Understanding the Manufacturing Process of Lipid Nanoparticles for mRNA Delivery Using Machine Learning.

Lipid nanoparticles (LNPs), used for mRNA vaccines against severe acute respiratory syndrome coronavirus 2, protect mRNA and deliver it into cells, making them an essential delivery technology for RNA medicine. The LNPs manufacturing process consists of two steps, the upstream process of preparing LNPs and the downstream process of removing ethyl alcohol (EtOH) and exchanging buffers. Generally, a microfluidic device is used in the upstream process, and a dialysis membrane is used in the downstream process. However, there are many parameters in the upstream and downstream processes, and it is difficult to determine the effects of variations in the manufacturing parameters on the quality of the LNPs and establish a manufacturing process to obtain high-quality LNPs. This study focused on manufacturing mRNA-LNPs using a microfluidic device. Extreme gradient boosting (XGBoost), which is a machine learning technique, identified EtOH concentration (flow rate ratio), buffer pH, and total flow rate as the process parameters that significantly affected the particle size and encapsulation efficiency. Based on these results, we derived the manufacturing conditions for different particle sizes (approximately 80 and 200 nm) of LNPs using Bayesian optimization. In addition, the particle size of the LNPs significantly affected the protein expression level of mRNA in cells. The findings of this study are expected to provide useful information that will enable the rapid and efficient development of mRNA-LNPs manufacturing processes using microfluidic devices.

Design of easily swallowable xerogel pill with enough physical strength through hardening-process under heating and humidification.

The xerogel pill has been developed as a novel dosage form with dose-adjusting and swallow-assisting functions by using drop freeze-drying (DFD) technique. It was double-structured small sphere composed of an inner drug core and an outer dried-gel layer, however, had problem of insufficient physical strength. In this study, it was attempted to use dextrin (DEX), one of oligosaccharides, to strengthen the xerogel pill. DEX was co-dissolved in the dropping fluid in the DFD process and co-loaded in the conventional pill, which was mainly composed of mannitol (MNT) as a filler, to prepare the rigid body. DEX-loaded pill could be successfully prepared with high recovery (>90 %) by optimizing the ratio of DEX and MNT. Further, the representative pills with and without DEX (P-DEX and P-MNT, respectively) were hardening-processed under humidification. The physical strength of P-DEX pill was significantly increased when humidified under severe condition, resulting in enough hardness (>5N) and friability (<1.0 %). Processed P-DEX was found to have dense structure covered with a thick outer shell, which would be formed by interparticle bridge of DEX. It was also found that processed P-DEX pill suppressed initial drug dissolution significantly and exhibited sustained dissolution behavior, suggesting the potential function of bitter taste masking. Processed P-DEX pill had excellent sliding behavior with low friction forces as a result of lubricant effect of xanthan gum (XG) surrounding the pills. Furthermore, the sliding test also suggested that processed P-DEX pill had hard candy-like texture, in contrast unprocessed P-DEX pill had orally disintegrating (OD) tablet-like texture. Various xerogel pills with such different swallowing texture would have a potential to accommodate the children's preferences when taking medication.

Lymph node macrophages drive innate immune responses to enhance the anti-tumor efficacy of mRNA vaccines.

mRNA vaccines are promising for cancer treatment. Efficient delivery of mRNAs encoding tumor antigens to antigen-presenting cells (APCs) is critical to elicit anti-tumor immunity. Herein, we identified a novel lipid nanoparticle (LNP) formulation, L17-F05, for mRNA vaccines by screening 34 ionizable lipids and 28 LNP formulations using human primary APCs. Subcutaneous delivery of L17-F05 mRNA vaccine encoding Gp100 and Trp2 inhibited tumor growth and prolonged the survival of mice bearing B16F10 melanoma. L17-F05 efficiently delivered mRNAs to conventional dendritic cells (cDCs) and macrophages in draining lymph nodes (dLNs). cDCs functioned as the main APCs by presenting antigens along with enhanced expression of co-stimulatory molecules. Macrophages triggered innate immune responses centered on type-I interferon (IFN-I) in dLNs. Lymph node (LN) macrophage depletion attenuated APC maturation and anti-tumor activity of L17-F05 mRNA vaccines. Loss-of-function studies revealed that L17-F05 works as a self-adjuvant by activating the stimulator of interferon genes (STING) pathway in macrophages. Collectively, the self-adjuvanticity of L17-F05 triggered innate immune responses in LN macrophages via the STING-IFN-I pathway, contributing to APC maturation and potent anti-tumor activity of L17-F05 mRNA vaccines. Our findings provide strategies for further optimization of mRNA vaccines based on the innate immune response driven by LN macrophages.

Participation of medical students in the medical team of sports events.

Including medical student volunteers in the medical team for the triathlon strengthens the medical team structure, and encourages medical students to involve in sports medicine. Family physicians who play a role in the medical team for sports events can adopt medical students as a member and can educate them.

Lipid nanoparticle-targeted mRNA formulation as a treatment for ornithine-transcarbamylase deficiency model mice.

Ornithine transcarbamylase (OTC) plays a significant role in the urea cycle, a metabolic pathway functioning in the liver to detoxify ammonia. OTC deficiency (OTCD) is the most prevalent urea cycle disorder. Here, we show that intravenously delivered human OTC (hOTC) mRNA by lipid nanoparticles (LNP) was an effective treatment for OTCD by restoring the urea cycle. We observed a homotrimer conformation of hOTC proteins produced by the mRNA-LNP in cells by cryo-electron microscopy. The immunohistochemistry revealed the mitochondria localization of produced hOTC proteins in hepatocytes in mice. In livers of mice intravenously injected with hOTC-mRNA/LNP at 1.0 mg/kg, the delivered hOTC mRNA levels steeply decreased with a half-life (t1/2) of 7.1 h, whereas the produced hOTC protein levels retained for 5 days and then declined with a t1/2 of 2.2 days. In OTCD model mice (high-protein diet-fed Otcspf-ash hemizygous males), a single dose of hOTC-mRNA/LNP at 3.0 mg/kg ameliorated hyperammonemia and weight loss with prolonged survival rate (22 days) compared with that of untreated mice (11 days). Weekly repeated doses at 0.3 and 1.0 mg/kg were well tolerated in wild-type mice and showed a dose-dependent amelioration of survival rate in OTCD mice, thus, showing the therapeutic potential of LNP-formulated hOTC mRNA for OTCD.

[Homogeneity of Two Semisolid Formulations Using Simple In-tube Mixing Method].

To accelerate therapeutic effects, the mixtures of two or more topical pharmaceutical products having different medicinal purposes are often applied in the medical field. In this study, we aimed to develop a simple mixing method/procedure to achieve excellent homogeneity in the mixture of two topical products, a steroidal ointment and a skin moisturizer. To assess an in-tube mixing method as a simple mixing procedure, we injected both topical products into an empty resin tube, a flexible hollow tube with an open end that can be closed on one side, and a closed end on the other, removed as less air as possible inside the tube, and then thermocompressed (sealed) the open end to close it. The two topical products were then mixed uniformly by repeated finger pressure along the longitudinal axis of the tube. The homogeneity of the two topical products in the tube was evaluated by measuring the content of methyl paraoxybenzoate (MP), an additive loaded in the skin moisturizer. In addition, the mixability was qualitatively evaluated from the distribution of white petrolatum, another additive loaded in the steroid ointment, using Raman spectroscopy. As a result, the measured value of MP relative to the label claim was in the range of 100±12%, and the coefficients of variation value was also less than 12%. These results indicate that the in-tube mixing method using two topical products is approximately hologenetic preparations that do not cause therapeutic problems.

Preparation of sustained-release tablets using a solventless-mixing tablet coating technique: Particle design of dry ammonioalkyl methacrylate copolymer latex with high coating performance using sodium lauryl sulfate.

The aim of this study was to produce sustained-release tablets by V-shaped blending of polymer and tablets without using solvents or heating, and we investigated the design of polymer particles with high coating performance by modifying the structure of the particles using sodium lauryl sulfate. Dry-latex particles of ammonioalkyl methacrylate copolymer were prepared by adding the surfactant into aqueous latex, followed by freeze drying. The resulting dry latex was mixed with tablets (1:10) using a blender and the resulting coated tablets were characterized. Tablet coating by the dry latex was promoted as the weight ratio of surfactant to polymer increased. At a surfactant ratio of 5%, deposition of the dry latex was most effective and the resulting coated tablets (annealed at 60 °C/75%RH for 6 h) exhibited sustained-release characteristics over a period of 2 h. The addition of SLS prevented coagulation of colloidal polymer in the freeze drying, resulting in a loose-structured dry latex. This latex was easily pulverized by V-shaped blending with tablets and the resulting fine particles with high adhesiveness were deposited on the tablets. However, at a surfactant ratio of 10%, the coating of dry latex decreased due to reduced adhesiveness.

Solventless amorphization and pelletization using a high shear granulator. Part II; Preparation of co-amorphous mixture-layered pellets using indomethacin and arginine.

The aim of this study was to investigate the preparation of co-amorphous mixture-layered pellets using solventless pelletization and amorphization using a high shear granulator (as suggested in the first part of this study) by high shear mixing of drug crystals and a crystalline co-former with inactive spheres. Mixtures of crystalline indomethacin and arginine at various molar ratios were mixed with microcrystalline cellulose spheres at a weight ratio of 1:10 using the granulator and the resulting particles were characterized using solid-state and particle analytical techniques as well as dissolution testing and physical stability. At jacket temperatures of 20 °C or more of the granulator, co-processing of indomethacin and arginine enhanced amorphization of indomethacin and provided a co-amorphous mixture due to homogenous mixing of indomethacin and arginine amorphous phases. The co-amorphous mixture was deposited on the surface of the spheres, yielding co-amorphous mixture-layered pellets. The co-amorphous mixtures at molar ratios of indomethacin to arginine of 2:1 and 1:1, deposited on the pellets, did not recrystallize for at least 4 weeks. The pellets exhibited higher dissolution characteristics as additional hypromellose could prevent precipitation. These findings demonstrate the potential of this technique as a solventless approach to prepare co-amorphous mixture-layered pellets through a one-step process.

Solventless amorphization and pelletization using a high shear granulator. Part I; feasibility study using indomethacin.

The aim of the current study was to investigate the feasibility of solventless amorphization and pelletization using a high shear granulator, to produce amorphous drug-layered pellets by simply mixing drug crystals and inactive spheres without using solvent and heating. Indomethacin crystals were mixed with microcrystalline cellulose spheres at a weight ratio of 1:10 using the granulator and the resulting particles were then characterized using solid-state and particle analytical techniques as well as pharmaceutically relevant tests. Amorphization of indomethacin crystals progressed with increasing processing time and decreasing jacket temperature. The amorphization rate increased as the spheres became larger and full amorphization was achieved using spheres of 414 and 649 µm in diameter. Indomethacin crystals were pulverized due to mechanical activation by the spheres and the resulting amorphous microparticles were then deposited on the spheres, yielding pellets with an amorphous layer. The pellets exhibited supersaturation characteristics and the dissolution rate was faster than that of quench-cooled indomethacin powder. However, the amorphous drug deposited on the pellets exhibited a lower physical stability than quench-cooled amorphous indomethacin, but recrystallization could be inhibited by co-processing with polyvinylpyrrolidone K-25 stabilizing the amorphous form. The findings suggest the feasibility of the solventless amorphization and pelletization technique.

Design and lyophilization of lipid nanoparticles for mRNA vaccine and its robust immune response in mice and nonhuman primates.

mRNA and lipid nanoparticles have emerged as powerful systems for the preparation of vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The emergence of novel variants or the necessity of cold chain logistics for approved mRNA vaccines undermines the investigation of next-generation systems that could preserve both potency and stability. However, the correlation between lipid nanoparticle composition and activity is not fully explored. Here, we screened a panel of ionizable lipids in vivo and identified lead lipid nanoparticles with a branched-tail lipid structure. Buffer optimization allowed the determination of lyophilization conditions, where lipid nanoparticle-encapsulated mRNA encoding SARS-CoV-2 spike protein could induce robust immunogenicity in mice after 1 month of storage at 5°C and 25°C. Intramuscularly injected lipid nanoparticles distributed in conventional dendritic cells in mouse lymph nodes induced balanced T helper (Th) 1/Th2 responses against SARS-CoV-2 spike protein. In nonhuman primates, two doses of 10 or 100 µg of mRNA induced higher spike-specific binding geometric mean titers than those from a panel of SARS-CoV-2-convalescent human sera. Immunized sera broadly inhibited the viral entry receptor angiotensin-converting enzyme 2 (ACE2) from binding to the spike protein in all six strains tested, including variants of concern. These results could provide useful information for designing next-generation mRNA vaccines.

Design of xerogel pill with good swallowing performance through wet milling and drop freeze-drying processes.

A novel dosage form with dose-adjusting and swallow-assisting functions, named "xerogel pills," was developed for pediatric or geriatric patients. It is a multiple-unit dosage form in which a single dose is divided into several pills. The pills are double-structured small spheres with an inner drug core and an outer dried-gel layer (xerogel shell). In this research, the preparing method (formulation and process) of the xerogel pills was established by using a combination of wet-milling and drop freeze-drying (DFD) techniques. Fexofenadine hydrochloride (FXF) was used as a poorly water-soluble model drug. Xanthan gum (XG), a gelling agent, was formulated to attain a smooth and soft mouth-feeling. The internal fluid consisting of the FXF nanosuspension prepared through the wet-milling process and the external fluid consisting of an XG solution were separately fed to a two-fluids nozzle composed of central and peripheral nozzles. Both fluids were concentrically dropped together into liquid nitrogen to construct double-structured droplets. After the freeze-drying process, the xerogel pills were formed. Mannitol (MNT), as a filler, was co-formulated with both fluids to strengthen the pills physically. The resultant pills were around 5-6 mm in diameter with a spherical shape, uniform size, and low density, enabling them to be easily swallowed, and quickly gelled upon contact with water. The FXF amount in one pill was 7-9 mg, depending on the XG loading in the external fluid. The relative standard deviation (RSD) of the FXF amount were varied in the range of around 3-10%, suggesting that the content uniformity would be acceptable as a composite dosage unit containing five or more pills. It was assumed that further improvements of the physical strength and drug content uniformity would be required to introduce the xerogel pills to practical use.

Family physicians can contribute to Olympic and Paralympic Games or other sports events.

Many sports physicians are from primary care backgrounds around the world but not in Japan. However, from the view of family physicians who contributed to Tokyo 2020 Olympic and Paralympic Games as medical staff, family physicians in Japan can play an active role in sporting events.

Development of Clinical Skills and Confidence Questionnaire for Triage and Action Minor Emergency Course: Test-Retest Exam.

AIM: The Triage and Action (T&A) minor emergency course was developed to improve the clinical skills of Japanese non-specialist physicians for minor emergent problems. Currently, the course quality is evaluated only by a self-reported satisfaction questionnaire. This study described a new clinical skills and confidence questionnaire to evaluate its validity and reliability. METHODS: The web-based questionnaire was evaluated by 103 physicians identified from a mailing list as having taken the T&A minor emergency course. The clinical experience and confidence (CEC) questionnaire was prepared, and its content and contextual validity were validated using a clinical sensibility test (CST). Reliability was assessed by the interclass correlation coefficient after two weeks via a follow-up CEC questionnaire. RESULTS:  Of the 103 physicians contacted 44 (42.7%) responded to the questionnaire, 36 (40.8%) to the follow-up CEC questionnaire, and 33 (32.0%) to both questionnaires; 28 (27.2%) participants took the clinical sensibility test. Five questions which asked the total number of patients treated within six months showed fair agreement on the reliability test. All answers to the questions in the CST were favorable. CONCLUSION: We removed every question which asked the total number of patients treated for various minor emergencies within six months from CEC. Consequently, the new questionnaire was shown to be contextually well validated and reliable. We will use the CEC questionnaire to improve our course, which we hope to demonstrate improved primary care for selected minor conditions.

Preparation of Fine-Drugs Layered Spherical Particles with Good Micromeritic and Dissolution Properties through Ultra Cryo-Milling and Mechanical Powder Processing.

The particles of phenytoin (Phe), a poorly water-soluble model drug, were bead-milled alone or co-milled with a hydrophilic waxy additive using an ultra cryo-milling technique in liquid nitrogen (LN2) to improve its dissolution properties. However, the micronized drug particles adhered and aggregated, resulting in poor handling in manufacturing processes such as blending or tableting. To improve the dissolution profile and powder properties of the drug simultaneously, the milled products were secondarily processed together with larger spherical particles by mechanical powder processing. These secondary products were composite particles with a core-shell structure, with fine drug particles adhered and deposited on the core, based on order mixing theory. As a core, three types/sizes of spherical pharmaceutical excipient particles were applied. The resultant composite particles produced much faster release profiles than just milled or co-milled mixtures. In addition, the composite particles showed good micromeritic properties depending on the size of the core particles. These results indicate that the ultra cryo-milling and subsequent dry composite mixing is a potential approach for developing drug particles with improved dissolution.

Design of taste-masked swellable drug particles using dry-coating technology with mechanical curing.

A novel dry coating technique for fine particles that does not require any liquids has been developed. Swellable ordered-mixed drug particles (Swell-OM-spheres, SOS), using a modified starch as the core particle and a drug coating layer have been previously developed. In the present work, SOS particles were further processed to generate 100-µm taste-masking particles using an all dry coating processes. SOS particles were coated with a gastric-soluble powder using a mechanical powder processor. The coated particles (CPs) were subsequently heated while rotating in the same powder processor, completing film formation by a process termed dynamic curing. As a control, conventional film formation (static curing) was performed using a drying oven. The CPs obtained by these two curing processes had distinct appearances, but exhibited equivalent dissolution suppression effects in a medium at pH 6.8 (the pH of the oral cavity). The suppression effect was further improved by adding a plasticizer to the coating powder, even though a lower heating temperature was required. Orally disintegrating tablets incorporating these CPs exhibited excellent taste-masking performance, i.e., suppressing taste in saliva while accelerating dissolution in gastric juice. The dissolution behavior indicated that the CPs can provide an ON/OFF switching function in drug release.

Analysis of disease model iPSCs derived from patients with a novel Fanconi anemia-like IBMFS ADH5/ALDH2 deficiency.

We have recently discovered Japanese children with a novel Fanconi anemia-like inherited bone marrow failure syndrome (IBMFS). This disorder is likely caused by the loss of a catabolic system directed toward endogenous formaldehyde due to biallelic variants in ADH5 combined with a heterozygous ALDH2*2 dominant-negative allele (rs671), which is associated with alcohol-induced Asian flushing. Phytohemagglutinin-stimulated lymphocytes from these patients displayed highly increased numbers of spontaneous sister chromatid exchanges (SCEs), reflecting homologous recombination repair of formaldehyde damage. Here, we report that, in contrast, patient-derived fibroblasts showed normal levels of SCEs, suggesting that different cell types or conditions generate various amounts of formaldehyde. To obtain insights about endogenous formaldehyde production and how defects in ADH5/ALDH2 affect human hematopoiesis, we constructed disease model cell lines, including induced pluripotent stem cells (iPSCs). We found that ADH5 is the primary defense against formaldehyde, and ALDH2 provides a backup. DNA repair capacity in the ADH5/ALDH2-deficient cell lines can be overwhelmed by exogenous low-dose formaldehyde, as indicated by higher levels of DNA damage than in FANCD2-deficient cells. Although ADH5/ALDH2-deficient cell lines were healthy and showed stable growth, disease model iPSCs displayed drastically defective cell expansion when stimulated into hematopoietic differentiation in vitro, displaying increased levels of DNA damage. The expansion defect was partially reversed by treatment with a new small molecule termed C1, which is an agonist of ALDH2, thus identifying a potential therapeutic strategy for the patients. We propose that hematopoiesis or lymphocyte blastogenesis may entail formaldehyde generation that necessitates elimination by ADH5/ALDH2 enzymes.

PEG shedding-rate-dependent blood clearance of PEGylated lipid nanoparticles in mice: Faster PEG shedding attenuates anti-PEG IgM production.

PEGylation-modification with polyethylene glycol (PEG)-is useful for stabilizing lipid nanoparticles (LNPs). However, such PEGylation can prevent small interfering RNA (siRNA) encapsulated in LNPs from exerting its gene-silencing effects by disrupting the interaction of LNPs with target cells and by inducing the accelerated blood clearance phenomenon via anti-PEG IgM. PEG-lipids with short acyl chains can be used to address these issues because they are quickly shed from LNPs after administration; however, there are few reports on the relationships among PEG shedding rate, anti-PEG IgM production, and the gene-silencing activity of siRNA upon repeated LNP administration. Here, in mice, we found that LNPs conjugated to a fast-shedding PEG-lipid (short acyl chain) induced less anti-PEG IgM compared with LNPs conjugated to a slow-shedding PEG-lipid (long acyl chain). Moreover, pretreatment of mice with LNPs conjugated to the slow-shedding PEG-lipid caused loss of RNA interference activity after subsequent LNP administration because the payload siRNA was delivered primarily to Kupffer cells rather than to hepatocytes. Together, these findings imply that manipulating PEG shedding rate and anti-PEG antibody production is enormously important in the development of RNA interference-based therapeutics utilizing LNP technology.

Solventless granulation and spheronization of indomethacin crystals using a mechanical powder processor: Effects of mechanically induced amorphization on particle formation.

Our aim was to reveal the effects of mechanically-induced amorphization on the solventless agglomeration and spheronization of drug crystals using a mechanical powder processor. This process can provide spherical particles comprising 100% drug. Indomethacin crystals were mechanically treated using various jacket temperatures and the resulting particles were characterized using particle and crystalline analyses. Also, the adhesive and mechanical properties of amorphous indomethacin were examined. At 20 °C, the indomethacin crystals fragmented and amorphized during processing, indicating that glassy-state indomethacin with no adhesiveness does not contribute to agglomeration or spheronization. At 40 °C, agglomeration occurred due to the transformation of mechanically-induced amorphous phases from non-adhesive glass to an adhesive supercooled liquid at around the glass transition temperature. However, at higher temperatures, the formation of agglomerates was suppressed by recrystallization of the amorphous surface. At 60 °C, the indomethacin crystals compacted and spheronized due to deformation of the particle surface, consistent with results showing that the stiffness of amorphous indomethacin decreased suddenly above 60 °C. The lifespan of the amorphous phase decreased due to enhanced recrystallization as the temperature increased, thereby reducing the degree of spheronization. In conclusion, agglomeration and spheronization are affected by the glass transition temperature and recrystallization of the mechanically-induced amorphous phase.

[Research on Gliding and Discharge Performance of Suspended Injection from Syringe -Effect of Diameter Ratio of Suspending Particle against Needle Hole on Needle Passageability].

Suspended injectable formulations such as sustained-release luteinizing hormone-releasing hormone (LH-RH) analogue loaded in polylactic acid-glycolic acid copolymer (PLGA) particles have been developed on market. Such formulations have potential issue of suspended particles blocking the injection needle. In this research, two types of injectability tests (gliding force, particles discharge) were developed to evaluate the needle passageability of suspended particles. The model suspension was newly designed using mono-dispersed polyethylene (PE) spheres and qualified dispersing fluid to enhance universality and validity of the test. The suspension-filled syringe, in which three sizes of spheres (L, M, S) were dispersed, was vertically fixed and pushed by auto-compression/tensile tester. The gliding force was continuously detected during testing time and all discharged PE spheres were collected and weighed. The combination of sphere (L, M, S) and injection needle were varied to evaluate the effect of the diameter ratio of sphere against needle hole (D/W) on passageability through needle. These injectability tests revealed that the blockage of a needle hole was occasionally observed when the D/W value increased up to 0.35-0.5, which was detected by jump-up of gliding force and drastic decrease of discharged sphere. In addition, the effect of the formulation properties (concentration of suspended spheres, viscosity of dispersing fluid) and operational factor (injection speed) on injectability was also investigated. The results from this study would be valuable in developing suspended injections and predicting injection trouble at the medical scene.