Michael R.L. Stratford

A Phase Ib trial of CA4P (combretastatin A-4 phosphate), carboplatin, and paclitaxel in patients with advanced cancer.

Background:The vascular disrupting agent combretastatin A4 phosphate (CA4P) causes major regression of animal tumours when given as combination therapy.Methods:Patients with advanced cancer refractory to standard therapy were treated with CA4P as a 10-min infusion, 20 h before carboplatin, paclitaxel, or paclitaxel, followed by carboplatin.Results:Combretastatin A4 phosphate was escalated from 36 to 54 mg m−2 with the carboplatin area under the concentration curve (AUC) 4–5, from 27 to 54 mg m−2 with paclitaxel 135–175 mg m−2, and from 54 to 72 mg m−2 with carboplatin AUC 5 and paclitaxel 175 mg m−2. Grade 3 or 4 neutropenia was seen in 17%, and thrombocytopenia only in 4% of 46 patients. Grade 1–3 hypertension (26% of patients) and grade 1–3 tumour pain (65% of patients) were the most typical non-haematological toxicities. Dose-limiting toxicity of grade 3 hypertension or grade 3 ataxia was seen in two patients at 72 mg m−2. Responses were seen in 10 of 46 (22%) patients with ovarian, oesophageal, small-cell lung cancer, and melanoma.Conclusion:The combination of CA4P with carboplatin and paclitaxel was well tolerated in the majority of patients with adequate premedication and had antitumour activity in patients who were heavily pretreated.

A Role for Cytosolic Fumarate Hydratase in Urea Cycle Metabolism and Renal Neoplasia

Summary The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH), predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.

Mechanistic studies of the inactivation of tyrosinase by resorcinol

The inactivation of tyrosinase by resorcinol (1,3-dihydroxybenzene) and seventeen simple derivatives has been investigated using combined spectrophotometry and oximetry together with hplc/ms examination of the oxidation products. The results are consistent with a Quintox mechanism, analogous to that proposed for catechol inactivation of tyrosinase, in which the resorcinol substrate is oxidised via the monooxygenase route leading to a hydroxy intermediate that undergoes deprotonation and results in irreversible elimination of Cu(0) from the active site. Hplc/ms evidence for formation of the resorcinol monooxygenase product (3-hydroxy-ortho-quinone) is presented and the relationship between the ring position of simple resorcinol substituents (H, Me, F, Cl) and tyrosinase inactivation is rationalised.

The influence of catechol structure on the suicide-inactivation of tyrosinase

3,6-Difluorocatechol, which cannot act as a monooxygenase tyrosinase substrate, is an oxidase substrate, and, in contrast to other catechols, oxidation does not lead to suicide-inactivation, providing experimental evidence for an inactivation mechanism involving reductive elimination of Cu(0) from the active site.

Regulation of O2 consumption by the PI3K and mTOR pathways contributes to tumor hypoxia

Background Inhibitors of the phosphatidylinositol 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) pathway are currently in clinical trials. In addition to antiproliferative and proapoptotic effects, these agents also diminish tumor hypoxia. Since hypoxia is a major cause of resistance to radiotherapy, we sought to understand how it is regulated by PI3K/mTOR inhibition. Methods Whole cell, mitochondrial, coupled and uncoupled oxygen consumption were measured in cancer cells after inhibition of PI3K (Class I) and mTOR by pharmacological means or by RNAi. Mitochondrial composition was assessed by immunoblotting. Hypoxia was measured in spheroids, in tumor xenografts and predicted with mathematical modeling. Results Inhibition of PI3K and mTOR reduced oxygen consumption by cancer cell lines is predominantly due to reduction of mitochondrial respiration coupled to ATP production. Hypoxia in tumor spheroids was reduced, but returned after removal of the drug. Murine tumors had increased oxygenation even in the absence of average perfusion changes or tumor necrosis. Conclusions Targeting the PI3K/mTOR pathway substantially reduces mitochondrial oxygen consumption thereby reducing tumor hypoxia. These alterations in tumor hypoxia should be considered in the design of clinical trials using PI3K/mTOR inhibitors, particularly in conjunction with radiotherapy.

The influence of hydroquinone on tyrosinase kinetics.

In vitro studies, using combined spectrophotometry and oximetry together with hplc/ms examination of the products of tyrosinase action demonstrate that hydroquinone is not a primary substrate for the enzyme but is vicariously oxidised by a redox exchange mechanism in the presence of either catechol, L-3,4-dihydroxyphenylalanine or 4-ethylphenol. Secondary addition products formed in the presence of hydroquinone are shown to stimulate, rather than inhibit, the kinetics of substrate oxidation.

Design, synthesis and evaluation of molecularly targeted hypoxia-activated prodrugs

Regions of insufficient oxygen supply—hypoxia—occur in diverse contexts across biology in both healthy and diseased organisms. The difference in the chemical environment between a hypoxic biological system and one with normal oxygen levels provides an opportunity for targeting compound delivery to hypoxic regions by using bioreductive prodrugs. Here we detail a protocol for the efficient synthesis of (1-methyl-2-nitro-1H-imidazol-5-yl)methanol, which is a key intermediate that can be converted into a range of 1-methyl-2-nitro-1H-imidazole–based precursors of bioreductive prodrugs. We outline methods for attaching the bioreductive group to a range of functionalities, and we discuss the strategy for positioning of the group on the biologically active parent compound. We have used two parent checkpoint kinase 1 (Chk1) inhibitors to exemplify the protocol. The PROCEDURE also describes a suite of reduction assays, of increasing biological relevance, to validate the bioreductive prodrug. These assays are applied to an exemplar compound, CH-01, which is a bioreductive Chk1 inhibitor. This protocol has broad applications to the development of hypoxia-targeted compounds.

Systemic inflammation alters central 5-HT function as determined by pharmacological MRI.

Considerable evidence indicates a link between systemic inflammation and central 5-HT function. This study used pharmacological magnetic resonance imaging (phMRI) to study the effects of systemic inflammatory events on central 5-HT function. Changes in blood oxygenation level dependent (BOLD) contrast were detected in selected brain regions of anaesthetised rats in response to intravenous administration of the 5-HT-releasing agent, fenfluramine (10 mg/kg). Further groups of rats were pre-treated with the bacterial lipopolysaccharide (LPS; 0.5 mg/kg), to induce systemic inflammation, or the selective 5-HT2A receptor antagonist MDL100907 prior to fenfluramine. The resultant phMRI data were investigated further through measurements of cortical 5-HT release (microdialysis), and vascular responsivity, as well as a more thorough investigation of the role of the 5-HT2A receptor in sickness behaviour. Fenfluramine evoked a positive BOLD response in the motor cortex (+ 15.9 ± 2%) and a negative BOLD response in the dorsal raphe nucleus (− 9.9 ± 4.2%) and nucleus accumbens (− 7.7 ± 5.3%). In all regions, BOLD responses to fenfluramine were significantly attenuated by pre-treatment with LPS (p < 0.0001), but neurovascular coupling remained intact, and fenfluramine-evoked 5-HT release was not affected. However, increased expression of the 5-HT2A receptor mRNA and decreased 5-HT2A-dependent behaviour (wet-dog shakes) was a feature of the LPS treatment and may underpin the altered phMRI signal. MDL100907 (0.5 mg/kg), 5-HT2A antagonist, significantly reduced the BOLD responses to fenfluramine in all three regions (p < 0.0001) in a similar manner to LPS. Together these results suggest that systemic inflammation decreases brain 5-HT activity as assessed by phMRI. However, these effects do not appear to be mediated by changes in 5-HT release, but are associated with changes in 5-HT2A-receptor-mediated downstream signalling pathways.

Efficient synthesis of 2-nitroimidazole derivatives and the bioreductive clinical candidate Evofosfamide (TH-302)

Hypoxia, regions of low oxygen, occurs in a range of biological environments, and is involved in human diseases, most notably solid tumours. Exploiting the physiological differences arising from low oxygen conditions provides an opportunity for development of targeted therapies, through the use of bioreductive prodrugs, which are selectively activated in hypoxia. Herein, we describe an improved method for synthesising the most widely used bioreductive group, 2-nitroimidazole. The improved method is applied to an efficient synthesis of the anti-cancer drug Evofosfamide (TH-302), which is currently in Phase III clinical trials for treatment of a range of cancers.

Validation of a method for the determination of the anticancer agent Combretastatin A1 phosphate (CA1P, OXi4503) in human plasma by HPLC with post-column photolysis and fluorescence detection.

A validated method for the determination of Combretastatin A1 phosphate (CA1P, OXi4503), a bisphosphate prodrug of the vascular disrupting agent Combretastatin A1 in human plasma has been developed using fluorescence detection after post-column photolysis. The separation used the ion-pairing agent tetrabutylammonium hydrogen sulphate, and this agent was also required to give consistently high recovery from plasma. Initially, the range was shown to be linear (r(2)>0.995) from the LOQ of 0.025 μM to 5 μM, but as the trial progressed to much higher doses, using a lower injection volume, the assay was subsequently subject to limited revalidation to cover the range from 0.05 to 50 μM. Intra-assay precision and accuracy ranged from 2.2 to 11.8% and 1.8 to 13% respectively, and for inter-assay from 4.4 to 14.9% and 1.7 to 6.5%. Mean recovery of OXi4503 from plasma was 80.2%.