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Original Studies |
INSERM U-403, Hôpital E. Herriot (P.G., P.D.D.), and Synarc (F.G.), 69437 Lyon, France; Institut de Recherches Internationales Servier (Y.T., C.V.), Courbevoie, France; MAV Hospital (I.M.), Budapest, Hungary; and Hotel Dieu Hospital (C.P.), Paris, France
Address all correspondence and requests for reprints to: Dr. Patrick Garnero, INSERM U-403, Hôpital E. Herriot, Pavillon F, 69437 Lyon Cedex 03, France. E-mail: patrick.garnero{at}synarc.com
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Abstract |
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We conclude that E2 administered by this new nasal route normalizes bone turnover to premenopausal levels. The delayed decrease in bone formation observed with intranasal E2 compared to oral E2 may be related to different effects on serum IGF-I levels.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Although oral and transdermal estrogen have demonstrated their efficacy in preventing postmenopausal bone loss (4, 16), both of them have some disadvantages: the significant intestinal and hepatic first pass effects for the oral formulation as outlined above and the large interindividual variation in bioavailability, variable adhesion, and local dermatological reactions of the transdermal route (17, 18, 19).
A new E2 spray formulation for nasal administration has been developed (S 21400, Aerodiol, Institut de Recherches Internationales Servier, France), avoiding the limitations of oral and transdermal estrogen. Intranasal absorption is facilitated by the highly vascularized, microvillous nature of the nasal mucosa (20). Intranasally absorbed estradiol has a very rapid uptake, achieving maximal plasma levels within 10–30 min. The plasma concentration returns rapidly to 10% of the maximal value approximately 2 h after administration and to the levels found in untreated postmenopausal women within 12 h (21). As a consequence, daily intranasal administration results in a pulse-like estrogen profile, rather than the relatively sustained serum levels attained with both oral and transdermal administration (22). However, no study to date has ascertained whether a sustained profile is a requirement of efficacy, and it has recently been shown that intranasal E2 increases serum estradiol exposure to levels similar to those achieved with oral E2 (1–2 mg) and with an efficacy similar to that of oral estrogens in reducing menopausal symptoms (23).
The aims of this double blind, placebo-controlled clinical study were to investigate the short and long term effects of intranasal E2 on bone turnover and serum IGF-I and to compare its effects to those of oral E2.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Four hundred and twenty Caucasian postmenopausal women, aged 45–60 yr (6 months to 5 yr postmenopause), with climacteric symptoms were included in a multinational European, double blind, placebo-controlled study. These women were randomly allocated to one of seven treatment groups: intranasal E2 (100, 200, 300, or 400 µg/day; delivered by one spray per nostril), each with a placebo capsule, or oral E2 valerate (1 or 2 mg/day), each with a placebo spray, or double placebo. E2 was administered double blind once daily throughout the study period of 0–12 weeks and then for a further 2 weeks supplemented each evening by medroxyprogesterone acetate (MPA; 5 mg) to promote endometrial shedding. Treatment was administered between 0600–1000 h, and women were asked to take treatment at the same time every day. No calcium supplement was administered. For all women a blood sample and a 2-h urine sample were collected fasting before nasal administration at baseline and 1 and 3 months before any MPA supplementation. After the 3-month, double blind, placebo-controlled trial, 112 of these women regardless of the treatment allocation during the first 3 months were further treated with intranasal E2 (300 µg/day), i.e. the dose that has been found to be optimal to reduce menopausal symptoms, as assessed by Kupperman index and incidence of hot flushes (23). During this second phase of the study, nonhysterectomized women received various cyclic progestative treatments, including MPA (5 mg/day), dydrogesterone (20 mg/day), chlormadinone acetate (10 mg/day), and promegestone (0.5 mg/day).
All subjects gave informed consent for their participation in the trial, which was conducted in accordance with the Declaration of Helsinki and local ethical committee approval.
Markers of bone turnover
Serum osteocalcin was measured with a human specific two-site
immunoradiometric assay (ELSA-OSTEO, CIS Bio International,
Gif-sur-Yvette, France), which recognizes a large N-terminal
midfragment in addition to the intact molecule (24). The intra- and
interassay coefficients of variation (CVs) are less than 4% and 6%,
respectively, and the sensitivity is 0.4 ng/mL (26). Serum N-terminal
extension propeptide of type I collagen (PINP) was measured by a new
RIA that recognizes the intact circulating form of PINP (Intact PINP,
Farmos Diagnostica, Uppsala, Sweden) (25). The intra- and interassay
CVs are below 5% and 8%, respectively, and the sensitivity is 1 ng/mL
(25). Serum bone alkaline phosphatase (BAP) was measured with a human
specific immunoradiometric assay using two monoclonal antibodies
directed against the human bone isoenzyme and BAP purified from human
SAOS-2 osteosarcoma cells as a standard (Ostase, Hybritech, Inc., San
Diego, CA). This assay cross-reacts only 16% with the circulating
liver isoenzyme. The intra- and interassay CVs are less than 10% (26).
Urinary type I collagen C-telopeptide breakdown products (CTX) were
measured by an enzyme-linked immunosorbent assay (CrossLaps ELISA, CIS
Bio International) based on an immobilized synthetic peptide with an
amino acid sequence specific for a part of the C-telopeptide of the
1-chain of type I collagen
(Glu-Lys-Ala-His-Asp-Gly-Gly-Arg; CrossLaps antigen) (27). The intra-
and interassay CVs are below 5% and 8%, respectively, and the
sensitivity is 0.05 µg/mL (28).
The premenopausal range for biochemical markers of bone turnover was determined in 134 healthy premenopausal women from 35–55 yr of age (mean, 40.6 ± 5.3 yr) belonging to a prospective population-based study (OFELY study) (29). Mean (±1 SD) values of premenopausal women were 18.3 ± 4.7 ng/mL, 42 ± 15 ng/mL, 8.7 ± 2.7 ng/mL, and 189 ± 88 µg/mmol creatinine for serum osteocalcin, PINP, BAP, and urinary CTX, respectively.
Serum IGF-I and IGFBP-3
Serum IGF-I was measured by a competitive RIA (IGF-I by extraction, Nichols Institute Diagnostics, San Juan Capistranao, CA) after acid-ethanol extraction. The intra- and interassay CVs are below 3% and 8%, respectively, and the sensitivity is 0.06 ng/mL.
Serum IGFBP-3 was measured by a two-site immunoradiometric assay (Diagnostic Systems Laboratories, Inc., Webster, TX). The intra- and interassay CVs are below 5%, and the detection limit is 0.5 ng/mL.
All biochemical measurements for both the postmenopausal women involved in the clinical trial and the premenopausal women used to establish normal ranges were performed in a central laboratory (Prof. Delmas, Synarc, Lyon, France).
Statistical analysis
All data are shown as the mean ± 1 SD.
Measurements of serum osteocalcin, serum BAP, and urinary CTX were
performed in all randomized women, whereas serum PINP, IGF-I, and
IGFBP-3 were assessed only in the per protocol population, including
the 326 women defined as those who received at least 56 days of study
treatment, who had confirmed postmenopausal status with centrally
verified baseline levels of serum FSH above 30 mIU/mL and serum
estradiol below 30 pg/mL and no major protocol deviations. Statistical
analysis was performed on the per protocol population. Comparison of
bone markers, serum IGF-I, and serum IGFBP-3 between treatment groups
was performed using a covariance analysis adjusted on baseline values.
In case of a significant group effect, pairwise comparison were
performed vs. placebo, where the risk is corrected
according to Bonferroni’s procedure. The within-group change with time
was studied using a two-way ANOVA (patient x time) followed in
case of significant time effect by a Newman-Keuls test. The correlation
coefficients (Pearson) among the percent change in serum IGF-I, serum
IGFBP-3, and biochemical markers at 3 months were obtained from linear
regression analysis.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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. Women in the different treatment
groups did not differ for history of previous hormone replacement
therapy and percentage with surgical menopause. Women in the oral
E2 (1 mg/day) group were slightly older than those in the
other groups.
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At baseline, there was no significant difference between groups
for biochemical markers of bone turnover (Table 2). Bone resorption, assessed by urinary
CTX, was significantly decreased compared to that after placebo
(P < 0.001) both by oral (-20% and -40% for 1 and
2 mg, respectively) and intranasal (-13% to -27%) E2
within 1 month of treatment (Table 2 and Fig. 1). Urinary CTX continued to decrease at
3 months for both routes of administration. At 3 months, a dose effect
was observed for intranasal E2, with a larger decrease for
the dose of 200 µg compared to 100 µg, but no additional effect
with doses of 300 and 400 µg.
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and Fig. 1). In contrast, neither osteocalcin, PINP, or
BAP levels changed significantly after 3 months of treatment with
intranasal E2. Interestingly, the higher doses of
intranasal E2 induced a significant increase in serum
osteocalcin (14% and 9% for 300 and 400 µg, respectively;
P < 0.05) and serum BAP (10%, 13%, and 7% for 200,
300, and 400 µg, respectively; P < 0.05) at 1 month,
an effect that was not observed with oral administration (Table 2 and
Fig. 1). Similar results at 1 and 3 months were observed in the
intention to treat population for urinary CTX and serum osteocalcin
(not shown). Short term effects of oral and intranasal E2 on serum IGF-I and IGFBP-3 levels: relationships with short term changes in bone turnover
To explain the different short term effects of oral and intranasal
E2 on bone formation markers, serum IGF-I and IGFBP-3 were
measured. Oral E2 induced a rapid and marked decrease in
serum IGF-I with declines of -23% and -28% at 1 month for the 1-
and 2-mg doses, respectively (P < 0.0001
vs. placebo). At 3 months, the decrease was slightly larger,
reaching -29% and -32% for 1 and 2 mg, respectively. In contrast,
with intranasal E2 at any dose, there was no
significant change in serum IGF-I at 1 month (Table 3 and Fig. 1) and only a slight decrease
at 3 months (
-15% for all doses), which was, however, not
significantly different from the effect of placebo. No significant
change from the placebo group of serum IGFBP-3 levels was observed for
both oral and intranasal E2 during the 3-month period
(Table 3). Baseline levels of serum IGF-I correlated with serum IGFBP-3
(r = 0.45; P < 0.001). No significant correlation
was observed between changes in IGF-I and changes in IGFBP-3 at both 1
and 3 months. Changes in serum IGF-I at 3 months, but not changes in
serum IGFBP-3, correlated significantly with changes in serum
osteocalcin, serum PINP (Fig. 2), and
serum BAP (r = 0.18; P = 0.006).
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After the first 3 months of treatment, 112 women continued
treatment with intranasal E2 (300 µg/day). As shown in
Table 4 and Fig. 3, regardless of the type of therapeutic
regimens and doses used during the first 3 months, 12 months of
treatment with intranasal E2 induced significant decreases
in urinary CTX (-45%), serum osteocalcin (-26%), serum PINP
(-31%), and serum BAP (-29%). Levels of bone markers returned to
the premenopausal range in 96%, 88%, 97%, and 91% of the patients
for urinary CTX, serum osteocalcin, serum PINP, and serum BAP,
respectively.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Both intranasal and oral E2 induced a significant and dose-dependent decrease in urinary CTX, a specific marker of bone resorption. The significant decrease in urinary CTX as soon as 1 month induced by intranasal E2 is similar to that observed in this study with oral E2 and with that previously reported for oral conjugated equine estrogen (30), in agreement with a direct inhibition of the osteoclastic activity. The 30–35% decrease in urinary CTX after 3 months of treatment observed with intranasal E2 at doses of 200, 300, and 400 µg was similar to that obtained with oral 1 mg E2 and slightly less than that with the 2-mg dose. This magnitude was also in the range of that previously reported for transdermal E2 (50 or 100 µg) (31, 32) and 0.625 mg oral conjugated equine estrogen (30). Thus, the short term antiresorptive effects of intranasal E2 are similar to those of other estrogen regimens that have been proven to prevent postmenopausal osteoporosis.
The decrease in bone formation observed after 3–6 months of estrogen
therapy has been generally ascribed to a decrease in bone turnover and
a loss of remodeling sites, rather than to any direct effect on
osteoblasts. Accordingly, for oral E2 we found no
significant changes of serum osteocalcin, serum PINP, and serum BAP
after 1 month of treatment, but there was a significant decrease in the
two former markers after 3 months. In contrast, intranasal
E2 at any dose did not decrease bone formation during the
first 3 months. More importantly, 1 month of treatment with the two
highest doses of intranasal E2 induced a slight (9–22%),
but significant, increase in formation markers, suggesting that in
addition to its antiresorptive effect, intranasal E2 could
directly stimulate bone formation. Several in vitro studies
have reported a direct stimulatory effect of E2 on rodent
and human osteoblastic cells, including an increase in 1 type I
collagen messenger ribonucleic acid (mRNA) and osteocalcin secretion
(33, 34, 35). However, the effects of E2 on bone formation have
been difficult to demonstrate in vivo, although some
histomorphometric studies in rodents and humans have reported an
increase in trabecular bone formation with high doses of estrogen
(36, 37, 38). This large study using specific and sensitive indexes of bone
turnover suggests for the first time that conventional doses of
E2 may induce a transient direct stimulation of bone
formation in humans when given by the intranasal route. Whether this
effect is specific for intranasal E2 or general to the
nonoral route of administration, such as transdermal patches, remains
to be investigated, as most previous randomized placebo-controlled
studies looking at the effects of transdermal E2 have not
assessed bone formation at 1 month.
To explain the different short term effects of oral and intranasal E2 on bone formation markers, we measured circulating IGF-I. More than 75% of plasma IGF-I circulates in a 150-kDa ternary complex with IGFBP-3 and an acid-labile subunit, whereas the remaining peptides are found in a 50-kDa complex with IGFBP-1 to -6 (39). Thus, to evaluate the effects of estrogens on bioavailable IGF-I, alterations in the concentrations of IGFBPs need to be taken into consideration. In that study, we found no significant change from placebo in serum IGFBP-3 levels over the 3-month period in women treated with either oral or transdermal E2 in agreement with previous smaller nonplacebo-controlled studies (40, 41, 42). Thus, the contrasting effects of oral and intranasal E2 on serum IGF-I levels, i.e. a marked decrease for the oral formulation and a nonsignificant change for intranasal administration, are likely to reflect differences in bioavailable serum IGF-I. However, we cannot exclude different effects of oral and intranasal E2 on the other IGFBPs. As E2 has been shown to inhibit hepatic IGF-I mRNA expression (43), the decrease in serum IGF-I during oral E2 treatment probably results from exposure of the liver, the major source of circulating IGF-I, to the high estrogen concentration that follows intestinal absorption. The link between circulating IGF-I levels and bone turnover, however, is unclear. Indeed, IGF-I in bone originates not only from the circulation, via trapping by hydroxyapatite (44), but also from the osteoblasts and the bone marrow stromal cells. Thus, whether serum concentration of IGF-I reflects skeletal tissue levels remains an open question. It has been shown, however, that the proportions of IGF-II/IGF-I in human skeletal tissue are nearly identical to the serum ratio (45) and that serum IGF-I correlated with spine, femoral neck, and radius BMD in women and men (46, 47), suggesting that circulating levels may be an adequate index of skeletal status. Whatever the major sources of IGF-I in bone, it is well recognized that this growth factor is a potent mitogen for osteoblastic cells in vitro (48, 49), stimulating alkaline phosphatase activity and increasing osteocalcin in rat calvaria (50) and human bone cells (51). Interestingly, E2 stimulates the expression of IGF-I mRNA in several rat osteoblastic cell lines, and its bone-forming effects are blocked by anti-IGF-I antibodies, suggesting that, at least in part, the stimulatory effects of E2 on osteoblastic activity in vitro could be mediated by IGF-I (52). We found that the changes in circulating IGF-I correlated with the changes in serum osteocalcin, serum PINP, and serum BAP, suggesting that the stimulatory effect of estrogen on bone formation may be mediated by IGF-I in vivo, although other mechanisms are likely to be involved. Taking into account these data, we can speculate that the increase in bone formation markers at 1 month with intranasal administration is likely to reflect the direct effect of E2 in stimulating osteoblastic activity. At 3 months, this effect could be balanced by the decrease in the overall bone turnover rate, resulting in an apparent lack of change in bone formation.
After 1 yr of treatment with intranasal E2 (300 µg), both bone resorption and bone formation markers decreased and returned to the premenopausal range, i.e. the levels in estrogen-repleted women, as previously reported after 1-yr treatment with 0.625 mg conjugated equine estrogen (30, 17), oral E2 (2 mg) (6), and transdermal E2 (50 µg) (17). These data indicate that although the decrease in bone formation is delayed for intranasal compared to oral E2, their long term effects on the overall rate of bone turnover are likely to be similar. However, as the main increase in BMD with antiresorptive therapy occurs during the first year, a sustained bone formation with a decreased bone resorption in the initial phase of treatment may result in a higher bone gain, an interesting hypothesis that needs further investigation.
In conclusion, this large study shows that intranasal administration of E2 normalizes bone turnover to premenopausal levels after 1 yr of treatment. Intranasal E2 does not alter serum IGF-I levels, contrasting with the dramatic decrease in this growth factor induced by oral administration. This results in a sustained bone formation during the first 3 months of treatment with intranasal, but not oral, E2 despite a decrease in bone resorption, which was similar for both routes of administration. Studies with BMD measurement are required to assess the long term effect of intranasal E2 on bone mass.
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Acknowledgments |
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Received September 30, 1998.
Revised March 22, 1999.
Accepted April 8, 1999.
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References |
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