Tatsuyuki Naruchi

1,24(R)-Dihydroxyvitamin D3, a Novel Active Form of Vitamin D3 with High Activity for Inducing Epidermal Differentiation but Decreased Hypercalcemic Activity

1α,25‐Dihydroxyvitamin D3 (1,25(OH)2D3) is known to be a hormonally active form of vitamin D3 in the regulation of intracellular and extracellular calcium levels and of differentiation of myeloid cells and epidermal keratinocytes. We found that 1α,24(R)‐dihydroxyvitamin D3 (1,24(OH)2D3), a novel synthetic derivative of vitamin D3, is also active in regulating the differentiation of epidermal keratinocytes. 1,24(OH)2D3 had the same affinity as 1,25(OH)2D3 for a receptor isolated from the epidermis of newborn mice. The incubation of mouse epidermal keratinocytes with 1,24(OH)2D3 induced their differentiation in a time‐ and dose‐dependent manner, as determined by the formation of a cornified envelope and an increase in the activity of transglutaminase. 1,24(OH)2D3 inhibited DNA synthesis of epidermal keratinocytes and also increased their cytosolic calcium level. These effects of 1,24(OH)2D3 were similar to, or rather more than, those of physiologically active 1,25(OH)2D3. However, 1,24(OH)2D3 was found to cause less hypercalcemia than 1,25(OH)2D3 when administrated intravenously to rats, suggesting its possible therapeutic value in psoriasis.

1,25-dihydroxyvitamin D3 and 1,24-dihydroxyvitamin D3 suppress invitro antibody response to T cell-dependent antigen

1,25-Dihydroxyvitamin D3 and 1,24-dihydroxyvitamin D3 suppressed an antibody response to sheep red blood cells (SRBC, T cell-dependent antigen) by murine splenocytes, in concentrations ranging from 10(-10)-10(-7)M. These suppressive effects were markedly abrogated when T cell-depleted lymphocytes were cultured in the presence of a supernatant of concanavalin A-stimulated spleen cells. On the contrary, neither of them suppressed antibody response to trinitrophenyl-lipopolysaccharide (T cell-independent antigen). These results suggest that the suppressive effect of active vitamin D3 on anti-SRBC response was mediated by the inhibition of T cells.

IMMUNOPHAFMACOLOGICAL PROFILE OF TEI-3096: A NEW IMMUNOMODULATOR

TEI-3096 [6-p-chlorobenzyl-5H-2,3,6,7-terahydro-5,7-dioxothiazolo (3,2-a) pyrimidine], a novel thiazolopyrimidine compound has been shown to suppress adjuvant arthritis in rats without any effect on conventional inflammation. We examined the immuno-pharmacological profile of TEI-3096 in murine lymphocytes. The blastformation induced by Con A or LPS was inhibited by addition of 50-500 microM concentrations of TEI-3096. This compound suppressed elevated plaque-forming cell (PFC) response against T cell-dependent antigen. However, TEI-3096 had no effect either on normal PFC response or on antibody formation against T cell independent antigen. Although orally administered TEI-3096 had no obvious effect on anti-SRBC PFC response in normal mice, it normalized the colchicine-induced enhancement of antibody formation against TNP-HGG. TEI-3096 also enhanced the delayed type hypersensitivity in mice and rats. These results suggests that TEI-3096 restores the abnormal immune response. Therefore, it may be useful for the treatment of auto-immune disease such as rheumatoid arthritis.

Intrinsic biological activities by 1α,24-dihydroxyvitamin D3 in the rat

Subcellular localization of [3H]1α,24(R)-dihydroxyvitamin D3 and [3H]1α,24(S)-dihydroxyvitamin D3 in rat intestinal mucosa was investigated in comparison with the [3H]1α-hydroxyvitamin D3. The 24(R) and 24(S) isomers of 1α,24-dihydroxyvitamin D3 were gradually transformed to 1α,24(R)25-trihydroxyvitamin D3 and 1α,24(S)25-trihydroxyvitamin D3, and the plasma concentrations of these metabolites were 10.30 and 1.36 pmol/ml, respectively. The major portions of the administered compounds distributed in the nuclear fraction of the intestinal mucosa remained unchanged, and the amounts of 1α,24(R)-dihydroxyvitamin D3 and 1α,24(S)-dihydroxyvitamin D3 were 4.25 and 0.306 pmol/g intestinal mucosa, respectively. No detectable amount of the metabolites, 1α,24(R)25-trihydroxyvitamin D3 and 1α,24(S)25-trihydroxyvitamin D3 were found in the same nuclear fractions. In the case with the [3H]1α-hydroxyvitamin D3, however, the compound was rapidly metabolized to 1α,25-dihydroxyvitamin D3. The metabolite, 1α,25-dihydroxyvitamin D3, was seen in the nuclear fraction of the intestinal mucosa at a concentration of 2.44 pmol/g intestinal mucosa.

A Convenient Synthesis of 1, 2, 5-Trisubstituted 3-Benzoylpyrroles

Abstract In the course of our investigation directing toward biologically active compounds, 2, 5-disubstituted 3-acylpyrrole derivatives were found to show a potent inhibiting activity of platelet aggregation.1,2,3 Thus, various acylation reactions were studied in order to achieve an efficient synthesis of 3-acylated pyrrole system. We wish to report here an effective method for the preparation of 2, 5-disubstituted 3-acylpyrroles dispensing from the formation of by-products such as 3, 4-diacylated pyrroles.

Studies on the mechanism of action of 1α,24-dihydroxyvitamin D3 III the specific binding of 1α,24-dihydroxyvitamin D3 to the receptor of chick parathyroid gland

Abstract Three protein fractions of the cytosol of the chick parathyroid glands, which had the sedimentation constants of 2.5 S, 3.7 S and 5.5 S, were found to bind with 1α, 25-dihydroxyvitamin D3. Among these proteins, the 3.7 S protein was assumed to be the specific receptor protein. The 3.7 S receptor protein was also capable of binding to 1α, 24-dihydroxyvitamin D3 but not 25-hydroxyvitamin D3. The binding affinity of 1α, 24(R)-dihydroxyvitamin D3 to the 3.7 S receptor protein was estimated to be 1.2 times greater than that of 1α,25-dihydroxyvitamin D3, while 1α,25-dihydroxyvitamin D3 bound to the receptor protein about 10 times stronger than 1α,24(S)-dihydroxyvitamin D3. The dissociation constant for, the receptor-1α, 25-dihydroxyvitamin D3 complex at 0°C was 2.7 × 10−11 M, the dissociation constants were calculated to be 2.2 × 10−11 M and 2.6 × 10−10 M for the complexes with 1α,24(R)-dihydroxyvitamin D3 and 1α,24(S)-dihydroxyvitamin D3, respectively.

Development of Radioimmunoassay for TG-51, a New Anti-Ulcer Drug, and Its Application

A sensitive and specific radioimmunoassay for a new anti-ulcer drug TG-51 has been developed and applied to the evaluation of its pharmacokinetics in humans. The antiserum was raised in rabbits against an immunogen of N-Acetyl-TG-51 coupled to human serum albumin. The radioactive compound was prepared by acetylating TG-51 with 3H-Acetic anhydride. The separation of free and antibody-bound N-Acetyl-TG-51 was performed by the dextran coated charcoal technique. TG-51 in biological fluids could be quantitatively converted to N-Acetyl-TG-51 prior to radioimmunoassay. This assay system made it possible to ascertain values of 3 ng/ml of TG-51 in plasma using 100 microliters of samples. Results were in good agreement with a high performance liquid chromatography method (HPLC), and the detection limit was raised 25 fold. The accuracy and repoducibility were also satisfactory. By use of this assay method, plasma levels of TG-51 could be determined after a single oral administration of clinical doses of human volunteers.

Effect of a new anti-ulcer drug, (3-[p-(trans-4-aminomethylcyclohexylcarbonyl)-phenyl] propionic acid hydrochloride, TEI-5103) on prostaglandin (PG) levels in rat gastric mucosa.

1. Effect of TEI-5103 on the levels of various prostaglandins (PGs) in rat gastric mucosa was studied by HPLC and RIA methods. 2. When administrated in vivo, TEI-5103 significantly increased the level of prostaglandin E2 (PGE2) but only slightly increased that of prostaglandin I2 (PGI2) or thromboxane A2 (TXA2). 3. In the water-immersion stress model, the PGE2 level in control animals was increased immediately after stress and then decreased gradually to the initial level. Indomethacin (5 mg/kg, s.c.) sustained a lower PGE2 level during the experiment, as well as aggravated the lesions. 4. TEI-5103 (400 mg/kg, p.o.) increased the PGE2 level just before stress, and it resulted in the prevention of lesion formation. 5. TEI-5103 inhibited the activity of 15-OH-PG-dehydrogenase from guinea pig lungs, but did not affect the activity of phospholipase A2 from porcine pancreas.

Effects of a novel acylindole derivative on experimental thrombotic models, ulceration of the gastric mucosa and prostacyclin production in the aorta

Abstract 3-Benzoyl-N-β-ethoxyisopropyl-2-methylindole (TEI-4120) strongly inhibited arachidonic acid- and collagen-induced aggregation of platelets of several animal species (IC 50 : 3.3×10 −6 M − 3.1×10 −8 M). It also inhibited the second phase of ADP- or epinephrine-induced human platelet aggregation (IC 50 : 2.0×10 −6 M − 1.0×10 −5 M). TEI-4120 at oral doses of 1–30 mg/kg was effective on aggregation of platelets removed from animals. In several experimental thrombotic models, TEI-4120 at doses of 1–30 mg/kg (p.o.) inhibited thrombus formation. It was less ulcerogenic than typical nonsteroidal antiinflammatory drugs such as indomethacin and caused only slight gastric lesions at a dose of 400 mg/kg (p.o.). It was also only a weak inhibitor of prostacyclin biosynthesis in rat aorta in vivo, a dose of about 100 mg/kg being required to inhibit prostacyclin biosynthesis. These results show that TEI-4120 has strong anti-platelet activity and suggest that it should be a good anti-thrombotic drug with weak side effects.

Inhibition of prostaglandin synthesizing enzymes by haptoglobin and plasma of rat with inflammation

ラットの足蹠にcarrageenin, compound48/80あるいはadjvantを注射すると, 一定時間後の血漿にprostaglandin E2生合成の阻害作用およびhaptoglobin値の増加が認められた.一方, 部分精製したhaptoglobinはarachidonic acidを基質とするprostaglandin E2生合成を用量依存性に阻害した.ウシ精嚢microsomeの精製酵素を用いてその作用機作を検討したところ, haptoglobinが活性化因子のhemeと相互作用して阻害作用を示すことが明らかになった.しかし, prostaglandin E2生合成に対して阻害作用を示すhaptoglobin濃度 (ID50) は, 炎症ラット血漿では11μg/mlであるのに対して, 部分精製したhaptoglobinでは175μg/mlであり, 炎症ラット血漿の阻害作用をhaptoglobinのみで説明できないことが示唆された.