In-line ATR-UV and Raman Spectroscopy for Monitoring API Dissolution Process During Liquid-Filled Soft-Gelatin Capsule Manufacturing

Complete dissolution of the active pharmaceutical ingredient (API) is critical in the manufacturing of liquid-filled soft-gelatin capsules (SGC). Attenuated total reflectance UV spectroscopy (ATR-UV) and Raman spectroscopy have been investigated for in-line monitoring of API dissolution during manufacturing of an SGC product. Calibration models have been developed with both techniques for in-line determination of API potency. Performance of both techniques was evaluated and compared. The ATR-UV methodology was found to be able to monitor the dissolution process and determine the endpoint, but was sensitive to temperature variations. The Raman technique was also capable of effectively monitoring the process and was more robust to the temperature variation and process perturbations by using an excipient peak for internal correction. Different data preprocessing methodologies were explored in an attempt to improve method performance.     

Development and Validation of a Luminescence-based,Medium-Throughput Assay for Drug Screening in Schistosoma mansoni

Background

Schistosomiasis, one of the world’s greatest neglected tropical diseases, is responsible for over 280,000 human deaths per annum. Praziquantel, developed in the 1970s, has high efficacy, excellent tolerability, few and transient side effects, simple administration procedures and competitive cost and it is currently the only recommended drug for treatment of human schistosomiasis. The use of a single drug to treat a population of over 200 million infected people appears particularly alarming when considering the threat of drug resistance. Quantitative, objective and validated methods for the screening of compound collections are needed for the discovery of novel anti-schistosomal drugs.

Methodology/Principal Findings

The present work describes the development and validation of a luminescence-based, medium-throughput assay for the detection of schistosomula viability through quantitation of ATP, a good indicator of metabolically active cells in culture. This validated method is demonstrated to be fast, highly reliable, sensitive and automation-friendly. The optimized assay was used for the screening of a small compound library on S. mansoni schistosomula, showing that the proposed method is suitable for a medium-throughput semi-automated screening. Interestingly, the pilot screening identified hits previously reported to have some anti-parasitic activity, further supporting the validity of this assay for anthelminthic drug discovery.

Conclusions

The developed and validated schistosomula viability luminescence-based assay was shown to be successful and suitable for the identification of novel compounds potentially exploitable in future schistosomiasis therapies.     

Development and Validation of a Discriminative Dissolution Test for Nimesulide Suspensions

The dissolution test for oral dosage forms has recently widened to a variety of special dosage forms such as suspensions. For class II drugs, such as nimesulide (NMS), this study is very important because formulation problems may compromise drug bioavailability. In the present work, tests with four brands of commercially available NMS (RA, TS, TB, and TC) have been performed in order to study their dissolution at different conditions. The suspensions have been characterized relatively to particle size, pH, and density besides NMS assay and the amount of drug in solution in the suspension vehicles. The dissolution study was conducted using the following media: simulated intestinal fluid, pH 6.8, containing polysorbate 80 (P80) or sodium lauryl sulfate (SLS); phosphate buffer, pH 7.4, with P80 and aqueous solution of SLS. Concerning the quantitative analysis, the UV–VIS spectrophotometry could have been used in substitution to high-performance liquid chromatography since the methodology had been adequately validated. The influence of the drug particle size distribution was significant on the dissolution profiles of NMS formulations, confirming to be a factor that should be strictly controlled in the development of oral suspensions.     

Development and Validation of a HPV-32 Specific PCR Assay

Background

The present work aims at determining HCV genotypes in patients with chronic HCV infection, in Gaza strip, Palestine. The most common risk factors for HCV transmission were also evaluated in conjunction with the genotyping data.

Results

The study shows that there are only two major genotypes of HCV in Gaza Strip: Genotype 1 (subtypes 1a and 1b) collectively contribute to 28.3% of the cases, and genotype 4 (subtypes 4a and 4c/d) collectively contribute to 64.1% of the cases. Mixed infection with the two genotypes was also present among 7.6% of the cases. In this study a statistically significant relationship was established between the distribution of these genotypes and the patients' living place, traveling history, history of blood transfusion and history of surgical operations.

Conclusion

The present study is the first to link HCV genotyping in Gaza strip with its possible roots of transmission. Traveling to endemic countries, especially Egypt; blood transfusion and surgical operations are major roots of HCV infection in Gaza strip. The results indicate that iatrogenic and nosocomial procedures may be responsible for the majority of HCV infections in Gaza strip.     

Development of a High-Throughput Three-Dimensional Invasion Assay for Anti-Cancer Drug Discovery

The lack of three-dimensional (3-D) high-throughput (HT) screening assays designed to identify anti-cancer invasion drugs is a major hurdle in reducing cancer-related mortality, with the key challenge being assay standardization. Presented is the development of a novel 3-D invasion assay with HT potential that involves surrounding cell-collagen spheres within collagen to create a 3-D environment through which cells can invade. Standardization was achieved by designing a tooled 96-well plate to create a precisely designated location for the cell-collagen spheres and by using dialdehyde dextran to inhibit collagen contraction, maintaining uniform size and shape. This permits automated readout for determination of the effect of inhibitory compounds on cancer cell invasion. Sensitivity was demonstrated by the ability to distinguish varying levels of invasiveness of cancer cell lines, and robustness was determined by calculating the Z-factor. A Z-factor of 0.65 was obtained by comparing the effects of DMSO and anti-β1-integrin antibody, an inhibitory reagent, on the invasion of Du145 cancer cells, suggesting this novel assay is suitable for large scale drug discovery. As proof of principle, the NCI Diversity Compound Library was screened against human invasive cancer cells. Nine compounds exhibiting high potency and low toxicity were identified, including DX-52-1, a compound previously reported to inhibit cell migration, a critical determinant of cancer invasion. The results indicate that this innovative HT platform is a simple, precise, and easy to replicate 3-D invasion assay for anti-cancer drug discovery.     

Development and Validation of a Discriminative Dissolution Method for Atorvastatin Calcium Tablets using in vivo Data by LC and UV Methods

A dissolution method to analyze atorvastatin tablets using in vivo data for RP and test pilot (PB) was developed and validated. The appropriate conditions were determined after solubility tests using different media, and sink conditions were established. The conditions used were equipment paddle at 50 rpm and 900 mL of potassium phosphate buffer pH 6.0 as dissolution medium. In vivo release profiles were obtained from the bioequivalence study of RP and the generic candidate PB. The fraction of dose absorbed was calculated using the Loo–Riegelman method. It was necessary to use a scale factor of time similar to 6.0, to associate the values of absorbed fraction and dissolved fraction, obtaining an in vivo–in vitro correlation level A. The dissolution method to quantify the amount of drug dissolved was validated using high-performance liquid chromatography and ultraviolet spectrophotometry, and validated according to the USP protocol. The discriminative power of dissolution conditions was assessed using two different pilot batches of atorvastatin tablets (PA and PB) and RP. The dissolution test was validated and may be used as a discriminating method in quality control and in the development of the new formulations.     

Development and Optimisation of Spironolactone Nanoparticles for Enhanced Dissolution Rates and Stability

Stable solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) formulations to enhance the dissolution rates of poorly soluble drug spironolactone (SP) were being developed. Probe ultra-sonication method was used to prepare SLNs and NLCs. All NLCs contained stearic acid (solid lipid carrier) and oleic acid (liquid lipid content), whereas, SLNs were prepared and optimised by using the solid lipid only. The particles were characterised in terms of particle size analysis, thermal behaviour, morphology, stability and in vitro release. The zeta sizer data revealed that the increase in the concentration of oleic acid in the formulations reduced the mean particle size and the zeta potential. The increase in concentration of oleic acid from 0 to 30% (w/w) resulted in a higher entrapment efficiency. All nanoparticles were almost spherically shaped with an average particle size of about ～170 nm. The DSC traces revealed that the presence of oleic acid in the NLC formulations resulted in a shift in the melting endotherms to a higher temperature. This could be attributed to a good long-term stability of the nanoparticles. The stability results showed that the particle size remained smaller in NLC compared to that of SLN formulations after 6 months at various temperatures. The dissolution study showed about a 5.1- to 7.2-fold increase in the release of the drug in 2 h compared to the raw drug. Comparing all nanoparticle formulations indicated that the NLC composition with a ratio of 70:30 (solid:liquid lipid) is the most suitable formulation with desired drug dissolution rates, entrapment efficiency and physical stability.     

Single-Injection HPLC Method for Rapid Analysis of a Combination Drug Delivery System

Developing combination drug delivery systems (CDDS) is a challenging but necessary task to meet the needs of complex therapy regimes for patients. As the number of multi-drug regimens being administered increases, so does the difficulty of characterizing the CDDS as a whole. We present a single-step method for quantifying three model therapeutics released from a model hydrogel scaffold using high-performance liquid chromatography (HPLC). Poly(ethylene glycol) dimethacrylate (PEGDMA) hydrogel tablets were fabricated via photoinitiated crosslinking and subsequently loaded with model active pharmaceutical ingredients (APIs), namely, porcine insulin (PI), fluorescein isothiocyanate-labeled bovine serum albumin (FBSA), prednisone (PSE), or a combination of all three. The hydrogel tablets were placed into release chambers and sampled over 21 days, and APIs were quantified using the method described herein. Six compounds were isolated and quantified in total. Release kinetics based on chemical properties of the APIs did not give systematic relationships; however, PSE was found to have improved device loading versus PI and FBSA. Rapid analysis of three model APIs released from a PEGDMA CDDS was achieved with a direct, single-injection HPLC method. Development of CDDS platforms is posited to benefit from such analytical approaches, potentially affording innovative solutions to complex disease states.Key words: combination drug delivery systems, combination therapy, controlled release, HPLC, hydrogel, PEGDMA, protein therapeutic     

Development of a New Dissolution Test Method for Soft Chewable Dosage Forms

Soft chewable dosage forms are a new approach to improve the compliance of medication for special patient populations. Based on their texture, they are chewed several times before they get swallowed. A suitable dissolution method based on in vivo chewing data was developed. The method covers parts of dissolution within the oral cavity (simulation of chewing) as well as the dissolution of the swallowed bolus within the gastrointestinal tract. Chewing was simulated by the help of a steel tooth assembled to a texture analyzer and wedge gliding on an inclined plane, imitating the occlusal glide. Chewing cycles of non-brittle, elastically deformable foods were predicted by a multiple linear regression (R _adj?=?0.985) using hardness, stickiness, fat/water content, and softening behavior as independent variables. Cross-validation of three sets of chewing data led to a root mean square error of prediction of 0.408 or 0.658 chewing cycles, respectively. The new method is able to distinguish between different soft chewable formulations which had been approved as similar by the dissolution method of the European Pharmacopoeia. Furthermore, it provides information about the drug content released within the time of chewing (7–15%).     

Development and Validation of a Discriminative Dissolution Test for Betamethasone Sodium Phosphate and Betamethasone Dipropionate Intramuscular Injectable Suspension

The intramuscular administration of the injectable suspension betamethasone sodium phosphate (BSP) and betamethasone dipropionate (BD) has immediate therapeutic activity due to solubilized BSP and prolonged activity resulting from the slow release of BD micro-crystals. The purpose of this study was to develop and validate a dissolution method for BD in intramuscular injectable suspensions with detection by high-performance liquid chromatography (HPLC) method. Five commercial products presented a distribution of particle sizes, ranging between 7.43 and 40.25 μm as measured by laser diffraction. It was also found that particle sizes differed between batches of the same product. The different products were tested using the paddle apparatus, with stirring speeds of 25 and 50 rpm in 300 mL of phosphate buffer; simulated body fluid, muscle fluid, and synovial fluid were used as biorelevant dissolution media at 37 ± 0.5°C. It was verified that not only does average particle size affect the dissolution rate, but also the mode and the polydispersity index of the particles. Discriminatory power was obtained using the in vitro dissolution method with 0.1 M sodium phosphate buffer pH 7.4 containing 0.1% sodium lauryl sulfate and a stirring speed of 50 rpm. The HPLC-method is linear, precise, selective, and accurate for the quantification of BSP and BD in dissolution profile testing. This dissolution method can be utilized as a method to control the quality of these injectable suspensions.Key words: dipropionate betamethasone, dissolution test, intramuscular injectable suspensions, simulated muscular fluid, sodium phosphate betamethasone     

天然产物在新药开发中的应用--HMG-CoA还原酶抑制剂新药的发现对天然产物研究与开发的启示

自1973年发现第一个作用于HMG-CoA还原酶的化合物mevastatin以来,多个具有HMG-CoA还原酶抑制活性的抗高血脂新药被开发成功,并成为治疗高血脂症的首选药物。在此简单介绍该类新药的发现与开发过程,并就天然产物的研究与开发提出一点个人见解。     

Development and Validation of a Next-Generation Sequencing Assay for BRCA1 and BRCA2 Variants for the Clinical Laboratory

The objective of this study was to design and validate a next-generation sequencing assay (NGS) to detect BRCA1 and BRCA2 mutations. We developed an assay using random shearing of genomic DNA followed by RNA bait tile hybridization and NGS sequencing on both the Illumina MiSeq and Ion Personal Gene Machine (PGM). We determined that the MiSeq Reporter software supplied with the instrument could not detect deletions greater than 9 base pairs. Therefore, we developed an alternative alignment and variant calling software, Quest Sequencing Analysis Pipeline (QSAP), that was capable of detecting large deletions and insertions. In validation studies, we used DNA from 27 stem cell lines, all with known deleterious BRCA1 or BRCA2 mutations, and DNA from 67 consented control individuals who had a total of 352 benign variants. Both the MiSeq/QSAP combination and PGM/Torrent Suite combination had 100% sensitivity for the 379 known variants in the validation series. However, the PGM/Torrent Suite combination had a lower intra- and inter-assay precision of 96.2% and 96.7%, respectively when compared to the MiSeq/QSAP combination of 100% and 99.4%, respectively. All PGM/Torrent Suite inconsistencies were false-positive variant assignments. We began commercial testing using both platforms and in the first 521 clinical samples MiSeq/QSAP had 100% sensitivity for BRCA1/2 variants, including a 64-bp deletion and a 10-bp insertion not identified by PGM/Torrent Suite, which also suffered from a high false-positive rate. Neither the MiSeq nor PGM platform with their supplied alignment and variant calling software are appropriate for a clinical laboratory BRCA sequencing test. We have developed an NGS BRCA1/2 sequencing assay, MiSeq/QSAP, with 100% analytic sensitivity and specificity in the validation set consisting of 379 variants. The MiSeq/QSAP combination has sufficient performance for use in a clinical laboratory.     

Oxidase-Peroxidase Method of Ethanol Assay in Fermented Musts and Wine Products

A new alcohol oxidase-peroxidase method of ethanol assay in fermented musts and wine products is described and compared to conventional methods routinely used in winemaking. The sensitivity, accuracy, and reliability of this method were determined. The results of ethanol determination in fermented musts and wines correlated well with the data obtained by refractometry (correlation coefficient R = 0.9595, p < 0.0001) and densitometry (correlation coefficient R = 0.9384, p < 0.0001). The proposed method is less time- and labor-consuming and allows simultaneous analysis of a series of wine samples.     

Development and Validation of a Discriminating <Emphasis Type="Italic">In Vitro</Emphasis> Dissolution Method for a Poorly Soluble Drug,Olmesartan Medoxomil: Comparison Between Commercial Tablets

A dissolution test for tablets containing 40 mg of olmesartan medoxomil (OLM) was developed and validated using both LC-UV and UV methods. After evaluation of the sink condition, dissolution medium, and stability of the drug, the method was validated using USP apparatus 2, 50 rpm rotation speed, and 900 ml of deaerated H₂O + 0.5% sodium lauryl sulfate (w/v) at pH 6.8 (adjusted with 18% phosphoric acid) as the dissolution medium. The model-independent method using difference factor (f ₁) and similarity factor (f ₂), model-dependent method, and dissolution efficiency were employed to compare dissolution profiles. The kinetic parameters of drug release were also investigated. The obtained results provided adequate dissolution profiles. The developed dissolution test was validated according to international guidelines. Since there is no monograph for this drug in tablets, the dissolution method presented here can be used as a quality control test for OLM in this dosage form, especially in a batch to batch evaluation.     

Roller Compaction, Granulation and Capsule Product Dissolution of Drug Formulations Containing a Lactose or Mannitol Filler, Starch, and Talc

This study investigated the influence of excipient composition to the roller compaction and granulation characteristics of pharmaceutical formulations that were comprised of a spray-dried filler (lactose monohydrate or mannitol), pregelatinized starch, talc, magnesium stearate (1% w/w) and a ductile active pharmaceutical ingredient (25% w/w) using a mixed-level factorial design. The main and interaction effects of formulation variables (i.e., filler type, starch content, and talc content) to the response factors (i.e., solid fraction and tensile strength of ribbons, particle size, compressibility and flow of granules) were analyzed using multi-linear stepwise regression analysis. Experimental results indicated that roller compacted ribbons of both lactose and mannitol formulations had similar tensile strength. However, resulting lactose-based granules were finer than the mannitol-based granules because of the brittleness of lactose compared to mannitol. Due to the poor compressiblility of starch, increasing starch content in the formulation from 0% to 20% w/w led to reduction in ribbon solid fraction by 10%, ribbon tensile strength by 60%, and granule size by 30%. Granules containing lactose or more starch showed less cohesive flow than granules containing mannitol and less starch. Increasing talc content from 0% to 5% w/w had little effect to most physical properties of ribbons and granules while the flow of mannitol-based granules was found improved. Finally, it was observed that stored at 40 °C/75% RH over 12 weeks, gelatin capsules containing lactose-based granules had reduced dissolution rates due to pellicle formation inside capsule shells, while capsules containing mannitol-based granules remained immediate dissolution without noticeable pellicle formation.     

Development and Validation of a Method for Profiling Post-Translational Modification Activities Using Protein Microarrays

Background

Post-translational modifications (PTMs) impact on the stability, cellular location, and function of a protein thereby achieving a greater functional diversity of the proteome. To fully appreciate how PTMs modulate signaling networks, proteome-wide studies are necessary. However, the evaluation of PTMs on a proteome-wide scale has proven to be technically difficult. To facilitate these analyses we have developed a protein microarray-based assay that is capable of profiling PTM activities in complex biological mixtures such as whole-cell extracts and pathological specimens.

Methodology/Principal Findings

In our assay, protein microarrays serve as a substrate platform for in vitro enzymatic reactions in which a recombinant ligase, or extracts prepared from whole cells or a pathological specimen is overlaid. The reactions include labeled modifiers (e.g., ubiquitin, SUMO1, or NEDD8), ATP regenerating system, and other required components (depending on the assay) that support the conjugation of the modifier. In this report, we apply this methodology to profile three molecularly complex PTMs (ubiquitylation, SUMOylation, and NEDDylation) using purified ligase enzymes and extracts prepared from cultured cell lines and pathological specimens. We further validate this approach by confirming the in vivo modification of several novel PTM substrates identified by our assay.

Conclusions/Significance

This methodology offers several advantages over currently used PTM detection methods including ease of use, rapidity, scale, and sample source diversity. Furthermore, by allowing for the intrinsic enzymatic activities of cell populations or pathological states to be directly compared, this methodology could have widespread applications for the study of PTMs in human diseases and has the potential to be directly applied to most, if not all, basic PTM research.