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.

Drug-DNA adducts as biomarkers for metabolic activation of the nitro-aromatic nitrogen mustard prodrug PR-104A.

PR-104A is a clinical-stage nitrogen mustard prodrug that is activated for DNA alkylation by reduction of a nitro group to the corresponding hydroxylamine (PR-104H) or amine (PR-104M). Metabolic reduction is catalysed by flavoreductases such as cytochrome P450 oxidoreductase (POR) under hypoxia, or by aldo-ketoreductase 1C3 (AKR1C3) independently of hypoxia. The unstable reduced metabolites are challenging to measure in biological samples, and biomarkers of the metabolic activation of PR-104A have not been used in the clinical evaluation of PR-104 to date. Here, we employ a selected reaction monitoring mass spectrometry assay for DNA crosslinks to assess the capacity of human cancer cells to bioactivate PR-104A. We also test whether the more abundant DNA monoadducts could be used for the same purpose. DNA monoadducts and crosslinks from PR-104A itself, and from its reduced metabolites, accumulated over 4 h in AKR1C3-expressing TF1 erythroleukaemia cells under hypoxia, whereas intracellular concentrations of unstable PR-104H and PR-104M reached steady state within 1 h. We then varied rates of PR-104A reduction by manipulating hypoxia or reductase expression in a panel of cell lines, in which AKR1C3 and POR were quantified by targeted proteomics. Hypoxia or reductase overexpression induced large increases in PR-104A sensitivity (inhibition of proliferation), DNA damage response (γH2AX formation), steady-state concentrations of PR-104H/M and formation of reduced drug-DNA adducts but not DNA adducts retaining the dinitro groups of PR-104A. The fold-change in the sum of PR-104H and PR-104M correlated with the fold-change in reduced crosslinks or monoadducts (R2 = 0.87 for both), demonstrating their potential for assessing the capacity of cancer cells to bioactivate PR-104A.

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.

Influence of a Basic Side Chain on the Properties of Hypoxia-Selective Nitro Analogues of the Duocarmycins: Demonstration of Substantial Anticancer Activity in Combination with Irradiation or Chemotherapy.

A new series of nitro analogues of the duocarmycins was prepared and evaluated for hypoxia-selective anticancer activity. The compounds incorporate 13 different amine-containing side chains designed to bind in the minor groove of DNA while spanning a wide range of base strength from pKa 9.64 to 5.24. The most favorable in vitro properties were associated with strongly basic side chains, but the greatest in vivo antitumor activity was found for compounds containing a weakly basic morpholine. This applies to single-agent activity and for activity in combination with irradiation or chemotherapy (gemcitabine or docetaxel). In combination with a single dose of γ irradiation 50 at 42 µmol/kg eliminated detectable clonogens in some SiHa cervical carcinoma xenografts, and in combination with gemcitabine using a well-tolerated multidose schedule, the same compound caused regression of all treated A2780 ovarian tumor xenografts. In the latter experiment, three of seven animals receiving the combination treatment were completely tumor free at day 100.

Measuring Quality of Healthcare Outcomes in Type 2 Diabetes from Routine Data: a Seven-nation Survey Conducted by the IMIA Primary Health Care Working Group.

Background: The Institute of Medicine framework defines six dimensions of quality for healthcare systems: (1) safety, (2) effectiveness, (3) patient centeredness, (4) timeliness of care, (5) efficiency, and (6) equity. Large health datasets provide an opportunity to assess quality in these areas. Objective: To perform an international comparison of the measurability of the delivery of these aims, in people with type 2 diabetes mellitus (T2DM) from large datasets. Method: We conducted a survey to assess healthcare outcomes data quality of existing databases and disseminated this through professional networks. We examined the data sources used to collect the data, frequency of data uploads, and data types used for identifying people with T2DM. We compared data completeness across the six areas of healthcare quality, using selected measures pertinent to T2DM management. Results: We received 14 responses from seven countries (Australia, Canada, Italy, the Netherlands, Norway, Portugal, Turkey and the UK). Most databases reported frequent data uploads and would be capable of near real time analysis of healthcare quality.The majority of recorded data related to safety (particularly medication adverse events) and treatment efficacy (glycaemic control and microvascular disease). Data potentially measuring equity was less well recorded. Recording levels were lowest for patient-centred care, timeliness of care, and system efficiency, with the majority of databases containing no data in these areas. Databases using primary care sources had higher data quality across all areas measured. Conclusion: Data quality could be improved particularly in the areas of patient-centred care, timeliness, and efficiency. Primary care derived datasets may be most suited to healthcare quality assessment.

Building a Privacy, Ethics, and Data Access Framework for Real World Computerised Medical Record System Data: A Delphi Study. Contribution of the Primary Health Care Informatics Working Group.

BACKGROUND: Privacy, ethics, and data access issues pose significant challenges to the timely delivery of health research. Whilst the fundamental drivers to ensure that data access is ethical and satisfies privacy requirements are similar, they are often dealt with in varying ways by different approval processes. OBJECTIVE: To achieve a consensus across an international panel of health care and informatics professionals on an integrated set of privacy and ethics principles that could accelerate health data access in data-driven health research projects. METHOD: A three-round consensus development process was used. In round one, we developed a baseline framework for privacy, ethics, and data access based on a review of existing literature in the health, informatics, and policy domains. This was further developed using a two-round Delphi consensus building process involving 20 experts who were members of the International Medical Informatics Association (IMIA) and European Federation of Medical Informatics (EFMI) Primary Health Care Informatics Working Groups. To achieve consensus we required an extended Delphi process. RESULTS: The first round involved feedback on and development of the baseline framework. This consisted of four components: (1) ethical principles, (2) ethical guidance questions, (3) privacy and data access principles, and (4) privacy and data access guidance questions. Round two developed consensus in key areas of the revised framework, allowing the building of a newly, more detailed and descriptive framework. In the final round panel experts expressed their opinions, either as agreements or disagreements, on the ethics and privacy statements of the framework finding some of the previous round disagreements to be surprising in view of established ethical principles. CONCLUSION: This study develops a framework for an integrated approach to ethics and privacy. Privacy breech risk should not be considered in isolation but instead balanced by potential ethical benefit.

Toxicology of freshwater cyanobacteria.

Many chemical contaminants in drinking water have been shown to cause adverse health effects in humans after prolonged exposure. Cyanobacteria are one of the most potent and diverse groups of photosynthetic prokaryotes. One key component of cyanobacterial success in the environment is the production of potent toxins as secondary metabolites, which have been responsible for numerous adverse health impacts in humans. Anthropogenic activities have led to the increase of eutrophication in freshwater bodies' worldwide, causing cyanobacterial blooms to become more frequent. The present article will discuss about harmful cyanobacteria and their toxicology with special references to microcystin, nodularin, and cylindrospermopsin.

Does Informatics Enable or Inhibit the Delivery of Patient-centred, Coordinated, and Quality-assured Care: a Delphi Study. A Contribution of the IMIA Primary Health Care Informatics Working Group.

BACKGROUND: Primary care delivers patient-centred and coordinated care, which should be quality-assured. Much of family practice now routinely uses computerised medical record (CMR) systems, these systems being linked at varying levels to laboratories and other care providers. CMR systems have the potential to support care. OBJECTIVE: To achieve a consensus among an international panel of health care professionals and informatics experts about the role of informatics in the delivery of patient-centred, coordinated, and quality-assured care. METHOD: The consensus building exercise involved 20 individuals, five general practitioners and 15 informatics academics, members of the International Medical Informatics Association Primary Care Informatics Working Group. A thematic analysis of the literature was carried out according to the defined themes. RESULTS: The first round of the analysis developed 27 statements on how the CMR, or any other information system, including paper-based medical records, supports care delivery. Round 2 aimed at achieving a consensus about the statements of round one. Round 3 stated that there was an agreement on informatics principles and structures that should be put in place. However, there was a disagreement about the processes involved in the implementation, and about the clinical interaction with the systems after the implementation. CONCLUSIONS: The panel had a strong agreement about the core concepts and structures that should be put in place to support high quality care. However, this agreement evaporated over statements related to implementation. These findings reflect literature and personal experiences: whilst there is consensus about how informatics structures and processes support good quality care, implementation is difficult.

Big Data Usage Patterns in the Health Care Domain: A Use Case Driven Approach Applied to the Assessment of Vaccination Benefits and Risks. Contribution of the IMIA Primary Healthcare Working Group.

BACKGROUND: Generally benefits and risks of vaccines can be determined from studies carried out as part of regulatory compliance, followed by surveillance of routine data; however there are some rarer and more long term events that require new methods. Big data generated by increasingly affordable personalised computing, and from pervasive computing devices is rapidly growing and low cost, high volume, cloud computing makes the processing of these data inexpensive. OBJECTIVE: To describe how big data and related analytical methods might be applied to assess the benefits and risks of vaccines. METHOD: We reviewed the literature on the use of big data to improve health, applied to generic vaccine use cases, that illustrate benefits and risks of vaccination. We defined a use case as the interaction between a user and an information system to achieve a goal. We used flu vaccination and pre-school childhood immunisation as exemplars. RESULTS: We reviewed three big data use cases relevant to assessing vaccine benefits and risks: (i) Big data processing using crowdsourcing, distributed big data processing, and predictive analytics, (ii) Data integration from heterogeneous big data sources, e.g. the increasing range of devices in the "internet of things", and (iii) Real-time monitoring for the direct monitoring of epidemics as well as vaccine effects via social media and other data sources. CONCLUSIONS: Big data raises new ethical dilemmas, though its analysis methods can bring complementary real-time capabilities for monitoring epidemics and assessing vaccine benefit-risk balance.

Mechanism of action of AminoCBIs: highly reactive but highly cytotoxic analogues of the duocarmycins.

Duocarmycins are highly cytotoxic natural products that have potential for development into anticancer agents. Herein we describe proposed but previously unidentified NH analogues of the DNA-alkylating subunit and characterise these by solvolysis studies, NMR and computational modelling. These compounds are shown to be the exclusive intermediates in the solvolysis of their seco precursors and to possess very similar structural features to the widely studied O-based analogues, apart from an unusually high basicity. The measured pKa of 10.5 implies that the NH compounds are fully protonated under physiological conditions. Remarkably, their extremely high reactivity (calculated hydrolysis rate 10(8) times higher for protonated NH compared to the neutral O analogue) is still compatible with potent cytotoxicity, provided the active species is formed in the presence of cells. These surprising findings are of relevance to the design of duocarmycin-based tumour-selective therapies.

Preparation and properties of clickable amino analogues of the duocarmycins: factors that affect the efficiency of their fluorescent labelling of DNA.

Herein we report the synthesis of three DNA-alkylating amino analogues of the duocarmycins that carry an alkyne functional group suitable for copper-catalysed click chemistry. The alkyne-containing substituents are connected via a side chain position which projects away from the minor groove, and have only a small effect on DNA alkylation and cytotoxicity. The efficiency of click reactions with fluorophore azides was studied using alkylated ctDNA by analysing the adenine adducts produced after thermal depurination. Click reactions "on DNA" were sensitive to steric effects (tether length to the alkyne) and, surprisingly, to the nature of the fluorophore azide. With the best combination of click partners and reagents, adducts could be detected in the nuclei of treated cells by microscopy or flow cytometry, provided that an appropriate detergent (Triton X-100 and not Tween 20) was used for permeabilisation. The method is sensitive enough to detect adducts at physiologically relevant concentrations, and could have application in the development of nitro analogues of the duocarmycins as hypoxia-activated anticancer prodrugs.

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.

The Evidence-base for Using Ontologies and Semantic Integration Methodologies to Support Integrated Chronic Disease Management in Primary and Ambulatory Care: Realist Review. Contribution of the IMIA Primary Health Care Informatics WG.

BACKGROUND: Most chronic diseases are managed in primary and ambulatory care. The chronic care model (CCM) suggests a wide range of community, technological, team and patient factors contribute to effective chronic disease management. Ontologies have the capability to enable formalised linkage of heterogeneous data sources as might be found across the elements of the CCM. OBJECTIVE: To describe the evidence base for using ontologies and other semantic integration methods to support chronic disease management. METHOD: We reviewed the evidence-base for the use of ontologies and other semantic integration methods within and across the elements of the CCM. We report them using a realist review describing the context in which the mechanism was applied, and any outcome measures. RESULTS: Most evidence was descriptive with an almost complete absence of empirical research and important gaps in the evidence-base. We found some use of ontologies and semantic integration methods for community support of the medical home and for care in the community. Ubiquitous information technology (IT) and other IT tools were deployed to support self-management support, use of shared registries, health behavioural models and knowledge discovery tools to improve delivery system design. Data quality issues restricted the use of clinical data; however there was an increased use of interoperable data and health system integration. CONCLUSIONS: Ontologies and semantic integration methods are emergent with limited evidence-base for their implementation. However, they have the potential to integrate the disparate community wide data sources to provide the information necessary for effective chronic disease management.

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.

Pharmacokinetic/pharmacodynamic modeling identifies SN30000 and SN29751 as tirapazamine analogues with improved tissue penetration and hypoxic cell killing in tumors.

PURPOSE: Tirapazamine (TPZ) has attractive features for targeting hypoxic cells in tumors but has limited clinical activity, in part because of poor extravascular penetration. Here, we identify improved TPZ analogues 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 the progression of 281 TPZ analogues 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, and 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 analogues with potential for targeting tumor hypoxia in humans. Novel SR-PKPD modeling approaches can be used for lead optimization during anticancer drug development.

Nitro-chloromethylbenzindolines: hypoxia-activated prodrugs of potent adenine N3 DNA minor groove alkylators.

Hypoxia represents an important therapeutic target in tumors because of the resistance of hypoxic cells to radiotherapy and chemotherapy and because it is more severe in many tumors than in normal tissues. Here, we describe a class of prodrugs, nitro-chloromethylindolines, which undergo hypoxia-selective activation by endogenous nitroreductases in tumor cells to form the corresponding amino compounds. The latter are chemically related to the cyclopropylindoline antitumor antibiotics and they share the same properties of sequence-selective DNA minor groove alkylation and high cytotoxic potency. Of three alkylating subunits investigated, the chloromethylbenzindoline (CBI) structure provided the most favorable prodrug properties: aerobic cytotoxic potency of the amines was approximately 90- to 3,000-fold higher than the corresponding nitro compounds, and the nitro compounds showed air/anoxia potency differentials of up to 300-fold. Selective alkylation of adenine N3 in calf thymus DNA by an amino-CBI was shown by characterization of the thermal depurination product; the same adduct was shown in hypoxic RIF-1 cells exposed to the corresponding nitro-CBI prodrug under hypoxic (but not oxic) conditions. The amino metabolite generated from a nitro-CBI by cells expressing Escherichia coli nfsB nitroreductase in multicellular layer cultures was shown to elicit bystander killing of surrounding cells. Nitro-CBI prodrugs were >500-fold less toxic to mice than amino-CBIs by i.p. administration and provided selective killing of hypoxic cells in RIF-1 tumors (although only at maximally tolerated doses). Nitro-CBIs are novel lead hypoxia-activated prodrugs that represent the first examples of hypoxia-selective generation of potent DNA minor groove alkylating agents.

Extravascular transport of drugs in tumor tissue: effect of lipophilicity on diffusion of tirapazamine analogues in multicellular layer cultures.

The extravascular diffusion of antitumor agents is a key determinant of their therapeutic activity, but the relationships between physicochemical properties of drugs and their extravascular transport are poorly understood. It is well-known that drug lipophilicity plays an important role in transport across biological membranes, but the net effect of lipophilicity on transport through multiple layers of tumor cells is less clear. This study examines the influence of lipophilicity (measured as the octanol-water partition coefficient P) on the extravascular transport properties of the hypoxic cytotoxin tirapazamine (TPZ, 1) and a series of 13 neutral analogues, using multicellular layers (MCLs) of HT29 human colon carcinoma cells as an in vitro model for the extravascular compartment of tumors. Flux of drugs across MCLs was determined using diffusion chambers, with the concentration-time profile on both sides of the MCL measured by HPLC. Diffusion coefficients in the MCLs (D(MCL)) were inversely proportional to M(r)(0.5) (M(r), relative molecular weight), although this was a minor contributor to differences between compounds over the narrow M(r) range investigated. Differences in lipophilicity had a larger effect, with a sigmoidal dependence of D(MCL) on log P. Correcting for M(r) differences, lipophilic compounds (log P > 1.5) had ca. 15-fold higher D(MCL) than hydrophilic compounds (log P < -1). Using a pharmacokinetic/pharmacodynamic (PK/PD) model in which diffusion in the extravascular compartment of tumors is considered explicitly, we demonstrated that hypoxic cell kill is very sensitive to changes in extravascular diffusion coefficient of TPZ analogues within this range. This study shows that simple monosubstitution of TPZ can alter log P enough to markedly improve extravascular transport and activity against target cells, especially if rates of metabolic activation are also optimized.

DNA-targeted 1,2,4-benzotriazine 1,4-dioxides: potent analogues of the hypoxia-selective cytotoxin tirapazamine.

Tirapazamine (TPZ, 1,2,4-benzotriazin-3-amine 1,4-dioxide) is a bioreductive hypoxia-selective cytotoxin, currently in phase II/III clinical trials in combination with radiotherapy and with cisplatin-based chemotherapy. We have prepared a series of 1,2,4-benzotriazine 1,4-dioxide (BTO) analogues of TPZ where a DNA-targeting chromophore is attached at the 3-position via a flexible linker. DNA binding affinity was modified through variation of the chromophore or the pK(a) of the linker chain. The association constants (K(DNA)) for calf thymus DNA ranged from 1 x 10(2) to 5.6 x 10(5) M(-1) (ionic strength of 0.01 M). DNA binding affinity was dependent on the presence of a positive charge, either in the linker chain or in the chromophore, and (for a series of 4-acridine carboxamide chromophore analogues) correlated strongly with linker chain pK(a). The efficacy of these BTOs in killing aerobic and hypoxic mouse SCCVII tumor cells in vitro was determined by clonogenic survival. Cytotoxicity was measured as the concentration required to reduce plating efficiency to 10% of controls (C(10)), and the hypoxic cytotoxicity ratio (HCR) for each BTO was calculated as C(10)(aerobic)/C(10)(hypoxic). BTOs bearing a positive charge showed increased hypoxic cytotoxicity (1.5-56-fold) compared to TPZ and mostly modest HCRs (8-51), but some excellent (>167 and 400) values. There was a strong correlation between K(DNA) and hypoxic cytotoxicity but no correlation between K(DNA) and HCR. Cytotoxicity in HT-29 human colon carcinoma cells, determined using IC(50) assays, showed similar relationships with a correlation between K(DNA) and hypoxic cytotoxicity but no correlation between K(DNA) and HCR. In this cell line, a higher proportion of compounds (7 of 11) had HCRs greater than or equal to that of TPZ. The data confirm that DNA targeting is a useful concept for increasing potency while maintaining hypoxic selectivity and provide a direction for the further development of DNA-targeted analogues of TPZ.

Clostridium beijerinckii and Clostridium difficile detoxify methylglyoxal by a novel mechanism involving glycerol dehydrogenase.

In contrast to gram-negative bacteria, little is known about the mechanisms by which gram-positive bacteria degrade the toxic metabolic intermediate methylglyoxal (MG). Clostridium beijerinckii BR54, a Tn1545 insertion mutant of the NCIMB 8052 strain, formed cultures that contained significantly more (free) MG than wild-type cultures. Moreover, BR54 was more sensitive to growth inhibition by added MG than the wild type, suggesting that it has a reduced ability to degrade MG. The single copy of Tn1545 in this strain lies just downstream from gldA, encoding glycerol dehydrogenase. As a result of antisense RNA production, cell extracts of BR54 possess significantly less glycerol dehydrogenase activity than wild-type cell extracts (H. Liyanage, M. Young, and E. R. Kashket, J. Mol. Microbiol. Biotechnol. 2:87-93, 2000). Inactivation of gldA in both C. beijerinckii and Clostridium difficile gave rise to pinpoint colonies that could not be subcultured, indicating that glycerol dehydrogenase performs an essential function in both organisms. We propose that this role is detoxification of MG. To our knowledge, this is the first report of targeted gene disruption in the C. difficile chromosome.