Production of matrix metalloproteinases and their inhibitors in osteoarthritic patients undergoing mud bath therapy.

Several studies have demonstrated that matrix metalloproteinases (MMPs) are frequently implicated in the destruction of articular cartilage in arthritis. The control of MMP activity is dependent on the local concentration of tissue inhibitors of metalloproteinases (TIMPs), and the imbalance of the enzyme-to-inhibitor ratios plays an important role in the remodeling of articular tissues. Some cytokines such as interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha which regulate leukocyte activities, promote MMP secretion and, as a consequence, cartilage degradation. The aim of the present study was to investigate whether a natural treatment is effective in reducing cartilage inflammation and degradation by influencing MMP and TIMP serum levels. Eighty patients with osteoarthritis (OA) were enrolled in the trial and were divided into group A (30 patients who did not undergo mud bath therapy), group B (28 patients repeating mud bath therapy more than 5 times and less than 10) and group C (22 patients repeating mud bath therapy more than 10 times). Blood samples were obtained from all the patients for assay of MMP-1, -2, -3, -8 and -9 and TIMP-1 and -2. The parameters were determined by an ELISA technique. Statistical indexes were calculated for each parameter and mean values were compared. The differences between mean values of MMP-3, -8 and -9 were statistically significant between group A and the treated groups (B and C). Analysis of variance established a significant difference (p < 0.05) between groups A and C in mean serum levels of MMP-8, MMP-9 showed a statistically significant difference (p < 0.05) in mean serum concentration between groups A and B. Regression analysis showed a very high R2 between MMP-2 and TIMP-2. One of the most interesting findings in this study was that MMP-3 serum levels were significantly lower in the treated groups, since this enzyme plays an important role in cartilage degradation, suggesting that mud bath therapy contributes to matrix integrity in OA cartilage. In contrast, MMP-8 and -9 were higher in the treated subjects and no correlation with TIMPs was evident. One possible explanation is that these enzymes are required for the efficient degradation and removal of already compromised cartilage matrix and that they operate as part of a matrix turnover and repair process. In conclusion, our data suggest that mud bath therapy alone is not able to influence chondrocyte metabolic activity in the advanced phases of OA. There could be a synergic and sequential association with pharmacologic therapy and/or interventions.

Both serum receptors of tumor necrosis factor are influenced by mud pack treatment in osteoarthrotic patients.

Several authors have demonstrated the pivotal role of proinflammatory cytokines in inducing progressive cartilage degradation and secondary inflammation of the synovial membrane in osteoarthritis (OA). It has recently been established that tumor necrosis factor (TNF)-alpha plays a well-defined role in the pathophysiology of inflammatory joint diseases and that binding to circulating soluble TNF-alpha receptors can inactivate it. We investigated the influence of mud pack treatment, which is able to diminish TNF-alpha serum values, on specific TNF receptor (sTNF-R) levels. Thirty-six patients with OA were enrolled and randomized into two groups. Group A underwent mud pack treatment and group B underwent thermal bath treatment. A group of 20 healthy untreated subjects was used as a control. Blood samples were collected at baseline and after treatment, and assays of sTNF-R55 and sTNF-R75 were performed in both groups. We found small changes in sTNF-Rs serum values but these were not statistically significant. sTNF-R55 serum values decreased by 0.4% after the therapy in group A, while in group B the decrease was -17.7%. sTNF-R75 was reduced by -21.17% in group A and by -10.6% in group B. In conclusion, through its thermic and ant/inflammatory activity mud pack treatment shows complex interaction with the most common factors of inflammatory and cartilage degradation. Our results suggest that the thermic component of this natural treatment is mainly involved in modulating inflammatory reaction and cartilage damage through binding of the circulating TNF, which controls the activation of the cells responsible for the production of proinflammatory cytokines.

In vitro antioxidant and ex vivo protective activities of green and roasted coffee.

The antioxidant properties of green and roasted coffee, in relation to species (Coffea arabica and Coffea robusta) and degree of roasting (light, medium, dark), were investigated. These properties were evaluated by determining the reducing substances (RS) of coffee and its antioxidant activity (AA) in vitro (model system beta-carotene-linoleic acid) and ex vivo as protective activity (PA) against rat liver cell microsome lipid peroxidation measured as TBA-reacting substances. RS of C. robustasamples were found to be significantly higher when compared to those of C. arabica samples (p < 0.001). AA for green coffee samples were slightly higher than for the corresponding roasted samples while PA was significantly lower in green coffee compared to that of all roasted samples (p < 0.001). Extraction with three different organic solvents (ethyl acetate, ethyl ether, and dichloromethane) showed that the most protective compounds are extracted from acidified dark roasted coffee solutions with ethyl acetate. The analysis of acidic extract by gel filtration chromatography (GFC) gave five fractions. Higher molecular mass fractions were found to possess antioxidant activity while the lower molecular mass fractions showed protective activity. The small amounts of these acidic, low molecular mass protective fractions isolated indicate that they contain very strong protective agents.

Mud bath therapy influences nitric oxide, myeloperoxidase and glutathione peroxidase serum levels in arthritic patients.

Nitric oxide (NO) has recently been proposed as an important mediator in inflammatory phases and in loss of cartilage. In inflammatory arthritis NO levels are correlated with disease activity and articular cartilage is able to produce large amounts of NO with the appropriate inducing factors such as cytokines and/or endotoxin. Neutrophils also play an important role in inflammatory reactions and the level of myeloperoxidase, a constituent of neutrophil granules, is related to the intensity of the inflammation. Because there is evidence that suggests that mud packs influence the main cytokines involved in cartilage damage, we tried to determine whether NO and myeloperoxidase are involved in the mechanisms of action of mud bath treatment. We enrolled 37 subjects and randomly assigned them to two groups: 19 patients underwent mud bath treatment (group A) while 18 patients underwent bath treatment alone. Blood samples were obtained before and after the treatment cycles to assay serum levels of NO, myeloperoxidase (MPO) and glutathione (GSH)-peroxidase. The results showed a statistically significant decrease in NO and myeloperoxidase serum values in groups A and B, while GSH-peroxidase was not significantly increase in either of the groups; no correlation was found between NO, myeloperoxidase and GSH-peroxidase serum values. Mud bath treatment can exert beneficial effects on cartilage homeostasis and inflammatory reactions, influencing NO and decreasing myeloperoxidase serum values. The increase in GSH-peroxidase was not correlated with the reduction of other biochemical markers, suggesting that mud bath treatment has different mechanisms of action.

Haloperidol-induced changes in glutathione and energy metabolism: effect of nicergoline.

The aim of this study was to evaluate the possible effects of nicergoline, a semisynthetic ergot derivative, on the biochemical changes observed during chronic treatment with haloperidol in male Sprague-Dawley rats. Chronic treatment with haloperidol induced a significant decrease in the cellular glutathione (GSH) content in selected areas of the brain (cerebellum, striatum and cortex) and in the liver. Prolonged nicergoline administration was able to antagonize the haloperidol-induced GSH decrease, maintaining the GSH concentration at levels comparable to those observed in the control group. Analysis of the energy charge revealed changes similar to those observed for GSH: haloperidol induced a significant decrease in ATP and energy charge that was completely reversed by repeated nicergoline administration. In conclusion, chronic treatment with the classical antipsychotic haloperidol induces profound biochemical changes in the brain and in the liver. Nicergoline treatment is able to counteract the haloperidol-induced decrease in GSH levels and energy charge, suggesting a potential role of the drug in the treatment of neuroleptic-induced side effects.

Toxicological evaluation of pidotimod.

This paper reports the toxicological evaluation of pidotimod ((R)-3-[(S)-(5-oxo-2-pyrrolidinyl) carbonyl]-thiazolidine-4-carboxylic acid, PGT/1A, CAS 121808-62-6). Its acute toxicity in mice, rats and dogs was very low after oral, i.v., i.m. and i.p. administration. The repeated administration studies in rats were performed for 4 months via the i.p. route and for 12 months via the oral route. Pidotimod did not show toxic effects at dosages up to 200 mg/kg i.p. and 800 mg/kg p.o. These dosages correspond to 32.5 times the maximum dosage intended for clinical use. The repeated administration studies in dogs were performed for 26 weeks via the i.m. route and for 52 weeks via the oral route. Pidotimod did not show toxic effects at dosages up to 300 mg/kg i.m. and 600 mg/kg p.o.. It did not affect male or female rat fertility at dosages up to 600 mg/kg by oral and 500 mg/kg by i.v. route. The compound was not teratogenic in rats (600 mg/kg p.o. and 1000 mg/kg i.v.), with no effects on subsequent embryofoetal development at dosages up to 1000 mg/kg/day, and in rabbits (300 mg/kg p.o. and 500 mg/kg. i.v.). There were no peri- and postnatal toxic effects in rats (600 mg/kg p.o. and 500 mg/kg i.v.). Local tolerability of pidotimod after i.m. administration was very good. In conclusion pidotimod is characterized by a high safety margin in all animal species.

Effects of thallium on primary cultures of testicular cells.

The objective of this in vitro study was to examine the response of mixed cultures of Sertoli and germ cells to treatment with thallium (Tl) at the range of concentrations that, in previous studies, was shown in vivo to affect reproduction. Cultures were prepared from the testis of Sprague-Dawley rats. Cultures containing approximately 3.75 x 10(6) cells/ml were treated with Tl concentrations corresponding to 35, 7, and 1.4 micrograms Tl/g testis, estimated from protein content of cultures. Observations at 24, 48, and 72 h after treatment showed a significant release of germ cells into the culture medium that was both concentration and time dependent. Cultures treated with 35 micrograms Tl/g testis showed a threefold increase in germ-cell detachment compared with controls after only 24 h of exposure. As the treatment time increased to 48 h of exposure, even cultures exposed at the lowest Tl concentration (1.4 micrograms Tl/g testis) showed significant loss of germ cells. After 48 h, cultures exposed to 7 micrograms Tl/g testis exhibited a 2.5-fold increase in germ-cell detachment, and those exposed to 35 micrograms Tl/g testis exhibited a 10-fold increase over controls. Morphological investigations of cell cultures showed evident loss of germ cells with significant reduction in prepachytene and pachytene spermatocytes and changes in the shape of Sertoli cells. These results are in agreement with in vivo studies, in which thallium treatment at comparable exposure levels manifested its earliest toxic testicular effects in Sertoli and germ cells. They also demonstrate the usefulness of this in vitro culture technique to assess toxic testicular damage rapidly.

Enhancement of specific biological activity of dipyridamole by complexation with beta-cyclodextrin.

Dipyridamole forms an inclusion complex with beta-cyclodextrin (dip-beta-CD) which shows better solubility and bioavailability than the uncomplexed compound. The present studies have demonstrated that dip-beta-CD is more effective than dipyridamole either as base or HCl (dispersed or not in lactose) on some important cardiovascular parameters when orally administered to conscious animals. In particular, dip-beta-CD causes a stronger and prompter coronary and carotid vasodilatation in dogs, at doses which weakly influence the systemic arterial pressure and the heart rate. In addition, platelets collected from treated rabbits at various intervals appear to be protected from sodium adenosine diphosphate-induced aggregation in vitro more effectively and rapidly by dip-beta-CD than by dipyridamole. Studies on tail bleeding time have confirmed that dip-beta-CD is more active than dipyridamole when given orally to mice. These biological findings are fully in agreement with other oral bioavailability studies in dogs and men indicating that dip-beta-CD gives quicker and higher blood levels with smaller interindividual variability than dipyridamole.

1,2-Dichloropropane (DCP) toxicity is correlated with DCP-induced glutathione (GSH) depletion and is modulated by factors affecting intracellular GSH.

Acute 1,2-dichloropropane (DCP) poisoning in humans is relatively frequent in Italy, where DCP is widely diffused as a constituent of commercial solvents and dry cleaners. In this study we have investigated the effects of DCP on intracellular glutathione (GSH) content in main target tissues of male Wistar rats, i.e. liver, kidney and blood, in order to establish if a correlation between DCP-induced GSH depletion and tissue damage exists. Administration of DCP (2 ml/kg body weight orally) caused a dramatic loss of tissue GSH occurring 24 h after DCP intoxication, followed by a slow restoration approaching physiological levels after 96 h. GSH depletion was associated with a marked increase in serum GOT, GPT, 5'-nucleotidase, gamma-glutamyl transpeptidase, alkaline phosphatase, urea and creatinine, and a significant degree of hemolysis. When animals were pretreated with a GSH depleting agent, buthionine-sulfoximine (BSO) (0.5 g/kg body weight) i.p. 4 h before DCP intoxication, an increase of overall mortality was found, significantly different from the group of animals treated with DCP alone. On the contrary, the administration of a GSH precursor, N-acetylcysteine (NAC) i.p. (250 mg/kg body weight) 2 and 16 h after DCCP intoxication prevented the dramatic loss of cellular GSH and reduced the extent of injury in target tissues, as demonstrated by laboratory indices. Furthermore, statistical analysis of the data revealed a correlation between: (1) depletion of liver GSH and increase in serum GOT, GPT, 5'-nucleotidase, (2) depletion of kidney GSH and increase in serum urea and creatinine and (3) depletion of blood GSH and the occurrence of hemolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

On the role of mitochondria in cell injury caused by vanadate-induced Ca2+ overload.

Incubation of isolated rat hepatocytes with vanadate (0.25, 0.5 and 1 mM) resulted in progressive accumulation of Ca2+ in the intracellular compartments. Vanadate- induced Ca2+ accumulation was related to inhibition of the plasma membrane Ca2+-extruding system, but did not involve either enhanced plasma membrane permeability to Ca2+ or the enhanced operation of a putative Na+/Ca2+ exchanger. After an initial rise in the cytosolic free Ca2+ concentration, as revealed by phosphorylase activation, Ca2+ was sequestered predominantly by the mitochondria with little contribution from the endoplasmic reticulum. As the amount of Ca2+ in the mitochondria increased, a progressive decrease in mitochondrial membrane potential occurred, together with an impairment of the ability of these organelles to further sequester Ca2+. Associated with this, there was a decrease in intracellular ATP level, formation of surface blebs and cytotoxicity. Addition of an uncoupler to vanadate-treated hepatocytes dramatically accelerated the appearance of plasma membrane blebs and toxicity. Our results demonstrate that under conditions in which the plasma membrane Ca2+ pump is inhibited, mitochondria play an important role in protecting hepatocytes against damage induced by Ca2+ overload.

Lack of influence of sulglycotide on naproxen bioavailability in healthy volunteers.

We explored the bioavailability and kinetics of naproxen (N) in 12 healthy volunteers treated orally with single doses of 500 mg and retreated after a washout period with the same dose of N plus sulglycotide (S) 200 mg. Naproxen blood levels were measured by high-performance liquid chromatography (HPLC) in samples collected at 0.5, 1, 2, 4, 8, 12, and 24 h of dosing with N or with N + S. No statistically significant difference in terms of naproxen blood levels emerged as the product was administered alone or concurrently with sulglycotide. Peak plasma concentrations and AUC values were 71 +/- 3.16 micrograms/ml and 685 +/- 27 micrograms/ml/h, respectively for N alone, and 72.5 +/- 2.85 micrograms/ml and 651 +/- 28 micrograms/ml/h, respectively for N + S. The difference was not significant. Similarly, the kinetic behavior of naproxen was not modified by the simultaneous presence of sulglycotide, as shown by the t1/2 beta-values obtained with N alone (8.39 +/- 0.31 h) and with N + S (7.93 +/- 0.30 h), and likewise by the distribution volumes at equilibrium (7.63 +/- 0.42 and 7.9 +/- 0.38, respectively), Cmax (63.3 +/- 2.86 and 60.4 +/- 2.9 micrograms/ml, respectively) and tmax (0.95 +/- 0.06 and 1.10 +/- 0.10 h, respectively). From these findings it seems legitimate to claim that sulglycotide can be administered concurrently with naproxen to prevent possible gastric injury by the anti-inflammatory agent thanks to its wellknown antiulcer and cytoprotective activity on the gastric mucosa, without any undue interference with the absorption (hence effectiveness) of naproxen.

Lack of effect of a single-dose of sulglicotide on the bioavailability of diclofenac.

The bioavailability of diclofenac (D) was assessed in 12 healthy volunteers treated orally with single doses of 100 mg (retard formulation) and subsequently retreated with the same dose of (D) plus sulglicotide (S) 200 mg. (D) blood levels were measured by GLC in samples collected after 1, 2, 4, 6, 8, 12, 24 h. No relevant difference was seen in (D) bioavailability after (S) administration; after 8 h plasma levels of (D) were slightly higher after (S) (p less than 0.05), but this difference can be considered incidental only. Thus, sulglicotide does not interfere with the bioavailability of diclofenac, and can be administered concurrently with the latter to prevent possible gastric injury by the antiinflammatory drug.

Clinical experience with two twice daily slow release theophylline preparations. Relation between serum theophylline concentrations, effectiveness, tolerability and compliance in elderly patients.

The relation between serum theophylline concentrations, effectiveness, tolerability and compliance were evaluated in 14 hospitalized elderly patients with broncho-obstructive pathology, using a new twice daily sustained release theophylline suspension compared to standard twice daily slow release tablets. The results showed that the mean theophylline serum concentration remains within the therapeutic range with both preparations. No significant difference exists between the two treatments with regard to effectiveness. Tolerability and compliance of the theophylline suspension, however, were higher.

Erythromycin estolate impairs the mitochondrial and microsomal calcium homeostasis: correlation with hepatotoxicity.

The effects of erythromycin estolate, a well known hepatotoxic macrolide antibiotic, on isolated rat hepatocyte viability and on subcellular Ca2+ transport have been investigated. Erythromycin estolate (0.5 mM), but not erythromycin base and erythromycin ethylsuccinate, induced 100% cell death after 60 min incubation, and caused maximal inhibition of mitochondrial and microsomal Ca2+ sequestration activities at 0.1 mM concentration. Sodium lauryl sulphate, which is the surfactant moiety of the erythromycin estolate molecule, caused effects similar to those exhibited by erythromycin estolate. Disorders of the intracellular calcium homeostasis seem to play a role in the lauryl sulphate-mediated hepatotoxic action of erythromycin estolate.

Effects of phenobarbitone on the distribution, metabolism and biliary excretion of erythromycin in rats.

The administration of phenobarbitone to the rat (8 mg/100 g BW) once daily for 3 days significantly decreased the serum and tissue levels of erythromycin administered intraperitoneally (5 mg/100 g BW). Furthermore, phenobarbitone stimulated the hepatic microsomal N-demethylation of erythromycin and increased the biliary concentration and the biliary excretion rate of the unmetabolized antibiotic. These effects were accompanied by augmented liver mass and bile flow. The possibility is discussed that erythromycin concentrates in the bile through a specialized hepatic drug transport system, activated by phenobarbitone.