The adverse skeletal effects of selective serotonin reuptake inhibitors

European Psychiatry 27 (2012) 156–169

Review

The adverse skeletal effects of selective serotonin reuptake inhibitors E.M. Tsapakis a,b, Z. Gamie c, G.T. Tran c, S. Adshead c, A. Lampard c, A. Mantalaris d, E. Tsiridis c,*,e a

Maudsley Hospital & Institute of Psychiatry, King’s College London, London, UK, SE5 8AF Aghios Charalambos Mental Health Centre, Heraklion, Crete, 71306 Greece Academic Unit of Trauma & Orthopaedics, Leeds School of Medicine, Leeds General Infirmary, Clarendon Wing A, Great George Street, Leeds, UK, LS1 3EX d Biological Systems Engineering Laboratory, Department of Chemical Engineering, South Kensington Campus, Imperial College London, London, UK, SW7 2AZ e Department of Surgery and Cancer, Division of Surgery, Imperial College London, B-block, Hammersmith Hospital, Du-Cane Road, London, UK, W12 0HS b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 6 June 2010 Received in revised form 5 October 2010 Accepted 17 October 2010 Available online 3 February 2011

Selective serotonin reuptake inhibitors (SSRIs) are a widely used group of antidepressants (ADs) with reported potential detrimental effects on bone mineral density (BMD) and increased fracture risk. Here, a comprehensive review of the in vitro, in vivo and clinical studies to date was carried out using the medical search engines MEDLINE (1950 to September 2010) and EMBASE (1980 to September 2010). Serotonin (5-HT) receptors have been identified on osteoclast, osteoblast and osteocyte cell lines. The effect of SSRIs on bone formation and resorption appears to be governed by the activation of a number of 5-HT receptors on osteoblasts and osteoclasts via endocrine, autocrine/paracrine and neuronal pathways. In vitro, in vivo and clinical collective data appears to indicate that SSRIs have a negative effect on bone at the therapeutic dose levels widely used for the treatment of depression in current clinical practice. Caution may therefore have to be employed with the use of SSRIs in patients at an increased risk of falls and osteoporosis. Further studies are needed in order to fully elicit the role of SSRIs in bone formation and their effects in the low oestrogen state. ß 2010 Elsevier Masson SAS. All rights reserved.

Keywords: Serotonin Reuptake Inhibitor Bone Resorption Formation

1. Introduction Selective serotonin reuptake inhibitors (SSRIs) represent a class of commonly used antidepressants (ADs). They act by preventing the reuptake of 5-hydroxytryptamine (5-HT) (serotonin) through the inhibition the 5-HT transporter (5-HTT) which is located on the presynaptic neurone, thereby increasing levels of 5-HT within the synaptic cleft and modulating neurochemical signalling [38]. Furthermore, SSRIs act on 5-HT receptors, thus modulating the release of 5-HT directly [38]. Recent research has also demonstrated a wider role for the SSRIs, as 5-HT plays an active role in numerous pathways including that of bone metabolism. Osteoblasts and osteoclasts express 5-HT receptors and can be exposed to 5-HT via autocrine, paracrine and endocrine pathways [52,57]. Clinical studies have shown a relationship between the use of SSRIs, reduced bone mineral density (BMD) and an increased risk of bone fracture [3,14,21,23,56]. However, there has been conflicting evidence regarding their effects on bone metabolism. Both an increase and decrease in BMD has been found, as well as a dose-dependent effect [21,41,49,51]. This paper aims to review the literature and

* Corresponding author. Direct line: +113 3922621; mob: +77 8685 8586; Secretary: Tel.: +113 3929901; fax: +113 3923290. E-mail address: [email protected] (E. Tsiridis). 0924-9338/$ – see front matter ß 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.eurpsy.2010.10.006

summarise the clinical evidence for the skeletal effects of SSRIs and the potential underlying mechanisms.

2. Clinical studies The effect of SSRIs and the risk of reduced BMD or fracture (vertebral or non-vertebral) has been addressed by few clinical studies. Longitudinal, cross-sectional and population-based cohort studies have either demonstrated a relationship between SSRIs and reduced BMD in both males and females [14,23,56] or no effect [27]. Indeed, SSRIs have been shown to affect BMD to the same extent as glucocorticoids [23]. Case-control studies have also found a relationship between the use of SSRIs and increased odds of hip fracture [8,31,49]. An odds ratio (OR) of 2.4 favoring hip fractures (95% CI 2.0–2.7) was found after adjusting for co-morbidity and previous drug exposure in a study investigating 891 patients taking SSRIs [31]. In this early study no dose relationship was found across the three dose categories used, low (< 0.5 defined daily dose [DDD]), medium (0.5–1.5 DDD), and high (> 1.5 DDD) [31]. Doses of ADs may be standardized through means of a DDD, which equates to the average dose a person uses per day according to the WHO and allows ease of comparison between different drugs [30]. Interestingly, a non-significant duration effect was demonstrated whereby hip fracture was greater but not significant in current new users of SSRIs when compared to those classed as continuous current SSRI

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users [31]. The study investigated new current users, defined as those who had a prescription within 30 days of the fracture, but no other prescriptions for that drug in the period 31–365 days before the fracture. New current users were compared with continuous current users, defined as those who had a prescription within 30 days of the fracture and at least one other prescription in the period 31–365 days before the fracture. [31]. In a study by Vestergaard et al., 2006 [48], a progressive increase in the risk of fractures at the wrist, hip and spine was shown with an increasing dose of the SSRI. However, when compared with other ADs, this increased fracture risk associated with SSRIs was only significant for fractures at the hip [48]. Similarly, in a further study by the same group, a population of 124,655 cases matched for age and gender was investigated, and a dose-dependent increase of fracture risk in patients taking ADs was found, more so with SSRIs than tricyclic ADs (TCAs) [49]. A significant dose response was observed with citalopram, sertraline and fluoxetine across the three dose categories investigated, low ( 0.25 DDD), medium (0.251–0.5 DDD), and high (> 0.5 DDD). Citalopram at any dose, however, including the lower dose categories was associated with an increased risk of any fracture, but for fluoxetine, paroxetine and sertraline, the effect was observed more so in higher dose categories. The increase in the risk of fracture was more evident with short, rather than extended, duration of use particularly for citalopram and fluoxetine, which the authors suggested was due to an increased risk of falls [49]. The findings have been further supported in a study investigating osteoporotic fracture risk (vertebral, wrist or hip) in 984 patients over the age of 50 taking SSRIs matched for age, sex, ethnicity, and co-morbidity. A significant increase in the risk of fracture was found in those currently taking SSRIs defined as at least one dispensation of the drug, 120 days preceding the date of the fracture (fully adjusted OR of 1.45 [1.32–1.59 95% CI]). A significant trend was found of increasing fracture risk with increasing dose across the three dose tertiles investigated; more so than other psychotropic medications investigated [8]. None of the psychotropic medications investigated were associated with an increased risk of fracture in individuals classified as past users, defined as at least one dispensation in the 121 to 365 days preceding the fracture. Therefore strengthening the possibility of a causal connection between fracture risk and proximity of medication exposure. Prospective cohort studies have also demonstrated that fracture risk is increased for those taking SSRIs. The Rotterdam cohort study investigated 18 individuals and found that there was a significant 2.35-fold increase in the Hazard Ratio (HR, 95% CI, 1.32–4.18) in non-vertebral fractures in users of SSRIs [62]. The results were only found to be significant in the current user category due to the small number of patients studied. Current use was defined as use of an AD on the date of the occurrence of the fracture. There was a clear duration effect before adjusting for confounding factors, with approximately a three-fold risk increase for those using SSRIs for at least 6 months than those not exposed at all. Interestingly, after accounting for confounding factors such as lower-limb disability, disability category, and the presence of depression, the duration of effect on risk of fracture was less evident, with no significant dosedependent relationship [62]. Data from the osteoporotic fracture in men cohort also demonstrated an increased risk of non-spine fracture [29]. In this study 275 of 5876 (4.7%) men experienced a fracture. After adjusting for age and BMD, the HR associated with the use of SSRIs was 1.65 (95% CI 0.92 to 2.94) and TCAs was 2.39 (95% CI 1.27 to 4.50) [29]. A 2003 cohort study assessed the incidence of non-spine fracture amongst 501 women. In this population 452 were daily users of ADs, 70% (n = 353) were prescribed a TCA (amitriptyline, clomipramine, doxepin, imipramine, trimipramine, desipramine,

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nortriptyline, or protriptyline) and 21% (n = 103) an SSRI (fluoxetine, paroxetine, or sertraline)[17]. It was reported that women taking any AD daily had a 1.7-fold increase in the risk of hip fracture (95% CI 1.05–2.57) and a 1.3-fold increase in the risk of non-spine fracture (95% CI 0.99–1.58). Although data concerning duration of treatment and daily dose was not available, women taking SSRIs had a 1.54-fold increase in the relative risk of hip fracture (95% CI 0.62–3.82), whilst those taking TCAs had an 1.83fold increase in hip fracture (95% CI 1.08–3.09). However, the relative risk was significant only for those women prescribed TCAs. Data from the Canadian Multicentre Osteoporosis Study (CaMOS) cohort, revealed that 137 patients over 50 taking SSRIs daily had reduced BMD at the hip and spine, and a doubled risk of fracture (hazard ratio [HR], 2.0; 95% CI, 1.3–3.1) compared to non-users, after adjusting for BMD [41]. Furthermore, this was a dosedependent relationship, with a HR of 1.5 (95% CI, 1.1–2.1) for each unit increase in SSRI daily dose. One unit was equivalent to 20 mg/ d of citalopram, 20 mg/d of fluoxetine, 50 mg/d of fluvoxamine, 20 mg/d of paroxetine, or 50 mg/d of sertraline, although the differential effects of SSRIs were not assessed. ADs have different affinities for the 5-HTT and the relationship between the individual affinity for the 5-HTT and the risk of osteoporotic fractures has recently been explored in a case-control study [47]. ADs with a high affinity for the 5-HTT such as citalopram, fluoxetine, paroxetine, sertraline and fluvoxamine have been associated with a significantly higher OR of osteoporotic fracture than ADs with medium or low affinity for the 5-HTT such as the TCAs [47]. However, clomipramine and imipramine are both TCAs that have a high affinity for the 5-HTT. Imipramine has been found to be associated with a three-fold increased fracture risk, an effect not found with clomipramine. This suggests that other pharmacological mechanisms of TCA use may account for the increased risk of fracture [47]. 3. Selective serotonin reuptake inhibitors and the risk of falls One case-control study has demonstrated an increased risk of hip fracture early in the course of SSRI treatment, with an OR of 6.30 (95% CI: 2.65, 14.97) in the first 2 weeks of SSRI administration. Meanwhile, a case series analysis showed an Incidence Ratio of 1.96 (95% CI: 1.35–2.83) [26]. The risk of fracture at 14 days or less was found to be greater than the risk between 15 and 42 days or at 43 days or over. Fluoxetine, in particular, was associated with a greater risk of hip fracture when commenced at 14 days or less prior to the fracture compared with paroxetine, which may be a result of an increased falls risk associated due to side-effects such as orthostatic hypotension and syncope. Results from the case series analysis were considered to be more valid due to potential selection and indication biases present in case-control studies, which could possibly overestimate the effects of SSRIs [26]. These results were corroborated in a further study where the use of SSRIs was shown to be associated with an increased risk of falls [41]. SSRIs exert cardiovascular effects such as dysrhythmias and bradycardias, which commonly lead to falls. SSRIs are hypothesized to inhibit cardiovascular Na+ and Ca2+ channels, which lead to dysrhythmic effects [39]. It must also be noted that depression has been associated with low bone density and agitation, which may increase the risk of fractures [37] (Table 1). 4. Sources of 5-HT and its effects on bone The source of 5-HT which influences bone is under investigation as 5-HT can be synthesised in gut [40], bone [21] and the CNS [13]. It has previously been suggested that indirect sources of skeletally relevant 5-HT such as the GI tract are unlikely [50] due to rapid

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Table 1 Summary of evidence from clinical studies. Design

Method

Outcome measures

Results

Conclusion

Case-control study of 8239 cases, aged 66 y+ with hip fracture Each matched for age and sex to five controls

Case data from discharge summaries from acute hospitals and controls data drawn from administrative healthcare database from Ontario, Canada Prescription data from pharmacy database

Defined daily dose and type of medication (TCA or SSRI)

Adjusted odds ratio for hip fracture was 2
4 (95% CI 2
0–2
7) for exposure to SSRIs, 2
2 (1
8– 2
8) for secondary-amine TCAs, and 1
5 (1
3–1
7) for tertiaryamine TCAs The odds ratios for hip fracture were higher for new current users than for continuous current users in all three drug classes No significant difference in estimated odds ratios across dose categories within any of the three drug classes

No significant difference in the risk of hip fracture between users of SSRIs and users of secondary-amine TCAs. Lower risk of fracture with tertiary-amine TCAs Risk of fracture highest in new users of antidepressants

Ensrud et al. 2003 [17] Central nervous system active medications and risk for fractures in older women

Prospective longitudinal analysis of 8127 community dwelling women aged 65y+ (SOF data) Average follow up 4.8 y

Questionnaire and series of interviews Self reporting of drug use Fractures confirmed by radiograph

Fracture incidence and type Defined daily dose of drug and duration

All antidepressants had increased fracture risk non spine (multivariate HR, 1.25; 95% CI, 0.99–1.58), hip (multivariate HR, 1.65; 95% CI, 1.05–2.570) after adjustment for confounders

TCA’s and SSRIs appeared to increase hip fracture risk compared to non users but this was only statistically significant in TCA users

Hubbard et al. 2003 [26] Exposure to Tricyclic and Selective serotonin reuptake inhibitor antidepressants and the risk of hip fracture

Case control analysis and a self-controlled caseseries analysis using 1987–1999 diagnosis data for 16,341 cases of hip fracture and 29,889 controls

Data drawn from the United Kingdom General Practice Research Database

Index date i.e., date of fracture Prior prescription for antidepressant Duration of depressant use

Odds ratios for fracture within the first 15 days of use: TCAs 4.76 (95% confidence interval (CI): 3.06, 7.41) SSRIs 6.30 (95% CI: 2.65, 14.97) Incidence ratios for same were 2.30 (95% CI: 1.82, 2.90) and 1.96 (95% CI: 1.35, 2.83) Fracture risk with TCAs peaks in first 15 days of use Fracture risk with SSRI use persistently high for 6 weeks

TCAs and SSRIs both associated with an independent increase in hip fracture incidence during the first weeks of treatment. Patients who commence antidepressant therapy will have a transient doubling of fracture risk

French et al. 2005 [18] Hip fracture linked with outpatient medications in the study group compared with matched control groups of patients with hospitalisations for an acute MI or pneumonia

Case-control evaluation of risk of hip fractures in three different drug classes (1) antiepileptics/ barbiturates, (2) antidepressants (selective serotonin reuptake inhibitors [SSRIs] and tricyclic antidepressants [TCAs]), and antiparkinson drugs

2212 hip fracture patients. 2212 control patients with acute MI or pneumonia

Controls matched for sex and age, no adjustment for confounding factors

2-fold increase in the risk of fracture in SSRI and TCA users vs control

SSRIs and TCAs are related to an increased risk in fall related hip fracture

Kinjo et al. 2005 [27] Bone mineral density in subjects using central nervous system-active medication

Cross sectional analysis of 14,646 adults aged 17y+ Data from NHANES III study

Interview and physical examination Self reporting of drug use, dosage and duration

DEXA bone scan to determine BMD of proximal femur Defined daily dose of drug

1.1% of those surveyed were antidepressant users (TCAs + SSRIs) No reduction in BMD in SSRI/TCA users when confounders are adjusted for

In this population (mean age 48 y) no effect on BMD was found

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Study Liu et al. 1998 [31] Use of selective serotonin-reuptake inhibitors or tricyclic antidepressants and risk of hip fractures in elderly people

Table 1 (Continued ) Method

Outcome measures

Results

Conclusion

Case control study of subjects sustaining any fracture during the year 2000 (n = 124.655). For each case, three controls (n = 373.962) All ages, male and female included

Using Danish National health service computerised records exposure to drugs identified

Defined daily dose of drug

Antidepressants, a dose–response relationship was observed for fracture risk (OR: increasing from 1.15, 95% CI: 1.11–1.19 at < 0.15 DDD/day to 1.40, 95% CI: 1.35– 1.46 for  0.75 DDD/day). The risk of fracture was higher with selective serotonin reuptake inhibitors than with tricyclic antidepressants

For antidepressants a dose– response relationship was found, with a higher fracture risk for SSRIs

Diem et al. 2007 [14] Use of antidepressants and rates of hip bone loss in older women. The study of osteoporotic fractures

2722 women aged 65y+ (mean age, 78.5 years) from the Study of Osteoporotic Fractures (SOF), a community based prospective cohort study Average follow up 4.9 years

Questionnaire based on serial visits as part of The Study of Osteoporotic fractures

BMD measurement by DEXA of 2 body regions on 2 separate occasions Self-reported antidepressant use (SSRIs + TCAs) Geriatric Depression Score (GDS) of 6+ considered indicative of depression.

Mean total hip BMD decreased 0.47% per year in nonusers compared with 0.82% in SSRI users (P_.001) and 0.47% in TCA users (P = .99). Higher rates of bone loss were also observed at the 2 hip subregions for SSRI users. Excluding those with GDS score > 6 did not significantly alter results

Use of SSRIs but not TCAs is associated with an increased rate of bone loss at the hip in this cohort of older women with depression

Haney et al. 2007 [23] Association of low bone mineral density with selective serotonin reuptake inhibitor use by older men

Cross sectional analysis of men 65+ n = 5995. Prospective cohort from MrOS study.

Questionnaire, interview and examination from study data

BMD by dual X-ray absorptiometry of lumbar spine and total hip at baseline Medication type, duration and dose

BMD in SSRI users 3.9% lower at total hip and 5.9% lower at lumbar spine than in non users Users of other antidepressants showed no significant difference in BMD at any site

SSRI users have lower BMD even after adjustment for depression using SF-12

Richards et al. 2007 [41] Effect of selective serotonin reuptake inhibitors on the risk of fracture

Prospective cohort study, randomly selected n = 5008, aged 50+. Population based on CaMos study. 5 yr follow up. Cross sectional and longitudinal analyses

Interviews, self reported drug use and fracture incidence. X-ray confirmation

BMD by dual X-ray absorptiometry of lumbar spine and hip at baseline Number of clinically incident fragility fractures i.e. Minimal trauma Medication type, duration and dosage

Daily SSRI use increased incidence of fragility fracture to hazard ratio of 2.1 (95%CI, 1.3– 3.4), increased odds ratio of falling (2.2, 95%CI, 1.4–3.5). BMD 4% lower in SSRI users at hip and 2.4% (not significant) at lumbar spine

Daily SSRI use was associated with a 2-fold increased risk of clinical fragility fracture after adjustment for confounders Risk of fracture in SSRI users remains higher even when adjusted for BMD and falls risk suggesting there may a further mechanism involved

Lewis et al. 2007 [29] Effect of selective serotonin reuptake inhibitors in nonvertebral fracture in elderly men.

Prospective, multicenter cohort evaluation of risk of nonspine fracture 5876 in men (MrOS Study) followed an average of 4.1 years

Fracture reporting via triannual questionnaire and varified by investigators

Fractures confirmed by radiographs

275 (4.7%) men experienced a fracture. Age- and BMD-adjusted HR SSRI: 1.65 (95% CI 0.92 to 2.94) TCA: 2.39 (95% CI 1.27 to 4.50)

Use of SSRI was associated with nearly a two-fold increase in risk of fracture

Ziere et al. 2008 [62] Selective serotonin reuptake inhibiting antidepressants are associated with an increased risk of non-vertebral fractures

Prospective population cohort study n = 7983 aged 55+ from Rotterdam Study. 9–12 yr follow up

Interview, computerised medical records and data of computerised drug dispensing pharmacies

Incidental non-vertebral fractures during follow up period Exposure defined as current, past or nonusers of drug Duration of drug use. Defined daily drug dosage

2.35-fold risk of non-vertebral # with SSRI compared to nonusers (95%CI, 1.32–4.18). Fracture risk with current SSRIs 2.07-fold (95% CI, 1.23–3.50) that of past users of SSRIs or TCAs. Greater than 3-fold increase risk if use SSRIs for more than 3 months (P = 0.0001) and 9% extra risk per month (P = 0.004) Greatest fracture risk with SSRI (2.35)>TCAs (1.60). No increased risk with ‘‘other’’ antidepressants (0.79)

Increased risk of nonvertebral fracture with SSRI use even when adjusted for confounding factors Effect is duration dependant

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Design

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Study Vestergaard et al. 2006 [48] Anxiolytics, sedatives, antidepressants, neuroleptics and the risk of fracture

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Table 1 (Continued ) Design

Method

Outcome measures

Results

Conclusion

Retrospective case control study based on population administrative database. Cases were 50 years+ with hip/wrist/vertebral fracture (n = 15,792) each matched to 3 controls (n = 47,289)

Computerised health and dispensary records. Controls matched randomly to cases by age, sex, ethnicity and comorbidity index

Index date fracture Antidepressant type classified. Exposure defined as current, past and non-use Duration and dosage of drug used

Fully adjusted odds ratio (and confidence interval) for osteoporotic fractures: SSRIs 1.45 (1.32–1.59) p < 0.01m Other MAOI 1.15 (1.07–1.24) Lithium 0.63 (0.43–0.93) Benzodiazepines 1.10 (1.04– 1.16) SSRIs showed dose dependant increased risk of fracture p < 0.05

SSRIs had the strongest positive association with fractures among all psychotropic medications, even after adjusting for physical and psychiatric diagnoses and other medications implicated in fracture pathophysiology

Vestergaard et al. 2008 [49] Selective serotonin reuptake inhibitors and other antidepressants and risk of fracture

Case control study with 124,655 fracture cases and 373,962 age and gendermatched controls All ages and both sexes included

Data from National Danish Hospital discharge registry and national prescription registry

Defined daily dose of drug exposed to

There was a dose–response relationship for several SSRIs (citalopram 2P = 0.01, fluoxetine 2P = 0.03, fluvoxamine 2P = 0.74, paroxetine 2P = 0.078, sertraline 2P = 0.01) The increase in relative risk of hip fractures was larger than the increase in other fracture types. The increased fracture risk with SSRIs peaks in initial period of use and though still present, declines with extended use, e.g. for fluoxetine 1.31 (1.05–1.65) at 0.5 year to 1.08 (1.02–1.14) at 2.5 years

SSRIs associated with increased fracture risk whilst TCAs and other antidepressants are not. The effect of antidepressants on the risk of fractures may be linked to their effect on the serotonin transporter system and BMD

Spangler et al. 2008 [44] Depressive symptoms, bone loss, and fractures in postmenopausal women

Prospective cohort study of 93,676 postmenopausal women aged 50–79y (WHI study) Subset of those who had BMD study n = 6,441 Av follow up 7.4 y

Data from Women’s Health Initiative study Note- excluded women with severe mental illness

DEXA bone scan at baseline and 3 years Self-reporting of fractures

8% taking antidepressants (4% SSRIs, n = 3,534) No association found between antidepressant use and change in BMD at 3 years, after adjustment for confounders Antidepressant therapy increased the risk of spine fracture (HR = 1.36; CI = 1.14 to 1.63) SSRI use associated with any fracture (OR = 1.3, 95%CI 1.2– 1.41) and wrist fracture (OR = 1.29, 95%CI 1.07–1.56) but not hip or spine

No significant associations observed between antidepressant therapy and changes in BMD Small associations with antidepressant use and some sites of fracture noted

Williams et al. 2008 [56] Selective serotonin reuptake inhibitor use and bone mineral density in women with a history of depression

Cross sectional analysis of 128 community-based women with lifetime or recurrent depression Geelong Osteoporosis Study Data

Structured clinical interview (DSM)-IV-TR research version, non-patient edition

BMD at spine, hip, total body and forearm using dual energy absorptiometry Self reporting of medication use

26 (20%) of women reported SSRI use BMD among SSRI users was 5.6% lower at the femoral neck (P = 0.03), 6.2% lower at the trochanter (P = 0.04) and 4.4% lower at the mid-forearm (P = 0.03) than nonusers after adjustment for confounders No differences in BMD were detected at other sites including spine

In women with depression, SSRI use is associated with reduced BMD at some anatomical sites, which equates to approximately 20% increase in fracture risk

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Study Bolton et al. 2008 [8] Fracture risk from psychotropic medications – a population based analysis

Conclusion

SSRIs particularly increased risk of hip fracture, whilst other drugs increased vertebral or other fractures. This may be due to increased fall risk with certain medications

Results

Several central nervous system active drugs found to increase fracture risk. Anti-depressants were used by 7.7% of control subjects. Antidepressants as a class were associated with an increased fracture occurrence (any fracture OR 1.3 [1.2–1.4], hip OR 1.5 [1.3– 1.6]) SSRI were associated with the following odds ratio of fracture; any 1.7 [1.6–1.9], hip 2.0 [1.8– 2.2], spine 1.2 [1.0–1.5])

Outcome measures

Defined daily dose of drugs taken within 5 years preceding fracture Fracture incidence and type

Method

Data from Danish National Hospital Discharge registry and national prescriptions database

Design

Case control study of 15,716 men aged 50+ who sustained fracture and 3 age and sex matched controls (n = 47,149) for each case

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5-HT uptake by platelets [34] and the metabolism of residual 5-HT by the liver and lungs [46]. Bone-modulating 5-HT seems to be produced in an autocrine/paracrine mode [50]. Several studies have indicated that bone may be capable of synthesising 5-HT, since osteoblasts (MC3T3-E1 cells) [6], osteocytes (MLO-Y4 cells) [6] and osteoclasts (RAW 264.7 and human peripheral blood monocytes derived) [21] have been found in RT-PCR studies to express mRNA for tryptophan hydroxylase 1 (TPH1), which is responsible for the rate limiting step of 5-HT production [60]. However, this has been brought to question as one study found no evidence of expression of mRNA for either TPH, or aromatic Lamino acid decarboxylase in RAW246.7 osteoclasts on RT-PCR analysis [3]. 5-HT receptors have been identified on osteoclasts (5HTR1A,1B,2A,2B,2C,4) [3,21], osteoblasts (5-HTR1A,1B,1D,2A,2B,2C) [5,21,24,32,54,57] and osteocytes (5-HTR1A,2A,2B) [6,54]. 5-HTR has also been found intracellularly in the somatodendritic and axonal locations of CNS in adult rats [12], but there is no evidence of internal 5-HTRs in osteoblasts or osteoclasts at present. Recent evidence suggests that gut-derived 5-HT may exert an effect on bone metabolism [57]. In a key study, a membrane protein found in enterochromaffin cells in the duodenum called low density lipoprotein receptor related protein-5 (LRP5) was shown to decrease 5-HT and to have an anabolic effect on bone. [57] TPH1gut ve mice have been shown to develop a high bone mass phenotype due to an increase in osteoblast proliferation with no associated bone resorption; however, mice deficient in osteoblast TPH (TPH1osb ve) have only a marginally increased bone mass phenotype, indicating the differential effect of gut- and bone-derived 5-HT in bone metabolism. It is of interest that brain-derived 5-HT has been shown to have a greater influence on bone- than gut-derived 5-HT [58] (Fig. 1). Recent evidence has suggested that bone resorption is increased by sympathetic tone [16,58]. It has been speculated that 5-HT may mediate, primarily through 5HTR2C in the hypothalamus, bone mass gain via a decrease in sympathetic activity [16,58]. Bone resorption has previously been shown to be regulated by a centrally-controlled leptin-dependent pathway acting on b2 adrenergic receptors (Adrb2) present on osteoblasts [16,20,43,45], and recent evidence has shown leptin to interfere with 5-HT production by reducing brain 5-HT synthesis and the activity of serotonergic neurons [58]. The regulation and breakdown of extracellular 5-HT occurs via 5-HTT. 5-HTT has been found in many tissues including the gut [19], as well as the synaptic junction and along the axonal membrane in the CNS [61]. 5-HTT mRNA has been shown to be present in osteoblasts [5], osteoclasts [21] and osteocytes, [6] and further studies have demonstrated the functionality of these proteins in bone cells [3,5,6].

5. 5-HT receptor pathways under investigation in bone

Study

Abrahamsen et al. 2009 [1] Mapping the prescription to fractures in men—a national analysis of prescription history and fracture risk

Table 1 (Continued )

5.1. Osteoblasts–The 5-HTR1 receptor family The 5-HTR1A, B and D receptors exert their effect by coupling to Gi/o protein, which then inhibits the formation of cAMP. Within the context of bone metabolism, inhibition of cAMP inhibits Protein Kinase A (PKA)-dependent phosphorylation pathways. This has been shown to reduce the phosphorylation of the transcription factor CREB, hence decreasing osteoblast proliferation [57] (Fig. 2). 5.2. Osteoblasts–The 5-HTR2 receptor family In contrast, 5HTR2A,B&C receptor activation increases osteoblast proliferation via the Phosphate Kinase C (PKC) pathway [21]. More specifically, the stimulation of 5HTR2A reduces osteoblastic

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Fig. 1. (1) Stimulation of receptor 5-HTR2C in the hypothalamus may increase bone mass via a decrease in sympathetic activity. Figure adapted from [58,16]. (2) Centrallycontrolled leptin dependent pathway acting on b2 adrenergic receptors (Adrb2) present on osteoblasts increasing bone resorption. Leptin can also reduce 5-HT synthesis in the brain as well as the activity of serotonergic neurons. Figure adapted from Figure adapted from [58,16].

Fig. 2. Stimulation of the 5-HTR1a receptor inhibits cAMP mediated inhibition of Protein Kinase A (PKA) dependent phosphorylation pathways. This in turn reduces the phosphorylation of the transcription factor CREB, therefore decreasing osteoblast proliferation. Figure adapted from [57].

signaling for the differentiation and activation of osteoclasts via increased osteoprotegerin (OPG) and reduced Receptor Activator for Nuclear Factor-kappaB (NF-kB) Ligand (RANKL) [21] (Fig. 3). The exact action of 5-HTR2B in osteoblasts is yet to be fully elucidated, but a putative mechanism may rely upon its interaction with 5-HTT. Binding of 5-HT to the 5-HTR2B receptor has been shown to initiate phosphorylation of the 5-HTT thus inactivating transport and reducing the reuptake of 5-HT via the PKC pathway [28]. In vivo evidence in mice demonstrating an increase in serum 5-HT upon activation of 5-HTR2B, possibly via its effect on 5-HTT, supports this finding [10]. Moreover, 5-HTR2B preferentially binds to Gq/11 protein, which enhances the hydrolysis of inositol phosphate and subsequently increases intracellular Ca2+ [11,25]. This increase in cytoplasmic Ca2+ has been postulated to induce osteoblast proliferation [59]. 5-HTR2B has been shown to mediate arachidonic acid (AA) production by activation of the phospholipase A2 (PLA2) pathway, which promotes prostaglandin E2 production through COX activation, thereby increasing mineralisation [32]. Finally, the 5-HTR2B receptor also regulates the production of nitric oxide (NO) and its inhibition results in a decrease of in vitro mineralisation [11,21,24,32] (Fig. 4). 5.2.1. Osteoclasts The 5-HTR1B,4 receptors have been shown to play a role in osteoclast differentiation, although the exact mechanism remains unknown [3]. Treatment of bone marrow cells with antagonists to these receptors were shown to significantly reduce osteoclast formation and also reduce the osteoclast markers tartrate-

Fig. 3. Stimulation of the 5-HTR2a receptor can increase osteoblast proliferation via the PKC pathway and also increase OPG and decrease RANKL production thereby reducing osteoclast proliferation. Figure adapted from [21].

resistant acid phosphatase (TRACP), cathepsin K, and Matrix Metalloproteinase 9 (MMP-9) suggesting their importance in the early phases of osteoclast differentiation [3]. A possible mechanism is the activation of transcription factors such as NF-kB and cFos, which may increase the proliferation and differentiation of osteoclasts [3,21]. 5.3. In vivo To date the results of such research in animal studies are conflicting, supporting both potentially detrimental and beneficial effects of 5-HT on bone [4,11,21,51,52,55]. 5.4. Selective serotonin reuptake inhibitors–do they have a direct effect on bone in vivo? In a number of studies, a significant reduction in cage activity was noted in both SSRI-treated mice and null mutation for 5-HTT mice, and it was postulated that this could be responsible for the decreased BMD [9,51]. However, when SSRI-treated mice were compared to other classes of ADs in mice, only the SSRI group had reduction in BMD, suggesting a direct role of SSRIs on bone

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high dose of SSRI, at 50 mM. It may be that smaller doses may have a different effect, and furthermore, it has been shown that the effect of 5-HT may be dependent on the maturity of the osteoblast [24]. 5.5.1. A dose-dependent effect A dose-dependent effect has also been shown on the administration of fluoxetine. When mice were treated with 5 mg/kg/day or 20 mg/kg/day delivered intraperitoneally, the higher dose alone lead to significantly lower bone mineral content [51]. It has been further shown that daily administration of 5 mg/ kg/day or 20 mg/kg/day resulted in a significant dose-dependent decrease in BMD [52]. Moreover, administration of 10 mg/kg/day of fluoxetine in mice resulted in similar plasma fluoxetine levels at the lower range of that found in humans taking 20–80 mg/day [15]. Administration of 18 mg/kg/day resulted in plasma levels roughly equivalent to that of the higher range found in humans taking 20– 80 mg/day.

Fig. 4. (1) Binding of 5-HT to 5-HTR2B receptor has been shown to initiate phosphorylation of the 5-HTT thus inactivating transport and reducing the reuptake of 5-HT via the PKC pathway as described by Launay, J.M., et al., 2006 [28]. (2) 5HTR2B also preferentially binds to Gq/11 protein, which enhances the hydrolysis of inositol phosphate and subsequently increases intracellular Ca2+. (3) This increase in cytoplasmic Ca2+ has been postulated to induce osteoblast proliferation. Figure adapted from [59]. 5-HTR2B mediates arachidonic acid (AA) production via phospholipase A2 (PLA2) activation, which promotes prostaglandin E2 production through COX activation, thereby increasing mineralisation. 5-HTR2B receptor also regulates the production of nitric oxide (NO) and its inhibition results in a decrease of in vitro mineralisation. Figure adapted from [32].

metabolism [9]. This is reinforced by the finding of reduced BMD even in non-weight bearing bones, such as the skull of mice treated with SSRIs [51]. Furthermore, 5-HT has been shown to exert skeletal effects irrespective of animal activity [53]. 5.5. Adverse effects on bone The overall effect of SSRI treatment on bone mass in vivo could be attributed to an effect on bone formation and bone resorption [9,51–53]. Treating mice with fluoxetine 10 mg/kg/ day delivered subcutaneously 5 days per week for 4 weeks, has demonstrated both reduced bone volume and trabecular thickness [9] as a result of decreased bone formation alone, rather than an increase in bone resorption. This was supported in a further study where both 5-HTT knockout mice (5-HTT / ) and administration of fluoxetine on growing mice resulted in reduced bone density and inferior mechanical properties secondary to reduced bone formation alone [51]. Histomorphometry also showed a decreased lower extremity BMD as a result of decreased bone formation in 4-week-old mice treated with 20 mg/kg/day of fluoxetine intraperitoneally (i.p.) for 4 weeks, without a concomitant increase in osteoclasts. In addition to reduced bone formation however, increased bone resorption has also been demonstrated for reasons that are not clearly delineated in the current literature [52]. In 4–18 month-old female mice exhibiting 5-HTR2B null mutations, decreased bone formation has also been shown [11]. Osteoblasts exhibited both reduced proliferation and differentiation and this resulted in trabecular and cortical osteopenia. Interestingly, another study was unable to demonstrate any effect on bone metabolism on 5-HTR2B-null mice [57]. However, this analysis was performed on primary osteoblasts with a relatively

5.5.2. Beneficial effects of selective serotonin reuptake inhibitors However, not all research in this area supports the negative effects of SSRIs on bone. Indeed, 5-HT may exert no effect or even have a beneficial effect on bone metabolism in animals [4,22,55]. When fluoxetine was administered at doses of 5 mg/kg once daily via gastric intubation to rats over six months, no significant effect on bone architecture or mechanical properties could be shown [55]. Furthermore, an overall significant increase in total body BMD resulting from a reduction in the rate of bone resorption was found when rats were administered daily subcutaneous injections of 5 mg/kg 5-HT over three months [22]. When aged 8–14 weekold mice were administered 10 mg/kg/day fluoxetine i.p. for 6 weeks, an increase in femoral trabecular and total bone volume took place [4]. It is difficult to explain such conflicting results, but it is possible that the administration of SSRIs via gastric intubation may affect its pharmacological properties. Furthermore, the animal models used may also contribute to variability in the findings. The use of SwissWebster mice [4] may offer different results compared to other studies where C57BL6J strains [9] or even rats [55] were used. This is not enough to explain the conflicting results, however, as contradictory findings were shown when the same animal models were employed [4,52]. 5.5.3. The role of oestrogen The effect of oestrogen and its role in 5-HT and bone metabolism has also been evaluated in vivo. Oestrogen appears to play an important role in SSRI and bone metabolism. Studies with ovariectomised mice have shown conflicting results, with the effect of SSRIs being either independent of oestrogen status [52] or dependent on oestrogen for its anabolic effect [4]. In addition, decreased duodenal synthesis of 5-HT has been shown to offer a protective effect on BMD in oestrogen depleted states [57]. Interestingly, no effect on 5-HTR2B-null mutations was seen in male mice, whereas decreased bone formation was seen in females [11]. Oestrogen has been shown to increase the expression of 5-HTT and alter 5-HTR expression in rat and monkey brains [33,35,36]. It can be postulated that such changes may lead to centrally mediated alteration in bone mass, although the exact interplay between oestrogen, 5-HT and bone mass remains unclear. Although these findings are difficult to reconcile, they may be partly explained by differences in the age of mice, with adult mice used in one study [52] and mice between the ages of 8–14 weeks used in the other [4]. In addition, the length of SSRI treatment varied, ranging from 4-week [52] to 6-week administration [4] (Table 2).

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Table 2 Summary of in vivo evidence from animal studies. Animal Model

Agent

Method

Measurements

Results

Conclusion

Warden et al. 2005 [51] Inhibition of the serotonin (5-hydroxytryptamine) transporter reduces bone accrual during growth

Mice

Fluoxetine (5mg/20mg)/kg

Model 1 5-HTT knockout mice (5-HTT / ) (n = 33) and wild type (5-HTT+/+) (n = 41) of both sexes were either 4 (young) or 19 (adult) weeks of age when killed Model 2 Virgin female mice (n = 30) were divided into 3 groups. 1; vehicle (sterile saline), 2; low-dose fluoxetine hydrochloride (5mg/ kg), 3; high-dose fluoxetine hydrochloride (20mg/kg). Each groups received intervention via intraperitoneal injection (6.7 ml/ kg) daily for 4 continuous weeks. All mice were euthanised at 8 weeks of age

Bone mineral content (BMC) assessment via dual X-ray (whole-cranial, spinal L2-L5 and femoral) or CT (localized femoral volumetric BMD) Biomechanical properties via 3point bending Histomorphometry of femur, L5 vertebrae and cranium from young mice and femur from adult Serum assays for creatinine, total calcium, phosphorus and albumin

Model 1 Whole body BMC (6.4–13.0%) distal femoral (17.6–20.2%) and midshaft femoral (2.8–10.6%) were all lower in the 5-HTT / mice. Force to break (9.8-13.2%), energy absorption (14.3–29.9%) and stiffness (11.3–16.8%) of femur were all lower in 5-HTT / mice. 5-HTT / mice had significantly reduced bone formation rates but bone resorption was similar in both groups. Serum marker did not differ between both groups. Model 2 High-dose group gained significantly less whole-body bone mineral than controls. Distal femoral (9.9%, p < 0.001) and midshaft (2.6%, p < 0.05) BMC lower in high-dose compared to controls. High-dose groups showed a 27.8% lower periosteal bone formation rate to bone surface ratio compared to control

Inhibition of the 5-HTT via either null mutation of the 5-HTT gene or pharmacological intervention results in reduced bone mineral accrual by reducing bone formation. This may be specific to the 5-HTT in bone as opposed to other tissues

Gustafsson et al. 2006 [21] Long-term serotonin administration leads to higher bone mineral density, affects bone architecture, and leads to higher femoral bone stiffness in rats

Rats

Serotonin 5mg/kg

Cases- 10, two month old rats injected subcutaneously with serotonin daily for 3 months Controls – injected with saline

Free circulating serotonin by microdialysis BMD assessment by DXA scans after 3 months of injections Micro-CT to determine bone architecture Histomorphometry Mechanical testing by loading and 3 point bending of femur

Long lasting hyperserotoninaemia with > 10fold increase in serotonin Total body BMD higher in serotonin treated group (0.1976  0.0015 vs. 0.1913  0.0012g/ cm+3) Cortical thickness higher in serotonin treated group, but trabecular bone volume lower to give similar MOI. 12.3% Higher femoral stiffness on 3 point bending in serotonin group

Serotonin reduces resorption and/or increases apposition of endosteal bone. Elevated extracellular serotonin may have beneficial effects on bone in vivo

Battaglino et al. 2007 [4] Fluoxetine treatment increases trabecular bone formation in mice (fluoxetine affects bone mass)

Mice

Fluoxetine

Ovarectomised and sham operated Swiss-Webster mice aged 8-14 weeks Systemic fluoxetine administered by SC injection daily for 6 weeks, 10 mg/kg/day

Histology and histomorphometrical analysis TRAP staining Micro-CT to determine bone architecture

Fluoxetine treated mice had increased trabecular (+50%) and total bone volume(+70%) in femurs and vertebrae(+22%) Fluoxetine treated mice still suffered bone loss (50%) after ovariectomy

Fluoxetine therapy leads to increased bone mass, but does not protect against bone loss in oestrogen deprived states

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Study

Table 2 (Continued ) Animal Model

Agent

Method

Measurements

Results

Conclusion

Westbroek et al. 2007 [55] Long-term fluoxetine administration does not result in major changes in bone architecture and strength in growing rats

Rats

Fluoxetine

Twenty-one, 8 weeks old Sprague-Dawley female rats 11 rats given fluoxetine (5 mg/ kg, once daily, via gastric intubation; 10 controls received tap water, also via gastric intubation

DEXA measurements performed at the beginning and after 4 and 6 months of treatment Leptin + adiponectin plasma concentration measured by competitive radioimmunoassay Bone architecture of femur studied by micro CT and histomorphometry Mechanical properties by 3 point bending studies

Reduced bone tissue strength compensated for by changes in bone geometry leading to similar overall strength on 3 point bending Femoral BMD was slightly lower in the fluoxetine-treated animals (P = 0.078) In femoral metaphyses fluoxetine treatment resulted in a lower trabecular thickness (P = 0.033) and higher endocortical bone volume (P = 0.043) Leptin and adiponectin levels were not significantly different between cases and controls

In clinically relevant doses fluoxetine does not have significant effect on bone architecture or mechanical properties in rats

Collet et al. 2008 [11] The serotonin 5-HT2B receptor controls bone mass via osteoblast recruitment and proliferation

Mice

N/A

5-HT2BR knockout mice aged 5 wk to 18 months used to investigate a putative bone phenotype in vivo

Radiography, DEXA and microCT of tibiae Histomorphometry of mice femurs Biochemical analysis of urine to assess osteoclastic bone resorption Calvarial osteoblast primary culture and binding assays

5-HT2BR_/_ female mice displayed reduced bone density of femur compared to WT mice from age 4 months ( 5.25%) and this intensified with age; at 18 months the difference was more pronounced and was detected both for the whole body ( 6.5%) and for the femur ( 10.6%) Mineral apposition rate was not affected, indicating that osteoblast function was normal in 5-HT2BR_/_ female mice Calcium incorporation was markedly reduced in osteoblasts after5-HT2BR depletion Low bone mass in 5-HT2BR_/_ mice is associated with a decreased number of boneforming cells that may account for the marked decrease in bone formation

Inactivation of the 5-HT2BR gene in mice (5-HT2BR_/_) leads to osteopenia and reduced bone formation in aging mice Osteoblast proliferation and recruitment are reduced in 5HT2BR-depleted primary cultures Shows for first time, that the 5HT2BR receptor is a physiological mediator of 5-HT in bone formation and, potentially, in the onset of osteoporosis in aging women

Warden et al. 2008 [52] Serotonin (5-hydroxytryptamine) transporter inhibition causes bone loss in adult mice independently of estrogen deficiency

Mice

Fluoxetine (5 or 20mg/kg)

60 Adult, female, Swiss Webster mice formed two groups; ovariectomised and sham surgery Both groups were treated with either fluoxetine (high or low dose) or control substance for 4 weeks

In vivo assessments of hind limb areal and tibial volumetric BMD at baseline and 4 weeks Ex vivo assessment of femur and lumbar vertebrae BMD, trabecular bone architecture and turnover

No interactions found between oestrogen deficiency and 5-HTT inhibition, therefore all effects seen considered independent Inhibition of 5-HTT by fluoxetine had negative effects on BMD (P = <0.01) and displayed a dose dependant response in reduction of BMD This effect is mediated by reduced bone formation and increased resorption

SSRIs have a dose dependant negative effect on the adult skeleton independent of oestrogen deficiency

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Study

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SSRIs have a negative effect on bone metabolism Significant lower ultimate force ( 10.5%, P = 0.10) in fluoxetine treated mice Osteocalcin levels lower in fluoxetine treated group SSRI group had a reduced trabecular bone thickness, trabecular number and cortical area Fluoxetine 10 mg/kg/day Mice

12 female C57BL6J mice used

Forced swimming and open field tests to determine antidepressant effect ELISA used to determine osteocalcin and CTx MicroCT used to demonstrate microarchitecture 3-point bending assessing mechanical properties

SSRIs negatively alter BMD, architecture and mechanical properties independent of animal activity Significant reduction in activity with SSRI administration No effect between drug intervention and activity on in vivo measures SSRI resulted in reduced aBMD and negatively altered spinal/ femoral mechanical properties and architecture SSRI reduced bone formation by 37.8% compared to control, with no effect on osteoclast numbers Physical activity measured by total distance travelled 30 minutes after drug given In vivo - DEXA and peripheral quantitative computed tomography of lower limbs and pelvis Ex vivo DEXA, micro-CT and mechanical properties of femur and L5 Histomorphometry of femur Fluoxetine (20 mg/kg) Mice

80 virgin Swiss Webster female mice were divided into cage and tail suspended mice for 4 weeks

Conclusion Agent Animal Model

Method

Results

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Measurements

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5.6. In vitro effects of 5-HT and selective serotonin reuptake inhibitors in cell cultures 5.6.1. Osteoblast 5-HT induces increased proliferation of murine MC3T3-E1, primary human osteoblasts and human mesenchymal stem cells (hMSCs) at concentrations ranging from 0.01 to 10 mM in a dose dependent manner [21]. For doses above 10 mM, the effect on proliferation is reduced. Similarly, fluoxetine at low doses between 0.001and 0.1 mM induced proliferation on all cell types but in higher concentrations (> 10 mM), fluoxetine reduced cell proliferation [21]. Small concentrations of 5-HT in the range of 0.1–10 mM have also been shown to reduce the release of RANKL and increase the release of OPG, suggesting that 5-HT in small concentrations reduces osteoblastic signaling for the differentiation and activation of osteoclasts. Fluoxetine, in contrast was found to inhibit OPG and to stimulate RANKL thus potentially increasing osteoclastic activity [21]. 5.6.2. Osteoclasts The murine preosteoclast cell line RAW264.7 and peripheral blood mononuclear cells (PBMC) have also been used to investigate the effects of 5-HT and SSRIs on osteoclast differentiation and proliferation. Both agents act in a highly dose-dependent manner. Doses between 0.01 and 1 mM for both 5-HT and fluoxetine increase murine osteoclast proliferation but higher concentrations above 1 mM, reduce proliferation maybe due to ineffective activation of NF-kB and c-Fos [21]. Fluoxetine concentrations in the order of 10 mM, also reduce human osteoclast and peripheral PBMC differentiation, an effect that is not observed with the same concentration of 5-HT [21]. Battaglino et al., demonstrated that 5HT promotes the differentiation of RAW264.7 cells and human bone marrow macrophages (BMMs) to mature osteoclasts in a dose-dependent manner in 5-HT-depleted serum supplemented with the addition of 3, 30, or 300ng/ml 5-HT [3]. Fluoxetine reduced BMM differentiation in the presence of RANKL and the differentiation of RAW264.7 cells at concentration levels of 1– 3 mM, and this was thought to be due to an inhibitory effect on NFkB activity and elevated inhibitory protein IkBa levels in RAW264.7 cells. Fluoxetine, however, did not decrease the level of preformed osteoclasts suggesting its actions primarily affect the differentiation of precursor cells [3]. Overall, in vitro studies have suggested that the effect of 5-HT and SSRIs are highly dose-dependent and may either promote or inhibit osteoblast and osteoclast proliferation and differentiation via a number of mechanisms. The therapeutic range for fluoxetine in human serum is 0.65–2.5 mM and bone marrow concentrations of fluoxetine have been reported to exceed 100 mM following longterm SSRI therapy [7]. Based on the current in vitro findings, these values are in the range of that reported to inhibit murine preosteoblasts, preosteoclasts and human osteoclasts, and may also decrease proliferation of human osteoblasts and MSCs [3,21] (Table 3).

Bonnet 2007 [9] Various effects of antidepressant drugs on bone microarchitectecture, mechanical properties and bone remodelling

Warden 2010 [53] Psychotropic drugs have contrasting skeletal effects that are independent of their effects on physical activity levels

Study

Table 2 (Continued )

6. Discussion Evidence from longitudinal, cross-sectional and prospective cohort studies suggests that therapeutic SSRI dosages are associated with reductions in BMD and increased fracture risk [8,31,41,48]. The risk of fracture has been demonstrated in several studies to be dependent on both the dose [41,48] and duration of exposure [8,31,62]. The individual affinity of the SSRI is also an important factor and SSRIs such as citalopram, fluoxetine and paroxetine have recently been associated with an increased risk of osteoporotic fracture [47].

Table 3 Summary of in vitro evidence. Materials and methods

Results

Conclusion

Battaglino et al. 2004 [3] Serotonin regulates osteoclast differentiation through its transporter

Fluoxetine

Gene microarrays used to detect 5-HTT expression in RANKL induced osteoclasts Osteoclasts generated by stimulating murine RAW264.7 macrophage like cells and mouse bone marrow with RANKL Functionality of osteoclast 5-HTT demonstrated by 5-HT uptake studies and inhibition by SSRI

Osteoclast 5-HTT exhibited typical 5-HT uptake activity that was inhibitable by fluoxetine Fluoxetine reduced osteoclast differentiation but did not inhibit the activation of preformed osteoclasts, whereas the addition of 5-HT itself enhanced differentiation. This suggested that its actions primarily affect the differentiation of precursor cells

5-HT system plays an important role in bone homeostasis through effects on osteoclast differentiation and implies that commonly used antidepressive agents may affect bone mass

Bliziotes et al. 2001 [5] Neurotransmitter action in osteoblasts: expression of a functional system for serotonin receptor activation and reuptake

Imipramine + fluoxetine

Reverse transcription-polymerase chain reaction (RTPCR) analysis of primary cultures of rat osteoblasts and a variety of clonal osteoblastic cell lines showed mRNA expression for 5-HTT as well as numerous 5-HT receptors Protein expression of 5-HT receptors were confirmed by immunoblot

Imipramine and fluoxetine, antagonists with specificity for 5-HTT, showed the highest potency to antagonize [125I]RTI-55 binding in ROS and UMR cells Functional down regulation of transporter activity was assessed after PMA treatment, which caused a significant 40% reduction in the maximal uptake rate of [3H]5-HT 5-HT potentiates the PTH-induced increase in AP1 activity in UMR cells

Results demonstrate that osteoblastic cells express a functional serotonin system, with mechanisms for responding to and regulating uptake of 5-HT

Bliziotes et al. 2006 [6] Serotonin transporter and receptor expression in osteocytic MLO-Y4 cells

Imipramine + fluoxetine

Whole rat bone sections used to demonstrate in situ expression of 5-HTT protein in osteoblasts and osteocytes Immunoblot and RTPCR analysis of receptor protein expression Immunohistochemistry using antibodies for the 5-HTT to identify receptors in rat tibiae 5-HTT binding, 5-HT uptake and prostaglandin assays

Immunohistochemistry using antibodies for the 5-HTT, and the 5-HT1A and 5-HT2A receptors reveals expression of all three proteins in both osteoblasts and osteocytes in rat tibia Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC50 values in the nanomolar range 5-HT rapidly stimulated a 3-fold increase of PGE2 from MLO-Y4 cells within 60 min indicating functional 5-HT receptor presence

The rate-limiting enzyme for serotonin synthesis, tryptophan hydroxylase, is expressed in osteocytes and osteoblasts First report that osteocytes, as well as osteoblasts, are capable of 5-HT synthesis, and express functional receptor and transporter components of the 5-HT signal transduction system Not found in osteoclasts in this study

Westbroek et al., 2001 [54] Expression of serotonin receptors in bone

N/A

mRNA expression of the 5-HT2B receptor in bone and other tissues of foetal chicken studied by RTPCR and Northern Blot analysis Bone cells retrieved from 18 day old foetal chicken calvariae and treated with collagenase to produce mixed population of osteocytes and osteoblasts To show that 5-HT2B receptor protein is also present in bone cells, effects of a 5-HT analogue, on the proliferation of periosteal fibroblasts studied Also studied the expression of serotonin receptors in isolated mouse osteoblasts to demonstrate that the expression of 5-HT2 receptors in bone is not restricted to chicken

5-HT2B receptor mRNA expression appeared to be the most pronounced in skeletal tissues, specifically osteocytes. The serotonin analogue a-methyl-5-HT, which preferentially binds to 5-HT2 receptors, decreased nitric oxide release by mechanically stimulated (fluid flow induced) mouse osteoblasts

Suggests that the 5-HT2B receptor and possibly other serotonin receptors play a role in the modulation of the mechanosensory function of osteocytes

Gustafsson et al., 2006 [21] Serotonin and fluoxetine modulate bone cell function in vitro

Serotonin + fluoxetine

Investigated the effects of serotonin and fluoxetine on human and murine osteoblast as well as osteoclast proliferation and differentiation RT-PCR on human osteoclasts studied OPG, RANKL release from serotonin and fluoxetine-treated murine preosteoblasts

Serotonin receptors are expressed in human osteoclasts and tryptophan hydroxylase is expressed in both osteoclasts and osteoblasts Serotonin stimulates osteoclast differentiation and proliferation Serotonin and Fluoxetine activate cFOS and NFkB in murine pre-osteoclasts Serotonin and Fluoxetine have a dose-dependent effect on osteoblast and bone marrow stem cell proliferation

Serotonin and fluoxetine may affect osteoblast and osteoclast formation both positively and negatively in vitro, via different mechanisms. Their effects are markedly concentrationdependent

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Agent

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Study summary of evidence from clinical studies

Results

5-HTR1B down regulates osteoblast proliferation through inhibition of CREB and Cyclin D1 expression. TPH1 / gut mice developed a low bone mass phenotype whereas TPH1 / osbdid not have a significant decrease in BMD

Materials and methods

HTR2A HTR1B TPH1 Gene knockout in mice gut and osteoblast cell lines

5-HTR1b receptors play a key role in the regulation of bone metabolism Gut derived serotonin is more significant in the control of bone turnover than osteoblast derived serotonin

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The risk of fracture also appears to be greater early in the course of treatment, particularly with fluoxetine [26]. SSRIs, however, have also been shown to exert cardiovascular effects [39] and increase the risk of falls [41]. This may contribute to the greater risk of fracture during the early phase of treatment. Despite this, there is evidence that SSRIs can directly impact upon bone metabolism. Data from in vivo studies have shown that lower dosages can stimulate bone formation and increase BMD; however, there is conflicting evidence regarding the overall effects on bone metabolism [4,9,22,51–53,55]. Data from in vitro studies suggests that lower dosages of SSRIs at the nanomolar level can stimulate both osteoclast and osteoblast proliferation with the potential to influence bone remodelling [3,21]. While some differences in vitro and in vivo may be explained considering the different methods employed, contrasting evidence remains. The limitations of in vitro studies include lack of circulating hormones, such as oestrogen, and neuronal effects. Furthermore, in vitro models may fail to replicate the seemingly paradoxical effects of central versus peripherally acting 5-HT which appear to have opposite effects on BMD [2], whereby former has been shown to increase BMD through decreased adrenergic tone [58], yet peripheral 5-HT appears to exert a negative effect on bone through the agonism of 5-HT receptors [11,21,24,32,57]. The serotonergic pathway has also been shown to interact with the hormonal pathways involved in bone metabolism, and these have yet to be fully elucidated. Furthermore, the delivery routes used in animal experiments (i.p. or s.c.) are dissimilar to the delivery method (po) in humans, a fact that may affect the pharmacological properties and the effects of 5-HT and SSRIs. Bone marrow concentration of SSRIs is currently under investigation; however, the evidence to date suggests that SSRI concentrations are greater than that expected to stimulate bone formation [7,21]. SSRI levels have been shown to remain steady in bone marrow for up to 3 months following cessation of therapy [7,21]. Furthermore, gut-derived circulating levels of free 5-HT may also be elevated in patients taking SSRIs possibly due to reduced transport in platelets and reduced 5-HT clearance from peripheral transporter-expressing target organs such as bone [42]. This may lead to a detrimental effect due to increased local bone concentrations of 5-HT. There is a growing body of evidence to suggest an increased, dose-dependent risk of fractures among patients taking SSRIs, and potential pathways exist for this to occur. When prescribing SSRIs the increased risk of fractures must be considered. Namely, the early problem with falls on initial administration, and the later problem of decreased BMD and its potential fracture risk. There is a further possibility of SSRIs affecting BMD in the low oestrogen state, but this needs to be evaluated further.

Agent

N/A

Study summary of evidence from clinical studies

Yadav et al. 2008 [57] Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum

Table 3 (Continued )

Conflict of interest statement All authors declare no conflict of interest or financial support for this study. There were no profit-sharing agreements, royalties, patents, and research or other grants from private industry or closely affiliated nonprofit funds. References [1] Abrahamsen B, Brixen K. Mapping the prescriptiome to fractures in men–a national analysis of prescription history and fracture risk. Osteoporos Int 2009;20(4):585–97. [2] Bab I, Yirmiya R, Depression. Selective serotonin reuptake inhibitors, and osteoporosis. Current Osteoporosis Reports 2010;8(4):185–91. [3] Battaglino R, et al. Serotonin regulates osteoclast differentiation through its transporter. J Bone Miner Res 2004;19(9):1420–31. [4] Battaglino R, et al. Fluoxetine treatment increases trabecular bone formation in mice. J Cell Biochem 2007;100(6):1387–94.

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