Roberta Merlanti

Sublethal effects of trimethoprim on four freshwater organisms.

Sublethal effects of trimethoprim (TMP) were evaluated in four freshwater organisms: Pseudokirchneriella subcapitata and Lemna minor (growth inhibition), Daphnia magna (reproduction and growth inhibition) and Poecilia reticulata (swimming activity inhibition). Cytochrome P4501A induction was also evaluated in P. reticulata. TMP showed varying levels of toxicity in the four test performed, with NOEC for the various endpoints in the range of 3.12-25 mg L(-1). The compound was active on P. reticulata at concentration ≥ 50 mg L(-1) causing inhibition of swimming activity. In the same organism an induction of CYP1A protein, mainly in kidney, gills and intestine, was also detected. L. minor was more sensitive than unicellular algae to TMP, with a NOEC of 12.5 mg L(-1). The lowest NOEC (3.12 mg L(-1)) was obtained in D. magna reproduction test and then a Risk Quotient of <0.03 was calculated by comparing the PNEC (31.2 μg L) and the TMP concentrations usually detected in freshwater (<1 μg L(-1)). However, based on recently reported data, it was concluded that while TMP concentrations normally detected in surface water are below those able to evoke appreciable biological effects in the various aquatic organisms, TMP concentrations in aquaculture and hospital effluents can be one to three orders of magnitude higher. Furthermore, the co-occurrence and additive effects of other antifolic agents should be taken into account for a cautious risk assessment of the drug.

Effects of enrofloxacin, ciprofloxacin, and trimethoprim on two generations of Daphnia magna.

Multigenerational tests on Daphnia magna were performed exposing two subsequent generation to enrofloxacin (EFX) and its metabolite ciprofloxacin (CPX), and to trimethoprim (TMP). Mortality rate of 100% and 50% was detected in F0 at concentrations of ≥ 13 mgL(-1) (EFX) and 50 mgL(-1) (TMP), respectively. In F1 with respect to F0, both for growth and reproduction, a worsening trend of the response with EFX, a similar response with CPX and an attenuating trend with TMP was observed. Furthermore, the lowest EC20 for reproduction inhibition (1.3 mgL(-1)) was calculated for F1 exposed to EFX. However, other experimentations, longer and more complex, are necessary in order to confirm that EFX is more hazardous to daphnids than CPX and TMP. EC50 measured for the three assayed antibacterials were in the 6.5-37 mgL(-1) range therefore environmental unrealistic, except in case of exceptional contaminations that may occur in relation to poorly controlled wastewaters from pharmaceutical factories or excessive use of prophylactic treatments in aquaculture.

High performance liquid chromatography determination of cytochrome P450 1A and 2C activities in bovine liver microsomes

This study reports fluorescence high performance liquid chromatography (HPLC) and UV-Vis HPLC methods for the determination of 7-ethoxyresorufin O-deethylase (EROD) and tolbutamide methylhydroxylase (TMH) activities, respectively, using bovine liver microsomes. The detection limits were 0.022 and 5.5 pmol on the column, respectively; intra-day and inter-day precisions (expressed as relative standard deviation) were <10%. Both methods showed enough sensitivity to allow for an accurate determination of enzyme kinetic parameters according to Michaelis-Menten plots and the results were: K(m)=0.23+/-0.051 microM, V(max)=0.488+/-0.035 nmol/min/mg protein for EROD activity, and K(m)=1010+/-155.7 microM, V(max)=0.089+/-0.006 nmol/min/mg protein for TMH activity. An Eadie-Hofstee plot analysis showed that in bovine liver microsomes, EROD and TMH activities followed a monophasic kinetic pattern. alpha-Naphthoflavone, a cytochrome P450 1A1/2 (CYP1A1/2) inhibitor, and sulfaphenazole, a cytochrome P450 2C9 (CYP2C9) inhibitor, decreased EROD and TMH activities, respectively. The sensitivity of the methods allowed the use of microsomes with low enzyme activity, such as those from veal calf liver. Thus, EROD and TMH activities may be adopted as markers for the evaluation of CYP1A and CYP2C9-like activities in liver microsomes from veal and beef cattle.

Acute toxicity evaluation of four antibacterials with Daphnia magna

Acute toxicity evaluation of four antibacterials with Daphnia magna G. Gallina & C. Poltronieri & R. Merlanti & M. De Liguoro Published online: 8 August 2008 # Springer Science + Business Media B.V. 2008

Pharmacokinetics of dexmedetomidine combined with methadone following oral-transmucosal and intramuscular administration in dogs

Oral-transmucosal (OTM) drug delivery refers to noninvasive and painless administration of medical preparations through any oral cavity membrane to achieve systemic effects (Sattar et al., 2014). Regarding sedative drugs, OTM administration is very attractive in veterinary medicine, especially for patients difficult to inject and restrain (Messenger et al., 2016). This study aims to compare the pharmacokinetics of dexmedetomidine after OTM and intramuscular (IM) administration combined with methadone. After obtaining Ethical Committee approval and owner’s written consent, eight dogs, were administered with dexmedetomidine (10 mg/kg) and methadone (0.4 mg/kg) by OTM and other 4 dogs by IM route. Blood samples were collected at prefixed times up to four hours. Dexmedetomidine was quantified by a validated HPLC-MS method. On dexmedetomidine concentrations, a pharmacokinetic analysis was carried out with a noncompartmental approach (Phoenix WinNonlin ® 7.0, Pharsight, Cary, NC). Mean ± SD terminal half-lives of dexmedetomidine were 187.42 ± 109.66 and 94.78 ± 34.08 min after OTM and IM administration, respectively. Maximum serum (C max ) concentrations were 0.83 ± 0.32 and 9.09 ± 2.46 ng/mL for OTM and IM administration, respectively. Time to maximum concentration (T max ) were 44.38 ± 32.16 and 21.25±11.39 min by OTM and IM administration, respectively. Area under the curve from 0 to the last measured concentration (AUC last ) were 103.75 ± 30.23 and 614.87 ± 77.15 min*ng/mL for OTM and IM administration, respectively. C max , T max and AUC last values by OTM route demonstrate a lower and delayed absorption of the drug compared to IM. To complete the study, the pharmacokinetic analysis of methadone is foreseen, so as a clinical trial to compare the clinical effects of the combination of dexmedetomidine and methadone by OTM and IM administration and to establish an effective dosage of oral-transumucosal route in dogs for this association.