Bioorganometallic fulvene-derived titanocene anti-cancer drugs

A review. 6-Substituted fulvenes are interesting and easily accessible starting materials for the synthesis of novel substituted titanocenes via reductive dimerization, carbolithiation or hydridolithiation reactions, which are followed by a transmetallation reaction with titanium tetrachloride in the latter two cases. Depending on the substitution pattern, these titanocenes prove to be bioorganometallic anticancer drugs, which have significant potential against advanced or metastatic renal-cell cancer. Patients bearing these stages of kidney cancer have a poor prognosis so far and therefore real progress in the area of metal-based anticancer drugs may come from this simple and effective synthetic approach. This tutorial review provides an insight into the synthesis of fulvene-derived titanocenes and their activity in preclin. expts.

Proliferative and antiproliferative effects in substituted titanocene anticancer drugs

Substituted titanocenes like ansa-titanocenes, diarylmethyl-substituted and benzyl-substituted titanocenes, are known for their cytotoxic potential and they can be synthesised using 6-arylfulvenes. Nevertheless, in the case of using 6-(4-morpholin-4yl-phenyl) fulvene (5a) or 6-{[bis-(2-methoxyethyl)amino]phenyl} fulvene (5b) the synthetic possibilities seem to be limited, but the morpholino and the bis-(2-methoxyethyl)amino substituent are in terms of an improved water solubility and drug availability in the cell very interesting groups. The corresponding benzaldehydes, which are the starting material for the synthesis of these fulvenes, were not commercially available and therefore, a modified synthetic approach had to be introduced. Nevertheless, the reactivity of the obtained fulvenes was unexpected and only the ansa-titanocene bis-[{[bis-(2-methoxyethyl)amino]phenyl}cyclopentadienyl] titanium(IV) dichloride (6b) and the benzyl-substituted titanocene [1,2-di(cyclopentadienyl)-1,2-di(4-morpholin-4yl-phenyl)-ethanediyl] titanium dichloride (8a) could be obtained and characterised. When the benzyl-substituted titanocene (8a) was tested against pig kidney cells (LLC-PK) an anti-proliferative effect, resulting in an IC50 value of 25 mu M, was observed. This IC50 value is in the lower range of the cytotoxicities evaluated for titanocenes up to now. The ansa-titanocene (6b) showed surprisingly, when tested on the same cell line, a proliferative effect.

Inhibition of synaptic transmission and G protein modulation by synthetic CaV2.2 Ca2+ channel peptides

Abstract: Modulation of presynaptic voltage-dependent Ca+ channels is a major means of controlling neurotransmitter release. The CaV 2.2 Ca2+ channel subunit contains several inhibitory interaction sites for Gβγ subunits, including the amino terminal (NT) and I–II loop. The NT and I–II loop have also been proposed to undergo a G protein-gated inhibitory interaction, whilst the NT itself has also been proposed to suppress CaV 2 channel activity. Here, we investigate the effects of an amino terminal (CaV 2.2[45–55]) ‘NT peptide’ and a I–II loop alpha interaction domain (CaV 2.2[377–393]) ‘AID peptide’ on synaptic transmission, Ca2+ channel activity and G protein modulation in superior cervical ganglion neurones (SCGNs). Presynaptic injection of NT or AID peptide into SCGN synapses inhibited synaptic transmission and also attenuated noradrenaline-induced G protein modulation. In isolated SCGNs, NT and AID peptides reduced whole-cell Ca2+ current amplitude, modified voltage dependence of Ca2+ channel activation and attenuated noradrenaline-induced G protein modulation. Co-application of NT and AID peptide negated inhibitory actions. Together, these data favour direct peptide interaction with presynaptic Ca2+ channels, with effects on current amplitude and gating representing likely mechanisms responsible for inhibition of synaptic transmission. Mutations to residues reported as determinants of Ca2+ channel function within the NT peptide negated inhibitory effects on synaptic transmission, Ca2+ current amplitude and gating and G protein modulation. A mutation within the proposed QXXER motif for G protein modulation did not abolish inhibitory effects of the AID peptide. This study suggests that the CaV 2.2 amino terminal and I–II loop contribute molecular determinants for Ca2+ channel function; the data favour a direct interaction of peptides with Ca2+ channels to inhibit synaptic transmission and attenuate G protein modulation. Non-technical summary: Nerve cells (neurones) in the body communicate with each other by releasing chemicals (neurotransmitters) which act on proteins called receptors. An important group of receptors (called G protein coupled receptors, GPCRs) regulate the release of neurotransmitters by an action on the ion channels that let calcium into the cell. Here, we show for the first time that small peptides based on specific regions of calcium ion channels involved in GPCR signalling can themselves inhibit nerve cell communication. We show that these peptides act directly on calcium channels to make them more difficult to open and thus reduce calcium influx into native neurones. These peptides also reduce GPCR-mediated signalling. This work is important in increasing our knowledge about modulation of the calcium ion channel protein; such knowledge may help in the development of drugs to prevent signalling in pathways such as those involved in pain perception.

Assembly of an injectable noncytotoxic peptide-based hydrogelator for sustained release of drugs

A new synthetic tripeptide-based hydrogel has been discovered at physiological pH and temperature. This hydrogel has been thoroughly characterized using different techniques including field emission scanning electron microscopic (FESEM) and high-resolution transmission electron microscopic (HR-TEM) imaging, small- and wide-angle X-ray diffraction analyses, FT-IR, circular dichroism, and rheometric analyses. Moreover, this gel exhibits thixotropy and injectability. This hydrogel has been used for entrapment and sustained release of an antibiotic vancomycin and vitamin B12 at physiological pH and temperature for about 2 days. Interestingly, MTT assay of these gelator molecules shows almost 100% cell viability of this peptide gelator, indicating its noncytotoxicity.

Diffusion of the synthetic pyrethroid permethrin into bed-sediments

Bed-sediments are a sink for many micro-organic contaminants in aquatic environments. The impact of toxic contaminants on benthic fauna often depends on their spatial distribution, and the fate of the parent compounds and their metabolites. The distribution of a synthetic pyrethroid, permethrin, a compound known to be toxic to aquatic invertebrates, was studied using river bed-sediments in lotic flume channels. trans/cis-Permethrin diagnostic ratios were used to quantify the photoisomerization of the trans isomer in water. Rates were affected by the presence of sediment particles and colloids when compared to distilled water alone. Two experiments in dark/light conditions with replicate channels were undertaken using natural sediment, previously contaminated with permethrin, to examine the effect of the growth of an algal biofilm at the sediment-water interface on diffusive fluxes of permethrin into the sediment. After 42 days, the bulk water was removed, allowing a fine sectioning of the sediment bed (i.e., every mm down to 5 mm and then 5-10 mm, then every 10 mm down to 50 mm). Permethrin was detected in all cases down to a depth of 5-10 mm, in agreement with estimates by the Millington and Quirk model, and measurements of concentrations in pore water produced a distribution coefficient (K-d) for each section, High K-d's were observed for the top layers, mainly as a result of high organic matter and specific surface area. Concentrations in the algal biofilm measured at the end of the experiment under light conditions, and increases in concentration in the top 1 mm of the sediment, demonstrated that algal/bacterial biofilm material was responsible for high K-d's at the sediment surface, and for the retardation of permethrin diffusion. This specific partition of permethrin to fine sediment particles and algae may enhance its threat to benthic invertebrates. In addition,the analysis of trans/cis-permethrin isomer ratios in sediment showed greater losses of trans-permethrin in the experiment under light conditions, which may have also resulted from enhanced biological activity at the sediment surface.

Proliferative and anti-proliferative effects of titanium- and iron-based metallocene anti-cancer drugs

In previous work we have found that Cp2TiCl2 and its corresponding deriv. of tamoxifen, Titanocene tamoxifen, show an unexpected proliferative effect on hormone dependent breast cancer cells MCF-7. In order to check if this behavior is a general trend for titanocene derivs. we have tested two other titanocene derivs., Titanocene Y and Titanocene K, on this cell line. Interestingly, these two titanocene complexes behave in a totally different manner. Titanocene K is highly proliferative on MCF-7 cells even at low concns. (0.5 .mu.M), thus behave almost similarly to Cp2TiCl2. This proliferative effect is also obsd. in the presence of bovine serum albumin (BSA). In contrast, Titanocene Y alone has almost no effect on MCF-7 at a concn. of 10 .mu.M, but exhibits a significant dose dependent cytotoxic effect of up to 50% when incubated with BSA (20-50 .mu.g/mL). This confirms the crucial role played by the binding to serum proteins in the expression of the in vivo, cytotoxicity of the titanocene complexes. From the hydridolithiation reaction of 6-p-anisylfulvene with LiBEt3H followed by transmetallation with iron dichloride [bis-[(p-methoxy-benzyl)cyclopentadienyl]iron(II)] (Ferrocene Y) was synthesized. This complex, which was characterized by single crystal X-ray diffraction, contains the robust ferrocenyl unit instead of Ti assocd. with easily leaving groups such as chlorine and shows only a modest cytotoxicity against MCF-7 or MDA-MB-231 cells.

Statistical methods for examining genetic influences of resistance to anti-epileptic drugs

It is generally accepted that genetics may be an important factor in explaining the variation between patients’ responses to certain drugs. However, identification and confirmation of the responsible genetic variants is proving to be a challenge in many cases. A number of difficulties that maybe encountered in pursuit of these variants, such as non-replication of a true effect, population structure and selection bias, can be mitigated or at least reduced by appropriate statistical methodology. Another major statistical challenge facing pharmacogenetics studies is trying to detect possibly small polygenic effects using large volumes of genetic data, while controlling the number of false positive signals. Here we review statistical design and analysis options available for investigations of genetic resistance to anti-epileptic drugs.

Sequential genome-wide association studies for monitoring adverse events in the clinical evaluation of new drugs

Pharmacovigilance, the monitoring of adverse events (AEs), is an integral part in the clinical evaluation of a new drug. Until recently, attempts to relate the incidence of AEs to putative causes have been restricted to the evaluation of simple demographic and environmental factors. The advent of large-scale genotyping, however, provides an opportunity to look for associations between AEs and genetic markers, such as single nucleotides polymorphisms (SNPs). It is envisaged that a very large number of SNPs, possibly over 500 000, will be used in pharmacovigilance in an attempt to identify any genetic difference between patients who have experienced an AE and those who have not. We propose a sequential genome-wide association test for analysing AEs as they arise, allowing evidence-based decision-making at the earliest opportunity. This gives us the capability of quickly establishing whether there is a group of patients at high-risk of an AE based upon their DNA. Our method provides a valid test which takes account of linkage disequilibrium and allows for the sequential nature of the procedure. The method is more powerful than using a correction, such as idák, that assumes that the tests are independent. Copyright © 2006 John Wiley & Sons, Ltd.

The comparative toxicity to soil invertebrates of natural chemicals and their synthetic analogues

The introduction of Registration, Evaluation and Authorisation of Chemicals (REACH), requires companies to register and risk assess all substances produced or imported in volumes of >1 tonne per year. Extrapolation methods which use existing data for estimating the effects of chemicals are attractive to industry, and comparative data are therefore increasingly in demand. Data on natural toxic chemicals could be used for extrapolation methods Such as read-across. To test this hypothesis, the toxicity of natural chemicals and their synthetic analogues were compared using standardised toxicity tests. Two chemical pairs: the napthoquinones, juglone (natural) and 1,4-naphthoquinone (synthetic); and anthraquinones, emodin (natural) and quinizarin (synthetic) were chosen, and their comparative effects on the survival and reproduction of collembolans, earthworms, enchytraeids and predatory mites were assessed. Differences in sensitivity between the species were observed with the predatory mite (Hypoaspis aculeifer) showing the least sensitivity. Within the chemical pairs, toxicity to lethal and sub-lethal endpoints was very similar for the four invertebrate species. The exception was earthworm reproduction, which showed differential sensitivity to the chemicals in both naphthoquinone and anthraquinone pairs. Differences in toxicity identified in the present study may be related to degree of exposure and/or subtle differences in the mode of toxic action for the chemicals and species tested. It may be possible to predict differences by identifying functional groups which infer increased or decreased toxicity in one or other chemical. The development of such techniques would enable the use of read-across from natural to synthetic chemicals for a wider group of compounds. (C) 2009 Elsevier Ltd. All rights reserved.

Fitting models for the joint action of two drugs using SAS(R)

Combinations of drugs are increasingly being used for a wide variety of diseases and conditions. A pre-clinical study may allow the investigation of the response at a large number of dose combinations. In determining the response to a drug combination, interest may lie in seeking evidence of synergism, in which the joint action is greater than the actions of the individual drugs, or of antagonism, in which it is less. Two well-known response surface models representing no interaction are Loewe additivity and Bliss independence, and Loewe or Bliss synergism or antagonism is defined relative to these. We illustrate an approach to fitting these models for the case in which the marginal single drug dose-response relationships are represented by four-parameter logistic curves with common upper and lower limits, and where the response variable is normally distributed with a common variance about the dose-response curve. When the dose-response curves are not parallel, the relative potency of the two drugs varies according to the magnitude of the desired effect and the models for Loewe additivity and synergism/antagonism cannot be explicitly expressed. We present an iterative approach to fitting these models without the assumption of parallel dose-response curves. A goodness-of-fit test based on residuals is also described. Implementation using the SAS NLIN procedure is illustrated using data from a pre-clinical study. Copyright © 2007 John Wiley & Sons, Ltd.

Hypotensive effects of hawthorn for patients with diabetes taking prescription drugs: a randomised controlled trial

Background Hawthorn (Crataegus laevigata) leaves, flowers and berries are used by herbal practitioners in the UK to treat hypertension in conjunction with prescribed drugs. Small-scale human studies support this approach. Aim To investigate the effects of hawthorn for hypertension in patients with type 2 diabetes taking prescribed drugs. Design of study Randomised controlled trial. Setting General practices in Reading, UK. Method Patients with type 2 diabetes (n = 79) were randomised to daily 1200 mg hawthorn extract (n = 39) or placebo (n = 40) for 16 weeks. At baseline and outcome a wellbeing questionnaire was completed and blood pressure and fasting blood samples taken. A food frequency questionnaire estimated nutrient intake. Results Hypotensive drugs were used by 71% of the study population with a mean intake of 4.4 hypoglycaemic and/or hypotensive drugs. Fat intake was lower and sugar intake higher than recommendations, and low micronutrient intake was prevalent. There was a significant group difference in mean diastolic blood pressure reductions (P = 0.035): the hawthorn group showed greater reductions (baseline: 85.6 mmHg, 95% confidence interval [Cl] = 83.3 to 87.8; outcome: 83.0 mmHg, 95% Cl = 80.5 to 85.7) than the placebo group (baseline: 84.5 mmHg, 95% Cl = 82 to 87; outcome: 85.0 mmHg, 95% Cl = 82.2 to 87.8). There was no group difference in systolic blood pressure reduction from baseline (3.6 and 0.8 mmHg for hawthorn and placebo groups, respectively; P = 0.329). Although mean fat intake met current recommendations, mean sugar intake was higher and there were indications of potential multiple micronutrient deficiencies. No herb-drug interaction was found and minor health complaints were reduced from baseline in both groups. Conclusions This is the first randomised controlled trial to demonstrate a hypotensive effect of hawthorn in patients with diabetes taking medication.