Experimental Gerontology, Vol. 25, pp. 349-356, 1990 Printedin the USA. All rightsreserved.

0531-5565/90 $3.00 + .00 Copyright© 1990PergamonPressplc

CALCITONIN AND POSTMENOPAUSAL BONE LOSS

J.Y. RECINSTER, R.

DEROISY,M.P. LECART,N. SARLET,M . A . FONTAINE,A. ALBERT and P. FRANCHIMONT

Bone Metabolism Unit, Rheumatology and Physical Medicine Department, University of Liege, Chu Bmll (+9), Quai Godefroid Kurth, 4020 Liege, Belgium

Abstract -- In order to establish the role of calcitonin (CT) in postmenopausal bone loss, we studied CT metabolism in 25 pre- and postmenopausal women. Postmenopausal women presented a highly significant reduction of CT basal levels compared to premenopausal females (p < 0.01). Also, production rates of CT in osteoporotics were significantly lower than in either young premenopausal (18-25 years old), older premenopausal (35--40 years old), or postmenopausal healthy subjects. In a study in rabbits, we found that injection of CT, along with equimolar amounts of antl-SCT antibodies extracted from serum of pagetic patients, did not inhibit the hypocalcemic response to the hormone, thus demonstrating that resistance to CT treatment cannot be accounted for by antibody production. In a subsequent clinical study in patients with Paget's disease of bone, we found that 200 IU/day of salmon CT (SCT), given by nasal spray, improved both clinically and biochemically the activity of the disease, as demonstrated by 37 - 4% decrease of serum alkaline phosphatase and 35 --5% fall of urinary excretion of hydroxyproline after six months of therapy. The effectiveness of CT as nasal spray was further tested in healthy women at an early stage of menopause. A 12-month course of intranasal SCT counteracted early postmenopausal bone loss, presumably by inhibiting bone resorption. In conclusion, intranasal CT seems to be a very attractive alternative to be considered for the prevention of postmenopausal osteoporosis. Key Words: caicitonin, osteoporosis, bone loss, menopause, estrogen

INTRODUCTION POSTMENOPAUSALOSTEOPOROSIS (OP) is the consequence o f a disordered bone remover characterized by an imbalance between resorption and formation (Riis et al., 1986). One of the most effective means of preventing postmenopausal bone loss, or at least of slowing its rate, is estrogen replacement therapy (ERT), either alone or in combination with a progestogen (Lindsay et al., 1980; Christiansen et al., 1980). However, the dose of estrogen required to produce a net gain of bone is much higher than that required to control other postmenopausal symptoms (Horsman et aL, 1983), hence the risk of adverse effects is such to make ERT insuitable for a large subset of postmenopausal women. Thus, an alternative to ERT as

Correspondence to: J.Y. Reginster. 349

350

LY. RI~GINSTER et al.

prevention of postmenopausat bone loss is needed. It has been known for nearly 25 years that calcitonin (CT) inhibits osteoclast activity (Copp et al., 1973; Lucht, 1973). For this reason, CT has been widely used in the treatment of bone diseases characterized by increased resorption (Reginster and Franchimont, 1985a), and a few studies have demonstrated its efficacy in treatment of postmenopausal OP (Gennari et al., t985; Mazzuoli et al., t986). The chronic nature of postmenopausal bone loss requires long-term treatment. This might create problems of patient compliance if the drug has to be given parenterally, as in the case of calcitonin. In addition, a number of annoying side effects (nausea, flush~ etc.) have been reported to occur after injections of calcitonin (Gennari et al.. 1983). A new form of synthetic salmon calcitonin (SCT) as nasal spray has been recently introduced. As it occurs for other hormones, such as vasopressin, luteinizing hormone, releasing hormone. insulin, and glucagon (Murlin et al.. 1940: Fink et al., 1974; Pontiroli et al., 1982; Pontiroli et al., 1983), SCT also appears to be absorbed through the mucosa of the respiratory system. Encouraging results have been obtained in a preliminary study conducted in our institution in the treatment of Paget's disease with intranasal SCT for 6 months (Reginster et ai., 1985b). However, before nasal SCT can be considered as an alternative to ERT in the prevention of postmenopausal bone loss, three major points will have to be elucidated: a) Despite the several studies published so far, the exact role of CT in the pathogenesis of OP remains controversial. It is still uncertain whether or not there is an age- or menopause-related decrease of CT secretory capacity, and whether osteoporotic patients have an absolute or relative CT deficiency. b) Since the amino acid sequence of SCT differs considerably from the human hormone (Niall et al., 1986), specific antibodies (Ab) develop in a significant proportion of treated patients (Singer et al.. 1972; Hosldng er al., 1979). Thus, it is crucial to assess if anti-SCT Ab may reduce SCT effectiveness. c) Is intranasal administration of SCT effective on biological markers of bone turnover in normal volunteers, and does its long-term use in disease characterized by high bone turnover result in the same therapeutic effect as that obtained by parenteral SCT treatment? IS CALCITONIN A PHARMACOLOGICAL OR AN ETIOPATHOGENIC ALTERNATIVE TO ESTROGEN THERAPY? Circulating CT concentrations have been reported to be lower in women than in men in several studies (Hillyard et al., 1978; Tiegs et al., 1986). Furthermore. some authors have suggested that calcitonin secretion declines at the menopause (Lore et al., 1984) and is stimulated by estrogen (Stevenson et al., 1983). However, this view is not shared by all the investigators (Tiegs et al., 1986; Body et al., 1989). To address this issue, we investigated 25 women, 9 eugonadal and 16 postmenopausal. Of these, 5 were healthy and t i osteoporotic. The experimental procedure was that described by Kanis et al. (1982), to assess basal levels of plasma calcitonin (iCT) and estrone (El), CT metabolic clearance rate (MCR), and 24-h CT production rate (PR). Mean basal calcitonin levels were not related to age in either premenopausal or postmenopausal women. However, the latter group showed a significant reduction (p < 0.001) of basal CT compared to premenopansal women (Table 1). MCR was substantially the same in the three groups, whereas PR was significantly lower in the osteoporotics than in both young eugonadal (18-25 years old), and elder eugonadal (35-40 years old) women. Also, there was a highly significant correlation between basal levels of CT

351

CALCITONININ OSTEOPOROSIS TABL~ 1. CT METABOUSM1N rmALTHYAND OS~OPOROaaCWO~mN(FROMRE~rNSVERet al., 1989)

Gr l Basal iCT

Gr H

84.2 ± 18.4 ~

74.2 +__ 24.7 a

99.3 +- 41.8 a 366 +_ 77.5 a

92.1 ± 31.7 a 335.7 ± 62.3 a

30.9 ± 9.9~

25.5 -+ 11.1a

Grill 47.5 ± 19.5 b

Gr W 32.3 ±

9.8 b

(pg/~) BasalE2 CTMCR

c'r PR

36 420

+ 21 b - 67 a

19.5 + 6.95"

32.2 ± 12.9 b 299 ± 33 a

9.8 ±

4b

a significantly higher than b (p < 0.001). Mean values - SD of plasma basal iCT, plasma basal E2, CT MCR, and CT PR in eugonadal women 18-25 years old (Gr I), eugonadal women 35--40 years old (Gr II), healthy postmenopausal women (Gr III), and postmenopausal osteoporotics (Gr IV). All groups were compared by means of a variance analysis.

and E1 values (r = 0173; p < 0.001), as well as between PR and E1 (r = 0.64; p < 0.001). From these data, it appears that in an normal female population, with eugonadal E1 circulating levels, CT secretory capacity is not affected by age. However, in both groups of postmenopausal women, healthy and osteoporotic, there was a fall in plasma circulating levels of CT, and a reduction of CT secretory capacity. It is therefore possible that in postmenopausal women a reduced CT secretion, in the presence of normal MCR, may lead to a reduction in CT basal levels. Furthermore, since osteoporotic patients have reduced CT production rates, compared to both eugonadal and healthy patients matched for age and E1 levels, it is likely that, in addition to the menopause effect, osteoporotic patients develop a specific and still unknown impairment of CT production in parafollicular cells.

ARE ANTIBODIES ABLE TO COUNTERACT THE ACTIVITY OF SALMON CALCITONIN? In Paget's disease of bone, resistance to SCT which sometimes develops during the treatment has been attributed to production of Ab (Singer et al., 1972; Singer et al., 1980). Conversly, other investigators found that the presence of significant Ab titers had no relationship with the development of resistance (Derose et al., 1974), since resistance to SCT occurs also in patients treated with human CT, which is not antigenic (Greenberg et al., 1974). The best way to determine if Ab to exogenous CT functionally block its hormonal activity is to study their effect on the acute hypocalcemic response to CT (Hosking et al., 1979). A highly sensitive method has been recently developed in animals. This is based on monitoring the serum concentration of ionized calcium after acute injection of CT (Azria et al., 1985). Using this technique we investigated the effect of anti-SCT Ab obtained from the blood of pagetic patients treated with nasal SCT on the hypocalcemic effect induced by the hormone in rabbits. The following injections were performed: -- SCT in acetic buffer, SCT with an equimolar amount of anti-SCT Ab extracted from the serum of pagetic patients, -- Anti-SCT Ab from pagetic patients without SCT, SCT with an equimolar amount of nonspecific human gammaglobulins, Nonspecific human gammaglobulins without SCT The occurrence of specific anti-SCT Ab in 65% of the pagetic patients treated by nasal SCT -

-

-

-

-

-

LY. REGINSTERet aI.

352

TABLE 2. CALCITONIN-INDUCEDHYPOCALCEMIAIN YouNG RABBITS

Time (minJ

CT Alone 1 U/kg Mean ~- SEM

CT (] U/kg) and Equimolar Amount of Specific Anti-SCT Gamma Globulins Mean ~_ SEM

00'

0

05'

0.19 -+ 0.07

-0.I7

0 z 0.19

10' 15'

- 6 . 5 6 - 0.58 1.96 ~ 0.73

-0.54 -1.75

_ 0 87 z 1.60

30' 60'

-10.37 -21.53

__ 1.65 -- 0.81

-7.03 -13.99

_ 1.95 z 2.38

90'

-26.06

-

0.60

-18.63

_~ 1 74

120' 150' 180'

-27.09 -25.43 -17.26

-- 0.48 -~ 0.02 - 0.53

-21.43 -19.71 -16.48

_~ J,07 _~ 0.79 ___ 0 53

210'

-2.61

-~ 0.74

-10.29

_~ 2,45

240' 270'

- 0 . 3 0 - 0.16 0.43 + 0.48

-5.11 -2.38

_~ 3.04 _~ 2.70

300'

0.57 --- 0.28

-1.19

_~ 2.60

64.03 -~ 0.50

-57.29

= 3.59

0--300' (%.rain)

Variations of blood-ionized calcium (%) in the young rabbit after i.v. administration of 1 IU/kg synthetic salmon calcitonin complexed (or notl with anticalcitonin antibodies.

confirms that SCT is absorbed in significant amounts when given by nasal route. Injection o f SCT to young rabbit produced a significant decrease in ionized calcium, as measured by a calcium selective electrode, between 60 and 180 min after the injection (Table 2). Treatment with SCT along with an equimolar amount of purified specific Ab did not decrease the intensity of the hypocalcemic response to SCT but apparently prolonged this effect, since Ca was still significantly decreased after 210 rain (Table 2). Injection of either anti-SCT Ab or nonspecific human gammaglobulins alone did not induce any statistically significant change in ionized calcium. These data do not indicate that specific anti-SCT Ab are able to neutralize SCT activity; they suggest, however, that Ab binding to SCT could result in an extension of SCT activity by slowing down its catabolism. DOES CALCITONIN GIVEN BY INTRANASAL ROUTE INDUCE THE SAME EFFECTS AS WHEN ADMINISTERED PARENTERALLY? Until recently, SCT has been administered via a parenteral route, either intramuscular or subcutaneous. Besides the discomfort associated with the injection itself, particularly in chronic treatments, in nearly 60% of cases CT therapy is associated with side effects, including vomiting, nausea, flushing and vertigo. These side effects may be severe enough to warrant discontinuation of treatment (Gennari et al., 1983). We previously showed that intranasaI administration of SCT was devoid of side effects, even in patients intolerant to intramuscular or subcutaneous SCT (Reginster and Franchimont, 1985c). For this reason, and because it overcomes the discomfort of repeated injections, the nasal spray would enhance the value of SCT in the treatment and prevention of chronic bone diseases provided that sufficient evidence

CALCITONIN IN OSTEOPOROSIS

353

TABLE 3. BIOLOgiCALEFFECTSOF SALMONCALCITONIN(FROM REGINSTERet al., 1987a)

T

Placebo

i.m.

Spray

Ca(mg~) mean± SD

0' 60' 240'

92.4 91.2 92.8

--- 1.2 - 1.2 ± 1.2

92.0 90.4 90.8

--- 0.8 + 2.0 a --- 0 . 8

91.6 88.4 89.2

± 0.8 ± 1.2 a ± 0.8

C a C (rag/L) m e a n ± SD

0' 60' 240'

87.9 87.0 87.9

-+ 0 . 6 ± 0.9 ± 1.0

87.7 86.0 85.3

... 0 . 9 ± 1.6 - 0~7

87.7 85.0 87.3

± 0.5 ± 0.5 b ± 0.7

P (mg/L) m e a n ± SD

0' 60' 240'

34.4 34.7 33.4

± 1.8 --- 1.8 ± 1.2

35.7 32.0 28.6

± 1.3 ± 1.1 a ± 0.9 a

33.2 30.5 30.5

- 1.5 ± 1.6 ± 1.2

iPTH ( m l U / m L ) m e a n --- SD

0' 60' 240'

1.75 ± 0 . 2 1.77 ± 0 . 2 5 1.65 --- 0 . 1 7

1.82 --- 0 . 1 7 1.87 ± 0.3 1.78 ± 0 . 2 6

1.71 ± 0 . 2 7 1.80 ± 0.23 1.63 ± 0.25

aStatisticallydifferentfrom TO (pretrialvalues) (p < 0.05). bStatisticallydifferentfrom TO (pretrialvalues) (p < 0.01). Values of serum (Ca), serumcalciumcorrectedfor serumalbumin(CaC), serumphosphorus(P), and serumparathormone(iPTH). is given to support its ability to intiibit osteoclastic bone resorption similarly to parenteral SCT. T o address this point, we conducted a study in men to compare the effects on mineral metabolism of 200 IU SCT nasal spray with that obtained using 80 ILl or a placebo administered parenterally (Reginster et al., 1987a). Intranasal administration of 200 IU SCT produced a significant fall in total serum calcium and in serum calcium corrected for serum albumin. This hypocalcemic response, occurring in all subjects investigated, was transient, since at 4 h after SCT administration, serum calcium levels were back to normal (Table 3). Probably in response to the induced hypocalcemia, parathyroid hormone levels tended to rise, but did not reach statistical significance. In conclusion, intranasal administration of 200 IU SCT to healthy volunteers causes a rapid and transient hypocalcemia, confirming that this administration route is effective. These results prompted us to investigate nasal SCT efficacy in long-term studies in : diseases characterized by high bone turnover. Paget's disease is a good model of extremely increased bone turnover disease, probably induced by a structural and/or functional abnormality of the osteoclasts (Meunier, 1975) and, therefore, it is suitable for investigating effectiveness of antiresorptive drugs (Kanis and Gray, 1987). Thus, in a subsequent study we investigated the effect of two doses (200 and 400 IU/day) of nasal SCT during a one-year treatment in pagetic patients (Reginster et al., 1988). SCT at both doses induced clinical and biochemical improvement of moderate Paget's disease of bone [mean initial serum alkaline phosphatases (SAP) levels being 334% of the normal range]. Both SAP, an index of bone formation, and urinary excretion of hydroxyproline (OHP), a marker of bone resorption, significaiatly decreased in 15 of 17 patients. For the whole group, the mean decrease in SAP was 37 ___ 4% after 6 months (p < 0.01) and 31 +_ 5% after one year (p < 0.01). The mean fall in OHP was 35 + 6% (19 < 0.01) and 37 --- 7% (p < 0,01) after 6 and 12 months, respectively. These findings are further evidence that intranasal SCT inhibits bone turnover rates. All these data convinced us to investigate the effect of intranasal SCT on bone turnover and bone mineral density (BMD) in healthy women who were in the early stages of their menopause

354

J.Y. REGINSTER et al. T A B L E 4.

Parameters

BMD L2-L4 (g/cm2) Ca (mM/L) TCT (pg/ml) OH/Cr (mg/g) Ca/Cr (mg/ 100 ml) SAP

Er~CT oF

NASAL CALCITONINON POSTMENOPAUSALWOMEN (FROM REGINSTERe t

al. 1987b)

dO

SCT + Ca Gr2

d360

0.84 + 0.09

0.90 - 0.14

NS

0.92 _ 0~4

NS

2.32 --_ 0.01

2.33 --+ 0.01

NS

2.32 ± 0.02

NS

49 +_- 25

a

59 + 4.4

55 ± 7

NS

60 ± 4

NS

12 ___ 10

18.6 _ 1.5

NS

18.9 --- 1.6

16.7 ± 0.9

NS

16.1 ___ 1.1

a

0.10 ± 0.05

0.14 + 0.01

NS

0.11 ___ 0.01

0.12 --- 0.01

NS

0.10 -+ 0.0t

NS

93 + 42

76 -~ 4

NS

83 z 3.8

79 - 3.9

NS

87 - 3.7

NS

50 --- 30

42.5 ~ 2

NS

46.7 -+ 1.9

42.8 -~ 2.6

NS

44.7 - 2.8

NS

34.2 ± 0.9 85.5 _ 7

NS NS

34.0 --- 0.7 78.3 _~ 6

35.3 - 0.9 84.6 --- 5

NS NS

34.1 --- 0.7 73.5 _+ 5,6

NS NS

Normal Range

dO

Ca Grl

d360

6.90 + 0.10

0.87 _ 0.09

~

2.35 -_+ 0.20

2.32 _+ 0.01

60 _ 50

Gr] /2 dO~d360

(IU/L) SAPb

(tU/L) P (rag/L) PTH (pg/ml)

33 - 7 100 --- 50

NS = nonsignificant. ap < 0.01. Changes occurring during the 12-month study in parameters reflecting bone mass and bone turnover. Grl received Ca and Gr2 received Ca - SCT. Grl/2 d0/360 reflects the comparison between the evolution of Grl and C_rff2during the one-year trial.

( R e g i n s t e r et al., 1987b). T h e r e s u l t s o f this i n v e s t i g a t i o n ( T a b l e 4) s u g g e s t that a 1 2 - m o n t h c o u r s e o f i n t r a n a s a l S C T c o u n t e r a c t s e a r l y p o s t m e n o p a u s a l b o n e loss b y i n h i b i t i n g b o n e resorption and (perhaps temporarily) uncoupling the mechanisms of resorption and formation, T h e l o n g - t e r m e f f e c t s as w e l l as the o p t i m a l d o s e o f S C T is, h o w e v e r , still to b e a s s e s s e d .

CONCLUSIONS F r o m o u r data, it a p p e a r s t h a t c a l c i t o n i n m a y p l a y a role in the d e v e l o p m e n t o f p o s t m e n o p a u s a l b o n e loss, s i n c e C T s e c r e t o r y c a p a c i t y is related to E1 c i r c u l a t i n g l e v e l s , a n d o s t e o p o r o f i c p a t i e n t s h a v e l o w e r C T p r o d u c t i o n r a t e s t h a n n o r m a l subjects. S a l m o n c a l c i t o n i n is, so far, t h e o n l y C T p r e p a r a t i o n a v a i l a b l e as n a s a l spray. T h i s a d m i n i s t r a t i o n r o u t e is as e f f e c t i v e in r e d u c i n g b i o c h e m i c a l i n d i c e s o f m i n e r a l m e t a b o l i s m i n h e a l t h y v o l u n t e e r s a n d in P a g e t ' s d i s e a s e o f b o n e as C T a d m i n i s t e r e d p a r e n t e r a l l y . N a s a l s p r a y not o n l y o b v i a t e s d i s c o m f o r t o f r e p o r t e d i n j e c t i o n s b u t it also p r e v e n t s t h e o c c u r r e n c e o f a n n o y i n g s i d e effects r e p o r t e d d u r i n g p a r e n t e r a l S C T t h e r a p y . T h e o c c u r r e n c e o f specific a n t i - S C T A b d o e s n o t b l o c k the a c t i v i t y o f S C T , t h e r e f o r e , t h e y s h o u l d n o t b e r e g a r d e d as a n e g a t i v e f a c t o r for c o n s i d e r i n g n a s a l S C T in l o n g - t e r m t r e a t m e n t s , as it is the c a s e o f p o s t m e n o p a u s a l o s t e o p o r o s i s . N a s a l C T s e e m s to b e a v e r y a t t r a c t i v e a l t e r n a t i v e f o r p r e v e n t i o n o f p o s t m e n o p a u s a l b o n e loss. S o m e c o n c e r n s , h o w e v e r , still n e e d to b e c l e a r e d a n d o n e s h o u l d k e e p i n m i n d t h a t e v e n

CALClTONININ OSTEOPOROSlS

355

if S C T is a v e r y active drug it cannot be considered for the therapy o f climateric complaints and therefore should be restricted to a subset o f p o s t m e n o p a u s a l w o m e n w h o are not eligible for E R T . REFERENCES AZRIA, M , ATTINGER, M., SCHUPFER, H., and TANNER, P. Comparison of the hypocalcaemic activities of three calcitonins using a new biological method. In: Calcitonin 1984, Doepfner, W. (Editor), pp. 104-110, Exerpta Medica, Amsterdam, Hong-Kong, Princeton, Sidney, Tokyo, 1985. BODY, J.J., STRUELENS, M., BORKOWSKI, A., and MANDART, G. Effects of estrogens and calcium on calcitonin secretion in postmenopausal women. J. Clin. Endocrinol. Metab. 68, 223-226, 1989. CHRISTIANSEN, C., CHRISTIANSEN, M.S., MAC NAIR, P., HAGEN, C., STOCKMLUND, K., and TRANSBOL, I. Prevention of early postmenopausal bone loss: controlled 2 year study in 315 normal females. Eur. J. Clin. Invest. 10, 273-279, 1980. COPP, D.H., CAMERON, E.C., CHENEY, B.A., DAVIDSON, A.G., and HENZE, K.G. Evidence for calcitonin, a new hormone from the parathyroid that lowers blood calcium. Endocrinology 70, 638--649, 1973. DEROSE, J., SINGER, F.R., AVRAMIDES, A., FLORES, A., DZIADIW, R., BAKER, R.K., and WALLACH, S. Response of Paget's disease to porcine and salmon calcitonin. Am. J. Med. 56, 850-856, 1974. FINK, G., GENNSER, G., LIEDHOLM, P., and THOREIL, J. Comparison of plasma levels of LH-RH in men after intravenous and intmnasal administration. J. Endocrinol. 63, 351-360, 1974. GENNARI, C., CECCHETTIN, M., PASSERI, M., FRANCINI, G., GONNELLI, S., NAMI, R., and BIGAZZI, S. Gli effetti collaterali di differenti calcitonine. In: Calcitonin in Man, Gennari, C. and Segre, G. (Editors), pp. 93-101, Masson, Milano, 1983. GENNARI, C., CHIERICHETrI, S.M., BIGAZZI, S., FUSI, C., GONNELLI, S., FERRARA, R., and ZACCHEI, F. Comparative effects on bone mineral content of calcium plus salmon calcitonin given in two different regimen in postmenopausal osteoporosis. Curr. Ther. Res. 38, 455-464, 1985. GREENBERG, P.B., DOYLE, F.M., FISHER, M.T., HILLYARD, CA., JOPLIN, G.F., PENNOCK, J., and MAC INTYRE, I. Treatment of Pagent's disease of bone with synthetic human calcitonin. Am. J. Med. 56, 867-870, 1974. H1LLYARD, C.J., STEVENSON, J.C., and MAC INTYRE, I. Relative deficiency of plasma calcitonin in normal women. Lancet ii, 961-962, 1978. HORSMAN, A., JONES, M., FRANCIS, R., and NORDIN, C. The effect of estrogen dose on postmenopausal bone loss. N. Engl. J. Med. 309, 1405-1407, 1983. HOSKING, J., DENTON, L.B., CADGE, B., and MARTIN, J.J. Functional significance of antibody formation after long-term salmon calcitonin therapy. Clin. Endocrinol. (Oxf.) 10, 243-252, 1979. KANIS, J.A., HEYNEN, G., CUNDY, T., CORNET, F., PATERSON, A., and RUSSEL, R.G.G. An estimate of the endogenous secretion rate of calcitonin in man. Clin. Sci. 63, 145-152, 1982. KANIS, J.A. and GRAY, R.E. Long-term follow-up observations on treatment in Paget's disease of bone. Clin. Orthop. Rel. Res. 217, 99-125, 1987. LINDSAY, R., HART, D.M., FORREST, C., and BAIRD, C. Prevention of spinal osteoporosis in oophorectomized women. Lancet ii, 1151-1153, 1980. LORE, F., GALLI, M., FRANCI, B., and MARTORELLI, M.T. Calcitonin levels in normal subjects according to age and sex. Biomed. Pharmacother. 38, 261-263, 1984. LUCHT, Y. Effects of calcitonin on osteoclasts in vivo. An ultrastructural and histochemical study. Z. Zellforsch 145, 75-87, 1973. MAZZUOLI, G.F., PASSERI, M., GENNARI, C., MINISOLA, S., ANTONELLI, R., VOLTORTA, C., PALUMMERI, E., CERVELLIN, G.F., GONNELLI, S., and FRANCINI, G. Effects of salmon calcitonin in postmenopausal osteoporosis: a controlled double blind clinical study. Calcif. Tissue Int. 38, 3-8, 1986. MEUNIER, P. J. La maladie osseuse de Paget. Histologie quantitative et prescription th6rapeutique. Lyon Med. 233, 839-853, 1975. MURL1N, J.R., GIBB, C.B.F., ROMANSKY, M.J., STEINHAUSEN, T.B., and TRUAX, F.L. Effectiveness of perioral insulin in human diabetes. Am. J. Physiol. 120, 709-722, 1940. NIALL, H.D., KEUTMANN, H.T., COPP, D.H., and POTTS, J.J. Amino-acid sequence of salmon ultimobranchial calcitonin. Proc. Natl. Acad. Sci. 64, 771-778, 1986. PONTIROLI, A.E., ALBERETTO, M., SECCHI, A., DOSSI, G., BOSI, I., and POZZA, G. Insulin given

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