Preformulation characterization and identification of excipients for nevirapine loaded niosomes.

Nevirapine (NVP) is used for the management of HIV/AIDS but must be dosed frequently, exhibits unpredictable bioavailability and a side effect profile that includes hepato- and dermo-toxicity. Niosomes are a colloidal drug delivery system that may be used to overcome the low bioavailability, side effect profile and frequent dosing needed when using conventional drug delivery systems. The compatibility of NVP with sorbitan esters, polysorbate, cholesterol and dihexadecyl phosphate (DCP) was investigated using Differential Scanning Calorimetry (DSC), Scanning Electron Microscopy (SEM), Fourier Transform Infra-red Spectroscopy (FTIR) and X-ray Powder Diffraction (XRPD). Screening studies were undertaken to identify potential excipients that would produce niosomes with target critical quality attributes (CQA) viz, a particle size (PS) < 1000 nm, a polydispersity index (PDI) < 0.500 and an entrapment efficiency >90%. The results revealed that sorbitan esters in combination with cholesterol and 5 µmol DCP produced niosomes with the best CQA and Zeta potential (ZP) < -30 mV which suggests good stability of the niosomes on storage. Sorbitan esters produced the smallest niosomes of < 400 nm diameter with a PDI < 0.400 and an entrapment efficiency > 78% without cholesterol. The addition of cholesterol and DCP was essential to form niosomes with target CQA.

Preformulation studies of efavirenz with lipid excipients using thermal and spectroscopic techniques.

Investigation and identification of potential lipids for the manufacture of efavirenz loaded solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) was undertaken. Polymorphic modification and characteristics of the lipids with the best solubilising potential for efavirenz was explored using Fourier Transform Infrared Spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC) and Wide-angle X-ray Scattering (WAXS). Lipid screening revealed that EFV is highly soluble in solid and liquid lipids, with glyceryl monostearate (GM) and Transcutol® HP (THP) exhibiting the best solubilising potential for EFV. GM exists in a stable ß-polymorphic modification prior to exposure to heat, but exists in an α-polymorphic modification following exposure to heat. However, it was established that the addition of THP to GM revealed the co-existence of the α- and ß'-polymorphic modifications of the lipid. EFV (60% w/w) exists in a crystalline state in a 70:30 mixture of GM and THP. Investigation of binary mixtures of EFV/GM and GM/THP, in addition to eutectic mixtures of EFV, GM and THP using FT-IR, DSC and WAXS revealed no potential interactions between EFV and the lipids selected for the production of the nanocarriers.

Development, manufacture and characterization of niosomes for the delivery for nevirapine.

Nevirapine (NVP), used for the treatment of HIV/AIDS, exhibits unpredictable oral bioavailability, has a poor side effect profile and requires frequent dosing. Niosomes are novel drug delivery systems that have the potential to overcome these challenges. A thin layer hydration approach was used to produce niosomes and optimisation was undertaken using design of experiments (DoE) and response surface methodology (RSM) establish and identify parameters that may affect the manufacture of niosomes. The impact of cholesterol and surfactant content, hydration time and temperature on manufacture was investigated. Critical quality attributes (CQA) in respect of particle size (PS), entrapment efficiency (EE), polydispersity index (PDI) and the amount of NVP released at 48 hours was also assessed. The optimised niosome composition was identified and manufactured and the CQA characterised prior to placing the batch on stability for 12 weeks at 4±2 °C and 22±2 °C. The PS, PDI, EE and % NVP released at 48 h was 523.36±23.16 nm, 0.386±0.054, 96.8 % and 25.3 % for niosomes manufactured with Span® 20. Similarly, the parameters were 502.87±21.77 nm and 0.394±0.027, 98.0 % and 25.0 % for mean PS, PDI, EE and %NVP released at 48 h for Span® 80 niosomes. All characterisation was undertaken on the day of manufacture. In conclusion, a simple, cheap, rapid and precise method of manufacture of NVP niosomes was developed, validated and optimised using DoE and RSM and the product exhibited the target CQA.

Design, evaluation and optimization of taste masked clarithromycin powder.

Clarithromycin (CLA) is an extremely bitter macrolide antibiotic used to treat paediatric and adult infections. The bitter taste affects patient adherence and may compromise therapy. This research developed a taste masked CLA resinate using Indion® 234, a weak acidic cation exchange resin. The factors affecting formation of the CLA-resin complex were assessed. Design of experiments was used to optimize response while evaluating input variables such as temperature, CLA-resin ratio,stirring time and pH. CLA loading efficiency was determined spectrophotometrically and CLA release using USP Apparatus II. Differential Scanning Calorimetry (DSC), Scanning Electron Microscop (SEM), Fourier Transform Infrared (FT-IR) Spectroscopy and X-ray Diffraction (XRD) were used to confirm complex formation. A spectrophotometric method was used to assess taste evaluation. The optimum CLA-resin ratio, temperature, and stirring time were 1:4, 80 °C, 3 hours, respectively, at pH 8. Characterization techniques revealed that CLA was crystalline and the complex amorphous in nature. FT-IR spectra of resinate revealed the absence of resonance due to the tertiary amine functional group that is responsible for the bitter taste of CLA. CLA was stable in simulated salivary fluid and was released within 3 hours in gastric fluid. All CLAresin batches revealed complete taste masking. Taste analysis highlighted the improvement of taste masking properties of the resinate as the CLA to resin ratio, increased.

The use of experimental design for the development and validation of an HPLC-ECD method for the quantitation of efavirenz.

A high performance liquid chromatography with electrochemical detection (HPLC-ECD) method for the quantitation of efavirenz (EFV) was developed, since traditional HPLC-UV methods may be inappropriate, given that EFV undergoes photolytic degradation following exposure to UV light. This work describes the use of response surface methodology (RSM) based on a central composite design (CCD) to develop a stability-indicating HPLC method with pulsed ECD in direct current (DC) mode at an applied potential difference and current of +1400 mV and 1.0 µA for the analysis of EFV. Separation of EFV and imipramine was achieved using a Nova-Pak®C18 cartridge column and a mobile phase of phosphate buffer (pH 4.5): acetonitrile (ACN) (55:45 v/v). Mobile phase pH, buffer molarity, ACN concentration and applied potential difference were investigated. The optimized method produced sharp well resolved peaks for imipramine and EFV with retention times of 3.70 and 8.89 minutes. The calibration curve was linear (R2 = 0.9979) over the range 5-70 µg/mL. Repeatability and intermediate precision ranged between 3.37 and 4.34 % RSD and 1.31 and 4.29 % RSD and accuracy between -0.80 and 4.71 % bias. The LOQ and LOD were 5.0 and 1.5 µg/mL. The method was specific for EFV and was used to analyse EFV in commercially available tablets. The HPLC-ECD method is more suitable for quantitative analysis of EFV than HPLC-UV.

The use of experimental design for the development of a capillary zone electrophoresis method for the quantitation of captopril.

A capillary zone electrophoresis (CZE) method for the quantitation of captopril (CPT) using UV detection was developed. Influence of electrolyte concentration and system variables on electrophoretic separation was evaluated and a central composite design (CCD) was used to optimize the method. Variables investigated were pH, molarity, applied voltage and capillary length. The influence of sodium metabisulphite on the stability of test solutions was also investigated. The use of sodium metabisulphite prevented degradation of CPT over 24 hours. A fused uncoated silica capillary of 67.5cm total and 57.5 cm effective length was used for analysis. The applied voltage and capillary length affected the migration time of CPT significantly. A 20 mM phosphate buffer adjusted to pH 7.0 was used as running buffer and an applied voltage of 23.90 kV was suitable to effect a separation. The optimized electrophoretic conditions produced sharp, well-resolved peaks for CPT and sodium metabisulphite. Linear regression analysis of the response for CPT standards revealed the method was linear (R2 = 0.9995) over the range 5-70 µg/mL. The limits of quantitation and detection were 5 and 1.5 µg/mL. A simple, rapid and reliable CZE method has been developed and successfully applied to the analysis of commercially available CPT products.

A stability-indicating high performance liquid chromatographic (HPLC) assay for the simultaneous determination of atorvastatin and amlodipine in commercial tablets.

A simple, rapid, precise and accurate isocratic reversed-phase stability-indicating HPLC method was developed and validated for the simultaneous determination of atorvastatin (AT) and amlodipine (AM) in commercial tablets. The method has shown adequate separation for AM, AT from their associated main impurities and their degradation products. Separation was achieved on a Perfectsil Target ODS-3, 5 microm, 250 mm x 4.6 mm i.d. column using a mobile phase consisting of acetonitrile-0.025 M NaH(2)PO(4) buffer (pH 4.5) (55:45, v/v) at a flow rate of 1 ml/min and UV detection at 237 nm. The drugs were subjected to oxidation, hydrolysis, photolysis and heat to apply stress conditions. The linearity of the proposed method was investigated in the range of 2-30 microg/ml (r=0.9994) for AT and 1-20 microg/ml (r=0.9993) for AM. The limits of detection were 0.65 microg/ml and 0.35 microg/ml for AT and AM, respectively. The limits of quantitation were 2 microg/ml and 1 microg/ml for AT and AM, respectively. Degradation products produced as a result of stress studies did not interfere with the detection of AT and AM and the assay can thus be considered stability-indicating.

Development and validation of a stability-indicating analytical method for the quantitation of oxytocin in pharmaceutical dosage forms.

A single stability-indicating assay for oxytocin (OT) in pharmaceutical dosage forms using gradient elution over 21 min has been reported in the literature. Furthermore, published and compendial methods for the analysis of OT containing dosage forms also involve using HPLC with gradient elution and complicated mobile phases that include hydrophobic ion pairing agents. A simple isocratic and stability-indicating assay was developed and validated. The conditions are as follows, column: Phenomenex C18 Hypersil, 5 microm packing, 4.6 mm x 150 mm with acetonitrile-phosphate buffer (pH 5; 0.08 M) (20:80) as the mobile phase with UV detection at 220 nm The method was found to be specific for OT in the presence of degradation products and chlorbutol (preservative) with an overall analytical run time of 16 min. Accuracy was determined to be 0.77-1.18% bias for all samples tested. Intra-assay precision (repeatability) was found to be 0.22-1.04%R.S.D. while the inter-day precision (intermediate precision) was found to be 1.27-1.68%R.S.D. for the samples studied. The calibration curve was found to be linear with the equation y = 1.81x + 0.02 and a linear regression coefficient of 0.9991 over the range 0.4-12.0 IU/ml. The LOD and the LOQ were determined to be 0.1 and 0.4 IU/ml, respectively. Syntocinon, a commercially available dosage form of OT was assayed resulting in 100.5-106.6% recovery of the label claim and an average of 10.04 IU/ml.

A stability-indicating high performance liquid chromatographic assay for the determination of orlistat in capsules.

A stability-indicating HPLC method was developed and validated for the quantitative determination of orlistat in capsule dosage forms. An isocratic separation was achieved using a Perfectsil target ODS-3, 250 mm x 4.6 mm i.d., 5 microm particle size column with a flow rate of 0.7 ml/min and using a UV detector to monitor the eluate at 210 nm. The mobile phase consisted of methanol:acetonitrile:trifluoroacetic acid (82.5:17.5:0.01, v/v/v). The drug was subjected oxidation, hydrolysis, photolysis and heat to apply stress conditions. Complete separation was achieved for the parent compound and all degradation products in an overall analytical run time of approximately 15 min with the parent compound orlistat eluting at approximately 9 min. The method was linear over the concentration range of 0.02-0.75 mg/ml (r = 0.9998) with a limit of detection and quantitation 0.006 and 0.02 mg/ml, respectively. The method has the requisite accuracy, selectivity, sensitivity and precision to assay orlistat in capsules. Degradation products resulting from the stress studies did not interfere with the detection of orlistat and the assay is thus stability-indicating.

Generic substitution: the use of medicinal products containing different salts and implications for safety and efficacy.

In their quest to gain early entry of new generic products into the market prior to patent expiration, one of the strategies pursued by generic drug product manufacturers is to incorporate different salts of an approved active pharmaceutical ingredient (API) in a brand company's marketed dosage form and subject such dosage forms to bioequivalence assessment. These initiatives present challenges to regulatory authorities where the decision to approve bioequivalent products containing such pharmaceutical alternatives must be considered in the light of safety and efficacy, and more particularly, with respect to their substitutability. This article describes the various issues and contentions associated with the concept of pharmaceutical alternatives, specifically with respect to the uses of different salts and the implications for safety, efficacy and generic substitution.

Exercise adaptation responses for gastric inhibitory polypeptide (GIP) and insulin in obese children. Possible extra-pancreatic effects.

Thirteen obese children and matched controls were fed a mixed meal, and responses were evaluated at fixed intervals for glucose, insulin, and gastric inhibitory polypeptide (GIP). The obese children were evaluated before and within 48 h after completion of a 5-mo exercise training program (ETP). The ETP included three aerobic exercise sessions per week and modest diet restrictions. Caloric expenditure was increased by approximately 300 kcal/exercise session. Weight gain was minimal over the 5 mo. An unexpected increase in GIP response and improved insulin tolerance were recorded for the obese children post-ETP. GIP values were higher (P less than 0.05) at 30 and 60 min and led to a highly significant elevation (P less than 0.01) of the integrated GIP response for post-ETP obese versus both pre-ETP and normal-weight controls. Insulin values were lower (P less than 0.05) at 30 and 60 min and led to a lower integrated insulin response (P less than 0.0585) for post-ETP obese children. However, the obese children continued to secrete more insulin (P less than 0.05) than normal-weight controls. Glucose tolerance, similar for pre-ETP obese subjects and controls, did not change in post-ETP children. Exercise-induced improvement in glucose utilization in these obese children was associated with an increase in GIP secretion. This contrasts with reports that calorie restriction will improve glucose utilization with decreased insulin and GIP secretion. The study demonstrates a previously unreported uncoupling of GIP and insulin secretion and suggests shifts in peripheral tissue sensitivity to insulin-induced glucose uptake. These shifts may, in part, be influenced by GIP.

An LC-MS-MS method for the determination of cyclizine in human serum.

Cyclizine is a piperazine derivative with anti-emetic activity that is useful in the prevention and treatment of nausea and vomiting associated with motion sickness. A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method is presented for the quantitation of cyclizine in serum. Sample pretreatment involved liquid-liquid extraction of 200 microl of serum with dichloromethane after the addition of 100 microl each of ammonium hydroxide and internal standard solutions. The extracts were analyzed by HPLC on a Luna C18 reversed-phase column and an ion-trap mass spectrometer with an electrospray interface. A limit of detection of 1 ng/ml was determined which allowed for the reliable measurement of cyclizine in the serum of human subjects. The method was found to be linear over the calibration range of 2.5-100 ng/ml. The applicability of this method was demonstrated by the analysis of serum obtained from a human volunteer following administration of a single 50 mg cyclizine hydrochloride tablet. The reported method was observed to have the necessary sensitivity, selectivity, precision and accuracy for monitoring cyclizine concentrations in human subjects following oral administration.

Nucleotide sequence of cDNA encoding the fire ant venom protein Sol i II.

For the first time the cDNA encoding a fire ant venom protein has been sequenced. Oligonucleotides were designed according to the amino acid sequence. The cDNA sequence was obtained by hybridizing these primers to mRNA and enhancement by the PCR technique. Comparison to the amino acid sequence of the venom protein shows a leader sequence 19 amino acids long.

Moderate diet control in children: the effects on metabolic indicators that predict obesity-related degenerative diseases.

Nine prepubertal obese boys ages 9 1/2 to 12 yr followed moderately restricted diets and moderate exercise routine for 31 wk. Foods were selected from the family's basic diet and the physical activities were tailored to the home environment. This dietary (approximate decrease of 600 kcal/day) and activity (approximate increase of 300 kcal/day) intervention program was sufficient to stop weight gain and normalize key metabolic indices for prediction of atherosclerosis, hypertension, and diabetes. Throughout the treatment period serum lipid responses included significantly lower (p less than 0.05) total cholesterol, low-density lipoprotein-cholesterol and triglycerides. High-density lipoprotein-cholesterol was constant throughout the period. Responses in carbohydrate metabolism included significantly lower (p less than 0.05) fasting insulin and glucose. Insulin and glucose levels were positively correlated with total caloric consumption and insulin was also positively correlated with sucrose consumption (p less than 0.05). Fasting insulin/glucose ratios and glycosylated Hb decreased throughout the treatment period, but serum glucagon levels remained constant. In response to a glucose load, insulin and glucose decreased significantly by wk 31 of treatment. A practical approach for normalizing metabolism in obese male children is presented.

Variability in an MHCMosa class II beta chain-encoding gene in striped bass (Morone saxatilis).

The major histocompatibility complex (MHC) class II B locus of the striped bass (Morone saxatilis) was found to contain multiple forms of the class II B gene. Seven complete MHC class II B cDNA clones were isolated and sequenced, identifying five unique allelic forms of a MHC class II B gene. Among three specimens, each representing a geographically distinct population (Chesapeake Bay, MD; Roanoke River, NC; and Santee-Cooper Reservoir, SC) extensive variability was detected in the beta 1 encoding domain, which corresponds with the functional peptide-binding region (PBR) of known MHC class II molecules. The location of variable amino acid residues in the beta 1 domains corresponds with polymorphic sites observed in other teleosts and higher vertebrates. The amino acid translated beta 2 domain encoding regions, transmembrane regions, and cytoplasmic regions of the five clones correlated well with those of known vertebrate MHC class II proteins. Seventy-one percent of the variability found within the presumed PBR encoded at the MHCMosa class II B locus corresponded with that of the PBR of a human MHC class II B gene. Overall, the Mosa sequences showed greatest similarity to the MHC class II B genes of cichlid fishes, as expected from phylogenetic relationships.

Quantitative analyses of the coexistence of gamma-aminobutyric acid in substance P-amacrine cells of the larval tiger salamander retina.

The present study was performed as part of a systematic examination of gamma-aminobutyric acid's (GABA) coexistence with other classical transmitters and neuropeptides in neuronal populations of the larval tiger salamander retina. Substance P immunocytochemistry was combined with either GABA immunocytochemistry or autoradiography of high-affinity GABA uptake to examine for the presence of GABA in substance P-amacrine cells of the larval tiger salamander retina. Double-label analyses revealed two populations of substance P-amacrine cells that express both markers of GABA activity. One population was situated in the innermost cell row of the inner nuclear layer, while the other population was located in the ganglion cell layer. In both cases, these double-labelled cells accounted for approximately 10% of substance P-amacrine cells in their respective layers. The present study demonstrates, therefore, that substance P-amacrine cells in the larval tiger salamander retina can be categorized on the basis of their coexisting/non-coexisting relationships with GABA and suggests a possible functional diversity in the population of substance P-amacrine cells.

Hyperthermic and anorectic effects of oxazolidines derived from L-ephedrine in rats.

Oxazolidines synthesized from (-) ephedrine have been proposed as potential pro-drugs, but no pharmacological data on these compounds has been yet reported. In this study, four such compounds are tested in rats for ephedrine-like activity using the hyperthermia and anorexia models. The compounds were synthesized by reaction of (-) ephedrine with salicylaldehyde, acetone, cyclohexanone, and benzaldehyde, respectively. The results showed that all of the compounds decreased food intake significantly, but only the acetone and the salicylaldehyde derivatives caused a significant elevation of body temperature. All of the compounds were less effective than (-) ephedrine in the anorexia model. The acetone and salicylaldehyde derivatives showed similar potency to (-) ephedrine in the hyperthermia model.

Analysis of macrolide antibiotics.

The following macrolide antibiotics have been covered in this review: erythromycin and its related substances, azithromycin, clarithromycin, dirithromycin, roxithromycin, flurithromycin, josamycin, rokitamycin, kitasamycin, mycinamycin, mirosamycin, oleandomycin, rosaramicin, spiramycin and tylosin. The application of various thin-layer chromatography, paper chromatography, gas chromatography, high-performance liquid chromatography and capillary zone electrophoresis procedures for their analysis are described. These techniques have been applied to the separation and quantitative analysis of the macrolides in fermentation media, purity assessment of raw materials, assay of pharmaceutical dosage forms and the measurement of clinically useful macrolide antibiotics in biological samples such as blood, plasma, serum, urine and tissues. Data relating to the chromatographic behaviour of some macrolide antibiotics as well as the various detection methods used, such as bioautography, UV spectrophotometry, fluorometry, electrochemical detection, chemiluminescence and mass spectrometry techniques are also included.

Exercise-induced bronchospasm in the young athlete: guidelines for routine screening and initial management.

Exercise induced bronchospasm (EIB) commonly occurs several minutes into or following an exercise event. Respiratory heat loss and respiratory water loss have been suspected as the precursor to exercise-induced bronchospasm. Obstructive EIB has been reported in elite Olympic athletes as well as the recreational athlete. Although exercise-induced bronchospasm presents as wheezing, chest tightness, or dizziness during or after exercise, cough post-exercise is a common and an easily detected characteristic of EIB. When exercise induced bronchospasm is suspected in the young athlete, an exercise challenge test should be performed. A 10% or more decrease in the peak expiratory flow rate in the post-exercise period is diagnostic of EIB. Once the diagnosis of EIB has been made, both nonpharmacological and pharmacological interventions are beneficial in reducing the airway responsiveness. Nonpharmacological measures include extensive education and cardiovascular fitness evaluation. Initial pharmacological management should consist of a trial of albuterol inhaler use 15 min prior to exercise. Early identification and treatment of EIB may enhance sports performance as well as enjoyment.