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The ciliate Paramecium species are giant eukaryotic cells of 100-250 μm in length and have been used in various researches as model organisms. Successful cryopreservation with DMSO (dimethyl sulfoxide) had been reported. However, due to unstable and low survival rate after the thawing, these methods could not be put into practical use in long-term preservation programs for large numbers of strains. Therefore, majority of Paramecium researchers have prevented senescence by culturing paramecia at low temperatures (about 10℃) to suppress the rate of the cell division, because the lifespan of Paramecium is determined by the number of the cell divisions after fertilization. However, even with this method, clonal death due to aging cannot be avoided. In our research, conventional cryopreservation methods using DMSO have been improved and are beginning to be put to practical use. This improved method consistently resulted in higher post-thaw survival than other previous methods. This method currently yielded viable cells after thawing in P. caudatum, P. bursaria, and P. woodruffi, and further improvements are expected to increase the number of species that can be cryopreserved. [ABSTRACT FROM AUTHOR]

Quantitative NMR (qNMR), particularly 1 H-qNMR, is useful for determining the absolute purity of organic molecules. However, identifying the target signal(s) for quantification is difficult, because of the overlap and complexity of organic molecules. Therefore, we focused on the 31 P nucleus, owing to the simplicity of its signals, and investigated the 31 P-qNMR absolute determination method by using organophosphorus drugs, water-soluble cyclophosphamide hydrate (CP), and water-insoluble sofosbuvir (SOF). The optimized and reproducible 31 P-qNMR conditions, such as qNMR sample preparation [i.e., selecting suitable deuterated solvents and a reference standard (RS) for 31 P-qNMR], hygroscopicity and solution stability of the analyte and RS, and qNMR measurements-such as acquisition time, relaxation delay time, and spectral width-were examined. The CP purities determined using 31 P-qNMR agreed well with those for the established 1 H-qNMR method in D 2 O. In contrast, the SOF purity determined using 31 P-qNMR was 1.6% higher than that for 1 H-qNMR in the protic solvent CD 3 OD. Therefore, using a protic solvent, such as CD 3 OD, was not suitable for 31 P-qNMR; the deuterium exchange with the RS for 31 P-qNMR (i.e., phosphonoacetic acid) resulted in a small integrated intensity. Consequently, the aprotic solvent DMSO-d 6 was employed to determine the SOF purity. The data revealed that the SOF purities determined using 31 P-qNMR agreed well with the established 1 H-qNMR values, indicating that the absolute quantification of SOF using both 31 P-qNMR and 1 H-qNMR is possible in DMSO-d 6 . Thus, we established an optimized and reproducible 31 P-qNMR method in validation study across multiple laboratories.

Young's modulus measurements and junction length estimates have been performed on poly(vinyl alcohol) (PVA) sample in mixtures of dimethyl sulfoxide (DMSO) and water with various mixing ratios to investigate the relationship between gel structure and mechanical strength. The interaction parameter between PVA and DMSO/water mixed solvent was also estimated from swelling experiments. The junction length ζ in number of vinyl alcohol units was estimated with the aid of the Tanaka and Stockmayer theory (TS theory), which is based upon the conjecture that junction points of a gel are aggregates comprised of ζ units in length and S sequences in cross section. The Young's modulus value of PVA gel increased with an increase in the DMSO volume fraction up to about 60 vol% and the further increased fractions led to a decrease in Young's modulus. Both χ12 and ζ were also dependent on the DMSO volume fraction, similar to Young's modulus. These results indicate that gelation of PVA in a poorer solvent provided the longer junction lengths, resulting in the PVA gel formation with high mechanical strength. [ABSTRACT FROM AUTHOR]

The new linear polyurethanes were synthesized by the reaction of methyl(S)-2,6-diisocyanatohexanoate (LDI) with bis(2-hydroxyethyl) terephthalate (BHET) and polycaprolactone diols (PCC2000 or PCL2000). The polymers were obtained in relatively high yields (90-95%) and possessed high inherent viscosities ([η] = 0.41-0.52). The polymers were soluble in dimethyl sulfoxide and dimethyl formamide. The mechanical properties such as tensile strength, adhesion strength, and hardness were better than the standard values. We have examined the biodegradability of the polymers by enzymes. The copolymers were found to have good enzymatic degradation (max. 80%, 30 days). The SEM micrographs, showed countless holes and hollow structures on the film surface. [ABSTRACT FROM AUTHOR]

Dimethyl sulfoxide (DMSO) is used as a cryoprotectant for peripheral blood stem cells (PBSC) preservation. Dimethyl sulfide (DMS) is a metabolite of DMSO secreted through patients' breath after PBSC infusion. It possesses malodor causing an unpleasant environment. We evaluated the efficacy of a photocatalyst environment purifier, which has the potential to lyse toxic substances, in reducing DMS malodor. High DMS concentration in the air after PBSC infusion rapidly decreased after operating the device. Our results suggest that photocatalytic reaction has the potential to reduce the DMS odor associated with PBSC infusion.

In this study, we attempted to improve the non-aqueous titration method using N,N-dimethylformamide (DMF) in the Japanese Pharmacopoeia seventeenth edition (JP XVII) for advancement in experimental safety. As an alternative solvent for DMF, we demonstrate titrations using dimethyl sulfoxide (DMSO), which has similar properties as and much higher safety than DMF. Five drugs (i.e., acetohexamide, glibenclamide, sulfamethoxazole, tranilast, and furosemide) listed in JP XVII use DMF as a solvent for titrations with sodium hydroxide standard solution. For these drugs, we examined whether DMF can be replaced with DMSO in quantitative analyses. As a result, a quantification similar to that of the Pharmacopoeia protocol is possible by simply replacing DMF with DMSO or using a mixed solvent of DMSO and water.

The mechanism of complexation between risperidone (RISP) and (-)-epigallocatechin gallate (EGCg) was clarified by ¹H-NMR and molecular modeling studies. RISP and EGCg formed an insoluble complex with a 1 : 1 stoichiometry in aqueous solution. In the ¹H-NMR spectra of RISP in DMSO-d₆, the chemical shifts of protons neighboring the N atom on the piperizine ring clearly moved downfield upon formation of the complex. In the molecular modeling study, the ¹H-chemical shifts for nine optimized structures of the complex were calculated to compare them with those of the experimental results. Only one conformer with the second minimum energy for the complex supported the downfield shifts of RISP protons. It was found from the structure of the complex that the two hydrogen bonds between hydroxyl groups of the galloyl ring in EGCg and N atoms in RISP, one of which was on the piperizine ring, were formed to stabilize the complex.

We introduce a method for measuring drug-metabolizing enzymes and transporters using real-time one-step RT-PCR with TaqMan probes. This method has the advantages of high sensitivity, simplicity, and linearity of quantification over a wide range of mRNA concentrations, making it particularly suitable for evaluating large numbers of samples, as required in expression profile determinations. We also introduce the use of primary cultures of cryopreserved human and cynomolgus monkey hepatocytes as an enzyme induction model. Furthermore, we introduce the combination of real-time one-step RT-PCR and hepatocytes for evaluating the potency of various drugs in inducing drug-metabolizing enzymes and transporters. We discuss the usefulness of evaluating the gene induction of drug-metabolizing enzymes and transporters following exposure to drugs in human and cynomolgus monkey hepatocytes. The combination of real-time one-step RT-PCR and primary cultures of cryopreserved hepatocytes is useful for preclinical drug evaluation and for screening to evaluate the induction potency of new drug candidates.

The application of combinatorial chemistry and high-throughput screening to biological targets has led to efficient identification of lead compounds in wide therapeutic areas. However, the physicochemical properties of some lead compounds are lipophilic with low water soluble. Since these parameters determine in vivo absorption, we established robust screening methods for solubility and Caco-2 membrane permeability which are applicable to our screening strategy based on the structure-pharmacokinetic parameter relationship (SPR). Of test compounds with different core structures, turbidimetric solubility and apparent solubility as determined by HPLC-UV analysis after dilution of aqueous media from DMSO stock solution was overestimated in comparison with the corresponding thermodynamic solubility obtained using a traditional shake-flask method. A new powder-dissolution method providing thermodynamic solubility similar to that in the traditional method was developed using 96-well plates for equilibrium dialysis. The throughput of the method was the almost the same as that using the apparent solubility method. In a conventional Caco-2 assay, membrane permeability (P(app)) of some lipophilic compounds was underestimated due to low solubility in the apical site and adhesion to the device, resulting in a poor relationship between the in vivo absorption fraction and the P(app) values. The addition of 0.1% Gelucire 44/14 into the apical site and 4% bovine serum albumin into the basolateral site improved the relationship. These newly developed methods are therefore useful to optimize lead compounds with less water solubility and high lipophilicity on the basis of SPR.

Metabolic screening using liver microsomes of rats and humans is an indispensable tool to optimize a lead structure and to select compounds for in vivo study. Elucidating the relationship between in vitro intrinsic clearance (CL(int, app)) and in vivo clearance (CL(b)) is a prerequisite for screening. We investigated the relationship between CL(int, app) in rat liver microsomes and CL(b) after intravenous administration in rats in eight projects. No relationship between these two parameters was found across all of the projects examined. However, there was a certain relationship in the same core structure of six projects, but not in the other two projects. The poor correlation in the projects was improved by considering serum protein binding or microsomal binding in the estimation of in vitro clearances. Although the binding assay was labor intensive, unlike metabolic screening, the introduction of the equilibrium dialysis method using a 96-well format increased the throughput. Optimization of metabolic stability was conducted on the basis of the structure-metabolic stability relationship (SMR) in one of the projects, showing a good correlation without the binding factors. The replacement of the piperazine with a homopiperazine moiety improved metabolic stability in the rat and human liver microsomes. The compound also showed a desirable in vivo pharmacokinetic profile in rats, suggesting that the SMR study on the confirmed in vitro and in vivo correlation is essential to the optimization.

We report on a 61-year-old woman with chronic myeloid leukemia (CML) who developed a hemolytic uremic syndrome (HUS)-like episode following nonmyeloablative allogeneic hematopoietic stem cell transplantation. Macrohematuria, hypertension, hemolytic anemia with red cell fragmentation, thrombocytopenia, and progressive renal insufficiency were observed after thawed peripheral blood stem cell (PBSC) infusion. Although transient systemic hemolysis is known to occur during dimethylsulfoxide (DMSO)-cryopreserved stem cell infusion, HUS caused by DMSO has not been described in the literature. We speculate that one of the triggers of the HUS-like episode could have been renal microangiopathy caused by the long-term administration of interferon-alpha before the stem cell transplantation.

It is generally agreed that the autogenous bone flap which has been removed at the time of external decompression would be superior to any artificial material if it can be used in cranioplasty. Cranioplasty using autogenous frozen bone graft has been reported and showed good results except for infection and severe bone absorption. We conducted 39 cases of cranioplasty with cryopreserved autogenous bone in the presence of 10% of Dimethylsulfoxide (DMSO), which has been reported as a cryoprotective agent. Although no remarkable histological effects have been recognized in frozen bone with or without DMSO, we have obtained excellent clinical results without bone absorption in 24 of the cases. These results suggest that DMSO is helpful for frozen bone preservation.

We developed two methods for solubility screening of drug candidates in drug discovery. The first is a solution-precipitation (SP) method, in which the sample solutions are prepared by adding the drug solution in dimethylsulfoxide (DMSO) to buffers followed by filtering off the precipitate using 96-well filterplate. The second is a powder-dissolution (PD) method, in which the solid samples are dissolved to the buffer in the HPLC vial equipped with the filter membrane in the HPLC autosampler. An HPLC equipped with a photodiode array detector is used to measure the concentration of the sample solutions in both methods. The SP method was used for high throughput screening the solvating process of the candidates in aqueous solutions with lower sample consumption, and the PD method was used for screening both inter-molecular interaction in solid state and solvation in aqueous solution with more sample amount than that of SP method. Therefore, the solubility screening from early to final stage of lead optimization process would be successfully accomplished by using both methods complementarily.

Tissue engineering (TE) is a new discipline that offers the potential to create replacement structures from autologous cells and biodegradable polymer scaffold. Various vascular and valvular grafts have been tried to create with this TE approach. In clinical use of this technique, harvested and cultured cells have to keep viability until implantation as tissue engineered tissue. But few research for cryopreservation of vascular mixed cells has been performed. So, we investigated the proper method for cryopreservation of vascular mixed cells harvested from femoral artery and vein of dogs. Cells were cultured and divide into three groups, A: cryopreserving in 5% dimethylsulfoxide (DMSO), hydroxyethyl starch (HES), and fetal bovine serum (FBS) with -80 degrees C freezer; B: cryopreserving in 10% DMSO and FBS with programmed freezer; C: control (continuous culture in media). After rapid thawing at 40 degrees C, group A showed higher viability than group B with flow cytometry. The results means that vascular mixed cells can be successfully cryopreserved in the DMSO/HES mixture simply and inexpensively, without rate controlled freezing.

We have developed a method using a laser flow meter for investigating the effect of compounds on peripheral blood flow. The differences of blood flow in several portions and areas of the probe of a laser flow meter in the mouse and the effect of solvents for dissolving compounds on the blood flow were observed. The results indicated that the invisible area of blood vessels at the back or hind paw of the mouse was the most suitable placement position for the probe. In these placement positions, none of the tested solvents, 0.9% physiological saline solution, dimethylsulfoxide and 50% ethanol, had any significant effect on the peripheral blood flow. Next, using this method, we determined how the peripheral blood flow is affected by several drugs active on the circulatory system. By comparison between the present results from mice and previously reported data from other animals, it was shown that the effects of these cardiovascular agents on the peripheral blood flow in mouse skin showed similar tendencies to those in the previous reports using larger animals and sites other than the skin. However, a few of the drugs used in this experiment showed characteristic action on the peripheral blood flow in mice. Consequently, it was demonstrated that this method using the laser flow meter was useful as a simple first screening method to evaluate the effect of a compound on the peripheral blood flow.