Integrins on joint cartilage chondrocytes and alterations by ofloxacin or magnesium deficiency in immature rats

 Recently, we showed that magnesium deficiency induces lesions in knee joint cartilage from 5-week-old rats that are very similar to ofloxacin-induced cartilage defects. We concluded that quinolone-induced arthropathy is probably due to chelation of magnesium and thus a deficit in functionally available magnesium in joint cartilage (Stahlmann et al. 1995). As magnesium deficiency in joint cartilage could impair chondrocyte-matrix interaction which is mediated by cation-dependent integrin receptors of the β₁-subfamily, we investigated integrin expression in joint cartilage from untreated, ofloxacin-treated and magnesium-deficient Wistar rats. With immunohistochemical methods using monoclonal and polyclonal antibodies, we showed that the integrin pattern in joint cartilage from rats corresponded largely to integrin expression described for human cartilage tissue: β₁, α₁, α₃ and α_ν subunits and the α₅β₁ and α_νβ₃ heterodimers were consistently expressed. Joint cartilage lesions were detected in ofloxacin-treated and magnesium-deficient rats. Lesions were more pronounced in the quinolone-treated group. Expression of several integrins was reduced in the vicinity of lesions after oral treatment with 2 × 600 mg ofloxacin/kg for 1 day. Gross-structural lesions (e. g., cleft formation, unmasked collagen fibres) in magnesium-deficient rats were very similar but changes in integrin expression were less pronounced. On the other hand, changes in cartilage matrix composition showed similar alterations in ofloxacin-treated and magnesium-deficient rats: fibronectin deposition in the cartilage matrix increased in both groups while glycosaminoglycan content decreased. In summary, similar defects occur in ofloxacin-treated and magnesium-deficient rats and with immunohistochemical methods subtle differences are demonstrable. Received: 8 June 1995 / Accepted: 21 August 1995     

In vitro evidence for a Donnan distribution of Mg 2+ and Ca2+ by chondroitin sulphate in cartilage

Fluoroquinolones are known for their ability to form chelate complexes with magnesium. Cartilage lesions observed in juvenile animals after quinolone treatment very probably are a consequence of the lack of functionally available magnesium. In cartilage, which contains high amounts of negatively charged proteoglycans, a Donnan distribution can be expected leading to an inhomogeneous distribution of ions (such as magnesium), which may support the toxic effects of magnesium deficiency or quinolone treatment of cartilage. We performed in vitro experiments using dialysis tubes to simulate the unequal distribution of proteoglycans in cartilage and measured the distribution of magnesium, calcium and ofloxacin. We found that the concentration of free magnesium is significantly reduced within the chondroitin sulphate-free solution due to a Donnan effect. For example, using a 3% chondroitin sulphate solution (outside the tubing) dialysed against a chondroitin sulphate-free solution (inside the tubing) the magnesium concentration decreased by 24% from 0.55 ± 0.02 to 0.42 ± 0.04 mmol/l inside the tubing during 48 h observation (P < 0.01). Under physiological conditions this unequal distribution of magnesium probably will be much more pronounced because chondroitin sulphate concentrations in cartilage are higher; nevertheless, magnesium concentration is sufficient for regular function of the tissue. During the sensitive phase of quinolone toxicity, magnesium in juvenile cartilage is lower than at other time points during postnatal development. Moreover, additional complexation by quinolones may further reduce the concentration of functionally available magnesium below the critical level. Received: 5 November 1996 / Accepted: 21 January 1997     

Ofloxacin in juvenile non-human primates and rats. Arthropathia and drug plasma concentrations

Arthropathia in juvenile animals is the most important toxic effect induced by quinolones. We conducted pharmacokinetic and morphological studies with ofloxacin on non-human primates (Callithrix jacchus, Marmosets) and rats. In the marmoset, electron microscopy and the application of immuno-morphological methods proved to be suitable for the detection of specific alterations in cartilage (e. g. loss of proteoglycanes and altered chondrocytes). Subsequently performed electron microscopic examinations in rats showed similar specific alterations of the femur cartilage surface after multiple oral applications of 600 mg ofloxacin/kg body wt. These results were correlated with pharmacokinetic data obtained for the same species. After single oral application of 100, 300 or 600 mg ofloxacin/kg body wt to 5 week-old rats peak plasma levels were achieved 15–45 min after administration indicating a rapid absorption of the drug. The following peak concentrations were measured for the three doses applied (mean±SD): 8.9±2.1, 22.6±7.5 mg/l and 33.5±9.8 mg/l, respectively. After 360 min the concentrations were 1.1±0.4, 5.9±2.5 and 15.9±5.1 mg/l, respectively. After subcutaneous injection of 100 mg ofloxacin/kg body wt the mean peak concentration was 27.7±2.6 mg/l after 45 min (0.5±0.2 mg/l after 360 min). In the marmoset higher plasma concentrations were measured with comparable doses. One, 3, and 6 h after the last of nine administrations of 200 mg ofloxacin/kg body wt, the mean (±SD) plasma concentrations were: 42.7±16.7, 40.6±9.5, and 26.5±3.6 mg ofloxacin/l plasma. Typical alterations of the joint cartilage of juvenile rats (e. g. opened chondrocyte cavities, swelling of rough endoplasmic reticulum and mitochondrial swelling in the chondrocytes) were induced by oral administration of ofloxacin at doses that were approximately 100 times higher than therapeutic ones, but led to peak plasma concentrations which were only approximately 10 times above the therapeutic level. Since we found corresponding cartilage alterations in marmosets and rats, this species provides a convenient model for additional studies on chondrotoxic effects of quinolones.     

Quinolone-induced cartilage lesions are not reversible in rats

 The reversibility of quinolone-induced cartilage lesions has not been studied in detail. We treated five groups of five to seven juvenile Wistar rats (male and female; age: 5 weeks) with 2×600 mg ofloxacin/kg by gastric intubation on 1 day only (9:00 a.m. and 5:00 p.m.) and studied the knee joints histologically 3 days, 1, 3, 8 and 17 weeks later. In addition, joint cartilage specimens from vehicle-treated control rats (n=21) at corresponding age were examined. Cartilage lesions such as matrix swelling, loss of proteoglycans and horizontal clefts were found in nearly all knee joints (26 of 27 joints; incidence: 96%) of the ofloxacin-treated rats. Within the observation period of 4 months the size of these lesions in knee joint cartilage did not decrease significantly. The diameter of the lesions at the time points of evaluation was 1146±535, 1713±309, 1250 ±585, 1406±356, and 1542±467 μm, respectively (mean values±sd). Chondrocyte clusters producing glycosaminoglycans were observed 3 weeks after dosing and at later time points. They are considered to reflect the onset of repair but chondrocyte organization did not normalize during the study period, thus indicating the irreversibility of the effect under the experimental conditions. In principle, long-term joint cartilage damage has to be taken into account when the use of quinolones in children is considered. More detailed pharmacokinetic data are necessary for a reasonable risk assessment approach. Received: 28 August 1995/Accepted: 14 November 1995     

Effects of fluoroquinolones and magnesium deficiency in murine limb bud cultures

Quinolone-induced arthropathy is probably caused by a lack of functionally available magnesium in immature joint cartilage. We used an in vitro assay to study the effects of fluoroquinolones on cartilage formation in mouse limb buds from 12-day-old mouse embryos in regular and in magnesium-deficient medium. Omission of magnesium from the medium had no adverse effect on the outcome of the culture: limb buds grew and differentiated well in regular and in magnesium-deficient Bigger's medium. Lack of calcium, however, severely impaired the development of the explants; this result was even more enhanced when both minerals (magnesium and calcium) were omitted. Electron microscopy revealed cell necrosis and deposition of electron-dense material in the vicinity of chondrocytes from limb buds after 6 days in a magnesium-free medium. A series of seven fluoroquinolones was tested at 30, 60, and 100 mg/l medium. At a concentration of 30 mg/l sparfloxacin only had a slight effect on limb development. At concentrations of 60 and 100 mg/l sparfloxacin, temafloxacin and ciprofloxacin impaired limb development in␣vitro concentration-dependently. The effects were enhanced in a magnesium-deficient medium (concentration of magnesium <10 μmol/l). Fleroxacin, lomefloxacin and ofloxacin impaired limb development only slightly; no significant differences were recognizable between the outcome in regular and in magnesium-deficient medium. Pefloxacin did not show any effect on limb development in both media. Using electron microscopy, very similar alterations as described above for the limbs cultured in magnesium-deficient medium were observed with ofloxacin at a concentration of 30 mg/l, which had no effect on the growth of the explants when evaluated macroscopically. The affinity of six fluoroquinolones to magnesium was determined by the use of a fluorescence assay. The affinity to magnesium correlated with the activity of the drugs in the limb bud assay. We conclude that fluoroquinolones have no effect on murine limb development in vitro at concentrations that are achieved under therapeutic conditions (peak concentrations approx. 1–5 mg/l in plasma). Effects at higher concentrations (60 and 100 mg/l) are slightly enhanced (factor 2) if the magnesium concentration in the medium is low. Macroscopically, limbs develop regularly in a magnesium-free medium, but ultrastructurally typical alterations are exhibited (e.g. cell necrosis and pericellular deposition of electron-dense material). Received: 7 October 1997 / Accepted: 25 February 1998     

Serum and urinary boron levels in rats after single administration of sodium tetraborate

The pharmacokinetics of boron was studied in rats by administering a 1 ml oral dose of sodium tetraborate solution to several groups of rats (n=20) at eleven different dose levels ranging from 0 to 0.4 mg/100 g body weight as boron. Twenty-four-hour urine samples were collected after boron administration. After 24 h the average urinary recovery rate for this element was 99.6 ± 7.9. The relationship between boron dose and excretion was linear (r=0.999) with a regression coefficient of 0.954. This result suggests that the oral bioavailability (F) of boron was complete. Another group of rats (n=10) was given a single oral injection of 2 ml of sodium tetraborate solution containing 0.4 mg of boron/100 g body wt. The serum decay of boron was followed and found to be monophasic. The data were interpreted according to a one-compartment open model. The appropriate pharmacokinetic parameters were estimated as follows: absorption half-life, t _1/2a=0.608±0.432 h; elimination half-life, t _1/2=4.64±1.19 h; volume of distribution, Vd=142.0±30.2 ml/100 g body wt.; total clearance, C _tot=0.359 ± 0.0285 ml/min per 100 g body wt. The maximum boron concentration in serum after administration (C _max) was 2.13 ± 0.270 mg/l, and the time needed to reach this maximum concentration (T _max) was 1.76 ± 0.887 h. Our results suggest that orally administered boric acid is rapidly and completely absorbed from the gastrointestinal tract into the blood stream. Boric acid in the intravascular space does not have a strong affinity to serum proteins, and rapidly diffuses to the extravascular space in proportion to blood flow without massive accumulation or binding in tissues. The main route of boron excretion from the body is via glomerular filtration. It may be inferred that there is partial tubular resorption at low plasma levels. The animal model is proposed as a useful tool to approach the problem of environmental or industrial exposure to boron or in cases of accidental acute boron intoxication. Received: 1 December 1997 / Accepted: 24 March 1998     

Toxicokinetics of p-tert-octylphenol in female DA/Han rats after single i.v. and oral application

Female DA/Han rats were administered p-tert-octylphenol [OP; p-(1,1,3,3-tetramethylbutyl)-phenol], either intravenously (5 mg/kg body wt.) or orally by gavage (50 or 200 mg/kg body wt.). After i.v. administration the blood concentration-time curve of OP was fitted to a tri-exponential model, resulting in a final half-life (γ-phase) of 36.1 h. This contrasts to much more rapid eliminations previously reported in male Wistar rats. The oral bioavailability of 50 mg/kg OP was 12.3% and of 200 mg/kg 8.4%. The higher dose (200 mg/kg) was absorbed slower than the smaller dose, probably due to low solubility of OP in aqueous media. Maximal OP blood levels in female DA/Han rats receiving 50 and 200 mg OP/kg body wt. were 4.5 and 3 times higher than previously reported in male Wistar rats. The blood concentration-time curves after oral administration of OP to female DA/Han rats revealed pronounced interindividual differences, indicating extensive enterohepatic circulation of OP in this rat strain. In contrast to male Wistar rats, after application of high doses of OP to female DA/Han rats the compound was not completely eliminated within 48 h; under these conditions some bioaccumulation might therefore occur. The experimental toxicokinetics of OP appears as a relevant subject to be integrated into extrapolation of toxicological data, from in vitro to in vivo, and into systems of risk assessment of endocrine modulating activity which are currently being developed. Received: 8 March 1999 / Accepted: 27 April 1999     

Interactive effect of combined exposure to glycol ethers and alcohols on toxicodynamic and toxicokinetic parameters

 Ethylene glycol monomethyl ether (EGME) exhibits testicular toxicity and ethylene glycol monobutyl ether (EGBE) is a solvent with haemolytic effects in rats. The study of the interaction of two glycol ethers (EGME and EGBE) and three alcohols (ethanol, n-propanol and n-butanol, 10 or 30 mmol/kg), orally co-administered in male rats, was carried out from a toxicodynamic and toxicokinetic point of view. Administered alone, EGME (10 mmol/kg) caused a 30- and 5-fold increase in the urinary creatine/creatinine ratio at 24 and 48 h, respectively, and 24 h urinary excretion of methoxyacetic acid was of 0.71± 0.042 mmol/24 h (mean±SE). The simultaneous administration of one of the three alcohols at either of the doses mentioned above did not significantly modify the urinary creatine/creatinine ratio (24 and 48 h), or the 24 h urinary excretion of methoxyacetic acid. Administered alone, EGBE (5 mmol/kg) caused an average decrease of 26% in the number of circulating red blood cells and a strong (250 times) increase in the level of plasma haemoglobin 4 h after treatment. Urinary excretion of butoxyacetic acid in rats treated with EGBE (1 mmol/kg) was 0.083±0.0039 mmol/24 h (mean±SE). The simultaneous injection of 30 mmol/kg alcohol (ethanol, n-propanol or n-butanol) almost totally inhibits the haemolytic effect of EGBE, and decreases the urinary excretion of butoxyacetic acid by 43–31%. A strong dose of alcohol (30 mmol/kg) decreases the haemolytic effect due to EGBE, and reduces the urinary excretion of butoxyacetic acid. In contrast, the coadministration of alcohol did not modify the testicular toxicity of EGME, or the 24 h urinary excretion of methoxyacetic acid. It is possible that competitive inhibition of alcohol dehydrogenase by alcohols results in the diversion of EGBE metabolism. Received: 3 July 1995/Accepted: 17 November 1995     

Chlorodibromomethane metabolism to bromide and carbon monoxide in rats

The chlorodibromomethane (CDBM) metabolites bromide and CO were analysed as bromide level in plasma and carboxyhaemoglobin (COHb) level in blood of rats, respectively. The mean basic levels of bromide in plasma of rats receiving vehicle were 0.075 ± 0.036 mmol/l (n = 27). After administration of CDBM at 0.4, 0.8, 1.6, and 3.1 mmol/kg p.o., the mean bromide levels rose to maximal values that were higher by a factor 27, 48, 69, and 135, respectively. Bromide elimination was slow and the plasma level was significantly increased following repeated administration in comparison to a single administration of CDBM. The CDBM concentrations in blood and in fat tissue 6 h after the last of 7 administrations of 0.8 mmol CDBM/kg p.o., once a day for 7 consecutive days, were significantly lower than 6 h after a single gavage of this CDBM dose. The mean normal level of 0.45 ± 0.32% COHb in rats (n = 30) was significantly increased following oral CDBM uptake. Initially higher COHb levels were measured after 7 consecutive applications of 0.8␣mmol/kg CDBM. After a single administration of CDBM the level of glutathione disulphide in the liver was significantly increased; this effect was reversible. The oxidative CDBM metabolism was influenced by the glutathione (GSH) concentration in the liver. The rate of COHb and bromide formation was decreased after GSH depletion due to pretreatment of rats with buthionine sulphoximine (BSO) and increased following enhancement of the GSH concentration due to pretreatment of the animals with butylated hydroxyanisole (BHA). CDBM is a substrate for cytochrome P-450 2E1 (CYP2E1), as demonstrated by the inhibition of bromide and COHb formation due to simultaneous administration of CDBM and the CYP2E1 inhibitor diethyldithiocarbamate (DDTC); also by the initially higher levels of bromide in plasma and COHb in blood after gavage of CDBM pretreated with isoniazid (INH), an inducer of CYP2E1. The increase of bromide formation after CDBM administration in phenobarbital (PB)-pretreated rats indicated that cytochrome P-450 2B1 and 2B2 (CYP2B1 and CYP2B2) play a role as catalysts of the CDBM biotransformation. It is shown that m-xylene pretreatment, which activates CYP2E1 as well as CYP2Bs, leads to a higher bromide level after CDBM administration than the INH or PB pretreatment. In liver microsomes of rats treated with CDBM (0.8 mmol/kg p.o., seven daily applications), the p-nitrophenol hydroxylase (p-NPH) activity, a marker of CYP2E1, was increased. It is concluded that CDBM may be an inducer of CYP2E1. These results combined with literature data demonstrate that the oxidation of CDBM was catalysed mainly by CYP2E1 and CYP2Bs and that there may be a risk of bromide accumulation following repeated uptake of the trihalomethane. Received: 15 May 1996 / Accepted: 5 September 1996     

Cardiotoxicity of chlorodibromomethane and trichloromethane in rats and isolated rat cardiac myocytes

The cardiovascular effects were investigated after acute and subacute treatment with chlorodibromomethane (CDBM; 0.4 to 3.2 mmol/kg p.o.), trichloromethane (TCM; 0.31 and 1.25 mmol/kg p.o.) and mixtures of CDBM and TCM (acute, 0.8 mmol CDBM/kg + 1.25 mmol TCM/kg p.o.; subacute, 0.4 mmol CDBM/kg+0.31 mmol TCM/kg p.o.) in conscious and urethane anaesthetized male Wistar rats (n=610 per treatment). Furthermore it was observed whether cardiovascular responses were modified in CDBM or TCM treated rats after administration of exogenous catecholamines (epinephrine, 1 μg/kg; norepinephrine, 2 μg/kg) and underpinned with in vitro alterations of Ca²⁺ dynamics in cardiac myocytes. The present findings demonstrated that single and subacute oral administration of CDBM or TCM and mixtures of CDBM and TCM resulted in arrhythmogenic and negative chronotropic and dromotropic effects in conscious and urethane anaesthetized rats. The atrioventricular conduction time and the intraventricular extension time were extended. A slight shortening of the repolarization velocity was observed. The myocardial contractility was depressed and the heart was sensitized to the arrhythmogenic effects of epinephrine. After catecholamine injection the adrenergic cardiovascular responses in urethane anesthetized rats were modified: increased hypertensive epinephrine and norepinephrine action as well as augmentation of negative chronotropic and negative dromotropic cardiac effects of catecholamines were observed. The positive inotropic adrenergic response was diminished. The present in vivo findings, myocardial depression after acute CDBM treatment, as determined by different indices of contractility, correlate well with the observed inhibitory actions of CDBM on Ca²⁺ dynamics in isolated cardiac myocytes. All cardiovascular alterations found after CDBM or TCM treatment were not intensified after treatment with mixtures of CDBM and TCM. The effects observed were distinctly stronger after TCM (1.25 and 0.31 mmol/kg) treatment compared to CDBM (0.8 and 0.4 mmol/kg) treatment. Received: 10 March 1997 / Accepted: 11 June 1997     

Acute myocardial infarction in streptozotocin-induced hyperglycaemic rats: protection by a carbon monoxide-releasing molecule (CORM-3)

Here, we have studied the effects of a carbon monoxide-releasing molecule (CORM-3, tricarbonylchloro(glycinato)ruthenium(II)) on acute myocardial ischemia/reperfusion (I/R) injury in hyperglycaemic streptozotocin-treated rats (STZ rats). Occlusion of the left descending coronary artery for 25 min followed by a 2-h reperfusion in STZ-induced hyperglycaemic rats was used as the model. CORM-3 and its inactive counterpart (iCORM-3) were administered 1 h prior to ischemia. The parameters measured included myocardial infarct size (IS) and a selection of inflammatory, oxidative markers and endothelial progenitor cells (CD34⁺ and CD117/c-kit⁺). In STZ-induced hyperglycaemic rats, occlusion of the left descending coronary artery caused injury of the myocardial tissue with an IS of ~70%, expressed as fraction of the area at risk. Given intraperitoneally 1 h prior to ischemia, CORM-3 (2–8 mg/kg) afforded significant dose-dependent cardio-protection. Specifically, pre-treatment with CORM-3 reduced infarct size by 14 ± 0.6%, 34 ± 1% and 53 ± 1.6% for doses of 2, 4 and 8 mg/kg, respectively. A negative control (iCORM-3) failed to prevent the cardiac damage induced by I/R. CORM-3 pre-treatment augmented cardiac heme oxygenase-1 (HO-1) protein levels and was associated with an increased number of CD34⁺- and CD117/c-kit⁺-positive immunostaining. Modulation of these markers was associated with augmented cardiac eNOS expression and levels of the cytokines TNF-α and IL-1 beta. CORM-3 afforded significant cardio-protection against acute myocardial infarction in STZ-induced hyperglycaemic rats through liberation of small amounts of CO. Of interest, CORM-3 promoted recruitment of the endogenous endothelial progenitor cells within the myocardium, possibly through modulation of cardiac HO-1 and eNOS expression and/or function.     

Safety of the novel atrial-selective K+-channel blocker AVE0118 in experimental heart failure

Congestive heart failure (CHF) is often associated with atrial fibrillation. The safety of many antiarrhythmic drugs in CHF is limited by proarrhythmic effects. We aimed to assess the safety of a novel atrial-selective K⁺-channel blocker AVE0118 in CHF compared to a selective (dofetilide) and a non-selective IKr blocker (terfenadine). For the induction of CHF, rabbits (n = 12) underwent rapid right ventricular pacing (330–380 bpm for 30 days). AVE0118 (1 mg/kg) dofetilide (0.02 mg/kg) and terfenadine (2 mg/kg) were administered in baseline (BL) and CHF. A six-lead ECG was continuously recorded digitally for 30 min after each drug administration. At BL, dofetilide and terfenadine significantly prolonged QTc interval (218 ± 30 ms vs 155 ± 8 ms, p = 0.001 and 178 ± 23 ms vs. 153 ± 12 ms, p =  0.01, respectively) while QTc intervals were constant after administration of AVE0118 (p = n.s.). In CHF, dofetilide and terfenadine caused torsades de pointes and symptomatic bradycardia, respectively, and prolonged QTc interval (178 ± 30 ms vs. 153 ± 14 ms, p = 0.02 and 157 ± 7 ms vs. 147 ± 10 ms, p = 0.02, respectively) even at reduced dosages, whereas no QTc-prolongation or arrhythmia was observed after full-dose administration of AVE0118. In conclusion, atrial-selective K⁺-channel blockade by AVE0118 appears safe in experimental CHF. H.-J. Schneider and O. Husser contributed equally.     

Hydroquinine pharmacokinetics after oral administration in adult patients with muscle cramps

Objective: This study was conducted to determine the pharmacokinetic properties of hydroquinine after oral administration in adult patients with muscle cramps. The main reason for this study was the poor availability of pharmacokinetic data, hindering the design of studies to explore the possible relationship between hydroquinine concentrations and effects. Methods: Sixteen adult patients with a clinical history of muscle cramps were given once-daily oral doses of 300 mg hydroquinine hydrobromide for 4 days. Serum and saliva samples were taken following a predefined schedule until 24 h after the last dose. Urine was collected during the study period. Hydroquinine concentrations were measured, and calculations were made of pharmacokinetic parameters using non-linear curve fitting. Results: Pharmacokinetics of hydroquinine could be best described using a one-compartment open model. After oral administration, hydroquinine was rapidly absorbed (mean ± SD: maximum concentration 2.43 ± 0.68 mg/l; time to maximum concentration 1.4 ± 1.2 h; lag time 0.54 ± 0.50 h). With an elimination half-life of 10.9 ± 6.1 h, steady-state was reached in several days. The distribution volume was 1.24 ± 0.29 l/kg, total clearance was 6.7 ± 3.2 l/h. The measured unbound hydroquinine fraction was 8.6 ± 3.0%. No correlation was found between saliva and serum concentrations. Cumulative urinary excretion of unchanged hydroquinine 24 h after the first dose was 35.5 ± 9.2 mg. Conclusion: Pharmacokinetic properties of hydroquinine are roughly similar to those of quinine. The unchanged fraction of hydroquinine excreted in urine is higher than that reported for quinine. Saliva hydroquinine concentrations could not be related to serum values. Steady-state trough or other fixed-time serum concentrations may prove useful for further optimisation of hydroquinine dosage. Received: 19 November 1999 / Accepted: 9 March 2000     

The protective effect of HMG-CoA reductase inhibitors against monocrotaline-induced pulmonary hypertension in the rat might not be a class effect: comparison of pravastatin and atorvastatin

Hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, so called statins, improve endothelial function and exert antiproliferative effects on vascular smooth muscle cells of systemic vessels. This study aimed at comparing the protective effects of two statins, pravastatin and atorvastatin, against monocrotaline (MC)-induced pulmonary hypertension in rats. Pravastatin or atorvastatin (PS or AS, 10 mg/kg per day) or vehicle were given orally for 28 days to Wistar male rats injected or not with MC (60 mg/kg intraperitoneally). At 4 weeks, MC-injected rats developed severe pulmonary hypertension, with an increase in right ventricular pressure (RVP) and right ventricle/left ventricle + septum weight ratio associated with a decrease in acetylcholine- or sodium-nitroprusside-induced pulmonary artery dilation observed in vitro. Hypertensive pulmonary arteries exhibited an increase in medial thickness and endothelial cell apoptosis and a decrease of endothelial nitric oxide synthase (eNOS) expression. MC-rat lungs showed a significant decrease of eNOS (P < 0.01) and increase of cleaved caspase-3 (P < 0.05) expression determined by Western blotting. PS (P = 0.02) but not AS (P = 0.30) significantly limited the development of pulmonary hypertension (RVP in mmHg: 30 ± 3, 36 ± 4 vs. 45 ± 4 and 14 ± 1 for MC + PS, MC + AS, MC, and control groups, respectively). Both statins significantly reduced MC-induced right ventricle hypertrophy [RV/left ventricular (LV) + S, in mg/g: 0.46 ± 0.04, 0.39 ± 0.03, 0.62 ± 0.05 and 0.29 ± 0.01 for MC + PS, MC + AS, MC, and control groups, respectively; P < 0.05),and reduced MC-induced thickening (61 ± 6 μm, 82 ± 5 μm, 154 ± 4 μm, and 59 ± 2 μm for MC + PS, MC + AS, MC, and control groups, respectively; P = 0.01) of small intrapulmonary artery medial wall, with MC + AS still being different from the control group. PS but not AS partially restored acetylcholine-induced pulmonary artery vasodilation in MC rats (E_max=65 ± 5%, 49 ± 6%, 46 ± 3%, and 76 ± 4% for MC + PS, MC + AS, MC, and control groups, respectively; P < 0.05 for MC + PS vs. other groups). Both statins prevented apoptosis and restored eNOS expression of pulmonary artery endothelial cells as well as in the whole lung with a more pronounced effect with PS compared with AS. In conclusion, despite its effects on eNOS expression, apoptosis, and medial wall thickening, AS was unable to significantly reduce pulmonary hypertension and to restore endothelium-dependent relaxation, suggesting intermolecular differences between the two HMG-CoA reductase inhibitors in the protection against MC-induced hypertension.     

Isolation and identification of metabolites of ofloxacin in rats,dogs and monkeys

1. Metabolites of (±)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid (ofloxacin) in excreta of rats, dogs and monkeys after oral administration of ¹⁴C-ofloxacin (20?mg/kg) were isolated and identified.

2. Three metabolites of ofloxacin were detected in the excreta of all three species, and identified by t.l.c., u.v., n.m.r. and mass spectrometry as follows: M-1, ester glucuronide of ofloxacin; M-2, unchanged ofloxacin; M-3, (±)-9-fluoro-2,3-dihydro-3-methyl-10-(1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid (desmethyl ofloxacin); M-4, (±)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de][1,4]-benzoxazine-6-carboxylic acid piperazine-4-oxide (ofloxacin N-oxide).

3. It is concluded that ofloxacin is metabolized by O-acyl glucuronidation, N-demethylation and N-oxidation.     

An iron-deficient diet stimulates the onset of the hepatitis due to hepatic copper deposition in the Long-Evans Cinnamon (LEC) rat

To study effects of dietary Cu and Fe levels on the onset of hepatitis in Long-Evans Cinnamon (LEC) rats, female rats (40 days old) were fed a semipurified diet containing 0.1 or 10 mg Cu/kg and 1.5 or 150 mg Fe/kg in a 2 × 2 factorial arrangement for 35 days. At 75 days after birth, LEC rats (+Cu−Fe) fed a Cu-sufficient but Fe-deficient diet (Cu, 10 mg/kg; Fe, 1.5 mg/kg) showed jaundice, with lethargy, anorexia, and malaise. The biochemical variables relating to liver function were significantly increased compared to three other groups, a Cu- and Fe-deficient (−Cu−Fe) group, a Cu-deficient but Fe-sufficient (−Cu+Fe) group, and a Cu and Fe sufficient (+Cu+Fe) group. Furthermore, the +Cu−Fe rat liver showed massive necrosis with huge nuclei. The other three groups presented no biochemical and histological findings of hepatitis. Hepatic Cu and metallothionein concentrations were 289 ± 87 (mean ± SD) μg/g liver and 8.7 ± 1.8 mg/g liver, respectively, in the +Cu−Fe rats. However, in the +Cu+Fe group the values were 196 ± 28 μg Cu/g liver and 10.8 ± 1.0 mg/g liver. Hepatic Fe deposition was not influenced significantly by the dietary Cu level. The +Cu−Fe group with jaundice showed the highest free Cu concentration in the liver among the four groups, but the hepatic free Fe concentration was similar to those in the −Cu+Fe and +Cu+Fe groups. Our results indicate that an Fe-deficient diet enhances the deposition of hepatic Cu due to increased absorption of Cu from the gastrointestinal tract. This deposition stimulated the onset of hepatitis. Received: 11 March 1999 / Accepted: 11 June 1999     

Enantioselective pharmacokinetic and pharmacodynamic properties of carvedilol in spontaneously hypertensive rats: focus on blood pressure variability

The cardiovascular effects and pharmacokinetics of carvedilol were assessed in spontaneously hypertensive (SH) and Wistar Kyoto (WKY) animals with special focus on short-term blood pressure variability (BPV). Male SH and WKY rats were acutely treated with vehicle or carvedilol 1 or 5 mg kg⁻¹ (i.v.), and effects on blood pressure (BP), heart rate (HR) and BPV were recorded. Plasma pharmacokinetics of R- and S-carvedilol was studied by traditional blood sampling. Relationship between carvedilol concentrations and their hypotensive and bradycardic effects was established by pharmacokinetic–pharmacodynamic (PK–PD) modelling. Short-term BPV was assessed by standard deviation of BP recording. Vascular sympatholytic activity of carvedilol was studied by estimation of drug effects on ratio between low frequency (LF) and high frequency (HF) BPV (LF/HF ratio). Although pharmacokinetic properties of carvedilol remained mainly unaffected in SH rats with regard to WKY rats, hypertensive animals showed a reduction in drug clearance of R- and S-carvedilol after administration of 1 mg kg⁻¹ compared with WKY rats. PK–PD analysis of HR changes induced by S-carvedilol showed a greater maximal bradycardic response to carvedilol in SH rats (E _max, −27.6 ± 3.9%; p < 0.05) compared with WKY group (E _max, −13.4 ± 2.5%). SH rats showed a greater hypotensive effect of racemic carvedilol (E _max, −45.5 ± 5.0%; p < 0.05) with regard to WKY group (E _max, −17.9 ± 4.5%). Carvedilol induced a greater reduction of LF/HF ratio in SH rats compared with WKY rats. Short-term BPV was markedly reduced by carvedilol in WKY and SH rats. In conclusion, as a consequence of an enhanced bradycardic response and a greater vascular sympatholytic activity, carvedilol exerts a greater hypotensive response in SH rats compared with WKY animals and dramatically reduces short-term BPV.     

Penetration of ciprofloxacin, norfloxacin and ofloxacin into the aqueous humour of patients by different topical application modes

Objectives: A prospective study was undertaken to determine the transcorneal penetration of three topically applied fluoroquinolones into aqueous humour. Methods: Two hundred and twenty-four patients undergoing cataract extraction received 0.3% ciprofloxacin, norfloxacin or ofloxacin eye drops by two different administration modes with different frequencies and intervals of application. At the beginning of cataract extraction (0.5–3 h after the last drop), 50–100 μl aqueous fluid was aspirated from the anterior chamber and immediately stored at −80 °C. Antibiotic concentrations were measured using high-performance liquid chromatography. Results: Generally, topical ofloxacin and ciprofloxacin yielded aqueous humour levels higher than topical norfloxacin. The highest concentrations of all tested fluoroquinolones were measured after using an application mode, in which one drop was given every 15 min between 0600 hours and 0800 hours, prior to operation. When applied by this mode, ciprofloxacin achieved a mean aqueous level of 0.380 (±0.328) μg · ml⁻¹ (range 0.033–1.388 μg · ml⁻¹), norfloxacin 0.182 (0.118) μg · ml⁻¹ (range 0.038–0.480 μg · ml⁻¹) and ofloxacin 0.564 (0.372) μg · ml⁻¹ (range 0.064–1.455 μg · ml⁻¹). These mean concentrations were above the minimum inhibitory concentration (MIC₉₀), concentrations required for inhibition of 90% of pathogen strains in vitro of gram-negative bacteria, such as Proteus mirabilis and Escherichia coli. Therapeutic values above the MIC₉₀ of Staphylococcus epidermidis, the pathogen causing eye infections most frequently, were reached by 67.5% of patients after ofloxacin and by 41% after ciprofloxacin, but never after norfloxacin treatment. Conclusion: Of the currently available topical fluoroquinolones, ofloxacin achieved the highest aqueous humour concentration. This fluoroquinolone may be an useful ophthalmic agent for topical antibacterial management, but it does not seem to be prophylactically effective against Streptococcus pneumoniae or Pseudomonas aeruginosa. Received: 22 April 1997 / Accepted: 8 June 1997     

Characterisation of colonic accommodation in Wistar Kyoto rats with impaired gastric accommodation

Defective colonic and gastric accommodations have been related to altered viscerosensitivity in irritable bowel syndrome and to functional dyspepsia, respectively. We assessed colonic accommodation in rats with impaired gastric accommodation to determine if altered accommodation can be regarded as a widespread pathophysiological alteration within the gastrointestinal (GI) tract. Colonic accommodation during colorectal distension (CRD) was assessed in Wistar Kyoto rats (WKY), an animal model of impaired gastric accommodation, and in Sprague–Dawley (SD) and Wistar rats, considered normal. CRD (10–80 mmHg)-induced visceral pain responses were also evaluated in the same strains of rats. During gastric distension, WKY rats had lower intra-gastric volume (0.96 ± 0.22 ml) than SD (1.85 ± 0.19 ml, P < 0.05) or Wistar rats (2.80 ± 0.26 ml, P < 0.05), indicating impaired gastric accommodation. In the same animals, pressure–volume curves were constructed during CRD as a measure of colonic accommodation. During short-lasting (1 min) phasic CRD (2–20 mmHg), the pressure–volume curve in WKY rats was displaced to the right compared with SD or Wistar rats, indicative of reduced colonic accommodation (maximal volume: SD, 1.22 ± 0.05 ml; Wistar, 1.07 ± 0.04 ml; WKY, 0.87 ± 0.07 ml; P < 0.01). Pre-treatment with atropine normalised the pressure–volume responses in WKY rats. No differences among strains were observed during the 2-min phasic or ramp-tonic CRD. Visceral pain responses during CRD (10–80 mmHg) were, overall, similar in the three strains, although WKY rats showed lower thresholds for pain (28.0 ± 4.9 mmHg) than SD (42.3 ± 6.6 mmHg, P = 0.072) or Wistar rats (48.3 ± 6.0 mmHg, P < 0.05). WKY rats, although having impaired gastric accommodation, have the ability to fully accommodate the colon to increasing pressures. In WKY rats, impaired accommodation of the smooth muscle might not be a widespread phenomenon along the GI tract but rather a local disturbance.