Characterization of Aptamer BC 007 Substance and Product Using Circular Dichroism and Nuclear Magnetic Resonance Spectroscopy.

Possible unwanted folding of biopharmaceuticals during manufacturing and storage has resulted in analysis schemes compared to small molecules that include bioanalytical characterization besides chemical characterization. Whether bioanalytical characterization is required for nucleotide-based drugs, may be decided on a case-by-case basis. Nucleotide-based pharmaceuticals, if chemically synthesized, occupy an intermediate position between small-molecule drugs and biologics. Here, we tested whether a physicochemical characterization of a nucleotide-based drug substance, BC 007, was adequate, using circular dichroism (CD) spectroscopy. Nuclear magnetic resonance confirmed CD data in one experimental setup. BC 007 forms a quadruplex structure under specific external conditions, which was characterized for its stability and structural appearance also after denaturation using CD and nuclear magnetic resonance. The amount of the free energy (ΔG0) involved in quadruplex formation of BC 007 was estimated at +8.7 kJ/mol when dissolved in water and +1.4 kJ/mol in 154 mM NaCl, indicating structural instability under these conditions. However, dissolution of the substance in 5 mM of KCl reduced the ΔG0 to -5.6 kJ/mol due to the stabilizing effect of cations. These results show that positive ΔG0 of quadruplex structure formation in water and aqueous NaCl prevents BC 007 from preforming stable 3-dimensional structures, which could potentially affect drug function.

Reinforcement of Aluminum Oxide Filler on the Flexural Strength of Different Types of Denture Base Resins: An In vitro Study.

INTRODUCTION: Acrylic resins have been used extensively for the fabrication of denture bases because of their aesthetic qualities, ease of manipulation and repairability. Flexural fatigue of the denture base has been shown to be a factor in the clinical failure of polymethyl methacrylate resin dentures. Also, the fracture can result from impact, fatigue or degradation of the base material. Hence, there is a need to increase the strength of denture base resins. AIM: To evaluate the effect of reinforcing alumina oxide filler on the flexural strength of different acrylic resins. MATERIALS AND METHODS: A total of 180 acrylic specimens were fabricated, which were divided into three groups self cure acrylic resin (SC), conventional heat cure resin (HC) and high strength heat cure resin (HI). Each group was divided into four subgroups i.e., control group and the specimens of the remaining three groups were reinforced with aluminum oxide (Al2O3) powder by 5%, 10% and 15% by weight. Specimens were stored in distilled water for one week; flexural strength was tested by universal testing machine. Results were analysed by one-way analysis of variance and post-hoc Tukey paired group comparison tests. RESULTS: Flexural strength of SC increased by 9%, 13% and 19%, Flexural strength of HC increased by 8%, 15% and 19% and that of HI increased by 21%, 26% and 29% compared to control group by adding 5%,10% and 15% of alumina filler (p-value <0.001). Addition of 15% of alumina powder to SC showed high flexure strength compared to control group of HC (p-value <0.001). Addition of 10% and 15% of alumina powder to HC showed high flexural strength compared to control group of HI (p-value <0.001). CONCLUSION: Addition of alumina to self cure acrylic resin, conventional heat cure acrylic resin and high strength heat cure acrylic resin increased the flexural strength. Increasing the flexural strength of the acrylic resin base material could lead to more clinical success.

Factors associated with poor adherence to antiviral treatment for hepatitis B.

Antiviral therapy for hepatitis B is effective and reduces the risk of progression to cirrhosis and liver cancer but is often required for an indefinite duration. Treatment adherence is important to prevent the development of resistance and optimize outcomes. Pharmacy adherence measures can be used to assess treatment adherence, with the medication possession ratio being less susceptible to bias than physician- or self-reported adherence. The aim of this study was to measure adherence in public hospital outpatients over a 3-year period and to examine factors associated with nonadherence. A retrospective study of pharmacy records of patients dispensed antiviral therapy for hepatitis B from four major hospitals in Melbourne between 2010 and 2013. Hospital record numbers were linked with and de-identified demographic information including age, sex, Indigenous status, country of birth, interpreter requirement, spoken language and postcode of residence. The medication possession ratio was the outcome measure with poor adherence defined <.90. Univariate logistic regression and multivariate logistic regression were performed to examine associations with nonadherence. Records of 1026 patients were included in the analysis. Twenty per cent of all participants met the definition of poor adherence. Significant factors affecting adherence included age <35 years (P=.002), hospital site and treatment by multiple doctors within shorter time periods. This is the largest study examining detailed factors associated with adherence to hepatitis B treatment. Understanding poor adherence in clinical settings, and the factors associated with lower adherence, is important to inform efforts towards promoting treatment adherence for hepatitis B.

Evaluation of the Various Drying Methods on Surface Hardness of Type IV Dental Stone.

BACKGROUND: Studies regarding the effect of various methods to increase the surface hardness of Type IV dental stone are not conclusive. Therefore, this study was carried out to evaluate the effect of air drying, micro oven drying and die hardener on surface hardness of Type IV dental stone. MATERIALS AND METHODS: A standard metal die was fabricated; polyvinyl siloxane impression material was used to make the molds of metal die. A total of 120 specimens were obtained from two different die stones and were grouped as Group A (kalrock) and Group B (pearl stone), and were subjected to air drying for 24 h, micro oven drying and application of die hardener. These models were then subjected to surface hardness testing using the knoop hardness instrument. The obtained data were subjected to statistical analysis. RESULTS: The hardness of Group A specimens was 64 ± 0.54 Knoop hardness number (KHN) after application of die hardener, 60.47 ± 0.41 KHN after 24 h air drying, 58.2 ± 0.88 after microwave oven drying and 24.6 ± 0.4 after 1 h air drying. The hardness of Group B specimens was 45.59 ± 0.63 KHN after application of die hardener, 40.2 ± 0.63 KHN after 24 h air drying, 38.28 ± 0.55 KHN after microwave oven drying and 19.91 ± 0.64 KHN after 1 h air drying. CONCLUSION: Group A showed better results than Group B at all times. Application of the die hardener showed highest hardness values followed in the order by 24 h air drying, microwave oven drying and 1 h air drying in both groups. The study showed that air drying the dies for 24 h followed by application of a single layer of the die hardener produced the best surface hardness and is recommended to be followed in practice.

Comparative analysis of postcementation hypersensitivity with glass ionomer cement and a resin cement: an in vivo study.

AIM: The aim of this clinical study was to compare the postoperative sensitivity of abutment teeth restored with full coverage restorations retained with either conventional glassionomer cement (GIC) or resin cement. MATERIALS AND METHODS: Fifty patients received full-coverage restorations on vital abutment teeth. Of these, 25 were cemented with GIC (GC Luting and Lining cement) and the other 25 using an adhesive resin cement (Smartcem 2). A randomized single blind study was undertaken for acquiring and evaluating the data. The teeth were examined before cementation, after cementation, 24 hours postcementation and 7 days postcementation. A visual analog scale was used to help the patient rate hypersensitivity. RESULTS: The statistical analysis of the result was done using students paired t-test. No statistically significant difference between Smartcem 2 and GIC was observed, when tested immediately and 24 hours after cementation. Statistically significant difference was seen between Smartcem 2 and GIC when tested 7 days postcementation with a significance level of 0.05. Higher postoperative sensitivity was seen with GIC when compared to resin cement. CONCLUSION: In this study, the incidence of postoperative hypersensitivity after cementation of full-crown restorations with GIC and resin cement was similar when tested immediately. However, 7 days postcementation, abutments with GIC showed higher response compared to resin cement. CLINICAL SIGNIFICANCE: A self-adhesive resin cement can be the material of choice for luting if presence of postoperative sensitivity is of prime consideration. In case GIC is being used, patient should be informed about the presence of sensitivity for a more prolonged period than with resin cement.

Occlusal plane determination using custom made broadrick occlusal plane analyser: a case control study.

Proper occlusal plane is an essential consideration when multiple long span posterior restorations are designed. The determination of the occlusal plane can have a profound effect on the short and long term success of a restorative case. Purpose of Study. (1) To determine the appropriate occlusal curve for individual patients. (2) To compare the deviation of the clinical occlusal curve with the ideal ones. Materials and Methods. A total of 20 subjects were examined and study models were made of their maxillary and mandibular dentition. Inter-occlusal records were made and the casts were articulated in semiadjustable articulator. An ideal occlusal plane was created. The distance of the farthest cusp tip from the Broadrick curve was measured along the long axis of the tooth for each individual. Paired t-tests were used to compare the findings between subjects and controls. Results. A statistically significant difference P < 0.05 was found in the deviation from the Broadrick curve between patients who have lost posterior teeth and the control group who had a full dentition with no missing teeth. Conclusion. Proper utilization of the broadrick flag on a semi-adjustable articulator will allow for a correct determination of the occlusal plane.

First-in-human mutation-targeted siRNA phase Ib trial of an inherited skin disorder.

The rare skin disorder pachyonychia congenita (PC) is an autosomal dominant syndrome that includes a disabling plantar keratoderma for which no satisfactory treatment is currently available. We have completed a phase Ib clinical trial for treatment of PC utilizing the first short-interfering RNA (siRNA)-based therapeutic for skin. This siRNA, called TD101, specifically and potently targets the keratin 6a (K6a) N171K mutant mRNA without affecting wild-type K6a mRNA. The safety and efficacy of TD101 was tested in a single-patient 17-week, prospective, double-blind, split-body, vehicle-controlled, dose-escalation trial. Randomly assigned solutions of TD101 or vehicle control were injected in symmetric plantar calluses on opposite feet. No adverse events occurred during the trial or in the 3-month washout period. Subjective patient assessment and physician clinical efficacy measures revealed regression of callus on the siRNA-treated, but not on the vehicle-treated foot. This trial represents the first time that siRNA has been used in a clinical setting to target a mutant gene or a genetic disorder, and the first use of siRNA in human skin. The callus regression seen on the patient's siRNA-treated foot appears sufficiently promising to warrant additional studies of siRNA in this and other dominant-negative skin diseases.

Biliary epithelial trefoil peptide expression is increased in biliary diseases.

AIMS: Maintenance of the cellular integrity of the biliary epithelium may involve the production of mucins and mucin-associated peptides. In the luminal gastrointestinal tract, mucins and the mucin-associated trefoil peptides (TFF) are integral to cytoprotection and cellular repair of the mucosa. METHODS AND RESULTS: Samples of normal and diseased human liver tissue were examined using histological and immunohistochemical techniques, for the expression of TFF and mucins. Bile ducts were classified as small, medium or large depending upon the number of biliary epithelial cells. TFF expression was demonstrated in biliary epithelial cells of both normal and diseased liver tissue. TFF expression was greatest in the large bile ducts. In normal liver tissue, expression of at least one TFF was demonstrated in 2-7% of small bile ducts, 5-31% of medium bile ducts and 31-85% of large bile ducts. Seventy-seven percent of large bile ducts secreted mucins and all three TFF concurrently, compared with 3% of medium bile ducts and no small bile ducts. Biliary disease resulted in an increased expression of TFF1 and TFF3 in the medium bile ducts. CONCLUSIONS: The biliary epithelial cells in normal and diseased human liver tissue express TFF, particularly in the larger bile ducts. TFF expression may be up-regulated or induced in biliary diseases as a response to injury, as is seen in epithelial damage elsewhere in the gastrointestinal tract.

Large-scale purification of antisense oligonucleotides by high-performance membrane adsorber chromatography.

Very high flux ion-exchange membranes were utilized for a novel purification of antisense oligonucleotides (20-mer). Strong anion-exchange membranes were produced by attaching polymeric ligands onto a microporous cellulosic matrix. The oligonucleotides purified were therapeutic single-stranded phosphorothioates deoxyribonucleotides. Although small-scale membrane devices (15 cm2) had similar resolution to traditional chromatographic columns; their throughputs were superior. Greater than a 1300-fold scale-up produced very similar purity and yields of the phosphorothionate product. Scale-up experiments were conducted with a 2 m2 surface area membrane module. These modules were easily capable of very high throughputs of 0.5 to 2 l/min. High purity and yields were achieved by both step and linear gradient elution.

Determination of free and encapsulated oligonucleotides in liposome formulated drug product.

Liposomes have been recognized as new delivery vehicles for peptide and oligonucleotide drugs, offering effective drug protection and influencing drug distribution from the circulation to tissues. To ensure consistent formulation behavior and drug distribution, the amounts of free and encapsulated active pharmaceutical ingredient in the liposome formulation must be determined. A simple and reliable method has been developed for the determination of free and encapsulated oligonucleotide drugs in liposomes. Capillary electrophoresis in entangled polyacrylamide solution was optimized for the analysis. Liposome samples were treated to release encapsulated oligonucleotide so that total concentration could be determined, and untreated liposomes were applied directly to capillary column to determine the fraction of free oligonucleotides. Recoveries are 96-105% and relative standard deviations are generally 2-3%. Multiple liposome types were analyzed with satisfactory results.

Analysis of internal (n-1)mer deletion sequences in synthetic oligodeoxyribonucleotides by hybridization to an immobilized probe array.

The purity of a drug substance can influence its toxicity and potency, so impurities must be specifically determined. In the case of synthetic oligodeoxyribonucleotide drugs, however, product complexity makes complete impurity speciation difficult. The goal of the present work was to develop a new analytical method for speciation of individual internal (n-1)mer impurities arising from formal nucleotide deletion in synthetic oligodeoxyribonucleotides. A complete series of oligodeoxyribonucleotide probes were designed, each complementary to an (n-1)mer deletion sequence of the drug in question. Glass plates were used as a solid support for individually immobilizing the entire probe array. The total mixture of internal (n-1) length impurities was isolated from a synthetic oligodeoxyribonucleotide by PAGE and labeled with 35S. Under stringently optimized conditions, only the perfectly sequence-matched oligodeoxyribonucleotide hybridized to each probe, while all other deletion sequences were removed by washing with buffer. The 35S signal intensity of the bound oligodeoxyribonucleotide was proportional to the concentration of each (n-1)mer deletion sequence in the analyte solution. This method has been applied to a number of synthetic phosphorothioate oligodeoxy-ribonucleotide lots and shown to be reliable for speciation and relative quantitation of the internal (n -1)mer deletion sequences present.

Selectivity of anion exchange chromatography and capillary gel electrophoresis for the analysis of phosphorothioate oligonucleotides.

The complementary nature of anion exchange chromatography and capillary gel electrophoresis for oligonucleotide analysis is demonstrated by evaluating a comprehensive series of authentic deletion sequences and partial phosphodiester analogs of five phosphorothioate oligonucleotides of different base composition and sequence. While anion exchange HPLC is sensitive to differences in backbone length of phosphorothioate oligonucleotides, oligomers with length difference of one base unit are not resolved. Capillary gel electrophoresis, on the other hand, has excellent single-base resolution while being relatively insensitive to phosphate in the phosphorothioate backbone. The data definitively establish the necessity of employing both separation techniques for adequate characterization of lower order process-related impurities potentially found in synthetic phosphorothioate oligonucleotides.

Sequence analysis of phosphorothioate oligonucleotides via matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Modification of the natural phosphodiester backbone of deoxyribooligonucleotides can impart increased biostability via nuclease resistance. Further, uniform incorporation of phosphorothioate linkages renders oligonucleotides highly resistant to reagents traditionally used in sequencing reactions. As a consequence, analytical tests crucial for establishing the identity of such oligonucleotide drugs are less informative. To circumvent this problem, chemical oxidation has been employed for converting the phosphorothioate to the uniform phosphodiester, thereby facilitating enzymatic degradation. Following oxidation, exonucleases which sequentially cleave individual bases from the 3' or 5' terminus of the oligonucleotide or base-specific cleavage chemicals were used to facilitate sequence identification of the oligonucleotide. Matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS), previously used to sequence natural phosphodiester DNA, was then used to sequence the chemically oxidized phosphorothioate. Sequential enzymatic cleavage of desulphurized phosphorothioates in combination with MALDI analysis not only provides a viable alternative to radiolabeling as used in conventional sequencing approaches (e.g. Maxam-Gilbert), but also enables rapid sequencing of phosphorothioate oligonucleotides, for routine drug analysis.

Development and validation of a method for routine base composition analysis of phosphorothioate oligonucleotides.

A method for routine base composition determination of phosphorothioate oligonucleotides containing as many as 21 nucleobases has been developed and systematically evaluated in terms of factors contributing to assay precision and accuracy. Phosphorothioate internucleotide linkages were oxidized with a mixture of tetrahydrofuran-water-methylimidazole (16:4:1, v/v/v) which has been shown to be 97.3% effective. This step was followed by enzymolysis and HPLC quantitation of individual nucleobases. RSD for inter-day base composition analysis ranged from 1.1 to 1.3%, and inter-lot variation was 0.6-2.0%. Accuracy of the determined nucleobase ratio was independently confirmed through sequencing of the oxidized oligomer by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS).

Quantitative capillary gel electrophoresis assay of phosphorothioate oligonucleotides in pharmaceutical formulations.

Quantitative capillary gel electrophoresis (QCGE) has been developed for the accurate quantitation of a 21-mer phosphorothioate oligonucleotide, ISIS 2922, and its degradation products in an intravitreal formulation. The electrokinetic mode of injection employed by CGE necessitates formulation of the external reference standard in a sample matrix similar to that of the drug product and the use of an internal standard for improved accuracy and precision. The analytical method detailed in this paper has demonstrated the necessary accuracy, precision, linearity, range, selectivity and ruggedness for use in routine drug product analysis and stability monitoring of phosphorothioate oligonucleotides.

Role for cyclic adenosine monophosphate in the synergistic interaction between oxytocin and corticotrophin-releasing factor in isolated human gestational myometrium.

OBJECTIVE: We wished to investigate the role of cAMP in the synergistic effect of corticotrophin-releasing factor and oxytocin on human myometrial contractility. DESIGN: Isolated human gestational myometrium obtained from Caesarean sections at term was studied in vitro. Static incubation techniques as well as tension recordings were applied to the tissue obtained. PATIENTS: The subjects were healthy pregnant women undergoing lower segment Caesarean section at term, prior to labour. MEASUREMENTS: Specimens obtained were immediately dissected into small strips and either incubated in multi-well trays (strip weight 2.75 mg) or superfused and used for tension recordings (strip weight 2.00 mg). cAMP accumulation was measured after incubation with oxytocin (0.1-10 nM), corticotrophin-releasing factor (1 nM) or a combination of both peptides. Tension generated by the muscle strips was recorded isometrically and response to oxytocin (0.01-10 nM), corticotrophin-releasing factor (1 nM) and forskolin (10 nM) expressed in force per gram wet tissue (N/g). RESULTS: Oxytocin (0.1 nM) causes a statistically significant dose-related decrease in cAMP when combined with 1 nM corticotrophin-releasing factor (P less than 0.001), as compared with cAMP stimulation by corticotrophin-releasing factor alone. Time course studies suggest a maximal effect at 1 minute. The hypothesis that an intracellular reduction of cAMP is a prerequisite for the synergistic response in contraction force was tested with tension recordings. Prevention of a decrease in cAMP in the tissue by addition of 10 nM forskolin to the superfusate abolished the potentiation between oxytocin and corticotrophin-releasing factor. CONCLUSIONS: These results indicate that a fall in cAMP concentration plays a vital mediating role in the synergistic interaction between oxytocin and corticotrophin-releasing factor.

Detection and identification of endogenous small molecules in ocular tissues by proton nuclear magnetic resonance spectroscopy.

Proton nuclear magnetic resonance (nmr) spectra of rabbit ocular tissue homogenates (corneal epithelium, conjunctiva and iris ciliary body) and aqueous humor have been recorded for the first time by incorporation of a spin-spin relaxation reagent and use of the CPMG pulse sequence. A number of endogenous species such as lactate, glucose, alanine and valine have been observed in these ocular homogenates and significant differences have been noted in the distribution of these small molecules in the ocular tissues studied. This technique has been used to study the hydrolysis of acetylcholine by ocular esterases in the iris ciliary body.