Supraclinical Doses Research Articles

Rat model of recalcitrant prosthetic joint infection using biofilm inocula.

Prosthetic joint infection (PJI) is a rare but devastating complication of joint arthroplasty. Biofilm formation around the prosthesis confers tolerance to antibiotics so that treatment is challenging. Most animal models of PJI use planktonic bacteria to establish the infection which fails to reproduce the pathology of chronic infection. We aimed to establish a rat model of Staphylococcus aureus PJI in male Sprague Dawley rats using biofilm inocula and demonstrate its tolerance to frontline antibiotics. Pilot studies indicated that infection could be introduced to the knee joint by a biofilm-coated pin but that handling the prosthetic without disturbing the biofilmwas difficult. We therefore developed a pin with a slotted end and used a miniature-biofilm reactor to develop mature biofilm in this niche. These biofilm-laden pins consistently produced infection of the bone and jointspace. Treatment with high dose cefazolin, 250mg/kg, starting the day of surgery reduced or cleared pin-adherent bioburden within seven days, however when escalation from 25mg/kg to 250mg/kg cefazolin treatment was delayed for 48h, rats were unable to clear the infection.To track infections, we used bioluminescentbacteria, however the bioluminescent signal did not accurately track the degree of infection in the bone and joint space as the signal did not penetrate the bone. In conclusion, we demonstrate that using a custom prosthetic pin, we can generate biofilm in a specific niche using a novel bioreactor setup and initiate a rat PJI that rapidly develops tolerance to supra-clinical doses of cefazolin. This article is protected by copyright. All rights reserved.

Randomized study of the effect of gadopiclenol, a new gadolinium-based contrast agent, on the QTc interval in healthy subjects.

We investigated the effect of gadopiclenol, a new gadolinium-based contrast agent, on the QTc interval at clinical and supraclinical dose, considering the relative hyperosmolarity of this product. This was a single centre, randomized, double-blind, placebo- and positive-controlled, 4-way crossover study. Forty-eight healthy male and female subjects were included to receive single intravenous (i.v.) administrations of gadopiclenol at the clinical dose of 0.1 mmol kg-1 , standard for current gadolinium-based contrast agents, the supraclinical dose of 0.3 mmol kg-1 , placebo and a single oral dose of 400 mg moxifloxacin. The largest time-matched placebo-corrected, mean change from-baseline in QTcF (ΔΔQTcF) was observed 3 hours after administration of 0.1 mmol kg-1 gadopiclenol (2.39 ms, 90% confidence interval [CI]: 0.35, 4.43 ms) and 5 minutes after administration of 0.3 mmol kg-1 (4.81 ms, 90%CI: 2.84, 6.78 ms). The upper limit of the 90% CI was under the threshold of 10 ms, demonstrating no significant effect of gadopiclenol on QTc interval. From 1.5 to 4 hours postdose moxifloxacin, the lower limit of the 90% CI of ΔΔQTcF exceeded 5 ms demonstrating assay sensitivity. Although there was a positive slope, the concentration-response analysis estimated that the values of ΔΔQTcF at the maximal concentration of gadopiclenol at 0.1 and 0.3 mmol kg-1 were 0.41 and 2.23 ms, respectively, with the upper limit of the 90% CI not exceeding 10 ms. No serious or severe adverse events or treatment discontinuations due to adverse events were reported. This thorough QT/QTc study demonstrated that gadopiclenol did not prolong the QT interval at clinical and supraclinical doses and was well tolerated in healthy volunteers. The positive slope of the QTc prolongation vs concentration relationship suggests that hyperosmolarity could be associated with QTc prolongation. However, the amplitude of this effects is unlikely to be associated with proarrhythmia.

Abstract P6-04-15: Oral selective estrogen receptor degrader AZD9496 demonstrated preclinical model specific differences to fulvestrant in estrogen receptor degradation, agonism and anti-tumour effects

Abstract Anti-hormonal therapy has been the mainstay for the treatment of estrogen receptor (ER) positive breast cancer for over 40 years. The selective ER degrader (SERD) fulvestrant (Faslodex) has been shown to provide clinical benefit over aromatase inhibitors and selective ER modulators. However, delivery of its maximum possible efficacy may be limited by the intra muscular route of administration. To overcome this, AstraZeneca developed the orally bioavailable SERD AZD9496. Here we report further preclinical characterisation of AZD9496 with regards to its ability to induce ERα degradation, antagonise and agonise ER in vitro and the impact on tumour xenografts. AZD9496 caused equivalent ERα degradation to fulvestrant in the MCF7 ER+ cell line. Interestingly, in other ER+ cell lines (e.g. CAMA1 and T47D), the maximal level of ERα degradation induced by AZD9496 was 54% of that induced by fulvestrant. RNAseq analysis of cell lines treated with AZD9496 or fulvestrant in the presence of estradiol did not reveal any differences in ER antagonism. Furthermore, no ER agonism was detected in ER+ breast cancer cell lines treated with AZD9496 in the absence of estradiol. However, unlike fulvestrant, AZD9496 was able to induce PR protein expression in the Ishikawa ER+ endometrial cancer cell line, demonstrating a potential to agonise ER. Also, in proliferation assays, while there was little difference in MCF7 cells between the maximal antiproliferative effect of fulvestrant and AZD9496, in CAMA1 and T47D cells the maximal antiproliferative effect of AZD9496 was 75% and 82% respectively of that caused by fulvestrant. In vivo, AZD9496 increased the uterine weight and induced PR expression in the uterus of juvenile rats, consistent with AZD9496 being a partial ER agonist albeit less than tamoxifen. AZD9496, at a maximally efficacious dose, did not induce the same degree of ER degradation or anti-tumour effect as a supraclinical dose of fulvestrant in CTC174, a patient derived xenograft that harbours a D538G ESR1 mutation and grows in the absence of supplemental estradiol. Taken together, these data suggest that, while AZD9496 is equivalent to fulvestrant in terms of ERα degradation and anti-proliferative/anti-tumour effects in some preclinical models, this is not the case in all models. However, it is not clear which, if any, model accurately reflects the clinical setting and how AZD9496 would compare to fulvestrant at exposures achievable in the clinic. Thus, testing AZD9496 vs fulvestrant in ER+ breast cancer patients is crucial to determine potential clinical benefit. Citation Format: Mandy Lawson, Natalie Cureton, Oona Delpuech, Pei Zhang, Azadeh Cheraghchi-Bashi-Astaneh, Sladjana Gagrica, Dawn Trueman, Gareth Maglennon, Daniel Sutton, Bairu Zhang, Jonathan Cairns, Teresa Klinowska, Christopher J Morrow. Oral selective estrogen receptor degrader AZD9496 demonstrated preclinical model specific differences to fulvestrant in estrogen receptor degradation, agonism and anti-tumour effects [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-15.

Effects of Clonidine on the Cardiovascular, Renal, and Inflammatory Responses to Experimental Bacteremia.

Supra-clinical doses of clonidine appear beneficial in experimental sepsis, but there is limited understanding of the effects of clonidine at clinically relevant doses. In conscious sheep, with implanted renal and pulmonary artery flow probes, sepsis was induced by infusion of live Escherichia coli. At 24 h, a high clinical dose of clonidine (HCDC) [1.0 μg/kg/h], a low clinical dose of clonidine (LCDC) [0.25 μg/kg/h] or vehicle, was infused for 8 h. Animals developed hyperdynamic, hypotensive sepsis with acute kidney injury. The HCDC decreased heart rate (153 ± 6 to 119 ± 7 bpm) and cardiac output (5.6 ± 0.4 to 5.0 ± 0.4 L/min), with no reduction in mean arterial pressure (MAP). In contrast, LCDC increased cardiac output with peripheral vasodilatation. Both doses induced a large transient increase in urine output, an increase in plasma osmolality and, with the high dose, an increase in plasma arginine vasopressin. Sepsis increased plasma interleukin-6 (IL-6) and IL-10 and clonidine further increased IL-10 (1.6 ± 0.1 to 3.3 ± 0.7 ng/mL), but not IL-6. Clonidine reduced rectal temperature. During recovery from sepsis, MAP returned to baseline values more rapidly in the HCDC group (P < 0.001). In hyperdynamic, hypotensive sepsis, the effects of clonidine at clinically relevant doses are complex and dose dependent. HCDC attenuated sepsis-related increases in heart rate and cardiac output, with little effect on arterial pressure. It also induced a water diuresis, increased AVP, reduced body temperature, and had an anti-inflammatory action. Low-dose clonidine had similar but less pronounced effects, except that it induced moderate vasodilatation and increased cardiac output.

Effects of short-term exposure to sevoflurane on the survival, proliferation, apoptosis, and differentiation of neural precursor cells derived from human embryonic stem cells.

Data from animal experiments suggest that exposure to general anesthetics in early life inhibits neurogenesis and causes long-term memory deficit. Considering short operating times and the popularity of sevoflurane in pediatric anesthesia, it is important to verify the effects of short-period exposure to sevoflurane on the developing brain. We measured the effects of short-term exposure (2h) to 3%, 6%, or 8% sevoflurane, the most commonly used anesthetic, on neural precursor cells derived from human embryonic stem cells, SNUhES32. Cell survival, proliferation, apoptosis, and differentiation on days 1, 3, 5, and 7 post treatment were analyzed. Treatment with 6% sevoflurane increased cell viability (P=0.046) and decreased apoptosis (P=0.014) on day 5, but the effect did not persist on day 7. Survival and apoptosis were not affected by 3% and 8% sevoflurane; there was no effect of proliferation at any of the tested concentrations. The differentiation of cells exposed to 6% or 8% sevoflurane decreased on day 1 (P=0.033 and P=0.036 for 6% and 8% sevoflurane, respectively) but was again normalized on days 3-7. Clinically relevant treatment with sevoflurane for 2h induces no significant changes in the survival, proliferation, apoptosis, and differentiation of human neural precursor cells, although supraclinical doses of sevoflurane do alter human neurogenesis transiently.

Class III antiarrhythmic drugs amiodarone and dronedarone impair KIR 2.1 backward trafficking.

Drug‐induced ion channel trafficking disturbance can cause cardiac arrhythmias. The subcellular level at which drugs interfere in trafficking pathways is largely unknown. KIR2.1 inward rectifier channels, largely responsible for the cardiac inward rectifier current (IK 1), are degraded in lysosomes. Amiodarone and dronedarone are class III antiarrhythmics. Chronic use of amiodarone, and to a lesser extent dronedarone, causes serious adverse effects to several organs and tissue types, including the heart. Both drugs have been described to interfere in the late‐endosome/lysosome system. Here we defined the potential interference in KIR2.1 backward trafficking by amiodarone and dronedarone. Both drugs inhibited IK 1 in isolated rabbit ventricular cardiomyocytes at supraclinical doses only. In HK‐KWGF cells, both drugs dose‐ and time‐dependently increased KIR2.1 expression (2.0 ± 0.2‐fold with amiodarone: 10 μM, 24 hrs; 2.3 ± 0.3‐fold with dronedarone: 5 μM, 24 hrs) and late‐endosomal/lysosomal KIR2.1 accumulation. Increased KIR2.1 expression level was also observed in the presence of Nav1.5 co‐expression. Augmented KIR2.1 protein levels and intracellular accumulation were also observed in COS‐7, END‐2, MES‐1 and EPI‐7 cells. Both drugs had no effect on Kv11.1 ion channel protein expression levels. Finally, amiodarone (73.3 ± 10.3% P < 0.05 at −120 mV, 5 μM) enhanced IKIR 2.1 upon 24‐hrs treatment, whereas dronedarone tended to increase IKIR 2.1 and it did not reach significance (43.8 ± 5.5%, P = 0.26 at −120 mV; 2 μM). We conclude that chronic amiodarone, and potentially also dronedarone, treatment can result in enhanced IK 1 by inhibiting KIR2.1 degradation.

Dexmedetomidine-Induced Neuroapoptosis Is Dependent on Its Cumulative Dose.

Dexmedetomidine (DEX) has inherent neuroprotective properties that have been attributed to the activation of prosurvival kinases. However, the impact of supraclinical doses of DEX on neuroapoptosis and neuronal viability has not been determined. Rat pups and primary neuronal cells were treated with DEX or ketamine (KET) alone or in combination. Neuroapoptosis was measured by cleaved-caspase-3 expression and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining in brain sections. Expression of prosurvival kinases was measured by Western blot. We measured the impact of DEX with and without α1-adrenergic receptor blockade on the viability of primary neuronal cell cultures. Increasing the cumulative dose of DEX resulted in elevated levels of neuroapoptosis in vivo. Low doses increased, whereas high dose decreased phosphorylation of the prosurvival kinases. KET alone and in combination with DEX produced a greater degree of apoptosis and reductions in expression of these protein kinases than DEX alone. Increasing concentrations of DEX decreased, while coadministration of an α1-adrenergic receptor blocker preserved neuronal viability in vitro. Although DEX is neuroprotective at clinical doses, high cumulative doses and concentrations induce neuroapoptosis, in vivo and in vitro, respectively. Because the current dosing schedules used in humans yield plasma levels that are substantially below concentrations that induce neurotoxicity, low-dose DEX should not be neurotoxic and has the potential to be a neuroprotective adjuvant.

Repositioning metformin in cancer: genetics, drug targets, and new ways of delivery

After sitting many years on the shelves of drug stores as a harmless antidiabetic drug, metformin comes back in the spotlight of the scientific community as a surprisingly effective antineoplastic drug. Metformin targets multiple pathways that play pivotal roles in cancer progression, impacting various cellular processes, such as proliferation, cell death, metabolism, and even the cancer stemness features. The biomolecular characteristics of tumors, such as appropriate expression of organic cation transporters or genetic alterations including p53, K-ras, LKB1, and PI3K may impact metformin's anticancer efficiency. This could indicate a need for tumor genetic profiling in order to identify patients most likely to benefit from metformin treatment. Considering that the majority of experimental models suggest that higher, supra-clinical doses of metformin should be used in order to obtain an antineoplastic effect, new ways of drug delivery could be developed, such as metformin-loaded nanoparticles or incorporation of metformin into microparticles used in transarterial chemoembolization, with the aim of obtaining higher intratumoral drug concentrations and a targeted therapy which will ultimately maximize metformin's efficacy.

Potent cardioprotection from ischemia–reperfusion injury by a two‐domain fusion protein comprising annexin V and Kunitz protease inhibitor

Considerable evidence suggests that coagulation proteases (tissue factor [TF]/activated factor VII [FVIIa]/FXa/thrombin) and their target protease activated receptors (PAR-1/PAR-2) play important roles in myocardial ischemia-reperfusion (I-R) injury. We hypothesized that localized inhibition of TF/FVIIa on the membrane surfaces of ischemic cells could effectively block coagulation cascade and subsequent PAR-1/PAR-2 cell signaling, thereby protecting the myocardium from I-R injury. We recently developed an annexin V-Kunitz inhibitor fusion protein (ANV-6L15) that could specifically bind to anionic phospholipids on the membrane surfaces of apoptotic cells and efficiently inhibit the membrane-anchored TF/FVIIa. In this study, we investigated the cardioprotective effect of ANV-6L15 in a rat cardiac I-R model in comparison with that of hirudin. Left coronary artery occlusion was maintained for 45min followed by 4h of reperfusion in anesthetized Sprague-Dawley rats. One minute before or 2min after coronary ligation, rats received an intravenous bolus injection of ANV-6L15 (2.5-250μgkg(-1) ), vehicle, or hirudin via bolus injection and continuous infusion. ANV-6L15 dose-dependently reduced infarct size by up to 87% and decreased plasma levels of cardiac troponin I, tumor necrosis factor-α, and soluble intercellular adhesion molecule-1, by up to 97%, 96%, and 66%, respectively, with little impact on the coagulation parameters. ANV-6L15 also ameliorated hemodynamic derangements, attenuated neutrophil infiltration and reduced Terminal deoxynucleotidyl transferase dUTP nick end labeling-positive apoptotic cardiomyocytes. Hirudin was less efficacious even at supraclinical dose. ANV-6L15 confers exceptionally potent cardioprotection and is a promising drug candidate for the prevention of myocardial I-R injury.

Effect of subclinical, clinical and supraclinical doses of calcium channel blockers on models of drug-induced hepatotoxicity in rats.

Drug-related hepatotoxicity is the leading cause of acute liver failure, and hepatic problems are responsible for a significant number of liver transplantations and deaths worldwide. Calcium has been associated with various metabolic processes that lead to cell death and apoptosis, and increased cytosolic Ca(2+) has been implicated in hepatotoxicity. This study was designed to investigate the effects of calcium channel blockers (CCBs) on isoniazid-rifampicin, zidovudine and erythromycin-induced hepatotoxicity in rats. Treatment groups comprised control, hepatotoxicant, hepatotoxicant along with each of silymarin, nifedipine, verapamil and diltiazem at subclinical, clinical and supraclinical doses. A day to the end of treatment for each model, rats were subjected to the hexobarbitone-induced hypnosis test. On the last days of treatment, blood samples were collected and serum was analyzed for relevant biochemical parameters. Animals were sacrificed after blood collection and livers were harvested, and samples obtained for in vivo antioxidant indices assay and histopathology. The hepatotoxicants significantly increased serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP), as well as duration of sleep in the hypnosis test. These drugs significantly reduced the hepatic levels of reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and increased the level of malondialdehyde (MDA). The CCBs at the various doses significantly reversed the effects of isoniazid-rifampicin, zidovudine and erythromycin. The results obtained in this study suggest that the CCBs possess hepatoprotective activity in drug-induced hepatotoxicity and may be beneficial at the subclinical and clinical doses.

Dexmedetomidine does not produce vasocontraction on human isolated gastroepiploic artery

BackgroundRecently, the addition of dexmedetomidine to sedation regimens after cardiac surgery had been reported and there is a possibility that dexmedetomidine can cause vasoconstriction. Vasopressin has been used as a prophylactic treatment for refractory vasodilatory shock during coronary artery bypass graft (CABG). Also, vasopressin may play an important role in initiating spasms at the graft artery. Here we evaluate the direct effect of dexmedetomidine on isolated human gastroepiploic arteries and the synergistic effect of dexmedetomidine and vasopressin.MethodsDiscarded gastroepiploic arteries from elective subtotal gastrectomy (n = 10) were used in this study. We measured the level of contraction in isolated human gastroepiploic arteries induced by increasing concentrations of dexmedetomidine (10-10 to 10-6 M) with or without vasopressin (10-10, 10-9 M). Arterial contractions caused by increasing concentrations of vasopressin (10-10 to 10-7.5 M) with or without dexmedetomidine (10-9, 10-7 M) were also measured in the tissue samples.ResultsSupraclinical concentrations of dexmedetomidine elicited contractions at concentrations of 10-7 M and 10-6 M (P < 0.05 versus resting tension). The same concentrations of dexmedetomidine (10-7, 10-6 M) significantly enhanced vasopressin-induced contractions (P < 0.05 versus vasopressin-induced contraction). Vasopressin produced concentration-dependent contractions and vasopressin (10-10, 10-9.5, 10-9 M) also increased the intensity of dexmedetomidine (10-7 M) induced contractions.ConclusionsThere was a synergistic effect between supraclinical doses of dexmedetomidine and vasopressin on the degree of contraction in isolated human gastroepiploic arteries. However, a sedative dose of dexmedetomidine (clinical dose: 0.2-0.7 µg/kg/hr, plasma concentration: 0.36-1.25 ng/ml) did not enhance vasopressin induced-contraction in isolated human gastroepiploic arteries.

The cardiorespiratory and anesthetic effects of clinical and supraclinical doses of alfaxalone in cats

ObjectiveTo determine the cardiorespiratory and anesthetic effects of 0, 5, 15, and 50 mg kg−1 intravenous (IV) alfaxalone in hydroxypropyl beta cyclodextrin (Alfaxan; Jurox Pty Ltd, Rutherford, NSW, Australia) in cats. Study designFour treatments of alfaxalone were administered in sequential order. AnimalsEight healthy adult cats (four male; four female) weighing between 3.71 and 5.91 kg. MethodsCats were instrumented for hemodynamic measurements. Four (0, 5, 15, and 50 mg kg−1) IV doses of alfaxalone were administered over one minute, with a 3-hour washout period between doses 0, 5, and 15 mg kg−1 on Day 0. The 50 mg kg−1 treatment was administered 24 hours later. Measurements of heart rate, aortic systolic, mean, and diastolic blood pressures, pulmonary arterial and right atrial mean pressures, cardiac output, respiratory rate, tidal and minute volumes, and arterial blood pH and blood gases (PaO2, PaCO2) were performed at pre-determined intervals. Systemic vascular resistance and rate pressure product were calculated. The quality of induction, maintenance, and recovery from anesthesia and the response to noxious stimulation were categorically scored. ResultsAlfaxalone administration resulted in dose-dependent cardiorespiratory depression. Decreases in arterial blood pressure and increases in heart rate occurred at higher doses. Most variables returned to baseline by 15-30 minutes. Respiratory rate, minute volume, and PaO2 decreased. Apnea was the most common side effect. Induction and maintenance quality were judged to be good to excellent at all doses and quality of recovery good to excellent at all but the 50 mg kg−1 dose. The duration of anesthesia and unresponsiveness to noxious stimulation increased with dose. The administration of the 50 mg kg−1 dose produced marked cardiorespiratory depression and apnea. Conclusions and clinical relevanceAlfaxalone produced dose-dependent anesthesia, cardiorespiratory depression and unresponsiveness to noxious stimulation in unpremedicated cats. Hypoventilation and apnea were the most common side effects.

The pharmacokinetics and pharmacodynamics of alfaxalone in cats after single and multiple intravenous administration of Alfaxan®at clinical and supraclinical doses

This study aimed to determine the pharmacokinetic parameters and pharmacodynamics of alfaxalone in a 2-hydroxypropyl-beta-cyclodextrin alfaxalone formulation (Alfaxan), Jurox Pty Ltd, Rutherford, NSW, Australia) in cats after single administration at clinical and supraclinical dose rates and as multiple maintenance doses. First, a prospective two-period cross-over study was conducted at single clinical and supraclinical doses. Second, a single group multiple dose study evaluated the effect of maintenance doses. Eight (five female and three male) domestic cats completed the cross-over experiment and six female cats completed the multiple dose study. In the first experiment, alfaxalone was administered intravenously (IV) at 5 or 25 mg/kg with a washout period of 14 days. In the second experiment, alfaxalone was administered IV at 5 mg/kg followed by four doses each of 2 mg/kg, administered at onset of responsiveness to a noxious stimulus. Blood was collected at prescribed intervals and analysed by LCMS for plasma alfaxalone concentration. Noncompartmental pharmacokinetics were used to analyse the plasma alfaxalone data. The plasma clearance of alfaxalone at 5 and 25 mg/kg differed statistically at 25.1 and 14.8 mL/kg/min respectively. The elimination half lives were 45.2 and 76.6 min respectively. Alfaxalone has nonlinear pharmacokinetics in the cat. Nevertheless, for cats dosed with sequential maintenance doses, a regression line through their peak plasma concentrations indicated that there was no clinically relevant pharmacokinetic accumulation. The duration of nonresponsiveness after each maintenance dose was similar at approximately 6 min, indicating a lack of accumulation of pharmacodynamic effect. The cardiovascular and respiratory parameters measured in cats after administration of the labelled doses of Alfaxan were stable. In conclusion, the pharmacokinetics of alfaxalone in cats are nonlinear. At clinical dose rates, however, neither alfaxalone nor its effects accumulated to a clinically relevant extent. Further, in the un-premedicated cat the induction and maintenance of surgical anaesthesia was free of untoward events after a dose of 5 mg alfaxalone/kg body weight followed by four sequential doses of 2 mg/kg as needed (i.e., approximately 7 to 8 mg/kg/h).

Cardiorespiratory and anesthetic effects of clinical and supraclinical doses of alfaxalone in dogs

ObjectiveTo determine the cardiorespiratory and anesthetic effects of 2, 6, and 20 mg kg−1 IV alfaxalone in hydroxypropyl beta cyclodextrin (Alfaxan) in dogs. Study designBlinded four-way crossover randomized by dose. AnimalsEight healthy adult purpose-bred mixed breed dogs (four male, four female) weighing between 12 and 28 kg. MethodsFour (0, 2, 6, 20 mg kg−1) IV treatments of alfaxalone were administered to each dog with a 3-hour washout period between doses. Measurements of heart rate, aortic systolic, mean, and diastolic blood pressures, pulmonary arterial and right atrial mean pressures, cardiac output, respiratory rate, tidal and minute volumes, and arterial blood pH, blood gases (PaO2, PaCO2) were performed prior to and at predetermined intervals after drug administration. Systemic vascular resistance and rate pressure product were calculated. The quality of induction, maintenance, and recovery from anesthesia were categorically scored as was the response to noxious stimulation. ResultsThe administration of alfaxalone resulted in dose-dependent changes in cardiovascular and respiratory parameters. Decreases in arterial blood pressure and increases in heart rate occurred at higher doses with most variables returning to baseline in 15–30 minutes. Respiratory rate, minute volume, and PaO2 decreased and apnea was the most common side effect. The duration of anesthesia increased with dose, and induction, maintenance, and recovery were judged to be good to excellent with all doses studied. Conclusions and clinical relevanceAlfaxalone produced good to excellent short-term anesthesia in unpremedicated dogs. Cardiorespiratory effects were minimal at lower doses. Anesthesia was judged to be good to excellent and associated with unresponsiveness to noxious stimulation for the majority of anesthesia. Hypoventilation and apnea were the most prominent and dose-dependent effects.

Inhibitory effect of tramadol on vasorelaxation mediated by ATP-sensitive K+ channels in rat aorta

Tramadol produces a conduction block similar to lidocaine by exerting a local anesthetic-like effect. The aims of this in vitro study were to determine the effects of tramadol on the vasorelaxant response induced by the adenosine triphosphate-sensitive K(+) (K(ATP)) channel opener, levcromakalim, in an endothelium-denuded rat aorta, and to determine whether this effect of tramadol is stereoselective. The effects of tramadol (racemic, R(-) and S(+): 10(-6), 10(-5), 5 x 10(-5) M), and glibenclamide on the levcromakalim dose-response curve were assessed in aortic rings that had been pre-contracted with phenylephrine. In the rings pretreated independently with naloxone, and glibenclamide, the levcromakalim dose-response curves were generated in the presence or absence of tramadol. The effect of tramadol on the dose-response curve of diltiazem was assessed. Racemic, R(-) and S(+) tramadol (10(-5), 5 x 10(-5) M) attenuated (P < 0.0001) levcromakalim-induced relaxation in the ring with or without naloxone in a dose-dependent manner. The magnitude of the R(-)-tramadol-induced attenuation of vasorelaxant response induced by levcromakalim was greater (P < 0.05) than that induced by S(+)-tramadol. Glibenclamide almost abolished the levcromakalim-induced relaxation. Tramadol, 5 x 10(-5) M, did not significantly alter the diltiazem-induced relaxation. These results suggest that a supraclinical dose (10(-5) M) of tramadol [racemic, R(-) and S(+)] attenuates the vasorelaxation mediated by the K(ATP) channels in the rat aorta. The R(-) tramadol-induced attenuation of vasorelaxation induced by levcromaklim was more potent than that induced by S(+) tramadol. This attenuation is independent of opioid receptor activation.

Alfentanil attenuates phenylephrine-induced contraction in rat aorta

Alfentanil was reported to relax the rat aorta by direct action on the vascular smooth muscle. The aims of this in vitro study were to examine the effect of alfentanil on phenylephrine-induced contractions in the rat aorta and to determine the cellular mechanism associated with this process. Endothelium-denuded aortic rings were suspended in order to record isometric tension. In the rings with or without 10(-6) mol naloxone or 10(-5) mol verapamil, the concentration-response curves for phenylephrine and potassium chloride were generated in the presence or absence of alfentanil (10(-6), 5 x 10(-5), 10(-4) mol). In the rings exposed to a calcium-free isotonic depolarizing solution, the contractile response induced by the addition of calcium was assessed in the presence or absence of alfentanil (5 x 10(-5), 10(-4) mol). Alfentanil (5 x 10(-5), 10(-4) mol) attenuated (P < 0.05) the phenylephrine-induced contraction in the ring with or without 10(-6) mol naloxone but had no effect on the phenylephrine-induced contraction in the rings pretreated with verapamil. Alfentanil (5 x 10(-5), 10(-4) mol) produced a significant rightward shift (P < 0.01) in the potassium chloride dose-response curve, and attenuated the contractile response (P < 0.001) induced by calcium in the calcium-free isotonic depolarizing solution in a dose-dependent manner. A supraclinical dose of alfentanil attenuates the phenylephrine-induced contraction via an inhibitory effect on calcium influx by blocking the l-type calcium channels in the rat aortic vascular smooth muscle.

A Supraclinical Dose of Tramadol Stereoselectively Attenuates Endothelium-Dependent Relaxation in Isolated Rat Aorta

Tramadol, a combination of R(-) and S(+) enantiomers, inhibits both the acetylcholine-mediated response of muscarinic receptors and the muscarine-induced accumulation of cyclic guanosine monophosphate. Our goals in this in vitro study were to investigate the effects of tramadol on endothelium-dependent relaxation induced by acetylcholine, to determine whether this effect of tramadol is stereoselective, and to elucidate the associated cellular mechanism in rat aorta. In endothelium-intact rings precontracted with phenylephrine with or without naloxone, dose-response curves for acetylcholine, histamine, and calcium ionophore A23187 were generated in the presence and absence of tramadol (racemic, R(-) and S(+)). Sodium nitroprusside dose-response curves were generated in the presence and absence of racemic tramadol. Racemic tramadol (5 x 10(-5) 10(-4) M) attenuated acetylcholine-induced relaxation in the rings with or without naloxone. R(-) tramadol, 5 x 10(-5) M, attenuated acetylcholine-induced relaxation, whereas S(+) tramadol, 5 x 10(-5) M, did not. Racemic tramadol (10(-4) M) had no effect on dose-response curves for calcium ionophore A23187 or sodium nitroprusside. Taken together, these results indicate that tramadol, at a supraclinical dose (5 x 10(-5) M), stereoselectively attenuates endothelium-dependent relaxation via an inhibitory effect at levels proximal to nitric oxide synthase activation on a pathway involving nonspecific endothelial receptor activation.

Plasma pharmacokinetics of alfaxalone in dogs after an intravenous bolus of Alfaxan-CD RTU

ObjectiveTo determine the pharmacokinetic parameters of alfaxalone in dogs after the intravenous (IV) administration of clinical and supra-clinical doses of a 2-hydroxypropyl-beta-cyclodextrin (HPCD) alfaxalone formulation (Alfaxan-CD RTU). Experimental designProspective two-period crossover design. AnimalsEight (four male and four female) young adult healthy Beagle dogs. MethodsThe steroid anaesthetic alfaxalone was administered IV at two doses in a crossover design (2 and 10 mg kg−1) with a washout period of 21 days. Blood samples were collected before and up to 8 hours after dosing. Plasma concentrations of alfaxalone were assayed using a liquid chromatograph/mass selective detector technique and analyzed to estimate the main pharmacokinetic parameters by noncompartmental analysis. Results were expressed as mean ± SD. ResultsThe mean duration of anaesthesia from endotracheal intubation to extubation was 6.4 ± 2.9 and 26.2 ± 7.5 minutes, for the 2 and 10 mg kg−1 doses, respectively. The plasma clearance of alfaxalone for the 2 and 10 mg kg−1 doses differed statistically at 59.4 ± 12.9 and 52.9 ± 12.8 mL kg−1 minute−1, respectively (p = 0.008) but this difference was deemed clinically unimportant; the harmonic mean plasma terminal half-lives (t1/2) were 24.0 ± 1.9 and 37.4 ± 1.6 minutes respectively. The volume of distribution was between 2 and 3 L kg−1 and did not differ between the two doses. No sex effect was observed. Conclusions and Clinical relevanceAlfaxalone, as an HPCD formulation (Alfaxan-CD RTU) administered in the dog provides rapid and smooth induction of anaesthesia, satisfactory conditions for endotracheal intubation and a short duration of anaesthesia. There was no clinically significant modification of the pharmacokinetic parameters between sexes and between the clinical (2 mg kg−1) and supra-clinical (10 mg kg−1) doses.

Maternal and fetal distribution of a phosphorothioate oligonucleotide in rats after intravenous infusion

Fetal uptake of an antisense oligonucleotide was evaluated after intravenous (i.v.) dosing of ISIS 2105, a 20-base phosphorothioate oligonucleotide, in timed-pregnant Sprague-Dawley rats. To maximize the potential for fetal exposure, ISIS 2105 was administered as a 3-hr infusion at 6.6 mg/kg/hr with a total dose of 20 mg/kg, or as a continuous 7-day infusion at 0.35 mg/kg/hr with a total dose of 59 mg/kg. This dosing regime is higher than a patient would be expected to receive in the clinical use of oligonucleotides. Infusions were delivered through a jugular vein cannula by syringe pump on gestation day (GD) 19 (3-hr exposure) or by osmotic pumps implanted subcutaneously (s.c.) starting on GD 12 (7-day exposures). After a 3-hr infusion, maternal and fetal plasma concentrations of ISIS 2105 were >100 microg/ml and <0.07 microg/ml, respectively with a maternal fetal ratio of >1,000. Maternal regions of the placenta had twice the oligonucleotide concentration compared to fetal regions of the placenta (6 microg/g vs. 3 microg/g). After this acute exposure the concentrations in fetal kidney and liver were approximately 140- and 500-fold less than the maternal kidney and liver respectively. After 7-day infusion maternal plasma concentrations were 0.82 microg/ml and fetal concentrations were <0.22 microg/ml. By capillary gel electrophoresis (CGE) only the fetal liver consistently had quantifiable oligonucleotide concentrations (range=1.01-4.95 microg/g) compared to a mean concentration of 50.11+/-1.71 microg/g in the maternal liver a maternal to fetal ratio of approximately 10:50 after 7 days of infusion. There was a low level of transfer from dam to fetus, consistent with a slow equilibrium but the permeability of placenta to this 6 kDa polyanionic compound seemed to be limited even at supraclinical doses.

Shockwave Lithotripsy: Dose-Related Effects on Renal Structure, Hemodynamics, and Tubular Function

Shockwave lithotripsy (SWL) predictably damages renal tissue and transiently reduces function in both kidneys. This study characterized the effects on renal function of a supraclinical dose of shockwaves (SWs) (8000) in porcine kidneys and tested the hypothesis that such excessive treatment would intensify and prolong the resulting renal impairment. Pigs aged 6 to 7 weeks were anesthetized and assigned to one of three groups. Groups 1 (N=8) and 2 (N=6) each received 8000 SWs at 24 kV (Dornier HM3) to the lower-pole calix of one kidney. Group 3 (7 pigs) received sham treatment. Renal function was monitored for the first 4 hours after SW treatment in Group 1 and for 24 hours in Group 2. Plasma renin activity was measured in Groups 2 and 3. The renal lesions produced by 8000 SWs comprised 13.8%+/-1.4% of the renal mass. In the 4-hour protocol, this injury was associated with marked reduction of the glomerular filtration rate (GFR), renal plasma flow (RPF), and urinary sodium excretion in both kidneys, although fractional sodium excretion was reduced only in the shocked kidneys. In the 24-hour protocol, GFR and RPF remained below baseline in shocked kidneys at 24 hours. Evidence of progressive ischemic injury was noted in shocked tissue at 24 hours after SW treatment. These findings support the hypothesis that the severity of the renal injury caused by SWL is related to the number of SWs administered and demonstrate the connection in this relation between renal structure and function.