Abstract
Purpose
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The aim of this study was to investigate the efficacy of calcitonin (CT) in animal models of experimental osteoarthritis (OA) and rheumatoid arthritis (RA), as new stabilized CT formulations are currently being introduced.
Methods
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A comprehensive and systemic literature search was conducted in PubMed/MEDLINE and Embase databases to identify articles with original data on CT treatment of preclinical OA and RA. Methodological quality was assessed using the Systematic Review Centre for Laboratory Animal Experimentation’s risk of bias tool for animal intervention studies. To provide summary estimates of efficacy, a meta-analysis was conducted for outcomes reported in four or more studies, using a random-effects model. Subgroup analyses were employed to correct for study specifics.
Results
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Twenty-six studies were ultimately evaluated and data from 16 studies could be analyzed in the meta-analysis, which included the following outcomes: bone mineral density, bone volume, levels of cross-linked C-telopeptide of type I collagen, histopathological arthritis score, and mechanical allodynia. For all considered outcome parameters, CT-treated groups were significantly superior to control groups (P = 0.002; P = 0.01; P < 0.00001; P < 0.00001; P = 0.04). For most outcomes, effect sizes were significantly greater in OA than in RA (P ≤ 0.025). High in-between study heterogeneity was detected.
Conclusion
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There is preclinical evidence for an antioxidant, anti-inflammatory, antinociceptive, cartilage- and bone-protective effect of CT in RA and OA. Given these effects, CT presents a promising agent for the treatment of both diseases, although the potential seems to be greater in OA.
Introduction
Rheumatoid arthritis (RA) and osteoarthritis (OA) are the most common forms of arthritis and affect large parts of the global population. While both diseases share some phenotypical similarities (joint swelling and stiffness), they differ fundamentally in disease origin, age at disease onset and progression patterns (1, 2). RA represents a systemic inflammatory autoimmune disease predominantly affecting the synovium of symmetrical small joints, which if left untreated, leads to progressive articular destruction (3). OA is a degenerative and inflammatory condition, characterized by low-grade intra-articular inflammation and extracellular matrix (ECM) degradation, which subsequently disrupt cartilage and subchondral bone integrity (4, 5).
Current pharmaceutical therapies for RA include glucocorticoids (GC), nonsteroidal anti-inflammatory drugs (NSAIDs) and disease-modifying antirheumatic drugs (DMARDs) (6, 7). While DMARD therapy can nowadays effectively contain overall disease progression, bone erosions are often not addressed adequately and available agents fail in repairing articular damage once it has occurred (8, 9). Additionally, most immunosuppressive RA therapies are accompanied by severe side effects, such as an increased risk of serious infections, hepatotoxicity, and osteoporosis (10, 11, 12). Therefore, new treatment approaches that specifically help to preserve cartilage and bone integrity and that may even initiate local repair processes are desperately needed.
In contrast to RA, pharmacological therapy for OA is far more limited and mainly comprises symptom-relieving strategies with oral analgesics and intra-articular injections of GC or hyaluronic acid (HA) (13, 14, 15). As there are no disease-modifying OA drugs available yet, halting structural degeneration in OA is currently impossible.
Calcitonin (CT), a 32-amino-acid-long peptide, was discovered in 1961 as a hormone that prevents renal calcium excretion in states of hypocalcemia (16). CT also retains calcium through the inhibition of bone resorption which is why it was previously used as a bone sparing agent in conditions of increased bone turnover, including osteoporosis (17). Synthetic salmon calcitonin (sCT) or eel calcitonin (eCT) exhibits a 50–100 times higher potency than human CT, making teleost CT the most common pharmacological CT application (18).
By modulating osteoclast and osteoblast activity and increasing the synthesis of type II collagen and proteoglycan in cartilage tissue, CT exerts a regenerative effect on bone and cartilage in arthritic joints, which is why it has been discussed as a treatment for RA and OA therapy (19, 20, 21). There is in vivo evidence for an antioxidant, anti-inflammatory, and antinociceptive role of CT in preclinical models of joint diseases (22, 23, 24, 25). Although CT has not yet been approved for RA or OA therapy, it was used for several decades to treat postmenopausal osteoporosis until the recommendation was withdrawn by the U.S. Food and Drug Administration (FDA) in 2013 due to an increased risk of malignancies together with a lack of efficacy (26, 27).
While teleost CT was shown to reduce immunoglobulin and rheumatoid factor levels in patients with RA (28), its bone preserving effect on the skeleton was inferior to that of alendronate (29). For OA, two large randomized-controlled trials failed to show a statistically significant therapeutic effect of sCT regarding reduction of joint space narrowing and although sCT reduced pain scores and biomarkers of catabolic bone and joint metabolism, these changes were not statistically significant (30).
To avoid the systemic side effects of CT and simultaneously profit from the beneficial actions of the hormone, new formulations are currently being investigated, which are supposed to be more stable, potent, and bone-specific compared to previous formulations (31, 32, 33). There is considerable preclinical evidence for CT-mediated effects in arthritis, which have, however, not yet been analyzed systematically. In light of new pharmacological developments, this systematic review and meta-analysis investigates the therapeutic potential of CT in preclinical OA and RA.
Methods
This systematic review and meta-analysis was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA; (34)) and Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) guidelines (35). The systematic review methodology was prospectively defined and documented using the Systematic Review Center for Laboratory Animal Experimentation’s (SYRCLE) protocol template (36). The protocol for this study was registered on March 20, 2022, on Open Science Framework (osf.io/yfpk2) with the PICO (Population, Intervention, Comparison, Outcome) question: ‘What is the effect of calcitonin compared to placebo, vehicle or untreated controls on bone, cartilage and inflammation in animal models of rheumatoid arthritis and osteoarthritis?’
Search strategy
The MEDLINE (via PubMed) and Embase databases were systematically searched in July 2022 to identify relevant animal studies reporting the effect of CT administration in RA and OA. The search was last updated on October 24, 2022. The following search terms and Medical Subject Headings were used: calcitonin, rheumatoid arthritis, and osteoarthritis. The full search strategy is available in Supplementary Table 1 (see section on supplementary materials given at the end of this article). No publication date or type restrictions were applied to the search. In addition, titles in reference lists of included articles were screened to locate additional studies not identified in the initial search.
Selection process
Inclusion criteria were prespecified as follows: (i) Experimental RA or OA was induced in female or male mammals at all ages by one of the following methods: antibody-based induction of RA, toxin-based induction of RA, toxin-based induction of OA, surgically induced OA or naturally occurring OA; (ii) CT or CT derivatives were administered (including altered or coupled formulations); (iii) presence of a vehicle, placebo, or untreated control group; (iv) Outcomes that reflect RA and OA progression were compared between the CT and control group.
We excluded papers if they presented nonoriginal (e.g. review), ex vivo, in vitro, or human data. Language restrictions were set to English, German, Spanish, and French. We only considered peer-reviewed full-length articles.
In a first selection phase, titles and abstracts were screened according to the predefined inclusion criteria. Matching studies were selected for a second screening and full texts were assessed for eligibility. Both screening phases were performed by two reviewers independently. If any discrepancies occurred, they were resolved by discussion or, when no agreement was met, by consensus with a third reviewer.
Data extraction
A standardized data extraction form was designed and data were extracted from text, tables and graphs. For each qualified study, the following information was retrieved: publication characteristics (title, first author, year of publication and journal), animal model characteristics (species, strain, sex, total number, age, body weight, disease model, disease induction, and features of the control group), intervention characteristics (type of agent, route of administration, dose, start and duration of intervention), and outcome data (type of outcome, methodology of measurement, and time point of outcome assessment). For all continuous outcome measures in the experimental and control groups, the following data were collected: mean values per outcome, s.d., and the number of animals per group (n). Where outcome measures were assessed repeatedly, we chose results from final assessment, unless there were data available for an intervention period ≤56 days and >56 days, in which case results for both periods were included in the meta-analysis. Where various CT doses were used, we included results for the highest dose. If outcomes were only presented graphically, WebPlotDigitizer (37) was utilized to extract data from the graphs. If results required for meta-analyses were lacking, studies were excluded from further analyses. All data were extracted by two independent reviewers.
Methodological quality
Two reviewers independently assessed methodological study quality of each included study. In case of discrepancies, consensus was reached by discussion with a third reviewer. Risk of bias was assessed using the SYRCLE’s risk of bias tool for animal intervention studies (38). Every domain was examined and evaluated for a low risk for bias, a high risk or a lack of information.
Meta-analyses
As we were specifically interested in the effect of CT on bone and cartilage, the following parameters were assessed in included studies: Bone mineral density (BMD), bone volume (BV/TV), cross-linked C-telopeptide of type I collagen (CTX-I), histopathological score, and mechanical allodynia. A meta-analysis for each outcome was conducted when at least four studies reported the respective parameters.
Data were compared between CT intervention group and vehicle, placebo or untreated control group and estimated as standardized mean differences (SMDs) with 95% CIs. When a single control group was compared to multiple treatment groups, the size of the control group was adjusted by dividing it by the number of intervention groups it served (39). A random effects model was applied for meta-analyses and the pooled effect size, and its standard error was calculated. Study heterogeneity was evaluated using the I2 statistic (40). Significance level was set to P < 0.05. To explore potential sources of heterogeneity, subgroup analyses were predefined for: disease model (RA vs OA), CT dosage, duration of CT treatment and animal species. Bonferroni adjustment was applied as a post hoc test. All analyses were performed in Review Manager (RevMan) Version 5.4, The Cochrane Collaboration, 2020.
Deviations from the intended review protocol
In addition to the search strategy described in the protocol (MEDLINE (via PubMed)), the Embase database was also included in the systematic literature search.
As subgroup analyses were only feasible for the disease model, we opted for the Bonferroni correction instead of the Holm–Bonferroni correction to adjust P-values for multiple comparisons.
Results
Search results
The initial search yielded 2651 unique publications (Fig. 1). After title and abstract screening, 39 studies were identified as potentially relevant. Of these 39 studies, one study was not retrievable (41). Of the remaining 38 studies retrieved for full-text screening, 13 were excluded for the following reasons: eight were not full-text articles (conference abstracts, posters) (42, 43, 44, 45, 46, 47, 48, 49), three were not describing RA or OA studies (50, 51, 52), one study did not administer CT (53) and one study was a clinical study (54). Thus, 25 studies fulfilled the inclusion criteria. Additionally, one further study was identified by checking reference lists, resulting in 26 publications ultimately included in this systematic review.
Selection process. Preferred items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram describing the selection of studies reporting on the effects of calcitonin (CT) administration in experimental rheumatoid arthritis (RA) and osteoarthritis (OA).
Citation: EFORT Open Reviews 9, 7; 10.1530/EOR-23-0133
As outcomes were only included in the meta-analysis if data from at least four studies were available, nine studies could not be included due to the reported outcomes that differed from the majority of included studies. Furthermore, one study was excluded because of insufficient data (55). Accordingly, data of 16 studies were included in the meta-analysis.
Study characteristics are described in Table 1. Of the 26 studies included, 17 investigated the effects of CT administration in animal models for OA and nine in RA models. Experimental groups received CT as an intramuscular (i.m.), subcutaneous (s.c.), intraperitoneal (i.p.), or intra-articular (i.a.) injection, as an intranasal spray or orally. Diverse formulations and dosages of CT were used and administration timing, duration, and time points for outcome assessments varied greatly among studies. Besides, the studies focused on different effects of CT and accordingly, a wide range of outcomes was reported.
Characteristics of included studies.
| Calcitonin administration | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Study | Species | Sex | Animals, n | Disease model | Model induction | Formulation | Day CT given* | Duration | Inj. method | Dose | Control group |
| Adeyemi & Olayaki (22) | Rat | M | 40 | OA | i.a. injection of MIA into the left knee joint | sCT | 9 | Daily for 28 days | i.m. | 2.5 IU/kg b.w. or 5 IU/kg b.w. | Untreated |
| Adeyemi & Olayaki (23) | Rat | M | 40 | OA | i.a. injection of MIA into the left knee joint | sCT | 9 | Daily for 28 days. | i.m. | 2.5 IU/kg b.w. or 5 IU/kg b.w. | Untreated |
| Adeyemi & Olayaki (64) | Rat | M | 49 | OA + DM | OA: i.a. injection of MIA into the left knee joint; | sCT | 9 | Daily for 4 weeks | i.m. | 2.5 IU/kg b.w. or 5 IU/kg b.w. | Untreated |
| DM: injection of streptozotocin and nicotinamide | |||||||||||
| Al-Kashi et al. (73) | Rat | F | NI | RA | CIA | eCT | 11 | Daily for 1 week | i.p. | 0.3-10 μg/kg b.w. | Vehicle |
| Badurski et al. (86) | Rabbit | NI | 40 | OA | Partial MNX of the right knee | sCT | NI | Every 3.5 days for 12 weeks | i.m. | 7 IU/kg b.w. | Untreated |
| Behets et al. (57) | Dog | NI | 12 | OA | ACLT of the right knee | sCT | 1 | Daily for 12 weeks | Nasal spray† | 400 U | Placebo |
| Bhandari et al. (31) | Rat | M | 16 | RA | AIA | sCT or sCT-BP or sCT-PEG-BP | NI | Daily for 21 days | s.c. | CT: 20 IU/kg b.w.; sCT-BP & sCT-PEG-BP: equivalent to 20 IU/ kg b.w. CT | Vehicle |
| Bobalik et al. (71) | Rat | M | NI | RA | AIA | sCT | 0 or 14 | Twice a day for 28, 35, or 49 days | s.c. | 10 IU/kg b.w. | Vehicle |
| Braga et al. (69) | Rat | M | NI | RA | AIA | sCT | NI | i.c.v. | 5, 10, 20, or 40 μg | Vehicle | |
| Cheng et al. (61) | Rat | M | 30 | OA | Bilateral ACLT | CT | 0 | Every 2 days for 12 weeks | s.c. | 5 IU/kg b.w. | Vehicle |
| Colombo et al. (56) | Rabbit | M | 7 | OA | MNX + dissection of the sesamoid and fibular collateral ligaments | sCT | 7 | 5 days/ week for 5 weeks | s.c. | 40 IU/day | Vehicle |
| El Hajjaji et al. (58) | Dog | NI | 18 | OA | ACLT of the right knee | sCT | 1 | Daily for 12 weeks | Nasal spray† | 100 IU or 400 IU | Placebo |
| Gou et al. (24) | Rat | M | 40 | OA | Collagenase (type II) injections in the right L3–L6 facet joints | sCT | 1 | Every 2 days for 8 weeks | s.c. | 16 IU/kg b.w./2 days | Vehicle |
| Katri et al. (25) | Rat | F | 50 | RA | CIA | KBP | 0 | Daily for 44 days | s.c. | 10 μg/kg b.w. | Untreated |
| Katri et al. (65) | Rat | F | 49 | OA | MNX of the right knee | KBP | 0 | Daily for 8 weeks | s.c. | 10 μg/kg b.w. | Untreated |
| Kyrkos et al. (55) | Rabbit | F | 18 | OA | MNX, ACLT and partial dissection of the medial collateral ligament in the right knee | sCT | 9 | Daily for 1, 2, or 3 months | s.c. | 10 IU | Placebo |
| Li et al. (63) | Rat | M | 70 | OA | MNX+ACLT in the right knee | CT | 1 | Daily until day 8, 11, 15, 22, 29, 43, or 57 | s.c. | 15 IU/kg b.w. | Untreated |
| Mancini et al. (70) | Rat | F | NI | RA | CIA | sCT | 13 | Daily for 5 days | i.p. | 2 μg/kg b.w. | Placebo |
| Manicourt et al. (59) | Dog | NI | 28 | OA | ACLT | sCT | 14 | Daily for 34 or 90 days | s.c. | 3 IU/kg b.w. | Untreated |
| Mero et al. (32) | Rabbit | M | 48 | OA | ACLT of the right knee | sCT or HA-sCT | 10 | At day 10, 17, and 24 | i.a. | sCT: 400 IU; HA-sCT: equivalent to 25, 100, and 400 IU of sCT | Placebo |
| Nielsen et al. (66) | Rat | F | 60 | OA | Bilateral MNX and/or ovariectomy (OVX) | sCT | NI | Twice a day for 56 days | Oral† | 2 mg/kg b.w. | Vehicle |
| Papaioannou et al. (60) | Rabbit | M | 30 | OA | CCL transection in the right knee | sCT | 1 or 56 | Daily from day 1 to week 8 or from week 8 to week 16 | i.m. | 7 IU | Placebo |
| Perrot et al. (68) | Rat | M | 26 | RA | AIA | Human CT | 21 | Daily for 1 week | s.c. | 0.125 mg | Vehicle |
| Ryan et al. (62) | Mouse | M | NI | RA | K/BxN serum-transfer arthritis | sCT or sCT-HA-NP | 1 | Once on day after model induction; animals were sacrificed on day 5 | s.c. | sCT: 0.2 μg | Vehicle |
| i.a. | NP: containing 0.2 μg sCT | ||||||||||
| Stuart et al. (72) | Rat | F | 40 | RA | CIA | sCT | 1 or 10 | Daily from day prior to model induction to day 29 or from day 10 to 36 | i.p. | 10 IU/kg b.w. | Vehicle |
| Wen et al. (67) | Rat | F | 35 | OA | ACLT of the right knee and bilateral OVX | sCT | 84 | Twice a week for 9 weeks | s.c. | 3 IU or 15 IU | Vehicle |
*Start of CT administration (days post model induction); †Not injected.
AIA, adjuvant-induced arthritis; ACLT, anterior cruciate ligament transection; b.w., body weight; BP, bisphosphonate; CCL, cranial cruciate ligament; CIA, collagen-induced arthritis; CT, calcitonin; DM, diabetes mellitus; eCT, eel calcitonin; HA, hyaluronic acid; HDF, high-fat diet; i.a., intra-articular; i.c.v., intracerebroventricular injection; Inj, injection; i.m., intramuscular; i.p., intraperitoneal; KBP, Key Bioscience peptides; MIA, monoiodoacetate; MNX, medial meniscectomy; NI, no information; NP, chitosan nanocomplexes; OA, osteoarthritis; PEG, polyethylene glycol; RA, rheumatoid arthritis; s.c., subcutaneous; sCT, salmon calcitonin.
Risk of bias
Figure 2 shows results of the risk of bias assessment. More than half of the studies reported random allocation to treatment group, whereas details of randomization and allocation concealment were not further specified. Blinded assessment of outcome was reported in 12 studies, but often it was not clearly stated whether this was applied to all outcomes. As shown in Fig. 2, many domains were scored ‘unclear’, indicating a lack of information on the reporting of measures for bias avoidance.
Quality assessment of included studies (22, 23, 24, 25, 31, 32, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 86). Green = low risk of bias; Red = high risk of bias; Orange = unclear risk of bias. (1) Random generation of allocation sequence, (2) similar baseline characteristics, (3) adequate allocation concealment, (4) random housing, (5) blinding of the caregivers and/or investigators, (6) random selection for outcome assessment, (7) blinding of the outcome assessor, (8) adequate addressing of incomplete outcomes, and (9) no selective outcome reporting.
Citation: EFORT Open Reviews 9, 7; 10.1530/EOR-23-0133
Systematic review – qualitative synthesis
Bone
Bone specific parameters were examined in all but five studies and included macroscopic, microscopic, biochemical, and radiological investigations.
The study of Colombo et al. was not able to show any bone-protective effects for CT in OA (56).
Macroscopically, four out of four studies investigating osteophyte formation showed an inhibitory effect of CT with smaller osteophytes in CT-treated animals compared to control groups (57, 58, 59, 60).
Three studies analyzed the effect of CT treatment on subchondral bone microarchitecture. Effects on trabecular thickness (Tb.Th) were reported in three studies. Two studies recorded a significantly higher Tb.Th (by 23% and 14.79%) in CT-treated groups compared to controls (24, 61), whereas the application of Key Bioscience peptides (KBPs), a novel CT formulation, did not show any difference between the two groups (25). In addition, two studies found a significant difference in trabecular separation (Tb.Sp) between the CT-treated group and control groups, with 28% and 13.36% reduced Tb.Sp in CT-treated animals (24, 61). Furthermore, Cheng et al. demonstrated a 17% higher trabecular number (Tb.N) in the CT-treated group (61), whereas no statistically relevant difference was found in the study of Gou et al. (24).
Cartilage
Cartilage-specific parameters were examined in 19 studies, where one study investigated CT in a model for RA (62).
Three studies presented data on the surface of cartilage ulcerations in OA models (57, 58, 59). Here, OA was induced by anterior cruciate ligament transection (ACLT) and treated by sCT. Considering the highest dose and an application duration of 12 weeks, studies showed 45.75% (57), 52.48% (58), and 75.17% (59) less surface ulcerations of the tibial plateau in the CT-treated groups compared to the control groups.
Preservation of cartilage ECM integrity by CT treatment was demonstrated in 11 studies using various serological and immunohistochemical markers. When compared to specimens from control groups, cartilage specimens from animals treated by CT showed lower expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 4 and matrix metalloproteinase 3 (24, 61, 63). Furthermore, expression of type II collagen was 51% higher in CT-treated animals (61). Consistent with these findings, serum levels of collagen type II degradation markers (CTX-II and C2M) were significantly lower in CT-treated animals compared to controls (22, 64, 65, 66).
Further biochemical and immunohistochemical analyses revealed higher cartilage proteoglycan (PG) and glycosaminoglycan (GAG) contents and lower serum levels of antigenic keratin sulfate (AgKS) and HA in CT-treated animals when compared to controls (24, 58, 59, 63). In addition, El Hajjaji et al. found that CT treatment also improved the quality of synthesized PGs by increasing AgKS content in PG molecules (58). The study of Colombo et al. was again not able to detect any cartilage-protective effects of CT in OA (56).
Nociception
Nociceptive behavior was assessed in a total of six studies and included tests for mechanical allodynia, cold hypersensitivity, weight bearing as well as electrophysiological investigations. Different formulations and doses were administered, and the treatment period ranged from 1 to 9 weeks.
CT was reported to effectively alleviate mechanical allodynia and cold hypersensitivity in models for OA (24, 65, 67), whereas this was not observed in RA models (25, 68). Nevertheless, in a model of RA, CT significantly inhibited evoked noxious firing of nociceptive thalamic neurons, when administered intracerebroventricularly (69).
In the OA study of Wen et al., CT was shown to reduce weight-distribution asymmetry between OA-affected and contralateral healthy hind paws.
Inflammation
Inflammation was quantified in six RA studies (25, 31, 70, 71, 72, 73) and one OA study (67) by measuring paw volume, paw width or knee width. Bobalik et al. were the first to provide evidence for an anti-inflammatory effect of sCT in rat adjuvant arthritis. When sCT was administered at a dose of 10 IU/kg body weight daily from the day of arthritis induction, paw volume was 50–80% lower compared to the control group. In addition, they found a synergistic effect in the combined administration of sCT with phenylbutazone, hydrocortisone acetate, adrenocorticotropic hormone, and calcium chloride (71). In several models of RA, the inhibitory effect on paw volume and width could not be replicated when CT was administered alone (25, 31, 70, 72), but a synergistic effect with NSAIDs and GCs was confirmed (25, 70). However, novel analogs of CT (sCT-BP and sCT-PEG-BP), which were developed to achieve a more joint-specific effect by coupling to bisphosphonate (BP), were able to improve the anti-inflammatory effect of sCT in experimental RA (31).
In contrast to these findings, the administration of native sCT alone resulted in significantly lower knee diameter in experimental OA (67).
Serum analyses in experimental OA revealed significantly lower IL-6 concentrations (by 52%) and white blood cell counts (by 32%) in CT-treated animals compared to controls (22, 64).
Immunohistochemistry showed lower macrophage infiltration and higher transforming growth factor (TGF)-β1 expression in RA and OA joints of CT-treated animals, indicating an immunosuppressive mode of action (25, 67).
Oxidative stress
Inflammation in RA and OA is accompanied by oxidative stress (74, 75). Adeyemi et Olayaki therefore investigated the effect of CT on oxidative and antioxidative stress markers in a sodium monoiodoacetate (MIA)-induced knee OA model (22) and a diabetic MIA-induced knee OA model (64). The authors found significantly higher malondialdehyde levels, a biomarker for oxidative stress (76) in untreated OA animals compared to healthy controls. Malondialdehyde was significantly lower in CT-treated compared to untreated OA animals. Furthermore, significantly lower antioxidative superoxide dismutase activity (SOD) and glutathione peroxidase (GPX) activity was observed in untreated OA animals compared to healthy controls. Both markers were significantly higher in CT-treated animals (22, 64).
Meta-analysis – quantitative synthesis
Bone
BMD was evaluated in eight comparisons from four studies, whereby different formulations and doses were administered and the treatment period ranged from 3 to 12 weeks (24, 31, 57, 61). A pooled analysis of all available data showed that BMD was 2.34-fold higher in the CT-treated group compared to the control group (95% CI: 0.82–3.85; P = 0.002) with high heterogeneity (I2 = 79%; Fig. 3 A).
Nine comparisons from five studies included complete data sets for bone volume (BV/TV). The pooled analysis showed that CT treatment was associated with a 1.69-fold increase in BV/TV compared to the control group (95% CI: 0.37–3.01; P = 0.01; I2 = 84%; Fig. 3 B).
Effects of CT on bone turnover in RA and OA was evaluated in five studies by measuring serum levels of CTX-I, a widely used marker for bone resorption (77). Treatment periods ranged from 1 to 8 weeks and two studies used a new CT formulation (KBP) (25, 47, 65). The pooled analysis showed that CTX-I levels were 3.95-fold lower in the CT-treated group than in the control group (95% CI: 5.42–2.49; P < 0.00001; I2 = 57%; Fig. 3C).
Cartilage
In 13 comparisons from 10 OA studies, articular cartilage lesions were graded using the Mankin`s histopathology grading system (78), a modified Mankin score (79) or the Osteoarthritis Research Society International (OARSI) OA cartilage histopathology assessment system (80). The pooled analysis showed that the score was 2.46-fold lower in the CT-treated group compared to the control group (95% CI: 3.35–1.57; P < 0.00001; I2 = 74%; Fig. 4A), indicating a cartilage protective effect of CT. Interestingly, the effect sizes of CT-treated groups differed greatly in the studies by Behets et al. and El Hajjaji et al., although study characteristics regarding species, agent, dose and route of administration were similar.
Effect of CT on cartilage integrity (24, 32, 57, 58, 58, 60, 61, 63, 65, 67) and nociception (24, 25, 65, 67, 68). Forest plots showing the impact of CT treatment on (A) histopathological score and (B) mechanical allodynia. CT-treated groups were compared to controls.
Citation: EFORT Open Reviews 9, 7; 10.1530/EOR-23-0133
Nociception
The pooled analysis of five comparisons from five studies for mechanical allodynia testing showed that paw withdrawal or vocalization thresholds were 2.73-fold higher in CT-treated groups compared to control groups (95% CI: 0.09–5.37; P = 0.04), with high heterogeneity I2 = 94%; Fig. 4B).
Subgroup analyses
Pooled random-effects meta-analysis for all outcomes showed medium to high levels of heterogeneity for BMD (I2 = 79%; P < 0.00001), BV/TV (I2 = 84%; P < 0.00001), CTX-I (I2 = 57%; P = 0.05), histopathological score (I2 = 74%; P < 0.00001), and mechanical allodynia (I2 = 94%; P < 0.00001). To explore this heterogeneity, we identified four study design variables that might explain some of the observed between-study variability: species (mouse or rat or other), CT dose (continuous variable), disease model (RA or OA), and intervention duration (≤56 days or >56 days).
Because only five of the 16 studies included in the meta-analysis used animal species other than rats and because CT dosage and duration of CT treatment varied widely among included studies, subgroup analyses for animal species and CT dose, and intervention duration were not viable for subgroup analysis.
To determine whether the disease model (RA vs OA) could explain the observed differences in the effects of CT, we conducted subgroup analyses on the outcomes BMD, BV/TV, CTX-I, and mechanical allodynia. The adjusted significance level (Bonferroni corrected P-value) for subgroup analyses for all outcomes was P ≤ 0.025. The subgroup analysis for disease model yielded that SMDs for BMD, BV/TV, and mechanical allodynia between CT and control groups, were significantly higher for OA models than for RA models (Fig. 5). The full dataset of subgroup analyses is available in Supplementary Figs. 1–4.
Subgroup analyses to determine the effect of CT on bone and nociception. Except for CTX-I, the effect of CT administration was significantly stronger in OA than that observed in RA (P < 0.025).
Citation: EFORT Open Reviews 9, 7; 10.1530/EOR-23-0133
Discussion
This study summarized and assessed the findings of 26 preclinical studies reporting the administration of CT for RA or OA treatment. The included studies provided evidence for five major effects of CT in preclinical RA and OA models: bone-protective, cartilage-protective, anti-inflammatory, antinociceptive, and antioxidative. Our meta-analysis revealed that CT improved results in all considered parameters (BMD, BV/TV, CTX-I, histopathological score, and mechanical allodynia), compared to vehicle, placebo or no treatment. It is important to note that CT not only inhibited bone and cartilage resorption, but also improved the quality of subchondral bone and articular cartilage, as indicated for bone by better results in Tb.Th, Tb.N, Tb.Sp and for cartilage by higher PG and GAG contents with higher AgKS levels in PG molecules (24, 58, 61). Nevertheless, the effect size of CT attributed effects varied between studies, especially when looking at the studies by Behets et al. and El Hajjaji et al., which illustrate markedly different effect sizes for histopathological scores, although study designs were comparable. A difference in animal numbers could possibly explain this phenomenon, yet this remains speculative. It should also be noted that cartilage-specific parameters were mainly investigated in OA and not RA models. Therefore, further scientific exploration of cartilage-protective effects of CT in preclinical RA models is warranted. The same applies to antioxidant effects.
Interestingly, subgroup analyses for disease models showed a greater effect of CT for OA compared to RA models regarding bone-specific parameters (BMD, BV/TV, and CTX-I), although this result was only significant for BMD and BV/TV. Unfortunately, cartilage-specific parameters could not be included in this analysis due to the limited data available. The greater effectiveness in OA compared to RA was also reported for antinociceptive and anti-inflammatory effects. This may in part be explained by the more aggressive nature of RA as a systemic pro-inflammatory disease, opposed to OA, which mainly affects local articular structures due to a chronic low-grade inflammation (81).
Despite the limited effectiveness of CT in RA, it may still be beneficial when used in combination with other agents. There is in vivo evidence for a synergistic effect of CT with NSAIDs (25, 71) and GCs (70, 71, 73). Combined administration of CT with GCs could thus allow a dose reduction of the latter to a subtherapeutic level, with effectiveness being comparable to high-dose GCs. In addition, CT also seems to diminish side effects associated with long-term GC administration, such as hyperglycaemia and osteoporosis (70, 73).
Due to the diverse tissue protective effects of CT in arthritic joints, it might be a promising agent for the treatment of OA and, when used in combination, also for RA. The effectiveness of CT administration in patients with OA was previously partially confirmed. In a phase I study, oral sCT was administered to patients with knee OA and shown to prevent degradation of cartilage and bone (82). Another study evaluated the effect of nasal sCT treatment in women with postmenopausal osteoporosis and knee OA and demonstrated a significant decreases in pain, joint stiffness and analgesic intake together with significant improvements in BMD, physical function and quality of life (83). However, data from two randomized-controlled trials showed no statistically significant therapy effect of sCT for joint space narrowing reduction, while pain and biomarker profiles were improved, but not statistically significant (30).
Systemic CT application has a low bioavailability and may be associated with relevant side effects, including an increased risk for malignancies (27, 31). These challenges could be resolved by new joint-specific formulations of CT, such as those used in the studies by Bhandari et al. (31), Mero et al. (32), and Ryan et al. (62). Coupling CT to HA or BP may increase joint specific availability while preventing systemic distribution and thus undesired systemic side effects. While the viscosity effect of HA reduces clearance from the joint space and improves residence time of CT (32), BP coupling may be useful to specifically target the subchondral bone, a central aspect of the joint, affected by OA (84).
Furthermore, it was recently shown that structural modifications of human CT such as phosphorylation led to a modified fibrillation behavior of the hormone (33), which increased its stability and may thus improve therapeutic efficacy of CT.
Following a report by the FDA outlining an increased risk of malignancy, which outweighed the benefits of the treatment for bone diseases such as osteoporosis and Paget’s disease, CT administration was restricted (26, 27). In the following years the biological plausibility of CT causing malignancies was questioned (85), yet safety concerns prevailed among clinicians. If CT will be reintroduced to clinical testing for chronic diseases such as OA, large patient numbers and closely monitored long-term safety and efficacy outcomes will be central to clinical trial designs.
This systematic review and meta-analysis has some limitations. First, measures for avoiding bias were only infrequently reported in the included studies. Second, the dataset is comparably small and data generalization should be avoided, especially due to the high in-between study heterogeneity. Third, study designs were heterogeneous in terms of dosage, route of administration, and CT formulation which was altered in some studies. Last, included studies did not report harmful (side) effects of CT, which would be of interest due to the history of the drug.
Despite these limitations, this study provides a robust framework for the design of future animal and clinical studies which investigate the therapeutic efficacy of new CT formulations in arthritis.
Conclusion
In conclusion, the results of our study indicate that CT is effective in protecting bone and cartilage and reducing inflammation, oxidative stress and hyperalgesia in preclinical animal models for both RA an OA. CT appears to have an exploitable potential for arthritis therapy, although we also found evidence suggesting that CT may be more beneficial for the treatment of OA than RA. Novel joint-specific formulations need to be preclinically and clinically tested to confirm efficacy and uncover potential side effects.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/EOR-23-0133.
ICMJE Conflict of Interest Statement
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the study reported.
Funding
This study was funded by the European Union under Grant Agreement No. 101095635 (PROTO). Views and opinions expressed are however those of the authors only and do not necessarily reflect those of the European Union or the European Health and Digital Executive Agency (HADEA). Neither the European Union nor the granting authority can be held responsible for them.
Author contribution statement
MMG, ABB, and TM designed the concept of the study. MMG and TM independently screened the studies for inclusion and exclusion criteria, assessed methodological study quality, extracted and analyzed the data. If any discrepancies occurred, they were resolved by discussion or, when no agreement was met, by consensus with RKZ. ABB provided further statistical expertise. TW, JK, and RKZ interpreted and curated the data. MMG and TM wrote the original manuscript and study protocol with help of ABB. All authors revised the manuscript critically for important intellectual content and approved the final version of the manuscript.
Acknowledgements
T Maleitzke is participant in the BIH Charité Clinician Scientist Program funded by the Charité – Universitätsmedizin Berlin, and the Berlin Institute of Health at Charité (BIH).
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