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An experimental design and response surface methodologies using Plackett-Burman and Box-Behnken designs were applied for selecting and optimizing the most appropriate parameters which significantly affect the separation and quantitative estimation of five skeletal muscle relaxants and four analgesic drugs (baclofen, methocarbamol, dantrolene sodium, orphenadrine citrate, cyclobenzaprine hydrochloride, ketoprofen, etoricoxib, ibuprofen, and mefenamic acid) with a relatively short duration of analysis in a single run. For the separation of the nine drugs, an INERTSIL ODS-V3-5 µm C18 column (250 × 4.6 mm I.D.) was used with the optimum mobile phase conditions (45.15 mM ammonium acetate buffer pH 5.56 adjusted with acetic acid, acetonitrile, and methanol in a ratio of 30.5:29.5:40, v/v/v with a flow rate of 1.5 mL/min) and UV-detection at 220 nm. The optimized method was successfully subjected to the validation steps as described in ICH guidelines for linearity, precision, accuracy, robustness, and sensitivity. The optimized and validated method was effectively applied to determine the content of the studied drugs in their pharmaceutical preparations and to expand its applicability to the counterfeit estimation of etoricoxib in different brands of tablet dosage forms.

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Botulinum neurotoxin type-A (BoNT-A) blocks the release of acetylcholine from peripheral cholinergic nerve terminals and is an important option for the treatment of disorders characterised by excessive cholinergic neuronal activity. Several BoNT-A products are currently marketed, each with unique manufacturing processes, excipients, formulation, and non-interchangeable potency units. Nevertheless, the effects of all the products are mediated by the 150 kDa BoNT-A neurotoxin. We assessed the quantity and light chain (LC) activity of BoNT-A in three commercial BoNT-A products (Dysport®; Botox®; Xeomin®). We quantified 150 kDa BoNT-A by sandwich ELISA and assessed LC activity by EndoPep assay. In both assays, we assessed the results for the commercial products against recombinant 150 kDa BoNT-A. The mean 150 kDa BoNT-A content per vial measured by ELISA was 2.69 ng/500 U vial Dysport®, 0.90 ng/100 U vial Botox®, and 0.40 ng/100 U vial Xeomin®. To present clinically relevant results, we calculated the 150 kDa BoNT-A/US Food and Drug Administration (FDA)-approved dose in adult upper limb spasticity: 5.38 ng Dysport® (1000 U; 2 × 500 U vials), 3.60 ng Botox® (400 U; 4 × 100 U vials), and 1.61 ng Xeomin® (400 U; 4 × 100 U vials). EndoPep assay showed similar LC activity among BoNT-A products. Thus, greater amounts of active neurotoxin are injected with Dysport®, at FDA-approved doses, than with other products. This fact might explain the long duration of action reported across multiple indications, which benefits patients, caregivers, clinicians, and healthcare systems.

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PURPOSE: There is a lack of studies to show localization of botulinum toxins (BoNT) within bladder wall and/or absorption rates. Our study examined the later distribution of BoNTA/gadolinium within the bladder wall by performing a delayed MRI scan after intravesical injection. This potentially may help to explain the level and mechanism at which BoNT may be producing its effect. METHODS: A prospective study enrolled 20 consecutive patients with neuropathic or idiopathic overactive bladders. The Aim of the study was to perform MRI 3 h post procedure. Botox 100-200 IU was reconstituted with 19 ml saline and 1 ml of gadolinium contrast. Intradetrusor injections were administered using a rigid 21F cystoscope with a total of 20 injections into bladder wall, including two into the trigone. The depth of injection was approximately 2 mm, without raising a bleb. One radiologist reviewed films and reported on the number of bladder walls with contrast, location, the presence of extravesical extravasation, contrast in distal ureter(s), and bladder wall thickness. RESULTS: Ninety percentage of patients had contrast within bladder wall. There was a variation in the number of bladder walls involved; 85 % had contrast seen in at least two walls. Also, a variation was noted in the extent of extravasation; 80 % showed some evidence. CONCLUSIONS: Diffusion of BoNT after intravesical injection is very common once bladder wall is breeched. Precise injection localization into muscle layer may not be as relevant to outcome as previously assumed. The assumption in our study that localization and diffusion of contrast also represents the localization of BoNT is open to critique as BoNT diffusion is potentially slower (Mehnert et al. in World J Urol 27(3):397-403, 2009). The absence of systemic symptoms after the injection in our series supports guidelines concerning the safety of procedure.

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Six simple, specific, accurate and precise spectrophotometric methods were developed and validated for the simultaneous determination of the analgesic drug; paracetamol (PARA) and the skeletal muscle relaxant; dantrolene sodium (DANT). Three methods are manipulating ratio spectra namely; ratio difference (RD), ratio subtraction (RS) and mean centering (MC). The other three methods are utilizing the isoabsorptive point either at zero order namely; absorbance ratio (AR) and absorbance subtraction (AS) or at ratio spectrum namely; amplitude modulation (AM). The proposed spectrophotometric procedures do not require any preliminary separation step. The accuracy, precision and linearity ranges of the proposed methods were determined. The selectivity of the developed methods was investigated by analyzing laboratory prepared mixtures of the drugs and their combined dosage form. Standard deviation values are less than 1.5 in the assay of raw materials and capsules. The obtained results were statistically compared with each other and with those of reported spectrophotometric ones. The comparison showed that there is no significant difference between the proposed methods and the reported methods regarding both accuracy and precision.

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The aqueous leaves extract of Prosopis cineraria (AEPC) is used traditionally for the treatment of various CNS disorder. The purpose of this study was to evaluate the extract for antidepressant and skeletal muscle relaxant activity. The antidepressant effect of the extract was evaluated using Forced swim test (FST). The immobility periods of control and treated mice were recorded. The antidepressant-like effect of tested compound was compared to that of imipramine (15 mg/kg. p.o). Muscle relaxant property was studied using rotarod apparatus and total fall off time for standard and control group was recorded. Phytochemical screening revealed the presence of saponins, flavonoids, alkaloids, glycosides, tannins and phenolic compounds. The leaf extract at doses of 200 mg/kg significantly decreased the duration of immobility time in FST. The efficacy of tested extract was found to be comparable to that of imipramine. Our results suggested that the aqueous extract of Prosopis cineraria leaves exerts antidepressant-like effect.

O extrato aquoso de folhas de Prosopis cineraria (AEPC) é utilizado, tradicionalmente, para o tratamento de várias disfunções do SNC. O propósito desse estudo foi avaliar o extrato quanto às atividades antidepressiva e relaxante muscular esquelética. O efeito antidepressivo do extrato foi avaliado usando o teste do nado forçado (FST). Registraram-se os períodos de imobilidade dos camundongos controle e dos tratados. O efeito antidepressivo do composto testado foi comparado com a imipramina ((15 mg/kg. p.o). A propriedade relaxante muscular foi estudada usando o cilindro giratório e o tempo total de queda para os grupos padrão e controle foram registrados. A triagem fitoquímica revelou a presença de saponinas, flavonoides, alcaloides, glicosídeos, taninos e compostos fenólicos. O extrato da folha em doses de 200 mg/kg diminui significativamente a duração do tempo de imobilidade no FST. A eficácia do extrato testado foi comparável àquela da imipramina. Nossos resultados sugeriram que o extrato aquoso das folhas da Prosopis cineraria exerce efeito semelhante ao antidepressivo.

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The estimation of paracetamol and orphenadrine citrate in a multicomponent pharmaceutical dosage form by spectrophotometric method has been reported. Because of highly interference in the spectra and the presence of non-linearity caused by the analyte concentrations which deviate from Beer and Lambert's law, partial least-squares (PLS) and artificial neural networks (ANN) techniques were used for the calibration. A validation set of spiked samples was employed for testing the accuracy and precision of the methods. Reasonably good recoveries were obtained with PLS for paracetamol and the use of an ANN allowed the estimation of orphenadrine citrate, a minor component which could not be adequately modeled by PLS. Three production batches of a commercial sample were analysed, and there was statistically no significant difference (P<0.05) between the results with the proposed method and those obtain with the official comparative method.

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Among the agents classified as "Category A" by the U.S. Centers for Disease Control and Prevention, botulinum neurotoxin (BoNT) is the most toxic protein known, with microgram quantities of the protein causing severe morbidity and mortality by oral or i.v. routes. Given that this toxin easily could be used in a potential bioterrorist attack, countermeasures urgently are needed to counteract the pathophysiology of BoNT. At a molecular level, BoNT exerts its paralytic effects through intracellular cleavage of vesicle docking proteins and subsequent organism-wide autonomic dysfunction. In an effort to identify small molecules that would disrupt the interaction between the light-chain metalloprotease of BoNT serotype A and its cognate substrate, a multifaceted screening effort was undertaken. Through the combination of in vitro screening against an optimized variant of the light chain involving kinetic analysis, cellular protection assays, and in vivo mouse toxicity assays, molecules that prevent BoNT/A-induced intracellular substrate cleavage and extend the time to death of animals challenged with lethal toxin doses were identified. Significantly, the two most efficacious compounds in vivo showed less effective activity in cellular assays intended to mimic BoNT exposure; indeed, one of these compounds was cytotoxic at concentrations three orders of magnitude below its effective dose in animals. These two lead compounds have surprisingly simple molecular structures and are readily amenable to optimization efforts for improvements in their biological activity. The findings validate the use of high-throughput screening protocols to define previously unrecognized chemical scaffolds for the development of therapeutic agents to treat BoNT exposure.

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A simple, accurate, precise and sensitive colorimetric method for the determination of some skeletal muscle relaxant drugs, namely orphenadrine citrate (I), baclofen (II), antihistaminic drugs as acrivastine (III) and fexofenadine hydrochloride (IV) is described. This method is based on the formation of charge transfer complex with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) in non-aqueous medium. The orange color products were measured at 472, 465, 475 and 469 nm for drugs I, II, III and IV, respectively. The optimization of various experimental conditions was described. Beer's Law was obeyed in the range (2.5-17.5), (5-70), (2.5-25) and (10-50)microg/ml for drugs I, II, III and IV, respectively. The molar absorptivity (epsilon), sandell sensitivity, detection((LOD)) and quantitation limits((LOQ)) are calculated. The procedure was favorably applied for determination of certain pharmaceutical dosage forms containing the studied drugs. The obtained results were compared with the official and reported methods. There were no significant differences between proposed, reported and the official methods.

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An analytical strategy is described for analyzing quaternary ammonium neuromuscular blocking agents in a wide variety of biological specimens in a forensic setting. Neuromuscular blocking agents such as succinylcholine, pancuronium, and tubocurarine, often used as paralytic agents during surgery, are occasionally suspected as paralytic poisoning agents involved in suspected homicide and suicide cases. Because suspicion in such cases can develop slowly, the age, nature, and quality of available specimens varies greatly. The compounds are challenging analytically because of their simultaneous precharged yet lipophilic character. An analytical strategy has been devised for extracting these compounds from complex matrices using a combination of a modified Bligh and Dyer liquid-liquid extraction (used in reverse) followed by reverse-phase ion pairing solid-phase extraction using heptafluorobutyric acid as an ion pairing reagent. Final analysis is by LC-MS/MS using a tandem quadrupole orthogonal acceleration time of flight instrument (Q-TOF) with repetitive product ion scanning at high resolution. Native and spiked specimens are compared for both quantitative and especially qualitative purposes. The method has been applied to a wide variety of fluid and tissue specimen types, including numerous specimens from exhumation autopsies. For most specimens, detection limits are in the 2 to 10 ng/g range. Succinylmonocholine has been demonstrated to be present at low levels in normal posthumous kidney and liver. The Q-TOF is an excellent platform for forensic analytical investigations. This analytical strategy should also be applicable to other problematic analytes and sample matrices.

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A capillary electrophoretic method with laser-induced fluorescence detection for baclofen (4-amino-3-p-chlorophenylbutyric acid) has been developed. 6-Carboxyfluorescein succinimidyl ester was used for precolumn derivatization of the non-fluorescent drug. Optimal separation and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH 9.5) and an air-cooled argon-ion laser (excitation at 488 nm, emission at 520 nm). Linearity (r > or = 0.99) over three orders of magnitude was generally obtained and the lowest derivatizable concentration limit for baclofen in aqueous solution was 10 nM (2 ng baclofen/ml). Coupled with a simple clean up procedure, the method can be applied to the analysis of baclofen in human plasma at micromolar level. Recovery of spiked baclofen in plasma was 95%. The relative standard deviation values on peak size (0.5 microM level) and migration time were 8.2 and 1.0% (n=7), respectively. The limit of detection of baclofen in plasma was 0.1 microM (21 ng/ml).

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A novel assay method based on the endopeptidase activities of the botulinum neurotoxins has been developed and applied to the detection of botulinum type A and B toxins. An assay system developed for the detection of botulinum type B neurotoxin (BoNT/B) is based on the cleavage of a synthetic peptide substrate representing amino acid residues 60 to 94 of the intracellular target protein for the toxin, VAMP (vesicle-associated membrane protein, or synaptobrevin). In this assay system, immobilized VAMP (60-94) peptide substrate is cleaved by BoNT/B at the Gln-76-Phe-77 bond, leaving the C-terminal cleavage fragment on the solid phase. This fragment is then detected by the addition of an antibody-enzyme reagent which specifically recognizes the newly exposed N terminus of the cleavage product. The developed assay was specific to BoNT/B, showing no cross-reactivity with other clostridial neurotoxins, and had a sensitivity for BoNT/B of 0.6 to 4.5 ng/ml, which could be increased to 0.1 to 0.2 ng/ml by using an assay amplification system based on catalyzed reporter deposition. Trypsin treatment of BoNT/B samples, which converts the single-chain toxin to the active di-chain form, was found to increase the sensitivity of the endopeptidase assay from 5- to 10-fold. An endopeptidase assay for BoNT/A, based on the cleavage of a peptide substrate derived from the protein SNAP-25 (synaptosome-associated protein), was also developed and characterized.

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