many years. They are antitumor prodrugs that require cytochrome P450 bioactivation leading to 4-hydroxy derivatives. In the case of ifosfamide (IFO), the bioactivation produces two toxic metabolites: acrolein, a urotoxic compound, concomitantly generated with the isophosphoramide mustard; and chloroacetaldehyde, a neurotoxic and nephrotoxic compound, arising from the oxidation of the side chains. To improve the therapeutic index of IFO, we have designed preactivated IFO derivatives with the covalent binding of several O- and S-alkyl moieties including polyisoprenoid groups at the C-4 position of the oxazaphosphorine ring to avoid cytochrome bioactivation favoring the release of the active entity and limiting the chloroacetaldehyde release. Thanks to the grafted terpene moieties, some of these new conjugates demonstrated spontaneous self-assembling properties into nanoassemblies when dispersed in water. The cytotoxic activities on a panel of human tumor cell lines of these novel oxazaphosphorines, in bulk form or as nanoassemblies, and the release of 4-hydroxy-IFO from these preactivated IFO analogues in plasma are reported.

The Disulfide Analogues of Isophosphoramide Mustard for Anticancer Therapy

Novel disulfide analogues of isophosphoramide mustard (iPAM) were designed and synthesised. All compounds were hydrolytically stable and underwent reduction by L-glutathione with different kinetic parameters. Based on the HPLC-MS analysis a mechanism of activation by glutathione obtained disulfide analogues of iPAM was proposed. The compounds were tested for cytotoxic activity against human promyelocytic leukaemia HL-60, human lymphoblastic leukaemia MOLT-4, human lymphoblastic leukaemia CCRF/CEM, human bladder cancer HCV29T, murine melanoma B16-F0 and murine fibroblasts Balb3T3 cell lines. The most promising anticancer activity was exhibited by compound 4, which proved to be more active than the reference cisplatin against all cancer cell lines. Keywords: Anticancer activity, disulfide, glutathione, isophosphoramide mustard.

Pyruvate induces transient tumor hypoxia by enhancing mitochondrial oxygen consumption and potentiates the anti-tumor effect of a hypoxia-activated prodrug TH-302.

BackgroundTH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302.Methodology/ResultsThe hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O2), with minimal effect under aerobic conditions (21% O2). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500–1500 mm3. Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼550 mm3), significantly delayed tumor growth.Conclusions/SignificanceOur in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.

Abstract 5583: Response biomarkers for the hypoxia-targeted drug TH-302: imaging, plasma, and tissue.

Abstract Biomarkers that assess the therapeutic response to anticancer treatments may aid in tailoring therapeutic regimens to individuals. Ideally, the relative contributory effect of each agent in a muti-agent regimen can be discerned. Hypoxia is a prevalent feature of solid tumors, associated with treatment failure, poor prognosis, and increased metastasis. TH-302, a hypoxia-activated prodrug of the DNA cross-linker bromo isophosphoramide mustard, has demonstrated broad efficacy in preclinical models and is exhibiting promising activity in multiple ongoing clinical trials, both as a monotherapy and in combination therapy regimens. Three fundamental biomarker platforms were assessed in this preclinical study: positron emission tomography (PET) imaging employing the tracer [18F]-HX4; immunohistochemistry of isolated tumor tissue employing antibodies directed against the exogenous hypoxia biomarker pimonidazole (PIMO) and the endogenous hypoxia biomarker carbonic anhydrase IX (CA-IX); and ELISA-based quantification of circulating plasma CA-IX. Both HX4 and PIMO utilize the same 2-nitroimidazole hypoxia-sensitive trigger for their activity and specificity as TH-302 utilizes. CA-IX is up-regulated by two distinct hypoxia sensing pathways: hypoxia-inducible factor 1; and the unfolded protein response. The human H460 non-small cell lung cancer human tumor ectopic xenograft model was employed for these studies. When the tumor size was 300-600 mm3, a single dose of TH-302 (150 mg/kg, ip) or vehicle was administered. This TH-302 treatment regimen yields a tumor growth inhibition of 58%, TGD1000 of 10 d, and maximum body weight loss of 2%. [18F]-HX4 uptake was assessed by PET/CT imaging and the tumor to muscle ratio (T/M) was determined. Animals were scanned pre- and post-treatment and the T/M ratio was significantly reduced 72 hours after TH-302 treatment (from 3.3 ± 0.3 to 2.1 ± 0.2, p<0.005). A slight increase in T/M was observed in the vehicle-treated animals (3.3 ± 0.4 to 3.6 ± 0.2, p>0.05). Blood was collected from the animals pre- and post- treatment via retro-orbital vein. Plasma CA-IX changed from baseline 165 ± 13 to 104 ± 9.3 pg/ml 72 hours after TH-302 dosing, a 37% reduction (p<0.05; n=10 per group). No change was observed in the vehicle-treated group. For tissue biomarker analysis, tumors were harvested 72 hours after TH-302 treatment, and tumor hypoxia was assessed by semi-quantitative morphometric analysis of the PIMO immunostaining at that time. The hypoxic fraction (HF) was 13 ± 1.7% in the vehicle group (n=5) and 5.8 ± 0.5% in the TH-302 treatment group (n=6, p<0.05). Strong co-localization of CA-IX and PIMO expression was observed in consecutive sections. These preclinical results highlight the promise of using TH-302 response biomarkers tested in the clinical setting for their ability to help guide TH-302 treatment decisions after the initiation of therapy. Citation Format: Jessica D. Sun, Qian Liu, Dharmendra Ahluwalia, Yan Wang, Anna-Katrin Szardenings, Luis F. Gomez, Hartmuth Kolb, Karen A. Pierce, Sheryl Brown-Shimer, Walter A. Carney, Charles P. Hart. Response biomarkers for the hypoxia-targeted drug TH-302: imaging, plasma, and tissue. [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 5583. doi:10.1158/1538-7445.AM2013-5583

Abstract 4766: The use of deuterium isotope effects to shift the partitioning of cyclophosphamide and ifosfamide among competing, P450-dependent, oxidative pathways

Abstract Prodrugs cyclophosphamide and ifosfamide are each ‘activated’ through a P450 oxidation at the C-4 position of the oxazaphosphorine ring. This reaction triggers a series of spontaneous (non-enzymatic) steps resulting in the formation of the ultimate DNA crosslinking agents, phosphoramide mustard and isophosphoramide mustard. Competing with reaction at C-4 are P450-catalyzed oxidations at the alpha positions of the chloroethyl side-chains; reactions at the side chains result in the production of the neurotoxic chloroacetaldehyde. Predominance of one pathway over another is isozyme-dependent and of greatest impact in ifosfamide therapy where about 50% of this drug is lost to side-chain oxidation (relative to an average of 10% for cyclophosphamide) and dose-limiting neurotoxicity. In this project, deuterium isotope effects are being explored as a means to effect ‘metabolic switching’ in the oxidations of cyclophosphamide and ifosfamide; that is, favoring oxidation at the C-4 position by disfavoring oxidations at the side chains. Cyclophosphamide and ifosfamide were synthesized with deuterium at the alpha and alpha’ positions of the chloroethyl chains. After optimizing reaction times, drug and enzyme concentrations, cyclophosphamide and ifosfamide (unlabeled and labeled) were incubated (20 min) with three, relevant, cDNA-expressed supersomes (human CYP2B6, CYP3A4, CYP3A5). The concentration of each metabolite produced by C-4 and the two possible side-chain oxidations was measured quantitatively using LC-MS-MS techniques developed for this project. For unlabeled cyclophosphamide, the fraction of products derived from C-4 oxidation was 73% (3A4), 90% (3A5) and 100% (2B6). Use of deuterium labeled cyclophosphamide gave 89% (3A4), 94% (3A5) and 100% (2B6); the amount of product produced by 2B6 and labeled drug was more than twice that given by unlabeled drug (56 micromoles versus 23 micromoles, respectively). For unlabeled ifosfamide, the fraction of products given by C-4 oxidation was 63% (3A4), 55% (3A5) and 36% (2B6). Use of deuterium labeled ifosfamide gave 82% (3A4), 75% (3A5) and 88% (2B6). Aside from the initial oxidation, the metabolic steps most likely to be impacted by deuterium isotope effects are those of the alkylation sequence by the metabolites phosphoramide mustard and isophosphoramide mustard. To determine the effect, if any, phosphoramide mustard and isophosphoramide mustard are being synthesized with deuterium at the alpha and alpha’ positions of the chloroethyl chains and their alkylation kinetics, relative to unlabeled compounds are being determined using 31P NMR spectroscopy. 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 4766. doi:1538-7445.AM2012-4766

Abstract B219: 4-Demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) and 4-hydroperoxyifosfamide (HOOI) as binary therapy for melanoma.

Abstract Introduction: 4-Demethyl-4-cholesteryloxycarbonyl-penclomedine (DM-CHOC-PEN) is a polychlorinated pyridine cholesteryl carbonate, which is in Phase I clinical trials in patients with advanced cancer - IND 68,876. DM-CHOC-PEN is active vs. intracranially (IC) implanted human xenograft models - U251 and D54 glioblastoma and MX-1 breast cancer and recently found to be active vs. B-16 melanoma - 142% ILS. DM-CHOC-PEN's MOA is via alkylation of DNA @ N7 - guanine, as well as converting melanoma cells into a melanotic Go phase with cell death. Not all cells were converted into Go phase and senescence, thus the interest in designing a binary drug approach for DM-CHOC-PEN, as an improvement in treatment for melanoma. A number of agents are cytotoxic vs. B-16 cells in vitro and in vivo; however, 4-hydroperoxyifosfamide (HOOI), which is converted to isophosphoramide mustard (IPM) in vivo, demonstrated the best %ILS. The latter drug has been chosen as a companion with DM-CHOC-PEN in the present binary drug study vs. B-16 mouse melanoma. Methods: B-16 melanoma cells were cultured using RPMI media with 10% FBS and pen/strep @ 37° C in a CO2 incubator. Drugs were added to the cells in a growth phase and removed after 8–12 h. Adult female C57BL mice in groups of 5–6 mice were implanted subcutaneously (SC) with B-16 mouse melanoma (106 cells) and when SC nodules were palpable the mice were dosed IP daily (200 mg/kg) for 5-days with DM-CHOC-PEN followed by HOOI administered IP @ varying doses and daily/weekly schedules and monitored daily until death. Mice with SC B-16 melanoma were dosed with single agents - DM-CHOC-PEN, HOOI, cis-platinum and temozolamide, which were used as controls. Tumor tissue was extracted with dichloromethane, and assayed per HPLC and NMR. Results: In vitro, DM-CHOC-PEN and HOOI, as single agents, had IC50 of 0.5 and 0.8 μg/mL vs. B-16 melanoma cells, resp. Mice bearing SC B-16 melanoma treated with DM-CHOC-PEN (200 mg/kg/d × 5d, IP) alone vs. saline controls demonstrated %ILS of 142%; thus supporting the in vitro observations. In vivo in the B-16 melanoma model, cis-platinum as a single agent had a %ILS = 0%, but for HOOI it was 85%. In 2-drug studies, DM-CHOC-PEN plus cis-platinum together (in theoretical therapeutic ranges) were too toxic in combination, however, the %ILS for DM-CHOC-PEN plus HOOI was 173%. Tumor tissue was removed within 2-days of treating mice with DM-CHOC-PEN, extracted and revealed 75 μg/g tumor tissue of DM-PEN, a metabolite. Discussion: HOOI is a S-phase alkylating agent that is an appropriate 2nd agent to kill cells escaping from the DM-CHOC-PEN - induced Go phase induction. To date, the best treatment regimen was DM-CHOC-PEN (200 mg/kg/d × 5d) followed by HOOI (90 mg/d × 3d) - %ILS = 173. The finding that melanoma tissue extracts resulted in μg of DM-PEN (the metabolite) is in agreement with the pharmacokinetic findings observed for DM-CHOC-PEN in rats and humans, which will be reviewed. The binary drug combination will be reviewed with the FDA. Supported in part by: NCI SBIR grants - R43/44CA85021. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B219.

Glufosfamide as a new oxazaphosphorine anticancer agent

Glufosfamide (β-D-glucose-isophosphoramide mustard, D-19575) belongs to the oxazaphosphorine class. Glufosfamide is a novel glucose conjugate of ifosfamide in which isophosphoramide mustard, the alkylating metabolite of ifosfamide, is glycosidically linked to the β-D-glucose molecule. Glufosfamide represents an attractive new agent for cancer therapy. Its mode of action on normal and pathological cells is still under experimental and clinical investigations. An assessment of the anticancer potential of glufosfamide is of key importance in therapy. The researchers reviewed the current knowledge available on glufosfamide tested in the preclinical studies/clinical trials, based on a collection of the original papers and conference abstracts published and relevant articles searched in the SCOPUS and MEDLINE database and websites.

Phase 1 Study of the Safety, Tolerability, and Pharmacokinetics of TH-302, a Hypoxia-Activated Prodrug, in Patients with Advanced Solid Malignancies

The objectives of this phase 1, first-in-human study were to determine the dose-limiting toxicities (DLT), maximum tolerated dose (MTD), safety, pharmacokinetics, and preliminary activity of the hypoxia-activated prodrug TH-302 in patients with advanced solid tumors. TH-302 was administered intravenously over 30 to 60 minutes in two regimens: three times weekly dosing followed by 1 week off (arm A) and every 3-week dosing (arm B). Fifty-seven patients enrolled (arm A: N = 37 and arm B: N = 20). The TH-302 dose was escalated from 7.5 to 670 mg/m(2) in arm A and from 670 to 940 mg/m(2) in arm B. The most common adverse events were nausea, skin rash, fatigue, and vomiting. Hematologic toxicity was mild and limited. Grade 3 skin and mucosal toxicities were dose limiting at 670 mg/m(2) in arm A; the MTD was 575 mg/m(2). In arm B, grade 3 fatigue and grade 3 vaginitis/proctitis were dose limiting at 940 mg/m(2); the MTD was 670 mg/m(2). Plasma concentrations of TH-302 and the active metabolite Br-IPM (brominated version of isophosphoramide mustard) increased proportionally with dose. Two partial responses were noted in patients with metastatic small cell lung cancer (SCLC) and melanoma in arm A at 480 and 670 mg/m(2). Stable disease was observed in arms A and B in 18 and 9 patients, respectively. The MTD of TH-302 was 575 mg/m(2) weekly and 670 mg/m(2) every 3 weeks. Skin and mucosal toxicities were DLTs. On the basis of responses in metastatic melanoma and SCLC, further investigations in these indications were initiated.

Abstract 3222: Pre-clinical toxicology and pharmacology of 4-hydroperoxyifosfamide (4-HOOI) and L-lysine salt

Abstract HOOI is a hydroperoxy-derivative of ifosfamide (IFOS) and a pro-drug of isophosphoramide mustard (IPM) [the active metabolite of IFOS] – a bi-functional alkylator that cross-links with G/C DNA base sequences resulting in irreparable inter-strand DNA cross-linking and cell death. HOOI has been an unstable laboratory curiosity for years; however, HOOI.L-lysine is a stable salt complex that has allowed the development of HOOI for clinical trials. HOOI spontaneously releases acrolein and chloroacetaldehyde in situ in cancer cells not extracellular in the general circulation (as does IFOS) and has not been associated with cystitis, renal tubular necrosis and/or CNS toxicity. HOOI has been screened in 20+ human xenograft tumor and murine tumor models and has significantly improved %ILS in intracranially implanted human xenografts – MX-1, U251; ZR-75-1 and in P399/CPA leukemia vs. IFOS and IPM. The U251 data (%ILS – 84+) is impressive considering – BCNU produced a 72% ILS. The ZR-75-1 breast cancer had a %ILS – 83. Bone marrow failure was the DLT in mice – LD10 100 mg/kg (for M/F). Dogs (M/F) were treated with 10, 15, 20 and 30 mg/kg. For both sexes, the LD10 was calculated to be 17.2, while the LD50 was calculated to be 17.3 mg/kg. PK values in dogs revealed the following profile for groups dosed with 30 mg/kg: AUC0- t = 1.53 (mg h/L), T1/2α = 0.93 (h), T1/2β = 6.1 (h) & CL = 19.5 (L/ h) [a two compartment model]. The AUC was linear for the 10 and 30 mg/kg doses. HOOI did not generate any detectable plasma chloroacetaldehyde vs. IFOS (2.12 µg/mL from 400 mg/kg) and 25% of the acrolein generated by the MTD of CPA & IFOS. No convulsions, neuropathies or renal dysfunction were observed in either specie. Unlike IFOS/IPM, HOOI is lipophilic, activated intracellular (with < extracellular acrolein and no chloroacetaldehyde released); no IFOS/IPM-associated CNS or GU toxicity was noted. HOOI may increase the safety and efficacy margins of this class of alkylators in advanced CPA- and IFOS-resistant cancers and broaden the target-range (CNS-gliomas). The L-lysine salt stability supports developing HOOI for clinical trials. Supported by grant R44 CA094566 from the NCI/SBIR program. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3222. doi:10.1158/1538-7445.AM2011-3222

Clinical benefits TH-302, a tumor-selective, hypoxia-activated prodrug, and gemcitabine in first-line pancreatic cancer (PanC).

265 Background: Gemcitabine (G) is the standard treatment for first- line PanC. PanC is one of the most hypoxic solid tumors. TH-302 is an inert prodrug of brominated isophosphoramide mustard and undergoes selective activation in deep hypoxia. As a single agent, tumor responses were reported in patients (pts) with metastatic melanoma, SCLC, and head/neck cancer at TH-302 weekly doses of 480-575 mg/m2. Methods: Eligible pts for the PanC expansion of this phase I/II study ( NCT00743379 ) had ECOG <1, locally advanced or metastatic PanC previously untreated with systemic chemotherapy other than adjuvant G, 5FU, and/or radiation. IV TH-302 was dosed at 240-575 mg/m2 (240 or 340 in expansion) with standard dose G (1000 mg/m2) on days 1, 8 and 15 of a 28-day cycle. Serum protein and microRNA hypoxia biomarkers were analyzed at baseline, start of cycle 3 and end of study. Results: 46 PanC subjects (12 locally advanced, 34 distant mets); median age: 63 (range 41-83); 24 male; ECOG 0/1 in 29/17 pts; RECIST response rate (RR) of 21%, median PFS of 6.1 mo (95%CI 4.8, 7.7) and median survival of 11.4 mo (95%CI 6.0, not reached) were observed. RR was 23% with median survival of 7.4 mo in pts with distant mets. 52% of pts had a >50% decrease in CA19-9. Common adverse events were skin or mucosal toxicity, nausea, fatigue and vomiting; most grade 1/2. Grade 3/4 neutropenia, thrombocytopenia and anemia in 68%, 64%, and 20% of pts respectively. The dose intensities at 240 mg/m2 and 340 mg/m2 were similar and related to hematologic toxicities. Skin toxicities were less common at 240 mg/m2. A TH-302 dose response was present with higher RR and PFS at 340 mg/m2. Initial serum hypoxia biomarkers did not identify a preferential pt population. Conclusions: The activity and clinical benefits of the combination of TH-302 with G in first line PanC are promising as compared to previous studies of G alone. TH-302 adds to the hematologic toxicity of G, but the regimen is well tolerated. The safety and activity provided rationale for comparing TH-302 plus G versus G alone in a randomized phase II trial ( NCT01144455 ) and indicate TH-302 may complement G by penetrating into the hypoxic regions of the PanC tumors where activation induces cytotoxicity. No significant financial relationships to disclose.