A highly sensitive LC-MS/MS method for simultaneous determination of glycyrrhizin and its active metabolite glycyrrhetinic acid: Application to a human pharmacokinetic study after oral administration.

A highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of glycyrrhizin (GL) and its active metabolite, glycyrrhetinic acid (GA), from human plasma was validated and applied to a human pharmacokinetic study. The analytes were extracted from human plasma using an Oasis MAX cartridge and chromatographic separation was performed on an Inertsil ODS-3 column. The detection was performed using an API 4000 mass spectrometer operating in the positive electrospray ionization mode. Selected ion monitoring transitions of m/z 823 → 453 for GL and m/z 471 → 149 for GA were obtained. The response was a linear function of concentration over the ranges of 0.5-200 ng/mL for GL and 2-800 ng/mL for GA (both R2 > 0.998). Using this method, the pharmacokinetics of GL after single oral administration of a clinical dose (75 mg) to six healthy male Japanese volunteers were evaluated. GL was detected in the plasma of all subjects and the average peak concentration was 24.8 ± 12.0 ng/mL. In contrast, peak concentration of GA was 200.3 ± 60.3 ng/mL, i.e. ~8-fold higher than that of GL. This is the first report clarifying pharmacokinetic profiles of GL and GA simultaneously at a therapeutic oral dose of a GL preparation.

Suppressive effects of glycyrrhetinic acid derivatives on tachykinin receptor activation and hyperalgesia.

Glycyrrhetinic acid (GA), an aglycone of glycyrrhizin, isolated from the licorice root (Glycyrrhizia), and its semi-synthetic derivatives have a wide range of pharmacological effects. To investigate whether GA derivatives may be used as a new class of analgesics, we examined the effects of these compounds on human tachykinin receptors expressed in CHO-K1 cells. Among the GA derivatives examined, the disodium salt of olean-11,13(18)-dien-3ß,30-O-dihemiphthalate inhibited the mobilization of [Ca(2+)](i) induced by substance P, neurokinin A, and neurokinin B in CHO-K1 cells expressing the human NK(1), NK(2), and NK(3) tachykinin receptors, respectively. In an inflammatory pain model, Compound 5 suppressed the capsaicin-induced flinching behavior in a dose-dependent manner. Compound 5 was also effective in suppressing pain-related behaviors in the late phase of the formalin test and reducing thermal hyperalgesia in the neuropathic pain state caused by sciatic nerve injury. Collectively, Compound 5 may be an analgesic candidate via tachykinin receptor antagonism.

Glycyrrhetinic acid prevents cutaneous scratching behavior in mice elicited by substance P or PAR-2 agonist.

Although glycyrrhetinic acid (GA) has been used for the prevention of itch in chronic dermatitis, the mechanism underlying the antipruritic effects of GA is still unclear. Recently, several mediators other than histamine, such as substance P and tryptase, were found to participate in chronic itch. Here, we investigated the effect of GA on pruritus induced by various pruritic agents including histamine in mice. We also determined the level of leukotriene (LT)B(4) in mouse skin injected with substance P in an effort to uncover part of the antipruritic mechanism of GA. Scratching events were counted for 10 min after intradermal injection of histamine, substance P (100 nmol per site each), protease-activated receptor-2 (PAR-2) agonistic peptide (50 nmol per site), or LTB(4) (0.03 nmol per site) with or without GA (4 nmol per site) into male ICR mice. Levels of LTB(4) in the skin after injection of substance P were determined by ELISA. GA did not suppress scratching behavior induced by histamine and LTB(4), but markedly and dose-dependently suppressed that induced by substance P and PAR-2 agonistic peptide. LTB(4) levels in skin elevated by substance P were lowered by GA. These data support the efficacy of GA in counteracting itch in chronic dermatitis because GA reduced scratching behavior induced by substance P and PAR-2 agonistic peptide. GA may exert antipruritic effects via inhibition of LTB(4) production in skin.

[No association between moyamoya disease and polymorphism of IGF2R].

Moyamoya disease is a cerebrovascular disorder of unknown etiology. Its high incidence in East Asia and accumulation in family members suggest a genetic background. A high incidence of maternal inheritance implicates genomic imprinting in this disorder. Based on this hypothesis, we studied the association between moyamoya disease and IGF2R gene on chromosome 6, but found no evidence for such association between them. On the other hand, heterogeneous expressions of IGF2R were confirmed in the lymphocytes. Some individuals showed monoallelic expression and others showed biallelic expression.

[Molecular screening for moyamoya disease by use of expressed sequence tag on chromosome 3p].

Moyamoya disease is a well-known cerebrovascular disorder of unknown pathogenesis affecting terminal portion of internal carotid arteries and causing ischemic attacks. Its familial occurrence suggests genetic background. We hypothesized that paternally imprinted gene might be associated with this disorder. To identify the expressed sequence tags (ESTs) with monoallelic expressions on chromosome 3, we used mouse A9 hybrid cells having human chromosome 3. Two ESTs showed only maternal expression in mouse A9 hybrid cells, and four showed non-expression in the lymphocytes derived from moyamoya patients. Although these ESTs are clustered on the same 150 kb region, we finally failed to identify cDNA in this region.