Formulation pH Research Articles

Probing the Conformation of an IgG1 Monoclonal Antibody in Lyophilized Solids Using Solid-State Hydrogen-Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS).

Therapeutic proteins are often formulated as lyophilized products to improve their stability and prolong shelf life. The stability of proteins in the solid-state has been correlated with preservation of native higher order structure and/or molecular mobility in the solid matrix, with varying success. In the studies reported here, we used solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS) to study the conformation of an IgG1 monoclonal antibody (mAb) in lyophilized solids and related the extent of ssHDX to aggregation during storage in the solid phase. The results demonstrate that the extent of ssHDX correlated better with aggregation rate during storage than did solid-state Fourier-transform infrared (ssFTIR) spectroscopic measurements. Interestingly, adding histidine to sucrose at different formulation pH conditions decreased aggregation of the mAb, an effect that did not correlate with structural or conformational changes as measured by ssFTIR or ssHDX-MS. Moreover, peptide-level ssHDX-MS analysis in four selected formulations demonstrated global changes across the structure of the mAb when lyophilized with sucrose, trehalose, or mannitol, whereas site-specific changes were observed when lyophilized with histidine as the sole excipient.

COMPARATIVE IN VITRO EVALUATION OF BRANDS OF CLOTRIMAZOLE CREAM FORMULATIONS MARKETED IN ETHIOPIA

The aim of present work was to undertake comparative in vitro quality evaluation of six marketed clotrimazole cream formulations in Ethiopia with respect to physico-chemical properties like viscosity, spreadability, extrudability, pH and drug content. In vitro clotrimazole release from cream formulations was also studied using synthetic cellulose acetate membrane at 37 ÂoC in a solvent containing methanol and PBS 7.4 in the ratio of 75:25 as receiver medium. The cumulative amounts of the drug released over 12 h (µg mm-2) were analyzed. All clotrimazole cream formulations showed good and smooth homogeneous appearance with white color. The pH of clotrimazole cream formulations ranged from 4-7, which is a physiologically acceptable pH range and in principle devoid of any skin irritation. Clotrimazole content ranged from 90-110%, ensuring the uniformity of the drug content in all formulations. The increase in diameter of clotrimazole cream formulations following the spreadability test was found to range from 4-6 cm. Cream formulation D (Clotri-Denk) exhibited highest viscosity values than other formulations, whereas formulation E (Chinese Clotrimazole BP) showed lowest viscosity value. Cream formulation F (Mycoril) showed better extrudability and spreadability as compared to other formulations. Drug release from all formulations was slow in the first 6 hrs. After the 6th hr, steady drug release continued for formulation D and E. Fast drug release was observed in formulations A (Candid) and B (Candigen), whereas for the formulations C (Canesten), D and E, steady drug release pattern was observed after the 6th hr. It can be concluded that all clotrimazole cream formulations fulfilled the quality criteria of in-house and pharmacopeias specifications. Keywords: In Vitro Evaluation, Clotrimazole, Cream, Spreadability, Extrudability, Ethiopia

A Small-Scale Process for Predicting Donnan and Volume Exclusion Effects During Ultrafiltration/Diafiltration Process Development

Achieving the desired final protein formulation using ultrafiltration/diafiltration (UF/DF) operations is an essential component of many protein purification processes. It is well documented that differences in the excipient and buffer concentrations exist between the DF and retentate solutions when they have achieved equilibrium. Several publications have proposed ways to calculate these differences. However, the accuracy of these methods has been limited by the use of an estimated protein charge value. In this article, a small-scale system is described, which can accurately determine the protein charge by making buffer and excipient concentration measurements and applying the determined values to the Donnan and volume exclusion equations. This information can be utilized to generate a standard curve, which in turn can be applied to at-scale UF/DF operations. For 2 different antibodies, the standard curve generated by the small-scale system yielded buffer concentrations and pH values that agreed well with those generated after UF/DF operations, whereas using the theoretical protein charge caused a departure from the measured results. This model also provides good estimates as to how the final formulation pH and buffer concentration vary as a function of the pH and buffer concentration in the DF buffer. This information is of important utility for the accurate formulation of high-concentration protein solutions (>100 mg/mL) where the coconcentration of buffers and the volume exclusion of certain excipients are amplified. The low material requirements of the small-scale system are a major benefit for early phase formulation and process development when sufficient time and material may not be available, in particular to ensure successful UF/DF operations for the development of high protein concentration formulations.

Design, formulation, and evaluation of a herbal gel contains melissa, sumac, licorice, rosemary, and geranium for treatment of recurrent labial herpes infections.

Background:The herpes simplex virus is a human pathogen which can cause skin or mucous membrane infections. Melissa, sumac, licorice, rosemary, and geranium have antimicrobial, antiviral, anti-inflammatory, and local analgesic effect. Shortening recovery period of recurrent herpes labialis and control of viral protein formation are the other effects of these herbs. The aim of this study is design, formulation, and evaluation of the gel containing extracts of these five herbs.Materials and Methods:In this experimental study after photochemical and macroscopic evaluation of these medicinal herbs, the semisolid concentrated extracts were incorporated in gel bases. Mucoadhesive gels were prepared using carbopol 940, sodium carboxymethylcellulose (Na CMC) and hydroxypropyl methylcellulose K4M as bioadhesive polymers. Physicochemical tests, viscosity, mucoadhesive strength measurement, and in vitro drug release study were carried out on formulations F10 (carbopol 940, 0.5% and Na CMC, 3%) and F11 (carbopol 940, 1% and Na CMC, 3%).Results:Polyphenol content of extracts mixture was measured 210.8 ± 13.68 mg GAE/g. pH of formulations was 6.0 ± 0.2. 14 gel formulations were prepared. Physical appearance, homogeneity, and consistency of F10 and F11 were good. Mucoadhesion and viscosity of F11 was more than F10. Study of release profiles in F10 and F11 formulations showed drug release from F11 was slower.Conclusion:The best formulation for treatment and shortening recovery period of recurrent labial herpes infections should exhibit high value of mucoadhesion, show controlled release of drug. F11 with the highest viscosity and mucoadhesion and the lowest release rate was considered as the best formulation.

Polyherbal formulation containing antioxidants may serve as a prophylactic measure to diabetic cataract: Preclinical investigations in rat model

Background: Cataract is a major cause of visual impairment in diabetic patients. Due to increasing numbers of type 1 and type 2 diabetics worldwide, the incidence of diabetic cataracts steadily rises. Cataract surgery is the best possible cure for patients suffering from this ailment. However, the elucidation of pathomechanisms to delay or prevent the development of cataract in diabetic patients remains a challenge. Objective: The aim of the present study was to develop a polyherbal eyedrops containing potent antioxidant herbal extract and study the effectiveness as prophylactic treatment against galactose-induced diabetic cataract. Materials and Methods: Formulations were prepared by using extracts of Ginkgo biloba leaves, beet root (Beta vulgaris), and amla (Emblica officinalis) fruits. The viscosity enhancers were used to increase the retention time. Formulations (F1–F5) were prepared by using carboxy methyl cellulose and poly ethylene glycol-400 as thickening agents and propylene glycol as a solubilizer. Preliminary evaluation showed that formulations have passed clarity, sterility, and eye irritancy tests. Viscosity and pH of formulation were within the normal range. Diabetic cataract was induced in Wistar rats by 10% galactose drink (for 30 days) and anticataract activity was evaluated. Formulation was installed in eye as a prophylactic treatment from day 1 of galactose drinking and continued for 30 days. Results: Slit-lamp photography of eyes of rats showed clear lens of rats without any trace of opacity. On the other hand, galactose-treated rats developed dense nuclear opacity in lens as an indication of diabetic cataract. Rats which received prophylactic treatment showed less percent opacity as compared to that of cataract control group and decreased vacuoles. Conclusion: We may conclude that polyherbal formulation containing extracts of Ginkgo biloba leaves, beet root (Beta Vulgaris), and amla (Emblica officinalis) fruits may prevent the development of cataract in diabetic patients. Abbreviations used: ARI: Aldose reductase inhibitors; GPx: Glutathione peroxidase; GR: Glutathione reductase; DTNB: Dithio-bis-nitrobenzoic acid; GSH: Thiol.

PH and Buffer Capacity of Topical Formulations.

The pH is an important physicochemical factor that plays a significant role in various metabolic, molecular and cell-regulating processes. In the epidermis, the pH affects the barrier function on different levels. In many dermatoses that come along with an impaired barrier, shifts in the pH can be observed, and this is a problem that definitely needs to be addressed by finding appropriate galenic formulas when prescribing barrier protective basic care. With this in mind, 66 cosmetic preparations have been chosen following German market analysis. These preparations have been investigated regarding phase relation of the emulsion, absolute pH value and buffer capacity. The results show that only 23 preparations have an appropriate pH of ≤5.5 and only 3 preparations show a buffer capacity of ≥1.0. This outcome demonstrates the fact that the significance of pH and buffer capacity as quality criteria for barrier-protective preparations is still highly underrated from the view point of manufacturers and users.

Effectiveness of three mouthwashes - Manuka honey, Raw honey, and Chlorhexidine on plaque and gingival scores of 12-15-year-old school children: A randomized controlled field trial.

Objectives:The aim of this study is to compare the effectiveness of three types of mouthwashes manuka honey (MH), raw honey (RH), and chlorhexidine (CHX) on plaque and gingival scores of 12–15-year-old government school children.Study Design:This study was a double-blind, randomized controlled field trial conducted in Belagavi city, India.Materials and Methods:One hundred and thirty-five government school children aged 12–15 years were randomly selected and allocated into three groups, RH, MH, and CHX mouthwash groups. Ten milliliters each of honey-based mouthwash formulation and CHX mouthwashes (0.2%) were administered according to the group allocation twice daily for 21 days. All the children were examined at baseline, 22nd day (after discontinuation of mouthwash) and 28th day (1 week after discontinuation of mouthwash) for Gingival (Loe and silness 1963) and Plaque Index (Silness and Loe, 1964).Results:Descriptive statistics was applied for distribution of study participants according to age and gender. One-way ANOVA followed by Tukey's post hoc test and repeated measures ANOVA test followed by Bonferroni's post hoc were applied for inter- and intragroup comparison, respectively. Statistically significant reductions (P < 0.001) in plaque and gingival scores were observed in all the three types of mouthwash groups at the end of the 22nd day and 28th day. MH and RH mouthwash demonstrated equal effectiveness, whereas CHX mouthwash showed the maximum reduction in clinical parameters.Conclusion:Honey-based mouthwash showed a promising antimicrobial effect on dental caries and plaque and gingival scores.

Intra Nasal In situ Gelling System of Lamotrigine Using Ion Activated Mucoadhesive Polymer.

Background:A novel drug delivery system for treating acute epileptic condition.Objective:To develop an intranasal mucoadhesive formulation of Lamotrigine (LTG) loaded insitu gel, for the treatment of epilepsy to avoid possible side effects and first pass metabolism associated with conventional treatment.Methods:Lamotrigine was loaded into different polymeric solutions of gellan and xanthan gum.Results:All formulations subjected to various evaluation studies were within their acceptable limits. The pH of formulation ranges between 5.8 ±.001 to 6.8 ±.005 indicating that no mucosal irritation is expected as pH was in acceptable range. Invitro drug release from the mucoadhesive insitu gel formulations showed immediate drug release pattern with a maximum drug release of 97.02 ±0.54% for optimized G5 formulation within 20min. Exvivo permeation studies of optimized formulation G5 and control formulation was estimated. Exvivo permeation studies of G5 insitu formulation done for a period of 12 h resulted in slow, sustained release and greater permeability significance(P <0.05) through nasal mucosa when compared to control. Histopathological studies showed that G5 formulation was safer for nasal administration without any irritation. The stability studies indicated that gels were stable over 45 days in refrigerated condition (4±2ºC).Conclusion:The intranasal insitu gelling system is a promising novel drug delivery system for an antiepileptic drug lamotrigine which could enhance nasal residence time with increased viscosity and mucoadhesive character and provided better release profile of drug for treating acute epileptic conditions.

The artificial membrane insert system as predictive tool for formulation performance evaluation

In view of the increasing interest of pharmaceutical companies for cell- and tissue-free models to implement permeation into formulation testing, this study explored the capability of an artificial membrane insert system (AMI-system) as predictive tool to evaluate the performance of absorption-enabling formulations. Firstly, to explore the usefulness of the AMI-system in supersaturation assessment, permeation was monitored after induction of different degrees of loviride supersaturation. Secondly, to explore the usefulness of the AMI-system in formulation evaluation, a two-stage dissolution test was performed prior to permeation assessment. Different case examples were selected based on the availability of in vivo (intraluminal and systemic) data: (i) a suspension of posaconazole (Noxafil®), (ii) a cyclodextrin-based formulation of itraconazole (Sporanox®), and (iii) a micronized (Lipanthyl®) and nanosized (Lipanthylnano®) formulation of fenofibrate. The obtained results demonstrate that the AMI-system is able to capture the impact of loviride supersaturation on permeation. Furthermore, the AMI-system correctly predicted the effects of (i) formulation pH on posaconazole absorption, (ii) dilution on cyclodextrin-based itraconazole absorption, and (iii) food intake on fenofibrate absorption. Based on the applied in vivo/in vitro approach, the AMI-system combined with simple dissolution testing appears to be a time- and cost-effective tool for the early-stage evaluation of absorption-enabling formulations.

Light Control of Protein Solubility Through Isoelectric Point Modulation.

We previously described the photoactivated depot or PAD approach that allows for the light control of therapeutic protein release. This approach relies on the ability to use light to change a protein's solubility. Traditionally this was accomplished by linking the protein to an insoluble but injectable polymer via a light cleaved linker. This allows the injected material to remain at the site of injection, until transcutaneous irradiation breaks the link between polymer and protein, permitting the protein to be absorbed. However, there are multiple problems associated with polymer based approaches: The polymer makes up a majority of the material, making it inefficient. In addition, after protein release, the polymer has to be cleared from the body, a significant design challenge. In this work, we create materials that form photoactivated depots of insulin without the need for polymers, by linking photolysis to an isoelectric point shift, which itself is linked to a solubility shift. Specifically, we linked basic groups to insulin via a light cleaved linker. These shift the normal pI of insulin from 5.4 to approximately 7. The result of this incorporation are materials that are completely soluble in mildly acidic solutions but precipitate upon injection into a pH 7 environment, i.e., the skin. We successfully synthesized four such modified insulins, demonstrating that their pI values were shifted in the expected manner. We then analyzed one of them, P2-insulin, in detail, demonstrating that it behaves as designed: It is soluble in a formulation pH of 4, but precipitates at pH 7.2, its approximate pI value. Upon irradiation, the photocleavable link to insulin is broken, and completely native and soluble insulin is released from the depot in a well behaved, first order fashion. These materials are 90% therapeutic, form completely soluble and injectable formulations in mildly acidic conditions, form insoluble depots at neutral pH, efficiently release soluble protein from these depots when irradiated, and leave behind only small easily absorbed molecules after irradiation. As such they approach ideality for photoactivated depot materials.

Effect of alginate size, mannuronic/guluronic acid content and pH on particle size, thermodynamics and composition of complexes with β-lactoglobulin

Alginate is an anionic polysaccharide capable of forming insoluble particles with proteins. Hence, alginate has potential as a protein carrier. However, the role of physical properties of the polysaccharide, such as degree of polymerization (DPn) and mannuronic/guluronic acid ratio, remains to be fully explored. Particle formation of a high and a low molar mass alginate (ALG) with β-lactoglobulin (BLG) at pH 2–8 depends on the average DPn (HMW-ALG: 1.59·103; LMW-ALG: 0.23·103) and the mannuronic/guluronic acid ratio (1.0; 0.6) as supported by using ManA6 and GulA6 as models. Dynamic light scattering (DLS) showed that particles of BLG with either of the two ALGs have essentially the same hydrodynamic diameter (DH) at pH 3 and 2, while at pH 4 particles of LMW-ALG/BLG have larger DH than of HMW-ALG/BLG. At pH 5–8 no significant particle formation was observed. ManA6 did not form insoluble particles at pH 2–8, while GulA6 formed insoluble particles, albeit only at pH 4. Kd was approximately 10-fold higher for LMW-ALG/BLG than HMW-ALG/BLG and 3 orders of magnitude higher for an alginate trisaccharide/BLG complexation as determined by isothermal titration calorimetry (ITC). The alginate trisaccharide did not form insoluble particles with BLG at pH 3 and 4, though interaction still occurred. ΔHapp and molar stoichiometry of BLG in the complexes with the two ALGs differed by a factor of 7, as did their DPn, which thus affected the interaction strength, but not the BLG content. At pH 4 the BLG content doubled in the particle due to BLG dimerization. The findings emphasize the importance of DPn, mannuronic/guluronic acid ratio and pH in formulations containing alginate/whey protein particles.

Putty-like bone fillers based on CaP ceramics or Biosilicate® combined with carboxymethylcellulose: Characterization, optimization, and evaluation.

Calcium phosphates and bioactive glass ceramics have been considered promising biomaterials for use in surgeries. However, their moldability should be further enhanced. We here thereby report the handling, physicochemical features, and morphological characteristics of formulations consisting of carboxymethylcellulose-glycerol and hydroxyapatite-tricalcium phosphate or Biosilicate® particles. We hypothesized that combining either material with carboxymethylcellulose-glycerol would improve handling properties, retaining their bioactivity. In addition to scanning electron microscopy, cohesion, mineralization, pH, and viscoelastic properties of the novel formulations, cell culture experiments were performed to evaluate the cytotoxicity and cell proliferation. Putty-like formulations were obtained with improved cohesion and moldability. Remarkably, mineralization in simulated body fluid of hydroxyapatite-tricalcium phosphate/carboxymethylcellulose-glycerol formulations was enhanced compared to pure hydroxyapatite-tricalcium phosphate. Cell experiments showed that all formulations were noncytotoxic and that HA-TCP60 and BGC50 extracts led to an increased cell proliferation. We conclude that combining carboxymethylcellulose-glycerol with either hydroxyapatite-tricalcium phosphate or Biosilicate® allows for the generation of moldable putties, improves handling properties, and retains the ceramic bioactivity.

Scaffold requirements for periodontal regeneration with enamel matrix derivative proteins

Periodontitis affects the attachment of natural teeth, and infection or inflammation associated with periodontitis may affect peri-implant tissues. Enamel matrix derivative (EMD) proteins provide stimulation for self-regeneration of the damaged tissue when applied to wide intrabony defects as part of a mixture with bone graft material. As a first step of the process enhancing cell proliferation and ligament formation, we demonstrated that EMD protein precipitation depends strongly on the physical and chemical characteristics of the bone grafts used in the mixture. To guarantee optimum protein-stimulated self-regulation, the pH of the initial EMD formulation must therefore be adjusted between 3.9 and 4.2 in order to compensate the change in pH induced by the bone graft. Moreover, the interaction between the two components resulted in precipitates of different shape and size differently covering the grafts. This outcome might potentially have clinical implications on cell attachment and periodontal ligament extension, which deserve further in vitro and in vivo tests.

Impact of Freeze/Thaw Process on Drug Substance Storage of Therapeutics

The storage of drug substance at subzero temperatures mitigates potential risks associated with liquid storage, such as degradation and shipping stress, making it the best solution for long-term storage. However, slower (generally uncontrolled) rates of freezing and thawing of drug substance in conventional large storage containers (>2L) can lead to greater cryoconcentration (exclusion of solute molecules) resulting in zones of higher protein and excipient concentrations and changes to the desired formulation pH and excipient concentration. These conditions can negatively impact product quality, thus changing the target product profile. Freeze/thaw studies can provide valuable knowledge on the molecule even when performed from an early formulation image. This study attempts to provide guidance and strategy for planning of drug substance freeze and thaw studies in early development using a scale-down model, evaluating the impact of the (1) freeze/thaw rate, (2) mode of freezing, (3) drug substance container, (4) drug substance concentration, and (5) formulation on the drug substance product quality. Data presented in this study showed no impact on drug substance product quality after undergoing the typical one freeze/thaw cycle process for the variables evaluated. These findings suggest that a qualified scale-down model is not required for early phases of process development and that existing small-scale models can be used for drug substance storage development studies. Based on our experience, a workflow is suggested with minimal experimental design to reduce the material requirement by >70% at early stages of product development to reduce constraints.

DEVELOPMENT AND EVALUATION OF TRANSDERMAL GEL OF LORNOXICAM

Objective: Transdermal drug delivery systems deliver the drug through the skin at controlled rate to the systemic circulation. It maintains the blood concentration of the drug within the therapeutic system window ensuring that drug levels neither fall below the minimum effective concentration nor exceed the minimum toxic dose. The objective of the present work was to formulate transdermal gel of Lornoxicam. It is a COX-1 and COX-2 inhibitor used in the treatment of inflammation, pain and edema, rheumatoid arthritis. Methods: Transdermal gel of Lornoxicam was formulated using triethanolamine as solvent, HPMC K100 and EC as polymers. Formulated gel was evaluated with respect to different physiochemical parameters such as pH, viscosity, spreadability. In-vitro release study was performed for 10 hrs. Selected formulation was subjected to stability testing at different temperatures. Results: There was good homogeneity in all formulations and no lumps were present. The pH of the gel formulations was in the range of 6.77 to 7.14. Viscosity of various formulated gels was found in the range of 2176.5 to 3468.4 centipoises. The cumulative percent drug release after 10 hrs in between 50.3 to 82.11%. Accelerated stability studies for 12 weeks revealed that the transdermal gel formulation were stable at up to 45oC. Conclusion: Study concludes Lornoxicam can be delivered in the form of transdermal gel in an efficient way. On basis of drug content, particle size morphology, in-vitro release and stability studies, it can be concluded that formulation LTG4 was an optimum formulation. Peer Review History: Received 7 February 2017; Revised 11 March; Accepted 13 March, Available online 15 March 2017 Academic Editor: Dr. Amany Mohamed Alboghdadly, Princess Nourah bint abdulrahman university, Riyadh, amalbgadley@pnu.edu.sa UJPR follows the most transparent and toughest ‘Advanced OPEN peer review’ system. The identity of the authors and, reviewers will be known to each other. This transparent process will help to eradicate any possible malicious/purposeful interference by any person (publishing staff, reviewer, editor, author, etc) during peer review. As a result of this unique system, all reviewers will get their due recognition and respect, once their names are published in the papers. We expect that, by publishing peer review reports with published papers, will be helpful to many authors for drafting their article according to the specifications. Auhors will remove any error of their article and they will improve their article(s) according to the previous reports displayed with published article(s). The main purpose of it is ‘to improve the quality of a candidate manuscript’. Our reviewers check the ‘strength and weakness of a manuscript honestly’. There will increase in the perfection, and transparency. Received file: Reviewer's Comments: Average Peer review marks at initial stage: 4.0/10 Average Peer review marks at publication stage: 7.5/10 Reviewer(s) detail: Prof. Dr. Syamsudin Abdillah, Pancasila University, Indonesia, syamsudin.abdillah@gmail.com Noha El Baghdady, MTI University, Cairo, Egypt, nohasalah21@yahoo.com Similar Articles: INVESTIGATION OF PRONIOSOMES GEL AS A PROMISING CARRIER FOR TRANSDERMAL DELIVERY OF GLIMEPIRIDE

Transdermal Delivery of Cimetidine Across Microneedle-Treated Skin: Effect of Extent of Drug Ionization on the Permeation

The objective of this work was to optimize a gel formulation of cimetidine to maximize its transdermal delivery across microporated skin. Specifically, the effect of extent of ionization in formulation on permeation of cimetidine across microporated skin was studied. Cimetidine was formulated into a gel using propylene glycol, water, and carbopol 980NF. Three strengths of gels (0.1% w/w, 0.5% w/w, and 0.8% w/w) were made and Tris base was used to adjust the pH of formulations to pH 5, pH 6.8, and pH 7.5. In vitro permeation testing was performed on vertical Franz cells with dermatomed porcine ear skin. Permeation studies suggested that pH 5 gels showed highest permeation through microchannels. This trend was more prominent with an increase in drug loading. The total amount of cimetidine delivered from 0.8% w/w gel at pH 5 at 24 h was 28.20 ± 4.63 μg, which was significantly higher than that from pH 6.8 (16.89 ± 3.56 μg) and pH 7.5 (12.03 ± 1.66 μg) gels. Cimetidine permeation across microporated skin was found to be pH dependent, with lower pH/highest ionization resulting in greatest permeation. The effect of ionization contributing to faster release was more pronounced when drug concentration was increased.

Characterization of aqueous formulations of tetra- and pentavalent forms of vanadium in support of test article selection in toxicology studies.

Tetravalent (VIV) and pentavalent (VV) forms of vanadium were selected for testing by the National Toxicology Program via drinking water exposure due to potential human exposure. To aid in the test article selection, drinking water formulations (125-2000mg/L) of vanadyl sulfate (VIV), sodium orthovanadate, and sodium metavanadate (VV) were characterized by ultraviolet/visible (UV/VIS) spectroscopy, mass spectrometry (MS), or 51V nuclear magnetic resonance (NMR) spectroscopy. Aqueous formulations of orthovanadate, metavanadate, and vanadyl sulfate in general were basic, neutral, and acidic, respectively. Changes in vanadium speciation were investigated by adjusting formulation pH to acidic, neutral, or basic. There was no visible difference in UV/VIS spectra of pentavalent forms. NMR and MS analyses showed that the predominant oxidovanadate species in both ortho- and metavanadate formulations at basic and acidic pH, respectively, were the monomer and decamer, while, a mixture of oxidovanadates were present at neutral pH. Oxidovanadate species were not observed in vanadyl sulfate formulations at acidic pH but were observed at basic pH suggesting conversion of VIV to VV. These data suggest that formulations of both ortho- and metavanadate form similar oxidovanadate species in acidic, neutral and basic pH and exist mainly in the VV form while vanadyl sulfate exists mainly as VIV in acidic pH. Therefore, the formulation stability overtime was investigated only for sodium metavanadate and vanadyl sulfate. Drinking water formulations (50 and 2000mg/L) of metavanadate (~pH 7) and vanadyl sulfate (~pH 3.5) were ≥92% of target concentration up to 42days at ~5°C and ambient temperature demonstrating the utility in toxicology studies.

Effect of pH and penetration enhancers on cysteamine stability and trans-corneal transport

Ocular cystinosis is a rare metabolic disorder characterized by the presence of insoluble cystine crystals inside the corneal stroma, with consequent photophobia, keratopathies and frequent corneal erosions. The current therapy consists in the lifetime ophthalmic administration of cysteamine, drug characterized by extremely high hydrophilicity, low molecular weight (77g/mol), and easy oxidization to disulfide. Ocular delivery of cysteamine is very challenging, for its poor permeability and stability in solution. The purpose of the present paper was to study the impact of formulation pH and composition on (1) the trans-corneal delivery and (2) the stability in solution of cysteamine, with particular focus on the use of alpha-cyclodextrin (α-CD), benzalkonium chloride (BAC) and disodium edetate (EDTA). Permeation experiments were performed ex vivo through freshly excised porcine cornea; stability was evaluated for six months at −20°, +4° and +25°C; irritation potential was evaluated using HET-CAM assay. The results showed that cysteamine trans-corneal diffusion is strictly dependent on both pH (7.4 preferred to 4.2) and buffering capacity, that negatively impact on the permeation; EDTA did not enhance the trans-corneal diffusion of cysteamine neither at pH 7.4 nor at pH 4.2, while benzalkonium chloride (BAC), antimicrobial agent present within commercial eye-drops, significantly enhanced it. Notably, α-CD was able to promote the trans-corneal diffusion of cysteamine and, at a 5.5%, a 4-fold higher penetration compared to the BAC-containing formulation was obtained. EDTA and acidic pH demonstrated to be essential for cysteamine stability. The formulation obtained by combining α-CD and EDTA was characterized by significant permeation, good stability profile, and no irritation potential, even if the tolerability should be further confirmed by in vivo test.

Methotrexate loading in chitosan nanoparticles at a novel pH: Response surface modeling, optimization and characterization

The aim of this study was to assess the feasibility of employing a novel but critical formulation pH (6.2) to encapsulate an anionic model drug (methotrexate, MTX) into chitosan(Cs)-tripolyphosphate nanoparticles(NPs). A response surface methodology using a three-level full factorial design was applied studying the effects of two independent variables namely; Cs concentration and MTX concentration. The responses investigated were the entrapment efficiency (EE%), mean hydrodynamic particle size (PS), polydispersity index (PDI) and zeta potential (ZP). In order to simultaneously optimize the series of models obtained, the desirability function approach was applied with a goal to produce high percent of MTX encapsulated into highly charged Cs-TPP NPs of homogenous optimum PS. MTX-loaded CsNPs were successfully prepared at the novel pH applied. The suggested significant models were found quadratic for EE, PS and ZP responses, while 2-factor interaction model for PDI. The optimization overlay graph showed that the maximum global desirability, D=0.856, was reached when the conditions were set at high Cs and MTX concentration. Thus, the use of such optimized conditions, at this novel pH, achieved a maximum drug EE% (73.38%) into NPs characterized by optimum PS (232.6nm), small PDI value (0.195) and highly surface charged (+18.4mV).

Critical processing parameters of carbon dioxide spray drying for the production of dried protein formulations: A study with myoglobin

The aim of this study was to gain fundamental insight into protein destabilization induced by supercritical CO2 spray drying processing parameters. Myoglobin was used as a model protein (5mg/ml with 50mg/ml trehalose in 10mM phosphate buffer, pH 6.2). The solution was exposed to sub- and supercritical CO2 conditions (65–130bar and 25–50°C), and CO2 spray drying under those conditions. The heme binding of myoglobin was determined by UV/Vis, fluorescence, and circular dichroism spectroscopy, while myoglobin aggregation was studied by using size-exclusion chromatography and flow imaging microscopy. It was found that pressure and temperature alone did not influence myoglobin’s integrity. However, when pressurized CO2 was introduced into myoglobin solutions at any condition, the pH of the myoglobin formulation shifted to about 5 (measured after depressurization), resulting in heme binding destabilization and aggregation of myoglobin. When exposed to CO2, these degradation processes were enhanced by increasing temperature. Heme binding destabilization and myoglobin aggregation were also seen after CO2 spray drying, and to a greater extent. Moreover, the CO2 spray drying induced the partial loss of heme. In conclusion, pressurized CO2 destabilizes the myoglobin, leading to heme loss and protein aggregation upon spray drying.