Masahiro Kawase

Iontophoretic pulsatile transdermal delivery of human parathyroid hormone (1-34).

Iontophoretic pulsatile transdermal delivery of hPTH(1–34) was examined in Sprague‐Dawley (SD) rats, hairless rats and beagle dogs. Application for 60 min (200 μ 0.1 mA cm−2) showed current‐responsive increases in serum hPTH(1–34) levels in all the animals. In SD rats, the area under the curves of serum hPTH(1–34) levels (AUCs) were proportional to the doses (40, 120, 200, 400 and 1000 μg) and current densities (0.05, 0.1 and 0.15 mA cm−2) applied. The absorption rates per 200‐μg dose, calculated by a deconvolution method, were 6.7, 2.4 and 3.7 μg h−1 for SD rats, hairless rats and beagle dogs, respectively. These values correlated well with the ratios of the skin porosity to the dermal thickness reported for these animals, which are believed to represent the reciprocal of the electrical resistance of the aqueous channels formed by the hair follicles. From this correlation, we suggested that absorption of hPTH(1–34) occurs mainly via the hair‐follicle route, and that the absorption rate in man might be intermediate between those in hairless rats and beagle dogs. Three‐fold repetitions of 30 min current with various rest intervals produced current‐responsive triple pulses in serum hPTH(1–34) levels in SD rats. Seven‐fold repetitions of current also produced similar current‐responsive pulsatile serum hPTH(1–34) levels. However, peak serum hPTH(1–34) levels tended to decrease gradually after the fourth current application, possibly due to consumption of the electrodes, suggesting that three‐fold repetitions of current might be optimal. These findings suggest that this iontophoretic administration system could create a repeated‐pulsatile pattern of serum hPTH(1–34) levels without the necessity for frequent injections, and may be useful for the treatment of osteoporosis with hPTH(1–34).

Enhancement of osteogenesis in vitro by a novel osteoblast differentiation-promoting compound, TAK-778, partly through the expression of Msx2.

TAK-778 [(2R,4S)-(-)-N-(4-Diethoxyphosphorylmethylphenyl)-1,2,4,5-tetrahydro-4-methyl-7,8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxamide: mw 505.52], a novel compound promoting osteoblast differentiation, promotes osteogenesis in vitro and enhances bone formation during skeletal repair in vivo. In this study, we further evaluated the effects of TAK-778 on the differentiation of cultured bone marrow stromal cells into osteoblasts in the presence of dexamethasone, paying particular attention to the expression of transcription factors involved in regulating osteoblast differentiation. Treatment of TAK-778 (10(-7)-10(-5) M) for 4 h resulted in an increase in the mRNA expression of Msx2, but not Cbfa1 or Dlx5. This transcriptional alteration preceded the changes in other markers related to the osteoblast phenotype, such as alkaline phosphatase and osteocalcin mRNA. The transfection of Msx2-antisense in the cells caused a significant reduction in the levels of alkaline phosphatase mRNA expression induced by TAK-778. These results suggest that TAK-778 promotes osteoblast differentiation partly through the expression of Msx2, a homeobox-related gene.

Acute effect of triiodothyronine on the dynamics of thyrotropin release from superfused anterior pituitary cells

The effects of triiodothyronine (T3) on the dynamics of thyrotropin (TSH) release induced by TSH-releasing hormone (TRH) were examined in the presence or absence of a protein synthesis inhibitor, cycloheximide (CX), in a superfusion system using primarily cultured cells of the rat anterior pituitary gland on microcarrier beads. When the cells were continuously stimulated with TRH (10 nM, 180 min), TSH release occurred in a biphasic manner and the profile of TSH release was characterized by an initial sharp peak (phase I), followed by a lower plateau form phase (phase II). Both phase-I and phase-II releases were significantly suppressed in the presence of T3 (1 ng/ml), which was added to the superfusion medium 1 h before initiation of TRH stimulation. The biphasic nature of the release profile was maintained in the presence of T3, suggesting that the site of the T3 action may be common between phase-I and phase-II release. We have already suggested that phase-I release is protein synthesis-independent and phase-II release protein synthesis-dependent using CX in TRH-stimulated cells. In the presence of CX, phase-I release was not suppressed by T3, while phase-II release was still suppressed by T3. The inability of CX to reverse the T3-induced suppression of phase-II release may be masked by the direct CX effect on phase-II release of TSH. The present study indicates that each component (phase I and phase II) of the biphasic release of TSH induced by TRH stimulation was acutely suppressed by T3 and suggests that the T3 action is mediated through protein synthesis.

Vitamin D independent anabolic action of PTHin vivo

We examined the involvement of endogenous calcium-regulating hormones in the anabolic action of PTH, especially in relation to vitamin D. When normal rats (4 weeks old) were given a subcutaneous injection of hPTH(1-34) everyday or one, two or three times a week for 4 weeks adjusting the total weekly dose to 350µg/kg, femoral dry weight increased in an injection frequency-dependent manner. Daily injection of hPTH(1-34) into normal rats for 12 days caused a dose-dependent increase in serum concentration of 1,25(OH)2D but not calcitonin, associated with a dose-dependent increase in femoral dry weight. Therefore, the effect of hPTH(1-34) was examined under the condition of vitamin D (VD)-deficiency. In VD-deficient rats (13 weeks old), the change in serum immunoreactive PTH after the injection of hPTH(1-34) was the same as that observed in normal rats, although the basal level of serum PTH was significantly higher. Furthermore, VD-deficient rats treated with hPTH(1-34) for 4 weeks showed a dose-dependent increase in femoral dry weight without affecting circulating 1,25(OH)2D. These results suggest the involvement of systemic or local factor(s) independent of the action of VD in the anabolic action of daily PTH treatment.

Marked impact of P-glycoprotein on the absorption of TAK-427 in rats.

The role of P‐glycoprotein (P‐gp, ABCB1) on the absorption process was investigated by drug–drug interaction studies of TAK‐427 with P‐gp inhibitors (erythromycin, ketoconazole or quinidine) in rats and by transport studies using rat multidrug resistance (MDR1) stably expressing cells and rat small intestine mounted in a Ussing‐type chamber. TAK‐427 showed high efflux activity with low permeability in rat MDR1a and MDR1b stably expressing cells and was revealed to be a typical substrate for P‐gps. Although TAK‐427 was mainly absorbed from the small intestine in rats, a large part of the dosed compound remained in the gastrointestinal tract. Orally co‐administered P‐gp inhibitors (50 mg/kg) increased the AUC of TAK‐427 after a 5 mg/kg oral dose 5.4‐ to 18.3‐fold, whereas orally administered P‐gp inhibitors had a minor effect on the increase in the AUC of TAK‐427 (1.3‐ to 2.2‐fold) after a 0.5 mg/kg intravenous dose. Thus, the bioavailability of TAK‐427 after oral administration in rats (7.3%) markedly increased when co‐administered with P‐gp inhibitors (28.6–57.6%). Moreover, the transport of TAK‐427 was predominantly secretory throughout the rat small intestine and was inhibited by P‐gp inhibitors. In conclusion, P‐gp can markedly reduce the absorption of a typical P‐gp substrate by its efflux activity throughout the absorption site. Copyright

Synthesis and biological activities of hPTH)1—34) analogues: modification of the middle part and C-terminal alkylamides

Human parathyroid hormone (hPTH) is a peptide hormone, consisting of 84 amino acid residues, which regulates calcium and phosphate ion homeostasis. The N-terminal fragment, hPTH(1–34), has been shown to possess full biological activity. The hormone is thought to have promise in the treatment of bone diseases, including osteoporosis, because of the anabolic effect which is elicited by its intermittent administration. We have already reported the recombinant synthesis of hPTH(1–84) and hPTH(1–34) [1], as well as some structureactivity relationships concerned with the chemically synthesized analogues [2]. In the present study, various hPTH(1–34) analogues were synthesized by recombinant or Fmoc-based chemical methods, and the in vitro (cAMP production in MC3T3-E1 cells) and in vivo (bone growth stimulation in 4 week old male rats) activities were evaluated.