Oral octreotide absorption in human subjects: comparable pharmacokinetics to parenteral octreotide and effective growth hormone suppression.

CONTEXT: Oral administration of a novel octreotide formulation enabled its absorption to the systemic circulation, exhibiting blood concentrations comparable to those observed with injected octreotide and maintaining its biological activity. OBJECTIVES: The aim of the study was to determine oral octreotide absorption and effects on pituitary GH secretion compared to sc octreotide injection. DESIGN: Four single-dose studies were conducted in 75 healthy volunteers. INTERVENTION: Oral doses of 3, 10, or 20 mg octreotide and a single sc injection of 100 µg octreotide were administered. MAIN OUTCOME MEASURE: We measured the pharmacokinetic profile of orally administrated octreotide and the effect of octreotide on basal and stimulated GH secretion. RESULTS: Both oral and sc treatments were well tolerated. Oral octreotide absorption to the circulation was apparent within 1 h after dose administration. Escalating oral octreotide doses resulted in dose-dependent increased plasma octreotide concentrations, with an observed rate of plasma decay similar to parenteral administration. Both 20 mg oral octreotide and injection of 0.1 mg sc octreotide resulted in equivalent pharmacokinetic parameters [mean peak plasma concentration, 3.77 ± 0.25 vs. 3.97 ± 0.19 ng/ml; mean area under the curve, 16.2 ± 1.25 vs. 12.1 ± 0.45 h × ng/ml); and median time ≥ 0.5 ng/ml, 7.67 vs. 5.88 h, respectively). A single dose of 20 mg oral octreotide resulted in basal (P < 0.05) and GHRH-stimulated (P < 0.001) mean GH levels suppressed by 49 and 80%, respectively. CONCLUSIONS: The results support an oral octreotide alternative to parenteral octreotide treatment for patients with acromegaly.

Reduced rates of axonal and dendritic growth in embryonic hippocampal neurones cultured from a mouse model of Sandhoff disease.

Sandhoff disease is a lysosomal storage disease in which ganglioside GM2 accumulates because of a defective beta-subunit of beta-hexosaminidase. This disease is characterized by neurological manifestations, although the pathogenic mechanisms leading from GM2 accumulation to neuropathology are largely unknown. We now examine the viability, development and rates of neurite growth of embryonic hippocampal neurones cultured from a mouse model of Sandhoff disease, the Hexb-/- mouse. GM2 was detected by metabolic labelling at low levels in wild type (Hexb+/+) neurones, and increased by approximately three-fold in Hexb-/- neurones. Hexb-/- hippocampal neurones were as viable as their wild type counterparts and, moreover, their developmental programme was unaltered because the formation of axons and of the minor processes which eventually become dendrites was similar in Hexb-/- and Hexb+/+ neurones. In contrast, once formed, a striking difference in the rate of axonal and minor process growth was observed, with changes becoming apparent after 3 days in culture and highly significant after 5 days in culture. Analysis of various parameters of axonal growth suggested that a key reason for the decreased rate of axonal growth was because of a decrease in the formation of collateral axonal branches, the major mechanism by which hippocampal axons elongate in culture. Thus, although the developmental programme with respect to axon and minor process formation and the viability of hippocampal neurones are unaltered, a significant decrease occurs in the rate of axonal and minor process growth in Hexb-/- neurones. These results appear to be in contrast to dorsal root ganglion neurones cultured from 1-month-old Sandhoff mice, in which cell survival is impaired but normal outgrowth of neurones occurs. The possible reasons for these differences are discussed.

The increased sensitivity of neurons with elevated glucocerebroside to neurotoxic agents can be reversed by imiglucerase.

We have recently demonstrated that incubation of cultured rat hippocampal neurons with conduritol beta-epoxide (CBE), an inhibitor of glucocerebrosidase, the enzyme defective in Gaucher disease, results in changes in intracellular morphology and in functional calcium stores. Changes in levels of functional calcium stores are directly related to neuronal cell death. We now show that neurons incubated with either CBE or a non-hydrolysable analogue of GlcCer (glucosylthioceramide), are more sensitive to the toxic effects of high concentrations of glutamate and of a variety of metabolic inhibitors. A linear relationship exists between level of accumulation of GlcCer and the extent of neuronal cell death. The deleterious effects of elevated GlcCer levels can be completely reversed by addition of human glucocerebrosidase (imiglucerase) to the culture medium. Imiglucerase is internalized to lysosomes, where it presumably degrades excess GlcCer. This suggests that the limited success of enzyme replacement therapy in neuronopathic forms of Gaucher disease is not due to lack of efficacy of glucocerebroside in degrading GlcCer in neurons of the central nervous system, and adds impetus to attempts to develop ways to efficiently deliver glucocerebrosidase to the brains of neurologically compromised Gaucher disease patients.

Autoantibodies to the glutamate receptor kill neurons via activation of the receptor ion channel.

Antibodies to the glutamate/AMPA receptor subunit 3 (GluR3), are found in a human epilepsy, Rasmussen's encephalitis [RE], and were hypothesized as the major cause for the neuronal loss, chronic inflammatory changes and epileptic seizures characteristic of the disease. To establish the pathogenic potential and mechanism of action of such antibodies, we raised murine antibodies against specific peptides of the GluR3 protein and studied their ability to bind, activate, and kill neurons. Mice were immunized with two GluR3 specific peptides: GluR3A (amino acids 245-274) and GluR3B (amino acids 372-395), and with a scrambled GluR3B peptide for control. High levels of antibodies to each of these peptides were obtained, with no cross reactivity between them. Antibodies to the GluR3B peptide were found to bind to cultured neurons, evoke GluR ion channel activity, and kill neurons. In contrast, antibodies against GluR3A peptide bound to neurons but failed to activate the receptor or kill neurons. Anti-scrambled-GluR3B antibodies had no effect. Both the activation of the GluRs and the neuronal death induced by anti-GluR3B antibodies were blocked by CNQX, a specific glutamate/AMPA receptor antagonist; killing was independent of complement. This indicates a mechanism of excitotoxicity-neuronal death due to over-activation of the receptor, a phenomenon known to be caused by excess of glutamate. Purified anti-GluR3B IgGs retained the neuronal killing capacity, and killing was completely and specifically blocked by preincubation with the GluR3B peptide. Excitotoxic neuronal death induced by anti-GluR3B antibodies took place primarily via apoptosis. Taken together, these results show that antibodies to a specific peptide of the GluR can kill neurons by an excitotoxic mechanism, thus mimicking the effects of excess of glutamate. This is the first example that antibodies can lead to neuronal death in a non-classical complement-independent manner, via activation of a membranal neurotransmitter receptor.

Elevation of intracellular glucosylceramide levels results in an increase in endoplasmic reticulum density and in functional calcium stores in cultured neurons.

Gaucher disease is a glycosphingolipid storage disease caused by defects in the activity of the lysosomal hydrolase, glucocerebrosidase (GlcCerase), resulting in accumulation of glucocerebroside (glucosylceramide, GlcCer) in lysosomes. The acute neuronopathic type of the disease is characterized by severe loss of neurons in the central nervous system, suggesting that a neurotoxic agent might be responsible for cellular disruption and neuronal death. We now demonstrate that upon incubation with a chemical inhibitor of GlcCerase, conduritol-B-epoxide (CBE), cultured hippocampal neurons accumulate GlcCer. Surprisingly, increased levels of tubular endoplasmic reticulum elements, an increase in [Ca(2+)](i) response to glutamate, and a large increase in [Ca(2+)](i) release from the endoplasmic reticulum in response to caffeine were detected in these cells. There was a direct relationship between these effects and GlcCer accumulation since co-incubation with CBE and an inhibitor of glycosphingolipid synthesis, fumonisin B(1), completely antagonized the effects of CBE. Similar effects on endoplasmic reticulum morphology and [Ca(2+)](i) stores were observed upon incubation with a short-acyl chain, nonhydrolyzable analogue of GlcCer, C(8)-glucosylthioceramide. Finally, neurons with elevated GlcCer levels were much more sensitive to the neurotoxic effects of high concentrations of glutamate than control cells; moreover, this enhanced toxicity was blocked by pre-incubation with ryanodine, suggesting that [Ca(2+)](i) release from ryanodine-sensitive intracellular stores can induce neuronal cell death, at least in neurons with elevated GlcCer levels. These results may provide a molecular mechanism to explain neuronal dysfunction and cell death in neuronopathic forms of Gaucher disease.

Abnormal purine and pyrimidine nucleotide content in primary astroglia cultures from hypoxanthine-guanine phosphoribosyltransferase-deficient transgenic mice.

Lesch-Nyhan syndrome is a pediatric metabolic-neurological syndrome caused by the X-linked deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The cause of the metabolic consequences of HGPRT deficiency has been clarified, but the connection between the enzyme deficiency and the neurological manifestations is still unknown. In search for this connection, in the present study, we characterized purine nucleotide metabolism in primary astroglia cultures from HGPRT-deficient transgenic mice. The HGPRT-deficient astroglia exhibited the basic abnormalities in purine metabolism reported before in neurons and various other HGPRT-deficient cells. The following abnormalities were found: absence of detectable uptake of guanine and of hypoxanthine into intact cell nucleotides; 27.8% increase in the availability of 5-phosphoribosyl-1-pyrophosphate; 9.4-fold acceleration of the rate of de novo nucleotide synthesis; manyfold increase in the excretion into the culture media of hypoxanthine (but normal excretion of xanthine); enhanced loss of label from prelabeled adenine nucleotides (loss of 71% in 24 h, in comparison with 52.7% in the normal cells), due to 4.2-fold greater excretion into the media of labeled hypoxanthine. In addition, the HGPRT-deficient astroglia were shown to contain lower cellular levels of ADP, ATP, and GTP, indicating that the accelerated de novo purine synthesis does not compensate adequately for the deficiency of salvage nucleotide synthesis, and higher level of UTP, probably due to enhanced de novo synthesis of pyrimidine nucleotides. Altered nucleotide content in the brain may have a role in the pathogenesis of the neurological deficit in Lesch-Nyhan syndrome.