Co-administration of a water-soluble polymer increases the usefulness of cyclodextrins in solid oral dosage forms

PURPOSE: The aim of this study was to investigate the effect of cyclodextrins (beta-CD, HP-beta-CD and (SBE)7m-beta-CD), and co-administration of a water-soluble polymer (HPMC) and cyclodextrins, on the oral bioavailability of glibenclamide in dogs. METHODS: Effects of cyclodextrins on the aqueous solubility of glibenclamide, with and without hydroxypropylmethylcellulose (HPMC), were determined by a phase-solubility method. Solid inclusion complexes were prepared by freeze-drying. Glibenclamide was administered orally and intravenously to beagle dogs. RESULTS: Aqueous solubility of glibenclamide increased as a function of cyclodextrin concentration, showing an AL-type diagram for beta-CD and an Ap-type diagrams for both of the beta-CD derivatives studied. HPMC enhanced the solubilising effect of cyclodextrins, but did not affect the type of phase-solubility diagram. Orally administered glibenclamide and its physical mixture with HP-beta-CD showed poor absolute bioavailability, while orally administered glibenclamide/cyclodextrin-complexes significantly enhanced the absolute bioavailability of glibenclamide. Orally administered glibenclamide/beta-CD/HPMC and glibenclamide/(SBE)7m-beta-CD/HPMC complexes showed similar absolute bioavailability compared to formulations not containing HPMC, even though 80% (in the case of (SBE)7m-beta-CD) or 40% (in the case of beta-CD) less cyclodextrin was used. CONCLUSIONS: The oral bioavailability of glibenclamide was significantly increased by cyclodextrin complexation. HPMC increased the solubilising effect of cyclodextrins and, therefore, the amount of cyclodextrin needed in the solid dosage form was significantly reduced by their co-administration. In conclusion, the pharmaceutical usefulness of cyclodextrins in oral administration may be substantially improved by co-administration of a water-soluble polymer.     

Oral low-dose magnesium gluconate preventing pregnancy induced hypertension

OBJECTIVE: To study the effects of oral low-dose magnesium gluconate in prevention of pregnancy induced hypertension (PIH) and its mechanism. METHODS: A prospective randomized double-blind study was carried out in 51 pregnant women as treatment group (including 22 cases as treatment group 1 and 29 cases as treatment group 2) and 51 pregnant women as controls (including 28 cases as controls group 1 and 23 cases as control group 2). Low-dose magnesium gluconate (3 g/day) or placebo was given from the 28th week of gestation to delivery consecutively. RESULTS: 4% of the pregnant women developed PIH after magnesium gluconate treatment, which was substantially lower than that in the control group (16%) (P < 0.05). In the treatment group 2, women showed higher concentration of 6-keto-prostaglandin F1 alpha (PGF1a) and 6-keto-/thromboxane B2(TXB2) (P/T) ratio than that of the control group 2. Moreover, TXB2 level was lower than that in the control group 2. In the treatment group 1 women showed higher ratio of P/T than that of the control group 1. There were no significant differences of serum magnesium concentration among all groups. CONCLUSION: Low-dose magnesium gluconate may efficiently prevent PIH in high risk women. The mechanism of action of magnesium gluconate probably involves to keep the balance of PGI2 and TXA2, but not associates with serum magnesium level.     

Thermal properties of adamantanol derivatives and their beta-cyclodextrin complexes in phosphatidylcholine bilayers

Differential Scanning Calorimetry (DSC) has been applied to study the thermal properties of the membrane perturbing antibacterial octyl- and dodecyl-bromide salts of quaternary dimethylamino adamantanol (ADM-8 and ADM-12 correspondingly) incorporated in free or complexed form with beta-cyclodextrin (beta-CD) into dipalmitoylphosphatidylcholine (DPPC) containing bilayers. The DSC results showed that the studied compounds exert pronounced thermotropic changes in DPPC bilayers when inserted as free molecules. These effects are reduced when are present in a complex form with beta-CD. Since the studied compounds exert destructive effects in membrane bilayers their insertion in membrane bilayers as complexes with cyclodextrin may result in differentiation of their activity. The obtained results suggest that their complexation with beta-CD may improve their biological profile. It also increases their aqueous solubility, a limited factor for their use as drugs.     

Structure of cyclodextrin glycosyltransferase complexed with a derivative of its main product beta-cyclodextrin

Crystals of the inactive mutant Glu257-->Ala of cyclodextrin glycosyltransferase were soaked with the cyclodextrin (CD) derivative S-(alpha-D-glucopyranosyl)-6-thio-beta-CD. The structural analysis showed its beta-CD moiety with no density indication for the exocyclic glucosyl unit. For steric reasons, however, the position of this unit is restricted to be at only two of the seven glucosyl groups of beta-CD. The analysis indicated that the enzyme can cyclize branched alpha-glucans. The ligated beta-CD moiety revealed how the enzyme binds its predominant cyclic product. The conformation of the ligated beta-CD was intermediate between the more symmetrical conformation in beta-CD dodecahydrate crystals and the conformation of a bound linear alpha-glucan chain. Its scissile bond was displaced by 2.8 A from the position in linear alpha-glucans. Accordingly, the complex represents the situation after the cyclization reaction but before diffusion into the solvent, where a more symmetrical conformation is assumed, or the equivalent state in the reverse reaction. Furthermore, a unifying nomenclature for oligosaccharide-binding subsites in proteins is proposed.     

Interactions of griseofulvin with cyclodextrins in solid binary systems

Griseofulvin/cyclodextrin interactions were investigated in aqueous environment and in solid state. Two cyclodextrin derivatives (beta-cyclodextrin and 2-hydroxypropyl beta-cyclodextrin) were used to prepare different physical mixtures and kneaded systems, and the drug/cyclodextrin ratios were 1:1 and 1:2 mol/mol. Scanning electron microscopy (SEM), hot-stage microscopy (HSM), differential scanning calorimetry (DSC), thermogravimetry (TG), and X-ray powder diffractometry were employed to characterize pure substances and their kneaded counterparts and all of the binary systems. The solubility of griseofulvin was increased in accord with the quantity of cyclodextrin added. HSM examination revealed that 2-hydroxypropyl-beta-cyclodextrin was dissolved in the droplets of melted griseofulvin, but did not show any interactions between melted griseofulvin and beta-cyclodextrin particles. The presence of griseofulvin endothermic peak in the DSC curves of all binary systems suggests the absence of any griseofulvin/cylcodextrin inclusion compound in the solid state. In TG, data of weight loss owing to the dehydration of cyclodextrins was similar for both kneaded systems and physical mixtures. X-ray diffraction patterns exhibited the amorphous nature of 2-hydroxypropyl-beta-cyclodextrin and the crystalline nature of griseofulvin and binary systems. Griseofulvin dissolution profiles from all binary systems showed an improvement in drug dissolution, which indicates that an "in situ" drug/cyclodextrin inclusion compound was formed in the aqueous dissolution medium.     

Retention mechanism study of imidazole derivatives on a beta-cyclodextrin-bonded stationary phase. Thermal analysis contributions

The high-performance liquid chromatography retention mechanism of a series of six imidazole derivatives was investigated over a wide range of mobile-phase compositions, pH, and column temperatures using a beta-cyclodextrin (beta-CD)-bonded chiral stationary phase. Thermodynamic constants for the transfer of a solute from the mobile to the beta-CD stationary phase were determined. Different van't Hoff plot shapes were observed with mobile-phase pH values, indicating a change in the retention mechanism. Enthalpy-entropy compensation revealed that the solute retention mechanism was independent of the compound molecular structure, the same at pH 7 and 7.5, but changed at pH 6.5, 8, and 8.5. Differential scanning calorimetry and thermogravimetric analysis were used to show different thermal features for the beta-CD stationary phase at pH 6.5, 8, and 8.5 and at pH 7 and 7.5. A new theory was presented to explain the beta-CD cavity structure balance between an ordered and disordered state. Variations of column temperature and mobile-phase pH tend to cause this phase transition between these two states, explaining the thermodynamic constant variations with pH and temperature.     

Effect of alkyl chain length and degree of substitution on the complexation of sulfoalkyl ether beta-cyclodextrins with steroids

This study was designed to test how the sulfoalkyl ether (SAE) modification of beta-cyclodextrin (beta-CD) affects the binding capacity of testosterone and progesterone, thereby enhancing their solubility. The SAE-beta-CD derivatives contain either sulfopropyl ether (SPE) or sulfobutyl ether (SBE) groups on the 2-, 3-, and 6-hydroxyl positions of the dextrose moieties. SAE-beta-CDs are a mixture of positional and regional isomers containing from one to as many as 12 SAE groups per CD. The effect of chain length and the degree of substitution on complexation behavior was investigated by the phase-solubility method. The results were compared with those obtained with beta-CD, where possible, and with hydroxypropyl-beta-CD (HP-beta-CD). To determine the effect of degree of substitution (DS) on the binding, mixtures of SAE-beta-CDs with multiple substitution levels and varying average degrees of substitution were studied as well as mixtures of SAE-beta-CDs that contained the same degree of substitution. Mixtures that contained SAE-beta-CDs of the same degree of substitution were isolated from the multiple substitution level mixtures by ion-exchange chromatography and purified for investigation. Unlike the parent beta-CD, linear increases in the apparent solubilities of testosterone and progesterone were observed, and the binding potentials were comparable to those of beta-CD or better. The results demonstrate that the binding potentials of the SAE-beta-CD derivatives were dependent on the guest molecule, the degree of substitution, and the alkyl ether chain length. Our previous study showed the inhibition of complexation by direct sulfonation of the beta-CD. However, in the present work, interferences with the charged sulfonate groups were avoided by repositioning them away from the cavity. Increasing the degree of substitution assisted in complex formation; however, its effects were limited. Reduction of the alkyl chain length, as in the case of SPE-beta-CD compared with SBE-beta-CD, decreased the complexation potential. This decrease in complexation potential was further suppressed with an increase in the number of substituents placed on the CD torus. Generally, the binding potential of SAE-beta-CD derivatives increased with increasing alkyl chain length. However, placement of more than an optimum number of SAE groups on the CD torus resulted in inhibition of complexation.     

In vitro corneal permeability of diclofenac sodium in formulations containing cyclodextrins compared to the commercial product voltaren ophtha

The influence of different cyclodextrin derivatives on the in vitro permeability of diclofenac sodium through pig cornea was investigated and compared to the commercial product Voltaren ophtha. (Hydroxypropyl)-beta-cyclodextrin (HP beta CD) and two amorphous methylated cyclodextrins with different degrees of substitution were used. In hemolysis studies on human erythrocytes, the hemolytic activity of the different cyclodextrins and the drug was assessed. It was shown that HP beta CD reveals the most favorable toxicological properties. A decrease in the hemolytic activity of diclofenac was yielded by adding HP beta CD. In the permeability experiments the dependency of the permeability coefficients and lag times on the type of cyclodextrin and pH of the solutions were examined. A solution containing HP beta CD buffered in the pH range 6.5 to 7 is proposed as a useful eye drop formulation. All cyclodextrin formulations showed advantages as compared to Voltaren ophtha.     

Micellar electrokinetic capillary chromatography (MECC) of UV-absorbing constituents in normal urine: a chemometric optimisation of the separation

A micellar electrokinetic capillary chromatography (MECC) method when compared to free solution capillary electrophoresis (CZE) was shown to offer improved selectivity and resolution for the separation of UV-absorbing components of human urine. Some of the factors affecting MECC separation e.g. methanol concentration, sodium dodecyl sulphate (SDS) concentration, beta-cyclodextrin (beta-CD) concentration, voltage, pH, temperature and electrolyte additives (urea, beta-CD and Brij 35) were optimised using chemometric techniques. Three-level three-factor (3(3)) factorial designs and simplex optimisation were used to achieve optimised conditions with the goal of obtaining the maximum number of peaks in the shortest possible analysis time. Using a TSP CE2000 instrument with detection from 195-300 nm and fitted with a 75 microns x 44 cm (37 cm effective length) fused silica capillary the final optimum conditions were found to be, an electrolyte consisting of 30 mM sodium tetraborate, pH 10, containing 75 mM SDS and 10 mM beta-CD, 15 degrees C, 20 kV, 4 s hydrodynamic injection of filtered urine. These conditions were capable of separating 70 peaks from a normal human urine pool in less than 12 min. The separation of components in urine using the optimised MECC was simpler, more reproducible, faster and gave better resolution than gradient reversed-phase high performance liquid chromatography.     

Evaluation of learning and memory dysfunction and histological findings in rats with chronic stage contusion and diffuse axonal injury

Enantiomer separations of various acidic racemates were performed by capillary electrophoresis (CE) using a commercial cationic beta-cyclodextrin (beta-CD), quaternary ammonium beta-CD (QA-beta-CD), and a commercial amphoteric beta-CD (AM-beta-CD) as chiral selectors. Eleven acidic racemates were successfully separated using QA-beta-CD by changing the CD concentration and the buffer pH. These enantiomer separations were compared with the results using five neutral CD derivatives. Although most racemates were separated with some of the neutral CDs, relatively high CD concentrations were required to obtain baseline separations. In contrast, when QA-beta-CD was employed, the enantiomer separations were successful at low concentrations below 5 mM. Enantiomers of five acidic racemates and ten dansylated amino acids (Dns-amino acids) were separated using AM-beta-CD. Although the baseline separation of racemic 4-chloromandelic acid was not achieved with either QA-beta-CD or five neutral CDs, AM-beta-CD showed complete resolution. Furthermore, the simultaneous enantiomer separation of eight Dns-amino acids was also achieved with AM-beta-CD. Both QA-beta-CD and AM-beta-CD were analyzed by CE and mass spectrometry (MS) in order to identify their compositions because they consisted of a mixture having different degrees of substitution. QA-beta-CD consisted of six components having from one to six quaternary ammonium groups. The composition of AM-beta-CD, however, was very complicated and could not be identified.     

Cyclodextrins as selectivity enhancers in capillary zone electrophoresis of proteins

The selectivity in the capillary zone electrophoresis (CZE) of a variety of acidic and basic proteins including alpha-chymotrypsinogen A, cytochrome c, lysozyme, ribonuclease A, ovalbumin, and beta-lactoglobulins A and B, was altered by adding 6-monodeoxy-6-monoamino-beta-cyclodextrin or carboxymethylated beta-cyclodextrin to the electrophoretic medium of aqueous 50 mM sodium phosphate, pH 2.5. On the other hand, no significant improvement was obtained in the separation upon addition of heptakis (2,6-di-O-methyl)-beta-cyclodextrin. Whereas protein adsorption on the wall of raw silica capillaries was significant in the absence of cyclodextrin, by addition of beta-cyclodextrin or its derivatives to the background electrolyte, wall adsorption was reduced with concomitant enhancement of the recovery. The results confirm that in various separation techniques, particularly those which employ microcolumns, certain cyclodextrin additives can be useful selectivity enhancers not only in the separation of small sample molecules but also in that of proteins.     

The equilibrium constant of beta-cyclodextrin-phenolphtalein complex; influence of temperature and tetrahydrofuran addition

The temperature influence on creation of a supramolecular complex in which beta-cyclodextrin (beta-CD) is the host molecule and phenolphtalein (PP) is the guest has been studied in aqueous solution by UV-visible absorption spectroscopy. The decrease of temperature of beta-cyclodextrin-phenolphtalein system resulted in a decrease in absorbance of the UV-vis spectrum. Under favourable conditions (0.1 mM beta-CD, 30 microM PP) the termochromic effect is very significant (approximately =0.1 U of absorbance/10 degrees C). The formation constant of inclusion complex was determined at various temperatures (from 10 to 70 degrees C) using Scott's equation. The association constants (K11) for the binding in 0.02 M sodium carbonate (pH 10.5) at 10 and 70 degrees C are 7.44 and 0.26 x 10(4) M(-1) respectively. The stoichiometric ratio of investigated complex was found to be 1:1 on wide range of beta-cyclodextrin:phenolphtalein concentration ratio (from 0.8:1 to 427:1). Additionally, strong interaction between cyclodextrin and tetrahydrofuran (THF) was observed and the inhibitory effect of tetrahydrofurane on the association of beta-CD PP complex was studied. From linear Van't Hoff plots thermodynamic parameters such as: the change of enthalpy (deltaH(o)) and change of entropy (deltaS(o)) were estimated and interpreted.     

Determination of association constants of dansyl-amino acids and beta-cyclodextrin in N-methylformamide by capillary electrophoresis

The use of nonaqueous background electrolytes in capillary electrophoresis (CE) is a promising new trend which should widen the scope of this technique. We demonstrate the chiral separation of dansyl-amino acids (Dns-AAs) in N-methylformamide (NMF) using beta-cyclodextrin (beta-CD) as chiral selector. The solubility of beta-CD is much better in NMF than in water, allowing high concentration of the chiral selector and successful enantioseparation despite the weak host-guest interaction between the Dns-AAs and beta-CD. The association constants for the complexation between Dns-AAs and beta-CD could be calculated from the electrophoretic mobilities, with attention paid to the change in viscosity of the electrolyte upon addition of beta-CD. The association constants ranged between 2 and 13 M(-1).     

Nucleotide sequence of DR-B exon of major histocompatibility complex from an Indian zebu cattle breed

Somatosensory evoked potential, locomotion and vocalisation upon tail pinch in rats was studied in order to determine whether intrathecal magnesium sulphate administration causes spinal anaesthesia. In Wistar rats with indwelling intrathecal catheters, cortical somatosensory evoked potential was recorded following stimulation via electrodes inserted into the hind paw under chloral hydrate anaesthesia before and after intrathecal administration of 10 microliters of either magnesium sulphate (12.3% or 24.6%) or lignocaine (4% or 8%). Locomotion and vocalisation after tail pinch were tested following intrathecal administration of the same two drugs in conscious rats. Somatosensory evoked potential amplitude was diminished after administration of lignocaine (p < 0.05) but did not change after magnesium sulphate. Latency of P1 was increased by lignocaine and by magnesium sulphate 12.3% (p < 0.05). Although lower extremity paralysis was observed in both groups, its duration with magnesium sulphate was much longer than with lignocaine. Vocalisation was recognised after magnesium sulphate 12.3%, but was not observed after lignocaine 8% during paralysis (p < 0.05). We believe that magnesium sulphate caused motor paralysis, but not complete analgesia.     

Magnesium sulphate versus diazepam in the management of eclampsia

This clinical trial was conducted to compare the anticonvulsant response of magnesium sulphate and diazepam in the management of eclampsia. The study was carried out at the eclampsia unit of Dhaka Medical College Hospital during the period from October, 1995 to January, 1996. Two hundred consecutive admitted patients were recruited for the study and randomly assigned to two treatment groups: magnesium sulphate and diazepam. One hundred patients received injection magnesium sulphate and another one hundred received injection diazepam. All patients of both the group were matched for baseline characteristics. Convulsion was controlled in 95% of the patients of magnesium sulphate group and 74% of the patients of diazepam group (p < .0005). The mean controlling time is also significantly lower in magnesium sulphate group than diazepam group (8.50 hours vs 9.39 hours). Patients of magnesium sulphate group regain consciousness much earlier (mean time 20.62 hrs.) than the patients of diazepam group (mean time 40.62 hrs.). No significant difference was observed in controlling blood pressure and foetal outcome. The study finding shows that magnesium sulphate has some advantage over diazepam in controlling convulsion and regaining consciousness. If magnesium sulphate can be made available in the market by local production it may be recommended to use this drug in the primary health care without any hazzard before referring to other hospital.     

Evaluation of a sulfobutyl ether beta-cyclodextrin as a solubilizing/stabilizing agent for several drugs

To evaluate the potential use of beta-cyclodextrin sulfobutyl ether, 7 sodium salt (SBE7-beta-CD) as a drug solubilizing and stabilizing agent, the solubilizing effects of SBE7-beta-CD on 22 different poorly water-soluble drugs were compared with those of intact beta-CD and heptakis-(2,6-di-O-methyl)-beta-CD (DMCD). SBE7-beta-CD was generally a more effective solubilizer for poorly water-soluble drugs than was intact beta-CD, but SBE7-beta-CD was not as effective as DMCD. The effects of SBE7-beta-CD on the acid hydrolysis rate of prostaglandin I2, the alkaline hydrolysis rate of indomethacin, the dehydration of prostaglandin E1, and the isomerization of prostaglandin A1 were also investigated and compared to those for intact beta-CD, DMCD, and 2,3,6 partially methylated-beta-CD (PMCD). The stabilizing effects of SBE7-beta-CD on chemically unstable drugs were generally higher than those of other CDs.     

Magnesium and gastric acid secretion

This study examined the modifications of acid gastric secretion caused by magnesium oxide, peroxide, silicate and sulphate in 805 patients with duodenal ulcers; The calcium-magnesium antagonism on the one hand and the acetazolamide - magnesium synergia on the other, were also investigated. Our results show that administration of magnesium, either oral or parenteral, does not significantly modify gastric acid secretion, either basal or stimulated by maximal histamine. Administration of un infusion of calcium gluconate 15 mg/kg body weight, significantly increases gastric acid secretion as compared to basal secretion. Addition of a dose of magnesium sulphate to the infusion antagonises the effect of gastric acid secretion caused by calcium. Administration of 1.5 gr magnesium oxide along with 25 mg acetazolamide per kg/body weight, strengthens the inhibitory effect of acetazolamide upon gastric acid secretion, increasing the proportion of significant inhibitory effects up to 98% of the cases under investigation. Addition of magnesium to the classical therapy antagonises the noxious effects of calcium compounds; the presence of magnesium in the composition of antacid powders proves necessary.     

Use of cyclodextrins for manipulating cellular cholesterol content

Previous studies from this laboratory have demonstrated that exposure of tissue culture cells to cyclodextrins results in rapid cholesterol depletion. In the present study, we have developed experimental systems for using solutions of cyclodextrins, either 2-hydroxypropyl beta-cyclodextrin or methylated beta-cyclodextrin, complexed with varying amounts of free cholesterol to manipulate cell cholesterol content. Cholesterol delivered via the cyclodextrin has been found to be metabolically active, as measured by the acyl-coenzyme A:cholesterol acyltransferase (ACAT)-mediated esterification of [3H]cholesterol in Fu5AH rat hepatoma cells and Chinese hamster ovary cells. The methylated beta-cyclodextrin was found to be a more efficient donor in all cell types studied, with an average cholesterol uptake of at least 100 microg cholesterol/mg protein within 6 h. By modifying the cyclodextrin:cholesterol molar ratio, it is possible to manipulate the cellular cholesterol content of cells, producing conditions ranging from net cholesterol enrichment to depletion. The use of cyclodextrins provides a convenient, precise and reproducible method for modulating the cholesterol content of tissue culture cells.     

Antibacterial activity of the antiinflammatory agent diclofenac sodium

Speed of sound and conductivity experiments have been done at 298.15 K to study the encapsulation process of sodium salicylate (NaSA) by beta-cyclodextrin (beta-CD) and 2,6-di-O-methyl-beta-cyclodextrin (DIMEB) in aqueous solutions. Since the concentration of the salicyclic form (HSA), coming from the hydrolysis of SA-, is negligible at biological pH, the binding process studied in this work is that of the SA- species. The stoichiometries of the complexes DIMEB: SA- and beta-CD:SA- have been found to be 1:1, as usually determined for most CD:drug complexes. Their association constants and their ionic molar conductivities at infinite dilution have been obtained by fitting the experimental conductivity data with a nonlinear regression method (NLR). For that purpose, a model based on that of Gelb and co-workers has been used. From the values of K beta-CD:SA- = (105 +/- 15)M-1 and KDIMEB:SA- = (140 +/- 20)M-1 obtained, the bioavailability of the salicylate drug in the complexed form has been discussed.     

Serotonin syndrome due to an overdose of moclobemide and clomipramine. A potentially life-threatening association

The speeds of onset of pancuronium, atracurium and vecuronium are increased by prior administration of magnesium sulphate. A prospective, randomized, double-blind, controlled, clinical study was performed to examine the effects of prior i.v. administration of magnesium sulphate 60 mg kg-1 on the neuromuscular blocking effects of rocuronium 0.6 mg kg-1 during isoflurane anaesthesia. Neuromuscular function was measured electromyographically (Relaxograph) in 30 patients who received either magnesium sulphate 60 mg kg-1 or normal saline, 1-min before rocuronium 0.6 mg kg-1. Mean onset times were similar in the two groups (magnesium sulphate 71 (SD 20) s; normal saline 75 (23) s), but times to initial, 10% and 25% recovery from neuromuscular block were significantly longer in the magnesium sulphate group (42.1 (16.3), 49.0 (12.4) and 56.5 (13.2) min, respectively) than in the saline group (25.1 (9.1), 33.0 (11.1) and 35.6 (13.2) min, respectively) (P < 0.05 in all three cases). Administration of magnesium sulphate was not associated with adverse haemodynamic effects. Prior administration of magnesium sulphate, under the study conditions described, prolonged rocuronium-induced neuromuscular block but did not increase speed of onset.