A Potent Neutralizing Monoclonal Antibody to Human Growth Hormone Suppresses Insulin-Like Growth Factor-1 in Female Rats.

Acromegaly and gigantism are disorders caused by hypersecretion of growth hormone (GH), usually from pituitary adenomas. Although somatostatin analogues (SSA), dopamine agonists, and GH receptor antagonists are important therapeutic agents, all of these have issues with their effectiveness, safety, and/or convenience of use. To overcome these, we developed a GH-specific potent neutralizing a mouse monoclonal antibody (mAb) named 13H02. 13H02 selectively bound both to human and monkey GH with high affinity, and strongly inhibited the biological activity of GH in the Nb2 rat lymphoma cell proliferation assay. In hypophysectomized/GH-supplemented rats, a single subcutaneous administration of 13H02 significantly and dose-dependently lowered the serum insulin-like growth factor-1 levels. To pursue the therapeutic potential of this antibody for acromegaly and gigantism, we humanized 13H02 to reduce its immunogenicity and applied a single amino acid mutation in the Fc region to extend its serum half-life. The resulting antibody, Hu-13H02m, also showed GH-specific neutralizing activity, similar to the parental 13H02, and showed improved binding affinity to human FcRn.

Region-specific loss of zinc in the brain in pentylentetrazole-induced seizures and seizure susceptibility in zinc deficiency.

The hippocampus is thought to be an epileptic focus in human temporal lobe epilepsy. Kainate-induced seizures decrease zinc concentrations in the hippocampus, which is also decreased in young mice fed a zinc-deficient diet for 4 weeks, and is enhanced by zinc deficiency. To understand zinc movement in the brain in epileptic seizures, zinc concentrations in the brain were measured in young mice after administration of pentylentetrazole, a GABAA receptor antagonist. Zinc concentration in the hippocampus and Timm's stain, with which histochemically reactive zinc in the presynaptic vesicle is detected, were decreased after the administration, suggesting that excessive excitation of zinc-containing glutamatergic neurons is induced in the hippocampus with pentylentetrazole. To clarify whether the decrease in zinc concentration in the hippocampus in zinc deficiency alter seizure susceptibility, furthermore, susceptibility to pentylentetrazole-induced seizures was examined in young mice fed the zinc-deficient diet for 4 weeks. The susceptibility, unlike susceptibility to kainate-induced seizures, was not appreciably enhanced by zinc deficiency. These results suggest that the decrease in zinc concentration in the hippocampus in zinc deficiency does not influence susceptibility to pentylentetrazole-induced seizures.

Elimination of zinc-65 from the brain under kainate-induced seizures.

On the basis of the previous evidence that 65Zn concentrations in the brain of EL (epilepsy) mice was affected by induction of seizures, 65Zn movement in the brain was quantitatively evaluated in ddY mice treated with kainate. Six days after intravenous injection of 65ZnCl2, mice were intraperitoneally injected with kainate (10 mg/kg x 6 times in 2 weeks). Myoclonic jerks were observed during treatment with kainate. Twenty days after 65Zn injection, 65Zn distribution in the brain was compared between the kainite-treated and control mice. 65Zn distribution in the brain of the kainate-treated mice was overall lower than in the control mice. 65Zn concentration was significantly decreased in the frontal cortex, hippocampal CA1, thalamus and hypothalamus by treatment with kainate. These results demonstrate that kainate-induced seizures are linked to decreased zinc concentrations in the brain.

Zinc movement in the brain under kainate-induced seizures.

On the basis of the evidence that elimination of 65Zn from the brain of epilepsy (EL) mice is facilitated by induction of seizures, zinc movement in the brain was studied in mice injected with kainate (12 mg/kg x 3), which exhibited status epilepticus within 120 min after the last injection of kainate. Zinc concentrations in the brain were determined 24 h after the last injection of kainate. Zinc concentrations in the hippocampus, amygdala and cerebral cortex, in which zinc-containing glutamatergic neuron terminals exist, were significantly decreased by the treatment with kainate, while that in the cerebellum was not decreased. Timm's stain in the brain was extensively attenuated 24 h after the last injection of kainate. These results indicate that zinc homeostasis in the brain is affected by kainate-induced seizures. In the hippocampus of rats injected with kainate (10 mg/kg), furthermore, the release of zinc and glutamate into the extracellular fluid was studied using in vivo microdialysis. The levels of zinc and glutamate in the perfusate were increased along with seizure severity after injection of kainate. It is likely that zinc concentration in the synaptic vesicles is decreased by the excess excitation of glutamatergic neurons. The present study suggests that the excessive release of zinc and glutamate from the neuron terminals under kainate-induced seizures is associated with the loss of zinc from the brain.

Susceptibility to kainate-induced seizures under dietary zinc deficiency.

Zinc homeostasis in the brain is altered by dietary zinc deficiency, and its alteration may be associated with the etiology and manifestation of epileptic seizures. In the present study, susceptibility to kainate-induced seizures was enhanced in mice fed a zinc-deficient diet for 4 weeks. When Timm's stain was performed to estimate zinc concentrations in synaptic vesicles, Timm's stain in the brain was attenuated in the zinc-deficient mice. In rats fed the zinc-deficient diet for 4 weeks, susceptibility to kainate-induced seizures was also enhanced. When the release of zinc and neurotransmitters in the hippocampal extracellular fluid of the zinc-deficient rats was studied using in vivo microdialysis, the zinc concentration in the perfusate was less than 50% of that of the control rats and the increased levels of zinc by treatment with kainate were lower than the basal level in control rats, suggesting that vesicular zinc is responsive to dietary zinc deficiency. The levels of glutamate in the perfusate of the zinc-deficient rats were more increased than in the control rats, whereas the levels of GABA in the perfusate were not at all increased in the zinc-deficient rats, unlike in the control rats. The present results demonstrate an enhanced release of glutamate associated with a decrease in GABA concentrations as a possible mechanism for the increased seizure susceptibility under zinc deficiency.

Release of glutamate and GABA in the hippocampus under zinc deficiency.

Zinc homeostasis in the brain is affected by dietary zinc deficiency, and its alteration may cause brain dysfunctions. On the basis of the previous evidence that hippocampal zinc was responsive to 12-week zinc deprivation, responsiveness of hippocampal zinc to dietary zinc deficiency was examined in rats fed a zinc-deficient diet for 4 weeks. Zinc concentration in the hippocampus was not decreased by zinc deprivation for 4 weeks. However, Timm's stain was extensively attenuated in the brain of the zinc-deficient rats. In the brain of the zinc-deficient rats, moreover, zinc concentration in the hippocampal extracellular fluid was approximately 30% of that of control rats. These results demonstrate that vesicular zinc is responsive to dietary zinc and may decrease easily under zinc deficiency. Zinc concentration in the hippocampal extracellular fluid during stimulation with high K(+) was significantly increased even in zinc-deficient rats, although the increased levels of zinc were lower than the basal levels of zinc in control rats. The basal glutamate concentration in the hippocampal extracellular fluid was not significantly different between the control and zinc-deficient rats. However, glutamate concentration in the hippocampal extracellular fluid during stimulation with high K(+) was more increased in the zinc-deficient rats than in the control rats. Gamma aminobutyric acid (GABA) concentration in the hippocampal extracellular fluid during stimulation with high K(+) was increased in the control rats, but not in the zinc-deficient rats. The present study suggests that the excitability of hippocampal glutamatergic neurons is enhanced by dietary zinc deficiency.

Distribution of trace elements in the brain of EL (epilepsy) mice.

The association of essential trace elements with epileptic seizures is poorly understood. On the basis of the evidences that the release of zinc from the brain of epilepsy (EL) mice, an animal model of genetically determined epilepsy, is enhanced by the induction of seizures and that alteration of zinc homeostasis is responsive to susceptibility to seizures, the distribution of trace elements in the brain was studied using EL mice and ddY mice, which form the genetic background for the inbred EL mice. The multitracer technique was applied to determine the distribution of trace elements. Twenty-four hours after intravenous injection of the multitracer, the concentration of 65Zn and 56Co in the brain of untreated EL mice was higher than in ddY mice, while the concentration of 65Zn and 56Co in the brain was decreased in seized EL mice. 75Se concentration in the hippocampus, cerebral cortex and cerebellum of untreated EL mice was lower than in ddY mice, while 75Se concentration in the hippocampus was increased in seized EL mice. 83Rb, an element of homologous series to potassium, concentration in the hippocampus and cerebral cortex of untreated EL mice was lower than in ddY mice, and 83Rb concentration in the cerebral cortex was decreased in seized EL mice. The movement of zinc, cobalt and selenium in the brain may be altered by enhancement of susceptibility to seizures. These results suggest that alteration of homeostasis of zinc, cobalt and selenium in the brain may be involved in the susceptibility, development or termination of seizures in EL mice.