Inhibition of inflammation but not ankylosis by glucocorticoids in mice: further evidence for the entheseal stress hypothesis

Kirsten Braem
Christophe M Deroose
Frank P Luyten
Rik J Lories
Inhibition of inflammation but not ankylosis by glucocorticoids in mice: further evidence for the entheseal stress hypothesis
R E S E A R C H A R T I C L E Open Access
Introduction: Studies in the spontaneous ankylosis model in aging male DBA/1 mice and in patients with ankylosing spondylitis provide evidence that inflammation and new tissue formation leading to joint or spine ankylosis are likely linked but largely uncoupled processes. We previously proposed the 'entheseal stress' hypothesis that defines microdamage or cell stress in the enthesis as a trigger for these disease processes. Here, we further investigated the relationship between inflammation and ankylosis by focusing on the early phase of the spontaneous arthritis model. Methods: Aging male DBA/1 mice from different litters were caged together at the age of ten weeks and studied for signs of arthritis. A group of DBA/1 mice were treated daily with dexamethasone (0.5 μg/g body weight). Severity of disease was assessed by histomorphology and by positron emission tomography (PET) using 2-[ 18 F] fluoro-2-deoxy-D-glucose ( 18 F-FDG) as a tracer. Bone loss in dexamethasone-treated or control mice was determined by in vivo dual-energy X-ray absorptiometry. Chemokine gene expression was studied ex vivo in dissected paws and in vitro in mesenchymal cells (periosteal and bone marrow stromal cells) by quantitative realtime PCR in the presence or absence of bone morphogenetic protein 2 (BMP2) and dexamethasone.Results: Dexamethasone treatment did not affect incidence or severity of ankylosis, but led to an expected reduction in inflammation in the paws at week 15 as measured by PET tracer uptake. Treatment with dexamethasone negatively affected bone mineral density. Chemokines attracting neutrophils and lymphocytes were expressed in affected paws. In vitro, BMP2 stimulation upregulated chemokines in different mesenchymal joint-associated cell types, an effect that was inhibited by dexamethasone. Conclusions: BMP signaling may be a trigger for both inflammation and ankylosis in the spontaneous model of ankylosing enthesitis. The lack of inhibition by glucocorticoids on new bone formation while causing systemic bone loss highlights the paradoxical simultaneous loss and gain of bone in patients with ankylosing spondylitis.

Introduction

Ankylosing spondylitis (AS) and related spondyloarthritides (SpA) are common chronic inflammatory joint diseases with severe impact on patients and society [1,2]. Inflammation is held responsible for many of the signs and symptoms of disease but the long-term prognosis for patients with AS is also determined by progressive ankylosis of the spine due to new cartilage and bone formation [3]. Remarkably, new bone formation at the bone borders occurs simultaneously with inflammation-induced loss of trabecular bone leading to osteoporosis [4,5].

Although traditionally viewed as a repair response, we hypothesized that ankylosis is a specific and primary aspect of AS and proposed the entheseal stress hypothesis which explains how inflammation and ankylosis are linked but largely uncoupled processes [6]. In this concept, microdamage or cell stress in the enthesis triggers both an inflammatory and a bone anabolic response leading to the clinical development of AS and the typical radiographic signs of disease. Specific environmental and genetic factors are suggested to influence chronicity of the inflammatory response and progression of ankylosis [6].

The introduction of anti-tumor necrosis factor (TNF) strategies has been a breakthrough for patients with AS and other SpAs [2]. These drugs have an unprecedented effect on symptoms of disease. However, recent data in mice and men suggest that control of inflammation with TNF blocking agents is not sufficient to prevent progressive ankylosis [7][8][9][10]. Therefore, in contrast to what is seen in patients with rheumatoid arthritis, anti-TNF appears to fail to inhibit radiographic progression of disease in AS [11,12].

DBA/1 mice spontaneously develop arthritis of the hind paws characterized by entheseal ankylosis upon grouped caging of males from different litters [13]. In this mouse model both anti-TNF treatment using human soluble receptor etanercept and anti-osteoclast strategies using zoledronic acid are not sufficient to inhibit new bone formation [7,14]. In contrast, overexpression of noggin, a bone morphogenetic protein (BMP) antagonist, reduced the incidence and severity of the murine arthritis [15].

The specific role of inflammation in this model remains unclear. We have demonstrated a short-lived inflammatory phase characterized by edema and neutrophil infiltration in the affected toes, likely preceding the remodeling phase [13]. Here we focused on these early inflammatory events, the interactions between BMPs and inflammation and the complex effects of glucocorticoids, drugs with strong anti-inflammatory and anti-bone anabolic effects, in the spontaneous model.

Materials and methods

Animal experiments

Male DBA/1 mice were obtained from Janvier (Le Genest St Isle, France). All experiments were approved by the Ethics Committee for Animal Research (KU Leuven, Belgium). Male mice from different litters were mixed and caged in groups of six mice at the age of ten weeks. Mice were treated daily with dexamethasone (0.5 mg/kg; Rotexmedica, Trittau, Germany) or phosphate buffered saline (PBS) by intraperitoneal injection from the age of 12 weeks onwards (n = 10 and 12 mice per group, respectively). Mice were scored blindly twice a week for clinical signs of arthritis [13,16]: 0 (no symptoms), 1 (redness and swelling in one toe), 2 (redness and swelling in more than one toe), 3 (toe stiffness), and 4 (deformity or ankle involvement). Hind paw forefeet were studied by histomorphology as described [13,16]: 0 (normal toe), 1 (acute inflammation including dactylitis), 2 (entheseal cell proliferation), 3 (cartilage formation), 4 (bone formation), and 5 (joint ankylosis). A cumulative score from all toes was calculated. In additional experiments, paws of control mice of 12 and 16-week-old DBA/1 mice were used for gene expression analysis.

The mice were imaged using a Focus 220 microPET ® scanner (Concorde Microsystems Inc., Knoxville, TN, USA). The mice were anesthetized with 2.5% isoflurane in 100% oxygen at a flow rate of 1 L/minute and 7.4 MBq of 2-[ 18 F]fluoro-2-deoxy-D-glucose ( 18 F-FDG) was administered by lateral tail vein injection. One hour after injection a ten minute static scan was acquired. Images were reconstructed using a maximum a posteriori algorithm from the vendor with 18 iterations and uniform resolution. The images were displayed in Amide's a Medical Imaging Data Examiner (AMIDE) [17] and three-dimensional spheroid regions of interest (ROIs) were drawn over the toes, the paws and within a reference region (liver). The mean radioactivity in the ROIs was converted to percent of the injected dose per gram of tissue (%ID/g) by a calibrated cylinder factor and after division with the injected dose (corrected for residual and decay). Standardized uptake values (SUV) were obtained by multiplying the % ID/g of the toe area by the weight of the animal.

Bone mineral content was assessed by dual X-ray absorptiometry using Piximus densitometer (Lunar, Madison, WI, USA) [18].

Cell culture

Human periosteum-derived cells (hPDCs) were isolated as described [19]. Human bone marrow cells (hBMCs) were isolated by standard aspiration from freshly isolated bone marrow as described previously [20]. hPDCs and hBMCs were expanded in complete culture medium consisting of high-glucose Dulbecco's Modified Eagle Medium (DMEM)/Glutamax (4.5 g/l of glucose) (Invitrogen, Merelbeke, Belgium) supplemented with 10% fetal bovine serum (Gibco, Merelbeke, Belgium), antibiotic-antimycotic solution (100 units/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B; Invitrogen) and sodium pyruvate (Gibco). hPDCs and hBMCs were seeded at a density of 12 × 10 4 c/ml in 24-well plates. After overnight adherence, cells were stimulated with 3 to 30 ng/ml BMP2 (R&D Systems, Minneapolis, MN, USA) with or without 1 μM dexamethasone (Rotexmedica). After a 24-hour incubation, samples were collected for RNA analysis.

RNA isolation and Real-Time RT-polymerase chain reaction (PCR)

For gene expression analysis of mouse tissues, hind paws of 12 and 16 week old mice were dissected just above the ankle joint and skin was removed. Total RNA was isolated using the standard Nucleospin RNAII kit (Macherey-Nagel, Düren, Germany) protocol. Complementary DNA was obtained by reverse transcription of 0.5 to 1 μg of total RNA using the RevertAid H Minus First Strand cDNA synthesis kit (Fermentas, Rockford, IL USA) following the manufacturer's instructions. For quantitative analysis, real-time RT-PCR was performed using Perfecta qPCR FastMix (Quanta Biosciences, Inc., Gaithersburg, MD, USA) carried out on a Corbett Rotor-Gene 6000 system (Corbett Research, Westburg, Leusden, The Netherlands). The expression of the following genes was examined: human chemokine (C-X-C motif) ligand 1 (CXCL1), granulocyte chemotactic protein 2 (GCP2), and inhibitor of DNA binding 1 (ID1) and mouse homolog Cxcl1 and Gcp2 and mouse chemokine (C-X-C motif) receptor 1 (Cxcr1) and Cxcr2. Human and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH and Gapdh, respectively) were used as housekeeping genes. Primer/probe sets for these genes were commercially available Assay-on-demands (Applied Biosystems, Lennik, Belgium).

Statistical analysis

For comparison between groups, Student's t-test was used. Results were considered statistically significant with two-sided P-values < 0.05.

Results

Severity of arthritis in mice treated with dexamethasone

Daily injection of dexamethasone (0.5 mg/kg) from week 12 onwards did not inhibit clinical incidence or severity of spontaneous arthritis in male DBA/1 mice as compared to placebo-treated mice ( Figure 1A-B). As the clinical evaluation of this model can not distinguish inflammatory and remodeling features, histomorphological analysis was performed. We found no difference in overall severity between dexamethasone-treated and control mice ( Figure 1C). In addition, there was no significant difference in pathological endochondral bone formation between both groups ( Figure 1D).

As expected dexamethasone treatment did significantly affect mouse weight ( Figure 2A) and total body bone mineral content ( Figure 2B), demonstrating that local new bone formation leading to joint ankylosis can occur simultaneously with systemic bone loss.

Control of inflammation by glucocorticoid treatment

Inflammation in the spontaneous arthritis model in DBA/1 mice has been described previously as short-lived and presenting as enthesitis or dactylitis. The process is characterized by infiltration of mostly neutrophils and to a lesser extent mononuclear inflammatory cells [13]. To quantify inflammation in the paws in a dynamic way, ten mice (four dexamethasone-and six placebo-treated) were injected with 18 F-FDG and uptake was measured by PETscan ( Figure 3A). 18 F-FDG has been shown to accumulate in activated inflammatory cells (neutrophils and macrophages) and serves as a marker of inflammation [21].

Mice treated with dexamethasone showed significantly lower tracer uptake at 15 weeks as compared to controls. By week 20 uptake of the tracer was similar in both groups ( Figure 3B) and lower than at 15 weeks.

Presence of chemokines in SpAD

As neutrophils are the most common cells in the inflammatory reaction in this mouse model, we measured expression of two chemokines (Cxcl1 and Gcp2) and their receptors (Cxcr1 and Cxcr2), well-known mediators of neutrophil migration to inflammatory sites, in male DBA/1 mice at different time points (Figure 4A

BMPs and chemokine upregulation

The entheseal stress hypothesis suggests that activation of entheseal cells may be the initiating event in attracting inflammatory cells [6]. Since earlier observation showed that inhibition of BMP signaling in this model prevented disease occurrence [15], we tested whether BMP2 could induce neutrophil chemokines. Human progenitor cells derived from either periost or bone marrow were stimulated with different concentrations of BMP2 and treated with 1 μM dexamethasone. Stimulation of hPDCs and hBMCs resulted in a dose dependent increase of ID1, a BMP target gene, and was not influenced by dexamethasone treatment ( Figure 5A). Twenty-four hours of BMP2 stimulation induced the expression of GCP2 in hPDCs and this induction was inhibited by dexamethasone (Figure 5B). A similar effect was suggested in hBMCs, although the difference was not statistically significant. CXCL1 was also significantly upregulated in hPDCs but not in hBMCs (data not shown). Dexamethasone treatment itself induced an increase in CXCL1 in vitro in both hPDCs and hBMCs, but did not further stimulate or inhibit the effect of BMP2.

Discussion

Patients who are suffering from AS show a remarkable paradox in the skeleton. Inflammation appears associated with osteoporosis of the trabecular bone, but also with new bone formation occurring at the edges of the bone, two contrasting features in close proximity. Targeted therapies antagonizing TNF result in substantial clinical improvement, reduction of inflammation and inhibition of bone loss in AS patients. In contrast, new bone formation leading to ankylosis does not appear to be inhibited by anti-TNF therapy [7][8][9][10]. The entheseal stress hypothesis challenges the paradigm that inflammation triggers ankylosis and proposes a concept in which both inflammation and new tissue formation in AS are secondary to a common trigger, for example, cell stress or microdamage in the enthesis [6]. In the study presented here, glucocorticoid treatment in mice developing spontaneous arthritis characterized by ankylosing enthesitis appears to affect inflammation without direct impact on joint remodeling. These observations parallel our earlier studies in which we demonstrated that neither anti-TNF therapy with etanercept nor osteoclast inhibition has a beneficial effect on progression of ankylosis [7,14].

Glucocorticoid use does have a negative effect on bone loss as demonstrated by DEXA analysis. Much like what is seen in AS patients, loss and gain of bone appear simultaneously and in close proximity. This observation provides support for the view that the molecular mechanisms of ankylosis are disconnected from inflammation or bone loss. These observations suggest that new bone formation as seen in this model and also in patients with ankylosing spondylitis is not dependent on the classical bone remodeling cycle essential for skeletal renewal and which is affected in osteoporosis. Therefore, molecular mechanisms leading to new bone formation and ankylosis are likely different from those guiding the physiological bone remodeling cycle. Such a difference suggests that specific targeting of ankylosis is feasible without necessarily interfering with homeostatic bone remodeling.

We previously demonstrated that inhibition of BMP signaling reduced the incidence and severity of spontaneous arthritis and ankylosis suggesting that BMP signals are an upstream event in this mouse model [15]. Inflammation in the spontaneous model is short-lived and the clinical evaluation of the mice does not distinguish between local joint swelling and redness caused by either inflammation, cell proliferation or differentiation [13]. In our experience, the inflammation phase of the model is often discrete and not readily recognized during routine clinical examination of the mice. We, therefore, used 18 F-FDG PET scans to quantify inflammation in the model. The lack of chronic inflammation is an intrinsic limitation of this model, but the current observation that inhibition of inflammation has no effect on ankylosis corroborates our hypothesis that inflammation and new tissue formation are largely independent events with a potential common trigger.

Further experiments identified upregulation of neutrophil chemokines in the early phase of the model. Since BMP inhibition was, in contrast to different anti-inflammatory strategies, capable of preventing clinical and histomorphological signs of arthritis [15], we further studied whether stromal cells could respond to BMPs by upregulating specific chemokines. BMP-induced GCP2 expression occurred late after stimulation. No significant GCP2 induction could be detected after an early timepoint (2-hour stimulation) as measured by gene expression analysis and ELISA by determination of GCP2 protein in the cell culture supernatant (data not shown). Also, dexamethasone treatment did not affect BMP2 induced ID1 expression. Together, these results suggest that the BMP-induced GCP2 expression is an indirect effect.

Our data suggest that at least in this model activation of the BMP signaling cascade can contribute to both joint inflammation and remodeling ( Figure 6). These observations further corroborate the entheseal stress hypothesis and provide support for a non-inflammatory  or non-specific primary cause of the arthritis with subsequent involvement of both inflammation and remodeling. In the specific entheseal micro-environment complex feedback loops may exist in which TNF and other cytokines stimulate the production BMP2. The BMP2 promoter contains TNF responsive elements and upregulation of BMP2 after TNF stimulation has been demonstrated in different skeletal cell types [22,23].

Taking into account the broad immune-modulating properties of glucocorticoids, we can not assume that the presented interaction with BMP-induced chemokine induction is the only or the main mechanism by which dexamethasone treatment affects the inflammatory disease of the mouse model. Nevertheless, the observed gene regulation provides an exciting link between signaling pathways involved in joint remodeling and signals contributing to inflammation. Such interactions are of great interest when a comprehensive or system-biology view of arthritis is developed.

A number of limitations of the current study need to be considered. First, we preferred to use human progenitor cells instead of mouse cells. Dissection and isolation procedures in the mouse can affect cell purity and it is also difficult to expand mouse periost derived cells into large amounts. hPDCs are periosteum derived progenitor cells involved in bone development and postnatal repair processes [19,24]. The periosteum, a fibrous tissue which covers the bone surface, is known to participate in the differentiation of chondrocytes and osteoblasts in fracture healing [25]. Progenitor cells derived from the bone marrow can be differentiated into osteoblasts and participate as well in bone fracture repair processes [26]. We preferred a broad non-specific anti-inflammatory approach with glucocorticoids to target inflammation in vivo rather than the inhibition of a specific chemokine as the inflammatory events are likely driven by different chemokines. A number of small molecules designed to inhibit chemokines are in clinical development but the short-lived pharmacokinetics of such molecules [27,28] are not suitable for the mouse model used here as we demonstrated earlier for p38 inhibition [29] and as suggested by human data using celecoxib in ankylosing spondylitis [30]. Glucocorticoid treatment allowed us to evaluate its effect on local bone formation and systemic bone loss simultaneously.

Conclusions

This study highlights the paradoxical simultaneous loss and gain of bone in a mouse model of AS and suggests that BMPs may trigger both inflammation and ankylosis. Further identification of factors that lead to the production of BMPs in the enthesis appears, therefore, critical to understand better the initiating events of the disease progress. Insights into these mechanisms based on functional and genomics studies may lead not only to the development of new therapeutic approaches to control and improve AS and related disorders but also to avoid radiographic progression.

-D). Both chemokines and their receptors are expressed in paws of DBA/1 mice, with a proportion of the paws showing high gene expression levels. The fold difference in gene expression level of Cxcr1, Cxcr2, Cxcl1 and Gcp2 between the four paws with the highest level compared to the four lowest levels was 5.0, 27.7, 7.2 and 2.5, respectively at 12 weeks and 7.4, 6.3, 3.4 and 3.9 at 16 weeks. Of note, clear clinical signs of disease in these mice were not apparent corroborating our previous observations that mild inflammatory changes in this model are not easily detected by clinical observation only.

Figure 1
1Corticosteroid treatment does not affect clinical signs of spontaneous arthritis. (A) Cumulative incidence of arthritis and (B) clinical severity score in dexamethasone-treated and control animals (n = 10 and 12 animals per group, respectively). (C) Dexamethasone treatment does not affect histological severity of arthritis (ns: P = 0.2). Data in (B) and (C) are shown as mean ± SEM. (D) Effect of dexamethasone treatment on different features of ankylosing enthesitis. Data in (D) are shown as percentage of affected paws exhibiting a specific treat compared to the total number of affected paws. ns, nonsignificant; SEM, standard error of the mean.

Figure 2
2Dexamethasone treatment affects body weight and whole body mineral content. (A) Body weights were measured after two weeks of dexamethasone treatment. (B) Bone mineral content was measured by DEXA at the end of the experiment (20 weeks). Data are shown as mean + SEM. (n = 12 control animals and 10 dexamethasone-treated animals; * P < 0.05). DEXA, dual energy X-ray absorptiometry; SEM, standard error of the mean.

Figure 3
3Dexamethasone treatment influences the inflammatory component of spontaneous arthritis as measured by 18 F-FDG PET scan. (A) Representative images of animals from both treatment groups. The 'toe' area used for measurements is indicated. (B) Standardized uptake values of the tracer demonstrating a reduction in the uptake after dexamethasone treatment at week 15. At week 20, the inflammatory component has largely disappeared as seen in the control group. (n = 4 animals in dexamethasone treated group and 6 control animals; * P < 0.05 dexamethasone treatment versus control group; # P < 0.05 week 20 versus week 15 in control group). 18 F-FDG, 2-[ 18 F]fluoro-2-deoxy-Dglucose; PET, positron emission tomography.

Figure 4
4Quantitative PCR analysis of neutrophil chemokines and receptors in paws of male DBA/1 mice aged 12 and 16 weeks. The expression levels of Cxcr1 (A), Cxcr2 (B), Cxcl1 (C) and Gcp2 (D) are shown. Y-axis displays gene expression values normalized to GAPDH and is presented as log-scale (* 10 -4 ). The average gene expression for each gene in both groups is presented by the horizontal line. PCR, polymerase chain reaction.

Figure 5
5Gene expression analyses of ID1 and chemokines in human periosteal and bone marrow stromal cells. Cells were cultured for 24 hours in the presence or absence of BMP2 (3 or 30 ng/ml) and 1 μM dexamethasone. Y-axis displays gene expression levels of (A) ID1 and (B) GCP2 normalized to housekeeping gene GAPDH. Values are expressed as fold increase relative to control condition (no BMP2, no dexamethasone) and are presented as log scale (n = 3; * P < 0.05). BMP2, bone morphogenetic protein 2.
Braem et al. Arthritis Research & Therapy 2012, 14:R59 http://arthritis-research.com/content/14/2/R59
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AcknowledgementsAuthors' contributions KB, FL and RL participated in the design of the study. Experiments were performed by KB. CMD participated in the analysis of PET scan data. The manuscript was drafted by KB and was commented on and revised by RL, FL and CMD. All authors read and approved the final manuscript for publication.Competing interestsKU Leuven holds a patent on behalf of FL and RL on the use of noggin for the treatment of spondyloarthritis. The other authors declare that they have no competing interests.
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Joint inflammation in the DBA/1 model is characterized by the presence of neutrophils which are attracted by BMP-induced chemokines. BMP-triggering of entheseal progenitor cells leads to new tissue formation and ankylosis in a complex cascade with multiple feedback mechanisms involving different molecular cascades. Pro-inflammatory cytokines such as TNF are known to stimulate BMPs in mesenchymal cell types and could provide another feedback system. Entheseal cell stress may also trigger other cascades leading to inflammation and tissue differentiation. BMP, bone morphogenetic protein; TNF, tumor necrosis factor. Braem, Arthritis Research & Therapy. 14As a reaction to local stress (damage, strain, and so on} BMP signaling can contribute to inflammation and ankylosisFigure 6 A model for the dual role of BMPs in the entheseal stress hypothesis. As a reaction to local stress (damage, strain, and so on} BMP signaling can contribute to inflammation and ankylosis. Joint inflammation in the DBA/1 model is characterized by the presence of neutrophils which are attracted by BMP-induced chemokines. BMP-triggering of entheseal progenitor cells leads to new tissue formation and ankylosis in a complex cascade with multiple feedback mechanisms involving different molecular cascades. Pro-inflammatory cytokines such as TNF are known to stimulate BMPs in mesenchymal cell types and could provide another feedback system. Entheseal cell stress may also trigger other cascades leading to inflammation and tissue differentiation. BMP, bone morphogenetic protein; TNF, tumor necrosis factor. Braem et al. Arthritis Research & Therapy 2012, 14:R59 http://arthritis-research.com/content/14/2/R59

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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution. Braem, Arthritis Research & Therapy. 1459Inhibition of inflammation but not ankylosis by glucocorticoids in mice: further evidence for the entheseal stress hypothesis. Submit your manuscript at www.biomedcentral.com/submitCite this article as: Braem et al.: Inhibition of inflammation but not ankylosis by glucocorticoids in mice: further evidence for the entheseal stress hypothesis. Arthritis Research & Therapy 2012 14:R59. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit


A pharmacy led program to review anti-psychotic prescribing for people with dementia

Anne Child
Amy Clarke
Chris Fox
Ian Maidment
A pharmacy led program to review anti-psychotic prescribing for people with dementia
R E S E A R C H A R T I C L E Open Access
Background: Anti-psychotics, prescribed to people with dementia, are associated with approximately 1,800 excess annual deaths in the UK. A key public health objective is to limit such prescribing of anti-psychotics. Methods: This project was conducted within primary care in Medway Primary Care Trust (PCT) in the UK. There were 2 stages for the intervention. First, primary care information systems including the dementia register were searched by a pharmacy technician to identify people with dementia prescribed anti-psychotics. Second, a trained specialist pharmacist conducted targeted clinical medication reviews in people with dementia initiated on anti-psychotics by primary care, identified by the data search. Results: Data were collected from 59 practices. One hundred and sixty-one (15.3%) of 1051 people on the dementia register were receiving low-dose anti-psychotics. People with dementia living in residential homes were nearly 3.5 times more likely to receive an anti-psychotic [25.5% of care home residents (118/462) vs. 7.3% of people living at home (43/589)] than people living in their own homes (p < 0.0001; Fisher's exact test). In 26 practices there was no-one on the dementia register receiving low-dose anti-psychotics. Of the 161 people with dementia prescribed low-dose anti-psychotics, 91 were receiving on-going treatment from local secondary care mental health services or Learning Disability Teams. Of the remaining 70 patients the anti-psychotic was either withdrawn, or the dosage was reduced, in 43 instances (61.4%) following the pharmacy-led medication review. Conclusions: In total 15.3% of people on the dementia register were receiving a low-dose anti-psychotic. However, such data, including the recent national audit may under-estimate the usage of anti-psychotics in people with dementia. Anti-psychotics were used more commonly within care home settings. The pharmacist-led medication review successfully limited the prescribing of anti-psychotics to people with dementia.

Background

Dementia has an estimated global prevalence of 35 million individuals [1]. In the UK 700,000 people live with dementia -a figure which will double over the next 30 years, reflecting global predictions [1][2][3]. Whilst BPSD (behavioural and psychological symptoms of dementia) are very common, both the point prevalence, the proportion having symptoms at any one time point, and the cumulative risk are unclear. A recent study concluded that BPSD affect nearly everyone with dementia [4]. Whereas the Cochrane review simply stated that 50% of people with dementia experience such symptoms [5]. Other studies have estimated the point prevalence between 60% and 80%, and the cumulative risk during the course of the illness at 90% [6].

BPSD are among the most difficult symptoms to treat, a primary source of caregiver stress, strongly associated with carer burden and depression and one of the main drivers for placement breakdown and admission to residential care facilities [7][8][9][10][11][12]. Behavioural symptoms have traditionally been treated with anti-psychotics, however their usage is implicated in approximately 1,800 annual deaths [2]. Recently the National Dementia Strategy (NDS) targeted a two-thirds reduction in such usage of anti-psychotics [2,[13][14][15]. However, there is limited information on the prevalence of anti-psychotic use in dementia and the NDS estimated that, as a conservative figure, up to 25% of people with dementia are likely to be receiving anti-psychotics [2]. Due to the lack of data, one of the initial recommendations of the NDS was to obtain baseline data on the extent of anti-psychotic usage [2]. Following collection of baseline data a target for the size and speed of the reduction should be set; the NDS also highlighted the potential role of pharmacists in improving medication management and limiting the inappropriate use of anti-psychotics in people with dementia [2,14,16].

Method

Setting

This pharmacy-led program was conducted within General Practitioner (GP) surgeries in Medway Primary Care Trust (PCT). Medway PCT covers the Medway towns in Kent with a total population of 256,700 and a 60 plus population of 51,500 [17]. Medway is a relatively deprived area, ranked 132rd most deprived of 325 UK boroughs in the 2010 Index of Deprivation, with pockets of significant deprivation; eight geographical units called 'Super Output Areas' (SOAs) were ranked in the 10% most deprived nationally [18].

Pharmaceutical support within Medway PCT is provided by a medication management team containing pharmacists, pharmacy technicians and support staff; the team has a history of a close working relationship with GPs and undertaking clinical projects within individual practices. Following the publication of the NDS one of the key strategic priorities of the PCT was to limit the prescribing of anti-psychotics to people with dementia. This project, which was conducted from January to December 2011, had two aspects. First, in line with the NDS, data were collected on anti-psychotic usage to identify people on the dementia register within Medway PCT prescribed anti-psychotics. Second, a specialist pharmacist intervened in an appropriate cohort of people with dementia commenced on anti-psychotics by primary care identified by the data search.

Patient identification

Every practice information system, both the electronic and paper format, was searched, by an experienced pharmacy technician to identify people with dementia currently prescribed a low dose anti-psychotic, on acute or repeat prescription. Dementia registers were established within the UK as part of the Quality Outcome Framework (QOF)an incentive scheme to reward good practice in primary care -to record people with dementia within the practice [19,20]. The registers were established within Medway PCT in 2006/07 and were used to identify people with a confirmed diagnosis of dementia.

The individual patient record including the medication history of every person on the dementia register was then searched to identify people prescribed low-dose anti-psychotics. Initially, this was restricted to the 6 most commonly prescribed anti-psychotics based on ePACT (electronic Prescribing Analysis and Cost Tabulation) data. However, to ensure completeness, patient's notes were hand searched to identify other, less frequently prescribed, anti-psychotics. For the six most commonly prescribed anti-psychotics, low dose was defined a priori (olanzapine up to 10 mg daily, risperidone up to 2 mg daily, quetiapine up to 100 mg daily, amisulpiride up to 50 mg daily, sulpiride up to 200 mg daily and haloperidol up to 1.5 mg daily). For the other less frequently prescribed anti-psychotics the standard British National Formulary (BNF) dosing schedules were used to inform the decision on whether the dose was low [21].

Finally, the document storage system, where key documents such as referral letters are stored, was searched to determine where treatment was commenced (primary care, secondary care mental health trust, acute trust or Learning Disability Team). If the anti-psychotic had been initiated by a secondary care organisation, the patient was not included in the medication review, because the anti-psychotic may have been used for under-lying psychosis or a complex mental health problem. The residential status (living at home or in a care home) was also collected.

Pharmacist-led medication review

After the cohort of people with dementia on long-term anti-psychotics initiated by primary care was identified, the intervention was delivered. Practices were ranked according to the frequency of prescribing to prioritise where the intervention should be delivered. The pharmacist delivering the intervention sent a copy of a report to the GP, which identified the cohort of people with dementia where anti-psychotic withdrawal should be attempted and that the pharmacist was able to give support to the GP in the process by providing advice on a suitable withdrawal scheme, advising on local and national guidance and linking with the care home [14,[22][23][24]. This initial communication was followed up either verbally or in writing.

The pharmacist collaborated with the GP in identifying, where withdrawal was potentially suitable and the level of pharmacy support including whether a pharmacist-led medication review, was required. Medication was withdrawn for clinical, not research reasons. Hence, consent was not required. All participants received usual care and any decision to alter medication was made by the GP based on individual clinical needs, following the pharmacist-led review. Withdrawal was generally considered if the patient was not under secondary care services, was receiving an anti-psychotic for non-acute behavioural problems, and the anti-psychotic had not been reviewed in the previous 12 months.

The clinical medication review (based on modified National Prescribing Centre Level 3 medication review criteria) was delivered by an experienced senior clinical PCT pharmacist, who had a further qualification in dementia with full access to medical and care home notes [25]. The review included an individual withdrawal plan, which took into account the length of time the person had been on the anti-psychotic. In general the dose was gradually reduced usually by 50% every two to four weeks following discussions with the prescriber. During the withdrawal patients continued to be monitored, followed up, and treated by their own physicians according to their individual clinical needs. Care home staff were encouraged to log behaviours using ABC (Antecedents, Behaviour, Consequences) charts and monitor residents for any change in general health [23].

Care staff discussed the project with next-of-kin and whenever possible the person with dementia; if required the pharmacist supported these discussions. Any decisions from these discussions incorporated the views of the person with dementia, next-of-kin and professional carers. The pharmacist also provided support and training to the care home, which was designed to help staff manage BPSD, and was available for further advice and follow-up. The training utilised treatment guidelines for use in primary care to treat BPSD that were developed by a multi-disciplinary team of clinicians from the local mental health trust and local PCTs [23]. The overall concept of the training was based upon a personcentred approach including the need to understand the causes of any behaviour and accurately record any behaviour, non-pharmacological treatments options, and the appropriate pharmacological management of behavioural problems in dementia [22,23].

This project was approved by Medway PCT. Ethics committee approval was not required because the project was a service evaluation and data were collected to evaluate this new service and not for the purposes of research. The decision to publish was made retrospectively and individual patient data was not identifiable to anyone in the team (IM, CF) conducting the secondary analysis of the results for publication.

Results

See Figure 1 for a diagrammatical representation of the results.

Anti-psychotic usage

Data were collected from 59 out of 60 (98.3%) practices (or groups of practices) within Medway PCT; one practice declined to take part. There were 1051 people on the dementia register in the 59 practices; of these 462 were in residential care and 589 lived in their own home. In total 161 people on the dementia register were receiving low-dose anti-psychotics; of these 118 were in residential care and 43 lived in their own home. People with  dementia living in residential homes were nearly 3.5 times more likely to receive an anti-psychotic; 25.5% (118/462) of people resident in care homes compared with 7.3% (43/589) of people living in their own homes (p < 0.0001; Fisher's exact test) [26].

On average (mean value) each practice treated 2.77 (range -0 to 26; +/− SD 4.88) people with dementia with low-dose anti-psychotics. In 26 practices no-one with dementia was receiving low-dose anti-psychotics. Put another way, if one assumes that the dementia register was accurate, approaching half (44.1%) of all practices in Medway PCT were not treating anyone with dementia with a low-dose anti-psychotic. Five practices accounted for over 50% (81/161) of the prescribing; 3 of these 5 practices were significantly larger (16,575, 13,805 and 8,866) than the mean practice in Medway PCT of 4,683 patients (report generated from http://www.epact. ppa.nhs.uk on the 13 th April 2012). The remaining two practices were responsible for large care homes.

The most commonly used anti-psychotic was amisulpride (52 out of 161; see Table 1 for full details of antipsychotics used).

Pharmacist-led medication review

Of the 161 people on the dementia register prescribed lowdose anti-psychotics, 87 were receiving on-going treatment from local secondary care mental health services and 4 from the local Learning Disability Teams. The remaining 70 patients were included in the pharmacy led review. Different surgeries accessed different levels of pharmaceutical support and in total, following the pharmacy-led intervention, anti-psychotics were withdrawn, or the dosage of the anti-psychotic was reduced, in 43 instances (61.4%). The pharmacist directly liaised with 11 practices to withdraw anti-psychotics in 33 people (47.1%). In a further two practices, one which supported a specialist dementia home, six patients identified during data collection were withdrawn from anti-psychotics. In one further practice, which supported a large EMI (elderly mentally ill) home, the pharmacist developed and provided withdrawal schedules for four patients.

Discussion

Over 15% of people on the dementia register were receiving low-dose anti-psychotics. This compares with the NDS, which estimated a point prevalence of 25% [14] and an observational study, involving a random sample of GP practices across five primary care trusts, which found that 26% of people with dementia were prescribed anti-psychotics [27]. The recent national audit calculated that in 2011, nationally 6.8% and within Medway PCT 10.5% [Personal Communication with Jonathan Hope] of people with dementia received an anti-psychotic [3]. However, only 45.7% of GP practices across England participated in the national audit due to technical issues or insufficient resources for data extraction [3]. Across Medway PCT 17.5% of practices participated in the national audit compared with 98.3% of practices in this project [3].

Both the national audit and this project relied upon the accuracy of the dementia registers. However, in many cases a formal diagnosis of dementia may not be recorded [3]. A typical GP practice would contain between 12 to 20 people with dementia and between three to five people with dementia on low-dose anti-psychotics [14,28]. In this project 26 practices failed to report anyone on the dementia register receiving low-dose antipsychotics. The dementia register was set-up in 2006/07 in Medway and should have been well-established. However, an Alzheimer's Society national survey identified significant under diagnosis; in Medway only 43.8% of the expected number of people with dementia received a diagnosis, which reflects the national picture [29]. This confirms findings elsewhere e.g. NHS Dumfries and Galloway estimated that the QOF dementia register only contained 32.9% of the expected dementia population [30]. Therefore, both the national audit and this project may under-estimate the number of people with dementia prescribed anti-psychotics.

To confirm the extent of the usage of anti-psychotics in dementia, an alternative approach to solely relying on the dementia register may be required. One such alternative option would be to initially identify everyone aged over 65 on low-dose anti-psychotics. A priori criteria for a diagnosis of dementia including a prescription for a cognitive enhancer, in addition to presence on the dementia register, could then be applied. This population identified by the alternative approach should be compared to that identified by solely relying upon the dementia registers, and if it captures the true magnitude of the issue used, as entry criteria for any future intervention. Prescribing was concentrated in a relatively small number of practices and a person with dementia in a residential home was nearly 3.5 times more likely to receive a low-dose anti-psychotic than someone with dementia living in their own home. This confirms earlier research, which highlighted that anti-psychotic use for people with dementia in nursing homes was becoming increasingly prevalent [31]. Therefore, to achieve maximum benefit, and in line with the NDS, and the approach adopted in this project, baseline data should be used to target where the intervention is delivered [14].

The most commonly used anti-psychotic was amisulpride, followed by risperidone and quetiapine. These three anti-psychotics were also identified by an expert group of old age consultant psychiatrists as the most appropriate treatments for challenging behaviour in dementia [32]. However, the efficacy of amisulpride for this indication is unproven, and although there may be less harm associated with quetiapine, there is no evidence that quetiapine is effective in people with dementia [22,[33][34][35]. Furthermore, neither amisulpride nor quetiapine are licensed to treat the behavioural and psychological symptoms of dementia. The licensed treatment, risperidone, which is recommended by local and national guidelines, was only used in 23.0% of instances [22,23]. Risperidone was one of the first antipsychotics highlighted by the Committee of Safety on Medicines to cause excess mortality and local and national guidelines, which highlight that all anti-psychotics carry a similar risk, appear not to have been fully communicated to prescribers [24,32,36].

There are various limitations with the project in addition to issues highlighted regarding the accuracy of the dementia register. The project was solely conducted within a single PCT, and whilst the PCT covered a very mixed area, the results are not necessarily generalisable to other locations. People initiated on anti-psychotics by secondary care were excluded and a joint pharmacist and GP review, linking with secondary care including experts in the non-pharmacological management of BPSD, might have a more significant impact in limiting the inappropriate prescribing of anti-psychotics [31].

In the short-term, the pharmacy-led intervention reduced the prescribing of anti-psychotics. However, the scope was relatively limited, and excluded other psychotropics, which might be used in place of anti-psychotics [37]. Further, patients were not formally followed-up to determine whether the anti-psychotic was stopped, or simply the dose reduced. The symptoms of BPSD fluctuate and continuing input is likely to be required. To demonstrate such a longterm sustainable quality improvement one would require a full-scale research project which was beyond the scope of this evaluation of a clinical service. An initial step would be a pilot randomised controlled trial (RCT) of a specialist pharmacist-led medication review of psychotropics based upon guidelines from the Alzheimer's Society [22]. Such a pilot should include a health economic assessment. A positive pilot, and subsequent full trial, could inform future service development.

Key Practice Implications -Whilst initial data is encouraging further research is required on the role of pharmacy-led interventions to reduce the prescribing of anti-psychotics and other psychotropics for people with dementia. -Any intervention should focus on care homes where the majority of anti-psychotic prescribing appears to occur. -The accuracy of the dementia register in identifying people with dementia prescribed low-dose antipsychotics is unclear. -Audit data including the recent national audit may under-estimate the usage of anti-psychotics in people with dementia.

Conclusion

This project identified that within one PCT approximately 15% of people on the dementia register were receiving low-dose anti-psychotics. However, this may be an under-estimate, because the dementia register may not capture everyone with dementia. Anti-psychotics were used much more commonly within care home settings and the most commonly used anti-psychotic was amisulpride. The project also identified that a pharmacist-led review could successfully limit the prescribing of anti-psychotics to people with dementia.

Figure 1
1Intervention Flow chart.

Table 1
1Details of low-dose anti-psychotics used in people with dementiaAnti-psychotic
N (%)

Amisulpride
52 (32.3%)

Risperidone
37 (23.0%)

Quetiapine
35 (21.7%)

Olanzapine
14 (8.7%)

Aripiprazole
8 (5.0%)

Haloperidol
7 (4.3%)

Chlorpromazine
2 (1.2%)

Flupentixol
2 (1.2%)

Benperidol
1 (0.6%)

Sulpride
1 (0.6%)

Trifluoperazine
1 (0.6%)

Zuclopenthixol
1 (0.6%)

Total number of anti-psychotics
161 (100%)

http://www.biomedcentral.com/1471-244X/12/155
Competing interests CF, AnC and IM have provided consultancy services to pharmaceutical companies marketing psychotropics. Lundbeck supported a series of Dementia Forums across Kent and Medway involving primary care, secondary care and care home staff to raise awareness about Dementia. Lundbeck had no direct involvement in the project.Authors' contributionsAnC conceived the project and delivered the intervention. AmC collected the data and assisted with data analysis. CF provided clinical input and interpreted the data. IM provided clinical supervision, led the data analysis and drafted the manuscript. All authors commented on earlier drafts, and read and approved the final manuscript.
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A pharmacy led program to review antipsychotic prescribing for people with dementia. Child, BMC Psychiatry. 12155Cite this article asCite this article as: Child et al.: A pharmacy led program to review anti- psychotic prescribing for people with dementia. BMC Psychiatry 2012 12:155.