Efficacy, safety and tolerability of an orally administered cannabis extract in the treatment of spasticity in patients with multiple sclerosis: a randomized, double-blind, placebo-controlled, crossover study.

OBJECTIVE: Cannabis may alleviate some symptoms associated with multiple sclerosis (MS). This study investigated the effect of an orally administered standardized Cannabis sativa plant extract in MS patients with poorly controlled spasticity. METHODS: During their inpatient rehabilitation programme, 57 patients were enrolled in a prospective, randomized, double-blind, placebo-controlled crossover study of cannabis-extract capsules standardized to 2.5 mg tetrahydrocannabinol (THC) and 0.9 mg cannabidiol (CBD) each. Patients in group A started with a drug escalation phase from 15 to maximally 30 mg THC by 5 mg per day if well tolerated, being on active medication for 14 days before starting placebo. Patients in group B started with placebo for seven days, crossed to the active period (14 days) and closed with a three-day placebo period (active drug dose escalation and placebo sham escalation as in group A). Measures used included daily self-report of spasm frequency and symptoms, Ashworth Scale, Rivermead Mobility Index, 10-m timed walk, nine-hole peg test, paced auditory serial addition test (PASAT), and the digit span test. RESULTS: In the 50 patients included into the intention-to-treat analysis set, there were no statistically significant differences associated with active treatment compared to placebo, but trends in favour of active treatment were seen for spasm frequency, mobility and getting to sleep. In the 37 patients (per-protocol set) who received at least 90% of their prescribed dose, improvements in spasm frequency (P = 0.013) and mobility after excluding a patient who fell and stopped walking were seen (P = 0.01). Minor adverse events were slightly more frequent and severe during active treatment, and toxicity symptoms, which were generally mild, were more pronounced in the active phase. CONCLUSION: A standardized Cannabis sativa plant extract might lower spasm frequency and increase mobility with tolerable side effects in MS patients with persistent spasticity not responding to other drugs.

Separation of inhibitory activity from biologically active parathyroid hormone in patients with pseudohypoparathyroidism type I.

Patients with pseudohypoparathyroidism type I have the symptoms of hypoparathyroidism despite elevated levels of immunoreactive parathyroid hormone (PTH). However, the circulating levels of bioactive PTH, as measured in a cytochemical bioassay, are generally within the normal range suggesting that the high levels of immunoreactive PTH are either due to the presence of biologically inactive fragments of parathyroid hormone or to the presence of an 'inhibitor' of PTH bioactivity. Gel-permeation chromatography has been used to fractionate plasma from patients with pseudohypoparathyroidism type I and revealed the presence of high levels of bioactive PTH and of an 'inhibitor'. This inhibitory activity was absent or much lower in plasma from control subjects. These results indicate, therefore, that in pseudohypoparathyroidism type I the expression of the biological activity of PTH at the level of the kidney is affected by the presence of a circulating inhibitor which can be separated from intact PTH by gel-permeation chromatography.

Distinct binding sites for calcitonin gene-related peptide and salmon calcitonin in rat central nervous system.

Calcitonin gene-related peptide (CGRP) binding sites have been identified in homogenates from the rat brain and spinal cord. Autoradiography with [125I]rat CGRP (rCGRP) revealed high grain density over the lateral hypothalamus, vestibular nuclei, colliculi, medial geniculate body, corpus mamillare and the molecular layer of the cerebellum which lacked binding sites for [125]salmon calcitonin (sCT). In contrast, no rCGRP labeling was seen over the anterior and dorsomedial hypothalamus which showed high sCT binding. The different regional distribution of rCGRP and sCT binding sites indicates that the structurally related peptides interact with separate receptors. The overlap between the localization of CGRP binding sites and endogenous CGRP in many regions of the central nervous system suggests that CGRP exerts unique physiological functions in the central nervous system.

Calcitonin gene-related peptide and its binding sites in the human central nervous system and pituitary.

Binding sites for synthetic human 125I-labeled calcitonin gene-related peptide (125I-CGRP) have been demonstrated in membranes of the human nervous system. Binding was high in the cerebellar cortex (1.35 +/- 0.27 fmol/mg of tissue; mean +/- SEM), spinal cord (1.06 +/- 0.27 to 1.27 +/- 0.23 fmol/mg), and nucleus dentatus (1.02 +/- 0.15 fmol/mg), intermediate in the inferior colliculus (0.80 +/- 0.14 fmol/mg) and substantia nigra (0.75 +/- 0.14 fmol/mg), low in the neocortex, globus pallidus, nucleus caudatus, hippocampus, amygdala, superior colliculus, thalamus, and hypothalamus (0.15-0.32 fmol/mg), and negligible in spinal and sympathetic ganglia and pituitary (less than 0.04 fmol/mg). Autoradiography showed distinct 125I-CGRP binding over the molecular and Purkinje cell layers of the cerebellar cortex and over the substantia gelatinosa posterior of the spinal cord. The highest levels of CGRP-like components were recognized in the dorsal part of the spinal cord and the pituitary gland. In the ventral part of the spinal cord as well as in the pituitary and thyroid glands, CGRP values were higher when measured by radioreceptorassay as compared to RIA, indicating that at least two CGRP-like components are present. The predominant CGRP-like peak on HPLC had the retention time of synthetic human CGRP. Immunohistochemistry revealed the presence of a dense plexus of CGRP immunoreactive nerve fibers in the dorsal horn of the spinal cord.

Immunoreactive calcitonin gene-related peptide and substance P coexist in sensory neurons to the spinal cord and interact in spinal behavioral responses of the rat.

Using immunohistochemistry evidence was obtained for the coexistence of calcitonin gene-related peptide (CGRP)- and substance P (SP)-like immunoreactivity in spinal sensory neurons. Analysis of caudally directed biting and scratching (CBS) behavior was carried out after intrathecal administration of CGRP and SP alone or in combination. Thus, SP (up to 20 micrograms) alone caused CBS only for a few minutes after injection, whereas SP (10 micrograms) plus CGRP (20 micrograms) caused a response with a duration up to 40 min. CGRP (20 micrograms) alone had no effects in this model. These findings provide support for a possible interaction of the two peptides at synapses in the dorsal horn of the spinal cord.

Calcitonin gene-related peptide in the human thyroid, pituitary and brain.

Alternative splicing of rat calcitonin (CT) gene transcripts resulting in the production of calcitonin gene-related peptide (CGRP) in neural tissue and of CT in the thyroid has been proposed by Rosenfeld et al. (1983b). We have recognized CGRP- and CT-like peptides in extracts of the human brain, pituitary and thyroid using a combination of gel filtration analysis and HPLC, and specific RIAs. Immunoreactive CGRP was estimated in a heterologous RIA using 125I-labelled rat CGRP as ligand and antibodies raised to the rat hormone, and human CT in a homologous RIA. The levels of CGRP and CT are measured against synthetic rat CGRP and monomeric human CT, respectively, and expressed in ng and micrograms equivalents (eq). The content of immunoreactive CGRP of the neocortex (n = 3), the cerebellar cortex (n = 6), the periventricular mesencephalic region (n = 3) and the thyroid (n = 5) was similar (mean +/- SE, 0.79 +/- 0.16 ng eq/g wet tissue, 1.51 +/- 0.14 ng eq/g, 1.84 +/- 0.12 ng eq/g and 5.0 +/- 0.9 ng eq/g, respectively), whereas pituitary glands (n = 21) and medullary thyroid carcinomas (n = 6) contained higher levels (31.3 +/- 5.1 ng eq/g and 7.66 +/- 5.42 micrograms eq/g, respectively). Immunoreactive CT was lowest in the neocortex, cerebellar cortex and the periventricular mesencephalon (0.31 +/- 0.07 ng eq/g, 0.30 +/- 0.09 ng eq/g and 0.26 +/- 0.09 ng eq/g, respectively), followed by the pituitary and thyroid (2.77 +/- 0.62 ng eq/g and 146 +/- 26 ng eq/g, respectively), and was highest in medullary thyroid carcinoma tissue (680 +/- 372 micrograms eq/g).(ABSTRACT TRUNCATED AT 250 WORDS)

Salmon and human calcitonin-like peptides in man.

Calcitonin-like peptides have been identified in the serum of normal subjects and of medullary thyroid carcinoma (MTC) patients. Using specific homologous radioimmunoassays (RIA) in combination with reversed-phase high performance liquid chromatography and gel permeation chromatography under denaturing conditions, we have recognized major components which coeluted with human calcitonin-(1-32), PDN-21, a carboxyl-terminal flanking peptide derived from the calcitonin mRNA sequence, and salmon calcitonin-(1-32). An additional 12000 molecular weight peak possibly represents a human calcitonin-PDN-21 polyprotein. In both the human calcitonin-(1-32) (normal value less than 0.043 ngEq/ml; MTC 140 +/- 80 ngEq/ml, mean value +/- SEM) and the PDN-21 (normal value less than 0.050 ngEq/ml; MTC 33.6 +/- 16.5 ngEq/ml) RIAs, serum levels were increased in MTC patients. Circulating levels of the salmon calcitonin-like peptide were indistinguishable between normal subjects (0.038 +/- 0.006 ngEq/ml) and MTC patients (0.037 +/- 0.011 ngEq/ml).

Pseudohypoparathyroidism: inheritance and expression of deficient receptor-cyclase coupling protein activity.

Pseudohypoparathyroidism, Type I (PSP-I) is a familial disorder characterized by secondary hyperparathyroidism, resistance of urinary cyclic adenosine-3', 5'-monophosphate (cAMP) excretion to exogenous parathyroid hormone (PTH), and by effects upon other hormones, including thyrotrophin (TSH) hyperresponsiveness to thyroliberin (TRH). In the present study, 12 PSP-I patients in five families exhibited partial deficiency of receptor-cyclase coupling protein (N protein) in blood cells, in association with the skeletal findings of Albright's hereditary osteodystrophy. In one father and six mothers of PSP-I patients, deficient N protein activity was associated with normal urinary cAMP responses to PTH. In this group of seven parents, five had Albright's osteodystrophy, two exhibited secondary hyperparathyroidism, and two had TSH hyperresponsiveness to TRH. In a sixth family with none of the features of Albright's osteodystrophy, N protein deficiency did not correlate with urinary cAMP responsiveness to PTH. In this kindred, one mother with N protein deficiency, but normal urinary cAMP responsiveness to PTH had raised serum levels of immunoreactive PTH. We conclude that in the majority of families with PSP-I the urinary cAMP response to PTH is an inadequate indicator of the genetic defect. In such families, deficiency of N activity more consistently points to metabolic defects, including secondary hyperparathyroidism and TSH hyperresponsiveness, even when urinary cAMP responses are normal.

Salmon and human calcitonin-like peptides coexist in the human thyroid and brain.

A salmon calcitonin-like material indistinguishable from synthetic salmon calcitonin-(1-32) on high performance liquid chromatography (HPLC) has been recognized in thyroid extracts of normal subjects and of patients with medullary carcinoma. The same peptide was detected in extracts of the periventricular mesencephalic region which included the periventricular dorsal thalamus, the subthalamus and the hypothalamus. Human calcitonin-(1-32)- and carboxyl-terminal adjacent peptide (CCAP)-like components were also found. The content of immunoreactive salmon calcitonin of the periventricular mesencephalic region (n = 6) and of normal thyroid glands (n = 6) was comparable (mean +/- SE, 0.34 +/- 0.17 ngeq/g wet tissue and 0.39 +/- 0.22 ngeq/g, respectively); and the levels were slightly, but not significantly higher in medullary thyroid carcinoma extracts (1.95 +/- 0.69 ngeq/g) (P less than 0.1). Immunoreactive human calcitonin and CCAP occurred in roughly equimolar concentrations. They were lowest in the periventricular mesencephalic region (0.26 +/- 0.09 ngeq/g and 0.46 +/- 0.10 ngeq/g, respectively), followed by normal thyroid glands (146 +/- 26 ngeq/g and 94 +/- 19 ngeq/g, respectively), and they were highest in medullary thyroid carcinoma tissue (680 +/- 372 mu geq/g and 144 +/- 125 mu geq/g, respectively).

Identification and characterization of calcitonin forms in plasma and urine of normal subjects and medullary carcinoma patients.

Different immunoreactive calcitonin (CT) forms were identified by a reversed phase high performance liquid chromatography gradient system in plasma and urine of normal subjects. The separated CT components were characterized by chemical and enzymatic methods and found to be identical in normal subjects and medullary thyroid carcinoma patients. Of seven major CT forms we identified monomeric human CT-(1-32), its sulfoxide form, and dimeric CT in plasma. The monomeric, but not the dimeric form, was also detected in urine. A predominant CT component in plasma with a molecular weight of about 12,000 may correspond to a biosynthetic precursor of the hormone.

Potassium stimulates parathyroid hormone release in the absence of extracellular calcium.

Potassium-stimulated release of many hormones requires the presence of extracellular calcium. At variance with this mechanism, potassium-evoked parathyroid hormone (PTH) release from perifused dispersed bovine parathyroid cells also occurred in calcium-free medium containing 1 mM EGTA. Tetraethylammonium, which presumably suppresses the efflux of potassium from parathyroid cells, also stimulated PTH secretion. Removal of sodium did not suppress potassium-stimulated PTH secretion, but inhibited the release of the hormone evoked by calcium removal and by isoproterenol. Ouabain, on the other hand, suppressed the release of PTH evoked by calcium removal and by potassium, but not by isoproterenol. Unlike high potassium and removal of calcium, isoproterenol caused a parallel increase in cAMP release. In conclusion, PTH secretion is reversibly stimulated by potassium in the absence of extracellular calcium. Our findings suggest that potassium stimulates PTH secretion by a unique mechanism the nature of which remains to be elucidated.

Inhibition of cytochemical bioactivity of parathyroid hormone by plasma in pseudohypoparathyroidism type I.

Despite the high circulating levels of immunoreactive PTH in patients with pseudohypoparathyroidism type I (PSPI) the levels of bioactive PTH (bioPTH) have been found to be close to the normal range. To elucidate this dissociation, we have studied the recovery of the biological activity of bovine PTH added to the plasma of patients with either PSPI, or with hypoparathyroidism (PTX), primary hyperparathyroidism (HPT) or of normal subjects. In PSPI (n = 10) the recovery of biological activity was 5.6% +/- 3.6 (mean +/- SEM) whereas in PTX (n = 7), in HPT (n = 4) and in normal subjects (n = 8) it was 79% +/- 5, 75% +/- 9 and 68% +/- 4, respectively. In another PSPI patient, who had undergone total parathyroidectomy, bioPTH was undetectable but the recovery from the plasma of added PTH was 84%. Thus we have found inhibition of PTH bioactivity by plasma of PSPI patients which was absent after parathyroidectomy.

The distribution of the glycosaminoglycans in the anatomic components of the lung and the changes in concentration of these macromolecules during development and aging.

The glycosaminoglycans of the normal human and bovine lungs and of the major structural components of these organs (pleura, 'alveoli', peripheral and central bronchi, arteries and veins) were investigated. To carry out this study, a micromethod for the separation and quantitative determination of these macromolecules, namely two-dimensional electrophoresis on cellulose acetate plates, was employed. This procedure made it possible to measure the content of each glycosaminoglycan present in the mentioned anatomic components. In the human lung the distribution of the glycosaminoglycans varies considerably from one component to another: dermatan sulfate was the predominant mucopolysaccharide of the pleura, chondroitin 6-sulfate that of the central bronchi, and heparan sulfate and chondroitin sulfate those of the alveoli. Heparin and keratan sulfate were not detected in any of the structural components. Significant changes in the mucopolysaccharide levels were found during maturation and aging. Further age-related changes were noted between 22 and 39 years. In the bovine lung significant changes in the glycosaminoglycan levels were also observed during growth and aging. Heparin appeared in the lung at an age between 1 and 16 months. Similarities and differences in the total contents and compositions of the glycosaminoglycans between the human and bovine lung were noted.

Synthesis of glycosaminoglycans by cultured rabbit smooth muscle cells.

Rabbit aortic smooth muscle cells were evaluated for their ability to synthesize and accumulate glycosaminoglycans (GAGs). Because of the sensitivity of the microtechniques utilized, it is possible to determine the specific radioactivity of the GAGs obtained after radioactive incorporation of [35S]SO4(2-) and [14C]glucosamine. Data obtained at various incubation times indicate that the distribution of the GAGs secreted by the cells into the medium is different from that retained by the cell layer. Hyaluronic acid was shown to be the most abundantly produced GAG, and much of this GAG does not appear to be incorporated into the extracellular matrix. Also, a high percentage of the total chondroitin sulfate B synthesized was secreted into the medium. On the other hand, most of the heparan sulfate and chondroitin sulfate C/A synthesized seems to be associated with the cell layer. These results are consistent with those found in whole rabbit aorta.