Mechanical release of homogenous proteins from supramolecular gels.

A long-standing challenge is how to formulate proteins and vaccines to retain function during storage and transport and to remove the burdens of cold-chain management. Any solution must be practical to use, with the protein being released or applied using clinically relevant triggers. Advanced biologic therapies are distributed cold, using substantial energy, limiting equitable distribution in low-resource countries and placing responsibility on the user for correct storage and handling. Cold-chain management is the best solution at present for protein transport but requires substantial infrastructure and energy. For example, in research laboratories, a single freezer at -80 °C consumes as much energy per day as a small household1. Of biological (protein or cell) therapies and all vaccines, 75% require cold-chain management; the cost of cold-chain management in clinical trials has increased by about 20% since 2015, reflecting this complexity. Bespoke formulations and excipients are now required, with trehalose2, sucrose or polymers3 widely used, which stabilize proteins by replacing surface water molecules and thereby make denaturation thermodynamically less likely; this has enabled both freeze-dried proteins and frozen proteins. For example, the human papilloma virus vaccine requires aluminium salt adjuvants to function, but these render it unstable against freeze-thaw4, leading to a very complex and expensive supply chain. Other ideas involve ensilication5 and chemical modification of proteins6. In short, protein stabilization is a challenge with no universal solution7,8. Here we designed a stiff hydrogel that stabilizes proteins against thermal denaturation even at 50 °C, and that can, unlike present technologies, deliver pure, excipient-free protein by mechanically releasing it from a syringe. Macromolecules can be loaded at up to 10 wt% without affecting the mechanism of release. This unique stabilization and excipient-free release synergy offers a practical, scalable and versatile solution to enable the low-cost, cold-chain-free and equitable delivery of therapies worldwide.

Unique ice dendrite morphology on state-of-the-art oil-impregnated surfaces.

Due to its multifaceted impact in various applications, icing and ice dendrite growth has been the focus of numerous studies in the past. Dendrites on wetting (hydrophilic) and nonwetting (hydrophobic) surfaces are sharp, pointy, branching, and hairy. Here, we show a unique dendrite morphology on state-of-the-art micro/nanostructured oil-impregnated surfaces, which are commonly referred to as slippery liquid-infused porous surfaces or liquid-infused surfaces. Unlike the dendrites on traditional textured hydrophilic and hydrophobic surfaces, the dendrites on oil-impregnated surfaces are thick and lumpy without pattern. Our experiments show that the unique ice dendrite morphology on lubricant-infused surfaces is due to oil wicking into the porous dendritic network because of the capillary pressure imbalance between the surface texture and the dendrites. We characterized the shape complexity of the ice dendrites using fractal analysis. Experiments show that ice dendrites on textured oil-impregnated surfaces have lower fractal dimensions than those on traditional lotus leaf-inspired air-filled porous structures. Furthermore, we developed a regime map that can be used as a design guideline for micro/nanostructured oil-impregnated surfaces by capturing the complex effects of oil chemistry, oil viscosity, and wetting ridge volume on dendrite growth and morphology. The insights gained from this work inform strategies to reduce lubricant depletion, a major bottleneck for the transition of micro/nanostructured oil-impregnated surfaces from bench-top laboratory prototypes to industrial use. This work will assist the development of next-generation depletion-resistant lubricant-infused ice-repellent surfaces.

Long-Acting Heterodimeric Paclitaxel-Idebenone Prodrug-Based Nanomedicine Promotes Functional Recovery after Spinal Cord Injury.

After spinal cord injury (SCI), successive systemic administration of microtubule-stabilizing agents has been shown to promote axon regeneration. However, this approach is limited by poor drug bioavailability, especially given the rapid restoration of the blood-spinal cord barrier. There is a pressing need for long-acting formulations of microtubule-stabilizing agents in treating SCI. Here, we conjugated the antioxidant idebenone with microtubule-stabilizing paclitaxel to create a heterodimeric paclitaxel-idebenone prodrug via an acid-activatable, self-immolative ketal linker and then fabricated it into chondroitin sulfate proteoglycan-binding nanomedicine, enabling drug retention within the spinal cord for at least 2 weeks and notable enhancement in hindlimb motor function and axon regeneration after a single intraspinal administration. Additional investigations uncovered that idebenone can suppress the activation of microglia and neuronal ferroptosis, thereby amplifying the therapeutic effect of paclitaxel. This prodrug-based nanomedicine simultaneously accomplishes neuroprotection and axon regeneration, offering a promising therapeutic strategy for SCI.

Quantification of Biopharmaceutically Relevant Nonionic Surfactant Excipients Using Benchtop qNMR.

Nonionic surfactant excipients (NISEs) are commonly added to biologics formulations to mitigate the effects of stress incurred by the active biotherapeutic during manufacturing, transport, and storage. During manufacturing, NISEs are added by dilution of a stock solution directly into a protein formulation, and their accurate addition is critical in maintaining the quality and integrity of the drug product and thus ensuring patient safety. This is especially true for the common NISEs, polysorbates 20 and 80 (PS20 and PS80, respectively) and poloxamer 188 (P188). With the increasing diversity of biologic modalities within modern pharmaceutical pipelines, there is thus a critical need to develop and deploy convenient and user-accessible analytical techniques that can rapidly and reliably quantify these NISEs under biopharmaceutically relevant conditions. We thus pursued 60 MHz benchtop quantitative NMR (qNMR) as a nondestructive and user-friendly analytical technique for the quantification of PS20, PS80, and P188 under such conditions. We demonstrated the ability of benchtop qNMR (1) to quantify simulated PS20, PS80, and P188 stock solutions representative of those used during the drug substance (DS) formulation step in biomanufacturing and (2) to quantify these NISEs at and below their target concentrations (≤0.025% w/v) directly in biologics formulations containing histidine, sucrose, and one of three biotherapeutic modalities (monoclonal antibody, antibody-drug conjugate, and Fc-fusion protein). Our results demonstrate that benchtop qNMR offers a fit-for-purpose, reliable, user-friendly, and green analytical route by which NISE of interest to the biopharmaceutical industry may be readily and reliably quantified. We conclude that benchtop qNMR has the potential to be applied to other excipient formulation components in the presence of various biological modalities as well as the potential for routine integration within analytical and QC laboratories across pharmaceutical development and manufacturing sites.

Probing the Protein-Excipient Interaction in the Orally Delivered Protein by Solid-State Hydrogen-Deuterium Exchange Mass Spectrometry and Molecular Dynamics.

The oral administration of protein therapeutics in solid dosage form is gaining popularity due to its benefits, such as improved medication adherence, convenience, and ease of use for patients compared to traditional parental delivery. However, formulating oral biologics presents challenges related to pH barriers, enzymatic breakdown, and poor bioavailability. Therefore, understanding the interaction between excipients and protein therapeutics in the solid state is crucial for formulation development. In this Letter, we present a case study focused on investigating the role of excipients in protein aggregation during the production of a solid dosage form of a single variable domain on a heavy chain (VHH) protein. We employed solid-state hydrogen-deuterium exchange coupled with mass spectrometry (ssHDX-MS) at both intact protein and peptide levels to assess differences in protein-excipient interactions between two formulations. ssHDX-MS analysis revealed that one formulation effectively prevents protein aggregation during compaction by blocking ß-sheets across the VHH protein, thereby preventing ß-sheet-ß-sheet interactions. Spatial aggregation propensity (SAP) mapping and cosolvent simulation from molecular dynamics (MD) simulation further validated the protein-excipient interaction sites identified through ssHDX-MS. Additionally, the MD simulation demonstrated that the interaction between the VHH protein and excipients involves hydrophilic interactions and/or hydrogen bonding. This novel approach holds significant potential for understanding protein-excipient interactions in the solid state and can guide the formulation and process development of orally delivered protein dosage forms, ultimately enhancing their efficacy and stability.

Poly(vinylpyridine-co-vinylpyridine N-oxide) Excipients Mediate Rapid Dissolution and Sustained Supersaturation of Posaconazole Amorphous Solid Dispersions.

The chemical structure of excipients molecularly mixed in an amorphous solid dispersion (ASD) has a significant impact on properties of the ASD including dissolution behavior, physical stability, and bioavailability. Polymers used in ASDs require a balance between hydrophobic and hydrophilic functionalities to ensure rapid dissolution of the amorphous dispersion as well as sustained supersaturation of the drug in solution. This work demonstrates the use of postpolymerization functionalization of poly(vinylpyridine) excipients to elucidate the impact of polymer properties on the dissolution behavior of amorphous dispersions containing posaconazole. It was found that N-oxidation of pyridine functionalities increased the solubility of poly(vinylpyridine) derivatives in neutral aqueous conditions and allowed for nanoparticle formation which supplied posaconazole into solution at concentrations exceeding those achieved by more conventional excipients such as hydroxypropyl methylcellulose acetate succinate (HPMCAS) or Eudragit E PO. By leveraging these functional modifications of the parent poly(vinylpyridine) excipient to increase polymer hydrophilicity and minimize the effect of polymer on pH, a new polymeric excipient was optimized for rapid dissolution and supersaturation maintenance for a model compound.

Parameterization of Physiologically Based Biopharmaceutics Models: Workshop Summary Report.

This Article shares the proceedings from the August 29th, 2023 (day 1) workshop "Physiologically Based Biopharmaceutics Modeling (PBBM) Best Practices for Drug Product Quality: Regulatory and Industry Perspectives". The focus of the day was on model parametrization; regulatory authorities from Canada, the USA, Sweden, Belgium, and Norway presented their views on PBBM case studies submitted by industry members of the IQ consortium. The presentations shared key questions raised by regulators during the mock exercise, regarding the PBBM input parameters and their justification. These presentations also shed light on the regulatory assessment processes, content, and format requirements for future PBBM regulatory submissions. In addition, the day 1 breakout presentations and discussions gave the opportunity to share best practices around key questions faced by scientists when parametrizing PBBMs. Key questions included measurement and integration of drug substance solubility for crystalline vs amorphous drugs; impact of excipients on apparent drug solubility/supersaturation; modeling of acid-base reactions at the surface of the dissolving drug; choice of dissolution methods according to the formulation and drug properties with a view to predict the in vivo performance; mechanistic modeling of in vitro product dissolution data to predict in vivo dissolution for various patient populations/species; best practices for characterization of drug precipitation from simple or complex formulations and integration of the data in PBBM; incorporation of drug permeability into PBBM for various routes of uptake and prediction of permeability along the GI tract.

Near UV Photodegradation Mechanisms of Amino Acid Excipients: Formation of the Carbon Dioxide Radical Anion from Aspartate and Fe(III).

Carbon dioxide radical anion (•CO2-) is a powerful reducing agent that can reduce protein disulfide bonds and convert molecular oxygen to superoxide. Therefore, the generation of •CO2- can be detrimental to pharmaceutical formulations. Iron is among the most prevalent impurities in formulations, where Fe(III) chelates of histidine (His) can produce •CO2- upon exposure to near-UV light (Zhang and Schöneich, Eur. J. Pharm. Biopharm. 2023, 190, 231-241). Here, we monitor by spin-trapping in combination with electron paramagnetic resonance spectroscopy and/or high-performance liquid chromatography-mass spectrometry analysis the photochemical formation of •CO2- for a series of common amino acid excipients, including arginine (Arg), methionine (Met), proline (Pro), glutamic acid (Glu), glycine (Gly), aspartic acid (Asp), and lysine (Lys). Our results indicate that in the presence of Fe(III), Asp, and Glu produce significant yields of •CO2- under photoirradiation with near-UV light. Notably, Asp demonstrates the highest efficiency of •CO2- generation compared with that of the other amino acid excipients. Stable isotope labeling indicates that •CO2- exclusively originates from the α-carboxyl group of Asp. Mechanistic studies reveal two possible pathways for •CO2- formation, which involve either a ß-carboxyl radical or an amino radical cation intermediate.

Impact of Excipient Extraction and Buffer Exchange on Recombinant Monoclonal Antibody Stability.

The foundation of a biosimilar manufacturer's regulatory filing is the demonstration of analytical and functional similarity between the biosimilar product and the pertinent originator product. The excipients in the formulation may interfere with characterization using typical analytical and functional techniques during this biosimilarity exercise. Consequently, the producers of biosimilar products resort to buffer exchange to isolate the biotherapeutic protein from the drug product formulation. However, the impact that this isolation has on the product stability is not completely known. This study aims to elucidate the extent to which mAb isolation via ultrafiltration-diafiltration-based buffer exchange impacts mAb stability. It has been demonstrated that repeated extraction cycles do result in significant changes in higher-order structure (red-shift of 5.0 nm in fluorescence maxima of buffer exchanged samples) of the mAb and also an increase in formation of basic variants from 19.1 to 26.7% and from 32.3 to 36.9% in extracted innovator and biosimilar Tmab samples, respectively. It was also observed that under certain conditions of tertiary structure disruptions, Tmab could be restabilized depending on formulation composition. Thus, mAb isolation through extraction with buffer exchange impacts the product stability. Based on the observations reported in this paper, we recommend that biosimilar manufacturers take into consideration these effects of excipients on protein stability when performing biosimilarity assessments.

Understanding the Roles of Excipients in Moisture Management in Solid Dosage Forms.

Excipients are ubiquitous in pharmaceutical products, and often, they can also play a critical role in maintaining product quality. For a product containing a moisture-sensitive drug, moisture can be deleterious to the product stability during storage. Therefore, using excipients that interact with moisture in situ can potentially alleviate product stability issues. In this study, the interactive behavior of starch with moisture was augmented by coprocessing maize starch with sodium chloride (NaCl) or magnesium nitrate hexahydrate [Mg(NO3)2·6H2O] at different concentrations (5 and 10%, w/w). The effect of the formulation on drug stability was assessed through the degradation of acetylsalicylic acid, which was used as the model drug. The results showed that coprocessing of the starch with either NaCl or Mg(NO3)2·6H2O impacted the number of water molecule binding sites on the starch and how the sorbed moisture was distributed. The coprocessed excipients also resulted in lower drug degradation and lesser changes in tablet tensile strength during post-compaction storage. However, corresponding tablet formulations containing physical mixtures of starch and salts did not yield promising outcomes. This study demonstrated the advantageous concomitant use of common excipients by coprocessing to synergistically mitigate the adverse effects of moisture and promote product stability when formulating a moisture-sensitive drug. In addition, the findings could help to improve the understanding of moisture-excipient interactions and allow for the judicious choice of excipients when designing formulations containing moisture-sensitive drugs.

Comparative Analysis of Chemical Descriptors by Machine Learning Reveals Atomistic Insights into Solute-Lipid Interactions.

This study explores the research area of drug solubility in lipid excipients, an area persistently complex despite recent advancements in understanding and predicting solubility based on molecular structure. To this end, this research investigated novel descriptor sets, employing machine learning techniques to understand the determinants governing interactions between solutes and medium-chain triglycerides (MCTs). Quantitative structure-property relationships (QSPR) were constructed on an extended solubility data set comprising 182 experimental values of structurally diverse drug molecules, including both development and marketed drugs to extract meaningful property relationships. Four classes of molecular descriptors, ranging from traditional representations to complex geometrical descriptions, were assessed and compared in terms of their predictive accuracy and interpretability. These include two-dimensional (2D) and three-dimensional (3D) descriptors, Abraham solvation parameters, extended connectivity fingerprints (ECFPs), and the smooth overlap of atomic position (SOAP) descriptor. Through testing three distinct regularized regression algorithms alongside various preprocessing schemes, the SOAP descriptor enabled the construction of a superior performing model in terms of interpretability and accuracy. Its atom-centered characteristics allowed contributions to be estimated at the atomic level, thereby enabling the ranking of prevalent molecular motifs and their influence on drug solubility in MCTs. The performance on a separate test set demonstrated high predictive accuracy (RMSE = 0.50) for 2D and 3D, SOAP, and Abraham Solvation descriptors. The model trained on ECFP4 descriptors resulted in inferior predictive accuracy. Lastly, uncertainty estimations for each model were introduced to assess their applicability domains and provide information on where the models may extrapolate in chemical space and, thus, where more data may be necessary to refine a data-driven approach to predict solubility in MCTs. Overall, the presented approaches further enable computationally informed formulation development by introducing a novel in silico approach for rational drug development and prediction of dose loading in lipids.

Understanding Glucagon Aggregation: In Silico Insights and Experimental Validation.

Peptide aggregation poses a significant challenge in biopharmaceutical development and neurodegenerative diseases. This study combines computational simulations and experimental validation to uncover the underlying mechanisms and countermeasures for the aggregation of glucagon, a peptide with a high tendency to aggregate. In silico simulations demonstrate that lactose and 2-hydroxypropyl-ß-cyclodextrin (2-HPßCD) influence glucagon aggregation differently: lactose stabilizes glucagon by increasing the α-helical content, while 2-HPßCD disrupts protein-protein interactions. According to the simulations, 2-HPßCD is particularly effective at preserving the monomeric form of glucagon. Experimental validation with microfluidic modulation spectroscopy (MMS) confirms these findings, showing that glucagon in the presence of 2-HPßCD remains structurally stable, supporting the antiaggregation effect of this excipient. This research provides essential insights into glucagon aggregation obtained through a new powerful tool for monitoring the critical properties of peptide aggregation, suggesting new strategies for addressing this challenge in therapeutic peptide development.

Investigation of the Solid-State Interactions in Lyophilized Human G-CSF Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) previously elucidated the interactions between excipients and proteins for liquid granulocyte colony stimulating factor (G-CSF) formulations, confirming predictions made using computational structure docking. More recently, solid-state HDX mass spectrometry (ssHDX-MS) was developed for proteins in the lyophilized state. Deuterium uptake in ssHDX-MS has been shown for various proteins, including monoclonal antibodies, to be highly correlated with storage stability, as measured by protein aggregation and chemical degradation. As G-CSF is known to lose activity through aggregation upon lyophilization, we applied the ssHDX-MS method with peptide mapping to four different lyophilized formulations of G-CSF to compare the impact of three excipients on local structure and exchange dynamics. HDX at 22 °C was confirmed to correlate well with the monomer content remaining after lyophilization and storage at -20 °C, with sucrose providing the greatest protection, and then phenylalanine, mannitol, and no excipient leading to progressively less protection. Storage at 45 °C led to little difference in final monomer content among the formulations, and so there was no discernible relationship with total deuterium uptake on ssHDX. Incubation at 45 °C may have led to a structural conformation and/or aggregation mechanism no longer probed by HDX at 22 °C. Such a conformational change was observed previously at 37 °C for liquid-formulated G-CSF using NMR. Peptide mapping revealed that tolerance to lyophilization and -20 °C storage was linked to increased stability in the small helix, loop AB, helix C, and loop CD. LC-MS HDX and NMR had previously linked loop AB and loop CD to the formation of a native-like state (N*) prior to aggregation in liquid formulations, suggesting a similar structural basis for G-CSF aggregation in the liquid and solid states.

A Lipophilic Salt Form to Enhance the Lipid Solubility and Certain Biopharmaceutical Properties of Lapatinib.

Lapatinib (LTP) commercially available as lapatinib ditosylate (LTP-DTS) salt is the only drug approved for the treatment of HER-positive metastatic breast cancer. A low and pH-dependent solubility results in poor and variable oral bioavailability, thus driving significant interest in molecular modification and formulation strategies of the drug. Furthermore, due to very high crystallinity, LTP and LTP-DTS have low solubility in lipid excipients, making it difficult to be delivered by lipid-based carrier systems. Thus, the present work reports a new salt form of LTP with a docusate counterion to enhance the pharmaceutical properties of the drug (LTP-DOC). NMR spectra showed a downfield shift of the methylene singlet proton from 3.83 and 4.41 ppm, indicating a lowering of electron density on the adjacent nitrogen atom and confirming the formation of amine-sulfonyl salt through the specified basic nitrogen center located adjacent to the furan ring. PXRD diffractograms of LTP-DOC indicated a reduced crystallinity of the prepared salt. The dissolution, equilibrium solubility, lipid excipient solubility, partitioning coefficient, distribution coefficient, tabletability, and in vitro cytotoxicity of the lipophilic salt of LTP were investigated. The equilibrium solubility data showed that LTP-DOC possesses a pH-independent solubility profile in the pH range of 3.5 to 7.4 with a 3.14 times higher permeability coefficient than commercial ditosylate salt. Furthermore, the prepared LTP-DOC salts showed twice higher log P than the free base and 8 times higher than LTP-DTS. The prepared LTP-DOC was found to have 4- to 9-fold higher solubility in lipid excipients like Capmul MCM C8 and Maisine CC compared to the ditosylate salt. The LTP-DOC salt was tabletable and showed approximately 1.2 times lower dissolution than commercial ditosylate salt, indicating extended-release behavior. A cytotoxicity study of LTP-DOC salt showed an approximately 2.5 times lower IC50 value than the LTP-free base and 1.7 times lower than commercial ditosylate salt with an approximately 3 times higher selectivity index. The investigations strongly indicate a high translational potential of the prepared salt form in maintaining solubility-lipophilicity interplay, enhancing the drug's bioavailability, and developing lipidic formulations.

Colyophilized Sugar-Polymer Dispersions for Enhanced Processing and Storage Stability.

Sucrose and trehalose pharmaceutical excipients are employed to stabilize protein therapeutics in a dried state. The mechanism of therapeutic protein stabilization is dependent on the sugars being present in an amorphous solid-state. Colyophilization of sugars with high glass transition polymers, polyvinylpyrrolidone (PVP), and poly(vinylpyrrolidone vinyl acetate) (PVPVA), enhances amorphous sugar stability. This study investigates the stability of colyophilized sugar-polymer systems in the frozen solution state, dried state postlyophilization, and upon exposure to elevated humidity. Binary systems of sucrose or trehalose with PVP or PVPVA were lyophilized with sugar/polymer ratios ranging from 2:8 to 8:2. Frozen sugar-PVPVA solutions exhibited a higher glass transition temperature of the maximally freeze-concentrated amorphous phase (Tg') compared to sugar-PVP solutions, despite the glass transition temperature (Tg) of PVPVA being lower than PVP. Tg values of all colyophilized systems were in a similar temperature range irrespective of polymer type. Greater hydrogen bonding between sugars and PVP and the lower hygroscopicity of PVPVA influenced polymer antiplasticization effects and the plasticization effects of residual water. Plasticization due to water sorption was investigated in a dynamic vapor sorption humidity ramping experiment. Lyophilized sucrose systems exhibited increased amorphous stability compared to trehalose upon exposure to the humidity. Recrystallization of trehalose was observed and stabilized by polymer addition. Lower concentrations of PVP inhibited trehalose recrystallization compared to PVPVA. These stabilizing effects were attributed to the increased hydrogen bonding between trehalose and PVP compared to trehalose and PVPVA. Overall, the study demonstrated how differences in polymer hygroscopicity and hydrogen bonding with sugars influence the stability of colyophilized amorphous dispersions. These insights into excipient solid-state stability are relevant to the development of stabilized biopharmaceutical solid-state formulations.

Dual Functionality of Poloxamer 188 in Freeze-Dried Protein Formulations: A Stabilizer in Frozen Solutions and a Bulking Agent in Lyophiles.

Poloxamer 188 (P188) was hypothesized to be a dual functional excipient, (i) a stabilizer in frozen solution to prevent ice-surface-induced protein destabilization and (ii) a bulking agent to provide elegant lyophiles. Based on X-ray diffractometry and differential scanning calorimetry, sucrose, in a concentration-dependent manner, inhibited P188 crystallization during freeze-drying, while trehalose had no such effect. The recovery of lactate dehydrogenase (LDH), the model protein, was evaluated after reconstitution. While low LDH recovery (∼60%) was observed in the lyophiles prepared with P188, the addition of sugar improved the activity recovery to >85%. The secondary structure of LDH in the freeze-dried samples was assessed using infrared spectroscopy, and only moderate structural changes were observed in the lyophiles formulated with P188 and sugar. Thus, P188 can be a promising dual functional excipient in freeze-dried protein formulations. However, P188 alone does not function as a lyoprotectant and needs to be used in combination with a sugar.

Predicting the Stability of Formulations Containing Lyophilized Human Serum Albumin and Sucrose/Trehalose Using Solid-State NMR Spectroscopy.

Stabilization of proteins by disaccharides in lyophilized formulations depends on the interactions between the protein and the disaccharide (system homogeneity) and the sufficiently low mobility of the system. Human serum albumin (HSA) was lyophilized with disaccharides (sucrose and/or trehalose) in different relative concentrations. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy 1H T1 and 1H T1ρ relaxation times were measured to determine the homogeneity of the lyophilized systems on 20-50 and 1-3 nm domains, respectively, with 1H T1 relaxation times also being used to determine the ß-relaxation rate. HSA/sucrose systems had longer 1H T1 relaxation times and were slightly more stable than HSA/trehalose systems in almost all cases shown. HSA/sucrose/trehalose systems have 1H T1 relaxation times between the HSA/sucrose and HSA/trehalose systems and did not result in a more stable system compared with binary systems. Inhomogeneity was evident in a sample containing relative concentrations of 10% HSA and 90% trehalose, suggesting trehalose crystallization during lyophilization. Under these stability conditions and with these ssNMR acquisition parameters, a 1H T1 relaxation time below 1.5 s correlated with an unstable sample, regardless of the disaccharide(s) used.

Effects of Polymers on Cocrystal Growth in a Drug-Drug Coamorphous System: Relations between Glass-to-Crystal Growth and Surface-Enhanced Crystal Growth.

Coamorphous and cocrystal drug delivery systems provide attractive crystal engineering strategies for improving the solubilities, dissolution rates, and oral bioavailabilities of poorly water-soluble drugs. Polymeric additives have often been used to inhibit the unwanted crystallization of amorphous drugs. However, the transformation of a coamorphous phase to a cocrystal phase in the presence of polymers has not been fully elucidated. Herein, we investigated the effects of low concentrations of the polymeric excipients poly(ethylene oxide) (PEO) and poly(vinylpyrrolidone) (PVP) on the growth of carbamazepine-celecoxib (CBZ-CEL) cocrystals from the corresponding coamorphous phase. PEO accelerated the growth rate of the cocrystals by increasing the molecular mobility of the coamorphous system, while PVP had the opposite effect. The coamorphous CBZ-CEL system exhibited two anomalously fast crystal growth modes: glass-to-crystal (GC) growth in the bulk and accelerated crystal growth at the free surface. These two fast growth modes both disappeared after doping with PEO (1-3% w/w) but were retained in the presence of PVP, indicating a potential correlation between the two fast crystal growth modes. We propose that the different effects of PEO and PVP on the crystal growth modes arose from weaker effects of the polymers on cocrystallization at the surface than in the bulk. This work provides a deep understanding of the mechanisms by which polymers influence the cocrystallization kinetics of a multicomponent amorphous phase and highlights the importance of polymer selection in stabilizing coamorphous systems or preparing cocrystals via solid-based methods.

Protecting Lyophilized Escherichia coli Adenylate Kinase.

Drying protein-based drugs, usually via lyophilization, can facilitate storage at ambient temperature and improve accessibility but many proteins cannot withstand drying and must be formulated with protective additives called excipients. However, mechanisms of protection are poorly understood, precluding rational formulation design. To better understand dry proteins and their protection, we examine Escherichia coli adenylate kinase (AdK) lyophilized alone and with the additives trehalose, maltose, bovine serum albumin, cytosolic abundant heat soluble protein D, histidine, and arginine. We apply liquid-observed vapor exchange NMR to interrogate the residue-level structure in the presence and absence of additives. We pair these observations with differential scanning calorimetry data of lyophilized samples and AdK activity assays with and without heating. We show that the amino acids do not preserve the native structure as well as sugars or proteins and that after heating the most stable additives protect activity best.

Synthon Approach in Crystal Engineering to Modulate Physicochemical Properties in Organic Salts of Chlorpropamide.

The formulation of drug with improved bioavailability is always challenging and indispensable in the field of pharmaceutics. The control of intermolecular interactions via crystal engineering approach and solid-state molecular recognition results in the formation of active drug molecules with modulated pharmacological benefits. Therefore, with the aim to improve the solubility and dissolution rate of the drug chlorpropamide (CPA), the mechanochemical liquid-assisted grinding (LAG) of the drug with several pharmaceutically accepted excipients was performed. This contributed to the discovery of six novel solid phases, namely salts, salt cocrystals and salt cocrystal hydrate─the salt of CPA with 3, 4-diaminopyridine (DAP); salt and salt cocrystal (SC) polymorph (Z″=3) with 1, 4-diazabicyclo [2.2.2] octane (DABCO); a salt, SC polymorph (Z″=9), and a SC hydrate (Z″=9) with piperazine (PIP). The formation of these salts and salt cocrystals are mainly guided by the strong hydrogen bonds with tunable strength having high electrostatic contribution. This attractive interaction brings the donor and the acceptor atoms close to each other for a facile proton transfer. Furthermore, the conformational constraints on the drug molecules, provided by the excipients via strong and directional hydrogen bonds, are quite impressive as this leads to the identification and characterization of "new conformational isomers" for the CPA molecules. The new crystalline phases exhibit enhanced intrinsic dissolution rate in comparison to that of the pure drug, the magnitude being 7, 131, and 120 folds for CPADAP, CPADABCO_II, and CPAPIP_III, respectively. Furthermore, it is interesting to note that the order of solubility is enhanced by 2.7-, 3-, and 7-fold, respectively, for the abovementioned salts. This also mirrors the trends in the magnitude of the binding energy, the higher magnitude being reflected in the lower solubility. Additionally, the in vivo experiments performed in SD rats results in the enhancement of the magnitude of the pharmacokinetic properties, when compared to the pristine drug. The concentration of the drug in CPADABCO_II and CPAPIP_III formulations exhibits 6- and 4-fold increments, respectively. Indeed, these results corroborate to the trends observed in the structural characterization, intermolecular energy calculations, solubility, and in vitro dissolution assessments.