A novel mutation in the RHD gene in Japanese individuals with weak D, encoding an amino acid change in the 11th transmembranous domain of the RhD protein.
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HLA-D Antigens↗
Biomedical subjects
F Hirayama
Publications and source records attributed to F Hirayama.
The effect of postoperative ataralgesia by manual therapy after pulmonary resection.
Muscle therapy, a form of manual therapy, was applied to control pain persisting for more than 1 week following posterolateral thoracotomy, and its efficacy for the alleviation of pain was investigated. Eight patients who underwent posterolateral thoracotomy and lung resection for cancer (n=7) or emphysema (n=1) received manual therapy to incised muscles and the muscles inserting into the ribs in the affected area for an average of 17 days postoperatively. Pressure-friction and stretching techniques were used. Treatment was continued until the intensity of the pressure-friction technique reached a level at which the patient complained of pain and a decrease in muscle tone was detected. Treatment was performed once a week for 3 weeks. Pain severity was measured using a visual analog scale (VAS) (0-10). Before the first treatment, the VAS was set at 10, and changes of the score were observed before and after the treatment as well as over time. After three sessions, all patients showed a decrease in pain from 10 to an average of 1.9 (range 1.3-2.6).
Adult↗
Protective effect of sulfobutyl ether beta-cyclodextrin on DY-9760e-induced hemolysis in vitro.
The hemolytic behavior of a novel cytoprotective agent, DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate) was investigated using rabbit erythrocytes. Further, the effects of water-soluble cyclodextrin derivatives, such as 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) and sulfobutyl ether of beta-cyclodextrin (SBE-beta-CyD), on the hemolytic activity of DY-9760e were studied. DY-9760e induced hemolysis at concentrations >0.2-0.3 mM in phosphate buffered saline (PBS) of pH 4.0 and 6.0, where DY-9760e is predominantly in dicationic and monocationic forms, respectively. The hemolytic activity of the monocationic DY-9760e was higher than that of the dicationic species, and the hemolysis at pH 4.0 involved the formation of methemoglobin. DY9760e induced the morphological change of erythrocytes towards membrane invagination at both pH 4.0 and 6.0. SBE7-beta-CyD significantly suppressed the DY-9760e-induced hemolysis and morphological change at both pH 4.0 and 6.0, as well as the formation of methemoglobin at pH 4.0. On the other hand, HP-beta-CyD suppressed only the hemolysis, but neither the morphological change nor the formation of methemoglobin. In addition, the inhibitory effect of SBE7-beta-CyD on the hemolysis was greater than that of HP-beta-CyD. The superior inhibitory effect of SBE7-beta-CyD on the DY-9760-induced hemolysis, the morphological change, and the formation of methemoglobin may be attributable to the formation of a stable inclusion complex with DY-9760e and to the weaker hemolytic activity of SBE7beta-CyD than HP-beta-CyD. These results suggest potential use of SBE7-beta-CyD as a parenteral carrier for DY-9760e.
Animals↗
Ex vivo expansion of human UC blood primitive hematopoietic progenitors and transplantable stem cells using human primary BM stromal cells and human AB serum.
BACKGROUND: In vitro maintenance and expansion of human hematopoietic stem cells is crucial for many clinical applications, and investigators have been using xenogeneic, especially murine, stromal cells for stem-cell expansion. In addition, many such culture systems utilize FCS-containing medium or serum-free medium that contains human- or animal-derived proteins. However, the possible transmission of infectious diseases has led to a debate about the safety of the delivery of grafts expanded in culture using cells and proteins of allogeneic or xenogeneic origin. Using primary human BM stromal cells, we have established an AB serum-based co-culture system to expand human primitive progenitors and transplantable stem cells. METHODS: Cord blood CD34+ cells were cultured on a monolayer of human BM-derived primary stromal cells with thrombopoietin (TPO), stem-cell factor (SCF) and flt3/flk2 ligand (FL) in the presence of either FCS or AB serum. One to three weeks later, cells were examined for total cells, CD34+ cells, CD34+ CD38- cells, and clonogenic progenitors. SCID mouse reconstituting cell (SRC) activity was also studied. RESULTS: Three weeks of culture with TPO, SCF, and FL supported more than a 250-fold expansion of CD34+ cells, CD34+ CD38- cells and CFU-C, regardless of the kind of serum used. SRC assay revealed that transplantable stem cells were moderately expanded as well. DISCUSSION: This ex vivo expansion system should prove valuable in clinical settings in which stromal cells and serum are available from recipients or stem-cell donors.
ADP-ribosyl Cyclase↗
Helicobacter pylori infection potentiates aspirin induced gastric mucosal injury in Mongolian gerbils.
BACKGROUND: Helicobacter pylori infection and non-steroidal anti-inflammatory drugs are two major causes of gastric ulceration but interactions between H pylori and these drugs in gastric mucosal injury are unclear. AIMS: We studied the influence of experimental H pylori infection on gastric mucosal injury induced by aspirin. SUBJECTS: Male Mongolian gerbils free of specific pathogens were used. METHODS: H pylori ATCC43504 culture broth was administered by oral gavage at seven weeks of age. After three weeks, acidified aspirin (400 mg/kg) was administered orally, and three hours later the total area of gastric erosions, myeloperoxidase (MPO) activity (an index of neutrophil accumulation), thiobarbituric acid reactive substances (TBARS, an index of lipid peroxidation), and KC/GRO (a chemoattractive cytokine in rodents) were measured in gastric mucosa. To determine the role of neutrophils in these circumstances, antigerbil neutrophil rabbit serum (ANS) was administered to some animals 18 hours before aspirin. RESULTS: Aspirin caused more extensive haemorrhagic erosions (33.1 (12.3) mm2) associated with greater MPO activity (1887.7 (598.5) microU/mg protein) and TBARS (0.33 (0.14) nmol/mg protein) and KC/GRO concentrations (28.3 (9.5) pg/mg protein) in infected than in uninfected gerbils (13.7 (2.3); 204.0 (68.9); 0.12 (0.06); 3.1 (0.8), respectively) Pretreatment with ANS inhibited the increases in gastric erosions, MPO activity, and TBARS but not KC/GRO concentration. The reduction in aspirin induced mucosal injury by administration of ANS was much greater in H pylori infected animals (65%) than in uninfected animals (31%). CONCLUSIONS: H pylori infection potentiates aspirin induced gastric mucosal injury by mechanisms that include accumulation of activated neutrophils.
Animals↗
Phagocytosis in vitro of polyethylene glycol-modified liposome-encapsulated hemoglobin by human peripheral blood monocytes plus macrophages through scavenger receptors.
Liposome-encapsulated hemoglobin (LEH), a candidate for red blood cell substitute, is cleared from circulation primarily by the phagocytic system, most likely after opsonization of the vesicles by immunoproteins, particularly complement components. Although modification of LEH by polyethylene glycol (PEG) derivatives prolongs its half-life by blocking the opsonization, the half-life is still short as compared with that of red blood cell components. Therefore, this study was performed to elucidate the opsonin-independent mechanisms that regulate phagocytosis of Neo Red Cell (NRC), a PEG-modified LEH, in culture. PKH67 was used as a fluorescence marker, allowing the quantitation of the phagocytosis of NRC by peripheral blood monocytes plus macrophages. The phagocytosis of PKH67-labeled NRC was inhibited by the addition of an excess of unlabeled NRC, indicating that the phagocytosis of PKH67-labeled NRC is specific to NRC, but not to PKH67. The phagocytosis of NRC was blocked about 70% by anti-CD14, 60% by anti-CD36 and 30% by anti-CD51/61 (vitronectin receptor, alpha(v)beta3). These results provided evidence of an opsonin-independent pathway for the phagocytosis of PEG-modified LEH.
Antigens, CD↗
Effect of peracylation of beta-cyclodextrin on the molecular structure and on the formation of inclusion complexes: an X-ray study.
The molecular structures of peracylated beta-cyclodextrins (CDs)--heptakis(2,3,6-tri-O-acetyl)-beta-CD (TA), heptakis(2,3,6-tri-O-propanoyl)-beta-CD (TP), and heptakis(2,3,6-tri-O-butanoyl)-beta-CD (TB)--have been determined by single crystal X-ray structure analysis. Due to the lack of O2...O3' hydrogen bonds between adjacent glucose units of the peracylated CDs, the macrocycles are elliptically distorted into nonplanar boat-shaped structures. The glucose units are tilted with respect to the O4 plane to relieve steric hindrance between adjacent acyl chains. In TB, all glucose units adopt the common (4)C(1)-chair conformation and one butanoyl chain intramolecularly penetrates the cavity, whereas, in TA and TP, one glucose unit each occurs in (O)S(2)-skew-boat conformation and one acyl chain closes the O6 side like a lid. In each of the three homologous molecules the intramolecular self-inclusion and lidlike orientation of acyl chains forces the associated O5-C5-C6-O6 torsion angle into a trans-conformation never observed before for unsubstituted CD; the inclusion behavior of TA, TP, and TB in solution has been studied by circular dichroism spectroscopy with the drug molsidomine and several organic compounds. No inclusion complexes are formed, which is attributed to the intramolecular closure of the molecular cavity by one of the acyl chains.
Acetylation↗
Improvement of some pharmaceutical properties of DY-9760e by sulfobutyl ether beta-cyclodextrin.
The interaction of DY-9760e, a novel cytoprotective agent, with sulfobutyl ether beta-cyclodextrin (SBE-beta-CyD) in phosphate buffered saline (PBS) at various pH and ionic-strengths was studied by spectroscopic methods and the solubility method, and the results were compared with that of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD). The circular dichroism (CD) spectroscopic studies suggested that both beta-CyDs form the inclusion complexes with DY-9760e in a molar ratio of 1:1, and the interaction of DY-9760e with SBE-beta-CyD is much stronger than that with HP-beta-CyD at any pH studied, in terms of a synergetic effect of hydrophobic and electrostatic interactions. The different intermolecular interaction between the SBE-and HP-beta-CyD complexes was clearly reflected in the stability constant (K'), e.g. the different dependence of K' value on pH and ionic strength of solutions. 1H- and 13C-NMR studies suggested that HP-beta-CyD interacts preferably with the benzene ring of DY-9760e, whereas SBE-beta-CyD interacts not only with the benzene ring via hydrophobic interaction but also with the piperazine ring of the drug via electrostatic interaction. The solubilizing ability of SBE-beta-CyD against DY-9760e was much greater than that of HP-beta-CyD at any pH studied. Furthermore, SBE-beta-CyD markedly suppressed the photo-degradation of DY-9760e in aqueous solution and reduced the adsorption of DY-9760e from PBS to polyvinyl chloride (PVC) tubes after incubation. The results suggest that SBE-beta-CyD is useful in preparing parenteral solutions of poorly water-soluble drugs with positive charge such as DY-9760e.
Adsorption↗
Liposome-encapsulated superoxide dismutase suppresses liposome-mediated augmentation of TNF-alpha production from peripheral blood leucocytes.
Liposome-encapsulated hemoglobin (LEH), a candidate for a red cell substitute, has been reported to be cleared from circulation primarily by the phagocytic system and modulate the production of inflammatory cytokines, such as TNF-alpha and IL-6, both in vivo and in vitro. In this study, we investigated the effects of liposome vesicles on the LPS-induced TNF-alpha production using a whole blood culture system. We also studied the effects of superoxide dismutase (SOD) encapsulated in liposome on the cytokine production. The pre-treatment of whole blood with liposome vesicles potentiated the LPS-induced TNF-alpha production. The encapsulation of SOD in the liposome vesicles suppressed the liposome-mediated augmentation of TNF-alpha production in a dose-dependent manner. These results suggest that encapsulation of SOD in LEH decreases the production of inflammatory cytokines from the phagocytic system which may be caused or augmented by LEH infusion in vivo.
Drug Carriers↗
Preparation of prednisolone-appended alpha-, beta- and gamma-cyclodextrins: substitution at secondary hydroxyl groups and in vitro hydrolysis behavior.
The carboxyl group of prednisolone 21-hemisuccinate was conjugated to one of the hydroxyl groups of alpha-, beta-, and gamma-cyclodextrins using a coupling agent, carbonyldiimidazole. The direct coupling produced prednisolone-appended cyclodextrin conjugates in which the drug is selectively introduced at one of the secondary hydroxyl groups of cyclodextrins through an ester linkage. The aqueous solubility (> 50% w/v at 25 degrees C) of these conjugates was much higher than those of prednisolone and its 21-hemisuccinate. Prednisolone was slowly released from the conjugate: the percents of prednisolone and its hemisuccinates released from the alpha-, beta-, and gamma-cyclodextrin conjugates were 49, 57, and 85%, respectively, for 24-h. The release pathway is proposed to be via two fast acyl migrations between the 2- and 3-hydroxyl groups of cyclodextrins and between the 21- and 17-hydroxyl groups of prednisolone. The slow release of prednisolone from the ester conjugates was in sharp contrast to the fast release of the prednisolone amide conjugate reported previously. Because of relatively slow and/or site-specific release properties, the present prednisolone-appended cyclodextrin conjugates may be of value as an orally administered delayed-release and/or colon-specific prodrug.
Cyclodextrins↗
Comparative studies of the enhancing effects of cyclodextrins on the solubility and oral bioavailability of tacrolimus in rats.
The enhancing effects of cyclodextrins (CyDs) on the solubility, the dissolution rate, and the bioavailability of tacrolimus after oral administration to rats were examined and compared with those after administration of a PROGRAF capsule containing the solid dispersion formulation of tacrolimus. Here we used natural CyDs and the hydrophilic beta-CyD derivatives; that is, randomly methylated-beta-cyclodextrin (RM-beta-CyD), heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CyD), 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD), and sulfobutyl ether beta-cyclodextrins (SBE-beta-CyDs). Of the natural CyDs, the solubility of tacrolimus increased in the addition of beta-CyD, indicating that the cavity of beta-CyD comfortably fits the drug. Of the beta-CyD derivatives, DM-beta-CyD had the greatest solubilizing activity and gave the A(p) type phase solubility curve as defined by Higuchi and Connors, suggesting the formation of higher-order complexes. The result of van't Hoff plot suggests that the enthalpy is dominant for the complexation of tacrolimus with DM-beta-CyD. The dissolution rate of tacrolimus was markedly augmented by the complexation with DM-beta-CyD, reflecting its solubilizing activity. An in vivo study revealed that DM-beta-CyD increased the bioavailability of tacrolimus with low variability in the absorption after oral administration of the tacrolimus suspension to rats. The present results suggest that DM-beta-CyD is particularly useful in designing oral preparations of tacrolimus with an enhanced bioavailability and a reduced variability in absorption.
Administration, Oral↗
Prednisolone-appended alpha-cyclodextrin: alleviation of systemic adverse effect of prednisolone after intracolonic administration in 2,4,6-trinitrobenzenesulfonic acid-induced colitis rats.
The titled compound is a cyclodextrin derivative in which prednisolone 21-succinate (PDsuc) is covalently bound to one of the secondary hydroxyl groups of alpha-cyclodextrin (alpha-CyD) via an ester linkage. In this study, the PDsuc-appended alpha-CyD ester conjugate (PDsuc/alpha-CyD conjugate) was intracolonically administered to rats with 2,4,6-trinitrobenzensulfonic acid-induced colitis, and its antiinflammatory and systemic adverse effects were compared with those of prednisolone (PD) alone and the PD/2-hydroxypropyl-beta-CyD complex (PD/HP-beta-CyD complex), which is a noncovalent inclusion complex. Colonic damage score, ratio of distal colon wet weight to body weight, and myeloperoxidase activity were evaluated as measures of the therapeutic effect of PD, whereas the ratio of thymus wet weight to body weight was evaluated as a measure of the side effect of PD. The local antiinflammatory activity increased in the order of PD alone approximately PDsuc/alpha-CyD conjugate < PD/HP-beta-CyD complex. As to systemic adverse effect, the PD/HP-beta-CyD complex and PD alone caused thymolysis at doses of 5-10 mg/kg. In contrast, the PDsuc/alpha-CyD conjugate showed no clear systemic adverse effect at the same doses. The low adverse effect of the conjugate may be ascribed to the slow release of PD in the colon, which keeps the local concentration in the colon at a low but constant level. The results suggest that the PDsuc/alpha-CyD conjugate can alleviate the systemic adverse effect of PD while maintaining the therapeutic activity of PD. This kind of knowledge will be useful in the rational design of steroid prodrugs for the colon-specific drug delivery system.
Administration, Rectal↗
Serum-free coculture system for ex vivo expansion of human cord blood primitive progenitors and SCID mouse-reconstituting cells using human bone marrow primary stromal cells.
OBJECTIVE: In an attempt to maintain and expand human stem cells, many investigators have used xenogeneic, especially murine, stromal cells and fetal calf serum. Because of the possible transmission of infectious diseases, however, the safety of the delivery of grafts expanded in culture using xenogeneic cells and serum has been debated. Using primary human marrow stromal cells, we established a novel serum-free culture system to expand human primitive progenitors and transplantable stem cells. MATERIALS AND METHODS: Cord blood CD34(+) cells were cultured on a monolayer of human primary marrow stromal cells in the presence of thrombopoietin (TPO), flt3/flk2 ligand (FL), and/or stem cell factor (SCF) under serum-free conditions. After 2 or 4 weeks of culture, cells were examined for clonogenic progenitors and severe combined immunodeficient disorder (SCID) mouse-reconstituting cells (SRC). RESULTS: In the presence of TPO, FL, and SCF, marrow stromal cells supported more than a 100- and 1,000-fold expansion of CD34(+) cells and colony-forming units in culture after 2 and 4 weeks of incubation, respectively. In addition, cobblestone area-forming cells were expanded more than 18- and 60-fold after 2 and 4 weeks of culture, respectively. Furthermore, SRC assay demonstrated augmented engraftment by cultured cells. CONCLUSION: This ex vivo expansion system should prove valuable in clinical settings in which stromal cells are available from recipients or stem cell donors.
Acute Disease↗
RHC/c genotyping based on polymorphism in the promoter region of the RHCE gene.
Designing of PCR tests for the RHC allele is difficult because of the high DNA sequence homology between RHC and RHD genes, which differ by only a one-nucleotide substitution at position 48 in exon 1 of the RHCE gene. We sequenced the promoter region of the RHCE gene, and compared our results with the reported sequence. Genomic DNA was prepared from blood samples collected from 656 Japanese donors. The DNA segment encompassing the promoter region and exon 1 of the RHCE gene from 30 donors was amplified by PCR and analyzed by DNA sequencing. Four nucleotide differences between RHC/c and RHD were found at positions -468, -304, -58, and -46. On the basis of the nucleotide differences at positions -468 (RHCE vs. RHD) and -292 (RHC vs. RHc), we then developed a novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for RHC/c genotyping. Analysis of the genomic DNA from the 656 donors revealed that this method could discriminate RHC from RHc, irrespective of the RHD genotype, with only a few exceptions. The combination of our system and the intron 2-based PCR-RFLP method previously reported may prove to be more accurate than either of the methods alone, and therefore, useful and valuable for RHC/c genotyping.
Journal Article↗
Inhibitory effects of novel hydrophilic cyclodextrin derivatives on nitric oxide production in macrophages stimulated with lipopolysaccharide.
PURPOSE: The objective of this study is to examine the effects of cyclodextrins (CyDs) on nitric oxide (NO) production in macrophages stimulated with lipopolysaccharide (LPS). METHODS: RAW264.7 cells, a mouse macrophage-like cell, were used. Cytotoxicity of CyDs was evaluated by WST-1 method. Nitrite, iNOS, and iNOS mRNA were determined by Griess method, Western blotting, and reverse transcription-polymerase chain reaction (RT-PCR) analysis, respectively. The interaction of LPS with CyDs was evaluated by utilizing a competitive inclusion phenomenon. The binding of FITC-labeled LPS to the surface of RAW264.7 cells was measured by a flow cytometry. RESULTS: Of 15 CyDs, 2,6-di-O-methyl-alpha-CyD (DM-alpha-CyD), and 2,6-di-O-methyl-3-O-acetyl-beta-cyclodextrin (DMA-beta-CyD) had greater inhibitory activity than did the other CyDs against NO production in RAW264.7 cells stimulated with LPS, without showing any cytotoxicity. DM-alpha-CyD and DMA-beta-CyD specifically inhibited the increase in iNOS and iNOS mRNA levels elicited by stimulation with LPS in RAW264.7 cells. DM-alpha-CyD and DMA-beta-CyD suppressed the binding of FITC-labeled LPS to the surface of cells, probably resulting in inhibitory effects on iNOS expression and NO production. DM-alpha-CyD had a greater interaction with RAW264.7 cells than did DMA-beta-CyD. The pretreatment of RAW264.7 cells with DM-alpha-CyD, not DMA-beta-CyD, decreased the LPS binding to the cell surface. The results suggested that the inhibitory mechanism of the LPS binding to the cell surface is different between DM-alpha-CyD and DMA-beta-CyD. CONCLUSIONS: The present results suggest that DM-alpha-CyD and DMAbeta-CyD attenuates NO production by inhibiting iNOS gene expression in RAW264.7 cells stimulated with LPS, probably due to the suppression of LPS binding to LPS receptors on the cells in the different way.
Blotting, Western↗
Stabilizing and solubilizing effects of sulfobutyl ether beta-cyclodextrin on prostaglandin E1 analogue.
PURPOSE: Parent cyclodextrins are known to accelerate the degradations such as dehydration and isomerization of E-type prostaglandins in neutral and alkaline solutions. The objective of this study was to attempt the stabilization and solubilization of E1-type prostaglandin analogue in aqueous solution by biocompatible cyclodextrin derivatives. METHODS: The interaction of an E1-type prostaglandin, methyl 7-[(1R,2R,3R)-3-hydroxy-2-[(E)-(3S)-3-hydroxy-4-(m-methoxymethylphenyl)1-butenyl]-5-oxocyclopentyl]-5-thiaheptanoate (MEester) with cyclodextrins (CyDs) was studied by spectroscopies and the solubility method. The degradation of MEester was monitored by high-performance liquid chromatography. RESULTS: 1H-nuclear magnetic resonance spectroscopic studies indicated that MEester forms 1:1 inclusion complexes with alpha-, beta-, and gamma-CyDs in solutions, where alpha-CyD interacts with the a-side chain containing methyl ester moiety of the drug, whereas beta- and gamma-CyDs preferentially include around the five-membered ring and both side chains of the drug. Parent alpha-CyD and hydrophilic derivatives, such as 2-hydoxypropyl-alpha- and -beta-CyDs, sulfobutyl ether beta-CyD (SBE-beta-CyD) and maltosyl beta-CyD showed higher solubilizing abilities against MEester over parent beta- and gamma-CyDs. SBE-beta-CyD and 2,6-dimethyl-beta-CyD (DM-beta-CyD) significantly decelerated the degradation of MEester, particularly the base-catalyzed dehydration, in neutral and alkaline solutions, whereas other CyDs accelerated the degradation. The acid-catalyzed degradation of MEester (pH < 3) was decelerated by the addition of CyDs, especially alpha-CyD. CONCLUSIONS: SBE-beta-CyD with low hemolytic activity and low toxicity is useful as a pharmaceutical carrier for the preparation of injectable MEester, because of its higher stabilizing and solubilizing effects on MEester. Furthermore, SBE-beta-CyD can be useful as a stabilizing agent for drugs, that are subject to base-catalyzed degradations, probably because of the electric repulsion between anionic charges of the sulfobutyl moiety and catalytic anionic species such as hydroxide ion.
Alprostadil↗
A moderate interaction of maltosyl-alpha-cyclodextrin with Caco-2 cells in comparison with the parent cyclodextrin.
The cytotoxicity of maltosyl-alpha-cyclodextrin (G2-alpha-CyD) and maltosyl-beta-cyclodextrin (G2-beta-CyD) toward Caco-2 cells was compared with that of natural alpha-cyclodextrin (alpha-CyD), beta-cyclodextrin (beta-CyD) and gamma-cyclodextrin (gamma-CyD). The degree of increase in cytotoxicity was dependent on the CyD's type and the concentration: the cytotoxicity of CyDs at the same concentration increased in the order of gamma-CyD<G2-beta-CyD<G2-alpha-CyD<<alpha-CyD, although beta-CyD could not be compared to other CyDs because of low solubility in water. Alpha-CyD decreased transepithelial electrical resistance (TEER) and increased the apical-to-basolateral (AP-to-BL) transport of [3H]mannitol, a paracellular transport marker, in a concentration-dependent manner, suggesting that alpha-CyD decreased the integrity of Caco-2 cell monolayers. In addition, alpha-CyD increased the AP-to-BL transport of rhodamine 123, a transcellular transport marker, under the experimental conditions being independent of P-glycoprotein. In contrast, G2-alpha-CyD, G2-betaCyD and gamma-CyD had slight effect on both TEER and the transport of mannitol and rhodamine 123 even at relatively high concentrations up to 150 mM. The inability of G2-alpha-CyD and G2-beta-CyD to effect TEER and the transport of mannitol and rhodamine 123 could be explained by the findings that these maltosylated CyDs released only a small amount of membrane constituents from Caco-2 cell monolayers and interacted only weakly with monolayers composed of L-alpha-dipalmitoylphosphatidylcholine (DPPC) formed on water. These results indicate that G2-alpha-CyD has less cytotoxicity and less disturbing ability toward Caco-2 cell monolayers than alpha-CyD, and G2-beta-CyD has, at least, comparable cytotoxicity to beta-CyD toward them. Thus, from the safety point of view, highly water-soluble G2-alpha-CyD and G2-beta-CyD may be particularly useful in various pharmaceutical formulations.
1,2-Dipalmitoylphosphatidylcholine↗
Analysis of the phase solubility diagram of a phenacetin/competitor/beta-cyclodextrin ternary system, involving competitive inclusion complexation.
The competitive inclusion complexations in the ternary phenacetin/competitors/beta-cyclodextrin (beta-CyD) systems were investigated by the solubility method, where m-bromobenzoic acid (m-BBA) and o-toluic acid (o-TA) were used as competitors. The solubility changes of the drug and competitors as a function of beta-CyD concentration in the ternary systems were formulated using their stability constants and intrinsic solubilities. The decrease in solubility of phenacetin by the addition of competitors could be quantitatively simulated by the formulation, when both drug and competitor give A(L) type solubility diagrams. On the other hand, when one of the guests gives a B(S) type solubility diagram, its solubility change was clearly reflected in that of the another guest, i.e., phenacetin gave an A(L) type solubility diagram in the binary phenacetin/beta-CyD system and o-TA gave a B(S) type diagram in the binary o-TA/beta-CyD system, but in the ternary phenacetin/o-TA/beta-CyD system, a new plateau region appeared in the original A(L) type diagram of phenacetin. This was explained by the solubilization theory of Higuchi and Connors. The solubility analysis of the ternary drug/competitor/CyD systems may be particularly useful for determination of the stability constant of a drug whose physicochemical and spectroscopic analyses are difficult, because they can be calculated by monitoring the solubility change of a competitor, without monitoring that of a drug. Furthermore, the present results suggest that attention should be paid to the type of the phase solubility diagram, as well as the magnitude of the stability constant and the solubility of the complex, for a rational formulation design of CyD complexes.
Cyclodextrins↗