J. A. Parsons

Anabolic effect of human parathyroid hormone fragment on trabecular bone in involutional osteoporosis: a multicentre trial.

After baseline studies, 21 patients with osteoporosis were treated with human parathyroid hormone fragment (PTH 1-34) given as once-daily subcutaneous injections for 6-24 months. The dose used did not cause hypercalcaemia even in the first few hours after injection. Calcium and phosphate balances improved in some patients, but there was no significant improvement in the group values. There were, however, substantial increases in iliac trabecular bone volume: the mean increase, confirmed by repeat blind measurements, was 70% above mean baseline volume. The new bone was histologically normal. Those patients who had the largest increases in 47Ca-kinetic and histomorphometric indices of new bone formation showed the greatest increases in trabecular bone volume, suggesting that treatment with human parathyroid hormone fragment caused a dissociation between formation and resorption rates that was confined to trabecular bone. Since vertebrae are four-fifths composed of trabecular bone, this hormone fragment may prove useful in treating patients with the crush fracture syndrome.

A SENSITIVE BIOASSAY OF PARATHYROID HORMONE IN PLASMA

Understanding of calcium metabolism in health and disease has been retarded by the lack of an adequately sensitive bioassay of parathyroid hormone. The problem of dissociation of bioactivity and immunoactivity, well recognized for other polypeptide hormones, is exaggerated in the case of parathyroid hormone by the disproportionately long half‐time in the circulation of the immunoreactive fragments. A new method of assaying the biological activity of parathyroid hormone in plasma has been developed, based on the cytochemical methods which have yielded highly sensitive bioassays of other polypeptide hormones. It depends on the stimulation of glucose 6‐phosphate dehydrogenase activity in the distal convoluted tubules of segments of guinea‐pig kidney maintained in vitro, and measured by microdensitometry. The limit of sensitivity of the assay is 5 fg/ml (bPTH); the index of precision is 0.09 ± 0.04 (mean ± SEM; n= 11).

Use of liposomes to aid intestinal absorption of entrapped insulin in normal and diabetic dogs

Abstract The effectiveness of liposomes in aiding intestinal absorption of entrapped insulin was studied in normal and diabetic dogs. Intraduodenal administration of free insulin (490 and 1630 U) or free insulin (88 U) plus empty liposomes to normal conscious dogs produced no change in plasma immunoreactive insulin or glucose Administration of 40–80 U insulin entrapped in liposomes composed of either phosphatidylcholine, distearoylphosphatidylcholine, or dipalmitoylphosphatidylcholine with cholesterol and dicetylphosophate ( in the ratio 10:2:1 by weight) to normal dogs produced substantial rises in peripheral plasma immunoreactive insulin after 45–60 min. However, the magnitude of these rises was neither reproducible nor dose-dependent. No fall in plasma glucose was observed. Intraduodenal administration of 50–100 U insulin entrapped in liposomes to diabetic dogs also produced rises in plasma immunoreactive insulin levels after 45–60 min but again these rises were not dose-related. However, unlike the results in normal dogs, a small fall in plasma glucose followed the plasma immunoreactive insulin rise in diabetic dogs. This glucose fall was not dose-dependent nor was it related to the magnitude of the rise in plasma immunoreactive insulin. In conclusion, it seems that administration of insulin in liposomes may allow absorption of partially degraded insulin into the circulation but the rise in plasma immunoreactive insulin observed in normal and diabetic dogs and the fall in plasma glucose in diabetic dogs are not influenced by the dose of insulin entrapped nor the lipid composition of the liposomes.

Prolonged Hypoglycemic Effect in Diabetic Dogs Due to Subcutaneous Administration of Insulin in Liposomes

SUMMARY The biologic action of insulin entrapped in liposomes (phospholipid vesicles) has been investigated following subcutaneous injection to dogs made diabetic with a combination of alloxan and streptozotocin. The fate of the liposomally entrapped material was determined by injecting rats subcutaneously with either 125l-insulin or the labeled polysaccharide 14C-inulin, incorporated in liposomes labeled with 3H-cholesterol. Injection of liposome insulin (0.75 U/kg) to five diabetic dogs resulted in a mean (± SEM) blood glucose fall from 16.4 ± 0.8 to 2.9 ± 0.4 mmol/L. The glucose level had still not returned to baseline after 24 h and, correspondingly, immunoreactive insulin (IRI) could still be detected in frozen and thawed plasma 24 h after injection. In contrast, the hypoglycemic effect of the same dose of free insulin with or without empty liposomes virtually ended within 8 h and IRI levels returned to baseline by 3 h after injection. In experiments on rats with liposomally entrapped 125l-insulin or 14C-inulin the proportion of the injected dose of tracer recoverable by excision of the injection site remained constant after about 1 h and 70% of the dose was still fixed in subcutaneous tissue for at least 5 h thereafter. When the plasma collected 3 h after subcutaneous injection of labeled liposomes containing 125I-insulin was passed through a column of Sepharose 6B, 50–75% of the 125I-activity was found in the fractions associated with intact liposomes. One possibility for the persistence of the hypoglycemic effect and of measurable IRI following injection of liposome insulin could be the presence of intact liposomes in the circulation for many hours after absorption had ceased.

In vivo STUDIES ON AN ANTAGONIST OF PARATHYROID HORMONE [Nle-8, Nle-18, Tyr-34]bPTH-(3–34)AMIDE

1 The actions of parathyroid hormone (PTH) are antagonized in vitro by the peptide [Nle‐8, Nle‐18, Tyr‐34]‐bPTH‐(3–34)amide, an analogue of PTH. In this paper, the actions of the inhibitory peptide were investigated in vivo. 2 Native parathyroid hormone (bPTH‐(1–84)), administered i.v. (0.17–1.51 nmol in a volume of 0.3 ml) to 7 day old chicks produced hypercalcemia but administration of the analogue in doses up to 173 nmol was ineffective in this respect. 3 The analogue failed to antagonize the hypercalcaemia produced by bPTH‐(1–34) when injected, in 10 fold molar excess, 2 min before or simultaneously with bPTH‐(1–34). 4 Normocalcaemia was restored in parathyroidectomized rats by intravenous infusion of bPTH‐(1–84) at 32 pmol kg−1 h−1. Addition of the analogue to the infusion fluid in a 200 fold molar excess did not affect the concentrations of calcium and phosphate in the plasma, cyclic adenosine 3′,5′‐monophosphate (cyclic AMP) in the urine or phosphate clearance but produced a significant (P< 0.05) rise in urinary calcium clearance. 5 The results suggest that the peptide [Nle‐8, Nle‐18, Tyr‐34]‐bPTH‐(3–34)amide does not antagonize the actions of PTH in vivo and demonstrate an important dichotomy between in vitro and in vivo biological properties of the PTH analogue.

Evidence that protease inhibitors reduce the degradation of parathyroid hormone and calcitonin injected subcutaneously.

1 Agents known to delay absorption from a subcutaneous site were tested in chicks for their ability to prolong the hypercalcaemic response to parathyroid hormone (PTH). 2 Polyvinylpyrrolidone was found to enhance the response but gelatine greatly reduced the 2 h hypercalcaemia. 3 The reduction by gelatine was reversed when the protease inhibitor aprotinin was added to the injection vehicle, and hypercalcaemia then persisted for more than 8 h. 4 Of other protease inhibitors studied, e‐aminocaproic acid was also found to enhance the hypercalcaemic response to subcutaneous PTH and its fragments but, unlike aprotinin, it was ineffective in the presence of gelatine. 5 By radioimmunoassay and bioassay respectively, it was confirmed that aprotinin raised circulating levels of PTH and also of another peptide hormone, calcitonin, injected subcutaneously. 6 Addition of calcium to the solutions injected subcutaneously abolished the hypercalcaemic response to PTH while injection of calcium and PTH simultaneously but at separate sites left the response unaltered. 7 The two protease inhibitors, e‐aminocaproic acid and aprotinin, each restored the response to subcutaneous PTH despite the presence of calcium at the injection site. 8 It was concluded that protease inhibitors injected subcutaneously with PTH and calcitonin in the chick reduced the rate of degradation of these hormones and that the proteases responsible for hormone degradation at the subcutaneous injection site may be released or activated by calcium ions.

An Homologous and Sensitive Radioimmunoassay for the Synthetic Amino-Terminal (1-34) Fragment of Human Parathyroid Hormone: Application to the Clearance of This Peptide Administered in Vivo

The synthetic 1-34 amino-terminal fragment of human parathyroid hormone (hPTH 1-34) is undergoing multicentre clinical trials to assess its long term therapeutic potential in the treatment of osteoporosis. An homologous radioimmunoassay (reagents prepared from the synthetic hPTH 1-34 peptide) has been developed to monitor the pharmacokinetics of hPTH 1-34 in man and in a dog model. The assay is rugged, sensitive (detection limit 1.75 x 10(-11) moles/litre) and precise (coefficient of variation 6%). Three different ampouled preparations of the native intact hPTH 1-84, of different degrees of purity (approximately 3%-90% pure) gave complete log dose response curves parallel to that of the ampouled synthetic hPTH 1-34 peptide, and were equipotent on a molar basis. Native intact bovine PTH 1-84 showed an incomplete non-parallel displacement curve; there was no recognition of synthetic hPTH 44-68 and 53-84 peptides. Preliminary application of the assay to the determination of the plasma disappearance of hPTH 1-34 in man and dog gave half-times (t1/2) of 3-8 minutes for a first exponential component and 12-18 minutes for the second; in the dog, metabolic clearance rate was calculated to be 9ml/kg/minute and the distribution space 160ml/kg.

Cat assay for the emetic action of digitalis and related glycosides (digitoxin, digoxin, lanatoside C, ouabain and calactin)

1 A titration assay with two end points is described for comparison of the emetic and lethal potencies of digitalis‐like drugs. 2 A drug was infused at constant rate to a conscious, unrestrained cat, through an indwelling venous cannula. At the moment of vomiting the cat was rapidly anaesthetized and infusion continued at the same rate until the moment of cardiac arrest. 3 With very slow and very fast infusions, the emetic and lethal doses tended to rise. In the range between these extremes (which varied from drug to drug) they were independent of time. 4 The observations could be accounted for by analogue computation, assuming that the drugs entered an initial pool and were distributed at finite rates to receptors in the CNS (vomiting centre) and heart. 5 Half times of metabolic loss derived from this computation for digitoxin, digoxin and ouabain (17, 9·9 and 1·8 h, respectively) were in the same ratio as the threefold longer half times reported for these drugs in man. 6 When measured with infusion rates in the time independent range, the ratio of lethal to emetic doses did not vary between the drugs studied. All caused vomiting at 40% of the lethal dose. 7 From a review of the literature, the emetic and cardiotoxic actions of digitalis‐like drugs appear inseparable and probably share a common biochemical mechanism. 8 It is concluded that foreseeable improvements in digitalis‐like drugs are small and would depend on the elimination of any local emetic effect on gut receptors which they may have.

A study on the mode of action and composition of a toxin from the female abdomen and eggs of Arctia caja (L.) (Lep. Arctiidae): An electrophysiological, ultrastructural and biochemical analysis

Abstract Cajin, the toxic principle extracted from the female abdomen and eggs of the aposematic moth Arctia caja, was investigated by means of column chromatography, and its effects on tissues were studied by electrophysiology and electron microscopy. Partial purification suggests that one fraction is a polypeptide with a molecular weight (mol. wt) of about 1000. It is lethal to both mammals and insects if injected into the body, but inactive via the oral route. Crude extracts applied to insect muscles produce a slow contracture which lasts for over 1 1 2 hr and is irreversible. Fine structural analyses of insect tissues after 5 min toxin treatment reveal disruption of both internal membranous systems and mitochondria. Unlike many other toxins, cajin appears to act by stimulating the muscle directly. The results of experiments involving Ca2+-free solutions plus cajin suggest that it may increase the permeability of the muscle membrane to Ca2+, perhaps behaving as a calcium ionophore, or may act as an analogue of an insect neurotransmitter.

Double isotope estimates of intestinal calcium absorptio in rats: Enhancement by parathyroid hormone and 1,25-dihydroxycholecalciferol

It is generally accepted that parathyroid hormone (PTH) acts on the intestine indirectly, by enhancing renal production of the active metabolite of vitamin D (1,25-DHCC; for reviews see Kodicek, 1972; DeLuca, 1974; Norman and Henry, 1974). However there is some evidence that it may also have a direct action (Olson et al., 1972), a possibility which would be favoured if it could be shown that the effects of the two agents were additive. This paper is a preliminary account of a method for the in vivo measurement of the absorption coefficient (a), using two isotopes of calcium in mature rats. The technique has proved suitable for experiments of bioassay design to make quantitative comparisons of the calcium absorpti0n-enhancing potency of hormones, and seems likely to be generally useful for further investigation of the pharmacology of other agents affecting intestinal calcium absorption.