Gantrez AN nanoparticles for ocular delivery of memantine: in vitro release evaluation in albino rabbits.

AIM: To prepare and evaluate the in vitro release of memantine-loaded poly(anhydride) (Gantrez®) nanoparticles (NPs). The clinical safety and retinal toxicity caused by unloaded NPs after sub-Tenon and intravitreal ocular injections were also evaluated. METHODS: Preparation and characterization of this type of NP as well as the in vitro release study are described. Twenty-three healthy New Zealand rabbits were used for clinical and histological assessment after sub-Tenon and intravitreal ocular injections of unloaded NPs. RESULTS: The amount of drug associated with NPs was 55 µg of memantine/mg of NP. The release profile of memantine from this type of NPs was characterized by an initial burst effect, followed by continuous release of the drug for at least 15 days. No relevant complications were found during the clinical follow-up. The histological evaluation suggested that Gantrez NPs are well tolerated after sub-Tenon ocular injection and that signs of inflammation during the first days after intravitreal ocular injections can be considered a normal reaction of the eye's defence mechanism.

Grepafloxacin concentration in ocular tissues after intravenous infusion in rabbits with intraocular inflammation.

PURPOSE: To determine the penetration of grepafloxacin into ocular tissues during experimental ocular inflammation. METHODS: 10 albino and 10 pigmented rabbits underwent a continuous intravenous infusion of the drug 24 h after injecting Salmonella typhimurium toxin intravitreously, creating ocular inflammation. The animals were killed and grepafloxacin levels were determined in plasma and ocular tissues using high performance liquid chromatography. RESULTS: Grepafloxacin levels achieved a steady-state plasma concentration of 1.5 microg/ml. The drug diffused more towards vascularized tissues (chorioretina and iris) in both albino and pigmented rabbits with a tissue/serum ratio higher than 1. Grepafloxacin showed more affinity to pigmented tissue, rising levels of 40,000-50,000 ng/g in the chorioretina and iris in pigmented animals. After inflammation, grepafloxacin intraocular penetration increased in albino animals with levels exceeding the minimum inhibitory concentration for the most common ocular pathogens. CONCLUSION: Grepafloxacin intraocular penetration is higher in pigmented tissues. Ocular inflammation increases the drug penetration into the vitreous.

Determination of enrofloxacin and its primary metabolite, ciprofloxacin, in pig tissues. Application to residue studies.

A simple and sensitive HPLC method has been developed for the simultaneous determination of enrofloxacin (ENR) and ciprofloxacin (CIP) in pig tissue using difloxacin (DIF) as internal standard. Tissue sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 m), followed by extraction with trichloromethane. Fluoroquinolones were separated on a reversed-phase column and eluted with aqueous buffer solution-acetonitrile (80:20, v/v). The concentrations of CIP, ENR and DIF eluted from the column, with retention times of 2.20, 2.73 and 4.38 min, respectively, were monitored by fluorescence detection at lambda(ex) 276 and lambda(em) 442 nm. The detection and quantitation limit were 8 and 25 ng/g, respectively, for both compounds. Standard curves were linearly related to concentration in the range 25-400 ng/g. The consequences of the introduction of minor reasonable variations (ruggedness studies) have also been analysed. Finally, the measurement of the tissue levels of ENR and CIP in the pig tissues after oral administration confirmed the utility of the proposed method.

Ocular penetration of grepafloxacin after intravitreal administration in albino and pigmented rabbits.

Ocular penetration of grepafloxacin into several ocular tissues was determined in albino and pigmented rabbits following a single intravitreal administration. After administration, grepafloxacin was detected in all ocular tissues studied in both breeds of rabbits. The superior mean penetration ratios were found in the chorioretina and lens of albino rabbits, and in the chorioretina, iris and lens of pigmented rabbits. A significantly greater penetration of grepafloxacin was found in the chorioretina and iris of the pigmented rabbits than in those of the albino rabbits. As a final conclusion, grepafloxacin detected in different ocular structures could attain therapeutic concentrations against a variety of ocular conditions.

Histological toxicity of grepafloxacin after intravitreal injection.

PURPOSE: To determine the intravitreal levels of grepafloxacin after intravitreal injection of 80 microg and to evaluate the retinal toxicity after intravitreal injection of different doses of grepafloxacin in rabbit eyes. METHODS: Fifteen female New Zealand white rabbits and 15 female pigmented 'Gigantes de España' rabbits were injected with 80 microg of grepafloxacin into the vitreous cavity. The grepafloxacin concentration was determined with HPLC after 2, 4, 8, 12 and 24 h. Eighteen female rabbits (9 New Zealand white rabbits and 9 pigmented 'Gigantes de España' rabbits) were used for a study of toxicity. The rabbits were divided into 6 treatment groups: group 1 (3 pigmented rabbits) received an intravitreal injection of 80 microg of grepafloxacin in 0.1 ml of saline solution, group 2 (3 white rabbits) 80 microg of grepafloxacin in 0.1 ml, group 3 (3 pigmented rabbits) 800 microg of grepafloxacin, group 4 (3 white rabbits) 800 microg of grepafloxacin, group 5 (3 pigmented rabbits) and group 6 (3 white rabbits) 0.1 ml of saline solution. Clinical examination was performed prior to injection and 24 h and 10 days after surgery. The animals were sacrificed 10 days after the injection, and the eyes were enucleated and fixed for histopathology. The specimens were stained with hematoxylin-eosin and toluidine blue. RESULTS: No relevant complications were found during the clinical follow-up. All the eyes showed no abnormalities in the histologic evaluation. CONCLUSION: Grepafloxacin can be considered as a safe alternative for intravitreal injection for the treatment of intraocular infections.

Pharmacokinetics and ocular penetration of grepafloxacin in albino and pigmented rabbits.

The pharmacokinetics of grepafloxacin were determined in albino and pigmented rabbits following a single 10 mg/kg intravenous administration. The penetration of grepafloxacin into various ocular tissues was also determined after continuous intravenous infusion in both types of animal. Grepafloxacin showed a bicompartmental model of distribution in both pigmented and albino rabbits with significant differences in the pharmacokinetics between the two types of animal. After continuous intravenous infusion, significantly greater penetration of grepafloxacin was found in the iris, cornea and chorioretina of pigmented rabbits compared with albino rabbits.

Penetration of diltiazem into breast milk and its pharmacokinetics in the lactating rabbit.

1. The aim was to investigate the milk transfer and pharmacokinetics of diltiazem (DTZ) in the lactating rabbit following DTZ intravenous (i.v.) administration. In addition, DTZ metabolism in mammary tissue and milk was also studied. 2. The pharmacokinetic parameters that largely determine drug disposition (AUC, VD, CL) showed no significant differences between the non-lactating and lactating rabbit. 3. When DTZ was administered to the lactating rabbit, the observed DTZ milk-to-blood AUC ratio (M/B) closely correlated with the calculated ratio, as predicted by a diffusional model, suggesting that DTZ passes into milk via non-ionic diffusion and that other factors which may affect the milk transfer seem to have limited relevance. 4. After a single intravenous dose of DTZ to the lactating rabbit, deacetyldiltiazem (M1) and demethyldiltiazem (MA) were observed in blood, but only M1 could be detected in milk. 5. In conclusion, DTZ seems to diffuse freely into milk after its i.v. administration to the lactating rabbit and should not be given to nursing mammals because of the potential risk for their young. This risk may be even higher because of the presence of M1 (a pharmacologically active metabolite) in milk after administration of the parent drug.

Development of diltiazem deacetylase and demethylase activities during ontogeny in rabbit.

1. Diltiazem (DTZ) undergoes extensive metabolism in hepatic and extrahepatic tissues. Deacetyldiltiazem (M1) and N-desmethyldiltiazem (MA) are two of the main basic metabolites of DTZ that retain pharmacological activity. The development of DTZ deacetylase and demethylase activities through ontogeny has not been addressed. In order to address this issue, in vitro studies have been carried out using the blood and several tissues of rabbit as enzyme sources. In addition, in vivo studies using a pharmacokinetic approach were carried out to support the in vitro findings. 2. DTZ was incubated with homogenates of selected tissues and in whole blood and DTZ, and its metabolites were assayed by HPLC. In addition, a pharmacokinetic study after intraperitoneal administration of DTZ in the 1-, 8-, 16-, 30-day-old and adult rabbit were also carried out. 3. DTZ deactylase activity was detected whatever the age and tissue examined (including blood). Except in gut homogenates, this activity was shown to be higher at earlier postnatal ages. DTZ demethylase activity was only detected in the liver and gut homogenates and in whole blood. This activity increases from the 1- to 30-day-old rabbit (except for blood), after which it decreases slightly to reach the adult level. 4. In vivo experiments showed a close pharmacokinetic profile throughout ontogeny (except for the 30-day-old rabbit) after DTZ intraperitoneal administration. 5. Extrahepatic metabolism may play a more significant role in the overall metabolism and pharmacokinetics of DTZ at earlier stages of development. 6. Finally, in vivo studies Suggest that age does not seem to modify DTZ disposition and, for this reason, dosage may not have to be taken into account as a function of age.

Altered diltiazem metabolism in the neonatal rabbit following intra-uterine chronic exposure to diltiazem.

1. Diltiazem undergoes extensive metabolism in hepatic and extrahepatic tissues. Deacetyldiltiazem (M1) and N-demethyldiltiazem (MA) are two of the main basic metabolites of diltiazem that retain pharmacological activity. This drug impairs its own metabolism after chronic administration in the adult patient. The study examines the possibility that intra-uterine exposure following chronic maternal therapy with DTZ from mid-gestation to term also impairs DTZ metabolism of its offspring. 2. DTZ was incubated in homogenates from liver, lung, brain and gut and in the whole blood of animals whose mothers were exposed to chronic treatment with diltiazem or unexposed (placebo). DTZ and its metabolites were assayed by HPLC. 3. DTZ deacetylase activity observed in liver, lung and brain homogenates from 1-, 8- and 16-day-old rabbits was significant lower in exposed animals. In gut homogenates, this age-dependent effect was not so clear. This inhibition could not be detected in any organ of 30-day-old rabbits. On the other hand, the activity observed in whole blood was not altered by intra-uterine chronic exposure to DTZ. 4. DTZ demethylase activity showed no differences in tissue homogenates and in whole blood from exposed compared with the unexposed rabbit. 5. In conclusion, the findings suggest that intra-uterine chronic exposure to DTZ has a large and prolonged effect on newborn metabolism deacetylase activity compared with the unexposed rabbit.

Pharmacokinetics of verapamil in New Zealand white rabbits during ontogeny.

The pharmacokinetics of verapamil during ontogeny in rabbits and in vitro verapamil demethylase activity have been investigated in the liver and whole blood. In vivo experiments revealed that the slope of the postdistributive phase as well as the area under the curve and clearance showed significant differences when newborn and adult rabbits were compared. Other pharmacokinetic parameters, such as volume of distribution and plasma protein binding did not show any statistical differences. Liver microsomal preparation samples from 1-, 8- and 16-day-old rabbits displayed approximately 20% of the activity observed in adults. A significant verapamil demethylase activity in the whole blood of rabbits was also noted. The in vivo results show that newborn rabbits have a capacity to eliminate verapamil that is similar or even higher than that of adults. These findings could not be explained with regard to the in vitro liver metabolism of verapamil, although they could with respect to blood metabolism.

Diltiazem blood pharmacokinetics in the pregnant and non-pregnant rabbit: maternal and foetal tissue levels.

1. The aim was to investigate the pharmacokinetics of diltiazem (DTZ) and its metabolites, deacetyldiltiazem (M1) and N-demethyldiltiazem (MA), in the pregnant rabbit following DTZ intravenous administration. In addition, DTZ tissue distribution in both the non-pregnant and pregnant rabbit and foetuses was also studied. 2. The slope of the alpha- and beta-phases increased slightly in six of the eight pregnant rabbits as compared with the non-pregnant animal, but the other pharmacokinetic parameters that largely determine drug disposition (AUC, V(n), CL) showed no significant differences. 3. MA blood disposition was unaltered by pregnancy. However, all the pharmacokinetic parameters calculated for the deacetylated metabolite of DTZ were significantly modified in the pregnant as compared with the non-pregnant rabbit. 4. DTZ tended to concentrate in most of the tissues examined. Significant differences were observed in the DTZ concentration in the uterus and kidney from the pregnant as compared with the non-pregnant rabbit. 5. The findings suggest that DTZ diffuses easily through the placenta, reaching DTZ blood concentrations equivalent to that observed in maternal blood. However, the concentration of DTZ and its metabolites in the selected foetal tissues was either higher (in brain and muscle) or lower than that observed in maternal tissues, suggesting a different tissue affinity and/or a different metabolic activity in the foetuses as compared with the mothers.

Development of a method for the determination of danofloxacin in plasma by HPLC with fluorescence detection.

A simple and sensitive HPLC method has been developed for the determination of danofloxacin (DAN) in plasma. Sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. DAN and the internal standard, sarafloxacin (SAR), were separated on a reversed-phase column, and eluted with aqueous solution-acetonitrile (80:20 v/v). The fluorescence of the column effluent was monitored at lambda(ex) = 338 and lambda(em) = 425 nm. The retention times were 2.80 and 4. 40 min for DAN and SAR, respectively. The method was shown to be linear from 1 to 1500 ng/mL (r(2) = 0.999). The detection and quantitation limit were 1 and 5 ng/mL, respectively. Mean recovery was determined as 80% by the analysis of plasma standards containing 150, 750 and 1500 ng/mL. Inter- and intra-assay precisions were 4.0% and 2.4%, respectively.

Comparison of the pharmacokinetics of verapamil in the pregnant and non-pregnant rabbit: study of maternal and foetal tissue levels.

1. The comparison of the pharmacokinetics of verapamil (VER) has been studied between the non-pregnant and pregnant rabbit following VER intravenous (i.v.) bolus administration. Also studied has been VER tissue distribution in the non-pregnant and pregnant rabbit and its foetuses following an i.v. infusion of VER. 2. When the pharmacokinetic variables were compared between the pregnant and non-pregnant rabbit, it was observed that t(1/2)lambda2 V1 and V(D) were significantly higher in the non-pregnant than in the pregnant rabbit. Moreover, lambda(z) was significantly lower in the non-pregnant than in the pregnant rabbit. However, AUC and CL showed no significant differences between the pregnant and non-pregnant rabbit. 3. When tissue concentrations were examined, it was found that in most of the tissues studied high concentrations of VER were found both in the pregnant and non-pregnant rabbit. Furthermore, VER concentrations in the uterus, heart, spleen and kidney were significantly higher in the non-pregnant than in the pregnant rabbit. 4. The results suggest that VER diffuses poorly through the placenta, given that VER blood concentrations were lower in blood foetuses than in maternal blood. Moreover, the concentrations of VER in the selected foetal tissues were either similar (brain and liver) or lower than those observed in maternal tissues.

Pharmacokinetics of verapamil in lactating rabbits. Prediction of verapamil distribution into rabbit milk.

In this work, we have studied the pharmacokinetics and milk penetration of verapamil following intravenous administration in lactating rabbits. Milk-to-serum drug concentration ratios (M/B(obs)) have been determined using area under the milk and serum concentration-time profiles, and the resulting values have then been compared with those obtained by theoretical classical diffusion milk transfer models that were described by Fleishaker et al. [J. Pharm. Sci. 76 (1987) 189.], Atkinson and Begg [Br. J. Clin. Pharmacol. 25 (1990) 495.], and Stebler and Guentert [Pharm. Res. 9 (1992) 1299.]. The pharmacokinetic profile of verapamil in lactating rabbits following endovenous administration is described in the form of a two-compartment model. Moreover, we detected an important milk transfer after endovenous administration of verapamil in lactating rabbits. M/B(obs) was near 15. The classical diffusional models mentioned were not able to predict this extensive transfer of verapamil into rabbit milk. However, when the classical Fleishaker equation was modified and a stepwise regression was carried out, we found that the M/B(obs) value could be predicted using the plasma and milk protein binding.

Comparative pharmacokinetics of enrofloxacin in mice, rats, rabbits, sheep, and cows.

OBJECTIVE: To compare pharmacokinetic variables of enrofloxacin (ENR) after IV administration in mice, rats, rabbits, sheep, and cows and to perform allometric analysis of ENR. ANIMALS: 47 mice, 5 rats, 5 rabbits, 5 sheep, and 5 cows. PROCEDURE: Serially obtained plasma samples were assayed for ENR concentration, using high-performance liquid chromatography. In vitro plasma protein binding was determined by ultrafiltration. Plasma ENR concentration versus time curves were fitted by use of nonlinear least-squared regression analysis. Pharmacokinetic variables were correlated further with body weight. RESULTS: In all species studied, the best fit was obtained for a two-compartment open model; ENR half-life ranged from 89 minutes in mice to 169 minutes in cows. Volume of distribution was large in all species studied, with values ranging from 10.5 L/kg in mice to 1.5 L/kg in sheep. Body clearance ranged from 68.1 ml/min/kg for mice to 4.6 ml/min/kg for sheep. Unbound ENR was found to be (mean +/- SD) 58+/-2, 50+/-6, 50+/-2, 31+/-2, and 40+/-3% in plasma of mice, rats, rabbits, sheep, and cows, respectively. The only pharmacokinetic variables that could be correlated with body weight were elimination half-life, clearance, and volume of distribution. Allometric exponents denoting proportionality of half-life, body clearance, and volume of distribution with body weight were 0.06, 0.82, and 0.90, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: An allometric approach could provide a suitable method for determining a scale for ENR pharmacokinetics among various mammalian species. This would faciliatate the administration of appropriate doses of ENR to all animals.

Determination of marbofloxacin in plasma samples by high-performance liquid chromatography using fluorescence detection.

A simple and sensitive HPLC method has been developed for the determination of marbofloxacin (MAR) in plasma. Sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. MAR and the internal standard, enrofloxacin (ENR), were separated on a reversed-phase column and eluted with aqueous solution-acetonitrile (80:20). The fluorescence of the column effluent was monitored at lambda(ex) = 338 and lambda(em) = 425 nm. The retention times were 2.20 and 3.30 min for MAR and ENR, respectively. The method was shown to be linear from 15 to 1500 ng/ml (r2 = 0.999). The detection limit was 15 ng/ml. Mean recovery was determined as 90% by the analysis of plasma standards containing 150, 750, and 1500 ng/ml. Inter- and intra-assay precisions were 3.3% and 2.7%, respectively.

Simultaneous determination of enrofloxacin and its primary metabolite, ciprofloxacin, in plasma by HPLC with fluorescence detection.

A simple and sensitive HPLC method has been developed for the simultaneous determination of enrofloxacin (ENR) and ciprofloxacin (CIP) in plasma. Plasma sample preparation was carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. ENR, CIP and the internal standard, sarafloxacin (SAR), were separated on a reversed-phase column, and eluted with aqueous acetonitrile (80:20). The fluorescence of the column effluent was monitorized at lambda(ex) 338 and lambda(em) 425 nm. The retention times were 2.28, 3.30 and 4.40 min for CIP, ENR and SAR, respectively. The detection limit for the two compounds was 10 ng/mL. Standard curves were linearly related to concentration in the range from 1 to 1500 ng/mL. The recovery was 93% for ENR and 75% for CIP.

Determination of orbifloxacin in rabbit plasma by high-performance liquid chromatography with fluorescence detection.

A simple and sensitive high-performance liquid chromatography method is developed for the determination of orbifloxacin (ORB) in rabbit plasma. Sample preparations are carried out by adding phosphate buffer (pH 7.4, 0.1 M) and extracting with trichloromethane. ORB and the internal standard, norfloxacin (NOR), are separated on a reversed-phase column using an aqueous phosphate buffer-acetonitrile (80:20, v/v) mobile phase. The concentrations of ORB and NOR eluting from the column with retention times of 2.16 and 3.09 min, respectively, are monitored by fluorescence detection at 338 (excitation) and 425 nm (emission). The method is shown to be linear from 4 to 1500 ng/mL (regression coefficient r2 = 0.999). The quantitation and detection limits are 4 and 9 ng/mL, respectively. Mean recovery is determined as 92% by the analysis of plasma standards containing 150, 750, and 1500 ng/mL. Inter- and intra-assay precisions were 4 and 3%, respectively.

A common method for the determination of several calcium channel blockers using an HPLC system with ultraviolet detection.

We report a common HPLC method for the single or simultaneous determination of four calcium channel blockers (CCB), namely diltiazem (DTZ), verapamil (VER), nifedipine (NIF) and nitrendipine (NIT) and their active metabolites demetildiltiazem and deacetildiltiazem (MA and M1), norverapamil (NOR), and dehydronifedipine (DHN). DHN was first synthesised in our laboratory and different pH values of the mobil phase were subsequently prepared and tested for chromatographic separation. The detection system and the environmental light conditions were optimised. The best separations of all analytes were obtained using a C(18) column and a mobile phase of methanol, 0.04 M ammonium acetate, acetonitrile and triethylamine (2:2:1:0.04 v/v). Quantitation was performed using imipramine (IMI) as the internal standard. For DTZ and its metabolites (M1 and MA), the wavelength chosen was 237 nm; for VER and its metabolite NOR, it was 210 nm; and, finally for NIF and its metabolite DHN and NIT it was 216 nm. When a simultaneous analysis was carried out the wavelength was of 230 nm. The optimum pH were 7.90 and 7.10 when the separation of NIT and DTZ or VER and NIF were carried out, respectively, and 7.90 when a simultaneous separation was carried out. The detection limit of the assay was less than 8 ng ml(-1) for all compounds, with coefficients of variation less than 7% (for inter- and intra-day) over the concentration range of 1-1000 ng ml(-1). The retention times were less than 11 min. When NIF or NIT were studied, it was necessary to use a sodium vapour lamp in order to avoid the photodegradation which takes place under daylight conditions.

Limited capacity of neonatal rabbits to eliminate enrofloxacin and ciprofloxacin.

The pharmacokinetics of enrofloxacin (ENR) and ciprofloxacin (CIP) in newborn and young rabbits were studied. Rabbits of different ages (1-, 8-, 16-, and 30-day-old) were administered, by the intraperitoneal route (i.p.), a dose of 7.5 mg of either drug/kg. In 1-, 8-, and 16-day-old rabbits, blood samples were drawn by cardiac puncture, under light ether anaesthesia, at predetermined times after drug administration. In 30-day-old rabbits, serial blood samples were drawn through an arterial catheter. Plasma was immediately obtained and analysed using an HPLC method. ENR and CIP plasma protein binding was also determined. The plasma pharmacokinetic profiles of ENR and CIP obtained for 30-day-old rabbits agreed with those reported in the literature for healthy adult rabbits. Nevertheless, significant differences were observed for the body clearance, the slope of the terminal phase, the volume of distribution, and the area under the curve when compared with those for younger animals (1-, 8-, and 16-day-old rabbits), indicating a limited capacity of neonatal rabbits to eliminate ENR and CIP. No differences were found when we compared the calculated values for ENR or CIP plasma protein binding as a function of the postnatal age, indicating that development does not seem to alter the free fraction of these drugs in the rabbit. Taking into account that extensive placental and milk transfer has been reported for these drugs after administration to pregnant or nursing rabbits, a cautious, attitude regarding their use in these animals must be adopted.