chemistry, physiology, and therapeutic applications of calcitonin

1139

CHEMISTRY, PHYSIOLOGY, AND THERAPEUTIC APPLICATIONS OF CALCITONIN

IAIN MacINTYRE, IMOGEN M. A. EVANS, HELMUT H. G. HOBITZ, GRAHAM F. JOPLIN, and JOHN C. STEVENSON

The chemistry and physiology of calcitonin are reviewed with particular emphasis on the evolution of the hormone and its modern role in humans. It seems likely that the relative deficiency of calcitonin in women may be important in postmenopausal bone loss. A major therapeutic application of calcitonin is in the treatment of Paget’s disease of bone, and current recommenda- tions for therapy are presented.

In 1961 Copp and colleagues postulated the existence of a calcium-lowering hormone which they named calcitonin (1). The existence of this new hormone was subsequently confirmed by Copp’s group and by Mac- Intyre’s group at the Hammersmith Hospital, London (2,3). Copp thought that calcitonin was secreted by the parathyroid gland but other evidence suggested a thyroid origin (4) and this was conclusively proved by MacIntyre and colleagues (5).

It is now firmly established that calcitonin is secreted by the parafollicular or C-cells of the thyroid (6- 9). These cells were first described over a century ago by Baber (10) and later by Nonidez (1 1). Although most of the C-cells are found in the thyroid in mammals, occasional cells may occur in the parathyroids and thymus (12). Carvalheira and Pearse demonstrated that these calcitonin-secreting cells migrate from the last ulti-

From the Endocrine Unit, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 OHS, Great Britain, and Ciba-Geigy AG, CH-400 2, Basel, Switzerland.

Supported in part by the following: Arthritis and Rheuma- tism Council, Ciba-Geigy, Laboratories Besins Iscovesco, Medical Research Council, Nuffield Foundation, Wellcome Trust.

Address reprint requests to Iain MacIntyre, D.Sc., FRCP., Professor, Endocrine Unit, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 OHS, Great Britain.

mobranchial pouch into the thyroid during embryologic development ( 13). In mammals, the ultimobranchial cells fuse with the thyroid, but in submammalian verte- brates they usually form a discrete ultimobranchial body (14,15). The ultimate embryologic origin of C- cells is the neural crest (16,17).

CHEMISTRY OF CALCITONIN Calcitonin is a peptide hormone composed of 32

amino acids. There is a disulfide bridge between the cysteine residues at positions 1 and 7, and a prolinamide at the carboxyl terminus. Major changes in the sequence can take place with retention of biologic activity; in- deed, only the prolinamide at the carboxyl-terminus, the glycine at position 28, and 7 of the 9 N-terminal residues remain constant (Figure 1). The sequences so far determined fall into 3 groups: the primate-rodent group (e.g. humans and rats), the teleost group (eg. eel and salmon), and the artiodactyl group (e.g. ox and sheep) ( 1 8). These differences in sequence necessitate the use of antisera prepared against hormones from the same group in order to obtain sufficient cross-reaction for ra- dioimmunoassay .

The entire calcitonin molecule is necessary for its hypocalcemic activity. Any shortening or modification at the carboxyl end leads to loss of this biologic activity (19). Structural modifications at the N-terminus have no effect (20), whereas opening of the disulfide ring abol- ishes the biologic activity (21).

Several circulating forms of calcitonin exist which differ immunochemically. The extraction and iso- lation of pure human calcitonin from a C-cell tumor initially revealed two forms of the hormone, the mono- meric calcitonin M and its antiparallel dimer, calcitonin

Arthritis and Rheumatism, Vol. 23, No. 10 (October 1980)

1140 MacINTYRE ET AL

H u m a n

Rat

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Salmon I I I

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Ovine

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Cys Gly A s n Leu Ser T h r Cys Met Leu Gly T h r Tyr T h r Gln Asp Phe Asn Lys Phe His T h r Phe Pro Gln T h r Ala I l e Gly Val Gly Ala Prc-C ’/

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Ala Val

A la Val

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A la T h r

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Figure 1. Amino acid sequences of the calcitonins of various species. The amino acids that differ from those of the human hormone are shown. (From MacIntyre I: J Endocrinol Invest 1:277, 1978, reproduced with permission.)

D (22,23). Since then, multiple immunoreactive forms ine), sometimes in high concentrations (Figure 2). The of calcitonin have been found in the plasma of patients molecule although very similar to human calcitonin is with medullary thyroid carcinoma and other tumors not identical (29). (24). More recently, five different immunoreactive In view of the marked differences in sequence forms of calcitonin have been demonstrated in the plasma of normal human subjects (25). Calcitonin M is a constant finding.

There are several reasons for this immunologic heterogeneity. Different antisera will describe different elution profiles of calcitonin in plasma. In humans, the immunodominant part of the molecule appears to be around position 17 but there are other immunoreactive sites (26). It seems possible that precursors and metabo- lites of the hormone are present among the various circulating immunochemical forms, especially since a pro- hormone has now been defined (27,28).

It is usually believed that calcitonin is not found in the agnatha but recent work has demonstrated the presence of a human calcitonin-like molecule in the brain of the hagfish (1 8). Furthermore, by means of re- fined immunochemical methods using several fully characterized specific antisera against human calcitonin, and by gel filtration and high performance liquid chro- matography, calcitonin-like immunoassayable activity has been found in several protochordates (Amphioxus, Ascidiella, Styella, and Ciona) and a cyclostome (Myx-

W I n e u r a l complex extract 6.3 12.5 25 50 100

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Figure 2. Human calcitonin-like activity in chordates as detected in an assay for human calcitonin. The immunoassay detects the substitution of one amino acid (leuI6) in human calcitonin as shown. (From Mo- lecular Endocrinology. I MacIntyre, M Szelke, eds. Amsterdam, Else- vier/North Holland Biomedical Press, 1979, pp 193-201. Reproduced with permission.)

CALCITONIN

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1141

between human and teleost calcitonin, these findings were initially surprising. It seems likely that the agnatha contained a similar molecule and that this molecule has been conserved in several mammalian groups including humans. The modem role of human calcitonin is probably quite different from its ancient parent, which may well function as a central neuromodulator or neuro- transmitter in protochordates.

PHYSIOLOGY OF CALCITONIN It is well established that the major action of cal-

citonin in humans is on bone where it directly inhibits bone resorption both in vivo and in vitro (30-32). Osteoclast activity is inhibited and, in the longer term, osteoclast number is reduced (33), leading to an overall decrease in the circulating levels of the products of resorption (34). Although this action may contribute to

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0

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treatment with

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Figure 4. Plasma calcitonin levels in postmenopausal women before, during, and after 12 weeks treatment with either percutaneous estra- diol (Oestrogel) or ethinylestradiol.

plasma calcium homeostasis, especially in children where bone turnover is high (35), its role in plasma calcium regulation in adults is probably not of as great importance. Rather it appears that a major function of calcitonin in adults is in the maintenance of the skeleton.

Normal women are relatively deficient in calcitonin compared to men (36,37). But the circulating levels of calcitonin in women show a sharp increase at times of increased physiologic need for calcium, such as during pregnancy and lactation (38) (Figure 3). At other times of physiologically disturbed calcium and skeletal homeostasis that occur in the fetus, neonate, and grow- ing child, calcitonin levels are again elevated (39). In all these situations the plasma levels of 1,25-dihydroxyvitamin D are elevated (40,41) to satisfy the enhanced need for calcium. It thus seems likely that calcitonin ensures that this function of 1,25-dihydroxyvitamin D is not ful- filled at the expense of the skeleton by opposing the re- sorptive action of the vitamin D metabolite.

There are further implications for this concept of the role of calcitonin. The relative deficiency of the hormone may explain why, for example, PTH-induced bone loss is much more severe in women (42). It may also be of importance in the pathogenesis of postmenopausal osteoporosis. In view of the sex difference it seems possible that the gonadal steroid hormones may

MacINTYRE ET AL 1142

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Figure 5. Serum alkaline phosphatase levels in patients who have required retreatment with synthetic human calcitonin after cessation of therapy.

be involved in the regulation of calcitonin secretion. In- deed, the administration of the combined estrogedpro- gestogen contraceptive pill causes a marked elevation in calcitonin levels (37). We have therefore recently stud- ied the effects of estrogen on calcitonin levels in postmenopausal women (43). The administration of estrogen causes a clear cut increase in plasma calcitonin levels (Figure 4) which is accompanied by a small fall in serum calcium and phosphate, and a small rise in plasma PTH which may be an homeostatic response.

The implications of these findings are significant. The loss of estrogen at menopause may exaggerate the decline of calcitonin that occurs with age (3944). Thus after menopause there will be a presumed decline in bone matrix formation due to loss of estrogen together with an inability to reduce bone resorption appropri- ately because of a lack of calcitonin. The overall result

would be loss of bone. It would follow that the known effect of estrogen to prevent bone loss (4547) may be achieved by a reversal of these two mechanisms.

There are pharmacologic actions of calcitonin on other organs such as kidney, gut, and pituitary which have been recently reviewed (48). Their true physiologic significance requires further elucidation but it is likely to be minor.

PAGET’S DISEASE OF BONE The action of calcitonin on bone has been ex-

ploited therapeutically and has proved of great benefit in the treatment of Paget’s disease of bone. The Ham- mersmith trial of long-term synthetic human calcitonin (C47 175 Ba) (HCT) treatment in Paget’s disease began in 1969. At the outset there were no guidelines for choice of dosage but by 1973 it had become apparent that a dose of 1.0 mg daily was necessary to check ra-

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Months Treatment Figure 6. Twenty-four hour urine hydroxyproline excretion in patients who have required retreatment with synthetic human calcitonin after cessation of therapy.

CALCITONIN 1143

diologic advance of the disease (49). From then on the majority of patients received this dose, which is equiva- lent to 100 MRC units of the hormone or to 0.5 mg of the highly purified human calcitonin (Cibacalcin) now available. Progress was monitored mainly by regular clinical assessment, relevant biochemical tests, and stan- dardized skeletal radiology.

It is now clear that calcitonin can alleviate the characteristic bone pain and other clinical features of Paget’s disease. The elevated levels of serum alkaline phosphatase (SAP) and urinary hydroxyproline (OHPr) are decreased and radiologic and histologic regression may be noted (50-52).

Side effects. Calcitonin is usually given by the subcutaneous route which tends to minimize any side effects and also enables patients to give their own injections easily. The side effect encountered most com- monly (20-30% of patients) is flushing and a feeling of warmth, predominantly in the face and hands. This occurs within minutes of an injection and usually lasts for about an hour (53). Paradoxically the occasional patient

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complains of feeling cold after the injection. Mild nausea is also common, and more rarely diarrhea, vomiting, and urinary frequency have been noted. The human hormone is nonantigenic (54).

Approach to treatment. Although the beneficial effect of calcitonin in Paget’s disease is now well established, there remain several outstanding questions:

1. Who needs treatment? 2. What is the best treatment regimen? 3. What happens when calcitonin is discontin-

In an attempt to answer the last question, HCT was stopped in a group of 27 patients who had received continuous (daily) treatment for 12-92 months. These patients were monitored at 3-6 month intervals during HCT treatment and after discontinuation of HCT the clinical and biochemical responses were assessed 6 and 12 months later together with radiologic response at 12 monthly intervals thereafter. (Some radiologic aspects of Paget’s disease are discussed by F. H. Doyle in this issue of the journal, pages 1205-1214.)

ued?


Figures 5-8 show the results of SAP and OHPr, plotted on a logarithmic scale, during treatment and for up to 32 months after withdrawal of treatment in 16 patients in remission and l l patients who have required retreatment. Each point represents the mean of 3 deter- minations of SAP and OHPr at any one time. The “lowest level” is defined as the lowest level recorded initially on treatment, regardless of the dose of HCT employed. In a few patients lower values were recorded at a much later date and after an increase in the dose of calcitonin. these values have not been used. SAP was estimated by a standard autoanalyzer technique and OHPr initially by the method of Kivirikko et a1 (55) and subsequently by amino acid analysis. The normal range for SAP is 30-130 international units (IU) and the upper limit of normal for OHPr is 45 mg/24 hours. At a mean value of 155 IU the coefficient of variation for SAP was 7% and at a mean of 286 IU it was 10%. For a mean OHPr of 180.4 mg/24 hours the coefficient of variation was 7%.

Both SAP and OHPr fell initially to attain the lowest level. Despite continued treatment the levels measured at the final on-treatment time had increased above the lowest level (P < 0.001 for SAP and P < 0.001 for OHPr) but this was not accompanied by a symptomatic deterioration. Six months after stopping HCT, SAP and OHPr had risen in all patients (P < 0.001 for SAP and P < 0.001 for OHPr) and clinically all remained well. However, on following these patients up to 18 months, it became necessary to restart treatment in 11 patients: 2 patients because of impending major orthopedic surgery and 9 patients with both clinical and radiologic relapse. Retreatment led to a fall in both SAP (P < 0.02) and OHPr (P < 0.01).

As a result we formulated an approach to retreatment: patients with a clinical relapse only are given 0.5 mg Cibacalcin twice weekly and patients with a radiologic relapse (with or without a clinical relapse) are given 0.5 mg Cibacalcin daily.

Who needs treatment and how? This brings us back to questions 1 and 2. In our opinion there are 5 situations that warrant calcitonin treatment, at least as a trial course: 1) bone pain, 2) immobilization hypercalcemia, 3) repeated fractures or nonunion, 4) neurologic complications, and 5) before and after major orthopedic surgery.

Bone pain remains the foremost indication for treatment. This requires painstaking assessment to es- tablish that the pain is attributable to the pagetic proc- ess and not to concomitant osteoarthritis which is very common in Paget’s disease patients. In patients with pri- marily sclerotic disease, daily calcitonin has been shown to be effective in relieving pain in most patients, but less

frequent doses may prove to be satisfactory in all but the most extreme cases. We suggest that a suitable regimen might be 6 months of twice weekly injections (0.5 mg Cibacalcin) after which, if symptoms have been re- lieved, treatment could be discontinued until pain re- curred. Higher dose regimens or more frequent injections e.g. 3 times weekly or daily may be necessary if the lower dose is ineffective.

If the patient has radiologic evidence of mechan- ically weak bones with osteolytic lesions (including resorption fronts), especially in weight-bearing long bones, a dose of 0.5 mg Cibacalcin daily is necessary, and treatment must be continued until the lesions have healed.

Clearly immobilization hypercalcemia should be readily avoidable but when it does occur, calcitonin is rapidly effective in normalizing the serum calcium (56). Complete fractures usually heal well in Paget’s disease (57) but if there are repeated fractures or if delayed union is the problem, calcitonin should be given to pro- mote healing. Fissure fractures are relatively common in Paget’s disease but they are a dubious indication for calcitonin because their response is highly variable (52).

Neurologic complications may warrant treatment with calcitonin. Improvement in cranial nerve pal- sies, ataxia, myelopathy with spastic paralysis, and paraplegia have all been noted during calcitonin treatment (53,58) but it must be remembered that only a small number of patients has been documented. Deaf- ness, the most common neurologic complication of Pa- get’s disease, does not usually respond to calcitonin therapy, although further hearing loss during treatment has not been observed (59,60).

Major orthopedic surgery such as hip replacement in patients with Paget’s disease has been associated with an increased risk of hemorrhage. Peri- operative treatment with calcitonin may improve hemostasis. It is suggested that calcitonin should be given for 3 months prior to the operation and then for 6- 12 months postoperatively.

The rare childhood disorder hereditary bone dysplasia with hyperphosphatasemia, also known as juvenile Paget’s disease (6 1,62), constitutes a definite indication for calcitonin to prevent the characteristic fractures and crippling deformities associated with this disease. In this instance the human hormone is probably essential since it is likely that treatment will be lifelong.

ACKNOWLEDGMENTS We are most grateful to the following for their assist-

ance in these studies: T.R. Arnett, L. Banks, Q. Bone, F.H. Doyle, F. Galan Galan, S.I. Girgis, C.J. Hillyard, J. Pennock, R.M. Rogers, P.T. Townsend, M.I. Whitehead, 0. Young.

CALCITONIN 1145

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CALCITONIN 1147

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DISCUSSION

Dr. Krane: Why was treatment with calcitonin started in

Dr. MacIntyre: Because of pain.

Dr. Krane: So you treated pain that was restricted to a lesion. But, in addition, you excluded patients who had, for example, joint disease?

Dr. MacIntyre: We did not exclude patients with joint disease, but selected patients on the basis of pain; I believe that in every case the pain was from a pagetic site. It is sometimes difficult to tell pagetic pain from osteoarthritis.

Dr. Krane: If treatment was for pain, why were the patients taken off therapy? Are you implying the pain did not come back?

Dr. MacIntyre: Twenty-seven people were able to dis- continue therapy. Of that 27, 11 patients had to restart because of pain. Treatment was withdrawn to determine what would happen.

these patients?

Dr. Altman: How long had you treated these patients?

Dr. MacIntyre: Up to 10 years before discontinuing the drug.

Dr. Singer: Some patients had excellent biochemical (50%) suppression with 0.5 mg/day. Yet you said that the radiologic lesions continued. Would you alter your treatment if you followed the x-ray, despite re- lief of pain? Would you increase the dose?

Dr. MacIntyre: Absolutely.

Dr. Doyle: Six were treated on 0.5 mg; 3 got better and 3 got worse.

Dr. MacIntyre: There is a further explanation of the problem with the 0.5 mg dosage. The preparation in the past was not the same as the one now in use. It was not as pure.

Dr. Bijvoet: If you were to start treating new patients now, how long would you treat them?

Dr. MacIntyre: I would treat a patient with pain but without a radiologic lytic lesion for 6 months to 1 year.

Dr. Doyle: A patient with a major lytic lesion would be treated for at least 1 year, probably not less than 2 years.

Dr. MacIntyre: One probably cannot form a judgment of how long someone will be on treatment without a strict and rigid radiologic assessment as conducted by Dr. Doyle.

Dr. Bijvoet: At what time does radiologic improvement occur?

Dr. Doyle: For the most part, in the first 2 years.

Dr. Canfield Do you have any idea of what causes the extreme nausea to calcitonin in some patients?

Dr. MacIntyre: Probably the peaking time of the drug is involved. We have had much less of a problem since we changed from an intramuscular to a subcutaneous injection.

chemistry, physiology, and therapeutic applications of calcitonin

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