Combination Of Excipients Research Articles

Preparation and Evaluation of Eprosartan Mesylate loaded PLGA Nanostructures

In modern medication, drug-loaded novel polymeric nanoparticles have potential advantages over conventional dosage forms. The present study focused on the formulation development and evaluation of newly introduced angiotensin receptor blocker (ARB) eprosartan mesylate loaded biodegradable acid terminated poly(lactide-co-glycolide) (50:50) nanoparticle using double emulsion solvent evaporation technique. This methodology was found to improve the therapeutic efficacy as Eprosartan Mesylate belongs to BCS class-II and water-insoluble antihypertensive drug with 13% bioavailability. In the earlier stage, screening was performed to find out the suitable combination of excipients (Polyvinyl alcohol, Dichloromethane and Ethanol) to formulate the nanoparticles. In this present study, the physical and chemical properties of polymer and drug were determined before and after the formulation of nanoparticles using experimental techniques, such as Differential Light Scattering (DLS), zeta potential, X-ray Deffractometry (XRD), Fourier-Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), and Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis. The eprosartan mesylate loaded PLGA nanoparticles showed fairly monodispersive, as determined from DLS (average particle diameter 136 nm, Poly Dispesity Index = 0.3). The zeta potential was found to be -10.8mV. This formulation showed a prolonged improved drug release (82.03%) for 360 hours in phosphate buffer (pH7.4) as compared to the conventional marketed dosage form. In-vitro release kinetics data of NPs (R2=0.8979) and diffusion exponent values (n=0.3328) suggested that optimized followed the Korsmeyer-Peppas model with Fickian mechanism.

Precise Dosing of Pramipexole for Low-Dosed Filament Production by Hot Melt Extrusion Applying Various Feeding Methods.

The aim of this research was the production of low-dosed filaments via hot-melt extrusion (HME) with the model drug pramipexole for the treatment of Parkinson’s disease. The active pharmaceutical ingredient (API) and one of the polymers polyvinyl alcohol (PVA) or basic butylated methacrylate copolymer (bPMMA) were fed by various dosing techniques with the aim of achieving the smallest deviation (RSD) from the target concentration of 0.1% (w/w) pramipexole. It was found that deviation from target pramipexole concentration occurred due to degradation products in bPMMA formulations. Additionally, material temperature above 120 °C led to the formation of the anhydrous form of pramipexole within the extruded filaments and need to be considered in the calculation of the recovered API. This study clearly shows that even if equilibrium state of the extrusion parameters was reached, equilibrium condition for drug content was reached relatively late in the process. In addition, the RSD calculated by the Stange–Poole equation was proposed by us to predict the final content uniformity considering the sample size of the analyzed filament. The calculated RSD, depending on sample size and drug load, can serve as upper and lower limits of variation from target concentration and can be used to evaluate the deviations of drug content in equilibrium conditions of the HME process. The lowest deviations from target concentration in equilibrium condition for drug content were obtained in filaments extruded from previously prepared granule mixtures (RSD = 6.00%, acceptance value = 12.2). These promising results can be transferred to other API–excipient combinations to produce low-dosed filaments, which can be used for, e.g., fused filament 3D printing. The introduced calculation of the RSD by Stange–Poole equation can be used for precise determination of the homogeneity of an extruded batch.

Substantiation of the composition of a semi-solid dosage form with a probiotic component for use in dermatology

One of the important issues in the pharmaceutical development of a semisolid preparation for dermal use is the scientific and experimental justification for choosing the base-carrier of active substances.
 The aim of this study was to experimentally substantiate the choice of rational combination of excipients in the development of a semisolid dosage form with a probiotic component for use in dermatology.
 Materials and methods. Hydrophilic gelling agents were used as excipients in the study: Sepiplus 400, Aristoflex AVC, Carbopol 934, hydroxyethylcellulose HEC, sodium alginate. Physical and chemical, pharmaco-technological and microbiological methods were used to select the optimal basis. Colloidal stability was determined using a laboratory centrifuge at a speed of 6000 rpm for 5 min, an electronic thermometer and a water bath laboratory. Thermostability was determined in the thermostat at (40±2) °C for 24 hours. Investigation of the rheological properties of the samples was performed using a Rheolab QC (Anton Paar, Austria) rheoviscometer using a system of coaxial cylinders C-CC27/SS. The kinetics of water absorption of the samples were studied by dialysis through a semipermeable Cuprophan membrane, Type 150 pm at a temperature of (34±2) °C. The number of viable lactobacilli cells was determined by surface seeding on Petri dishes with dense MRS medium.
 Research results. The results of the study of the number of viable lactobacilli cells showed that the lowest number of viable cells was in sample based on HEC. Tests for thermostability and colloidal stability showed that for sample No. 5 based on sodium alginate, stratification was observed in the experiment, which indicates its instability. These samples were excluded. Samples on Sepiplus 400 and Aristoflex AVC on a set of rheological features have advantages both consumer and technological; and sample on t Aristoflex AVC has the best indicators of osmotic activity and the viability of cells in the dynamics for this study.
 Conclusions. As a result of physical and chemical (organoleptic, colloidal and thermostability), pharmaco-technological (osmotic and structural and mechanical properties) and microbiological studies (number of viable lactobacilli cells) studies it was found that further work should be carried out with a sample based on gelling agent Aristoflex AVC, which has the most optimal performance in this development for a semisolid dosage form with a probiotic component for use in dermatology

Engineering of levodopa inhalable microparticles in combination with leucine and dipalmitoylphosphatidylcholine by spray drying technique

The aim of this work was to study the effect of concomitant use of leucine and dipalmitoylphosphatidylcholine, in different ratios, on aerosolization performance of levodopa. Three-component formulations were selected based on a central composite design using percentages of leucine and dipalmitoylphosphatidylcholine as the independent variables. Particle size, surface roughness index, surface phosphorus and fine particle fraction were considered as dependent variables in the model. The spray dried samples were also characterized to determine their particle shape and solid state nature. levodopa was spray dried with 10–40% w/w of the excipients to prepare two- or three-component formulations. A crystalline nature was determined for levodopa in all samples spray dried from water:ethanol (30:70 v/v). Roughness in surface of the processed particles increased with increasing total concentration of the excipients, specially above 25% w/w. Analysis of phosphorus on the surface demonstrated that three-component formulations prepared with combination of 12.5% w/w leucine had the highest amount of dipalmitoylphosphatidylcholine in the surface, regardless of its percentage used in the initial feed. A combination of 12.43% w/w of leucine and 9.80% w/w of dipalmitoylphosphatidylcholine used in formulation exhibited the highest fine particle fraction (72.63%). It can be concluded that spray drying of levodopa with a suitable combination of both excipients leads to production of a three-component formulation of crystalline levodopa, with an aerosolization performance which is significantly higher than two-component formulations composed of the drug with either leucine or dipalmitoylphosphatidylcholine.

Design of Biopharmaceutical Formulations Accelerated by Machine Learning.

In addition to activity, successful biological drugs must exhibit a series of suitable developability properties, which depend on both protein sequence and buffer composition. In the context of this high-dimensional optimization problem, advanced algorithms from the domain of machine learning are highly beneficial in complementing analytical screening and rational design. Here, we propose a Bayesian optimization algorithm to accelerate the design of biopharmaceutical formulations. We demonstrate the power of this approach by identifying the formulation that optimizes the thermal stability of three tandem single-chain Fv variants within 25 experiments, a number which is less than one-third of the experiments that would be required by a classical DoE method and several orders of magnitude smaller compared to detailed experimental analysis of full combinatorial space. We further show the advantage of this method over conventional approaches to efficiently transfer historical information as prior knowledge for the development of new biologics or when new buffer agents are available. Moreover, we highlight the benefit of our technique in engineering multiple biophysical properties by simultaneously optimizing both thermal and interface stabilities. This optimization minimizes the amount of surfactant in the formulation, which is important to decrease the risks associated with corresponding degradation processes. Overall, this method can provide high speed of converging to optimal conditions, the ability to transfer prior knowledge, and the identification of new nonlinear combinations of excipients. We envision that these features can lead to a considerable acceleration in formulation design and to parallelization of operations during drug development.

Influence of excipients in Protein A chromatography and virus inactivation

The purification of monoclonal antibodies and Fc fusion proteins consist of several unit operations operated commonly as a platform approach, starting with Protein A chromatography. The first capture step, the following low pH virus inactivation, and subsequent ion exchange chromatography steps are mostly able to remove any impurities, like host cell proteins, aggregates, and viruses. The changes in pH and conductivity during these steps can lead to additional unwanted product species like aggregates. In this study, excipients with stabilizing abilities, like polyols, were used as buffer system additives to study their impact on several aspects during Protein A chromatography, low pH virus inactivation, and cation exchange chromatography. The results show that excipients, like PEG4000, influence antibody elution behavior, as well as host-cell protein elution behavior in a pH-gradient setup. Sugar excipients, like Sucrose, stabilize the antibody during low pH virus inactivation. All excipients tested show no negative impact on virus inactivation and dynamic binding capacity in a subsequent cation exchange chromatography step. This study indicates that excipients and, possibly excipient combinations, can have a beneficial effect on purification without harming subsequent downstream processing steps.

Live vaccine preserved at room temperature: Preparation and characterization of a freeze-dried classical swine fever virus vaccine

The heat-stable live-attenuated classical swine fever virus (CSFV) vaccine is an urgent need in many countries of Asia, Europe and Latin America. In this study, the thermostability of lyophilized live-attenuated CSFV vaccine formulations were investigated using accelerated stability at 37 °C for 10 days. The freeze-dried heat-stable formulation ST16, containing excipient combinations of trehalose, glycine, thiourea and phosphate buffer shows the superior thermostability. Moreover, the lyophilized vaccine with formula ST16 kept loss of viral activity less than 0.5 log10 during 24 months at storage temperatures of 2–8 °C. In thermal study, ST16 stabilized the vaccine within 1.0 log10 loss after storage at up to 25 °C for 6 months and room temperature for 7 months. Even under the harshest storage conditions of 37 °C for 25 days and 45 °C for 2 weeks, the virus titer dropped less than 1.0 log10 using ST16. Besides, it is notable that ST16 excluded gelatin and exogenous proteins, which might cause allergic reactions, thus avoiding immune side effects. The vaccine formulated ST16 proved to be safe and effective when immunized to piglets in vivo. The characteristics of dried vaccines were analyzed by X-ray powder diffraction, residual water measurements, differential scanning calorimetry and it was found that vaccine antigen were preserved in an amorphous matrix with high glass transition temperature above 60 °C and low residual water content below 2%, which made the vaccine more stable during storage.

Enabling Lyophilized Pneumococcal Conjugate Vaccines Through Formulation Design and Excipient Selection Suitable for A Multivalent Adjuvanted Vaccine

Despite a consistent benefit of existing pneumococcal conjugate vaccine (PCV) on invasive pneumococcal disease and pneumonia across different epidemiological settings a tremendous gap exists towards global PCV coverage. Currently, no lyophilized dosage form exists in the PCV global vaccine marketplace and currently licensed vaccines target some, but not all relevant serotypes of Streptococcus pneumoniae. The development of lyophilized presentations of an adjuvanted multivalent vaccine formulation that aligns with the evolving epidemiological assessment of the pneumococcal disease offers broader coverage with distinct cold chain and thermostability advantages. To make progress towards this goal, we evaluated the feasibility of developing new formulation to enable a lyophilized adjuvanted PCV vaccine containing 15 different serotypes. Our findings successfully demonstrate a formulation design space that enables enhanced physical stability which controls vaccine agglomeration, preserves in-vitro vaccine potency, maintains PCV antigen adsorption, and yields elegant lyophilized cakes with acceptable clinically relevant reconstitution times. This research also demonstrates the benefit of utilizing specific vaccine formulation excipients and the effectiveness of excipient combinations that may be beneficial for other multivalent adjuvant containing vaccines to enable novel lyophilized formulations necessary for improved global vaccine access.

Development of a Self-Emulsifying Drug Delivery System for Optimized Topical Delivery of Clofazimine.

A quality-by-design and characterization approach was followed to ensure development of self-emulsifying drug delivery systems (SEDDSs) destined for topical delivery of the highly lipophilic clofazimine. Solubility and water-titration experiments identified spontaneous emulsification capacity of different excipient combinations and clofazimine. After identifying self-emulsification regions, check-point formulations were selected within the self-emulsification region by considering characteristics required to achieve optimized topical drug delivery. Check-point formulations, able to withstand phase separation after 24 h at an ambient temperature, were subjected to characterization studies. Experiments involved droplet size evaluation; size distribution; zeta-potential; self-emulsification time and efficacy; viscosity and pH measurement; cloud point assessment; and thermodynamic stability studies. SEDDSs with favorable properties, i.e., topical drug delivery, were subjected to dermal diffusion studies. Successful in vitro topical clofazimine delivery was observed. Olive oil facilitated the highest topical delivery of clofazimine probably due to increased oleic acid levels that enhanced stratum corneum lipid disruption, followed by improved dermal clofazimine delivery. Finally, isothermal microcalometric experiments studied the compatibility of excipients. Potential interactions were depicted between argan oil and clofazimine as well as between Span®60 and argan-, macadamia- and olive oil, respectively. However, despite some mundane incompatibilities, successful development of topical SEDDSs achieved enhanced topical clofazimine delivery.

(Invited) Engineering Single Wall Carbon Nanotubes for Sub-Cellular Drug Delivery

Single wall carbon nanotubes (SWCNTs) have been rigorously studied and engineered for a myriad of applications including drug delivery due to their unique, desirable properties. Since SWCNTs bundle in aqueous media and the bundling substantially diminishes their desirable properties along with inducing cytotoxicity, strategies to disperse individually SWCNTs with therapeutic molecules and biologic excipients have been heavily investigated. However, a handful of studies has been performed to understand the interactions between therapeutic molecules and excipients with SWCNT interface as well as the mechanism of intracellular release for therapeutic effects. Additionally, little attention has been paid to how cell type and cell metabolic activity level influence internalization of SWCNTs with different lengths. In this talk, I will discuss cell type, cell metabolic activity, and SWCNT length dependent cellular processing of SWCNTs dispersed with diverse biocompatible dispersing agents. Our results demonstrate that proper choice of dispersing agent can allow for interaction with F-actin, accumulation in the endoplasmic reticulum for eventual expulsion, or accumulation in cell nucleus. Further, macrophages with relatively high cell metabolic activity level compared to other cell types such as fibroblasts have shown to internalize significantly higher amount of SWCNTs per cell. The total mass of SWCNTs internalized per cell increases with a decrease in SWCNT length. Lastly, I will discuss our recent effort to create a ternary complex of SWCNTs, drug, and protein for intracellular delivery and release of model drugs. To overcome the limitation of the existing methods being only plausible for model drugs that are soluble in aqueous solutions, we developed a facile schema to assemble stepwise various combinations of therapeutic molecules and excipients on SWCNTs in different solvents using a preformed SWCNT network. These ternary complexes allow dialing in delivery of therapeutic molecules and excipients to achieve desired cell viability reduction. These findings allow for the development of new SWCNT-based biological applications as they establish important parameters of biomolecule adsorption on SWCNTs, SWCNT internalization and subcellular processing, which are crucial for applications ranging from drug delivery to cell type-specific modulation.

The Stabilizing Excipients in Dry State Therapeutic Phage Formulations.

Phage therapy has gained prominence due to the increasing pathogenicity of "super bugs" and the rise of their multidrug resistance to conventional antibiotics. Dry state formulation of therapeutic phage is attractive to improve their "druggability" by increasing their shelf life, improving their ease of handling, and ultimately retaining their long-term potency. The use and selection of excipients are critical to stabilize phage in solid formulations and protect their viability from stresses encountered during the solidification process and long-term storage prior to use. Here, this review focuses on the current classes of excipients used to manufacture dry state phage formulations and their ability to stabilize and protect phage throughout the process, as discussed in the literature. We provide perspective of outstanding challenges involved in the formulation of dry state phage. We suggest strategies to improve excipient identification and selection, optimize the potential excipient combinations to improve phage viability during formulation, and evaluate new methodologies that can provide greater insight into phage-excipient interactions to improve design criteria to improve formulation of dry state phage therapeutics. Addressing these challenges opens up new opportunities to re-design and re-imagine phage formulations for improved efficacy as a pharmaceutical product.

The Comparison of Two Challenging Low Dose APIs in a Continuous Direct Compression Process.

Segregation is a common problem in batch-based direct compression (BDC) processes, especially with low-dose tablet products, as is the preparation of a homogenous mixture. The scope of the current work was to explore if a continuous direct compression (CDC) process could serve as a solution for these challenges. Furthermore, the principle of a platform formulation was demonstrated for low dose tablets. The combination of filler excipients and the API in the formulation used was suitable for direct compression, but also prone to induce segregation in BDC process. The CDC process was found to be very promising; it was shown that tablets with the desired quality parameters could be manufactured successfully with both of the APIs studied. Powder analysis indicated that the APIs display some fundamental differences in their physical properties, which was also reflected in powder mixture properties and, hence, eventually in processing. However, process parameters, especially mixer impeller speed, were not found to have any significant influence on end product quality. The study suggests that a CDC process can be a viable solution to resolve the challenges described. Moreover, manufacturing by using a universal platform formulation seems to be a feasible way for producing low-dose tablets.

ВИБІР СКЛАДУ БУФЕРНОЇ СИСТЕМИ ДЛЯ ОЧНИХ КРАПЕЛЬ З ТЕТРАГІДРОЗОЛІНУ ГІДРОХЛОРИДОМ

The purpose of this article is developing the optimal formulation of excipients for eye drops with tetrahydrosoline hydrochloride and researching of the locally irritating effect of the selected formulation of eye drops. The objects of the research are experimental solutions of eye drops with tetrahydrosoline hydrochloride using different combinations of excipients. Evaluation of test results in the selection of the formulation was performed according to the physico-chemical parameters of the prepared eye drops solution along with researching of stability. Assessment of the local irritation was performed on Chinchilla rabbits, particularly on the mucous membrane of the rabbit’s eye. The optimal formulation of the eye drops with tetrahydrozoline hydrochloride of a medicinal product using citrate buffer solution was developed. The result solution represents a clear homogeneous aqueous solution to physical and chemical properties. The developed formulation contains (in grams): tetrahydrozoline hydrochloride - 0.05; benzalkonium chloride - 0.01; disodium edetate - 0.01; sodium chloride - 0.84; citrate buffer - to a pH of 6.2 to 6.5; injection water - up to 100 ml. Samples of the experimental series of eye drops meet the requirements of HFC on ophthalmic drugs in terms of physico-chemical quality indicators as description, transparency, osmolality, pH of the solution, quantitative content of tetrahydrazoline hydrochloride. The results obtained in the research of stability of the eye drops experimental series throughout a long-term and accelerated storage during 6-months observation indicate the invariance of critical quality indicators, such as description, transparency, pH of the solution and the quantitative content of tetrahydrazoline hydrochloride. The results of determining the locally irritant effect on the mucous membrane of the rabbit’s eye showed that developed formulation of the eye drops does not cause any local irritation, neither visual reaction from the mucous membrane of the eye. The damaging effect level was 0 points in comparison with the other drugs. It is proved that the proposed formulation of eye drops meets the requirements of SPhU, stable and comfortable to use. The results of this experiment have a practical importance for development of new generic drugs.

Towards an improved understanding of drug excipient interactions to enable rapid optimization of nanosuspension formulations.

Suspensions of drug nanoparticles known as nanosuspensions have emerged as a successful enabling formulation approach for poorly soluble drug candidates. These nanoparticles typically require stabilization with specific polymer or surfactant excipients to prevent aggregation from occurring. This study demonstrates the necessity of formulating drug nanosuspensions with amphiphilic excipients possessing long hydrophobic alkyl or polymer block chains to produce stable nanoparticles. 28 different excipients and excipient combinations at various loadings were screened across the 3 drug compounds and their effectiveness, as characterized by the lowest excipient loading needed to stabilize a monodisperse drug suspension, is quantified as a function of various excipient parameters such as molecular weight, HLB value, CMC, H-bond donors and acceptors, and the length of the hydrophobic alkyl chains and polymer blocks within their molecular structure. Traditional characterization parameters (molecular weight, HLB value, and CMC) fail to predict excipient effectiveness. The conformational flexibility and length of the hydrophobic regions of amphiphilic excipients appears to be critical for effectiveness. This hypothesis was supported by molecular modeling studies to better understand the interactions between the excipients with the drug nanoparticle surface.

A Novel Testing Approach for Oxidative Degradation Dependent Incompatibility of Amine Moiety Containing Drugs with PEGs in Solid-State.

Reactive impurities originating from excipients can cause drug stability issues, even at trace amounts. When produced during final dosage form storage, they are especially hard to control, and often, factors inducing their formation remain unidentified. Oxidative degradation dependent formation of formaldehyde and formic acid is responsible for N-methylation and N-formylation of amine-moiety-containing drug substances. A very popular combination of polyethylene glycols and iron oxides, used in more than two-thirds of FDA-approved tablet formulation drugs in 2018, was found to be responsible for increased concentrations of N-methyl impurity in the case of paroxetine hydrochloride. We propose a novel testing approach for early identification of potentially problematic combinations of excipients and drug substances. The polyethylene glycol 6000 degradation mechanism and kinetics in the presence of iron oxides is studied. The generality of the proposed stress test setup in view of the susceptibility of amine-moiety-containing drug substances to N-methylation and N-formylation is evaluated.

Experimental design approach for development of novel microemulsion system and immediate release self microemulsifying tablet of nebivolol HCl

The objective of this study was to determine specific combination of pharmaceutical excipients that lead to formulation of efficient nebivolol hydrochloride SMEDDS and its subsequent formulation into IR-SET (Immediate release- Self emulsifying tablet) which will enhance its solubility and dissolution. Solubility and Pseudo-ternary phase studies were carried out to identify the excipients showing highest solubility and to identify the zone of microemulsion with selected ingredients. Liquid-SMEDDS (L-SMEDDS) were optimized for Concentration of oil(X1) and Smix(X2) and formulated using a combination of Kollisolv GTA as oil, Tween 80 as surfactant and propylene glycol as co-surfactant which gave smaller droplet size(Y1) 55.98nm , Emulsification time (Y2) 16±1.5 s,% transmittance (Y3) 99.94±0.47%. Neusilin US2 was used as solid carrier for solidification of L-SMEDDS in to Solid-SMEDDS (S-SMEDDS) by adsorption technique. IR-SET of nebivolol were formulated with S-SMEDDS and optimized for the concentration of binder (X1) (PVP K30) and superdisintegrant (X2) (KOLLIDON CL) which showed low Disintegration time (Y1) (92±0.5s) and low Friability(Y2)(0.424±0.03%). Also the DSC and XRD data revealed the molecular state of the drug in S-SMEDDS. The extent of in-vivo drug release and ex-vivo diffusion values from L-SMEDDS and IR-SET was much higher than pure drug and marketed tablet. In conclusion, the results showed potential of SMEDDS to improve solubility and thus the bioavailability.

Probiotic goat milk tablets: Formulation optimization and stability evaluation

In this study, a D-optimal mixture design was adopted to evaluate the effect of different excipient combinations of xylitol, erythritol, mannitol, sodium alginate, and microcrystalline cellulose on the quality of probiotic goat milk tablets. Four indicators, namely, viable cell counts, friability, hardness, and sensory evaluation, were used to determine the optimal formulation with xylitol of 9.00%, erythritol of 2.85%, mannitol of 3.00%, microcrystalline cellulose of 0.15%, goat milk powder of 84.90%, and mixed bacteria powder of 0.10%, which can well protect probiotics. In addition, the viability of the probiotics was measured during the storage and in the gastrointestinal tract. The storage stability test showed that the viable counts of probiotics goat milk tablets could still remain at 106 CFU/g after storage at room temperature (25 °C) for 210 days. The simulation experiment in vitro verified that probiotics can tolerate gastrointestinal fluids, reach the intestine smoothly under the protection of the goat milk tablet formula, and exert its probiotic effect. The research demonstrates that the optimal mixture design is reliable and the tablets exhibit high viable counts, have good sensory characteristics, and offer ideal stability, which provides a basis for the development of industrial production of probiotic goat milk tablets.

Improved stability of polyclonal antibodies: A case study with lyophilization-conserved antibodies raised against epitopes from the malaria parasite Plasmodium falciparum

Antibodies can be produced as polyclonal (pAb) or monoclonal (mAb) liquid formulations with limited shelf-life. For pAbs, unlike mAbs, only little is known about excipients and lyophilization affecting antibody stability upon reconstitution. We used a model pAb directed against Plasmodium falciparum (Pf) pyridoxal 5′-phosphate synthase 2 (Pdx2) to systemically study effects of bulking agents (amino acids, phosphate buffers, salt solutions), sugar(alcohols), surfactants and protein additions (bovine serum albumin, BSA) in liquid pAb formulations (isolated or in combinations) on the activity to detect the antigen in Pf extracts by Western blots. Repeated freeze-thaw cycles (20x) and extended room temperature storage markedly compromised pAb stability, the former being ameliorated by addition of cryoprotectants (glycerol, sucrose). Lyophilization of pure liquid pAb formulation markedly decreased antibody reactivity upon reconstitution which was not preserved by most bulking agents tested (e.g., histidine, arginine, acetate). Among the tested salt solutions (NaCl, Ringer, PBS), phosphate buffered saline had the largest lyoprotective potential, alongside sucrose, but not trehalose or maltitol. Among combinations of excipients, PBS, sucrose, low concentration BSA and Tween potently preserved PfPdx2 stability. Results for PBS were transferable to PfEnolase pAb, indicating that some of the formulations investigated here might be a low-cost solution for more general applicability to pAbs.

Stabilization and formulation of a recombinant Human Cytomegalovirus vector for use as a candidate HIV-1 vaccine

Live attenuated viral vaccine/vector candidates are inherently unstable and infectivity titer losses can readily occur without defining appropriate formulations, storage conditions and clinical handling practices. During initial process development of a candidate vaccine against HIV-1 using a recombinant Human Cytomegalovirus vector (rHCMV-1), large vector titer losses were observed after storage at 4 °C and after undergoing freeze-thaw. Thus, the goal of this work was to develop candidate frozen liquid formulations of rHCMV-1 with improved freeze-thaw and short-term liquid stability for potential use in early clinical trials. To this end, a virus stability screening protocol was developed including use of a rapid, in vitro cell-based immunofluorescence focus assay to quantitate viral titers. A library of ∼50 pharmaceutical excipients (from various known classes of additives) were evaluated for their effect on vector stability after freeze-thaw cycling or incubation at 4 °C for several days. Certain additives including sugars and polymers (e.g., trehalose, sucrose, sorbitol, hydrolyzed gelatin, dextran 40) as well as removal of NaCl (lower ionic strength) protected rHCMV-1 against freeze-thaw mediated losses in viral titers. Optimized solution conditions (e.g., solution pH, buffers and sugar type) slowed the rate of rHCMV-1 titer losses in the liquid state at 4 °C. After evaluating various excipient combinations, three new candidate formulations were designed and rHCMV-1 stability was benchmarked against both the currently-used and a previously reported formulation. The new candidate formulations were significantly more stable in terms of reducing rHCMV-1 titer losses after 5 freeze-thaw cycles or incubation at 4 °C for 30 days. This case study highlights the utility of semi-empirical design of frozen liquid formulations of a live viral vaccine candidate, where protection against infectivity titer losses due to freeze-thaw and short-term liquid storage are sufficient to enable more rapid initiation of early clinical trials.

EXPERIMENTAL STUDY OF ANTIMICROBIAL PROPERTIES AND ACUTE TOXICITY OF THE PEO-BASED COMBINED SUPPOSITORIES

Aim. The research of antimicrobial and toxicological parameters of a promising pharmaceutical composition with indole-3-carbinol and meloxicam in the form of rectal suppositories. Materials and methods. The research of antimicrobial activity was carried out in vitro by diffusion in nutrient agar in the modification of "holes" on the reference strains of common pathogens Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Candida albicans. Adult rats were used for the study of acute toxicity. Suppository mass were administrated in the largest possible volume rectally or orally. The animals were periodically monitored according to the experimental plan – the assessment of physiological parameters before administration and after 6, 12, 18, 24 hours, at 3, 7 and 14 days. The animals were removed from experiment and necropsy provided after 1, 3, 7, and 14 days. Results. The sizes of zones of inhibition of the microorganisms growth were most significant (from 19.27±0.61 mm of E. coli to 40.80±0.42 mm of S. aureus) near sample of the combined composition suppository compared with other combination of active substances and excipients. During the observation of animals for 14 days and the study of internal organs after autopsy, deviations in physiological (weight, temperature, activity, respiratory rate) and macroscopic morphological indicators of animals from reference values were not detected. Conclusion. According to the results of microbiological studies, a moderate antimicrobial effect of suppositories of combined composition in relation to all the studied pathogens was revealed. The absence of manifestations of acute toxicity allows us to conclude that the pharmaceutical composition can be classified as practically non-toxic substances. The obtained results allow us to recommend a pharmaceutical composition with indole-3-carbinol and meloxicam on a polyethylene oxide basis in the form of suppositories for further preclinical studies of specific pharmacological effects as a prostate protective agent.