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.

Prediction of Human Hepatic Clearance for Cytochrome P450 Substrates via a New Culture Method Using the Collagen Vitrigel Membrane Chamber and Fresh Hepatocytes Isolated from Liver Humanized Mice.

In drug discovery, hepatocytes have been widely utilized as in vitro tools for predicting the in vivo hepatic clearance (CL) of drug candidates. However, conventional hepatocyte models do not always reproduce in vivo physiological function, and CYP activities in particular decrease quite rapidly during culture. Furthermore, conventional in vitro assays have limitations in their ability to predict hepatic CL of metabolically stable drug candidates. In order to accurately predict hepatic CL of candidate drugs, a new method of culturing hepatocytes that activates their functional properties, including CYP activities, is in high demand. In the previous study, we established a novel long-term culture method for PXB-cells® using a collagen vitrigel membrane (CVM) chamber, which can maintain CYP activity and liver specific functions at high levels for several weeks. In this study, the vitrigel culture method was applied to predictions of hepatic CL for 22 CYP typical substrates with low to middle CL, and the prediction accuracy by this method was assessed by comparing CL data between predicted (in vitro intrinsic CL using the dispersion model) and observed (in vivo clinical data) values. The results of this study showed that in vitro CL values for approximately 60% (13/22) and 80% (18/22) of the compounds were predicted within a 2- and 3-fold difference with in vivo CL, respectively. These results suggest that the new culture method using the CVM chamber and PXB-cells is a promising in vitro system for predicting human hepatic CL with high accuracy for CYP substrates, including metabolically stable drug candidates.

A long-term culture system based on a collagen vitrigel membrane chamber that supports liver-specific functions of hepatocytes isolated from mice with humanized livers.

During drug discovery, in vitro models are used to predict the in vivo pharmacokinetic and toxicological properties of drug candidates in humans. However, the conventional method of culturing human hepatocytes as monolayers does not necessarily replicate biologic reactions and does not support liver-specific functions, such as cytochrome P450 (CYP) activities, for prolonged periods. To remedy these problems and thus increase and prolong hepatic functions, we developed a culture system comprising a collagen vitrigel membrane (CVM) chamber and PXB-cells®, fresh hepatocytes isolated from liver-humanized chimeric mice (PXB-mice®). To quantitatively assess our new system, we evaluated the activities of 5 major CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A), albumin secretion, and urea synthesis. First, between Days 14 and 21, the activities of all CYP isoforms tested in vitrigel culture were equal to or higher than in conventional monolayer culture system. Second, the activities of CYP3A, CYP2C9, and CYP2C19 during Days 10 through 17 were higher in vitrigel culture than in suspended PXB-cells prepared on Day 0 (suspension assay). Third, albumin secretion and urea synthesis were higher in vitrigel culture than in conventional monolayer culture. Fourth, the vitrigel-cultured PXB-cells showed the characteristic morphology of parenchymal hepatocytes and were almost all alive in monolayer. These results indicate that our vitrigel culture method is superior to the conventional monolayer method in terms of diverse liver-specific functions, including CYP activity. Our findings suggest that the vitrigel culture method could be a powerful in vitro tool for predicting the pharmacokinetic and toxicological properties of drug candidates in humans.

Investigation of utility of cerebrospinal fluid drug concentration as a surrogate for interstitial fluid concentration using microdialysis coupled with cisternal cerebrospinal fluid sampling in wild-type and Mdr1a(-/-) rats.

In drug discovery, the cerebrospinal fluid (CSF) drug concentration (CCSF) has been used as a surrogate for the interstitial fluid (ISF) concentration (CISF). However, the CCSF-to-CISF gradient suggested for P-glycoprotein (P-gp) substrates in rodents causes uncertainty in CISF estimations and subsequent pharmacokinetic-pharmacodynamic analyses. To evaluate the utility of CCSF as a surrogate for CISF, this study directly compared the CCSF with the CISF of 12 compounds, including P-gp substrates, under steady-state conditions in wild-type and Mdr1a(-/-) rats using microdialysis coupled with cisternal CSF sampling. In wild-type rats, the ISF-to-unbound plasma (Kp,uu,ISF) and CSF-to-unbound plasma (Kp,uu,CSF) concentration ratios of the P-gp substrates, except for metoclopramide, were lower than those of the non-P-gp substrates, and the Kp,uu,CSF values were within or close to 3-fold of the Kp,uu,ISF values for all the compounds examined. The Kp,uu,CSF values of the selected P-gp substrates increased in Mdr1a(-/-) rats with a similar magnitude to the Kp,uu,ISF values, resulting in the Kp,uu,CSF-to-Kp,uu,ISF ratios being unchanged. These results suggested that P-gp-mediated active efflux at the blood-brain barrier is a major determinant not only for CISF, but also for CCSF, and that CCSF can be used as a surrogate for CISF even for P-gp substrates in rats.

Overexpression in yeast, photocycle, and in vitro structural change of an avian putative magnetoreceptor cryptochrome4.

Cryptochromes (CRYs) have been found in a wide variety of living organisms and can function as blue light photoreceptors, circadian clock molecules, or magnetoreceptors. Non-mammalian vertebrates have CRY4 in addition to the CRY1 and CRY2 circadian clock components. Though the function of CRY4 is not well understood, chicken CRY4 (cCRY4) may be a magnetoreceptor because of its high level of expression in the retina and light-dependent structural changes in retinal homogenates. To further characterize the photosensitive nature of cCRY4, we developed an expression system using budding yeast and purified cCRY4 at yields of submilligrams of protein per liter with binding of the flavin adenine dinucleotide (FAD) chromophore. Recombinant cCRY4 dissociated from anti-cCRY4 C1 mAb, which recognizes the C-terminal region of cCRY4, in a light-dependent manner and showed a light-dependent change in its trypsin digestion pattern, suggesting that cCRY4 changes its conformation with light irradiation in the absence of other retinal factors. Combinatorial analyses with UV-visible spectroscopy and immunoprecipitation revealed that there is chromophore reduction in the cCRY4 photocycle and formation of a flavosemiquinone radical intermediate that is likely accompanied by a conformational change in the carboxyl-terminal region. Thus, cCRY4 seems to be an intrinsically photosensitive and photoswitchable molecule and may exemplify a vertebrate model of cryptochrome with possible function as a photosensor and/or magnetoreceptor.

Light-dependent structural change of chicken retinal Cryptochrome4.

Animals have several classes of cryptochromes (CRYs), some of which function as core elements of circadian clockwork, circadian photoreceptors, and/or light-dependent magnetoreceptors. In addition to the circadian clock genes Cry1 and Cry2, nonmammalian vertebrates have the Cry4 gene, the molecular function of which remains unknown. Here we analyzed chicken CRY4 (cCRY4) expression in the retina with in situ hybridization and found that cCRY4 was likely transcribed in the visual pigment cells, cells in the inner nuclear layer, and retinal ganglion cells. We further developed several monoclonal antibodies to the carboxyl-terminal extension of cCRY4 and localized cCRY4 protein with immunohistochemistry. Consistent with the results of in situ hybridization, cCRY4 immunoreactivity was found in visual pigment cells and cells located at the inner nuclear layer and the retinal ganglion cell layer. Among the antibodies, one termed C1-mAb had its epitope within the carboxyl-terminal 14-amino acid sequence (QLTRDDADDPMEMK) and associated with cCRY4 in the retinal soluble fraction more strongly in the dark than under blue light conditions. Immunoprecipitation experiments under various light conditions indicated that cCRY4 from the immunocomplex formed in the dark dissociated from C1-mAb during blue light illumination as weak as 25 µW/cm(2) and that the release occurred with not only blue but also near UV light. These results suggest that cCRY4 reversibly changes its structure within the carboxyl-terminal region in a light-dependent manner and operates as a photoreceptor or magnetoreceptor with short wavelength sensitivity in the retina.