Toshihiko Kaminuma

Prevention of cerebral vasospasm by calcitonin gene-related peptide slow-release tablet after subarachnoid hemorrhage in monkeys.

OBJECTIVE The goal of this work was to investigate the efficacy of a calcitonin gene-related peptide (CGRP) slow-release tablet (CGRP tablet) for the prevention of cerebral vasospasm after subarachnoid hemorrhage (SAH). METHODS Experimental SAH was produced in 10 cynomolgus monkeys by placing a clot around the internal carotid artery bifurcation (Day 0). In five animals, CGRP tablets (1200 micrograms of CGRP) were then placed in the cerebrospinal fluid space (CGRP group). In two animals, placebo tablets were similarly placed (placebo group). The remaining three animals were treated with no tablets after SAH (SAH group). A series of angiographic analyses were performed, before SAH and on Days 7 and 14, to examine changes in the diameters of the ipsilateral internal carotid artery, middle cerebral artery, and anterior cerebral artery. The CGRP concentration in the cerebrospinal fluid taken before each angiogram was also determined. RESULTS In the SAH and placebo groups, cerebral vasospasm developed on Day 7 (54.8% of the pre-SAH value at the internal carotid artery, 62.3% at the middle cerebral artery, 51.3% at the anterior cerebral artery, and 56.1% as an average of the three arteries). In the CGRP group, vasospasm was significantly ameliorated at the middle cerebral artery, at the anterior cerebral artery, and on average (81.7, 81.1, and 75.7%, P < 0.05, 0.03, and 0.02, respectively). The CGRP concentration was positive only on Day 7 for the CGRP group (6.5 nmol/L). CONCLUSION The CGRP tablet prevented cerebral vasospasm after SAH and may have significant potential for the treatment of patients with SAH.

Effect of calcitonin gene-related peptide on delayed cerebral vasospasm after experimental subarachnoid hemorrhage in rabbits.

BACKGROUND Calcitonin gene-related peptide (CGRP) is an intrinsic vasodilatory substance contained in perivascular nerve fibers of intracranial arteries. It is suggested that CGRP plays a role in cerebral vasospasm after subarachnoid hemorrhage (SAH). METHOD An experimental SAH was produced by intracisternal injection of arterial blood in rabbits. The animals were treated with intrathecal administration of CGRP solution 3 days after SAH. The degree of vasospasm and the effect of CGRP were evaluated angiographically by measuring the basilar artery diameter. RESULTS The basilar artery constricted to 73.0% of the pre-SAH values 3 days after SAH. Fifteen minutes after injection of 10(-10) mol/kg CGRP, the basilar artery dilated to 117.1% (n = 8), which was significantly larger than 67.1% in the vehicle group (n = 8) (p < 0.01). The significant vasodilatory effect of CGRP, compared with the vehicle group, lasted for 6 hours. CONCLUSIONS Intrathecal administration of CGRP has therapeutic potential for treating cerebral vasospasm.

High-level synthesis in Escherichia coli of recombinant human calcitonin: Collagenase cleavage of the fusion protein and peptidylglycine α-amidation

Abstract A synthetic gene encoding glycine-extended human calcitonin (hCT-Gly) was inserted between the tac -promoter and the lacZ gene. A synthetic DNA with collagenase recognition sites was placed just downstream at the hCT-Gly gene. In induced cultures, the E. coli cells harboring the gene produced about 0.8 g of the fusion protein (22 mg of hCT-Gly) per liter of culture. The fusion protein was easily and precisely cleaved with a bacterial collagenase to obtain the hCT-Gly. The hCT-Gly was converted with a peptidylglycine α-amidating enzyme to mature hCT, which had hypocalcemic activity similar to that of authentic hCT. These results demonstrate that expression of the fusion gene containing collagenase recognition sites is an effective method for the high-level production of intact peptide hormones.

Prevention of cerebral vasospasm by vasodilatory peptide maxadilan following subarachnoid hemorrhage in rabbits.

Maxadilan is a vasodilatory peptide isolated from the blood-feeding sand fly Lutzomyia longipalpis. Its vasodilatory activity, estimated by the formation of erythema on rabbit skin, is greater than those of calcitonin gene-related peptide, vasoactive intestinal polypeptide and pituitary adenylyl cyclase activating polypeptide (PACAP). We have recently demonstrated that maxadilan is a specific agonist for the PACAP type I receptor, which is widely distributed in brain. Therefore, we were interested in the vasodilatory effect of maxadilan on cerebral arteries and the possibility of its clinical use for the delayed cerebral vasospasm following subarachnoid (SAH). In the first experiment, 10(-10) mol/kg of maxadilan (in sterile water) was injected into the cisterna magna three days after the induction of experimental SAH in rabbits (n = 6). Maxadilan dilated spastic basilar arteries within 30 min of the injection, but not at 6 h. In the second experiment, to prolong the vasodilatory effect of maxadilan, tablets containing stearic acid, hydrogenated oil, lactose, hydroxypropylcellulose and 15 mg of maxadilan were prepared. In vitro testing showed that 60% of maxadilan could be released slowly within the initial five days. In vivo experiments were performed to implant the maxadilan tablet (n = 7) and the placebo tablet (n = 6) into the cisterna magna after the induction of experimental SAH in rabbits. The spastic response of the basilar artery was maximum on day three in the placebo-treated groups. In contrast, we observed no significant change in the arterial diameter until day five in the rabbits treated with maxadilan tablet. These data suggest that maxadilan may have therapeutic potency in treating cerebral vasospasm.

Expression of Human Calcitonin in Escherichia coli

Calcitonin, which effects skeletal maintenance, is a 32 amino acid thyroid hormone containing a 1-7 disulfide bridge and a C-terminal amide [1]. The use of plasmids is able to overexpress in E. coli via recombinant DNA and may be an effective means for the mass production of hCT1. However, the production of hCT is directly impossible, because the E. coli does not have α -amidating enzyme. We have performed the following strategies.