RP-HPLC Estimation of Imipramine Hydrochloride and Diazepam in Tablets

A simple and rapid reversed phase-high performance liquid chromatographic method was developed for simultaneous determination of imipramine hydrochloride and diazepam in pharmaceutical formulations. The elution was done in isocratic mode utilizing a mobile phase consisting of methanol:water:0.1M sodium acetate (30:50:20 v/v/v) on Chromosil C18 column with a flow rate of 1.0 ml/min and with detection at 243 nm. The measured retention time was 3.33±0.02 min for imipramine hydrochloride and 4.64±0.02 min for diazepam. Linearity was measured in the range 25-150 μg/ml for imipramine hydrochloride (r²=0.999) and in the range 5-30 μg/ml for diazepam (r²=0.9994), respectively. The limits of detection and quantitation were 0.03 and 0.1 μg/ml for imipramine hydrochloride and 0.02 and 0.07 μg/ml for diazepam. Satisfactory validation was also obtained from recovery (100.95-101.52% for imipramine hydrochloride and 99.47-100.33% for diazepam) studies, intraday and interday precision (<2%) and robustness results. The reported method was the first study of these drugs in combination and could be employed for routine quantitative determination of imipramine hydrochloride and diazepam in tablets.     

Development and Validation of a Reversed-Phase High-performance Liquid Chromatography Method for Determination of Desmopressin in Chitosan Nanoparticles

A simple isocratic reversed-phase high performance liquid chromatographic method was developed for determination of released desmopressin from chitosan nanoparticles in the in vitro media. The chromatographic separation was achieved with acetonitrile/water (25:75, v/v), in which water contained 0.1% v/v trifluoroacetic acid with pH=2.5 as mobile phase, a Chromolith^® Performance RP-18e column (150×4.6 mm; 5 μm) kept at 40° and ultraviolet detection at 220 nm. The compound was eluted isocritically at a constant flow rate of 1.6 ml/min. The method was validated according to the International Conference on Harmonisation guidelines. The validation characteristics included accuracy, precision, linearity rang, selectivity, limit of detection, limit of quantitation and robustness. The calibration curve was linear (r>0.9999) over the concentration rang 0.5-100 μg/ml. The limit of detection and limit of quantitation in the release media were 0.05 and 0.5 μg/ml, respectively. The proposed method had an accuracy of and intra- and inter-day precision <4.2. Furthermore, to evaluate the performance of the proposed method, it was used in the analysis of desmopressin level in real samples containing chitosan nanoparticles in the in vitro media.     

Determination of stress-induced degradation products of cetirizine dihydrochloride by a stability-indicating RP-HPLC method

Background

A new, simple and accurate stability-indicating reverse phase high performance liquid chromatography method was developed and validated during the early stage of drug development of an oral lyophilizate dosage form of cetirizine dihydrochloride.

Methods

For RP-HPLC analysis it was used an Eclipse XDB C8 column 150 mm × 4.6 mm, 5 μm (Agilent columns, Barcelona, Spain) as the stationary phase with a mobile phase consisted of a mixture of 0.2 M K₂HPO4 pH 7.00 and acetonitrile (65:35, v/v) at a flow rate of 1 mL min ⁻¹. Detection was performed at 230 nm using diode array detector. The method was validated in accordance with ICH guidelines with respect to linearity, accuracy, precision, specificity, limit of detection and quantification.

Results

The method results in excellent separation between the drug substance and its stress-induced degradation products. The peak purity factor is >950 for the drug substance after all types of stress, which confirms the complete separation of the drug substance peak from its stress induced degradation products.Regression analysis showed r² > 0.999 for cetirizine dihydrochloride in the concentration range of 650 μg mL ⁻¹ to 350 μg mL⁻¹ for drug substance assay and a r² > 0.999 in the concentration range of 0.25 μg mL⁻¹ to 5 μg mL⁻¹ for degradation products. The method presents a limit of detection of 0.056 μg mL ⁻¹ and a limit of quantification of 0.25 μg mL⁻¹. The obtained results for precision and accuracy for drug substance and degradation products are within the specifications established for the validation of the method.

Conclusions

The proposed stability-indicating method developed in the early phase of drug development proved to be a simple, sensitive, accurate, precise, reproducible and therefore useful for the following stages of the cetirizine dihydrochloride oral lyophilizate dosage form development.     

Histamine mediates behavioural and metabolic effects of 3-iodothyroacetic acid,an endogenous end product of thyroid hormone metabolism

BACKGROUND AND PURPOSE

3-Iodothyroacetic acid (TA1) is an end product of thyroid hormone metabolism. So far, it is not known if TA1 is present in mouse brain and if it has any pharmacological effects.

EXPERIMENTAL APPROACH

TA1 levels in mouse brain were measured by HPLC coupled to mass spectrometry. After i.c.v. administration of exogenous TA1 (0.4, 1.32 and 4 μg·kg⁻¹) to mice, memory acquisition-retention (passive avoidance paradigm with a light-dark box), pain threshold to thermal stimulus (51.5°C; hot plate test) and plasma glucose (glucorefractometer) were evaluated. Similar assays were performed in mice pretreated with s.c. injections of the histamine H₁ receptor antagonist pyrilamine (10 mg·kg⁻¹) or the H₂ receptor antagonist zolantidine (5 mg·kg⁻¹). TA1 (1.32 and 4 μg·kg⁻¹) was also given i.c.v. to mice lacking histidine decarboxylase (HDC^−/−) and the corresponding WT strain.

KEY RESULTS

TA1 was found in the brain of CD1 but not of HDC mice. Exogenous TA1 induced amnesia (at 0.4 μg·kg⁻¹), stimulation of learning (1.32 and 4 μg·kg⁻¹), hyperalgesia (0.4, 1.32 and 4 μg·kg⁻¹) and hyperglycaemia (1.32 and 4 μg·kg⁻¹). All these effects were modulated by pyrilamine and zolantidine. In HDC^−/− mice, TA1 (1.32 and 4 μg·kg⁻¹) did not increase plasma glucose or induce hyperalgesia.

CONCLUSIONS AND IMPLICATIONS

Behavioural and metabolic effects of TA1 disclosed interactions between the thyroid and histaminergic systems.     

Determination of Ochratoxin A in Wheat and Maize by Solid Bar Microextraction with Liquid Chromatography and Fluorescence Detection

Solid bar microextraction (SBME), followed by liquid chromatography with fluorescence detection (HPLC-FLD), for the quantification of ochratoxin A in wheat and maize was developed. Ground wheat and maize grains were extracted with acetonitrile-water-acetic acid (79:20:1, v/v/v), followed by defatting with cyclohexane, and subjected to SBME-LC-FLD analysis. SBME devices were constructed by packing 2 mg sorbent (C18) into porous polypropylene micro-tubes (2.5 cm length, 600 μm i.d., and 0.2 μm pore size). SBME devices were conditioned with methanol and placed into 5 mL stirred sample solutions for 70 min. After extraction, OTA was desorbed into 200 μL of methanol for 15 min, the solution was removed in vacuum, the residue was dissolved in 50 μL of methanol-water (1:1, v/v) and ochratoxin A content was determined by HPLC-FLD. Under optimized extraction conditions, the limit of detection of 0.9 μg·kg⁻¹ and 2.5 μg·kg⁻¹ and the precision of 3.4% and 5.0% over a concentration range of 1 to 100 μg·kg⁻¹ in wheat and maize flour, respectively, were obtained.     

Development and Validation of an RP-HPLC Method for Quantitative Estimation of Eslicarbazepine Acetate in Bulk Drug and Tablets

A convenient, simple, accurate, precise and reproducible RP-HPLC method was developed and validated for the estimation of eslicarbazepine acetate in bulk drug and tablet dosage form. Objective was achieved under optimised chromatographic conditions on Dionex RP-HPLC system with Dionex C18 column (250×4.6 mm, 5 μm particle size) using mobile phase composed of methanol and ammonium acetate (0.005 M) in the ratio of 70:30 v/v. The separation was achieved using an isocratic elution method with a flow rate of 1.0 ml/ min at room temperature. The effluent was monitored at 230 nm using diode array detector. The retention time of eslicarbazepine acetate is found to be 4.9 min and the standard calibration plot was linear over a concentration range of 10-90 μg/ml with r²=0.9995. The limit of detection and quantification were found to be 3.144 and 9.52 μg/ml, respectively. The amount of eslicarbazepine acetate in bulk and tablet dosage form was found to be 99.19 and 97.88%, respectively. The method was validated statistically using the percent relative standard deviation and the values are found to be within the limits. The recovery studies were performed and the percentage recoveries were found to be 98.33± 0.5%.     

Formulation and Evaluation of Irinotecan Suppository for Rectal Administration

Irinotecan suppository was prepared using the moulding method with a homogeneous blend. A sensitive and specific fluorescence method was developed and validated for the determination of irinotecan in plasma using HPLC. The pharmacokinetics of intravenous administered and rectal administered in rabbits was investigated. Following a single intravenous dose of irinotecan (50 mg/kg), the plasma irinotecan concentration demonstrated a bi-exponential decay, with a rapid decline over 15 min. C_max, t_1/2, AUC_0–30h and AUC_0-∞ were 16.1 ± 2.7 g/ml, 7.6 ± 1.2 h, 71.3 ± 8.8 μg·h/ml and 82.3 ± 9.5 μg·h/ml, respectively. Following rectal administration of 100 mg/kg irinotecan, the plasma irinotecan concentration reached a peak of 5.3 ± 2.5 μg/ml at 4 h. The AUC_0–30h and AUC_0-∞ were 32.2 ± 6.2 μg·h/ml and 41.6 ± 7.2 μg·h/ml, respectively. It representing ∼50.6% of the absolute bioavailability.     

The acute cardiovascular actions of intravenous thyrotrophin releasing hormone (TRH) in man are mediated by non-catecholaminergic mechanisms

1. Intravenous bolus doses of thyrotrophin releasing hormone (TRH, 50–1000 μg) caused statistically significant, non-dose dependent and transient rises in blood pressure, heart rate and plasma catecholamines in healthy young males.
2. Mean peak incremental rises in systolic blood pressure (mean ± s.e. mean) following 50, 200 and 500 μg TRH were 14.3 ± 2.9 mmHg, 15.7 ± 3.2 mmHg and 17.1 ± 3.9 mmHg respectively (all P < 0.05 vs placebo). Mean incremental rises in heart rate for the three doses of TRH were 8.2 ± 2.2 beats min⁻¹, 7.1 ± 1.8 beats min⁻¹, and 1O.7 ± 2.9 beats min⁻¹ respectively (all P < 0.05 vs placebo).
3. Following the 50 μg and 1000 μg doses of TRH, plasma noradrenaline and adrenaline rose significantly (P < 0.05) between 4 and 8 min. Mean ± s.e. mean incremental plasma noradrenaline rise following 50, 200 and 1000 μg TRH were 0.4 ± O.13 nmol 1⁻¹, 0.37 ± 0.21 nmol 1⁻¹ and 0.41 ± 0.18 nmol 1⁻¹ respectively. Mean ± s.e. mean incremental rise in adrenaline for the 50, 200 and 1000 μg dose were 0.13 ± 0.04 nmol 1⁻¹, 0.08 ± 0.03 nmol 1⁻¹, and 0.11 ± 0.05 nmol l⁻¹ respectively.
4. Following administration of the ganglion blocking drug pentolinium (5 mg) the incremental systolic blood pressure and heart rate rises following 500 μg TRH alone 16.6 ± 2.8 mmHg and 1O.4 ± 3.1 beats min⁻¹ respectively.
5. The rises in plasma noradrenaline and adrenaline following TRH were attenuated by prior ganglion blockade.
6. α-adrenoceptor blockade with thymoxamine (0.3 mg kg⁻¹ bolus + 0.3 mg kg⁻¹ h⁻¹ infusion), singly and combined with intravenous propranolol (10 mg i.v. over 10 min), did not alter the pressor or tachycardic effects of 500 μg TRH.
7. In conclusion, although plasma noradrenaline rises following i.v. TRH, suggesting activation of the sympathetic nervous system, this effect is not responsible for the pressor response to TRH, which appears to be due to either a direct vasoconstrictive effect on the peripheral resistance vessels or a direct inotropic/chronotropic effect on the heart.

     

Development and Validation of a Stability-Indicating RP-HPLC Method for the Assay of Pristinamycin in Bulk and Tablet Dosage Form

Pristinamycin is an antibiotic used mainly in the treatment of Staphylococcus infections. The aim of this study was to develop a rapid and simple stability-indicating RP-HPLC method for the determination of pristinamycin in tablet dosage form. Pristinamycin was eluted on the ACE-5, C₁₈-HL, 250 x 4.6 mm, 5 µm analytical column with a mobile phase consisting of 0.2% orthophosphoric acid and acetonitrile 63:37 v/v, pumped at 1.5 ml/min flow rate. The column was maintained at 40°C and 10 μl of the solutions were injected. UV detection was performed at 206 nm. The procedure separated pristinamycin and its potential degradation products in an overall analysis time of less than 10 min with pristinamycin eluting at about 3 min. The method was validated according to the regulatory guidelines with respect to specificity, precision, accuracy, linearity, and robustness. Forced degradation studies were also performed for pristinamycin bulk drug samples to demonstrate the stability-indicating power of the HPLC method. The % RSD of system precision and method precision was found to be 0.64 and 1.49%, respectively. The procedure provided a linear response over the concentration range 25–150 μg/ml (r = 0.9998). Finally, the applicability of the method was evaluated in the tablet dosage form as well as in stability samples.     

RP-HPLC method validation for fast extraction and quantification of Levonorgestrel drug from silicone based intrauterine device intended for in-process and finished formulation

BackgroundTo develop and validate a simple and consistent reversed phase high performance liquid chromatography (RP-HPLC) method for the estimation of Levonorgestrel (LNG) drug from silicone based intrauterine device.MethodsSample solution was prepared using tetrahydrofuran (THF) as solvent for the drug extraction, and RP-HPLC analysis was performed using Luna C18 analytical column (150 × 4.6 mm, 5 μm, 100 Å – Phenomenex), with a mobile phase consisting of a mixture of acetonitrile and water (50:50, v/v) at a flow rate of 1.0 ml/min and injection volume of 20 μl. Detection was carried out at 241 nm in PDA detector, with a total run time of 15 min. The method was validated in accordance with ICH guidelines. Method applicability was tested for optimizing formulation using quality-by-design approach, to check the stability and content uniformity of levonorgestrel-silicone mixture (core blend), and quantifying the amount of LNG from commercially available silicone based formulation.ResultsThe retention time for LNG drug was obtained at 8.5 min (± 0.3 min). A linear relationship was observed over the concentration range of 2.6–15.6 μg/ml with the correlation coefficient (r) value 0.9999. The method was found to be precise within the acceptable limit (RSD < 2%) and the drug recovery from the intrauterine device was found in the range 99.78–100.0%. Content uniformity for different prototypes developed was observed in the range of 91.6–101.4%, and assay of optimized core blend was in the range of 97.78–106.79% during the 10 days of retention period for stability studies.ConclusionThe validated method is found to be a simple, accurate, precise, reproducible, and hence can be used for the routine analysis of LNG such as in-process, quality control and stability assays of silicone based intrauterine devices by RP-HPLC.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s40199-021-00396-7.     

Development and Validation of Stability-indicating High Performance Liquid Chromatographic Method for the Estimation of Everolimus in Tablets

The present study depicts the development of a validated reversed-phase high performance liquid chromatographic method for the determination of the everolimus in presence of degradation products or pharmaceutical excipients. Stress study was performed on everolimus and it was found that it degrade sufficiently in oxidizing and acidic conditions but less degradation was found in alkaline, neutral, thermal and photolytic conditions. The separation was carried out on Hypersil BDS C18 column (100×4.6 mm, 5 μ) column having particle size 5 μ using acetate buffer:acetonitrile (50:50 v/v) with pH 6.5 adjusted with orthophosphoric acid as mobile phase at flow rate of 1 ml/min. The wavelength of the detection was 280 nm. A retention time (R_t) nearly 3.110 min was observed. The calibration curve for everolimus was linear (r²=0.999) from range of 25-150 μg/ml with limit of detection and limit of quantification of 0.036 μg/ml and 0.109 μg/ml, respectively. Analytical validation parameters such as selectivity, specificity, linearity, accuracy and precision were evaluated and relative standard deviation value for all the key parameters were less than 2.0%. The recovery of the drug after standard addition was found to be 100.55%. Thus, the developed RP-HPLC method was found to be suitable for the determination of everolimus in tablets containing various excipients.     

Development and validation of a stability indicating UPLC method for determination of ticlopidine hydrochloride in its tablet formulation

The objective of the current study was the development of a simple, precise and accurate isocratic reversed-phase stability indicating Ultra Performance Liquid Chromatography [UPLC] assay method and validated for determination of ticlopidine hydrochloride in solid pharmaceutical dosage forms. Isocratic separation was achieved on a Zorbax SB-C18 (50 mm × 4.6 mm, 1.8 μm) column using mobile phase of methanol–0.01 M ammonium acetate buffer, pH 5.0 (80:20, v/v) at a flow rate of 0.8 ml min⁻¹, the injection volume was 4.0 μl and the detection was carried out at 235 nm by using photo-diode array detector. The drug was subjected to oxidation, hydrolysis, photolysis and heat to apply stress condition. The method was validated for specificity, linearity, precision, accuracy, robustness and solution stability. The method was linear in the drug concentration range of 62.5–375 μg ml⁻¹ with a correlation coefficient of 0.9999. The precision (relative standard deviation – RSD) of six samples was 1.31% for repeatability and the intermediate precision [RSD] among six-sample preparation was 0.77%. The accuracy (recovery) was between 98.80% and 101.50%. Degradation products produced as a result of stress studies did not interfere with detection of ticlopidine hydrochloride and the assay can thus be considered stability indicating.     

Development and Validation of Stability-indicating RP-HPLC Method for Estimation of Pamabrom in Tablets

The present study depicts the development of a validated RP-HPLC method for the determination of the pamabrom in presence of degradation products or other pharmaceutical excipients. Stress study was performed on pamabrom and it was found that it degrade sufficiently in acidic, alkali and oxidative condition but less degradation was found in thermal and photolytic condition. The separation was carried out on Enable G 120 A⁰ (250×4.6 mm, 5 μ) column having particle size 5 μ using methanol: water (75:25 v/v) with pH 4.0 adjusted with ortho phosphoric acid as mobile phase at flow rate of 1 ml/min. The wavelength of the detection was 280nm. A retention time (R_t) nearly 3.9 min was observed. The calibration curve for pamabrom was linear (r² = 0.9997) from range of 10-60 μg/ml with limit of detection and limit of quantification of 1.41 μg/ml and 4.28 μg/ml, respectively. Analytical validation parameter such as selectivity, specificity, linearity, accuracy and precision were evaluated and relative standard deviation value for all the key parameters were less than 2.0%. The recovery of the drug after standard addition was found to be 101.35%. Thus, the developed RP-HPLC method was found to be suitable for the determination of pamabrom in bulk as well as stability samples of tablets containing various excipients.     

Development and Validation of RP-HPLC Method for Simultaneous Estimation of Cefpodoxime Proxetil and Dicloxacillin Sodium in Tablets

A simple, accurate, rapid and precise reversed-phase high-performance liquid chromatographic method has been developed and validated for simultaneous determination of cefpodoxime proxetil and dicloxacillin sodium in tablet. The chromatographic separation was carried out on kromasil C₁₈ analytical column (250×4.6 mm; 5 μm) with a mixture of acetonitrile:methanol:trifloroacetic acid (0.001%) with pH 6.5 (30:50:20, v/v/v) as mobile phase; at a flow rate of 1.0 ml/min. UV detection was performed at 235 nm. The dicloxacillin sodium and cefpodoxime proxetil were eluted at 1.92 and 3.35 min, respectively. The peaks were eluted with better resolution. Calibration plots were linear over the concentration range 0.5-20 μg/ml for cefpodoxime proxetil (r²=0.9996) and 5-50 μg/ml for dicloxacillin sodium (r²=0.9987). The method was validated for accuracy, precision, linearity and specificity. The method was very sensitive with limit of detection 0.0726, 0.3685 μg/ml and limit of quantification 0.220, 1.116 μg/ml for cefpodoxime proxetil and dicloxacillin sodium, respectively. The high recovery and low relative standard deviation confirm the suitability of the method for routine determination of cefpodoxime proxetil and dicloxacillin sodium in bulk drug and tablet dosage form.     

Comparison of the acute cardiotoxicity of the antimalarial drug halofantrine in vitro and in vivo in anaesthetized guinea-pigs

1. Several unrelated drugs have pro-arrhythmic activity associated with an ability to prolong the QT interval of the ECG. The aim of this work was to examine the effects of the antimalarial drug halofantrine in vivo and in vitro.
2. In anaesthetized guinea-pigs consecutive bolus doses of halofantrine (0.3, 1, 3, 10 and 30 mg kg⁻¹, i.v.) at 25 min intervals caused dose-dependent prolongation of the rate corrected QTc interval and bradycardia. The change in heart rate became significant after administration of 10 mg kg⁻¹ halofantrine (−23±9 beats min⁻¹) whereas the increase in QTc was significant with only 1 mg kg⁻¹ halofantrine (22±10 ms). It was only with the highest dose of halofantrine that the PR interval was increased (from 52±3 to 67±4 ms) and second degree atrioventricular (AV) block (type 1 Mobitz) occurred in all animals. No changes were observed in any parameters in a separate group of guinea-pigs which received vehicle (dimethylacetamide 60% propylene glycol 40%) at equivalent time points.
3. The blood concentrations of halofantrine ranged from 0.26±0.17 μM after administration of 0.3 mg kg⁻¹ to 2.79±0.87 μM after 30 mg kg⁻¹, i.v. There was a significant correlation between the blood concentrations of halofantrine and the changes in QTc interval.
4. In guinea-pig left papillary muscles the effective refractory period was increased significantly 60 min after addition of halofantrine; from 161±4 to 173±6 ms with 10 μM, 156±8 to 174±6 ms with 30 μM and 165±6 to 179±5 ms with 100 μM halofantrine. However, the vehicle (0.1% Tween 80 in DMSO; final concentration of vehicle in Krebs, 1%) also increased the effective refractory period from 164±5 to 173±6 ms. Similar results were obtained in right ventricular strips but left atrial effective refractory periods were not altered by either the vehicle or halofantrine.
5. The results of these experiments suggest that any direct effects that halofantrine may have had on the effective refractory period of cardiac muscle cannot be separated from those of the vehicle. The prolongation of QTc and consistent observation of AV block with halofantrine in anaesthetized guinea-pigs suggest that in vivo models may be more useful for further studies investigating the mechanisms underlying the cardiotoxicity of halofantrine.

     

Anti-inflammatory and bronchodilator properties of RP 73401, a novel and selective phosphodiesterase type IV inhibitor

1 We have investigated the effects of RP 73401, a novel, potent and highly selective cyclic nucleotide phosphodiesterase (PDE) type IV inhibitor, in guinea-pig and rat models of bronchoconstriction and allergic inflammation. In some models, the effects of RP 73401 have been compared with those of the standard PDE type IV inhibitor, rolipram.

2 RP 73401 (0.4-400 μg kg^-1, intratracheally (i.t.) on lactose) inhibited antigen-induced bronchospasm in previously sensitized conscious guinea-pigs (ID₅₀: 7±1 μg kg^-1) and in anaesthetized rats (ID₅₀: 100±25 μg kg^-1). Rolipram inhibited the antigen-induced bronchospasm in guinea-pigs with an ID₅₀ of 5±1 μg kg^-1. In guinea-pig bronchoalveolar lavage (BAL) fluid, total inflammatory cell and eosinophil numbers were reduced by RP 73401 (ID₅₀S: 3.9±0.8 μg kg^-1 and 3.2±0.7 μg kg^-1, respectively). In the rat, inflammatory cell numbers are less affected. Only the highest dose of RP 73401 (400 μg kg^-1) significantly inhibited eosinophil influx (41±16% inhibition).

3 RP 73401 (0.02-100 μg kg^-1, i.v.) inhibited PAF-induced bronchial hyperreactivity to bombesin in the anaesthetized guinea-pig (ID₅₀: 0.09±0.03 μg kg^-1) and inhibited (0.4-40 μg kg^-1, i.t.) histamine-induced airway microvascular leakage in the anaesthetized guinea-pig by approximately 60% at all doses.

4 RP 73401 relaxed guinea-pig isolated trachea under basal tone (EC₅₀: 9 nM) and when precontracted with histamine (IC₅₀: 2 nM), methacholine (IC₅₀: 29 nM) or leukotriene D₄ (LTD₄, IC₅₀: 4 nM).

5 RP 73401 (0.4-100 μg kg^-1, i.t.) inhibited bronchospasm induced by histamine (ID₅₀: 34±6 μg kg^-1), methacholine (ID₅₀: 66±12 μg kg^-1) and LTD₄ (ID₅₀: <4 μg kg^-1) in the anaesthetized guinea-pig. Against these same bronchoconstrictors, rolipram (i.t.) had ID₅₀ values of 44±4, 72±18 and <4 μg kg^-1 respectively. RP 73401 (4 and 40 μg kg^-1, i.t.) increased the magnitude and duration of bronchodilatation produced by salbutamol in the anaesthetized guinea-pig. At doses producing significant bronchodilatation, RP 73401 was without effect on heart rate or blood pressure in the anaesthetized guinea-pig. RP 73401 (0.01-0.25 mg kg^-1, i.v.) did not affect heart rate and produced only a small fall in blood pressure in the anaesthetized rat.

6 These data demonstrate that RP 73401 and rolipram inhibit antigen- and mediator-induced bronchospasm in guinea-pigs with the same potency. Furthermore, RP 73401 administered directly into the airways, protects against allergic airway inflammation. These results indicate the importance of PDE IV in regulating smooth muscle and inflammatory cell activity. At doses suppressing the inflammatory response in the lung, RP 73401 had little effect in the cardiovascular system. RP 73401 may have a role as a bronchodilator and, more importantly, as a prophylactic anti-inflammatory agent in the treatment of asthma.

     

Antithrombotic effect of Z4A5 on coronary thrombosis in a canine model of acute unstable angina

Background and Purpose

The glycoprotein IIb/IIIa receptor is the final common pathway of platelet aggregation, regardless of the agonist, and thus represents an ideal therapeutic target for blocking coronary thrombosis. In this study, the anti-platelet and antithrombotic actions of Z4A5, a new glycoprotein IIb/IIIa receptor inhibitor, were evaluated in a canine model of acute unstable angina.

Experimental Approach

Z4A5 was given i.v. as a bolus followed by 60 min of continuous infusion at doses of 30 μg·kg⁻¹ + 1 μg·kg⁻¹·min⁻¹, 30 μg·kg⁻¹ + 5 μg·kg⁻¹·min⁻¹ or 300 μg·kg⁻¹ + 5 μg·kg⁻¹·min⁻¹. Its antithrombotic effect was evaluated in a model of coronary thrombosis, the injured, stenosed left circumflex coronary artery, in which platelet-dependent cyclic flow reductions (CFRs) were induced by vascular compression and constriction to simulate clinical acute unstable angina. Platelet aggregation and coagulation parameters were determined in platelet-rich plasma and platelet poor plasma respectively.

Key Results

The Z4A5 infusion induced a dose-dependent reduction in CFR frequency, which returned to baseline levels after the termination of the infusion at low doses. At medium dose that inhibited most part of platelet aggregation, it increased tongue bleeding time marginally with no dramatic changes in haemodynamic and coagulation parameters. Furthermore, the inhibition of ADP-induced platelet aggregation and prolonged bleeding time observed during Z4A5 infusion reverted to baseline levels after the termination of the infusion.

Conclusions and Implications

Z4A5 is an effective antithrombotic agent for coronary artery thrombosis with a rapid-on and rapid-off pharmacological profile, and could be used as an alternative treatment of coronary artery ischaemic syndromes.     

A Validated RP-HPLC Method for the Estimation of Lapatinib in Tablet Dosage form using Gemcitabine Hydrochloride as an Internal Standard

A simple, selective, rapid, precise and economical reverse-phase high-performance liquid chromatography method has been developed for the determination of lapatinib in tablet using gemcitabine hydrochloride as an internal standard. Chromatography was carried out on an ODS C-18 RP column (4.6 mm i.d. ×250 mm) using a mixture of acetonitrile and water (50:50 v/v) as the mobile phase at a flow rate of 1.0 ml/min. The drug was monitored at 232 nm. The retention times for lapatinib and gemcitabine hydrochloride were found to be 4.25±0.05 and 6.10±0.05 min, respectively. The method produced linear responses in the concentration range of 2-60 μg/ml of lapatinib. The limit of detection and limit of quantitation were 0.265 and 0.884 μg/ml, respectively.     

Passage of gallamine from blood into the liquor space in man and in dog

1. Patients receiving an intravenous injection of 2-3·8 mg/kg gallamine showed gallamine-like activity in their lumbar c.s.f. collected 15 and 70-100 min after the injection. The activity assayed on acetylcholine contractions of frog rectus muscle was equivalent to between 0·1 and 0·75 μg/ml gallamine.

2. In anaesthetized dogs an intravenous injection as well as an intravenous infusion of gallamine led to the appearance of gallamine-like activity in the cisternal c.s.f. and, on perfusion of the cerebral ventricles, in the effluent collected from the cisterna magna.

3. After an intravenous injection of 1 mg/kg the activity in the cisternal c.s.f. corresponded to between 0·2 and 1 μg/ml and in the effluent to between 130 and 175 ng/min during the first 15 min perfusion and then declined.

4. On intravenous infusion of gallamine at a rate of 10 (μg/kg)/min for 2 h the cisternal c.s.f. showed a uniform gallamine-like activity corresponding to between 0·4 and 0·67 μg/ml during the infusion. In the cisternal effluent the gallamine-like activity rose initially to between 20 and 90 ng/min but declined before the infusion was ended.

5. The intravenous injection of gallamine caused respiratory paralysis but did not affect arterial blood pressure; its intravenous infusion caused no respiratory paralysis and did not affect arterial blood pressure.

     

High-Performance Liquid Chromatographic and High-Performance Thin-Layer Chromatographic Method for the Quantitative Estimation of Dolutegravir Sodium in Bulk Drug and Pharmaceutical Dosage Form

Simple, sensitive, precise, and specific high-performance liquid chromategraphic (HPLC) and high-performance thin-layer chromatographic (HPTLC) methods for the determination of dolutegravir sodium in bulk drug and pharmaceutical dosage form were developed and validated. In the HPLC method, analysis of the drug was carried out on the ODS C₁₈ column (150 × 4.6 mm, 5 μm particle size) using a mixture of acetonitrile: water (pH 7.5) in the ratio of 80:20 v/v as the mobile phase at the flow rate 1 mL/min at 260 nm. This method was found to be linear in the concentration range of 5–35 μg/mL. The peak for dolutegravir sodium was observed at 3.0 ± 0.1 minutes. In the HPTLC method, analysis was performed on aluminum-backed plates pre-coated with silica gel G60 F₂₅₄ using methanol: chloroform: formic acid in the proportion of 8:2:0.5 v/v/v as the mobile phase. This solvent system was found to give compact spots for dolutegravir sodium with the Rf value 0.77 ± 0.01. Densitometric analysis of dolutegravir sodium was carried out in the absorbance mode at 265 nm. Linear regression analysis showed good linearity with respect to peak area in the concentration range of 200–900 ng/spot. The methods were validated for precision, limit of detection (LOD), limit of quantitation (LOQ), accuracy, and specificity. Statistical analysis showed that both of the methods are repeatable and specific for the estimation of the said drug. The methods can be used for routine quality control analysis of dolutegravir sodium.