Tyrosine kinase inhibitors. 15. 4-(Phenylamino)quinazoline and 4-(phenylamino)pyrido[d]pyrimidine acrylamides as irreversible inhibitors of the ATP binding site of the epidermal growth factor receptor.

A series of 6- and 7-acrylamide derivatives of the 4-(phenylamino)quinazoline and -pyridopyrimidine classes of epidermal growth factor receptor (EGFR) inhibitors were prepared from the corresponding amino compounds by reaction with either acryloyl chloride/base or acrylic acid/1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. All of the 6-acrylamides, but only the parent quinazoline 7-acrylamide, were irreversible inhibitors of the isolated enzyme, confirming that the former are better-positioned, when bound to the enzyme, to react with the critical cysteine-773. Quinazoline, pyrido[3,4-d]pyrimidine, and pyrido[3,2-d]pyrimidine 6-acrylamides were all irreversible inhibitors and showed similar high potencies in the enzyme assay (likely due to titration of the available enzyme). However the pyrido[3,2-d]pyrimidine analogues were 2-6-fold less potent than the others in a cellular autophosphorylation assay for EGFR in A431 cells. The quinazolines were generally less potent overall toward inhibition of heregulin-stimulated autophosphorylation of erbB2 (in MDA-MB-453-cells), whereas the pyridopyrimidines were equipotent. Selected compounds were evaluated in A431 epidermoid and H125 non-small-cell lung cancer human tumor xenografts. The compounds showed better activity when given orally than intraperitoneally. All showed significant tumor growth inhibition (stasis) over a dose range. The poor aqueous solubility of the compounds was a drawback, requiring formulation as fine particulate emulsions.     

Nitracrine N-oxides: effects of variations in the nature of the side chain N-oxide on hypoxia-selective cytotoxicity

The tertiary amine N-oxide (nitracrine-N-oxide, 1b) of the 1-nitroacridine nitracrine is a bis-bioreductive agent showing very high hypoxic selectivity (approximately 1000-fold) against tumour cells in culture, but only modest activity against the hypoxic subfraction of tumours in vivo. Because the hypoxic selectivity of 1b was considered to depend significantly on the rate of enzyme-mediated reduction of the N-oxide group, this paper reports the preparation and evaluation of a series of analogues in which the environment of this group was modified. Three analogues contained more weakly basic N-oxides, while two others had varying degrees of steric bulk around the N-oxide. In all but one case (an aromatic N-oxide), the N-oxides were much less cytotoxic (10- to 300-fold) than the corresponding tertiary amines towards AA8 Chinese hamster cells under aerobic conditions. Both the N-oxides and the corresponding amines were more cytotoxic to an ERCC-1 mutant defective in nucleotide excision repair, indicating that DNA alkylation was the cytotoxic event. However, there was no apparent correlation of these parameters with structure. All of the aliphatic N-oxides, with the exception of the aromatic N-oxide example, showed substantial (70- to 800-fold) hypoxic selectivity against AA8 cells in a clonogenic assay. While the weakly basic derivatives were the least selective, there was no apparent relationship between hypoxic selectivity and the steric environment of the N-oxide. Selectivity for hypoxic cells in culture is shown to depend on the hypoxic selectivity of the corresponding tertiary amine (reflecting O2-inhibitable reduction of the 1-nitro group) and the differential in aerobic toxicity between amine and N-oxide (a measure of the potential toxicity increase achievable by reducing the N-oxide). Four analogues whose structures fairly represented the range of steric and electronic modifications of the N-oxide site were evaluated against the hypoxic subfraction of cells in KHT tumours in vivo, but were inactive. These results suggest that either such modifications do not exert significant effects on N-oxide reduction, or that the rate of such reduction is not a factor limiting the in vivo activity of the parent analogue 1b.     

Bis-bioreductive agents as hypoxia-selective cytotoxins: nitracrine N-oxide.

Drugs with two reducible centers, both of which must be metabolized by oxygen-inhibitable processes for full activation ("bis-bioreductive agents"), offer potential for the development of hypoxia-selective cytotoxins with improved oxygen sensitivity. The sidechain N-oxide (1-NCO) of the (mono)bioreductive agent nitracrine (1-NC) has been synthesized and evaluated as a potential example of such an approach. The association constant for reversible DNA binding of 1-NCO was 15-fold lower than that of 1-NC, as measured by equilibrium dialysis in a low ionic strength buffer, indicating that the N-oxide has the potential to act as a less toxic pro-drug of 1-NC. Cell uptake and aerobic cytotoxicity of 1-NCO were much lower than for 1-NC whereas its hypoxic selectivity as a cytotoxin was greatly increased. In stirred suspension cultures of AA8 cells, pure (less than 0.02% 1-NC) 1-NCO was 1000-1500 times more potent under hypoxia than in 20% O2. For 1-NC the corresponding ratio was 10 +/- 1. 1-NCO had greater hypoxic selectivity in this system than misonidazole (ratio 11), RSU 1069 (ratio 25), 8Me-5NQ (ratio 60), or SR 4233 (ratio 80). Studies of 1-NCO metabolism indicate rapid, O2-inhibited reduction to 1-NC. The data are consistent with a two-step bioactivation mechanism, with reduction of the N-oxide generating a DNA intercalator of increased binding affinity, followed by reduction of the nitro group of this DNA-targeted cytotoxin to form reactive cytotoxic metabolites.     

Investigating the Effects of a Multinutrient Supplement on Cognition,Mood and Biochemical Markers in Middle-Aged Adults with ‘Optimal’ and ‘Sub-Optimal’ Diets: A Randomized Double Blind Placebo Controlled Trial

Background: Previous randomized controlled trials examining cognitive and mood effects of combination multivitamin supplements in healthy, non-clinical adults have reported mixed results. One purported explanation for this is that the dietary status of participants at the start of supplement interventions may influence the magnitude of the effect of supplementation. Methods: In this study, we evaluated the effect of a multinutrient formula containing B group vitamins, Bacopa monniera and Ginkgo biloba on memory, attention, mood and biochemical markers of nutrient status in middle-aged adults (M = 52.84 years, n = 141) with ‘optimal’ and ‘sub-optimal’ diets over 12 weeks. We hypothesised that active supplementation would differentially improve memory and attention in those with a ‘sub-optimal’ diet. Results: Mixed model, repeated measures analysis revealed that, in comparison to placebo, active treatment was associated with significant increases in B vitamin status (B1, B6, B12). Regarding behavioural outcomes there was no significant benefit to memory (F(1, 113.51) = 0.53, p = 0.470) nor attention (F(1,113.77) = 1.89, p = 0.171) in the whole cohort. Contrary to our hypothesis, there was a significant beneficial effect of supplementation on attentional performance in individuals with an ‘optimal’ diet prior to supplementation (F(1,57.25) = 4.94, p = 0.030). In the absence of a main effect of supplementation across the entire cohort, there were also a number of significant three-way interactions (treatment by time by diet group) detected in secondary outcomes including lower state anxiety and mental fatigue in those with an ‘optimal’ diet. Conclusion: These findings suggest that the cognitive benefit of B vitamin and herbal supplementation may be dependent on diet quality, supporting the concepts of ‘co-nutrient optimisation’ and interdependency of nutrients. This warrants further investigation. This study advocates characterising the diet of participants prior to supplementation as it may influence the effect of a nutraceutical intervention.     

Origin of DNA damage in ejaculated human spermatozoa

The molecular basis of many forms of male infertility is poorly defined. One area of research that has been studied intensely is the integrity of the DNA in the nucleus of mature ejaculated spermatozoa. It has been shown that, in men with abnormal sperm parameters, the DNA is more likely to possess strand breaks. However, how and why this DNA damage originates in certain males and how it may influence the genetic project of a mature spermatozoon is unknown. Two theories have been proposed to describe the origin of this DNA damage in mature spermatozoa. The first arises from studies performed in animal models and is linked to the unique manner in which mammalian sperm chromatin is packaged, while the second attributes the nuclear DNA damage in mature spermatozoa to apoptosis. One of the factors implicated in sperm apoptosis is the cell surface protein, Fas. In this review, we discuss the possible origins of DNA damage in ejaculated human spermatozoa, how these spermatozoa arrive in the ejaculate of some men, and what consequences they may have if they succeed in their genetic project.     

Evaluation of Vascular Delivery Methodologies to Enhance rAAV6-mediated Gene Transfer to Canine Striated Musculature

A growing body of research supports the development of recombinant adeno-associated viral (rAAV) vectors for delivery of gene expression cassettes to striated musculature as a method of treating severe neuromuscular conditions. However, it is unclear whether delivery protocols that achieve extensive gene transfer in mice can be adapted to produce similarly extensive gene transfer in larger mammals and ultimately patients. Consequently, we sought to investigate methodological modifications that would facilitate rAAV-mediated gene transfer to the striated musculature of canines. A simple procedure incorporating acute (i) occlusion of limb blood flow, (ii) exsanguination via compression bandage, and (iii) vector “dwell” time of <20 minutes, markedly enhanced the transduction of limb muscles, compared with a simple bolus limb infusion of vector. A complementary method whereby vector was infused into the jugular vein led to efficient transduction of cardiomyocytes and to a lesser degree the diaphragm. Together these methods can be used to achieve transgene expression in heart, diaphragm, and limb muscles of juvenile dogs using rAAV6 vectors. These results establish that rAAV-mediated gene delivery is a viable approach to achieving systemic transduction of striated musculature in mammals approaching the dimensions of newborn humans.     

Novel Linker Variants of Antileishmanial/Antitubercular 7-Substituted 2-Nitroimidazooxazines Offer Enhanced Solubility

Antitubercular 7-substituted 2-nitroimidazo[2,1-b][1,3]oxazines were previously shown to exhibit potent antileishmanial and antitrypanosomal activities, culminating in a new clinical investigational drug for visceral leishmaniasis (DNDI-0690). To offset development risks, we continued to seek further leads with divergent candidate profiles, especially analogues possessing greater aqueous solubility. Starting from an efficacious monoaryl derivative, replacement of the side chain ether linkage by novel amine, amide, and urea functionality was first explored; the former substitution was well-tolerated in vitro and in vivo but elicited marginal alterations to solubility (except through a less stable benzylamine), whereas the latter groups resulted in significant solubility improvements (up to 53-fold) but an antileishmanial potency reduction of at least 10-fold. Ultimately, we discovered that O-carbamate 66 offered a more optimal balance of increased solubility, suitable metabolic stability, excellent oral bioavailability (100%), and strong in vivo efficacy in a visceral leishmaniasis mouse model (97% parasite load reduction at 25 mg/kg).     

Comparison of hematin-targeting properties of pynacrine,an acridine analog of the benzonaphthyridine antimalarial pyronaridine

The hematin-targeting properties of pynacrine, an acridine analog of the schizontocidal antimalarial drug, pyronaridine, were evaluated to probe the role of the latter's benzonaphthyridine moiety. Pynacrine was as active as pyronaridine in inhibiting glutathione-induced hematin degradation and in enhancing hematin-mediated membrane lysis. It formed a 1:2 complex with hematin but was 50-fold less effective in inhibiting β-hematin formation. However, pynacrine was as potent as pyronaridine in inhibiting intra-erythrocytic Plasmodium falciparum growth in culture, suggesting that it has other off-target(s) effects.