Vehicle effects on in vitro skin permeation of thiocolchicoside.

Thiocolchicoside, a semi-synthetic derivative of colchicoside, is used in topical formulations for its anti-inflammatory and muscle-relaxant properties. The objective of this study was to evaluate the effect of a (propylene glycol diperlagonate) DPPG and (propylene glycol) PG mixture present in an innovative foam formulation (Miotens) on the flux of thiocolchicoside through excised human skin. Furthermore, the in vitro permeation behaviour of this new formulation (Miotens foam) was compared to another commercial product (Muscoril ointment) and to a control gel formulation (thiogel), both enhancer free. The best permeation profile was obtained from the foam formulation (Miotens) which was able to increase the thiocolchicoside flux about three fold compared to control formulation (thiogel) and about two fold compared to the commercial formulation Muscoril ointment.

In vitro permeation screening of a new formulation of thiocolchicoside containing various enhancers.

Thiocolchicoside, a muscle relaxant agent with anti-inflammatory and analgesic actions, also is used topically for the treatment of muscular spasms and for rheumatologic, orthopedic, and traumatologic disorders. In this study, thiocolchicoside was formulated to use as foam to avoid contact with the afflicted area during the spreading phase. To enhance drug penetration, various enhancers were added to the base formulation. The tested enhancers were ethoxyethylendiglycol (Transcutol), highly purified phosphatidylcholine (Lipoid S20), capsaicin, propylene glycol dipelargonate (DPPG), and glycolysed ethoxylated glycerides (Labrafil M1944 CS). The transdermal absorption of the tested formulations containing enhancers, in comparison with base formulation, was evaluated in vitro through rat skin using standard Franz diffusion cells. Base formulation was found to have a higher permeation profile than the simple aqueous and hydroalcoholic solutions of the drug, meaning that the base formulation by itself enhances the drug permeation. Among the tested formulations, only the formulation containing DPPG/ethanol was found to be statistically different, showing an enhancement factor of 3.58. In the same experimental session, Muscoril ointment, the commercially available pharmaceutical product containing the same thiocolchicoside concentration (0.25%), also was tested. The formulation containing DPPG/ethanol showed a 4.86 times increase of permeability constant in comparison with Muscoril ointment. The formulation containing DPPG/ethanol as an enhancer could be a good candidate for a new topical foam, considering its good characteristics of permeability and compliance.

Efficacy of a new topical gel-spray formulation of ketoprofen lysine salt in the rat: percutaneous permeation in vitro and in vivo and pharmacological activity.

The aim of this study was to evaluate the percutaneous permeation of a new topical Gel-Spray formulation, containing 15% of ketoprofen lysine salt (KLS), both in vitro, using the Franz-type diffusion cells and in vivo, by evaluating urinary recovery after topical administration and to correlate the absorption data with KLS pharmacological activity in the rat. Concentrations of ketoprofen free acid (KFA) were determined by HPLC in the receptor compartment (in vitro), or in urine (in vivo). The permeation of ketoprofen evaluated in vitro after the application of KLS Gel-Spray was higher than that observed with the marketed formulation Profénid gel (containing KFA at 2.5%). The same evidence was found in vivo, except when the ratio between the administered dose and the area treated was higher than 1 mg cm-2. Thus, the difference between the two formulations seems to be the resultant of two opposing components: a positive gradient of concentration that favours the absorption of ketoprofen from KLS Gel-Spray and the presence of the enhancer ethanol that could favour the efficacy of Profénid gel. Under our conditions the former prevailed. As for the efficacy, evaluated in the carrageenan-induced oedema and hyperalgesia model, KLS Gel-Spray confirmed the data obtained for in vivo absorption, being more efficient than the reference standard Profénid gel. The observed inhibitory effects were due only to dermal absorption, oral absorption was excluded by an Elizabethan collar applied around the neck of the rat. In these experimental conditions, no significant damage of the rat stomach mucosa was observed. These results indicate that KLS Gel-Spray, due to its high KLS concentration, allows a very high efficiency in delivering ketoprofen to the inflamed area using a minimal amount of formulation, even in the absence of permeation enhancers.

Tartronates: a new generation of drugs affecting bone metabolism.

In the search for a new class of bone-sparing agents for treating osteopenic disorders, we hypothesized that tartronic acid derivatives, sharing the chemical characteristics both of bisphosphonates and of Gla residues contained in matrix proteins such as osteocalcin, could positively affect bone metabolism. A series of tartronates was therefore tested for their ability to affect bone metabolism. In vitro resorption tests were performed examining pit formation by freshly isolated rat and rabbit osteoclasts plated onto bone slices and exposed to the drugs for 48 h. Tartronates bearing a linear side-chain (DF 1222 and DF 1363A) were the most effective in inhibiting pit excavation in the pM-nM range. Tartronates did not affect osteoclast viability, number, adhesion, or tartrate resistant acid phosphatase activity. Transient cell retraction was observed in osteoclasts plated onto glass and exposed to DF 1222. The maximal effect was seen in cells treated for 4 h at a concentration of 1 pM. DF 1222 accelerated mineralization in cultures of periosteal cells without affecting other osteoblast-like functions. This product was therefore tested in vivo in ovariectomized mice. Bone mass in femur was evaluated, by ash gravimetry, 21 days after ovariectomy. Unfortunately, DF 1222, the most active of tartronates in vitro, was inactive in this test because of its high hydrophilicity and the subsequent too short residence time. On the contrary, its tetrahydropyranyl ether derivative, DF 1363A, endowed with a significantly higher lipophilicity, showed a dose-dependent bone-sparing effect when administered subcutaneously at 10, 30, and 100 mg/kg/die, thus confirming the activity seen in in vitro tests. Because of their feasible parallel effect on both bone resorption and formation, tartronate derivatives may be tested to candidate this class of products for clinical studies.

Protective activity of ketoprofen lysine salt against the pulmonary effects induced by bradykinin in guinea-pigs.

We investigated the capacity of ketoprofen lysine salt (KLS) to counteract the pulmonary effects of some mediators of airway inflammation. The protective effect of KLS and its R-isomer against bradykinin (BK) induced plasma extravasation in the airways and bronchoconstriction was evaluated in anaesthetized guinea-pigs, in parallel with the capacity of KLS to inhibit the production of thromboxane A2 (TXA2). Moreover, we studied the ability of KLS to modulate leukotriene C4 (LTC4) and acetylcholine (ACH) induced bronchoconstriction and the associated production of TXA2. Nimesulide (NIM) was used as the reference compound. KLS dose-dependently inhibited the bronchoconstriction and the associated production of TXA2 induced by BK, with closely related ID50 values of 31.2 and 34.0 micrograms/kg i.v., respectively. The protection was evident 10 min after KLS administration and, at 100 micrograms/kg i.v., lasted up to 2h, Moreover, KLS dose-dependently inhibited the increase in capillary permeability induced by BK, with a potency (ID50 23.4 micrograms/kg i.v.) slightly higher than that shown against the bronchoconstriction. KLS also prevented the bronchoconstriction and TXA2 production triggered by LTC4, but not ACH induced bronchoconstriction. In all the models studied, KLS was about 10 times more potent than NIM. These data demonstrate the capacity of KLS to counteract the bronchoconstriction induced by BK and LTC4 and to a large extent the airway inflammation induced by BK. Blockade of prostanoid production is likely to account for this protective effect, since the R-isomer of KLS was devoid of significant activity.

Ketoprofen lysine salt inhibits disuse-induced osteopenia in a new non-traumatic immobilization model in the rat.

Immobilization and the consequent unloading can cause osteopenia both in humans and in animals due to an increased bone resorption and a parallel reduction in bone formation. Non-steroidal anti-inflammatory drugs (NSAIDs), and in particular the aryl propionic acids, are described to prevent bone loss by inhibiting the cyclo-oxygenase activity. In this study we evaluated the role of a classical aryl propionic acid, ketoprofen lysine salt (KLS), in a new model of disuse-induced osteoporosis in the rat. Tail immobilization evoked a time-dependent bone loss in the caudal vertebral bodies, measured densitometrically as a reduction of bone mineral density (BMD) and content (BMC). KLS was administered once daily for 10 days by subcutaneous route at 0.5 mg kg-1, a dose lower than that effective to elicit an anti-inflammatory response. In these conditions, KLS completely abolished BMD and BMC decrease observed in the caudal vertebrae after 10-day immobilization, without affecting bone mass in normal (non-immobilized) rats. These results suggest that KLS can exert, besides to its anti-inflammatory effect, an anti-resorptive activity on bone that could be useful in the prevention of disuse-induced osteopenia.

Effectiveness of carbocysteine lysine salt monohydrate on models of airway inflammation and hyperresponsiveness.

We investigated the possible effects of the mucoactive drug Carbocysteine lysine salt monohydrate (CLS.H2O) on experimentally-induced airway inflammation and hyperresponsiveness. CLS.H2O given by the oral route (300 mg kg(-1)) significantly reduced neutrophil infiltration into the airway lumen induced by intratracheal injection of IL-1 beta in rats. In addition, CLS.H2O inhibited dose-dependently (100-300 mg kg(-1) p.o.) the formation of pleural exudate and leukocyte recruitment induced by intrapleural injection of carrageenan in rats. Because of the close interaction between the inflammatory process and the development of airway hyperresponsiveness we also tested CLS.H2O on cigarette-smoke-induced inflammation and hyperreactivity in anaesthetized guinea-pigs. The drug, given either by oral (300 mg kg(-1)) or aerosol route (30-100 mg ml(-1)), was able to reduce the increase in airway responsiveness induced by smoke and the associated cell recruitment detected in the bronchoalveolar lavage (BAL) fluids. These results suggest that CLS.H2O can exert an anti-inflammatory action in addition to its mucoregulatory activity. The anti-inflammatory and anti-hyperreactivity effect of the drug within the airways may be of advantage in the treatment of inflammatory lung diseases where mucus secretion together with airway inflammation and hyperreactivity contribute to airway obstruction.

Effects of a new foam formulation of ketoprofen lysine salt in experimental models of inflammation and hyperalgesia.

The anti-inflammatory and analgesic profile of a new topical foam formulation of ketoprofen lysine salt (CAS 57469-78-0, Artrosilene Schiuma, KLS-foam) was characterized in comparison with marketed gel formulations containing KLS (KLS-gel) or diclofenac diethylammonium salt (DCF-gel). KLS-foam dose-dependently inhibited oedema formation and hyperalgesia induced by subplantar injection of carrageenan or substance P, being more potent than KLS-gel. At equieffective anti-inflammatory doses, KLS-foam provided a more pronounced analgesic effect than DCF-gel. KLS-foam also markedly inhibited exudate formation and prostaglandin production induced by subcutaneous implantation of carrageenan soaked sponges. In carrageenan induced paw inflammation, KLS-foam provided the same anti-inflammatory effect as orally administered KLS, but induced significantly less gastric damages. Oral administration of KLS resulted in sustained systemic absorption of ketoprofen, whereas after topical application of KLS-foam no appreciable ketoprofen plasma levels were detected. These data support the anti-inflammatory and particularly the analgesic effectiveness of the new foam formulation of KLS, a finding that, together with the high gastric tolerability, further emphasizes the usefulness of KLS-foam in the treatment of localized flogistic diseases and associated pain.

Free solution capillary electrophoresis of calcitonins and calcitonin tryptic digests.

The aim of the present work was the development of a simple capillary electrophoretic strategy, complementary to high-performance liquid chromatography, for the separation of different calcitonins (CTs) and calcitonin tryptic digests. Capillary electrophoresis was carried out with a manual capillary electropherograph with "on column" UV absorbance detection at 200 nm. The separation was accomplished in a 70 cm x 50 microns I.D. bare silica capillary. About 6 nl was loaded into the capillary by means of a split-flow system. Except in particular cases, electric fields of 300 V/cm were used at constant voltage. Separations were carried out in 0.05 M citrate buffer pH 2.5 or, alternatively, in 0.05 M borate buffer pH 9.5. A complete resolution of salmon, ASU1,7-eel, and human calcitonins was obtained in citrate and borate buffers. Other CT analogues could be separated only in one of the two buffers. Capillary electrophoresis in citrate buffer was also successful in the separation of the four final trypsin cleavage fragments of salmon calcitonin and, at least tentatively, of the nine intermediate cleavage products.

Antitussive effects of levodropropizine in the dog.

The antitussive activity of levodropropizine (S(-)3-(4-phenyl-piperazine-1-yl)-propane-1,2-diol, DF 526, CAS 99291-25-5) was evaluated after oral administration to the conscious dog. Levodropropizine had a good antitussive activity, comparable with, but having a longer duration of action than dropropizine, the racemate from which it is derived. The antitussive activity of levodropropizine in the dog was approximately 1/20 of that of codeine phosphate.

Effectiveness of levodropropizine against cigarette smoke-induced airway hyperreactivity: possible mechanism.

We verified the possible effect of the new antitussive drug levodropropizine on airway hyperreactivity and lung inflammation induced by cigarette smoke exposure in anaesthetized guinea-pigs. Levodropropizine, administered by aerosol at 25 mg/ml for 30 s completely prevented smoke induced airway hyperreactivity. The protective effect was early in onset (3 min) and lasted up to 30 min. The same dose of codeine, administered in the form of an aerosol, decreased the increase in airway responsiveness induced by smoke inhalation slightly but not significantly. In parallel with the functional results, levodropropizine also inhibited the recruitment of inflammatory cells triggered by smoke exposure within the airway lumen. When levodropropizine was administered i.v. to anaesthetized guinea-pigs, it reduced the bronchocontractile effect of capsaicin dose-dependently, whereas it was without effect against substance P-induced bronchoconstriction. These data demonstrate the ability of levodropropizine to counteract the hyperreactive phenomenon and the associated inflammatory event induced by cigarette smoke exposure, an effect which might depend on its capacity to modulate the activation of the peptidergic system.

Peripheral site of action of levodropropizine in experimentally-induced cough: role of sensory neuropeptides.

The mechanism of action of levodropropizine has been investigated in different models of experimentally-induced cough in guinea-pigs. In particular it has been demonstrated that the antitussive drug has a peripheral site of action by injecting the drug intracerebroventricularly (i.c.v.). In these experiments levodropropizine (40 micrograms/50 microliters i.c.v.) did not prevent electrically-induced cough. On the other hand, codeine (5 micrograms/50 microliters i.c.v.) markedly prevented coughing. A difference in the potency ratio of levodropropizine and codeine has been demonstrated in capsaicin-induced cough; after oral administration, codeine was about two to three times more potent than levodropropizine. However, after aerosol administration the two compounds were equipotent. These data might suggest a peripheral site of action for levodropropizine which is related to sensory neuropeptides. Further support for the role of sensory neuropeptides in the mechanism of action of levodropropizine comes from the results obtained in capsaicin-desensitized animals. In this experimental model levodropropizine failed to prevent the vagally elicited cough in neuropeptide-depleted animals, whereas codeine did not differentiate between control and capsaicin-treated animals. In conclusion, our results support the suggestion that levodropropizine has a peripheral site of action. In addition, the interference with the sensory neuropeptide system may explain, at least in part, its activity in experimentally-induced cough.

Pharmacological studies on the new mucoactive agent (-)-6(S)-hydroxy-4(R)-(1-hydroxy-1-methylethyl)-1-cyclohexene-1-ethanol .

The pharmacological activity and acute toxicity of (-)-6(S)-hydroxy-4(R)-(1-hydroxy-1-methylethyl)-1-cyclohexene-1-ethanol (CO/1408, CAS 103079-06-7), a new mucoactive drug, were evaluated. After oral and intravenous administration CO/1408 increased the pulmonary secretion of fluorescein in rats, as an index of bronchosecretogogue activity. In addition, CO/1408 markedly increased the mucociliary transport rate. In in vitro study CO/1408 did not modify the viscosity of pig gastric mucin. Acute toxicity studies showed a very low toxicity after single dose indicating a high safety level for the doses used in this report. The results obtained point out the potential usefulness of CO/1408 to ameliorate the symptoms observed in some obstructive pulmonary diseases.

Heterogeneity of [3H]-mepyramine binding sites in guinea pig cerebellum and lung.

The nature of histamine receptors in peripheral tissues is still controversial. However, evidence of heterogeneous classes of binding sites for [3H]-mepyramine are reported in the literature. The aim of our study was, therefore, to investigate the nature of this heterogeneity by comparing [3H]-mepyramine binding in a central tissue (cerebellum) and in a peripheral tissue (lung) obtained from guinea pigs and to assess its dependence upon the temperature of incubation. The results revealed that the [3H]-mepyramine interaction in both tissues is temperature-dependent. At 25 degrees C, the interaction between [3H]-mepyramine and the receptors was biphasic in the lung while only a single class of binding site was found in the cerebellum. At 0 degrees C, [3H]-mepyramine interacted with three binding sites in the lung and two in the cerebellum. The behaviour of the reference compounds (clemastine, promethazine and histamine) also supported this temperature-dependence. Moreover, two new compounds (DF 11062 and DF 11113), synthesized in our laboratories and endowed with antihistamine activity, can differentiate between the low affinity site seen at 25 degrees C in the lung and that seen in the cerebellum at 0 degrees C.

Determination of nuvenzepine in human plasma by a sensitive [3H]pirenzepine radioreceptor binding assay.

A sensitive method for the quantitation of small amounts of nuvenzepine, a new M1-selective antimuscarinic drug, in plasma is described. The analytical method involves the use of a radioreceptor binding assay based on [3H]pirenzepine displacement in rat cerebral cortex homogenates; no previous extraction is required. The method is reliable, with an interassay CV ranging from 5 to 10%, and allows the analysis of greater than 100 samples/experiment. The limit of detection is approximately 0.1 ng/assay. Using this method we have determined the plasma levels of nuvenzepine in eight healthy volunteers treated PO with 15 or 25 mg of nuvenzepine.HCl. The pharmacokinetic parameters obtained were (for 15 and 25 mg): Cmax, 64 and 131 ng/mL; AUC0-infinity, 851 and 1379 ng.h/mL; t1/2, 8.6 and 7.2 h. These values are in good agreement with those obtained using an HPLC method. Therefore, this radioreceptor binding assay proved to be simple, rapid, and specific for the determination of low levels of nuvenzepine in human plasma.

Prostaglandins and gastric mucosal protection by esaprazole in rats.

Esaprazole, N-cyclohexyl-1-piperazineacetamide monohydrochloride, was studied for its activity to prevent gastric mucosal damage induced by several necrotizing agents in the rat. Its effects on acid gastric secretion and the role of gastric mucosal prostaglandin generation were also investigated. Esaprazole, given orally, dose dependently prevented the formation of mucosal damage induced by absolute ethanol, 0.2 N NaOH or 0.6 N HCl. This activity occurred at doses lower than the antisecretory doses. Esaprazole was also found to increase the gastric mucosal prostaglandin content but at doses that exceeded the cytoprotective doses. The failure of indomethacin to impair the gastric mucosal protection provided by esaprazole suggests that mechanisms other than mobilization of endogenous prostaglandins may be involved.

Passive cigarette smoke exposure induces airway hyperreactivity to histamine but not to acetylcholine in guinea-pigs.

We have investigated the changes in airway reactivity induced in guinea-pigs by passive cigarette smoke exposure. In particular, we studied the development of airway hyperresponsiveness both in vivo and in vitro after passive exposure of the animals to cigarette smoke in a plexiglass box. Passive smoke exposure significantly (p less than 0.01) increased histamine induced bronchoconstriction. The dose-ratio calculated between histamine dose-response curves constructed in control and smoke exposed animals was 2.13 (95% confidence limits: 1.46-3.09). Acetylcholine induced bronchoconstriction was not potentiated after the same smoke exposure, indicating some specificity of the mediators involved in the airway hyperresponsiveness triggered by passive smoke exposure. The airway hyperreactivity induced by smoke exposure was associated with a specific increase in histamine contraction induced in vitro in lung parenchymal strips but not in tracheae or pulmonary arteries. Maximal histamine contraction in parenchymal strips taken from smoke exposed animals was significantly (p less than 0.01) enhanced by approximately 110% when compared to control. There was a significant (p less than 0.01) increase in the number of macrophages and eosinophils in the bronchoalveolar lavage fluid of animals passively exposed to cigarette smoke, but no mucus hypersecretion was evident. Our data demonstrate the occurrence of airway hyperreactivity induced by passive cigarette smoke exposure, which involves increased smooth muscle reactivity and cell recruitment.

Esaprazole, a new antiulcer agent, stimulates gastric mucus output in the rat.

The effect of esaprazole, a new antiulcer compound endowed with gastro-protective properties, on rat gastric mucus was investigated after acute oral administration. Both soluble (luminal) and insoluble (parietal) mucus were examined for their content of: acidic and neutral glycoproteins; N-acetylneuraminic acid by colorimetric techniques; and fucose by HPLC methods. One hour after dosing (50-200 mg/kg) a 2-15-fold increase in soluble mucus and a 2-4-fold increase in insoluble mucus output were observed. Under the same experimental conditions, carbenoxolone (200 mg/kg, p.o.) caused a similar increase in gastric mucus output. These results demonstrate that quantitative changes in both luminal and parietal mucus occurred after orally dosed esaprazole: it is tempting to speculate that this effect might be responsible, at least in part, for the gastro-protective action of the drug.

A rapid and highly predictive in vitro assay for non-steroidal anti-inflammatory agents.

The inhibition of the production of malonyldialdehyde (MDA) in guinea-pig lung homogenates, incubated in the presence of 50 microM arachidonic acid and 1.4 mM adrenaline, has been exploited as a simple and reliable assay to test in vitro non-steroidal anti-inflammatory agents (NSAIA). The inhibitory potencies of a series of reference NSAIA, which correlated fairly well with in vivo anti-inflammatory activity as determined by carrageenin oedema, are herewith reported. The specificity of the assay was also evaluated by testing up to forty miscellaneous drugs: none of these significantly reduced the MDA production.

Synthesis and pharmacological evaluation of imidazopyridinium derivatives as neuromuscular blocking agents.

A series of new imidazo[1,2-a]pyridinium derivatives have been prepared and studied as neuromuscular blocking agents in rats. These new substances are structurally related to fazadinium whose heterocyclic moieties were retained. All the new synthetized compounds are endowed with competitive neuromuscular blocking activity both in vitro and in vivo. Some were found to be equieffective with fazadinium with similar cardiovascular effects while some others possess more favorable therapeutic indexes.