A phase I study on pharmacokinetics and pharmacodynamics of higenamine in healthy Chinese subjects

Aim:

To investigate the pharmacokinetics, pharmacodynamics, and safety of higenamine, an active ingredient of Aconite root, in healthy Chinese volunteers.

Methods:

Ten subjects received continuous, intravenous infusion of higenamine at gradually escalating doses from 0.5 to 4.0 μg·kg⁻¹·min⁻¹, each dose was given for 3 min. Blood and urine samples were collected at designated time points to measure the concentrations of higenamine. Pharmacodynamics was assessed by measuring the subject''s heart rate. A nonlinear mixed-effect modeling approach, using the software Phoenix NLME, was used to model the plasma concentration-time profiles and heart rate.

Results:

Peak concentrations (C_max) of higenamine ranged from 15.1 to 44.0 ng/mL. The half-life of higenamine was 0.133 h (range, 0.107–0.166 h), while the area under concentration-time curve (AUC), extrapolated to infinity, was 5.39 ng·h·mL⁻¹ (range, 3.2-6.8 ng·h·mL⁻¹). The volume of distribution (V) was 48 L (range, 30.8–80.6 L). The total clearance (CL) was 249 L/h (range, 199-336 L/h). Within 8 h, 9.3% (range, 4.6%–12.4%) of higenamine was recovered in the urine. The pharmacokinetics of higenamine was successfully described using a two-compartment model with nonlinear clearance. In the pharmacodynamic model, heart rates were related to the plasma drug concentrations using a simple direct effect model with baseline. The E₀, E_max, and EC₅₀ were 68 bpm, 73 bpm and 8.1 μg/L, respectively.

Conclusion:

Higenamine has desirable pharmacokinetic and pharmacodynamic characteristics. The results provide important information for future clinical studies on higenamine.     

The plasma–occupancy relationship of the novel GABAA receptor benzodiazepine site ligand, α5IA,is similar in rats and primates

Background and purpose:

α5IA (3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-yl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine) is a triazolophthalazine with subnanomolar affinity for α1-, α2-, α3- and α5-containing GABA_A receptors. Here we have evaluated the relationship between plasma α5IA concentrations and benzodiazepine binding site occupancy in rodents and primates (rhesus monkey).

Experimental approach:

In awake rats, occupancy was measured at various times after oral dosing with α5IA (0.03–30 mg·kg⁻¹) using an in vivo {[³H]flumazenil (8-fluoro 5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester)} binding assay. In anaesthetized rhesus monkeys, occupancy was measured using {[¹²³I]iomazenil (ethyl 5,6-dihydro-7-iodo-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester)} γ-scintigraphy and a bolus/infusion paradigm. In both rat and rhesus monkey, the plasma drug concentration corresponding to 50% occupancy (EC₅₀) was calculated.

Key results:

In rats, α5IA occupancy was dose- and time-dependent with maximum occupancy occurring within the first 2 h. However, rat plasma EC₅₀ was time-independent, ranging from 42 to 67 ng·mL⁻¹ over a 24 h time course with the average being 52 ng·mL⁻¹ (i.e. occupancy decreased as plasma drug concentrations fell). In rhesus monkeys, the EC₅₀ for α5IA displacing steady-state [¹²³I]iomazenil binding was 57 ng·mL⁻¹.

Conclusions and implications:

Rat plasma EC₅₀ values did not vary as a function of time indicating that α5IA dissociates readily for the GABA_A receptor in vivo. These data also suggest that despite the different assays used (terminal assays of [³H]flumazenil in vivo binding in rats and [¹²³I]iomazenil γ-scintigraphy in anaesthetized rhesus monkeys), these techniques produced similar plasma α5IA EC₅₀ values (52 and 57 ng·mL⁻¹ respectively) and that the plasma–occupancy relationship for α5IA translates across these two species.     

Stimulation of angiotensin AT2 receptors by the non-peptide agonist,Compound 21, evokes vasodepressor effects in conscious spontaneously hypertensive rats

Background and purpose:

Angiotensin type 2 receptor (AT₂ receptor) stimulation evokes vasodilator effects in vitro and in vivo that oppose the vasoconstrictor effects of angiotensin type 1 receptors (AT₁ receptors). Recently, a novel non-peptide AT₂ receptor agonist, Compound 21, was described, which exhibited high AT₂ receptor selectivity.

Experimental approach:

Functional cardiovascular effects of the drug candidate Compound 21 were assessed, using mouse isolated aorta and rat mesenteric arteries in vitro and in conscious spontaneously hypertensive rats (SHR).

Key results:

Compound 21 evoked dose-dependent vasorelaxations in aortic and mesenteric vessels, abolished by the AT₂ receptor antagonist, PD123319. In vivo, Compound 21 administered alone, at doses ranging from 50 to 1000 ng·kg⁻¹·min⁻¹ over 4 h did not decrease blood pressure in conscious normotensive Wistar-Kyoto rats or SHR. However, when given in combination with the AT₁ receptor antagonist, candesartan, Compound 21 (300 ng·kg⁻¹·min⁻¹) lowered blood pressure in SHR only. Further analysis in separate groups of conscious SHR revealed that, at a sixfold lower dose, Compound 21 (50 ng·kg⁻¹·min⁻¹) still evoked a significant depressor response in adult SHR (∼30 mmHg) when combined with different doses of candesartan (0.01 or 0.1 mg·kg⁻¹). Moreover, the Compound 21-evoked depressor effect was abolished when co-infused (50 µg·kg⁻¹·min⁻¹ for 2 h) with the AT₂ receptor antagonist PD123319.

Conclusion and implications:

Collectively, our results indicate that acute administration of Compound 21 evoked blood pressure reductions via AT₂ receptor stimulation. Thus Compound 21 can be considered an excellent drug candidate for further study of AT₂ receptor function in cardiovascular disease.     

Determination of mirtazapine in spiked human plasma and tablets by first derivative spectrofluorimetric method

A sensitive first derivative spectrofluorimetric method (¹D-spectrofluorimetry) was developed for the determination of mirtazapine. Calibration graph for mirtazapine determination was established using the first derivative amplitudes of the mirtazapine emission spectrum (λ_ex = 314 nm) in 0.1 M sulphuric acid measured at 375–435 nm from peak to peak, as the analytical signals. Moreover, the ratio of ¹D-spectrophotometric peak amplitudes at these wavelengths was calculated and used for the detection of the presence of interferences. Linearity range was found to be between 1 and 40 ng ml⁻¹ with correlation coefficient (r) = 0.9999. The limit of quantitation (LOQ) was 1.0 ng ml⁻¹ and the limit of detection (LOD) was 0.2 ng ml⁻¹. The proposed method was validated according to ICH; and it has been applied for the drug determination in human plasma without prior extraction and in tablets. The proposed method’s accuracy, reproducibility, selectivity and simplicity suggest its application in quality control analysis of the drug.     

Simultaneous Effect of Temperature and Irradiance on Growth and Okadaic Acid Production from the Marine Dinoflagellate Prorocentrum belizeanum

Benthic marine dioflagellate microalgae belonging to the genus Prorocentrum are a major source of okadaic acid (OA), OA analogues and polyketides. However, dinoflagellates produce these valuable toxins and bioactives in tiny quantities, and they grow slowly compared to other commercially used microalgae. This hinders evaluation in possible large-scale applications. The careful selection of producer species is therefore crucial for success in a hypothetical scale-up of culture, as are appropriate environmental conditions for optimal growth. A clone of the marine toxic dinoflagellate P. belizeanum was studied in vitro to evaluate its capacities to grow and produce OA as an indicator of general polyketide toxin production under the simultaneous influence of temperature (T) and irradiance (I₀). Three temperatures and four irradiance levels were tested (18, 25 and 28 °C; 20, 40, 80 and 120 µE·m⁻²·s⁻¹), and the response variables measured were concentration of cells, maximum photochemical yield of photosystem II (PSII), pigments and OA. Experiments were conducted in T-flasks, since their parallelepipedal geometry proved ideal to ensure optically thin cultures, which are essential for reliable modeling of growth-irradiance curves. The net maximum specific growth rate (µ_m) was 0.204 day⁻¹ at 25 °C and 40 µE·m⁻²·s⁻¹. Photo-inhibition was observed at I₀ > 40 μEm⁻²s⁻¹, leading to culture death at 120 µE·m⁻²·s⁻¹ and 28 °C. Cells at I₀ ≥ 80 µE·m⁻²·s⁻¹ were photoinhibited irrespective of the temperature assayed. A mechanistic model for µ_m-I₀ curves and another empirical model for relating µ_m-T satisfactorily interpreted the growth kinetics obtained. ANOVA for responses of PSII maximum photochemical yield and pigment profile has demonstrated that P. belizeanum is extremely light sensitive. The pool of photoprotective pigments (diadinoxanthin and dinoxanthin) and peridinin was not able to regulate the excessive light-absorption at high I₀-T. OA synthesis in cells was decoupled from optimal growth conditions, as OA overproduction was observed at high temperatures and when both temperature and irradiance were low. T-flask culture observations were consistent with preliminary assays outdoors.     

Selectivity of action of 8-alkylamino analogues of N6-cyclopentyladenosine in vivo: haemodynamic versus anti-lipolytic responses in rats

1. A₁ adenosine receptor agonists with reduced intrinsic activity may be therapeutically useful as result of an increased selectivity of action. In this study the tissue selectivity of three 8-alkylamino substituted analogues of N⁶-cyclopentyladenosine (CPA) was investigated for haemodynamic and anti-lipolytic effects using an integrated pharmacokinetic-pharmacodynamic approach.
2. Chronically instrumented male Wistar rats received intravenous infusions of 4.0 mg kg⁻¹ 8-methylaminoCPA (8MCPA), 12.0 mg kg⁻¹ 8-ethylaminoCPA (8ECPA), 20.0 mg kg⁻¹ 8-butylaminoCPA (8BCPA) or vehicle during 15 min. During experimentation, serial arterial blood samples were drawn for the determination of agonist concentrations and plasma non-esterified fatty acid (NEFA) levels. Blood pressure and heart rate were monitored continuously. In addition to the CPA analogues, each rat received a rapid bolus infusion of CPA to determine the maximal effects of the full agonist.
3. The concentration-time profiles of the CPA analogues could be described by a bi-exponential function. Values for clearance, volume of distribution at steady state and elimination half-life were 44±5, 48±6 and 39±2 ml min⁻¹ kg⁻¹, 0.97±0.09, 0.84±0.10 and 1.05±0.07 1 kg⁻¹ and 25±2, 28±2 and 40±2 min for 8MCPA, 8ECPA and 8BCPA, respectively (mean±s.e.mean, n=6–8).
4. Different models were used to derive the concentration-effect relationships for heart rate and NEFA, yielding estimates of potency (EC₅₀) and instrinsic activity (E_max) for both effects of the compounds in vivo. On heart rate the compounds acted as partial agonists, with E_max values of −173±14, −131±11 and −71±6 beats min⁻¹ for 8MCPA, 8ECPA and 8BCPA, respectively. These E_max values were significantly lower than the maximal effect of CPA (−208±8 beats min⁻¹). With regard to the anti-lipolytic effect all three compounds were full agonists and lowered NEFA levels to the same extent as CPA (69%). The estimated E_max values were 63±5, 63±4 and 68±2%, respectively.
5. Furthermore, the compounds were more potent in causing anti-lipolytic than cardiovascular effects. The EC₅₀ values for the NEFA and heart rate lowering effects were 37±15, 68±22 and 659±108 ng ml⁻¹ and 164±22, 341±76 and 975±190 ng ml⁻¹ for 8MCPA, 8ECPA and 8BCPA, respectively (mean±s.e.mean, n=6–8).
6. This study demonstrates that partial agonists for the A₁ adenosine receptor have increased selectivity of action in vivo. The 8-alkylamino analogues of CPA may be useful anti-lipolytics with less pronounced haemodynamic side effects.

     

Pharmacokinetic-pharmacodynamic modelling of the anti-lipolytic and anti-ketotic effects of the adenosine A1-receptor agonist N6-(p-sulphophenyl)adenosine in rats

1. The purpose of this study was to develop and validate an integrated pharmacokinetic-pharmacodynamic model for the anti-lipolytic effects of the adenosine A₁-receptor agonist N⁶-(p-sulphophenyl)adenosine (SPA). Tissue selectivity of SPA was investigated by quantification of haemodynamic and anti-lipolytic effects in individual animals.
2. After intravenous infusion of SPA to conscious normotensive Wistar rats, arterial blood samples were drawn for determination of blood SPA concentrations, plasma non-esterified fatty acid (NEFA) and β-hydroxybutyrate levels. Blood pressure and heart rate were monitored continuously.
3. The relationship between the SPA concentrations and the NEFA lowering effect was described by the indirect suppression model. Administration of SPA at different rates and doses (60 μg kg⁻¹ in 5 min and 15 min, and 120 μg kg⁻¹ in 60 min) led to uniform pharmacodynamic parameter estimates. The averaged parameters (mean±s.e., n=19) were E_max: −80±2% (% change from baseline), EC₅₀: 22±2 ng ml⁻¹, and Hill factor: 2.2±0.2.
4. In another group, given 400 μg kg⁻¹ SPA in 15 min, pharmacodynamic parameters for both heart rate and anti-lipolytic effect were derived within the same animal. The reduction in heart rate was directly related to blood concentration on the basis of the sigmoidal E_max model. SPA inhibited lipolysis at concentrations lower than those required for an effect on heart rate. The EC₅₀ values (mean±s.e., n=6) were 131±31 ng ml⁻¹ and 20±3 ng ml⁻¹ for heart rate and NEFA lowering effect, respectively.
5. In conclusion, the relationship between blood SPA concentrations and anti-lipolytic effect was adequately described by the indirect suppression model. For SPA a 6 fold difference in potency was observed between the effects on heart rate and NEFAs, indicating some degree of tissue selectivity in vivo.

     

Metabolism and Disposition of Tribendimidine and Its Metabolites in Healthy Chinese Volunteers

Background: Tribendimidine is a new anthelmintic agent synthesized by Chinese scientists. It is a broad spectrum agent with high activity against parasites. However, its disposition and metabolism remain unknown.Objective: To investigate the metabolism, disposition, and metabolites of tribendimidine in healthy human volunteers.Methods: Twelve healthy Chinese volunteers were chosen after clinical assessment of health status and laboratory tests. They received single oral doses of tribendimidine 400mg enteric-coated tablets. Blood and urine samples were collected at scheduled timepoints. Samples were qualitatively and quantitatively analyzed by liquid chromatography-mass spectrometric (LC-MS) and high performance liquid chromatography (HPLC) methods, respectively.Results: Tribendimidine was rapidly and completely broken down to p-(1-dimethylamino ethylimino) aniline (dADT) and terephthalaldehyde (TPAL). Furthermore, dADT was partially transformed to acetylated dADT, and TPAL completely transformed to terephalic acid (TPAC). The main pharmacokinetic parameters (± SD) of dADT were as follows: elimination half life (t_1/2) 4.74 ± 1.80 h; elimination rate constant (K_e) 0.16 ± 0.06 h⁻¹; apparent volume of distribution (Vd/F) 12.23 ± 8.69L • kg⁻¹; apparent total clearance of the drug from plasma (CL/F) 1.63 ± 0.58L • h⁻¹ • kg⁻¹; area under the plasma concentration-time curve (AUC) from time 0 to time 24 hours (AUC₂₄) 4.29 ± 1.88 μg • mL⁻¹ • h; AUC from time zero to infinity (AUC_∞) 4.45 ± 1.81 μg • mL⁻¹ • h; maximum plasma drug concentration (C_max) 0.64 ± 0.27 μg • mL⁻¹; and time to C_max (t_max) 4.20 ± 0.71 h. A total of 35.28% dADT and 28.50% TPAC were excreted through the urine within 24 hours after tribendimidine administration.Conclusion: These results reveal the disposition, metabolism, and main metabolites of tribendimidine in healthy Chinese volunteers.     

Development of an Enzyme-Linked Immunosorbent Assay Method Specific for the Detection of G-Group Aflatoxins

To detect and monitor G-group aflatoxins in agricultural products, we generated class-specific monoclonal antibodies that specifically recognized aflatoxins G₁ and G₂. Of the final three positive and stable hybridomas obtained, clone 2G6 produced a monoclonal antibody that had equal sensitivity to aflatoxins G₁ and G₂, and did not cross-react with aflatoxins B₁, B₂, or M₁. Its IC₅₀ values for aflatoxins G₁ and G₂ were 17.18 ng·mL⁻¹ and 19.75 ng·mL⁻¹, respectively. Using this new monoclonal antibody, we developed a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA); the method had a limit of detection of 0.06 ng·mL⁻¹. To validate this CI-ELISA, we spiked uncontaminated peanut samples with various amounts of aflatoxins G₁ and G₂ and compared recovery rates with those determined by a standard HPLC method. The recovery rates of the CI-ELISA ranging from 94% to 103% were comparable to those of the HPLC (92% to 102%). We also used both methods to determine the amounts of G-group aflatoxins in five peanut samples contaminated by aflatoxin B₁-positive, and their relative standard deviations ranged from 8.4% to 17.7% (under 20%), which demonstrates a good correlation between the two methods. We further used this CI-ELISA to assess the ability of 126 fungal strains isolated from peanuts or field soils to produce G-group aflatoxins. Among these, seven stains producing different amounts of G-group aflatoxins were identified. Our results showed that the monoclonal antibody 2 G6-based CI-ELISA was suitable for the detection of G-group aflatoxins present in peanuts and also those produced by fungi.     

Antioxidant and anti-inflammatory effects of Marrubium alysson extracts in high cholesterol-fed rabbits

The antioxidant and anti-inflammatory effects of hexane (HEXA), chloroform (CHLORO), ethyl acetate (EA) and total alcoholic (T. ALCOH) extracts of Marrubium alysson in hypercholesterolemic-fed rabbits were evaluated. Hypercholesterolemia was induced in male rabbits by high cholesterol diet (HCD) (350 mg/kg) for 8 weeks. Hypercholesterolemic rabbits were allocated into groups, treated with simvastatin (SIM 5 mg/kg), different extracts of M. alysson at two doses of 250, 500 mg/kg. A normal control group and an HCD control one were used for comparison. Lipid profile, as well as oxidized low density lipoprotein-cholesterol (ox-LDL-C), myeloperoxidase activity (MPO) and superoxide anion production (O₂•⁻), C-reactive protein (CRP) and monocyte chemoattractant protein-1 (MCP-1) were also evaluated. In addition, histological examination of ascending aorta was performed. We found dyslipidemia associated with significant increases in ox-LDL-C 123.5 ± 9.8 nmol MDA/mg non-HDL, MPO activity 0.08 ± 0.05 U/100 mg tissue and O₂•⁻ production 3.5 ± 0.3 nmol cytochrome C reduced/min/g tissue × 10⁻⁴ in hypercholerterolemic rabbits. In addition, there was a significant increase in CRP 6.6 ± 0.49 μmol/L and MCP-1 190.9 ± 6.4 pg/ml and its mRNA expression in HCD. Intima appeared thick with thick plaques surrounding the intima and luminal narrowing. SIM, EA and HEXA extracts of M. alysson had lipid lowering effect, decrease in ox-LDL-C, MPO, O₂•⁻, CRP and MCP-1 mRNA expression with improvement of the pathological picture. M. alysson enhanced the stability of plaque, had lipid lowering, anti-inflammatory and antioxidant activities.     

Effects of moexiprilat on oestrogen-stimulated cardiac fibroblast growth

1. The effects of 2-2-(1-(ethoxycarbonyl)-3-phenylpropyl)-[amino-oxopropyl]−6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-3 carboxylic acid (moexiprilat), 17β-oestradiol (E₂), oestrone (ES) and angiotensin II (AII) on growth and activation of oestrogen receptors and the immediate-early gene egr-1 were investigated in neonatal rat cardiac fibroblasts of female and male origin.
2. In BrdU proliferation assays, oestrone (10⁻⁷–10⁻⁹ M) stimulated cardiac fibroblast growth in a concentration-dependent fashion (maximum at 10⁻⁷ M, 4.0 fold±0.14 in female and 3.1 fold±0.06 in male cells, n=9, P<0.05), while E₂ (10⁻⁷–10⁻⁹ M) had no effect. Moexiprilat (10⁻⁷ M) completely inhibited oestrone-induced cardiac fibroblast growth.
3. Angiotensin II (10⁻⁷ M) induced cardiac fibroblast growth (female 4.1 fold±0.1/male 3.9 fold±0.2; n=9, P<0.05). Angiotensin II induced oestrogen receptor (maximum 21.8 fold at 60 min) and egr-1 (maximum 47.5 fold at 60 min) expression in a time-dependent fashion.
4. In immunoblot experiments, oestrogen activated oestrogen receptor (ES: 12.8 fold±2.0; E₂: 14.7 fold±4.9; n=3, P<0.05) and egr-1 (ES: 5.1 fold,±0.24; E₂: 3.8 fold,±0.25; n=3, P<0.05) expression. The induction of oestrogen receptor and egr-1 protein expression was time-dependent and inhibited by moexiprilat.
5. Our results show that oestrone and 17β-oestradiol reveal a significant difference in their potential to activate cardiac fibroblast growth in female and male cells and that oestrone-stimulated growth is inhibited by moexiprilat. The inhibition of oestrone-stimulated cardiac fibroblast growth by moexiprilat may contribute to the beneficial effects seen in postmenopausal women with hypertensive heart disease treated with ACE inhibitors.

     

Pharmacokinetics of the Melanocortin Type 1 Receptor Agonist PL8177 After Subcutaneous Administration

Background and ObjectivePL8177 is a selective melanocortin 1 receptor agonist in development for the treatment of various immunologic and inflammatory conditions. Here we describe the pharmacokinetics of PL8177 after subcutaneous (sc) delivery in animals and humans.MethodsMice, rats, and dogs were administered sc PL8177 at single doses of 1.0 and 3.0 mg/kg (mice); 1.0, 5.0, and 25.0 mg/kg/day (rats); or 1.5, 8.0, and 40.0 mg/day (dogs). Blood was collected over 24 h (mice) or 28 days (rats and dogs). Safety and pharmacokinetics of single and multiple sc doses were also examined in human volunteers. Two dose levels were tested in two dosing cohorts of 1.0 and 3.0 mg/day for 7 days. Blood samples were collected through Day 1 and on Days 2 to 6 at peak and trough times based on analysis of the first two single-dose cohorts.ResultsIn mice, 3 mg/kg PL8177 resulted in an area under the plasma concentration–time curve from 0 to infinity (AUC_∞) of 1727 ng·h/mL, a maximum plasma concentration (C_max) of 2440 ng/mL, an elimination half-life (t_½) of 0.5 h, and a time to maximum concentration (t_max) of 0.25 h. Results for the 1-mg/kg dose were generally proportional. In rats, mean t_max values were independent of dose and ranged from 0.25 to 1.0 h for single and multiple dosing. C_max values ranged from 516 to 695 ng/mL (1-mg/kg dose) and from 666 to 1180 ng/mL (25-mg/kg dose). In dogs, mean t_max values ranged from 0.4 to 1.3 h for single and multiple dosing. Values for t_max decreased with increasing dose and mean plasma C_max increased less than dose proportionally (96–129 ng·h/mL [1.5 mg], 275–615 ng·h/mL [8.0 mg], and 633–1280 ng·h/mL [40.0 mg]). In humans, PL8177 was observed in the plasma within 15 min after a single dose and persisted for up to 48 h at higher doses. The t_max was 30–45 min (single dose) and 15–45 min (multiple doses). In multiple-dose studies, maximum steady-state plasma concentration (C_max,ss) and AUC_∞ increased with dose. Geometric mean C_max,ss values were 20.1 ng/mL (1.0 mg) and 57.2 ng/mL (3.0 mg). AUC_∞ values were 54.3 ng·h/mL (1.0 mg) and 199 ng·h/mL (3.0 mg). Unchanged PL8177 excreted in the urine was ≤ 1%, and accumulation was minimal.ConclusionPL8177 administration resulted in a consistent pharmacokinetic profile. The measured exposure levels resulted in pharmacologically active PL8177 concentrations at the targeted MC1R. Rapid absorption was seen in healthy volunteers, and multiple-dose administration over 7 days resulted in pharmacokinetic characteristics similar to those observed after single-dose administration. Results support the continued development of PL8177 to treat immunologic and inflammatory conditions.     

An effect of moderate hepatic impairment on the pharmacokinetics and safety of darapladib

Aim/MethodsThis was a phase 1, open label, non-randomized study designed to assess the pharmacokinetics and safety/tolerability of 10 consecutive once daily 40 mg oral doses of darapladib in subjects with moderate hepatic impairment (n = 12) compared with matched healthy volunteers (n = 12).ResultsFor total darapladib, a small increase in total and peak exposure was observed in the subjects with moderate hepatic impairment compared with the subjects with normal hepatic function. The area under the plasma concentration−time curve during a dosing interval of duration τ (AUC(0,τ), geometric mean 223 ng ml⁻¹ h [90% CI 158, 316 ng ml⁻¹h], in moderate hepatic impaired subjects, vs. geometric mean 186 ng ml⁻¹h [90% CI 159, 217 ng ml⁻¹h], in healthy subjects) and maximum concentration (C_max) were 20% and 7% higher, respectively, in the subjects with moderate hepatic impairment than in the healthy control subjects and there was no change in time to maximum concentration (t_max). Protein binding was performed to measure the amount of unbound drug vs. bound. Steady-state was achieved by day 10 for darapladib and its metabolites (M4, M3 and M10). Darapladib was generally well tolerated, with adverse events (AEs) reported by seven subjects in the hepatic impairment group and three subjects in the healthy matched group (five and one of which were drug-related AEs, respectively). The most common AEs were gastrointestinal. These AEs were mostly mild to moderate and there were no deaths, serious AEs or withdrawals due to AEs.ConclusionsThe results of this phase 1 study show that darapladib (40 mg) is well tolerated and its pharmacokinetics remain relatively unchanged in patients with moderate hepatic impairment.     

Effects of prostaglandins and nitric oxide on the renal effects of angiotensin II in the anaesthetized rat

1. The potential influences of nitric oxide (NO) and prostaglandins on the renal effects of angiotensin II (Ang II) have been investigated in the captopril-treated anaesthetized rat by examining the effect of indomethacin or the NO synthase inhibitor, N^ω-nitro-L-arginine methyl ester (L-NAME), on the renal responses obtained during infusion of Ang II directly into the renal circulation.
2. Intrarenal artery (i.r.a.) infusion of Ang II (1–30 ng kg⁻¹ min⁻¹) elicited a dose-dependent decrease in renal vascular conductance (RVC; −38±3% at 30 ng kg⁻¹ min⁻¹; P<0.01) and increase in filtration fraction (FF; +49±8%; P<0.05) in the absence of any change in carotid mean arterial blood pressure (MBP). Urine output (Uv), absolute (UNaV) and fractional sodium excretion (FENa), and glomerular filtration rate (GFR) were unchanged during infusion of Ang II 1–30 ng kg⁻¹ min⁻¹ (+6±17%, +11±17%, +22±23%, and −5±9%, respectively, at 30 ng kg⁻¹ min⁻¹). At higher doses, Ang II (100 and 300 ng kg⁻¹ min⁻¹) induced further decreases in RVC, but with associated increases in MBP, Uv and UNaV.
3. Pretreatment with indomethacin (10 mg kg⁻¹ i.v.) had no significant effect on basal renal function, or on the Ang II-induced reduction in RVC (−25±7% vs −38±3% at Ang II 30 ng kg⁻¹ min⁻¹). In the presence of indomethacin, Ang II tended to cause a dose-dependent decrease in GFR (−38±10% at 30 ng kg⁻¹ min⁻¹); however, this effect was not statistically significant (P=0.078) when evaluated over the dose range of 1–30 ng kg⁻¹ min⁻¹, and was not accompanied by any significant changes in Uv, UNaV or FENa (−21±12%, −18±16% and +36±38%, respectively).
4. Pretreatment with L-NAME (10 μg kg⁻¹ min⁻¹ i.v.) tended to reduce basal RVC (control −11.8±1.4, +L-NAME −7.9±1.8 ml min⁻¹ mmHg⁻¹×10⁻²), and significantly increased basal FF (control +15.9±0.8, +L-NAME +31.0±3.7%). In the presence of L-NAME, renal vasoconstrictor responses to Ang II were not significantly modified (−38±3% vs −35±13% at 30 ng kg⁻¹ min⁻¹), but Ang II now induced dose-dependent decreases in GFR, Uv and UNaV (−51±11%, −41±14% and −31±17%, respectively, at an infusion rate of Ang II, 30 ng kg⁻¹ min⁻¹). When evaluated over the range of 1–30 ng kg⁻¹ min⁻¹, the effect of Ang II on GFR and Uv were statistically significant (P<0.05), but on UNaV did not quite achieve statistical significance (P=0.066). However, there was no associated change in FENa observed, suggesting a non-tubular site of interaction between Ang II and NO.
5. In contrast to its effects after pretreatment with L-NAME alone, Ang II (1–30 ng kg⁻¹ min⁻¹) failed to reduce renal vascular conductance in rats pretreated with the combination of L-NAME and the selective angiotensin AT₁ receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"GR117289","term_id":"238364164","term_text":"GR117289"}}GR117289 (1 mg kg⁻¹ i.v.). This suggests that the renal vascular effects of Ang II are mediated through AT₁ receptors. Over the same dose range, Ang II also failed to significantly reduce GFR or Uv.
6. In conclusion, the renal haemodynamic effects of Ang II in the rat kidney appear to be modulated by cyclooxygenase-derived prostaglandins and NO. The precise site(s) of such an interaction cannot be determined from the present data, but the data suggest complex interactions at the level of the glomerulus.

     

Down-regulation of aortic and cardiac AT1 receptor gene expression in transgenic (mRen-2) 27 rats

1. Transgenic(TG) (mRen-2) rats overexpressing the mouse renin gene develop fulminant hypertension and cardiac hypertrophy. Since the activation of AT₁ receptor by angiotensin II is involved in blood pressure regulation, cardiac performance and myocardial growth, we investigated the biological effects of angiotensin II and the regulation of the AT₁ receptor in the heart and aorta of TGR (mRen-2)27 rats in comparison to control animals.
2. Contraction studies on isolated cardiac muscle strips reveal that angiotensin II exerts no positive inotropic effect on the left ventricular myocardium of both, transgenic and control rats. In contrast, angiotensin II leads via AT₁ receptor activation in the left atrium of control rats to a significant contraction (130±5% of basal contraction) which is not detectable in left atrium preparations of the transgenic animals. Furthermore, AT₁ receptor activation causes a profound contraction of aortic rings isolated from control rats amounting to 1.39±0.2 mN mg⁻¹ wet weight, whereas aortic rings from TGR (mRen-2)27 rats contract only minimally upon angiotensin II stimulation (0.2±0.02 mN mg⁻¹ wet weight).
3. These altered physiological responses of angiotensin II in the transgenic rats are in part due to a marked down-regulation of the AT₁ receptor in atrial, ventricular and aortic tissue of these transgenic animals in comparison to control Sprague-Dawley rats, as shown by radioligand binding assays and quantitative polymerase chain reaction (PCR) experiments. The AT₁ receptor density B_max in the left atrium was 1.3±0.08 fmol mg⁻¹ protein in control rats (K_D 1.1±0.18 nmol l⁻¹) and 0.94±0.15 fmol mg⁻¹ protein (K_D 2.1±0.3 nmol l⁻¹. In the aorta B_max values were 15.1±0.5 fmol mg⁻¹ protein (K_D 1.9±0.27 nmol l⁻¹) for control rats and 11.3±0.76 fmol mg⁻¹ protein (K_D 1.9±0.27 nmol l⁻¹) for the TGR(mRen-2)27 rats AT₁ receptor mRNA was reduced in the transgenic animals to 46±3% in the left atrium, 50±11% in the left ventricle and 40±3% in the aorta, respectively.
4. Together, the AT₁ receptor is down-regulated in TGR (mRen-2)27 rats in comparison to wildtype Sprague Dawley rats leading to a profoundly decreased response of cardiac and aortic tissue upon stimulation with angiotensin II.

     

Catecholamine synthesis and metabolism in the central nervous system of mice lacking α2-adrenoceptor subtypes

Background and purpose:

This study investigates the role of α₂-adrenoceptor subtypes, α_2A, α_2B and α_2C, on catecholamine synthesis and catabolism in the central nervous system of mice.

Experimental approach:

Activities of the main catecholamine synthetic and catabolic enzymes were determined in whole brains obtained from α_2A-, α_2B- and α_2C-adrenoceptor knockout (KO) and C56Bl\7 wild-type (WT) mice.

Key results:

Although no significant differences were found in tyrosine hydroxylase activity and expression, brain tissue levels of 3,4-dihydroxyphenylalanine were threefold higher in α_2A- and α_2C-adrenoceptor KO mice. Brain tissue levels of dopamine and noradrenaline were significantly higher in α_2A and α_2CKOs compared with WT [WT: 2.8 ± 0.5, 1.1 ± 0.1; α_2AKO: 6.9 ± 0.7, 1.9 ± 0.1; α_2BKO: 2.3 ± 0.2, 1.0 ± 0.1; α_2CKO: 4.6 ± 0.8, 1.5 ± 0.2 nmol·(g tissue)⁻¹, for dopamine and noradrenaline respectively]. Aromatic L-amino acid decarboxylase activity was significantly higher in α_2A and α_2CKO [WT: 40 ± 1; α_2A: 77 ± 2; α_2B: 40 ± 1; α_2C: 50 ± 1, maximum velocity (V_max) in nmol·(mg protein)⁻¹·h⁻¹], but no significant differences were found in dopamine β-hydroxylase. Of the catabolic enzymes, catechol-O-methyltransferase enzyme activity was significantly higher in all three α₂KO mice [WT: 2.0 ± 0.0; α_2A: 2.4 ± 0.1; α_2B: 2.2 ± 0.0; α_2C: 2.2 ± 0.0 nmol·(mg protein)⁻¹·h⁻¹], but no significant differences were found in monoamine oxidase activity between all α₂KOs and WT mice.

Conclusions and implications:

In mouse brain, deletion of α_2A- or α_2C-adrenoceptors increased cerebral aromatic L-amino acid decarboxylase activity and catecholamine tissue levels. Deletion of any α₂-adrenoceptor subtypes resulted in increased activity of catechol-O-methyltransferase. Higher 3,4-dihydroxyphenylalanine tissue levels in α_2A and α_2CKO mice could be explained by increased 3,4-dihydroxyphenylalanine transport.     

Development and Characterization of Pharmacokinetic Parameters of Fast-Dissolving Films Containing Levocetirizine

A fast-dissolving film containing levocetirizine, a non-sedative antihistamine drug, was developed using pullulan, xanthan gum, propylene glycol, and tween 80 as the base materials. The drug content of the prepared films was within an acceptable limit as prescribed by the USP. The film exhibited excellent stability for four months when stored at 40 °C and 75% humidity. In vitro dissolution studies suggested a rapid disintegration, in which most of levocetirizine (93.54 ± 3.9%) dissolved within 90 seconds after insertion into the medium. Subsequently, Sprague–Dawley rats were used to compare the pharmacokinetic properties of the film preparation administered to the oral cavity, to those with oral administration of the pure drug solution. The pharmacokinetic parameters were similar between the two groups in which AUC_0–t (ng h/ml), AUC_0–∞ (ng h/ml) C_max (ng/ml), T_max (min), K_el (h⁻¹), and t_1/2 (h) of the reference were 452.033 ± 43.68, 465.78 ± 48.16, 237.16 ± 19.87, 30, 0.453 ± 0.051, and 1.536 ± 0.118, respectively, for the film formulation 447.233 ± 46.24, 458.22 ± 46.74, 233.32 ± 17.19, 30, 0.464 ± 0.060, and 1.496 ± 0.293, respectively. These results suggest that the present levocetirizine containing fast-dissolving film is likely to become one of the choices to treat different allergic conditions.     

The Effect of Pioglitazone on Pharmacokinetics of Carbamazepine in Healthy Rabbits

Introduction

Drug–drug interactions can lead to serious and potentially lethal adverse events. In recent years, several drugs have been withdrawn from the market due to interaction-related adverse events. The objective of this study was to evaluate the pharmacokinetic interaction between pioglitazone (PG) and carbamazepine (CBZ) in healthy male rabbits.

Methods

A randomized, two-crossover design study was conducted in six healthy male rabbits. The study consisted of two periods: period one, when each rabbit received a single dose of 70 mg CBZ-suspension. Period two, when each rabbit received a single dose of 70 mg CBZ-suspension co-administered with a single dose of 1.5 mg PG with a washout period of one week between the two periods. Serial blood samples were collected over a period of 48 h. Chemiluminescent enzyme immunoassay (CLEIA) was used to measure CBZ in serum. Pharmacokinetic (PK) parameters C_max, T_max, t _1/2, AUC_0-t, AUC _0-∞, and k_e were determined for the two periods using non-compartmental analysis.

Results

In the two periods of treatment, C_max, T_max, AUC_0-t, AUC_0-∞, t _½ and k_e for CBZ were administered alone and in combination with PG. C_max, the mean peak plasma concentration was 4.33 ± 2.4 μg/mL versus 4.76 ± 2.1 μg/ml, t_max, time taken to reach, was 2.91 ± 1.11 h versus 3.6 ± 1.83 h, total area under the curve AUC_0-t was 64.90 ± 43.6 μg·h/ml versus 102.90 ± 66.9 μg·h/ml, AUC_0-∞ was 74.0 ± 52.6 μg·h/ml versus 124.3 ± 85 μg·h/mL, t _½ was 14.10 ± 2.5 h versus 16.43 ± 6.43 h and elimination rate constant k_e was 0.050 ± 0.009 h⁻¹ versus 0.057 ± 0.049 h⁻¹, respectively. No statistical differences were found in pharmacokinetic of CBZ in both cases (P > 0.05).

Conclusion

The result of the study demonstrated that PG does not affect pharmacokinetic parameters of CBZ. Therefore, no cautions regarding dose or administration pattern of CBZ with PG should be taken.     

Flavocoxid,a dual inhibitor of cyclooxygenase and 5-lipoxygenase,blunts pro-inflammatory phenotype activation in endotoxin-stimulated macrophages

Background and purpose:

The flavonoids, baicalin and catechin, from Scutellaria baicalensis and Acacia catechu, respectively, have been used for various clinical applications. Flavocoxid is a mixed extract containing baicalin and catechin, and acts as a dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (LOX) enzymes. The anti-inflammatory activity, measured by protein and gene expression of inflammatory markers, of flavocoxid in rat peritoneal macrophages stimulated with Salmonella enteritidis lipopolysaccharide (LPS) was investigated.

Experimental approach:

LPS-stimulated (1 µg·mL⁻¹) peritoneal rat macrophages were co-incubated with different concentrations of flavocoxid (32–128 µg·mL⁻¹) or RPMI medium for different incubation times. Inducible COX-2, 5-LOX, inducible nitric oxide synthase (iNOS) and inhibitory protein κB-α (IκB-α) levels were evaluated by Western blot analysis. Nuclear factor κB (NF-κB) binding activity was investigated by electrophoretic mobility shift assay. Tumour necrosis factor-α (TNF-α) gene and protein expression were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. Finally, malondialdehyde (MDA) and nitrite levels in macrophage supernatants were evaluated.

Key results:

LPS stimulation induced a pro-inflammatory phenotype in rat peritoneal macrophages. Flavocoxid (128 µg·mL⁻¹) significantly inhibited COX-2 (LPS = 18 ± 2.1; flavocoxid = 3.8 ± 0.9 integrated intensity), 5-LOX (LPS = 20 ± 3.8; flavocoxid = 3.1 ± 0.8 integrated intensity) and iNOS expression (LPS = 15 ± 1.1; flavocoxid = 4.1 ± 0.4 integrated intensity), but did not modify COX-1 expression. PGE₂ and LTB₄ levels in culture supernatants were consequently decreased. Flavocoxid also prevented the loss of IκB-α protein (LPS = 1.9 ± 0.2; flavocoxid = 7.2 ± 1.6 integrated intensity), blunted increased NF-κB binding activity (LPS = 9.2 ± 2; flavocoxid = 2.4 ± 0.7 integrated intensity) and the enhanced TNF-α mRNA levels (LPS = 8 ± 0.9; flavocoxid = 1.9 ± 0.8 n-fold/β-actin) induced by LPS. Finally, flavocoxid decreased MDA, TNF and nitrite levels from LPS-stimulated macrophages.

Conclusion and implications:

Flavocoxid might be useful as a potential anti-inflammatory agent, acting at the level of gene and protein expression.