Contribution of a C-Terminal Extension to the Substrate Affinity and Oligomeric Stability of Aldehyde Dehydrogenase from Thermus thermophilus HB27.

Aldehyde dehydrogenase enzymes (ALDHs) are widely studied for their roles in disease propagation and cell metabolism. Their use in biocatalysis applications, for the conversion of aldehydes to carboxylic acids, has also been recognized. Understanding the structural features and functions of both prokaryotic and eukaryotic ALDHs is key to uncovering novel applications of the enzyme and probing its role in disease propagation. The thermostable enzyme ALDHTt originating fromThermus thermophilus, strain HB27, possesses a unique extension of its C-terminus, which has been evolutionarily excluded from mesophilic counterparts and other thermophilic enzymes in the same genus. In this work, the thermophilic adaptation is studied by the expression and optimized purification of mutant ALDHTt-508, with a 22-amino acid truncation of the C-terminus. The mutant shows increased activity throughout production compared to native ALDHTt, indicating an opening of the active site upon C-terminus truncation and giving rationale into the evolutionary exclusion of the C-terminal extension from similar thermophilic and mesophilic ALDH proteins. Additionally, the C-terminus is shown to play a role in controlling substrate specificity of native ALDH, particularly in excluding catalysis of certain large and certain aromatic ortho-substituted aldehydes, as well as modulating the protein's pH tolerance by increasing surface charge. Dynamic light scattering and size-exclusion HPLC methods are used to show the role of the C-terminus in ALDHTt oligomeric stability at the cost of catalytic efficiency. Studying the aggregation rate of ALDHTt with and without a C-terminal extension leads to the conclusion that ALDHTt follows a monomolecular reaction aggregation mechanism.

Continuous Microfluidic Antisolvent Crystallization as a Bottom-Up Solution for the Development of Long-Acting Injectable Formulations.

A bottom-up approach was investigated to produce long-acting injectable (LAI) suspension-based formulations to overcome specific limitations of top-down manufacturing methods by tailoring drug characteristics while making the methods more sustainable and cost-efficient. A Secoya microfluidic crystallization technology-based continuous liquid antisolvent crystallization (SCT-CLASC) process was optimized and afterward compared to an earlier developed microchannel reactor-based continuous liquid antisolvent crystallization (MCR-CLASC) setup, using itraconazole (ITZ) as the model drug. After operating parameter optimization and downstream processing (i.e., concentrating the suspensions), stable microsuspensions were generated with a final solid loading of 300 mg ITZ/g suspension. The optimized post-precipitation feed suspension consisted of 40 mg ITZ/g suspension with a drug-to-excipient ratio of 53:1. Compared to the MCR-CLASC setup, where the post-precipitation feed suspensions contained 10 mg ITZ/g suspension and had a drug-to-excipient ratio of 2:1, a higher drug concentration and lower excipient use were successfully achieved to produce LAI microsuspensions using the SCT-CLASC setup. To ensure stability during drug crystallization and storage, the suspensions' quality was monitored for particle size distribution (PSD), solid-state form, and particle morphology. The PSD of the ITZ crystals in suspension was maintained within the target range of 1-10 µm, while the crystals displayed an elongated plate-shaped morphology and the solid state was confirmed to be form I, which is the most thermodynamically stable form of ITZ. In conclusion, this work lays the foundation for the SCT-CLASC process as an energy-efficient, robust, and reproducible bottom-up approach for the manufacture of LAI microsuspensions using ITZ at an industrial scale.

Development of long-acting injectable suspensions by continuous antisolvent crystallization: An integrated bottom-up process.

Our present work elucidated the operational feasibility of direct generation and stabilization of long-acting injectable (LAI) suspensions of a practically insoluble drug, itraconazole (ITZ), by combining continuous liquid antisolvent crystallization with downstream processing (i.e., centrifugal filtration and reconstitution). A novel microchannel reactor-based bottom-up crystallization setup was assembled and optimized for the continuous production of micro-suspension. Based upon the solvent screening and solubility study, N-methyl pyrrolidone (NMP) was selected as the optimal solvent and an impinging jet Y-shaped microchannel reactor (MCR) was selected as the fluidic device to provide a reproducible homogenous mixing environment. Operating parameters such as solvent to antisolvent ratio (S/AS), total jet liquid flow rates (TFRs), ITZ feed solution concentration and the maturation time in spiral tubing were tailored to 1:9 v/v, 50 mL/min, 10 g/100 g solution, and 96 h, respectively. Vitamin E TPGS (0.5% w/w) was found to be the most suitable excipient to stabilize ITZ particles amongst 14 commonly used stabilizers screened. The effect of scaling up from 25 mL to 15 L was evaluated effectively with in situ monitoring of particle size distribution (PSD) and solid-state form. Thereafter, the suspension was subjected to centrifugal filtration to remove excess solvent and increase ITZ solid fraction. As an alternative, an even more concentrated wet pellet was reconstituted with an aqueous solution of 0.5% w/w Vitamin E TPGS as resuspending agent. The ITZ LAI suspension (of 300 mg/mL solid concentration) has the optimal PSD with a D10 of 1.1 ± 0.3 µm, a D50 of 3.53 ± 0.4 µm and a D90 of 6.5 ± 0.8 µm, corroborated by scanning electron microscopy (SEM), as remained stable after 548 days of storage at 25 °C. Finally, in vitro release methods using Dialyzer, dialysis membrane sac were investigated for evaluation of dissolution of ITZ LAI suspensions. The framework presented in this manuscript provides a useful guidance for development of LAI suspensions by an integrated bottom-up approach using ITZ as model API.

Controlling the Polymorphism of Indomethacin with Poloxamer 407 in a Gas Antisolvent Crystallization Process.

The polymorphic control of active pharmaceutical ingredients (APIs) is a major challenge in the manufacture of medicines. Crystallization methods that use supercritical carbon dioxide as an antisolvent can create unique solid forms of APIs, with a particular tendency to generate metastable polymorphic forms. In this work, the effects of processing conditions within a gas antisolvent (GAS) crystallization method, such as pressure, stirring rate, and temperature, as well as the type of solvent used and the presence of an additive, on the polymorphism of indomethacin were studied. Consistent formation of the X-ray powder diffraction-pure α polymorphic form of indomethacin by GAS was only achieved when a polymer, poloxamer 407, was used as an additive. Using the GAS method in combination with poloxamer 407 as a molecular additive enabled full control over the polymorphic form of indomethacin, regardless of the processing conditions employed, such as pressure, temperature, stirring rate, and type of solvent. A detailed molecular modeling study provided insight into the role of poloxamer 407 in the polymorphic outcome of indomethacin and concluded that it favored the formation of the α polymorph.

Solid-state and particle size control of pharmaceutical cocrystals using atomization-based techniques.

Poor bioavailability and aqueous solubility represent a major constraint during the development of new API molecules and can influence the impact of new medicines or halt their approval to the market. Cocrystals offer a novel and competitive advantage over other conventional methods with respect towards the substantial improvement in solubility profiles relative to the single-API crystals. Furthermore, the production of such cocrystals through atomization-based methods allow for greater control, with respect to particle size reduction, to further increase the solubility of the API. Such atomization-based methods include supercritical fluid methods, conventional spray drying and electrohydrodynamic atomization/electrospraying. The influence of process parameters such as solution flow rates, pressure and solution concentration, in controlling the solid-state and final particle size are discussed in this review with respect to atomization-based methods. For the last decade, literature has been attempting to catch-up with new regulatory rulings regarding the classification of cocrystals, due in part to data sparsity. In recent years, there has been an increase in cocrystal publications, specifically employing atomization-based methods. This review considers the benefits to employing atomization-based methods for the generation of pharmaceutical cocrystals, examines the most recent regulatory changes regarding cocrystals and provides an outlook towards the future of this field.

Production and isolation of pharmaceutical drug nanoparticles.

Nanosizing of pharmaceutical drug particles is one of the most important drug delivery platforms approaches for the commercial development of poorly water-soluble drug molecules. Though nanosizing of drug particles has been proven to greatly enhance drugs dissolution rate and apparent solubility, nanosized materials have presented significant challenges for their formulation as solid dosage forms (e.g. tablets, capsules). This is due to the strong Van der Waals attraction forces between dry nanoparticles leading to aggregation, cohesion, and consequently poor flowability. In this review, the broad area of nanomedicines is overviewed with the primary focus on drug nanocrystals and the top-down and bottom-up methods used in their fabrication. The review also looks at how nanosuspensions of pharmaceutical drugs are generated and stabilised, followed by subsequent strategies for isolation of the nanoparticles. A perspective on the future outlook for drug nanocrystals is also presented.

Pharmaceutical nanoparticle isolation using CO2-assisted dynamic bed coating.

Poor solubility of new chemical entities (NCEs) is a major bottleneck in the pharmaceutical industry which typically leads to poor drug bioavailability and efficacy. Nanotechnologies offer an interesting route to improve the apparent solubility and dissolution rate of pharmaceutical drugs, and processes such as nano-spray drying and supercritical CO2-assisted spray drying (SASD) provide a route to engineer and produce solid drug nanoparticles. However, dried nanoparticles often show poor rheological properties (e.g. flowability, tabletability) and their isolation using these methods is typically inefficient and leads to poor collection yields. The work presented herein demonstrates a novel production and isolation method for drug nanoparticles using a 'top spray dynamic bed coating' process, which uses CO2 spray as the fluidizing gas. Nanoparticles of three BCS class II Active Pharmaceutical Ingredients (APIs), namely carbamazepine (CBZ), ketoprofen (KET) and risperidone (RIS), were produced and successfully coated onto micron-sized microcrystalline cellulose (MCC) particles. The size distribution of the API nanoparticles was in the range of 90-490 nm. The stable forms of CBZ (form III), KET (form I), and the metastable form of RIS (form B) were produced and coated onto MCC carrier microparticles. All the isolated solids presented optimal rheological properties along with a 2-6 fold improvement in the dissolution rate of the corresponding APIs. Hence, the 'top spray dynamic bed coater' developed in this work demonstrates to be an efficient approach to produce and coat API nanoparticles onto carrier particles with optimal rheological properties and improved dissolution.

Cortisone and cortisol break hydrogen-bonding rules to make a drug-prodrug solid solution.

Multidrug products enable more effective therapies and simpler administration regimens, provided that a stable formulation is prepared, with the desired composition. In this view, solid solutions have the advantage of combining the stability of a single crystalline phase with the potential of stoichiometry variation of a mixture. Here a drug-prodrug solid solution of cortisone and cortisol (hydrocortisone) is described. Despite the structural differences of the two components, the new phase is obtained both from solution and by supercritical CO2 assisted spray drying. In particular, to enter the solid solution, hydrocortisone must violate Etter's rules for hydrogen bonding. As a result, its dissolution rate is almost doubled.

Solubility and thermodynamic analysis of ketoprofen in organic solvents.

The solubility of the racemic solid phase of ketoprofen (KTP) in methanol, ethanol, isopropanol, butanol, acetonitrile, ethyl acetate, 1,4-dioxane and toluene has been determined between 273 and 303 K by a gravimetric method. FTIR and Raman spectroscopy, SEM and PXRD, have been used to characterise the solid phase. The melting data and heat capacity of solid and melt have been determined by DSC, and used to estimate fusion thermodynamics and the activity of the solid phase as functions of temperature. Empirical and semi-empirical models have been fitted to experimental solubility data. The solution activity coefficients reveal positive deviation from ideality in all solvents except for in dioxane, and very close to ideality in methanol. The solubility is fairly high in the alcohols but decrease with increasing hydrocarbon chain. Generally and due to the presence of the carboxylic acid group, KTP is more readily dissolved in polar protic solvents, followed in order by polar aprotic and non-polar solvents. However, the highest solubility is found in dioxane, classified as a non-polar solvent, but notably though the molecule having two strong hydrogen bond accepting functionalities, and no hydrogen bond donation capability.

Amorphous solid dispersion of ibuprofen: A comparative study on the effect of solution based techniques.

Amorphous solid dispersion (ASD) is one of the most promising strategies for improving the solubility of active pharmaceutical ingredients (APIs) with low aqueous solubility. Solvent-based techniques such as electrospinning (ES), spray-drying (SD) and rotary evaporation (RE), have all previously been shown to be effective techniques for formulating ASDs. To date however, the effect of these processing techniques on the physicochemical properties and ASD homogeneity or "quality of ASD" produced remains largely unexplored. This work uses ibuprofen (IBU) as a model BCS class II API with two cellulosic excipients, HPMCAS and HPMCP-HP55 to produce ASDs by employing ES, SD and RE processing techniques. The physicochemical, morphological and dissolution properties of each sample were evaluated and the ASD forming strengths of each of the polymers were assessed using Differential Scanning Calorimetry (DSC). Principal |Component Analysis (PCA) of Raman spectra of crystalline and amorphous IBU was employed for qualitative analysis of ASD homogeneity and subsequent ASD stability during long-term storage. Results show that while ASD formation is predominantly dependent on API:excipient ratio, the ASD homogeneity is highly dependent on processing technique. Dissolution studies show that electrospun samples had the highest API release rate due to their fibrous morphology and higher specific surface area. However, these samples were the least homogenous of all ASDs produced thereby potentially influencing sample stability during long term storage. In addition, the higher melting point depression, higher Tg, and increased abundance of functional groups suitable for hydrogen bonding, show HPMCAS to be a significantly better ASD co-former when compared with HPMCP-HP55.

From batch to continuous - New opportunities for supercritical CO2 technology in pharmaceutical manufacturing.

Poor solubility and bioavailability of new chemical entities is a major challenge that keeps plaguing the pharmaceutical industry and jeopardizes their away to the market. Nanotechnologies hold a great promise to overcome these chemical barriers. In particular, for supercritical CO2 technologies, the scientific community has seen significant development of these types of processes over the last 15-20 years, however these techniques are still waiting to see the daylight in the industrial environmental. Continuous operation of supercritical processes and their adaptation to existing industrial facilities opens new doors for their success in the pharmaceutical arena. This commentary paper aims to discuss the current status of supercritical CO2 techniques and the major future opportunities for their implementation in the pharmaceutical industry in the coming years.

Spray drying of pharmaceuticals and biopharmaceuticals: Critical parameters and experimental process optimization approaches.

Spray drying is increasingly becoming recognized as an efficient drying and formulation technique for pharmaceutical and biopharmaceutical processing. It offers significant economic and processing advantages compared to lyophilisation/freeze-drying techniques even though the optimisation of process parameters is often a costly and time-consuming procedure. Spray Drying has primarily been used in formulating small molecule drugs with low solubility however it is increasingly being applied to the processing of large biomolecules and biopharmaceuticals. This review examines the basics of spray drying process, current technology and various components used in spray drying process. Moreover, it is focused on introducing critical formulation and processing factors in spray drying of small molecule drugs and large biomolecules, their similarities and differences. Finally, it provides an overview of the experimental optimisation strategies designed to achieve optimum spray drying results in the shortest possible timeframe while utilising minimum product.

Supercritical carbon dioxide-based technologies for the production of drug nanoparticles/nanocrystals - A comprehensive review.

Low drug bioavailability, which is mostly a result of poor aqueous drug solubilities and of inadequate drug dissolution rates, is one of the most significant challenges that pharmaceutical companies are currently facing, since this may limit the therapeutic efficacy of marketed drugs, or even result in the discard of potential highly effective drug candidates during developmental stages. Two of the main approaches that have been implemented in recent years to overcome poor drug solubility/dissolution issues have frequently involved drug particle size reduction (i.e., micronization/nanonization) and/or the modification of some of the physicochemical and structural properties of poorly water soluble drugs. A large number of particle engineering methodologies have been developed, tested, and applied in the synthesis and control of particle size/particle-size distributions, crystallinities, and polymorphic purities of drug micro- and nano-particles/crystals. In recent years pharmaceutical processing using supercritical fluids (SCF), in general, and supercritical carbon dioxide (scCO2), in particular, have attracted a great attention from the pharmaceutical industry. This is mostly due to the several well-known advantageous technical features of these processes, as well as to other increasingly important subjects for the pharmaceutical industry, namely their "green", sustainable, safe and "environmentally-friendly" intrinsic characteristics. In this work, it is presented a comprehensive state-of-the-art review on scCO2-based processes focused on the formation and on the control of the physicochemical, structural and morphological properties of amorphous/crystalline pure drug nanoparticles. It is presented and discussed the most relevant scCO2, scCO2-based fluids and drug physicochemical properties that are pertinent for the development of successful pharmaceutical products, namely those that are critical in the selection of an adequate scCO2-based method to produce pure drug nanoparticles/nanocrystals. scCO2-based nanoparticle formation methodologies are classified in three main families, and in terms of the most important role played by scCO2 in particle formation processes: as a solvent; as an antisolvent or a co-antisolvent; and as a "high mobility" additive (a solute, a co-solute, or a co-solvent). Specific particle formation methods belonging to each one of these families are presented, discussed and compared. Some selected amorphous/crystalline drug nanoparticles that were prepared by these methods are compiled and presented, namely those studied in the last 10-15 years. A special emphasis is given to the formation of drug cocrystals. It is also discussed the fundamental knowledge and the main mechanisms in which the scCO2-based particle formation methods rely on, as well as the current status and urgent needs in terms of reliable experimental data and of robust modeling approaches. Other addressed and discussed topics include the currently available and the most adequate physicochemical, morphological and biological characterization methods required for pure drug nanoparticles/nanocrystals, some of the current nanometrology and regulatory issues associated to the use of these methods, as well as some scale-up, post-processing and pharmaceutical regulatory subjects related to the industrial implementation of these scCO2-based processes. Finally, it is also discussed the current status of these techniques, as well as their future major perspectives and opportunities for industrial implementation in the upcoming years.

Spray drying ternary amorphous solid dispersions of ibuprofen - An investigation into critical formulation and processing parameters.

A design of experiment (DoE) approach was used to investigate the critical formulation and processing parameters in spray drying ternary amorphous solid dispersions (ASDs) of ibuprofen. A range of 16 formulations of ibuprofen, HPMCP-HP55 and Kollidon VA 64 were spray dried. Statistical analysis revealed the interrelation of various spray drying process conditions and formulation factors, namely solution feed rate, inlet temperature, Active Pharmaceutical Ingredient (API)/excipients ratio and dichloromethane (DCM)/methanol (MeOH) ratio. Powder X-ray diffraction analysis (PXRD) showed that all the samples with the lowest API/excipient ratio (1:4) were amorphous, while others were crystalline. Moreover, differential scanning calorimetry (DSC) analysis was employed to investigate ASD formulation more in-depth. The glass transition temperatures (Tg) of all ASDs were in the range 70-79°C, while crystalline formulations displayed an endothermic peak of melting of crystalline ibuprofen in the range of 50-80°C. The high Tg of ASDs was an indication of highly stable ASD formulations as verified via PXRD at zero day and afterward at 1, 1.5, 3 and 6month intervals. The intermolecular interactions between ibuprofen molecule and excipients were studied by Fourier transform infrared spectroscopy (FTIR) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy. FTIR and Carbon-13 ssNMR analysis indicated that hydrogen bond formation involving the carboxyl group in ibuprofen within the ASDs is likely. More importantly, the solubility of ibuprofen in ASD formulations is improved compared to pure ibuprofen. This was due to both the amorphous structure of ibuprofen and of the existence of amphiphilic excipient, Kollidon VA 64, in the formulation.

New thermoresistant polymorph from CO2 recrystallization of minocycline hydrochloride.

PURPOSE: To prepare and thoroughly characterize a new polymorph of the broad-spectrum antibiotic minocycline from its hydrochloride dehydrate salts. METHODS: The new minocycline hydrochloride polymorph was prepared by means of the antisolvent effect caused by carbon dioxide. Minocycline recrystallized as a red crystalline hydrochloride salt, starting from solutions or suspensions containing CO2 and ethanol under defined conditions of temperature, pressure and composition. RESULTS: This novel polymorph (ß-minocycline) revealed characteristic PXRD and FTIR patterns and a high melting point (of 247 ºC) compared to the initial minocycline hydrochloride hydrates (α-minocycline). Upon dissolution the new polymorph showed full anti-microbial activity. Solid-state NMR and DSC studies evidenced the higher chemical stability and crystalline homogeneity of ß-minocycline compared to the commercial chlorohydrate powders. Molecular structures of both minocyclines present relevant differences as shown by multinuclear solid-state NMR. CONCLUSIONS: This work describes a new crystalline structure of minocycline and evidences the ability of ethanol-CO2 system in removing water molecules from the crystalline structure of this API, at modest pressure, temperature and relatively short time (2 h), while controlling the crystal habit. This process has therefore the potential to become a consistent alternative towards the control of the solid form of APIs.

Development of a novel mucosal vaccine against strangles by supercritical enhanced atomization spray-drying of Streptococcus equi extracts and evaluation in a mouse model.

Strangles is an extremely contagious and sometimes deadly disease of the Equidae. The development of an effective vaccine should constitute an important asset to eradicate this worldwide infectious disease. In this work, we address the development of a mucosal vaccine by using a Supercritical Enhanced Atomization (SEA) spray-drying technique. Aqueous solutions containing the Streptococcus equi extracts and chitosan were converted into nanospheres with no use of organic solvents. The immune response in a mouse model showed that the nanospheres induced a well-balanced Th1 and Th2 response characterized by a unitary ratio between the concentrations of IgG2a and IgG1, together with IgA production. This strategy revealed to be an effective alternative for immunization against S. equi, and therefore, it may constitute a feasible option for production of a strangles vaccine.

Powder X-ray diffraction method for the quantification of cocrystals in the crystallization mixture.

CONTEXT: The solid state purity of cocrystals critically affects their performance. Thus, it is important to accurately quantify the purity of cocrystals in the final crystallization product. OBJECTIVE: The aim of this study was to develop a powder X-ray diffraction (PXRD) quantification method for investigating the purity of cocrystals. The method developed was employed to study the formation of indomethacin-saccharin (IND-SAC) cocrystals by mechanochemical methods. MATERIALS AND METHODS: Pure IND-SAC cocrystals were geometrically mixed with 1:1 w/w mixture of indomethacin/saccharin in various proportions. An accurately measured amount (550 mg) of the mixture was used for the PXRD measurements. The most intense, non-overlapping, characteristic diffraction peak of IND-SAC was used to construct the calibration curve in the range 0-100% (w/w). This calibration model was validated and used to monitor the formation of IND-SAC cocrystals by liquid-assisted grinding (LAG). RESULTS: The IND-SAC cocrystal calibration curve showed excellent linearity (R(2) = 0.9996) over the entire concentration range, displaying limit of detection (LOD) and limit of quantification (LOQ) values of 1.23% (w/w) and 3.74% (w/w), respectively. Validation results showed excellent correlations between actual and predicted concentrations of IND-SAC cocrystals (R(2) = 0.9981). DISCUSSION: The accuracy and reliability of the PXRD quantification method depend on the methods of sample preparation and handling. The crystallinity of the IND-SAC cocrystals was higher when larger amounts of methanol were used in the LAG method. CONCLUSION: The PXRD quantification method is suitable and reliable for verifying the purity of cocrystals in the final crystallization product.

Formation of indomethacin-saccharin cocrystals using supercritical fluid technology.

The main objective of the present work is to check the feasibility of supercritical fluid (SCF) technologies in the screening and design of cocrystals (novel crystalline solids). The cocrystal formation tendencies in three different SCF techniques, focusing on distinct supercritical fluid properties - solvent, anti-solvent and atomization enhancer - were investigated. The effect of processing parameters on the cocrystal formation behaviour and particle properties in these techniques was also studied. A recently reported indomethacin-saccharin (IND-SAC) cocrystalline system was our model system. A 1:1 molar ratio of indomethacin (gamma-form) and saccharin was used as a starting material. The SCF techniques employed in the study include the CSS technique (cocrystallization with supercritical solvent), the SAS technique (supercritical anti-solvent), and the AAS technique (atomization and anti-solvent). The resulting cocrystalline phase was identified using differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), and Fourier transform-Raman (FT-Raman). The particle morphologies and size distributions were determined using scanning electron microscopy (SEM) and aerosizer, respectively. The pure IND-SAC cocrystals were obtained from SAS and AAS processes, whilst partial to no cocrystal formation occurred in the CSS process. However, no remarkable differences were observed in terms of cocrystal formation at different processing conditions in SAS and AAS processes. Particles from CSS processes were agglomerated and large, whilst needle-to-block-shaped and spherical particles were obtained from SAS and AAS processes, respectively. The particle size distribution of these particles was 0.2-5microm. Particulate IND-SAC cocrystals with different morphologies and sizes (nano-to-micron) were produced using supercritical fluid techniques. This work demonstrates the potential of SCF technologies as screening methods for cocrystals with possibilities for particle engineering.