Amides and formamidines with antinociceptive activity: note II

Forty amides, formamidines and trifluoromethylsulfonylamides bearing on the nitrogen a cyclohexyl residue, eventually 2-substituted, were prepared and tested for analgesic activity against a chemical stimulus. Good activity was exhibited by amides 9, 11 and 28, by formamidine 34, as well as by triflyamide 40. Eleven additional compounds exhibited a moderate activity

Novel LC-MS/MS method for CJ-023423 (Grapiprant) determination in rabbit plasma

Grapiprant is a new anti-inflammatory drug that preferentially targets the EP4 receptor of prostaglandin E2 limiting the wide range of adverse effects caused by the classical non steroideal anti-inflammatory drugs. The aim of this study was to develop and validate a new, simple, sensitive and rapid Liquid Chromatography tandem Mass Spectrometry method (LC-MS/MS) in order to quantify this novel drug in plasma. The method involved a simple liquid extraction followed by a gradient elution with formic acid 0.2% in water and acetonitrile in reverse phase chromatography. The method was validated according to international guidelines determining selectivity, linearity, sensitivity, recovery, matrix effect and precision. Linearity was obtained over a range of 5-1000 ng mL−1. The obtained Limit of Quantitation (LOQ) and of Determination (LOD) were of 5 and 1.5 ng mL−1respectively. Extraction recovery was >73% for all the tested concentrations. Matrix effect, expressed as ion suppression, was ≤9%. The intraday and inter-day precision results showed good RSD values. All the validation parameters were satisfactory making this new method an interesting tool for scientists to further investigations on pharmacokinetics parameters. This validated method was applied to assess the pharmacokinetic of grapiprant in one rabbit administered with 0.5 mg kg-1

Pk/PD evaluations of the novel atypical opioid tapentadol in red-eared slider turtles

The aim of this study was to evaluate the pharmacokinetics and pharmacodynamics of tapentadol (TAP) in red-eared slider turtles after a single intramuscular injection of 5 mg/kg. Turtles (n = 9) were randomly assigned to two treatment groups, according to an open, single-dose, single-treatment, unpaired, two-period crossover design. Group A (n = 5) received a single IM dose of TAP (5 mg/mL) at 5 mg/kg. Group B (n = 4) received a single IM injection of saline (equivalent to the opioid in volume). After a one-month wash-out period, the groups were rotated, and the experiment was repeated. TAP plasma concentrations were evaluated by a validated HPLC-FL method, while an infrared thermal stimulus was applied to the plantar surface of the turtles' hind limbs to evaluate the thermal withdrawal latency (TWL). TAP plasma concentrations were detectable between 1 and 24 h (2141 42 ng/mL, respectively). The TAP-treated group showed a dramatic increase in TWL one hour after drug administration (15.31 +/- 4.73 s). Subsequently, TWL decreased with time. Significant differences between the treatment and control groups were apparent up to 10 h following treatment. A linear relationship (r(2) = 0.98) between the TAP plasma concentration and effect was found. Given these qualities, TAP appears to be an attractive option for antinociception in turtles because of its rapid onset and acceptable duration of effect

Microcystins Presence in Mussels (M. galloprovincialis) and Water of Two Productive Mediterranean’s Lagoons (Sardinia, Italy)

Microcystins (MCs) are hepatotoxins harmful for animal and human health. The most toxic type between them is MC-LR whose presence has been investigated in different reservoirs all around the world. In this work microcystins were monitored in spring and summer in water and mussels (Mytilus galloprovincialis) of two Sardinia lagoons: Cabras and Calich lagoons. A Solid Phase Extraction method was developed to clean and concentrate samples before the Enzyme Linked Immunosorbent Assay (ELISA) and the following Mass Spectrometry detection. MCs presence was detected using the screening ELISA test in both lagoons. MCs peak was revealed in July for water and mussels belonging to Cabras lagoon (0.75±0.07 ng/L in water and 0.12±0.04 ng/g ww in mussels). In water of Calich lagoon there was a constant trend in the concentration of MCs during the considered months, while there was a MCs peak in July (0.6±0.5 ng/g ww) in mussels. The following LC-MS/MS analysis did not reveal MC-LR presence in all analyzed samples. These results can be useful to enrich knowledge on public health and consumer’s safeguard

A Kallikrein-like enzyme in the aorta of normotensive and hypertensive rats

We evaluated whether vascular kallikrein is altered in rats with genetic or experimental hypertension. Group 1 was infused intraperitoneally with angiotensin II (Ang II) or vehicle for 4 weeks; group 2 was injected subcutaneously with deoxycorticosterone (75 mumol/kg once a week) or vehicle for 4 weeks; group 3 consisted of uninephrectomized rats on high sodium intake given deoxycorticosterone or vehicle; and group 4 consisted of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. Active and total kallikrein activity was measured in abdominal aortic homogenates using an amidolytic assay. Ang II increased systolic blood pressure at a dose of 400 nmol/kg per day (146 +/- 6 versus 123 +/- 3 mm Hg in controls, P < .01) but not at 80 nmol/kg per day. Deoxycorticosterone did not increase blood pressure except in uninephrectomized rats on high salt (173 +/- 6 versus 135 +/- 4 mm Hg in controls, P < .01). Blood pressure averaged 194 +/- 2 mm Hg in SHR and 123 +/- 3 mm Hg in WKY rats. Vascular kallikrein was similar in rats given Ang II or vehicle. In deoxycorticosterone-treated rats total kallikrein was higher than in controls (9.2 +/- 0.8 versus 3.5 +/- 0.1 pkat/mg protein, P < .05), whereas active kallikrein did not differ (0.09 +/- 0.04 versus 0.09 +/- 0.03 pkat/mg protein, P = NS). A similar pattern was observed in uninephrectomized deoxycorticosterone-treated rats (active, 0.09 +/- 0.03 versus 0.10 +/- 0.04, P = NS; total, 7.4 +/- 0.7 versus 4.1 +/- 0.2 pkat/mg protein, P < .05). Kallikrein activity was higher in SHR compared with WKY rats (active, 0.34±0.04 versus 0.10±0.03; total, 9.5 + 1.2 versus 4.6±0.3 pkat/mg protein, P<.05). In conclusion, a kallikrein-like enzyme is present in rat aorta. The activity of this enzyme is elevated in rats with genetic hypertension, and it may be regulated by mineralocorticoids

Different response of intracerebroventricular cadmium administration on blood pressure in normal and low urinary kallikrein rats

Cadmium intracerebroventricular (i.c.v.) administration, at definite concentrations, induces a dose-dependent increase in the systemic blood pressure. Kallikreins are suggested to be important regulators of cardiovascular function. We evaluated the effects of 10 μg i.c.v. cadmium on mean blood pressure (MBP) and several urinary parameters such as 24 h urine volume, sodium and potassium excretion and osmolality in a rat strain inbred for low urinary kallikrein and in normal-kallikrein Wistar rats. Low-kallikrein rats (LKR) showed an increase in MBP that, after an initial peak (27% from baseline), persisted higher than basal levels (10%) over 24 h. In normal-kallikrein rats (NKR) a different reaction of blood pressure to cadmium was observed, causing a temporary increase (26% from baseline) of the systemic blood pressure, that returned to normal values within 2 h. In addition, LKR showed a considerable reduction in the urinary volume (UV; 43.0±20 ml/24 h versus 13.2±6 ml/24 h, P < 0.006), with an increase in the urinary osmolality (UOsm; 500±210 mOsm/l versus 1391±245 mOsm/l, P < 0.0002). Sodium (UNa; 1761±432 μEq/24 h versus 1156±522 μEq/24 h, P < 0.03) and potassium excretion (UK; 2186±482 μEq/24 h versus 936±299 μEq/24 h, P < 0.0006) were both significantly reduced. No changes in UV, UOsm and UK were observed in normal urinary kallikrein rats with the exception of UNa excretion that was significantly increased (667±274 μEq/24 h versus 1725±300 μEq/24 h, P < 0.03). These results suggest that a genetically determined defect in urinary kallikrein excretion leads to a different hypertensive response to i.c.v. cadmium and to a different renal excretion of electrolytes. Perhaps the differences of blood pressure response could be due, at least in part, to a different sensitivity of LKR to cadmium: this implies a complex and articulate hypertensive effect of cadmium involving more systems than those supposed so far

An LC—MS—MS method for quantitation of four new phenethylamines (BOX series) in plasma: in vivo application

The appearance of new “designer drugs” in the illicit market poses a serious health risk because they have unknown safety profiles, have a high potential for abuse, high potency, and can lead to devastating health consequences. For this reason, it is desirable to develop validated and reliable analytical screening tests that allow detection of amphetamines and related designer drugs in biological samples. We report a method for separation and quantitation of four new phenethylamines, 4-bromo-2,5-beta-trimethoxyphenethylamine (BOB), 4-methyl-2,5-beta-trimethoxyphenethylamine (BOD), 3,4-methylenedioxy-beta-methoxyphenethylamine (BOH), and 4-methyl-2,5-dimethoxy-beta-hydroxyphenethylamine (BOHD), in plasma. Quantitation was achieved via liquid chromatography—tandem mass spectrometry (LC—MS—MS) in the multiple reaction monitoring mode, using 2,3-dimethoxyphenethylamine-d 3 as internal standard. The method was validated according to international guidelines. The parameters determined were selectivity, sensitivity, matrix effect, linearity, precision, recovery, and stability. All parameters were satisfactory. To remove matrix interference, solid-phase extraction was introduced in the method as clean-up step. The same method was applied in a pharmacokinetic study to monitor the target compounds in rat plasma after a single oral administration. The developed and validated LC—MS—MS method is the first available for quantitation of BOB, BOH, BOD, and BOHD in a biological matrix. This method is recommended for use in forensic and clinical toxicology, because of its sensitivity, selectivity, and simplicity. An important extension of this method could involve its application to other complex matrices

Verapamil prevents the acute hypertensive response to intracerebroventricular cadmium in conscious normotensive rats

Cadmium, a trace element from natural and industrial sources, may contribute to the pathogenesis of arterial hypertension. We evaluated the effect induced by acute intracerebroventricular (icv) administration of cadmium on mean blood pressure of normotensive conscious rats. Intracerebroventricular cadmium (1 to 10 micrograms) produced a dose-dependent, sustained increase in mean blood pressure. The hypertensive response to icv cadmium was significantly (P < .01) prevented by icv pretreatment with verapamil (10 to 100 micrograms). A preventive effect was exerted also by icv nifedipine (100 micrograms); however, this result was attributable, at least in part, to the antihypertensive action of the vehicle, polyethylene glycol. The hypertensive response to icv cadmium was blunted by icv administration of 10 ng clonidine, 10 micrograms vasopressin antagonist, or 10 micrograms bradykinin antagonist (P < .05), but it was not altered by icv enalaprilat (100 micrograms). These results indicate that brain calcium channels play a role in the hypertensive action induced by icv cadmium. Accumulation of cadmium in the brain caused by prolonged exposure to this heavy metal might lead to chronic arterial hypertension

Pharmacokinetic/pharmacodynamic assessments of 10 mg/kg tramadol intramuscular injection in yellow-bellied slider turtles (<i>Trachemys scripta scripta</i>)

In reptiles, administration of opioid drugs has yielded unexpected results with respect to analgesia. The aims of this study were to assess the pharmacokinetic/pharmacodynamic (PK/PD) properties of tramadol and its active metabolite M1 and to evaluate the effect of the renal portal system on the PK/PD parameters in yellow-bellied slider turtles. Turtles (n = 19) were randomly assigned to four treatment groups, according to a masked, single-dose, four-treatment, unpaired, four-period crossover design. Group A (n = 5) received a single i.m. dose of tramadol (50 mg/mL) at 10 mg/kg in the proximal hindlimb. Group B (n = 5) received the same i.m. dose but in the forelimb. Groups C (n = 5) and D (n = 4) received a single i.m. injection of saline (NaCl 0.9%) of equivalent volume to the volumes of tramadol injected in the hind- and forelimb, respectively. Groups were rotated (1-month washout period) until the completion of the crossover study. Tramadol plasma concentrations were evaluated by a validated HPLC-FL method. An infrared thermal stimulus was applied to the plantar surface of the turtles' hindlimbs to evaluate the thermal withdrawal latency (TWL). The two PK profiles of tramadol differed in the first 2 h following administration, but overlapped in the elimination phases. The metabolite M1 was formed in both the treatment groups, showing similar pharmacokinetic trends, although the amount of M1 was significantly higher (20%) in the hindlimb vs. forelimb group. Turtles given tramadol in the hind- and forelimb showed a significant increase in TWL over the periods of 0.5–48 and 8–48 h, respectively. The calculated % maximal possible response (% MPR) was low (about 24%). The PK/PD correlations between M1 plasma concentrations vs. % MPR appeared to show a counterclockwise hysteresis loop shape

Contaminants of Emerging Concern: Antibiotics Research in Mussels from the Coasts of the Tyrrhenian Sea (Sardinia, Italy)

Contaminants of emerging concern (CECs) are compounds found in several environmental compartments whose ubiquitous presence can cause toxicity for the entire ecosystem. Several personal care products, including antibiotics, have entered this group of compounds, constituting a major global threat. It is essential to develop simple and reliable methods by which to quantify these contaminants in several matrices. In this work, mussels were chosen as sentinel organisms to assess environmental pollution and the safety of bivalve mollusk consumption according to the “One Health perspective”. A liquid chromatographic tandem mass spectrometry method (LC-MS/MS) was developed for the quantification of two macrolides, erythromycin (ERY) and azithromycin (AZI), in mussels. This new method was validated according to international guidelines, showing high selectivity, good recoveries (>60% for both of them), sensitivity, and precision. The method was successfully applied for ERY and AZI research in mussels farmed along the Sardinian coasts (Italy), demonstrating itself to be useful for routine analysis by competent authorities. The tested macrolides were not determined in the analyzed sites at concentrations above the limits of detection (LODs). These results demonstrate the food safety of mussels (as concerns the studied antibiotics) and a negligible amount of pollution derived from these drugs in the studied area