Statin-induced myopathy in the rat: relationship between systemic exposure, muscle exposure and myopathy.

Rare instances of myopathy are associated with all statins, but cerivastatin was withdrawn from clinical use due to a greater incidence of myopathy. The mechanism of statin-induced myopathy with respect to tissue disposition was investigated by measuring the systemic, hepatic, and skeletal muscle exposure of cerivastatin, rosuvastatin, and simvastatin in rats before and after muscle damage. The development of myopathy was not associated with the accumulation of statins in skeletal muscle. For each statin exposure was equivalent in muscles irrespective of their fibre-type sensitivity to myopathy. The low amount of each statin in skeletal muscle relative to the liver does not support a significant role for transporters in the disposition of statins in skeletal muscle. Finally, the concentration of cerivastatin necessary to cause necrosis in skeletal muscle was considerably lower than rosuvastatin or simvastatin, supporting the concept cerivastatin is intrinsically more myotoxic than other statins.

Liposomal encapsulation enhances the antitumour efficacy of the vascular disrupting agent ZD6126 in murine B16.F10 melanoma.

Vascular disrupting agents (VDAs) are able to affect selectively tumour endothelial cell morphology resulting in vessel occlusion and widespread tumour cell necrosis. However, single-agent antitumour activity of VDAs is typically limited, as tumour regrowth occurs rapidly following drug treatment. To improve the therapeutic effectiveness of VDAs, we investigated liposomal targeting using ZD6126 as a model VDA. ZD6126 is a phosphate-prodrug of the tubulin-binding vascular disrupting agent ZD6126 phenol. ZD6126 was encapsulated into long circulating PEG-liposomes for passive targeting and PEG-liposomes conjugated with peptide ligands containing the RGD-motif for active targeting to alpha(v)-integrins on tumour endothelial cells. ZD6126 could be stably encapsulated, and liposomes displayed minimal leakage in vitro (<10% in 3 weeks). In vivo, upon intravenous injection, free ZD6126 was rapidly converted into ZD6126 phenol, which was cleared from the circulation within minutes. In contrast, ZD6126 encapsulated into either RGD-targeted or PEG liposomes showed prolonged blood circulation times (t(1/2)=10 h), and ZD6126 phenol exposure was also prolonged (t(1/2)=8 h). Both liposomal formulations displayed tumour accumulation plus hepatosplenic uptake by local macrophages. The altered pharmacokinetics and tissue distribution profiles of both liposomal ZD6126 formulations resulted both in single-dose and multiple-dose regimes, in improved therapeutic efficacy in established murine B16.F10 melanomas compared with free ZD6126. The passively and actively targeted liposomes showed equal antitumour efficacy, indicating that delivery of ZD6126 to the tumour tissue may suffice to disrupt tumour blood vessels without the need for specific targeting to the tumour endothelium.

Some aspects of rat femorotibial joint microanatomy as demonstrated by high-resolution magnetic resonance imaging.

High-resolution magnetic resonance images (MRI) of the right femorotibial joint of normal Han:Wistar rats were acquired using a 4.7 Tesla magnet and a single-turn solenoid radio frequency coil (built in-house). Some anatomical findings of the rat femorotibial joint, which have not been reported previously using MRI, are described. The separation of patellar ligament and crural fascia was feasible on MRI. This separation would not be seen on images of lower resolution and its presence on high-resolution images could be mistaken for artefact due to the magic angle effect. Band-like fibrous structures exist in the infra-patellar fat pad, which might be mistaken as ligaments within the femorotibial joint. On sagittal MRI a vessel was seen inserted on the central part of the caudal surface of the patellar ligament. Subcutaneous fascia/cutaneous muscles (panniculus carnosus) could also be demonstrated with MRI in the femorotibial joint area.

Fluid collection within the synovial sheath of the tendon of the flexor hallus longus muscle in the tarsal joint of rats: an anatomic variant detectable with magnetic resonance imaging.

Magnetic resonance (MR) images of the right tarsal joint of 22 normal male Han:Wistar rats were acquired using a 4.7 T magnet. An intermediate-high signal area associated with the tendon of the flexor hallus longus muscle was noticed in three rats on T2-weighted images. These areas appeared as an intermediate-high signal on lightly T2-weighted images, but appeared as an iso-signal to muscle structure on proton density weighted images. Histology preparations showed that such areas were caused by a sizable fluid collection within the synovial sheath of the tendon of the flexor hallus longus muscle, with all other joint structures appearing normal. This anatomic variant could be potentially regarded as a lesion on T2-weighted MR images, such as inflamed tissue with oedema, especially when the spatial resolution and/or signal-to-noise ratio are not optimal.

Metabonomics with 1H-NMR spectroscopy and liquid chromatography-mass spectrometry applied to the investigation of metabolic changes caused by gentamicin-induced nephrotoxicity in the rat.

The model nephrotoxin gentamicin was administered to male Wistar-derived rats daily, for 7 days, at 60 mg kg-1 day-1, subcutaneously, twice daily. Conventional clinical chemistry urinalysis showed a significant increase in N-acetyl-beta-D-glucosaminidase (NAG) activity from day 3. At necropsy on day 9, clear histological damage to the kidney was noted with all animals showing a generally severe nephropathy primarily focused on the proximal convoluted tubules. The urinary excretion pattern of endogenous metabolites over the time course of the study was studied using a combination of 1H-NMR spectroscopy and HPLC-TOF-MS/MS using electrospray ionization (ESI). Changes in the pattern of endogenous metabolites as a result of daily administration of gentamicin were readily detected by both techniques with significant perturbations of the urinary profile observed from day 7 onwards. The findings by 1H-NMR included raised glucose and reduced trimethylamine N-oxide (TMAO). Changes in metabonomic profiles were observed by HPLC-MS in both positive and negative ESI. The MS data showed reduced xanthurenic acid and kynurenic acid, whilst neutral loss experiments also revealed a changed pattern of sulphate conjugation on gentamicin administration.

Characterisation of the guinea pig model of osteoarthritis by in vivo three-dimensional magnetic resonance imaging.

OBJECTIVE: To characterise longitudinal changes in joint integrity and cartilage volume in vivo in the guinea pig spontaneous osteoarthritis (OA) model by magnetic resonance imaging (MRI). METHODS: Guinea pigs knee were imaged in vivo by high-resolution three-dimensional (3D) MRI between the ages of 3 and 12 months. Image analysis was performed to assess qualitative knee joint changes between 3 and 12 months (n=16) and quantitative volumetric changes of the medial tibial cartilage between 9 and 12 months (n=7). After imaging, animals were killed and knees were assessed macroscopically and histologically. RESULTS: From 3 to 6 months qualitative observation by MRI and histopathology indicated localised cartilage swelling on the medial tibial plateau. At 6 months, bone cysts had developed in the epiphysis. At 9 months, we observed by MRI and histopathology, fragmentation of the medial tibial cartilage in areas not protected by the meniscus. Cartilage degeneration had intensified at 12 months with evidence of widespread loss of cartilage throughout the tibial plateau. Segmentation of the MR cartilage images showed a 36% loss of volume between 9 and 12 months. CONCLUSIONS: We have achieved 3D image acquisition and segmentation of knee cartilage in a guinea pig model of chronic OA, which permits measurements previously only possible in man. High resolution and short acquisition time allowed qualitative longitudinal characterisation of the entire knee joint and enabled us to quantify for the first time longitudinal tibial cartilage volume loss associated with disease progression.

The synovial membrane, liver, and tongue: target organs for a ricin A-chain immunotoxin (ZD0490).

ZD0490 is an immunotoxin consisting of a mouse monoclonal antibody conjugated to recombinant ricin A-chain (rRAC). It was developed at Zeneca Pharmaceuticals as a treatment for certain antigen-bearing tumors. During safety evaluation studies in rats, a number of reversible inflammatory changes were seen. The synovial membranes of articular joints showed a marked degeneration and necrosis with an associated inflammation. When of mild severity only the synovial membrane was involved, but when more severe many adjacent tissues including the surface of the articular cartilage were affected. Some nonspecific skeletal muscle toxicity occurred. However, tongues from the intravenously (tail) dosed rats consistently showed inflammation specifically located in the ventral subepithelial area with myocyte degeneration and necrosis. Also, hepatic peliosis primarily located in the subcapsular areas was induced. Studies with rRAC alone indicated that ricin A-chain (RAC) is the component responsible for these findings. It is suggested that cells of a macrophage type with the ability to specifically bind RAC may at least in part determine the location and nature of the changes seen.

Disturbance of macrophage and monocyte function in the dog by a thromboxane receptor antagonist: ICI 185,282.

ICI 185,282 is a specific thromboxane receptor antagonist developed by ZENECA Pharmaceuticals for potential use in the treatment of inflammatory disease. During safety evaluation in dogs, multifocal granulomatous infiltrates occurred in multiple organs at high dose levels. These consisted predominantly of enlarged histiocytic cells. We reported a 28-day investigative study in which Millipore filters overlaid with carbon were implanted subcutaneously. Histological assessment of the developing foreign body granulomatous tissue response and evaluation of in vitro migration of peripheral blood monocytes were performed. The development of epithelioid macrophages with altered behavior, modification of fibroplasia, and increased monocyte infiltration at the implant site resulted from administration of ICI 185,282. This was accompanied by enhanced migration of isolated peripheral blood monocytes in vitro. We believe that the granulomatous infiltrates that occurred during toxicological assessment in dogs may be a result of a drug-induced disturbance in macrophage response to concurrent subclinical inflammations or alteration in the normal disposition of tissue macrophages, i.e., they were a result of an atypical response to a concurrent stimulus for macrophage activity.

Crescentic cataracts in Alderley Park rats.

The ophthalmoscopic appearances and the clinical evolution of a previously poorly documented form of crescentic cataract was studied in over 2,000 untreated Alderley Park rats between 4 and 110 weeks of age. In addition, 401 ex-breeder females, 54 gonadectomized males, and 56 gonadectomized females of the same strain were studied. The light microscopic features of the lenticular changes were also examined in detail. The cataracts, crescentic in shape and originating dorsally at the lens equator, were mostly unilateral. They appeared from about 30 weeks of age and affected 4% of intact males and 14% of intact females by about 2 years of age. Fewer cataracts were found in ex-breeder females, and none were found in male or female gonadectomized rats up to 2 years of age. The cataracts developed rapidly and became fully formed in 3 months. Subsequently, most cataracts scarcely progressed. Only a few extended to involve the entire lens. Some appeared to regress completely. Histologically, affected lens fibers showed swelling, globular disintegration, and liquefaction, most marked in the cortex medial to the lens bow area and extending along the direction of the lens fibers in an anterior and posterior direction but only occasionally reaching the lens capsule. The cause of these age-related lenticular changes in the Alderley Park rat is uncertain, but these changes are important during the conduct of long-term studies with this strain because their incidence can be modulated by agents that alter sex hormone status.

Neuromyoblastoma in the rat.

We report three tumours arising in the brain stem or adjacent cranial nerves of the Alderly Park rat. Light microscopy with special stains, immunocytochemistry and electron microscopy supported the conclusion that both neuronal and myoid differentiation occurred in all neoplasms. This was shown by the presence in neural cells of axon-like cell processes and in myoblastic cells of striated myofibrils. Although medulloblastomas occur in man and rats the tumours reported here were not related to the cerebellum. We have termed this tumour neuromyoblastoma.

Long-term effects of an inotropic phosphodiesterase inhibitor (ICI 153,110) on the rat salivary gland, harderian gland, and intestinal mucosa.

The inotropic vasodilator, ICI 153,110, a phosphodiesterase inhibitor intended for the treatment of congestive heart failure, was administered to Alderley Park Wistar-derived rats for periods of up to 182 days. Treatment produced hypertrophy of salivary glands, hyperplasia of intestinal mucosa, and dacryoadenitis of the harderian gland. As the functions of these glandular tissues can be modified by factors which alter cyclic nucleotide metabolism, it is postulated that the glandular alterations produced by ICI 153,110 occurred as a result of phosphodiesterase inhibition.

Pathologic changes in blood vessels following administration of an inotropic vasodilator (ICI 153,110) to the rat.

ICI 153,110 is an inotropic vasodilator compound intended for the treatment of congestive heart failure. It was administered to rats at dose levels of 5, 10, and 250 mg/kg/day for up to 6 months as part of its preclinical development program. Detailed clinical investigations were conducted during the course of the study and histopathological examination took place after 28 days and 182 days of treatment as well as 42 days following cessation of dosing. Changes were identified in blood vessels in the greater proportion of animals from the high dose group, although some of the changes were also observed at lower dose levels. Vascular tissues from a variety of sites were affected, particularly those of the mesentery, splanchnum, heart, testis, and the pampiniform plexus. Early changes characteristic of acute injury such as arterial medial necrosis and inflammation occurred, which were distinguishable from those following chronic administration of the compound where there was a pronounced arterial and venous wall thickening and accompanying plexiform vasculopathy. The essential components contributing to the thickening were a smooth muscle hypertrophy and hyperplasia of the media. At the end of the period following withdrawal of dosing, vascular thickening was still present and arteritis showed an increased incidence relative to that seen at termination of the main test. Systemic hypertension was not detected during these studies. Vasodilation occurring at or near normal blood pressure, resulting in breakdown of vascular autoregulation and excessive critical wall tension, may have been the cause of the pathological changes. Our findings indicate that medial necrosis is an early component in a sequence of adaptive, destructive, and reparative changes not only following a chemically induced perturbation of the hemodynamic status in arteries and veins but also following a shift back to the "normal state" on withdrawal of compound.

Analysis of cell cultures of 3,4-benzpyrene-treated subcutis and subsequent growth in semi-solid medium.

An in vivo-in vitro implantation model has been used to investigate further the early stages of chemically induced s.c. neoplasia in the mouse. Cell cultures of implant-site tissues from control and 3,4-benzpyrene (BP)-treated animals were found to mirror the in vivo tissue reactions occurring at the time of explantation (Westwood et al., 1979). Cells were classified into 6 different types. The most abundant cell type in later control cultures was of a typical fibroblast morphology. However, a suppression of growth of fibroblast-like cells occurred when BP-treated tissues were explanted, and a selection of growth in favour of the large polygonal Type 5 cells was observed. When grown from BP-treated tissues Type 5 cells were found to be capable of growth in a semi-solid agar medium. Quantitative studies showed that cells capable of growth in agar reached a peak about 4 weeks after implantation, followed by a decline in numbers until the formation of tumours. This observation may result from the parameters regulating the development of chemically induced neoplasia in the subcutis.

Cellular progression of neoplasia in the subcutis of mice after implantation of 3,4-benzpyrene.

An implantation model has been used to investigate the cellular progression of chemically induced subcutaneous neoplasia in the mouse. Implantation of 3,4-benzpyrene induced persistent changes in the normal process of connective tissue formation around the implant. Light-microscope and autoradiographic studies have shown a temporal progression from aberrant filter- or muscle-associated cells through proliferative foci to large invasive sarcoma. Electron microscopy revealed that presarcomatous cell foci consisted of one of two different cell types. These were either spindle cells with ultrastructural characteristics similar to foreign-body-induced sarcoma, or cells with the ultrastructural features of rhabdomyosarcoma. The subsequent appearance of two histological groups of sarcoma that were ultrastructurally similar to the cells of the early proliferative foci indicated that both elements may progress to form tumours. However, the constituent cells of both groups of tumours displayed a broad histological and ultrastructural spectrum and the marked similarity between the undifferentiated cells of each suggested that both may have arisen from diverse differentiation of a common pluripotential cell such as the pericyte.

An evaluation of 6 short-term tests for detecting organic chemical carcinogens.

A number of tests have been described which are thought to be capable of identifying carcinogens without using the actual induction of cancer as an endpoint. This study compared the performance of 6 such tests on a selection of 120 organic chemicals. The tests studies were: (1) mutation of Salmonella typhimurium; (2) cell transformation; (3) degranulation of endoplasmic reticulum; (4) sebaceous gland suppression; (5) tetrazolium reduction and (6) subcutaneous implant. A further 4 tests were examined briefly, but were not included in the complete evaluation. The chemicals were classified into carcinogens (58) and non-carcinogens (62) on the basis of published experimental data, and into 1 of 4 broad chemical classes. There was considerable variation between tests in their ability to predict carcinogenicity, with the cell-transformation test and the bacterial-mutation test being the most accurate (94% and 93% accurate respectively). These 2 tests were considered to be of general use in screening, since they were clearly more accurate than the others. Statistical consideration of various combinations of these tests showed that the use of cell transformation and bacterial mutation together, provide an advantage over the use of either test alone. The inclusion of the other 4 tests in a screening battery predictably resulted in a great increase in overall inaccuracy and loss of discrimination, even though the detection of carcinogens is improved. All the tests were shown to generate both false positive and false negative results, a situation which may be controlled by the use, where possible, of appropriate chemical-class controls, to identify the test which is optimal for the class of chemical under test. Structural analogy may have a part to play in the rapid detection of environmental carcinogens, and some general guidelines for its use are given.

Evaluation of six short term tests for detecting organic chemical carcinogens and recommendations for their use.

Six short term tests for detecting carcinogenicity have been evaluated using 120 compounds, of which half were carcinogens and the rest non-carcinogens. The results obtained indicate that the Ames test and a "cell transformation" assay are both sufficiently sensitive to carcinogenicity, or the lack of it, in the compounds studied to enable them to be employed for detecting potential carcinogens. The consequences of using short term tests under various screening conditions have been explored. In order to have confidence in the results obtained for new or previously untested compounds it is important to use such tests in a carefully controlled manner.

Stimulation of cellular ingestion by basic proteins in vitro.

The ingestion of carbon and benzpyrene particles in vitro by rat peritoneal macrophages, baby hamster kidney fibroblasts (BHK-21) and mouse L-cells has been shown to be significantly stimulated by the inclusion of histone or polylysine in the culture medium. Parallel studies using methylated bovine albumin did not significantly stimulate carbon or benzpyrene uptake relative to untreated control cultures. Incubation of carbon particles with histone before inclusion in the culture medium of macrophages resulted in the same degree of uptake as in the cultures where carbon and histone were added independently of each other. The implications of these findings to in vivo chemical carcinogenesis are examined.