Preclinical Activity and Pharmacokinetic/Pharmacodynamic Relationship for a Series of Novel Benzenesulfonamide Perforin Inhibitors.

Perforin is a key effector of lymphocyte-mediated cell death pathways and contributes to transplant rejection of immunologically mismatched grafts. We have developed a novel series of benzenesulfonamide (BZS) inhibitors of perforin that can mitigate graft rejection during allogeneic bone marrow/stem cell transplantation. Eight such perforin inhibitors were tested for their murine pharmacokinetics, plasma protein binding, and their ability to block perforin-mediated lysis in vitro and to block the rejection of major histocompatibility complex (MHC)-mismatched mouse bone marrow cells. All compounds showed >99% binding to plasma proteins and demonstrated perforin inhibitory activity in vitro and in vivo. A lead compound, compound 1, that showed significant increases in allogeneic bone marrow preservation was evaluated for its plasma pharmacokinetics and in vivo efficacy at multiple dosing regimens to establish a pharmacokinetic/pharmacodynamic (PK/PD) relationship. The strongest PK/PD correlation was observed between perforin inhibition in vivo and time that total plasma concentrations remained above 900 µM, which correlates to unbound concentrations similar to 3× the unbound in vitro IC90 of compound 1. This PK/PD relationship will inform future dosing strategies of BZS perforin inhibitors to maintain concentrations above 3× the unbound IC90 for as long as possible to maximize efficacy and enhance progression toward clinical evaluation.

In situ gelling system for sustained intraarticular delivery of bupivacaine and ketorolac in sheep.

Suboptimal control of postoperative pain following knee arthroplasty can slow recovery and reduce patient satisfaction. Intraarticular (IA) administration of bupivacaine and ketorolac offers efficient pain control and minimizes opioid consumption. However, the clinical benefits of this approach are short lived due to rapid clearance of drugs from the joint cavity. Here, we describe a poloxamer based thermoresponsive in situ gelling system for the sustained IA delivery of bupivacaine hydrochloride (BH) and ketorolac tromethamine (KT) following knee surgery in an ovine model. Drug loaded formulations were prepared using poloxamer 407, poloxamer 188 and sodium chloride. In vitro characterization was conducted, followed by in vivo evaluation of sustained drug release and safety in an ovine model of knee joint surgery. Rheological studies revealed a Newtonian-like flow of the developed formulation at room temperature, confirming its injectability, followed by a transition to a viscous gel as temperature approached body temperature. The developed formulation successfully sustained the in vivo release of BH for 72 h and KT for 48 h, as determined by circulating drug levels, compared to 24 and 8 h for marketed drug solutions. The concentrations of BH and KT in the synovial fluids at 72 h were 11.5 and 1.8 times that of marketed products, suggesting a significant increase in the IA residence time. The developed formulation induced a comparable inflammatory response compared to the marketed drug solutions, however a significantly higher chondrotoxicity was observed following administration of the gel formulations. Poloxamers based in situ gelling systems are promising delivery platforms for the sustained and localised IA delivery of BH and KT, with potential clinical benefits in managing the postoperative pain following knee arthroplasty.

Spatially-resolved pharmacokinetic/pharmacodynamic modelling of bystander effects of a nitrochloromethylbenzindoline hypoxia-activated prodrug.

PURPOSE: Hypoxia-activated prodrugs (HAPs) have the potential for eliminating chemo- and radiation-resistant hypoxic tumour cells, but their activity is often compromised by limited penetration into hypoxic zones. Nitrochloromethylbenzindoline (nitroCBI) HAPs are reduced in hypoxic cells to highly cytotoxic DNA minor groove alkylating aminoCBI metabolites. In this study, we investigate whether a lead nitroCBI, SN30548, generates a significant bystander effect through the diffusion of its aminoCBI metabolite and whether this compensates for any diffusion limitations of the prodrug in tumour tissue. METHODS: Metabolism and uptake of the nitroCBI in oxic and anoxic cells, and diffusion through multicellular layer cultures, was characterised by LC-MS/MS. To quantify bystander effects, clonogenic cell killing of HCT116 cells was assessed in multicellular spheroid co-cultures comprising cells transfected with cytochrome P450 oxidoreductase (POR) or E. coli nitroreductase NfsA. Spatially-resolved pharmacokinetic/pharmacodynamic (PK/PD) models, parameterised by the above measurements, were developed for spheroids and tumours using agent-based and Green's function modelling, respectively. RESULTS: NitroCBI was reduced to aminoCBI by POR under anoxia and by NfsA under oxia, and was the only significant cytotoxic metabolite in both cases. In spheroid co-cultures comprising 30% NfsA-expressing cells, non-metabolising cells were as sensitive as the NfsA cells, demonstrating a marked bystander effect. Agent-based PK/PD models provided good prediction of cytotoxicity in spheroids, while use of the same parameters in a Green's function model for a tumour microregion demonstrated that local diffusion of aminoCBI overcomes the penetration limitation of the prodrug. CONCLUSIONS: The nitroCBI HAP SN30548 generates a highly efficient bystander effect through local diffusion of its active metabolite in tumour tissue.

Efficacy of Providing the PI3K p110α Inhibitor BYL719 (Alpelisib) to Middle-Aged Mice in Their Diet.

BYL719 (alpelisib) is a small molecule inhibitor of PI3K p110α developed for cancer therapy. Targeted suppression of PI3K has led to lifespan extension in rodents and model organisms. If PI3K inhibitors are to be considered as an aging therapeutic, it is important to understand the potential consequences of long-term exposure, and the most practical way to achieve this is through diet administration. Here, we investigated the pharmacokinetics of BYL719 delivered in diet and the efficacy of BYL719 to suppress insulin signaling when administered in the diet of 8-month-old male and female mice. Compared to oral gavage, diet incorporation resulted in a lower peak plasma BYL719 (3.6 vs. 9.2 µM) concentration but similar half-life (~1.5 h). Consuming BYL719 resulted in decreased insulin signaling in liver and muscle within 72 h, and mice still showed impaired glucose tolerance and insulin sensitivity following 6 weeks of access to a diet containing 0.3 g/kg BYL719. However, consuming BYL719 did not affect food intake, body mass, muscle function (rotarod and hang time performance) or cognitive behaviors. This provides evidence that BYL719 has long-term efficacy without major toxicity or side effects, and suggests that administering BYL719 in diet is suitable for studying the effect of pharmacological suppression of PI3K p110α on aging and metabolic function.

The CSF1 receptor inhibitor pexidartinib (PLX3397) reduces tissue macrophage levels without affecting glucose homeostasis in mice.

BACKGROUND AND OBJECTIVES: Excessive adipose tissue macrophage accumulation in obesity has been implicated in mediating inflammatory responses that impair glucose homeostasis and promote insulin resistance. Colony-stimulating factor 1 (CSF1) controls macrophage differentiation, and here we sought to determine the effect of a CSF1 receptor inhibitor, PLX3397, on adipose tissue macrophage levels and understand the impact on glucose homeostasis in mice. METHODS: A Ten-week-old mice were fed a chow or high-fat diet for 10 weeks and then treated with PLX3397 via oral gavage (50 mg/kg) every second day for 3 weeks, with subsequent monitoring of glucose tolerance, insulin sensitivity and assessment of adipose tissue immune cells. RESULTS: PLX3397 treatment substantially reduced macrophage numbers in adipose tissue of both chow and high-fat diet fed mice without affecting total myeloid cell levels. Despite this, PLX3397 did not greatly alter glucose homeostasis, did not affect high-fat diet-induced increases in visceral fat cytokine expression (Il-6 and Tnfa) and had limited effect on the phosphorylation of the stress kinases JNK and ERK and macrophage polarization. CONCLUSIONS: Our results indicate that macrophage infiltration of adipose tissue induced by a high-fat diet may not be the trigger for impairments in whole body glucose homeostasis, and that anti-CSF1 therapies are not likely to be useful as treatments for insulin resistance.

Inhibition of the Cytolytic Protein Perforin Prevents Rejection of Transplanted Bone Marrow Stem Cells in Vivo.

Perforin is a key effector protein in the vertebrate immune system and is secreted by cytotoxic T lymphocytes and natural killer cells to help eliminate virus-infected and transformed target cells. The ability to modulate perforin activity in vivo could be extremely useful, especially in the context of bone marrow stem cell transplantation where early rejection of immunologically mismatched grafts is driven by the recipient's natural killer cells, which overwhelmingly use perforin to kill their targets. Bone marrow stem cell transplantation is a potentially curative treatment for both malignant and nonmalignant disorders, but when the body recognizes the graft as foreign, it is rejected by this process, often with fatal consequences. Here we report optimization of a previously identified series of benzenesulfonamide-based perforin inhibitors for their physicochemical and pharmacokinetic properties, resulting in the identification of 16, the first reported small molecule able to prevent rejection of transplanted bone marrow stem cells in vivo by blocking perforin function.

Schedule-dependent potentiation of chemotherapy drugs by the hypoxia-activated prodrug SN30000.

Hypoxia-activated prodrugs (HAPs) are hypothesized to improve the therapeutic index of chemotherapy drugs that are ineffective against tumor cells in hypoxic microenvironments. SN30000 (CEN-209) is a benzotriazine di-N-oxide HAP that potentiates radiotherapy in preclinical models, but its combination with chemotherapy has not been explored. Here we apply multiple models (monolayers, multicellular spheroids and tumor xenografts) to identify promising SN30000/chemotherapy combinations (with chemotherapy drugs before, during or after SN30000 exposure). SN30000, unlike doxorubicin, cisplatin, gemcitabine or paclitaxel, was more active against cells in spheroids than monolayers by clonogenic assay. Combinations of SN30000 and chemotherapy drugs in HCT116/GFP and SiHa spheroids demonstrated hypoxia-and schedule-dependent potentiation of gemcitabine or doxorubicin in growth inhibition and clonogenic assays. Co-administration with SN30000 suppressed clearance of gemcitabine in NIH-III mice, likely due to SN30000-induced hypothermia which also modulated extravascular transport of gemcitabine in tumor tissue as assessed from its diffusion through HCT116 multicellular layer cultures. Despite these systemic effects, the same schedules that gave therapeutic synergy in spheroids (SN30000 3 h before or during gemcitabine, but not gemcitabine 3 h before SN30000) enhanced growth delay of HCT116 xenografts without increasing host toxicity. Identification of hypoxic and S-phase cells by immunohistochemistry and flow cytometry established that hypoxic cells initially spared by gemcitabine subsequently reoxygenate and re-enter the cell cycle, and that this repopulation is prevented by SN30000 only when administered with or before gemcitabine. This illustrates the value of spheroids in modeling tumor microenvironment-dependent drug interactions, and the potential of HAPs for overcoming hypoxia-mediated drug resistance.

An agent-based model for drug-radiation interactions in the tumour microenvironment: Hypoxia-activated prodrug SN30000 in multicellular tumour spheroids.

Multicellular tumour spheroids capture many characteristics of human tumour microenvironments, including hypoxia, and represent an experimentally tractable in vitro model for studying interactions between radiotherapy and anticancer drugs. However, interpreting spheroid data is challenging because of limited ability to observe cell fate within spheroids dynamically. To overcome this limitation, we have developed a hybrid continuum/agent-based model (ABM) for HCT116 tumour spheroids, parameterised using experimental models (monolayers and multilayers) in which reaction and diffusion can be measured directly. In the ABM, cell fate is simulated as a function of local oxygen, glucose and drug concentrations, determined by solving diffusion equations and intracellular reactions. The model is lattice-based, with cells occupying discrete locations on a 3D grid embedded within a coarser grid that encompasses the culture medium; separate solvers are employed for each grid. The generated concentration fields account for depletion in the medium and specify concentration-time profiles within the spheroid. Cell growth and survival are determined by intracellular oxygen and glucose concentrations, the latter based on direct measurement of glucose diffusion/reaction (in multilayers) for the first time. The ABM reproduces known features of spheroids including overall growth rate, its oxygen and glucose dependence, peripheral cell proliferation, central hypoxia and necrosis. We extended the ABM to describe in detail the hypoxia-dependent interaction between ionising radiation and a hypoxia-activated prodrug (SN30000), again using experimentally determined parameters; the model accurately simulated clonogenic cell killing in spheroids, while inclusion of reversible cell cycle delay was required to account for the marked spheroid growth delay after combined radiation and SN30000. This ABM of spheroid growth and response exemplifies the utility of integrating computational and experimental tools for investigating radiation/drug interactions, and highlights the critical importance of understanding oxygen, glucose and drug concentration gradients in interpreting activity of therapeutic agents in spheroid models.

Cellular pharmacology of evofosfamide (TH-302): A critical re-evaluation of its bystander effects.

Evofosfamide (TH-302) is a clinical-stage hypoxia-activated prodrug with proven efficacy against hypoxic cells in preclinical tumour models. TH-302 is designed to release the DNA crosslinking agent bromo-isophosphoramide mustard (Br-IPM) when reduced in hypoxic tissue. Br-IPM is considered to diffuse locally from hypoxic regions, eliciting additional tumour cell killing, but the latter 'bystander effect' has not been demonstrated directly. Previous studies with multicellular co-cultures that included cells expressing the E. coli nitroreductase NfsA as a model TH-302 reductase have provided clear evidence of a bystander effect (which we confirm in the present study). However, NfsA is an oxygen-insensitive two-electron reductase that is not expected to generate the nitro radical intermediate that has been demonstrated to fragment to release Br-IPM. Here, we use mass spectrometry methods to characterise TH-302 metabolites generated by one-electron reduction (steady-state radiolysis by ionising radiation and cellular metabolism under hypoxia, including HCT116 cells that overexpress P450 oxidoreductase, POR) or by NfsA expressed in HCT116 cells under oxic conditions, and investigate the stability and cytotoxicity of these products. Br-IPM is shown to have very low cytotoxic potency when added to extracellular culture medium and to be rapidly converted to other hydrophilic products including dichloro-isophosphoramide mustard (IPM). Only traces of Br-IPM or IPM were detected in the extracellular medium when generated by cellular metabolism of TH-302. We identify, in NfsA-expressing cells, the hydroxylamine metabolite of TH-302, and downstream products resulting from rearrangement or hydration of the imidazole ring, and demonstrate that formation of these candidate bystander effect mediators is suppressed by hypoxia. This characterisation of the cellular pharmacology of TH-302 implies that bystander effects from hypoxic activation of TH-302 are unlikely to contribute to its anticancer activity.

Nanocarrier mediated retinal drug delivery: overcoming ocular barriers to treat posterior eye diseases.

Effective drug delivery to the retina still remains a challenge due to ocular elimination mechanisms and complex barriers that selectively limit the entry of drugs into the eye. To overcome these barriers, frequent intravitreal injections are currently used to achieve high drug concentrations in vitreous and retina. However, these repetitive injections may result in several side effects. Recent advancements in the field of nanoparticle-based drug delivery could overcome some of these unmet needs and various preclinical studies conducted to date have demonstrated promising results of nanotherapies in the treatment of retinal diseases. Compared to the majority of commercially available ocular implants, the biodegradable nature of most nanoparticles (NPs) avoids the need for surgical implantation and removal after the release of the payload. In addition, the sustained drug release from NPs over an extended period of time reduces the need for frequent intravitreal injections and the risk of associated side effects. The nanometer size and highly modifiable surface properties make NPs excellent candidates for targeted ocular drug delivery. Studies have shown that nanocarriers enhance the intravitreal half-life and thus bioavailability of a number of drugs including proteins and peptides. In addition, they have shown promising results in delivering genetic material to the retinal tissues by protecting it from possible intravitreal degradation. This review covers the various challenges associated with drug delivery to the posterior segment of the eye, particularly the retina, and highlights the application of nanocarriers to overcome these challenges in context with recent advances in preclinical studies. WIREs Nanomed Nanobiotechnol 2018, 10:e1473. doi: 10.1002/wnan.1473 This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Implantable Materials and Surgical Technologies > Nanomaterials and Implants.

Reductive Metabolism Influences the Toxicity and Pharmacokinetics of the Hypoxia-Targeted Benzotriazine Di-Oxide Anticancer Agent SN30000 in Mice.

3-(3-Morpholinopropyl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine 1,4-dioxide (SN30- 000), an analog of the well-studied bioreductive prodrug tirapazamine (TPZ), has improved activity against hypoxic cells in tumor xenografts. However, little is known about its biotransformation in normal tissues. Here, we evaluate implications of biotransformation of SN30000 for its toxicokinetics in NIH-III mice. The metabolite profile demonstrated reduction to the 1-N-oxide (M14), oxidation of the morpholine side-chain (predominantly to the alkanoic acid M18) and chromophore, and subsequent glucuronidation. Plasma pharmacokinetics of SN30000 and its reduced metabolites was unaffected by the presence of HT29 tumor xenografts, indicating extensive reduction in normal tissues. This bioreductive metabolism, as modeled by hepatic S9 preparations, was strongly inhibited by oxygen indicating that it proceeds via the one-electron (radical) intermediate previously implicated in induction of DNA double strand breaks and cytotoxicity by SN30000. Plasma pharmacokinetics of SN30000 and M14 (but not M18) corresponded closely to the timing of reversible acute clinical signs (reduced mobility) and marked hypothermia (rectal temperature drop of ∼8°C at nadir following the maximum tolerated dose). Similar acute toxicity was elicited by dosing with TPZ or M14, although M14 did not induce the kidney and lung histopathology caused by SN30000. M14 also lacked antiproliferative potency in hypoxic cell cultures. In addition M14 showed much slower redox cycling than SN30000 in oxic cultures. Thus a non-bioreductive mechanism, mediated through M14, appears to be responsible for the acute toxicity of SN30000 while late toxicities are consistent with DNA damage resulting from its one-electron reduction. A two-compartment pharmacokinetic model, in which clearance of SN30000 is determined by temperature-dependent bioreductive metabolism to M14, was shown to describe the non-linear PK of SN30000 in mice. This study demonstrates the importance of non-tumor bioreductive metabolism in the toxicology and pharmacokinetics of benzotriazine di-oxides designed to target tumor hypoxia.

Nanoparticle-loaded biodegradable light-responsive in situ forming injectable implants for effective peptide delivery to the posterior segment of the eye.

Diseases affecting the posterior segment the eye, such as age-related macular degeneration (AMD), are the leading cause of blindness worldwide. Conventional dosage forms, such as eye drops, have to surmount several elimination mechanisms and complex barriers to achieve therapeutic concentrations at the target site often resulting in low anterior segment bioavailability (ca. 2-5%) with generally none of the drug reaching posterior segment tissues. Thus, frequent intravitreal injections are currently required to treat retinal conditions which have been associated with poor patient compliance due to pain, risk of infection, hemorrhages, retinal detachment and high treatment related costs. To partially overcome these issues, ocular implants have been developed for some posterior segment indications; however, the majority require surgical implantation and removal at the end of the intended treatment period. The transparent nature of the cornea and lens render light-responsive systems an attractive strategy for the management of diseases affecting the back of the eye. Light-responsive in situ forming injectable implants (ISFIs) offer various benefits such as ease of application in a minimally invasive manner and more site specific control over drug release. Moreover, the biodegradable nature of such implants avoids the need for surgical removal after release of the payload. Incorporating drug-loaded polymeric nanoparticles (NPs) into these implants may reduce the high initial burst release from the polymeric matrix and further sustain drug release thus avoiding the need for frequent injections as well as minimizing associated side effects. However, light-responsive systems for ophthalmic application are still in their early stages of development with limited reports on their safety and effectiveness. We hypothesize that the innovative design and properties of NP-containing light-responsive ISFIs can serve as a platform for effective management of ocular diseases requiring long term treatment.

Substituted arylsulphonamides as inhibitors of perforin-mediated lysis.

The structure-activity relationships for a series of arylsulphonamide-based inhibitors of the pore-forming protein perforin have been explored. Perforin is a key component of the human immune response, however inappropriate activity has also been implicated in certain auto-immune and therapy-induced conditions such as allograft rejection and graft versus host disease. Since perforin is expressed exclusively by cells of the immune system, inhibition of this protein would be a highly selective strategy for the immunosuppressive treatment of these disorders. Compounds from this series were demonstrated to be potent inhibitors of the lytic action of both isolated recombinant perforin and perforin secreted by natural killer cells in vitro. Several potent and soluble examples were assessed for in vivo pharmacokinetic properties and found to be suitable for progression to an in vivo model of transplant rejection.

Benzenesulphonamide inhibitors of the cytolytic protein perforin.

The pore-forming protein perforin is a key component of mammalian cell-mediated immunity and essential to the pathway that allows elimination of virus-infected and transformed cells. Perforin activity has also been implicated in certain auto-immune conditions and therapy-induced conditions such as allograft rejection and graft versus host disease. An inhibitor of perforin activity could be used as a highly specific immunosuppressive treatment for these conditions, with reduced side-effects compared to currently accepted therapies. Previously identified first-in-class inhibitors based on a 2-thioxoimidazolidin-4-one core show suboptimal physicochemical properties and toxicity toward the natural killer (NK) cells that secrete perforin in vivo. The current benzenesulphonamide-based series delivers a non-toxic bioisosteric replacement possessing improved solubility.

Development of a novel stability indicating RP-HPLC method for quantification of Connexin43 mimetic peptide and determination of its degradation kinetics in biological fluids.

Connexin43 mimetic peptide (Cx43MP) has been intensively investigated for its therapeutic effect in the management of inflammatory eye conditions, spinal cord injury, wound healing and ischemia-induced brain damage. Here, we report on a validated stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) method for the quantification of Cx43MP under stress conditions. These included exposure to acid/base, light, oxidation and high temperature. In addition, the degradation kinetics of the peptide were evaluated in bovine vitreous and drug-free human plasma at 37 °C. Detection of Cx43MP was carried out at 214 nm with a retention time of 7.5 min. The method showed excellent linearity over the concentration range of 0.9-250 µg/mL (R2 ≥ 0.998), and the limits of detection (LOD) and quantification (LOQ) were found to be 0.90 and 2.98 µg/mL, respectively. The accuracy of the method determined by the mean percentage recovery at 7.8, 62.5 and 250 µg/mL was 96.79%, 98.25% and 99.06% with a RSD of < 2.2%. Accelerated stability studies revealed that Cx43MP was more sensitive to basic conditions and completely degraded within 24 h at 37 °C (0% recovery) and within 12 h at 80 °C (0.34% recovery). Cx43MP was found to be more stable in bovine vitreous (t1/2slow= 171.8 min) compared to human plasma (t1/2slow = 39.3 min) at 37 °C according to the two phase degradation kinetic model. These findings are important for further pre-clinical development of Cx43MP.

Evidence that phospholipase C is involved in the antitumour action of NSC768313, a new thieno[2,3-b]pyridine derivative.

BACKGROUND: The thieno[2,3-b]pyridines were discovered by virtual high throughput screening as potential inhibitors of phospholipase C (PLC) isoforms and showed potent growth inhibitory effects in National Cancer Institute's human tumour cell line panel (NCI60). The mechanism of the anti-proliferative activity of thieno[2,3-b]pyridines is explored here. OBJECTIVES: We aimed to investigate the basis for the anti-proliferative activity of these thieno[2,3-b]pyridines and to determine whether the cellular inhibition was related to their inhibition of PLC. METHODS: Four breast cancer cell lines were used to assess the anti-proliferative effects (IC50 values) of six representative thieno[2,3-b]pyridines. The most potent compound (derivative 3; NSC768313), was further studied in MDA-MB-231 cells. DNA damage was examined by γH2AX expression level, and cell cycle arrest by flow cytometry. Cell morphology was examined by tubulin antibody staining. The growth inhibitory effect of combination treatment with derivative 3 and paclitaxel (tubulin inhibitor), doxorubicin (topoisomerase II inhibitor) or camptothecin (topoisomerase I inhibitor) was evaluated. A preliminary mouse toxicity assay was used to evaluate the pharmacological properties. RESULTS: Addition of the thieno[2,3-b]pyridine derivative 3 to the MDA-MB-231 cells induced G2/M growth inhibition, cell cycle arrest in G2-phase, membrane blebbing and the formation of multinucleated cells. It did not induce DNA damage, mitotic arrest or changes in calcium ion flux. Combination of derivative 3 with paclitaxel showed a high degree of synergy, while combinations with doxorubicin and camptothecin showed only additive effects. A mouse pharmacokinetic study of derivative 3 showed that after intraperitoneal injection of a single does (10 mg/Kg), the Cmax was 0.087 µmol/L and the half-life was 4.11 h. CONCLUSIONS: The results are consistent with a mechanism in which thieno[2,3-b]pyridine derivatives interact with PLC isoforms (possibly PLC-δ), which in turn affect the cellular dynamics of tubulin-ß, inducing cell cycle arrest in G2-phase. We conclude that these compounds have novelty because of their PLC target and may have utility in combination with mitotic poisons for cancer treatment.

Diarylthiophenes as inhibitors of the pore-forming protein perforin.

Evolution from a furan-containing high-throughput screen (HTS) hit (1) resulted in isobenzofuran-1(3H)-one (2) as a potent inhibitor of the function of both isolated perforin protein and perforin delivered in situ by intact KHYG-1 NK cells. In the current study, structure-activity relationship (SAR) development towards a novel series of diarylthiophene analogues has continued through the use of substituted-benzene and -pyridyl moieties as bioisosteres for 2-thioxoimidazolidin-4-one (A) on a thiophene (B) -isobenzofuranone (C) scaffold. The resulting compounds were tested for their ability to inhibit perforin lytic activity in vitro. Carboxamide (23) shows a 4-fold increase over (2) in lytic activity against isolated perforin and provides good rationale for continued development within this class.

Photodegradation of the benzotriazine 1,4-Di-N-oxide hypoxia-activated prodrug SN30000 in aqueous solution.

SN30000 is a benzotriazine di-N-oxide which is selectively toxic to radio-resistant hypoxic cells in tumours. Given the complex photochemistry of some aromatic N-oxides, we evaluated the potential for photodegradation of SN30000 solutions. Initial studies demonstrated significant oxygen-insensitive degradation under normal laboratory lighting conditions. The kinetics of photodegradation showed marked concentration dependence of the form predicted by Beer's law, with a quantum yield of 0.016. The photoproducts could be rationalised as arising from an oxaziridine intermediate. The major stable product (cmpd 6; yield ∼50% of SN30000 loss under either UV or visible light) was characterised as resulting from intra-molecular oxygen transfer to the morpholine side chain of SN30000. This mechanism is consistent with lack of formation of the corresponding morpholine N-oxide from an analogue (SN29751) in which the proposed six-membered-ring transition state cannot form. Cmpd 6 was less cytotoxic than SN30000 to human tumour cells in culture, under either hypoxic or aerobic conditions, and was not toxic when administered intra-peritoneally to NIH-III nude mice at a dose (750 µmol/kg) above the maximal tolerated dose of SN30000 itself. In conclusion, SN30000 solutions are significantly photosensitive at low concentration, requiring protection from light, but the major photoproduct is less toxic than the parent.

The flavoprotein FOXRED2 reductively activates nitro-chloromethylbenzindolines and other hypoxia-targeting prodrugs.

The nitro-chloromethylbenzindoline prodrug SN29428 has been rationally designed to target tumour hypoxia. SN29428 is metabolised to a DNA minor groove alkylator via oxygen-sensitive reductive activation initiated by unknown one-electron reductases. The present study sought to identify reductases capable of activating SN29428 in tumours. Expression of candidate reductases in cell lines was modulated using forced expression and, for P450 (cytochrome) oxidoreductase (POR), by zinc finger nuclease-mediated gene knockout. Affymetrix microarray mRNA expression of flavoreductases was correlated with SN29428 activation in a panel of 23 cancer cell lines. Reductive activation and cytotoxicity of prodrugs were measured using mass spectrometry and antiproliferative assays, respectively. SN29428 activation under hypoxia was strongly attenuated by the pan-flavoprotein inhibitor diphenyliodonium, but less so by knockout of POR suggesting other flavoreductases contribute. Forced expression of 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR), as well as POR, increased activation of SN29428 in hypoxic HCT 116 cells. SN29428 activation strongly correlated with expression of POR and also FAD-dependent oxidoreductase domain containing 2 (FOXRED2), in cancer cell lines. This association persisted after removing the effect of POR enzyme activity using first-order partial correlation. Forced expression of FOXRED2 increased SN29428 activation and cytotoxicity in hypoxic HEK293 cells and also increased activation of hypoxia-targeted prodrugs PR-104A, tirapazamine and SN30000, and increased cytotoxicity of the clinical-stage prodrug TH-302. Thus this study has identified three flavoreductases capable of enzymatically activating SN29428, one of which (FOXRED2) has not previously been implicated in xenobiotic metabolism. These results will inform future development of biomarkers predictive of SN29428 sensitivity.

Preparation and antitumour properties of the enantiomers of a hypoxia-selective nitro analogue of the duocarmycins.

Racemic 2-{[1-(chloromethyl)-5-nitro-3-{5-[2-(dimethylamino)ethoxy]indol-2-carbonyl}-1,2-dihydro-3H-benzo[e]indol-7-yl]sulfonyl}aminoethyl dihydrogen phosphate, a synthetic nitro derivative of the duocarmycins, is a hypoxia-selective prodrug active against radiation-resistant tumour cells at nontoxic doses in mice. An intermediate in the synthesis of this prodrug was resolved by chiral HPLC and the absolute configuration assigned by X-ray crystallography. The intermediate was used to prepare the prodrug's enantiomers, and also the enantiomers of the active nitro and amino metabolites. In vitro analysis in the human cervical carcinoma cell line SiHa showed that both nitro enantiomers are hypoxia-selective cytotoxins, but the "natural" S enantiomer is at least 20-fold more potent. Examination of extracellular amino metabolite concentrations demonstrated no enantioselectivity in the hypoxia-selective reduction of nitro to amino. Low levels of amino derivative were also found in aerobic cell suspensions, sufficient to account for the observed oxic toxicity of the nitro form. At an equimolar dose in SiHa-tumour bearing animals, the (-)-R enantiomer of the prodrug was inactive, while the (+)-S enantiomer caused significantly more hypoxic tumour cell kill than the racemate. At this dose, the combination of (+)-S-prodrug and radiation eliminated detectable colony-forming cells in four out of five treated tumour-bearing animals.