The preclinical toxicology of salmeterol hydroxynaphthoate.

An extensive toxicology programme on salmeterol hydroxynaphthoate (Serevent), a marketed long-acting beta(2)-adrenoceptor agonist, has been carried out. The studies evaluated both the local (respiratory tract) and systemic tolerance to single and repeated dosing, effects on all stages of reproduction, as well as the genotoxic and oncogenic potential. High acute doses were well tolerated and caused no specific target organ toxicity. In repeat dose studies, animals tolerated salmeterol very well both locally and systemically. No significant effects on the respiratory tract of dogs were seen and only minor laryngeal changes, typical of those occurring with many inhaled medicines, were noted in rats. The high systemic concentrations achieved resulted in a number of changes that are considered to be the result of excessive and prolonged beta( 2)-adrenoceptor stimulation. These included tachycardia, skeletal muscle hypertrophy and minor haematological and blood biochemical changes in general toxicity studies, foetal effects in rabbit organogenesis studies and increased incidences of smooth muscle tumours of the mesovarium in the rat and of the uterus in the mouse oncogenicity studies. Salmeterol showed no evidence of any genotoxic potential. Results of the extensive toxicology programme provide good assurance of the safety for the inhaled use of salmeterol in patients; this has ben confirmed by many years of clinical experience during its development and marketing.

Assessment of effects of lanthanum carbonate with and without phosphate supplementation on bone mineralization in uremic rats.

AIMS: Previous studies have indicated that impaired bone mineralization in 5/6 th nephrectomized rats given high doses of lanthanum carbonate is due to phosphorus depletion caused by excessive binding to, and reduced absorption of, dietary phosphate. This study aimed to test this hypothesis by: 1) directly comparing the effects of a supratherapeutic dose of lanthanum carbonate or dietary phosphorus restriction on bone mineralization in a rodent model of chronic renal failure (CRF); and 2) investigating whether phosphorus supplementation would prevent the bone mineralization defect associated with lanthanum carbonate treatment. METHODS AND MATERIALS: Male Sprague-Dawley rats were subjected to sham surgery or a two-step 5/6th nephrectomy to induce CRF and randomized across five treatment groups: sham, CRF, CRF + dietary phosphorus deficiency, CRF + lanthanum carbonate (1000 mg/kg/ day), and CRF + lanthanum carbonate + parenteral phosphorus repletion. RESULTS: Rats with 5/6th nephrectomy had elevated serum creatinine, blood urea concentration, and urine volume and protein, consistent with impaired renal function, and increased urinary phosphorus and serum parathyroid hormone, consistent with hyperparathyroidism. Lanthanum carbonate and dietary phosphate insufficiency induced parallel changes in serum and urine markers of phosphate homeostasis and increased osteoid formation. These changes induced by lanthanum carbonate were normalized by systemic phosphate supplementation. CONCLUSIONS: These findings provide further support for the concept that supratherapeutic doses of lanthanum carbonate induce effects on bone mineralization in uremic rats via an indirect pharmacological mechanism (phosphate depletion) and not via direct bone toxicity.

Evaluation of the potential genotoxicity of the phosphate binder lanthanum carbonate.

Lanthanum was evaluated for potential genotoxicity using a range of in vitro assays (as the carbonate) in the presence and absence of post-mitochondrial fraction (S9) and in vivo in three independent tests for mutagenicity and clastogenicity (as the carbonate and chloride). The drug was devoid of mutagenic activity in bacterial assays (maximum concentration 5000 microg/plate) using a range of test strains (Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 and TA102 and Escherichia coli WP2 uvrA and WP2 uvrA pkm101). No effects were seen in the hgprt gene mutation assay in Chinese hamster ovary cells in the presence of S9. In the absence of S9, sporadic increases in revertant numbers were not dose-related or reproducible in subsequent experiments and hence were concluded to be chance events. In an in vitro chromosome aberration assay using Chinese hamster ovary cells, chromosome damage in the presence and absence of S9 (concentration 200-5000 microg/ml) was attributed to overt cell toxicity. To confirm this, a comprehensive in vivo evaluation of the drug was performed. Negative results were obtained in two independent rodent micronucleus tests. In the first mice were given oral doses (of carbonate) up to 2000 mg/kg, in the second rats were given a single i.v. bolus injection (of chloride) up to 0.1 mg/kg. Negative results were also obtained in a rat liver unscheduled DNA synthesis assay after treatment for 28 days with i.v. bolus injections (of chloride) up to 0.1 mg/kg/day. In these in vivo studies lanthanum plasma concentrations were >3000 times higher than the steady-state peak plasma concentration observed in dialysis patients given therapeutic doses of lanthanum carbonate. It can be concluded that lanthanum is not genotoxic and that lanthanum carbonate is unlikely to present a latent hazard in therapeutic use.

Metabonomic investigations into hydrazine toxicity in the rat.

The systemic biochemical effects of oral hydrazine administration (dosed at 75, 90, and 120 mg/kg) have been investigated in male Han Wistar rats using metabonomic analysis of (1)H NMR spectra of urine and plasma, conventional clinical chemistry, and liver histopathology. Plasma samples were collected both pre- and 24 h postdose, while urine was collected predose and daily over a 7 day postdose period. (1)H NMR spectra of the biofluids were analyzed visually and via pattern recognition using principal component analysis. The latter showed that there was a dose-dependent biochemical effect of hydrazine treatment on the levels of a range of low molecular weight compounds in urine and plasma, which was correlated with the severity of the hydrazine induced liver lesions. In plasma, increases in the levels of free glycine, alanine, isoleucine, valine, lysine, arginine, tyrosine, citrulline, 3-D-hydroxybutyrate, creatine, histidine, and threonine were observed. Urinary excretion of hippurate, citrate, succinate, 2-oxoglutarate, trimethylamine-N-oxide, fumarate and creatinine were decreased following hydrazine dosing, whereas taurine, creatine, threonine, N-methylnicotinic acid, tyrosine, beta-alanine, citrulline, Nalpha-acetylcitrulline and argininosuccinate excretion was increased. Moreover, the most notable effect was the appearance in urine and plasma of 2-aminoadipate, which has previously been shown to lead to neurological effects in rats. High urinary levels of 2-aminoadipate may explain the hitherto poorly understood neurological effects of hydrazine. Metabonomic analysis of high-resolution (1)H NMR spectra of biofluids has provided a means of monitoring the progression of toxicity and recovery, while also allowing the identification of novel biomarkers of development and regression of the lesion.

Investigation of the metabolite variation in control rat urine using (1)H NMR spectroscopy.

An exploratory statistical analysis has been undertaken of 640 (1)H NMR spectra of rat urine, obtained from predose and control animals during the course of eight separate toxicology studies. The aim was to determine the degree and type of variation between (1)H NMR spectra from such control animals and to investigate the variations in the spectral descriptors based on averaged peak intensities. The results showed that many of the spectral descriptors had skew and/or multimodal distributions, and that it was possible to distinguish between samples of urine collected at different times of day with a success rate of (89%) and to classify 90% of the predose spectra into their correct study group using principal component and linear discriminant analyses. The results show that successful classification can be achieved of NMR spectra of control rat urine, which exhibit more subtle changes than those previously reported when treated and control animals were compared. The results presented here suggest that it will be possible to identify very subtle toxicological changes if care is taken to standardize the experimental conditions used during toxicity screens.

Prospects for reducing and refining the use of dogs in the regulatory toxicity testing of pharmaceuticals.

A workshop was held to critically discuss the need for a nonrodent species and the role of the dog in regulatory toxicity testing of pharmaceuticals; to discuss opportunities to reduce and refine the use of dogs in preclinical toxicology; and to identify a number of specific recommendations which could be feasibly achieved to move the process forward. To facilitate a preliminary evaluation of the contribution of dog studies to the risk assessment process, anonymised, unpublished data were provided from fully evaluated, repeat-dose toxicity studies in the rat and dog. Results of the International Life Sciences Institute (ILSI) Human Toxicity Project were also presented and discussed. Analysis of the data demonstrated that the dog can provide additional toxicity information, which, in some cases, was shown to be predictive for humans. Discussions indicated that there is potential for achieving a reduction in dog use and several possible approaches were identified. To further the progress of this initiative, there is a need to collate the results of pharmacology, toxicology, and clinical studies to address some of the proposed approaches. One of the outcomes of the workshop will be the establishment of a steering group to co-ordinate data collation for further analysis.

Chemometric models for toxicity classification based on NMR spectra of biofluids.

1H NMR spectroscopic and pattern recognition (PR)-based methods were used to investigate the biochemical variability in urine obtained from control rats and from rats treated with a hydrazine (a model hepatotoxin) or HgCl(2) (a model renal cortical toxin). The 600 MHz (1)H NMR spectra of urine samples obtained from vehicle- or toxin-treated Han-Wistar (HW) and Sprague-Dawley (SD) rats were acquired, and principal components analysis (PCA) and soft independent modeling of class analogy (SIMCA) analysis were used to investigate the (1)H NMR spectral data. Variation and strain differences in the biochemical composition of control urine samples were assessed. Control urine (1)H NMR spectra obtained from the two rat strains appeared visually similar. However, chemometric analysis of the control urine spectra indicated that HW rat urine contained relatively higher concentrations of lactate, acetate, and taurine and lower concentrations of hippurate than SD rat urine. Having established the extent of biochemical variation in the two populations of control rats, PCA was used to evaluate the metabolic effects of hydrazine and HgCl(2) toxicity. Urinary biomarkers of each class of toxicity were elucidated from the PC loadings and included organic acids, amino acids, and sugars in the case of mercury, while levels of taurine, beta-alanine, creatine, and 2-aminoadipate were elevated after hydrazine treatment. SIMCA analysis of the data was used to build predictive models (from a training set of 416 samples) for the classification of toxicity type and strain of rat, and the models were tested using an independent set of urine samples (n = 124). Using models constructed from the first three PCs, 98% of the test samples were correctly classified as originating from control, hydrazine-treated, or HgCl(2)-treated rats. Furthermore, this method was sensitive enough to predict the correct strain of the control samples for 79% of the data, based upon the class of best fit. Incorporation of these chemometric methods into automated NMR-based metabonomics analysis will enable on-line toxicological assessment of biofluids and will provide a tool for probing the mechanistic basis of organ toxicity.

Development of a model for classification of toxin-induced lesions using 1H NMR spectroscopy of urine combined with pattern recognition.

Pattern recognition approaches were developed and applied to the classification of 600 MHz 1H NMR spectra of urine from rats dosed with compounds that induced organ-specific damage in either the liver or kidney. Male rats were separated into groups (n = 5) and each treated with one of the following compounds; adriamycin, allyl alcohol, 2-bromoethanamine hydrobromide, hexachlorobutadiene, hydrazine, lead acetate, mercury II chloride, puromycin aminonucleoside, sodium chromate, thioacetamide, 1,1,2-trichloro-3,3,3-trifluoro-1-propene or dose vehicle. Urine samples were collected over a 7 day time-course and analysed using 600 MHz 1H NMR spectroscopy. Each NMR spectrum was data-reduced to provide 256 intensity-related descriptors of the spectra. Data corresponding to the periods 8-24 h, 24-32 h and 32-56 h post-dose were first analysed using principal components analysis (PCA). In addition, samples obtained 120-144 h following the administration of adriamycin and puromycin were included in the analysis in order to compensate for the late onset of glomerular toxicity. Having established that toxin-related clustering behaviour could be detected in the first three principal components (PCs), three-quarters of the data were used to construct a soft independent modelling of class analogy (SIMCA) model. The remainder of the data were used as a test set of the model. Only three out of 61 samples in the test set were misclassified. Finally as a further test of the model, data from the 1H NMR spectra of urine from rats that had been treated with uranyl nitrate were used. Successful prediction of the toxicity type of the compound was achieved based on NMR urinalysis data confirming the robust nature of the derived model.