Optimization of a Self-microemulsifying Drug Delivery System for Oral Administration of the Lipophilic Drug, Resveratrol: Enhanced Intestinal Permeability in Rat.

Purpose: This study aimed to formulate Resveratrol, a practically water-insoluble antioxidant in a self-microemulsifying drug delivery system (SMEDDS) to improve the solubility, release rate, and intestinal permeability of the drug. Methods: The suitable oil, surfactant, and co-surfactant were chosen according to the drug solubility study. Utilizing the design of experiment (DoE) method, the pseudo-ternary phase diagram was plotted based on the droplet size. In vitro dissolution study and the single-pass intestinal perfusion were performed for the investigation of in vitro and in-situ permeability for drugs formulated as SMEDDS in rat intestine using High-Performance Liquid Chromatography. Results: Castor oil, Cremophor® RH60, and PEG 1500 were selected as oil, surfactant, and co-surfactant. According to the pseudo-ternary phase diagram, nine formulations developed microemulsions with sizes ranging between 145-967 nm. Formulations passed the centrifuge and freeze-thaw stability tests. The optimum formulation possessed an almost 2.5-fold higher cumulative percentage of in vitro released resveratrol, in comparison to resveratrol aqueous suspension within 120 minutes. The results of the in-situ permeability study suggested a 2.6-fold higher intestinal permeability for optimum formulation than that of the resveratrol suspension. Conclusion: SMEDDS can be considered suitable for the oral delivery of resveratrol according to the observed increased intestinal permeability, which could consequently enhance the bioavailability and therapeutic efficacy of the drug.

Enhancing the Neuroprotection Potential of Edaravone in Transient Global Ischemia Treatment with Glutathione- (GSH-) Conjugated Poly(methacrylic acid) Nanogel as a Promising Carrier for Targeted Brain Drug Delivery.

Ischemic stroke is the most common among various stroke types and the second leading cause of death, worldwide. Edaravone (EDV) is one of the cardinal antioxidants that is capable of scavenging reactive oxygen species, especially hydroxyl molecules, and has been already used for ischemic stroke treatment. However, poor water solubility, low stability, and bioavailability in aqueous media are major EDV drawbacks. Thus, to overcome the aforementioned drawbacks, nanogel was exploited as a drug carrier of EDV. Furthermore, decorating the nanogel surface with glutathione as targeting ligands would potentiate the therapeutic efficacy. Nanovehicle characterization was assessed with various analytical techniques. Size (199 nm, hydrodynamic diameter) and zeta potential (-25 mV) of optimum formulation were assessed. The outcome demonstrated a diameter of around 100 nm, sphere shape, and homogenous morphology. Encapsulation efficiency and drug loading were determined to be 99.9% and 37.5%, respectively. In vitro drug release profile depicted a sustained release process. EDV and glutathione presence in one vehicle simultaneously made the possibility of antioxidant effects on the brain in specific doses, which resulted in elevated spatial memory and learning along with cognitive function in Wistar rats. In addition, significantly lower MDA and PCO and higher levels of neural GSH and antioxidant levels were observed, while histopathological improvement was approved. The developed nanogel can be a suited vehicle for drug delivery of EDV to the brain and improve ischemia-induced oxidative stress cell damage.

Pulmonary Delivery of Levamisole Nanoparticles as an Immunomodulator Affecting Th and a Potential ADAM10 Inhibitor to Ameliorate Severe Allergic Asthma.

Asthma is a common chronic lung disease without absolute treatment, and hypersensitivity reactions and type 2 immune responses are responsible for asthma pathophysiology. ADAM10 as a metalloproteinase transmembrane protein is critical for development of Th2 responses, and levamisole as an anthelmintic drug has immunomodulatory effects, which not only regulates ADAM10 activity but also can suppress the bone marrow and neutrophil production. Therefore, in the present study, nanoparticles were used as a levamisole delivery system to reduce bone marrow suppression, and the immunomodulatory and ADAM10 inhibitory effects of levamisole were studied in allergic asthma. Asthmatic mice were treated with PLGA-levamisole nanoparticles. Then, AHR, BALF, and blood cell counts, levels of the IgG1 subclass, total and OVA-specific IgE, IL2, IL-4, IL-5, IL-10, IL-13, IL-17, IL-25, IL-33, INF-γ, and TNF-α, gene expression of FoxP3, T-bet, RORγt, PU.1, GATA3, FcεRII, CysLT1R, eotaxin, and ADAM10, and lung histopathology were evaluated. PLGA-LMHCl with considered characteristics could control airway hyper-responsiveness, eosinophils in the BALF, levels of immunoglobulins, Th2-, Th9-, and Th17-derived cytokines and pivotal genes, eosinophilic inflammation, hyperplasia of the goblet cell, and hyperproduction of mucus and could increase Th1- and Treg-derived cytokines and also pivotal genes. It could also modulate the ADAM10 activity and had no effect on the number of neutrophils in the bloodstream. The novel safe nanodrug had no side effect on the bone marrow to produce neutrophils and could control the allegro-immuno-inflammatory response of asthma.

Oral delivery of solid lipid nanoparticles: underlining the physicochemical characteristics and physiological condition affecting the lipolysis rate.

INTRODUCTION: Lipid-based nano-drug delivery systems (LBNDDSs) have gained widespread attention in oral drug delivery due to their tunable and versatile properties such as biocompatibility and biodegradability, which makes them promising delivery systems for a variety of therapeutics. Currently, different types of LBNDDSs including liposomes, micelles, nanoemulsions, and solid lipid nanoparticles (SLNs) are developed for drug delivery applications. SLNs can be used as a controlled drug delivery system for oral delivery applications. However, its lipidic context makes that susceptible to lipolysis. The lipolysis rate of SLNs is affected by many factors that raise many questions for developing a more efficient delivery system. AREAS COVERED: In the present work, we highlighted different factors affecting the digestion rate/level of SLNs in the gastrointestinal tract. This paper can be most useful for those researchers who are keen to develop a properly controlled drug delivery system based on SLNs for oral delivery applications. EXPERT OPINION: SLNs can be used as a controlled drug delivery system for oral delivery applications. However, its lipidic context makes that susceptible to lipolysis. The lipolysis rate of SLNs is affected by many factors that raise many questions for developing a more efficient delivery system.

Effect of anionic macromolecules on intestinal permeability of furosemide.

CONTEXT: Furosemide is an anionic molecule and has very low absorption in gastro intestinal tract. OBJECTIVE: The aim of this study was to investigate the effect of anionic macromolecules on the intestinal permeability of Furosemide. MATERIALS AND METHODS: The intestinal permeability of Furosemide was determined using single-pass intestinal perfusion technique in rats. Briefly a jejunal segment of ∼10 cm was isolated and cannulated in both ends for inlet and outlet solution. The perfusate was collected every 10 min and samples were analyzed using the RP-HPLC method. Test samples containing furosemide and two anionic macromolecules, sodium carboxy methyl cellulose and sodium alginate, at different concentrations were used. RESULTS: The obtained data showed that existence of Sodium carboxy methyl cellulose significantly increased the Peff values in all three investigated concentrations (p < 0.05) but sodium alginate only in concentrations <0.1% increased drug permeability. DISCUSSION: It is concluded that the anionic macromolecules at specific concentrations could alter the permeability of anionic drugs across the biological membranes. CONCLUSIONS: Donnan phenomenon and chelating property of macromolecules could be attributed to the observed effect.

Development and physicochemical characterization of sirolimus solid dispersions prepared by solvent evaporation method.

PURPOSE: The aim of the present investigation was preparation and characterization of sirolimus solid dispersions by solvent evaporation technique to improve its dissolution properties. METHODS: Polyvinylpyrrolidone (PVP), Poloxamer 188 and Cremophore RH40 were used to prepare the solid dispersions of sirolimus. In vitro dissolution study using USP type I apparatus, were performed in distilled water (containing SLS 0.4%) for pure sirolimus, physical mixtures, Rapamune and prepared solid dispersions. The characterization of solid dispersions was performed using Fourier Transform Infrared (FTIR) Spectroscopy and Differential Scanning Calorimetry (DSC). RESULTS: More than 75% of sirolimus was released within 30 minutes from all prepared solid dispersions. The dissolution rate of all prepared solid dispersion powders were more than physical mixtures. The absence of sirolimus peak in the DSC spectrum of solid dispersions indicated the conversion of crystalline form of sirolimus into amorphous form. The results from FT-IR spectroscopy showed that there was no significant change in the FT-IR spectrum of solid dispersions indicating absence of well-defined interaction between drug and carriers. CONCLUSION: It was concluded that solid dispersion method, using PVP, Poloxamer 188 and Cremophore RH40 can improve dissolution rate of sirolimus.

A study on enhanced intestinal permeability of clarithromycin nanoparticles

The main objective of the present study was to determine the permeability of clarithromycin (CLA)-PLGA nanoparticles using single-pass intestinal perfusion technique in rats. Clarithromycin nanoparticles were prepared by nano-precipitation according to the modified quasi emulsion solvent diffusion technique and evaluated for their physicochemical characteristics. Permeability coefficients (Peff) in anaesthetized rats were determined at 3 different concentrations. Drug solution or suspensions in PBS was perfused through a cannulated jejunal segment and samples were taken from outlet tubing at different time points up to 90 min. Microbiological assay of CLA and phenol red in the samples were analyzed using an agar well diffusion procedure and HPLC method respectively. The average particle size of prepared nanoparticles was 305 ± 134 nm. The mean Peff of CLA solution in concentrations of 150, 250 and 400 µg/mL was found to be 1.20 (±0.32) ×10-3, 9.62 (±0.46) ×10-4, and 1.36 (±0.95) ×10-3 cm/sec, respectively. The corresponding values for the same concentration of nanoparticles were found to be 2.74 (±0.73) ×10-3, 2.45 (±0.88) ×10-3, and 3.68 (±0.46) ×10-3 cm/s, respectively. The two-tailed Student’s t-test showed that the intestinal permeability of CLA nanoparticle suspensions in prepared concentrations were significantly increased in comparison with its solution.

O objetivo principal do presente estudo foi determinar a permeabilidade de nanopartículas de claritromicina (CLA)-PLGA, utilizando a técnica de perfusão intestinal de passo único em ratos. As nanopartículas de claritromicina foram preparadas por nanoprecipitação, de acordo com a técnica modificada de difusão de solvente quase-emulsão, e suas características físico-químicas avaliadas. Os coeficientes de permeabilidade (Peff) em ratos anestesiados foram determinados em três concentrações diferentes. A solução, ou suspensões, do fármaco em PBS foi perfundida através do segmento de jejuno canulado e as amostras foram tomadas do tubo externo em diferentes tempos até 90 minutos. Os ensaios microbiológico de CLA e de vermelho de fenol das amostras foram realizados, utilizando-se o procedimento de difusão em poço de ágar e de CLAE, respectivamente. O tamanho médio das partículas das nanopartículas preparadas foi de 305 ± 134 nm. O Peff médio da solução de CLA em concentrações de 150, 250 and 400 µg/mL foi de 1.20(±0.32)×10-3, 9.62(±0.46)]×10-4 e de 1.36(±0.95)×10-3 cm/s, respectivamente. O valor correspondente para a mesma concentração de nanopartículas foi de 2.74 (±0.73)×10-3, 2.45(±0.88)×10-3 e de 3.68 (±0.46)×10-3 cm/s, respectivamente. O teste t de Student com duas variáveis mostrou que a permeabilidade intestinal das suspensões de nanopartículas de CLA nas concentrações preparadas foram significativamente aumentadas em comparação com sua solução.

Design and in vitro evaluation of a novel polymeric excipient for buccal applications.

BACKGROUND: The objective of this study was to develop and evaluate a more effective mucoadhesive thiomer for buccal drug-delivery systems. METHODS: 2-iminothiolane was covalently attached to a chitosan backbone. A preactivation step followed, mediated by 6,6´dithionicotinamide, thiol groups were modified by disulfide bonds formation. Mucoadhesion studies were performed on buccal mucosa. In addition, water-uptake capacity, disintegration, release and cytotoxicity studies were completed. RESULTS: The obtained chitosan-thiobutylamidine conjugate displayed 647.4 ± 85.23 µmol/g immobilized thiol groups. Due to the preactivation, approximately 60% of thiol groups were modified by formation of disulfide bonds. Chitosan-thiobutylamidine-mercaptonicotinamide displayed 1.8-fold higher stability and 1.6-fold higher mucoadhesive properties, respectively. The release study demonstrated a 1.4-fold more prolonged drug release compared with corresponding thiomers. CONCLUSION: According to these results, preactivated thiomers demonstrate an improved stability and increased mucoadhesive properties. Therefore, they seem to be advantageous for buccal administration over corresponding thiomers and chitosan.

Sleep paralysis in medieval Persia - the Hidayat of Akhawayni (?-983 AD).

Among the first three manuscripts written in Persian, Akhawayni's Hidayat al-muta'allemin fi al-tibb was the most significant work compiled in the 10th century. Along with the hundreds of chapters on hygiene, anatomy, physiology, symptoms and treatments of the diseases of various organs, there is a chapter on sleep paralysis (night-mare) prior to description and treatment of epilepsy. The present article is a review of the Akhawayni's teachings on sleep paralysis and of descriptions and treatments of sleep paralysis by the Greek, medieval, and Renaissance scholars. Akhawayni's descriptions along with other early writings provide insight into sleep paralysis during the Middle Ages in general and in Persia in particular.

Development and validation of an HPLC method for the analysis of sirolimus in drug products.

PURPOSE: The aim of this study was to develop a simple, rapid and sensitive reverse phase high performance liquid chromatography (RP-HPLC) method for quantification of sirolimus (SRL) in pharmaceutical dosage forms. METHODS: The chromatographic system employs isocratic elution using a Knauer- C18, 5 mm, 4.6 × 150 mm. Mobile phase consisting of acetonitril and ammonium acetate buffer set at flow rate 1.5 ml/min. The analyte was detected and quantified at 278nm using ultraviolet detector. The method was validated as per ICH guidelines. RESULTS: The standard curve was found to have a linear relationship (r(2) > 0.99) over the analytical range of 125-2000ng/ml. For all quality control (QC) standards in intraday and interday assay, accuracy and precision range were -0.96 to 6.30 and 0.86 to 13.74 respectively, demonstrating the precision and accuracy over the analytical range. Samples were stable during preparation and analysis procedure. CONCLUSION: Therefore the rapid and sensitive developed method can be used for the routine analysis of sirolimus such as dissolution and stability assays of pre- and post-marketed dosage forms.

Rapid HPLC determination of pioglitazone in human plasma by protein precipitation and its application to pharmacokinetic studies.

A simple and rapid HPLC method with UV detection was developed for the determination of pioglitazone in human plasma. The method was based on protein precipitation using perchloric acid on an ODS column. The mobile phase consisted of a mixture of phosphate buffer, methanol, acetonitrile, and 12 M perchloric acid (54 + 33 + 12 + 1, v/v/v/v). The UV detector was set at 269 nm. Under these conditions, the retention time of pioglitazone was 5.2 min. The standard curve was linear over the range of 50-2000 ng/mL pioglitazone in human plasma. The within-day and between-day precision studies showed high reproducibility, with CV less than 5%. The LOQ was 44.2 ng/mL. The method has been applied to a bioequivalence study after administration of pioglitazone as 30 mg tablets to 12 healthy volunteers.

Pharmacokinetic and bioequivalence study of two brands of valsartan tablets in healthy male volunteers.

Valsartan (CAS 137862-53-4) is an antihypertensive drug belonging to the family of angiotensin II receptor antagonists acting at the AT1 receptor, which mediates all known effects of angiotensin II on the cardiovascular system. In the present study, the pharmacokinetic parameters of two oral formulations of valsartan tablets were compared in a randomized, single oral dose, two-treatment crossover design in 24 healthy male volunteers under fasting conditions. After an overnight fast, the volunteers received 80 mg valsartan. Blood samples were collected up to 48 h and drug concentrations were determined by a reverse-phase HPLC method with fluorescence detection. Various pharmacokinetic parameters were determined from the plasma concentration-time curves of both formulations. The obtained values for test and reference products were 3067.7 +/- 1,281.7 and 3,304.3 +/- 1,196.4 ng/ml for Cmax; 17,834.4 +/- 7,083.8 and 18,319.1 +/- 7,800.7 ng x h/ml for AUC0-48; 18,825.7 +/- 7,553.2 and 19,172.2 +/- 8,307.2 ng x h/ml for AUC0-infinity, respectively. The 90% confidence intervals obtained by analysis of variance were 86.84-100.87% for Cmax and 93.43-115.54% for AUC0-t, which are within the acceptance range of 80-125%. Therefore it can be concluded that both products are bioequivalent in terms of rate and extent of drug absorption and therefore interchangeable.

The utility of rat jejunal permeability for biopharmaceutics classification system.

PURPOSE: The biopharmaceutical classification system has been developed to provide a scientific approach for classifying drug compounds based on their dose/solubility ratio and human intestinal permeability. Therefore in this study a new classification is presented, which is based on a correlation between rat and human intestinal permeability values. METHODS: In situ technique in rat jejunum was used to determine the effective intestinal permeability of tested drugs. Then three dimensionless parameters--dose number, absorption number, and dissolution number (D(o), A(n), and D(n))--were calculated for each drug. RESULTS: Four classes of drugs were defined, that is, class I, D(0) < 0.5, P(eff(rat)) > 5.09 x 10(-5) cm/s; class II, D(o) > 1, P(eff(rat)) > 5.09 x 10( -5) cm/s; class III, D(0) < 0.5, P(eff(rat)) < 4.2 x 10(-5) cm/s; and class IV, D(o) > 1, P(eff(rat)) < 4.2 x 10(-5) cm/s. A region of borderline drugs (0.5 < D(o) < 1, 4.2 x 10(-5) < P(eff(rat)) < 5.09 x 10(-5) cm/s) was also defined. CONCLUSION: According to obtained results and proposed classification for drugs, it is concluded that drugs could be categorized correctly based on dose number and their intestinal permeability values in rat model using single-pass intestinal perfusion technique. This classification enables us to remark defined characteristics for intestinal absorption of all four classes using suitable cutoff points for both dose number and rat effective intestinal permeability values.

Bioequivalence of fexofenadine tablet formulations assessed in healthy Iranian volunteers.

BACKGROUND AND OBJECTIVE: Many drug products containing the same amount of active drug are made and marketed by more than one pharmaceutics manufacturer. Since the quality of final drug product is affected by the source of ingredients, type and amount of excipients and manufacturing process, bioequivalence studies are used to determine the bioavailability and characterize the pharmacokinetics of the new formulation relative to a reference formulation. In the present study the bioavailability of a new capsule formulation of fexofenadine (CAS 153439-40-8) was compared to a reference formulation in 12 healthy male volunteers. METHODS: The blood samples were collected at different time points. After centrifugation and decanting the plasma, the drug was extracted using a mixture of diethyl ether/isopropyl alcohol (5:95% v/v). Then the samples were dried at 45 degrees C under nitrogen and finally, after dissolving the dried sample in mobile phase, the plasma drug concentrations were determined using HPLC. The pharmacokinetic parameters (Cmax, AUCt0, AUCinfinity0) were statistically compared by analysis of variance (ANOVA) for test and reference formulations and no statistical differences were observed. RESULTS AND DISCUSSION: The maximum plasma concentration (Cmax) of fexofenadine was 1206.3 +/- 619.0 ng/ml for the test and 1172.6 +/- 493.7 ng/ml for the reference formulation. The mean AUC0-infinity of fexofenadine was 8911.4 +/- 3870.0 and 9363.9 +/- 2668.0 ng x h/ml for the test and reference formulation, respectively. The calculated 90% confidence intervals for the mean test/reference ratios of mentioned parameters were 90.0-113.9, 86.9-109.5 and 80.8-102.8, respectively, which are in the bioequivalence range. CONCLUSION: Based on the obtained results the two fexofenadine formulations are considered to be equivalent.

Investigation of the intestinal permeability of ciclosporin using the in situ technique in rats and the relevance of P-glycoprotein.

The primary endpoint of this study was to determine the intestinal permeability of ciclosporin (cyclosporine A, CsA, CAS 59865-13-3) using the single-pass intestinal perfusion technique (SPIP) and a range of concentrations in rats. The second objective was to assess the quantitative contribution of P-glycoprotein (P-gp)-mediated efflux in limiting the oral bioavailability of CsA using erythromycin (Ery, CAS 114-07-8) as an inhibitor of P-gp efflux transporter. A solution containing CsA and phenol red either in the presence or in the absence of Ery as a P-gp inhibitor was perfused through a cannulated jejunal segment in rats. Outlet samples were collected every 10 min in micro tubes up to 90 min. Samples were analyzed using a modified reverse phase HPLC method. The mean effective permeability coefficients (Peff) of CsA in concentrations of 5, 10, 15 and 20 micromol/L in the perfusion solution were found to be 2.21 (+/- 0.26) x 10(-4) cm/s, 3.34 (+/- 1.29) x 10(-4) cm/s, 3.12 (+/- 0.23) x 10(-4) cm/s and 2.73 (+/- 0.28) x 10(-4) cm/s, respectively. The corresponding values in the presence of Ery were found to be 3.96 (+/- 1.04) x 10(-4) cm/s, 5.34 (+/- 1.29) x 10(-4) cm/s, 3.72 (+/- 0.21) x 10(-4) cm/s and 4.41 (+/- 0.89) x 10(-4) cm/s, respectively. The two-tailed Student's t-test showed that the intestinal permeability of CsA was significantly increased by Ery in all four CsA concentrations used (P < 0.05). However, there was no significant difference between the Peff values of CsA in different concentrations, indicating that the CsA permeation was independent of the concentration. Therefore it is concluded that at least some part of the observed clinical interaction between Ery and CsA is due to the interaction in absorption level.

Comparative bioavailability study of two cefixime formulations administered orally in healthy male volunteers.

The bioavailability of a new cefixime ((6R,7R)-7-[(Z)-2-(2-amino-4-thiazolyl)-2-(carboxymethoxyimino) acetamido]-8-oxo-3-vinyl-5-thia-1-azabicyclo-[4,2,0]-oct-2-ene-2-carboxylic acid, CAS 79350-37-1) tablet preparation (Loprax) was compared with that of a reference preparation of the drug in 24 healthy male volunteers. The trial was designed as an open, randomized, single-blind, two-sequence, two-period crossover study. Under fasting conditions, each subject received a single oral dose of 400 mg cefixime tablet as a test or reference formulation on 2 treatment days. The treatment periods were separated by a one-week washout period. The plasma concentrations of the drug were analyzed by a rapid and sensitive HPLC method with UV detection. The pharmacokinetic parameters included AUC0-24h, AUC0-infinity, Cmax, t1/2, and Ke. The mean AUC0-infinity of cefixime was 45008.7 +/- 10989.9 and 45221.3 +/- 2155.7 n x h/ml for the test and reference formulation, respectively. The maximum plasma concentration (Cmax) of cefixime was on average 4746.9 +/- 1284 ng/ml for the test and 4726.3 +/- 1206.9 ng/ml for the reference product. No statistical differences were observed for Cmax and the area under the plasma concentration-time curve for test and reference tablets. The calculated 90% confidence intervals based on the ANOVA analysis for the mean test/reference ratios of Cmax, AUC0-infinity and AUC0-24h of cefixime were in the bioequivalence range (94%-112%). Therefore, the two formulations were considered to be bioequivalent.

A simple and rapid high-performance liquid chromatography method for determining furosemide, hydrochlorothiazide, and phenol red: applicability to intestinal permeability studies.

A simple reversed-phase high-performance liquid chromatography (HPLC) method with ultraviolet detection at 280 nm was developed for simultaneous quantitation of furosemide and hydrochlorothiazide along with phenol red as a nonabsorbable marker for in situ permeability studies in anaesthetized rats. A jejunal segment of approximately 10 cm was isolated and cannulated in both ends for inlet and outlet solution. The perfusate was collected every 10 min, and samples were analyzed using the developed method. The mobile phase was acetonitrile-water-triethylamine-glacial acetic acid (41.5 + 57.4 + 0.1 + 0.9, adjusted to pH 5.6) at a flow rate of 1 mL/min; the run time was 9 min. The calibration graphs were linear for all 3 compounds (r > 0.999) across the concentration range of 7.93-125 microg/mL for phenol red and 6.25-100 microg/mL for hydrochlorothiazide and furosemide. The limits of quantitation were 7.2, 8.9, and 6.8 microg/mL for furosemide, hydrochlorothiazide, and phenol red, respectively. The coefficients of variation for intraassay and interassay precision were less than or equal to 7.6%, and the accuracy was between 93.2-103.4%. Using the single pass intestinal perfusion technique and the suggested HPLC method for sample analysis, mean values of 0.25 x 10(-4) (+/-0.16) cm/s and 0.22 x 10(-4) (+/-0.13) cm/s were obtained for furosemide and hydrochlorothiazide, respectively.