Balicatib, a cathepsin K inhibitor, stimulates periosteal bone formation in monkeys.

UNLABELLED: Balicatib, an inhibitor of the osteoclastic enzyme cathepsin K, was tested in ovariectomized monkeys, a model for osteoporosis. As expected, ovariectomy-induced bone mass changes were partially prevented by balicatib treatment. Bone turnover was significantly decreased at most sites, but unlike most bone resorption inhibitors, periosteal bone formation rates were increased. INTRODUCTION: Selective inhibitors of the osteoclastic enzyme cathepsin K have potential in osteoporosis treatment. This study evaluated the efficacy of balicatib (AAE581), a novel inhibitor of human cathepsin K, on bone mass and dynamic histomorphometric endpoints in ovariectomized monkeys. METHODS: Eighty adult female Macaca fascicularis underwent bilateral ovariectomies and were dosed twice daily by oral gavage with balicatib at 0, 3, 10, and 50 mg/kg for 18 months (groups O, L, M, H, respectively). Approximately 1 month after treatment initiation, the 50 mg/kg dose was decreased to 30 mg/kg. Twenty animals underwent sham-ovariectomies (group S). Bone mass was measured at 3-6 month intervals. At 18 months, vertebra and femur were collected for histomorphometry. RESULTS: In both spine and femur, group O animals lost BMD and all other groups gained BMD between 0 and 18 months. In balicatib-treated animals, BMD change in the spine was intermediate between group S and O, with groups L and M significantly different from group O. In femur, all three doses of balicatib significantly increased BMD gain relative to group O and group mean values were also higher than group S. Most histomorphometric indices of bone turnover in vertebra and femoral neck were significantly lower than group O with balicatib treatment, except that periosteal bone formation rates (Ps.BFR) were significantly higher. Ps.BFR in mid-femur was also significantly increased by treatment. CONCLUSIONS: Balicatib partially prevented ovariectomy-induced changes in bone mass, inhibited bone turnover at most sites, and had an unexpected stimulatory effect on periosteal bone formation.

Balicatib, a cathepsin K inhibitor, stimulates periosteal bone formation in monkeys.

UNLABELLED: Balicatib, an inhibitor of the osteoclastic enzyme cathepsin K, was tested in ovariectomized monkeys, a model for osteoporosis. As expected, ovariectomy-induced bone mass changes were partially prevented by balicatib treatment. Bone turnover was significantly decreased at most sites, but unlike most bone resorption inhibitors, periosteal bone formation rates were increased. INTRODUCTION: Selective inhibitors of the osteoclastic enzyme cathepsin K have potential in osteoporosis treatment. This study evaluated the efficacy of balicatib (AAE581), a novel inhibitor of human cathepsin K, on bone mass and dynamic histomorphometric endpoints in ovariectomized monkeys. METHODS: Eighty adult female Macaca fascicularis underwent bilateral ovariectomies and were dosed twice daily by oral gavage with balicatib at 0, 3, 10, and 50 mg/kg for 18 months (groups O, L, M, H, respectively). Approximately 1 month after treatment initiation, the 50 mg/kg dose was decreased to 30 mg/kg. Twenty animals underwent sham-ovariectomies (group S). Bone mass was measured at 3-6 month intervals. At 18 months, vertebra and femur were collected for histomorphometry. RESULTS: In both spine and femur, group O animals lost bone mineral density (BMD), and all other groups gained BMD between 0 and 18 months. In balicatib-treated animals, BMD change in the spine was intermediate between group S and O, with groups L and M significantly different from group O. In femur, all three doses of balicatib significantly increased BMD gain relative to group O, and group mean values were also higher than group S. Most histomorphometric indices of bone turnover in vertebra and femoral neck were significantly lower than group O with balicatib treatment, except that periosteal bone formation rates (Ps.BFR) were significantly higher. Ps.BFR in mid-femur was also significantly increased by treatment. CONCLUSIONS: Balicatib partially prevented ovariectomy-induced changes in bone mass, inhibited bone turnover at most sites, and had an unexpected stimulatory effect on periosteal bone formation.

Antiemetic action of 5-HT3 receptor antagonists: review of preclinical and clinical results with ICS 205-930.

These results thus indicate, that ICS 205-930 is a very effective antiemetic for cancer chemotherapy-induced emesis in man. Given as a single dose prior to the chemotherapeutic agent, ICS 205-930 inhibits emesis and nausea for at least 24 hr. Together with the lack of extrapyramidal side-effects, these properties of ICS 205-930 indicate a clear superiority to the current therapeutic standard metoclopramide.

The serotonin 5-HT4 receptor. 1. Design of a new class of agonists and receptor map of the agonist recognition site.

The design and synthesis of a new class of potent and selective 5-HT4 receptor agonists containing an indole nucleus linked to a carbazimidamide are presented. A conformational study of the 5-HT4 receptor agonists serotonin and zacopride led to the identification of an initial pharmacophore and to the definition of a three-dimensional map of the 5-HT4 agonist recognition site. 1, a representative member of our new class of 5-HT4 receptor agonists, incorporates all reference structural features and matched perfectly with these models. 1 is a highly potent, full agonist at 5-HT4 receptors present in the isolated electrically stimulated guinea pig ileum preparation, with a pD2 value of 8.8, displaying selectivity (ranging from 40- to over 10,000-fold) versus other members of the serotonin receptor family.

The serotonin 5-HT4 receptor. 2. Structure-activity studies of the indole carbazimidamide class of agonists.

A number of substituted indole carbazimidamides were prepared and evaluated as 5-HT4 receptor agonists by using the isolated field-stimulated guinea pig ileum preparation. Their selectivity for the 5-HT4 receptor was established by examining their affinity for other 5-HT receptors using radioligand-binding techniques. Several selective and highly potent full as well as partial agonists emerged from this study. For example, 1b,d were found to be the most potent, full 5-HT4 receptor agonist described so far (EC50 = 0.5 and 0.8 nM, respectively), being 6 and 4 times more potent than serotonin itself. On the other hand, 5b and 1h appeared as partial 5-HT4 receptor agonists in the nonstimulated guinea pig ileum preparation with potencies, evaluated against serotonin action, respectively similar (5b, Ki = 12 nM) to and 300-fold higher (1h, Ki = 0.04 nM) than serotonin.

Capsaicin induced release of multiple tachykinins (substance P, neurokinin A and eledoisin-like material) from guinea-pig spinal cord and ureter.

The release of tachykinins from isolated slice preparations of the guinea-pig spinal cord and ureter was studied in vitro. Capsaicin (10 microM) caused release of substance P, neurokinin A and an eledoisin-like component from both the spinal cord and ureter. The release of tachykinins induced by capsaicin or potassium (60 mM) was calcium dependent. No detectable release of neurokinin B or neuropeptide K, an N-terminally extended form of neurokinin A, was induced by capsaicin. No detectable release of tachykinins could be demonstrated after exposure to agents which are known to activate C-fibre afferents, such as histamine, bradykinin, serotonin, prostaglandins E1, E2 or acetylcholine. Protein extravasation in the ureter, as determined by the Evans Blue extravasation technique was used as a functional correlate to the tachykinin release. Protein extravasation was induced in vivo by local intraluminal injections of capsaicin at several hundred-fold lower concentrations than those required to induce a detectable release of tachykinins in vitro. The difference may, however, partly depend on the experimental conditions and the detection limit of the tachykinin assay used. The protein extravasation response to capsaicin was absent after systemic capsaicin pretreatment, which causes a marked depletion of tachykinins in the ureter. In conclusion, capsaicin evokes release of several tachykinins from both central and peripheral endings of primary afferent neurons. The peptides released from sensory nerves in the periphery may induce effects such as protein extravasation and smooth muscle contraction.

Decrease of substance P in primary afferent neurones and impairment of neurogenic plasma extravasation by capsaicin.

1 Rats were pretreated with capsaicin (50 mg/kg, s.c.) on the 2nd, 10th, or 20th day of life. Three months later immunoreactive substance P (I-SP) was determined in skin, sensory nerves and the central nervous system. Neurogenic plasma extravasation was also examined.2 Pretreatment at the age of 2 or 10 days resulted in a decrease (26 to 69%) of I-SP in skin, saphenous and vagus nerve, dorsal roots, dorsal half of the spinal cord, and medulla oblongata. The I-SP content of the ventral half of the spinal cord, of midbrain, hypothalamus, striatum, cortex, and cerebellum remained unchanged. Neurogenic plasma extravasation was inhibited by more than 80%.3 In contrast to this irreversible effect of capsaicin on newborn rats, pretreatment of 20 day old rats led to reversible depletion of I-SP and to reversible impairment of neurogenic plasma extravasation.4 Capsaicin pretreatment of adult rats caused a marked depletion of I-SP in the skin of the hind paw and an impairment of neurogenic plasma extravasation. A similar decrease of I-SP was seen after chronic denervation of the skin.5 Intra-arterial infusion of substance P (threshold dose 5 x 10(-13) mol/min) or physalaemin induced dose-dependent plasma extravasation. Somatostatin, vasoactive intestinal polypeptide, caerulein and the enkephalin-analogue FK 33-824 were ineffective in doses 100 fold higher.6 The results indicate that the action of capsaicin on substance P neurones is restricted to primary sensory neurones. Since in every case a decreased substance P content of the skin was associated with impaired neurogenic plasma extravasation, it is suggested that release of substance P is involved in neurogenic plasma extravasation.

5-HT3 receptor antagonists inhibit sensory neuropeptide release from the rat spinal cord.

5-HT3 receptors may be present on primary afferent neurons containing substance P (SP), neurokinin A (NKA) or calcitonin gene-related peptide (CGRP). We investigated the release of SP-, NKA- and CGRP-immunoreactivities (IR) from rat spinal cord slices. Thirty mM potassium chloride caused an increased outflow of all three peptides, i.e. 140-190% of spontaneous release. This release was slightly enhanced in the presence of 3 x 10(-5) M 5-hydroxytryptamine (5-HT). In contrast, a significant inhibition of potassium-evoked, but not of basal NKA-IR and CGRP-IR release was observed when 10(-7) M BRL 43694 or ICS 205-930, two specific 5-HT3 receptor antagonists, were superfused together with 5-HT. In conclusion, 5-HT may facilitate the evoked release of peptides from central terminals of primary sensory neurons via 5-HT3 receptors.

Characterization of substance P-like immunoreactivity in submammalian species by high performance liquid chromatography.

Substance P-like immunoreactivity (SP-LI) as measured by RIA was found to be present in a variety of submammalian species and invertebrates. We analyzed this SP-LI in extracts from submammalian species by high performance liquid chromatography. The following species were investigated for the presence of SP-LI (RIA) which was further characterized by subsequent HPLC (investigated areas in parentheses): Hagfish (brain plus spinal cord), (brain, intestine, skin), frog (brain, intestine), turtle (brain, intestine), lizard (brain, intestine, skin) and mouse (spinal cord). RIA alone was performed in extracts from branchiostoma and cricket. The concentrations of SP-LI in brain, spinal cord and intestine of different submammalian species except branchiostoma brain and intestine and turtle brain, were in a similar range (2.1-5.3 fmol/mg in the brain, 0.2-2.0 fmol/mg in the spinal cord, 0.3-4.2 fmol/mg in the intestine). In the turtle brain, extremely high SP-LI concentrations (210 fmol/mg) were found, whereas brain and intestine of branchiostoma contained very little SP-LI (0.1 fmol/mg). In the skin of different species, SP-LI concentrations varied from 0.04 fmol/mg (trout) to 2.0 fmol/mg (lizard). In the cricket, high SP-LI concentrations were found in the cerebral ganglion (15 fmol/mg protein) and in the subesophageal ganglion (27 fmol/mg protein). HPLC analysis of extracts showed that all tissues investigated contained a substance which co-eluted with synthetic SP, and in most tissues a peak was present which co-eluted with SP sulfoxide. Only in mouse spinal cord, trout brain and hagfish brain were these the only peaks.(ABSTRACT TRUNCATED AT 250 WORDS)

Substance P in tumors: pheochromocytoma and carcinoid.

Immunoreactive substance P (I-SP) was measured in two pheochromocytomas and a carcinoid of the caecum. The pheochromocytomas contained 1,5 and 50 pmol/g I-SP respectively. An HPLC analysis I-SP and catecholamines were enriched in the chromaffin granule fraction. The carcinoid contained 2900 pmol/g I-SP. About 40% of the immunoreactivity behaved like substance P on gel filtration, HPLC and in bioassays (fall in rabbit blood pressure and contraction of the guinea-pig ileum which was abolished by specific tachyphylaxis to substance P). These results indicate that authentic substance P is present in carcinoids and pheochromocytomas and that in the latter it is most likely stored in chromaffin granules.

Capsaicin applied to peripheral nerve inhibits axoplasmic transport of substance P and somatostatin.

Capsaicin was applied locally to the sciatic or saphenous nerve, and the effects on axoplasmic transport, neurogenic plasma extravasation, and thermal pain were studied. Capsaicin (10 mg/ml) led to a complete block of axoplasmic transport of immunoreactive substance P (I-SP) and somatostatin (I-SRIF) in rat sciatic nerve without affecting the transport of noradrenaline or acetylcholinesterase. Inhibition of I-SP transport was also found in sciatic nerves of guinea-pig, cat and rabbit. In contrast, one or two weeks after systemic capsaicin treatment (125 mg/kg s.c.), orthograde transport of I-SP was the same in control and capsaicin-treated rats. After local capsaicin application to the sciatic nerve, a decrease of I-SP was found not only in skin and sciatic nerve distal to the site of application, but also in dorsal root ganglia, dorsal roots and the dorsal half of the spinal cord segments L 4-5. This was accompanied by a loss of acid phosphatase activity in the substantia gelatinosa supplied by sciatic nerve afferents. Plasma extravasation by mustard oil was reduced in the skin of the hind paw with a time course identical to the I-SP depletion. The response to noxious heat (hot plate test) was, however, abolished earlier. These results indicate that capsaicin applied to a peripheral nerve inhibits axoplasmic transport in sensory but not in adrenergic or cholinergic neurons, which leads to long-term biochemical and functional changes of the entire sensory neuron. In addition, capsaicin appears to inhibit impulse propagation in certain populations of sensory neurons.

Reduced neurogenic inflammation in streptozotocin-diabetic rats due to microvascular changes but not to substance P depletion.

The effect of streptozotocin treatment (65 mg/kg i.v.) on plasma protein extravasation, nociception, and the content of substance P immunoreactivity (SP-IR) and somatostatin immunoreactivity (SOM-IR) was investigated in the rat. Twelve days after treatment, the neurogenic plasma extravasation induced by 5% mustard oil was reduced by 67% in the skin of the hind paw. Extravasation caused by SP, a putative mediator of neurogenic inflammation, was also reduced by 61% in the abdominal skin. While calcitonin gene-related peptide (CGRP) potentiated the SP-induced extravasation in control rats, no potentiation was observed in diabetic rats. Thermonociception or chemonociception was unchanged after streptozotocin treatment. The content of SP-IR and SOM-IR in sensory nerves or spinal ganglia was also not altered. These results indicate that the impairment of neurogenic inflammation in diabetic rats is not the result of depletion of neurogenic mediators like SP. Changes of the microvasculature at the leakage site appear to account for the effects observed.

The spinal cord contains multiple factors causing plasma protein extravasation in the skin.

Nervous tissue was analyzed for possible mediators of neurogenic inflammation. Acid extracts of spinal cord or spinal roots contained activity causing plasma protein extravasation when injected into the rat abdominal skin. The activity was more than 1000-fold higher than could be attributed to the content of substance P (SP). It was not depleted from spinal cord after destruction of afferent C fibers by capsaicin and was resistant to proteolytic enzymes. The activity was clearly separated from SP or neurokinins by HPLC or gel filtration and was due to compounds of high polarity and low molecular weight. Further HPLC separated at least 6 peaks, two of which were found to contain adenosine and AMP, respectively, as active substances. The activity of these compounds and of the peaks was reduced by antihistaminics. A further compound identified was 5-HT. Thus, while several active non-peptidergic compounds were found, no clear evidence for a new mediator of neurogenic inflammation was obtained.

Potentiation of tachykinin-induced plasma protein extravasation by calcitonin gene-related peptide.

The effect of neuropeptides on plasma protein extravasation was investigated in the abdominal skin of rats. Substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) induced extravasation with a threshold dose of about 1 pmol. Calcitonin gene-related peptide (CGRP) was ineffective up to 6 pmol. However, when CGRP was injected together with either of the tachykinins extravasation was potentiated. A dose of 6 pmol CGRP shifted the dose-response curve of SP to the left by a factor of about 100. The vasoconstrictor neuropeptide Y (NPY, 12 pmol) reduced the extravasation caused by SP or SP plus 6 pmol CGRP. These results indicate that all 3 tachykinins currently known to be present in sensory neurons induce plasma protein extravasation, i.e. mimic one sign of neurogenic inflammation. This activity is potentiated in the presence of CGRP which coexists with SP and NKA indicating that neurogenic inflammation may be augmented by these interactions.

Distribution of substance P in the rat gastrointestinal tract--lack of effect of capsaicin pretreatment.

A new method for extraction of immunoreactive substance P (I-SP) from rat intestine including pulverization of tissue frozen in liquid nitrogen and extraction with acid acetone is described. Using this method, amounts of I-SP in the rat intestine were found to be higher than previously reported. The highest concentrations of I-SP were found in the small intestine. Capsaicin pretreatment of newborn or adult rats had no effect on intestinal I-SP concentrations indicating that intrinsic SP neurones are capsaicin-insensitive.

Nociceptive threshold after neonatal capsaicin treatment.

The nociceptive threshold as determined by the reaction time in the hot-plate and tail-flick tests was measured 3 to 4 months after pretreatment of young rats with capsaicin (50 mg kg-1 s.c.). The reaction time in the tail-flick test was prolonged in rats pretreated with capsaicin on the 2nd day of life. Capsaicin pretreatment up to the 10th day of life also resulted in a prolonged reaction time in the hot-plate test whereas capsaicin pretreatment after the 10th day of life was without effect. The elevation of the nociceptive threshold after neonatal capsaicin pretreatment most likely follows from degeneration of afferent nerve fibres activated by noxious stimuli.

Inhibition of 5-hydroxytryptamine- and enterotoxin-induced fluid secretion by 5-HT receptor antagonists in the rat jejunum.

The effects of cholera toxin and heat stable Escherichia coli (E. coli) enterotoxin on intestinal fluid secretion are commonly considered to be mediated by cyclic nucleotides. It was demonstrated recently, by using the 5-hydroxytryptamine (5-HT)2 receptor antagonist ketanserin and the 5-HT3 receptor antagonist tropisetron, that 5-HT acts as an important mediator in cholera toxin- and heat stable E. coli enterotoxin-induced fluid secretion. In the present investigation ketanserin and tropisetron were compared with the newer 5-HT3 receptor antagonists ondansetron and granisetron versus 5-HT-, cholera toxin- and heat stable E. coli enterotoxin-induced fluid secretion in the rat jejunum in vivo. Both ondansetron and granisetron dose-dependently inhibited 5-HT- and enterotoxin-induced fluid secretion. Ketanserin blocked 5-HT-induced fluid secretion, but only diminished enterotoxin-induced effects even at higher doses. Tropisetron inhibited 5-HT- and cholera toxin-induced effects at high dose but only diminished heat stable E. coli enterotoxin-induced effects. We conclude that 5-HT3 receptors, located on enterochromaffin cells and nervous structures, are more important in mediating fluid secretion than 5-HT2 receptors, located on the epithelial cells.

Release of neurotensin from rat spinal cord in vitro.

The release of immunoreactive neurotensin (I-NT) from the dorsal half of the rat spinal cord was studied in vitro. A basal release in the order of 0.03-0.06% per min of tissue content was found. Depolarization with K+ caused a dose-dependent increase, with 60 mM K+ causing a 7.7-fold stimulation of release. This K+-evoked release was strictly calcium-dependent. Veratridine (50 microM) produced a 4.9-fold increase which was inhibited by 0.3 microM tetrodotoxin. Noradrenaline, 5-hydroxytryptamine, glutamate, kainic acid, N-methyl-D-aspartate, or quisqualate did not stimulate release of I-NT. The results support a role of NT as a neurotransmitter or modulator in the spinal cord.

Nociceptive behavior after intrathecal injections of substance P, neurokinin A and calcitonin gene-related peptide in mice.

Intrathecal injections of substance P (SP) or neurokinin A (NKA) in the mouse caused dose-dependent reciprocal hindlimb scratching, licking and biting responses directed to the caudal part of the body. NKA decreased the latency in the tail flick assay but like SP, did not alter the reactions in the hot plate test or the hypertonic saline assay. Although immunoreactivity of calcitonin gene-related peptide (CGRP) was detected in mouse spinal cord, CGRP caused no behavioral reactions, nor did it significantly affect thermo- or chemonociception or the scratching induced by SP. Since NKA-like immunoreactivity was found to be present in sensory neurons, NKA as well as SP are likely transmitters of nociceptive primary afferent neurons.

Behavioral and neurochemical changes after intracisternal capsaicin treatment of the guinea pig.

Guinea pigs were treated with 125-150 micrograms capsaicin intracisternally (i.c.) or intraperitoneally (i.p.). Tested up to one week later, i.c.-treated animals showed reduced behavioral responses to the irritating effects of capsaicin applied to the eye or nose, to ether vapor, cigarette smoke and to hot water (50 degrees C) applied to the forepaw or ear. The concentrations of substance P (SP)- and neurokinin-immunoreactivity were decreased in the medulla oblongata but not in the trigeminal root of i.c.-treated animals as compared to controls or i.p.-treated guinea pigs. These results suggest that i.c. capsaicin causes a degeneration of the central terminals of chemo- and thermonociceptive afferent neurons, some of which contain the putative neurotransmitters SP and neurokinin A.