Potential effects of teriparatide (PTH (1-34)) on osteoarthritis: a systematic review.

Osteoarthritis (OA) is a common and prevalent degenerative joint disease characterized by degradation of the articular cartilage. However, none of disease-modifying OA drugs is approved currently. Teriparatide (PTH (1-34)) might stimulate chondrocyte proliferation and cartilage regeneration via some uncertain mechanisms. Relevant therapies of PTH (1-34) on OA with such effects have recently gained increasing interest, but have not become widespread practice. Thus, we launch this systematic review (SR) to update the latest evidence accordingly. A comprehensive literature search was conducted in PubMed, Web of Science, MEDLINE, the Cochrane Library, and Embase from their inception to February 2022. Studies investigating the effects of the PTH (1-34) on OA were obtained. The quality assessment and descriptive summary were made of all included studies. Overall, 307 records were identified, and 33 studies were included. In vivo studies (n = 22) concluded that PTH (1-34) slowed progression of OA by alleviating cartilage degeneration and aberrant remodeling of subchondral bone (SCB). Moreover, PTH (1-34) exhibited repair of cartilage and SCB, analgesic, and anti-inflammatory effects. In vitro studies (n = 11) concluded that PTH (1-34) was important for chondrocytes via increasing the proliferation and matrix synthesis but preventing apoptosis or hypertrophy. All included studies were assessed with low or unclear risk of bias in methodological quality. The SR demonstrated that PTH (1-34) could alleviate the progression of OA. Moreover, PTH (1-34) had beneficial effects on osteoporotic OA (OPOA) models, which might be a therapeutic option for OA and OPOA treatment.

Teriparatide prevented synovial inflammation and cartilage destruction in mice with DMM.

AIM: Emerging data have demonstrated that low-grade inflammation in osteoarthritis, a long-held degenerative disease. The inflamed synovium produces various cytokines that induce cartilage destruction and joint pain. A previous study showed that teriparatide, an FDA approved anti-osteoporotic drug, may enhance cartilage repair. Our study focuses on its role in OA synovitis. MATERIALS AND METHODS: Primary mouse articular chondrocytes were used to determine the most potent cytokines involved in OA inflammation and cartilage destruction. A destabilization of the medial meniscus mouse model was established to investigate the effect of teriparatide in OA, particularly, on synovial inflammation and cartilage degradation. RESULTS: In vitro experiments showed that TNF-α was the most potent inducer of cartilage matrix-degrading enzymes, and that teriparatide antagonized the TNF-α of effect. Consistently, articular cartilage samples from TNF-α transgenic mice contained more MMP-13 positive chondrocytes than those from wild type mice. In addition, more type II collagen was cleaved in human OA cartilage than in normal cartilage samples. CONCLUSIONS: Teriparatide can prevent synovitis and cartilage degradation by suppressing TNF-α mediated MMP-13 overexpression. Together with its chondroregenerative capability, teriparatide may be the first effective disease modifying osteoarthritis drug.

Solution design to extend the pH range of the pH-responsive precipitation of a CspB fusion protein.

Cell surface protein B (CspB) from Corynebacterium glutamicum has been developed as a reversible pH-responsive tag for protein purification. CspB fusion proteins precipitate at acidic pH, after that they completely dissolve at neutral pH. This property has been used in a non-chromatographic protein purification method named pH-responsive Precipitation-Redissolution of CspB tag Purification (pPRCP). However, it is difficult to apply pPRCP to proteins that are unstable under acidic conditions. In an effort to shift the precipitation pH to a milder range, we investigated the solution conditions of CspB-fused Teriparatide (CspB50TEV-Teriparatide) during the process of pH-responsive precipitation using pPRCP. The purified CspB50TEV-Teriparatide in buffer without additives precipitated at pH 5.3. By contrast, CspB50TEV-Teriparatide in buffer with 0.5 M Na2SO4 precipitated at pH 6.6 because of the kosmotropic effect. Interestingly, the pH at which precipitation occurred was independent of the protein concentration. The precipitated CspB50TEV-Teriparatide was fully redissolved at above pH 8.0 in the presence or absence of salt. The discovery that proteins can be precipitated at a mild pH will allow pPRCP to be applied to acid-sensitive proteins.

Teriparatide with Octacalcium Phosphate Collagen Composite Stimulates Osteogenic Factors.

Octacalcium phosphate and collagen composite (OCPcol) promotes osteogenic differentiation and angiogenesis, thereby enhancing bone regeneration. Although a newly developed freeze-dried composite of OCPcol and teriparatide (OCPcolTPTD) reinforced bone regeneration more than OCPcol, the mechanism of bone regeneration remains unresolved. In this study, disks containing OCPcolTPTD, OCPcol, or ß-tricalcium phosphate (ß-TCP) col were inserted into rodents with calvarial bone defects, before euthanasia 4 weeks later. Immunohistochemical and histochemical analyses were performed on bone samples to evaluate bone matrix development, angiogenesis, and osteoclast and osteoblast localization. In the OCPcolTPTD and OCPcol groups, bone regeneration was observed at the surface of calvarial dura mater and around acidophilic granular cells with abundant collagenous fiber-containing cells. Furthermore, the newly formed bone in the OCPcolTPTD group showed a larger total area and individual separated area than the other groups. Various osteogenic proteins were detected in the regenerated bone and peri-bone tissues by histochemistry and immunohistochemistry. Although the expression of several osteogenic biomarkers in the OCPcolTPTD group after 4 weeks of implantation was significantly lower than that in the OCPcol group, new bone formation by OCPcolTPTD in the center of the defect, where bone regeneration is difficult, tended to be superior to that by OCPcol. These results suggest that OCPcolTPTD enhanced bone regeneration more evenly and homogenously than OCPcol. Impact statement Our study suggests that octacalcium phosphate and collagen (OCPcol) together with a TPTD enhances bone regeneration in rodents with calvarial bone defects. Furthermore, we believe that composite of OCPcol and teriparatide (OCPcolTPTD) could be developed into novel clinical technique for the regeneration or repair of bone.

Effect of Clp protease from Corynebacterium glutamicum on heterologous protein expression.

The protease present in a host may reduce the yield and biological activity of heterologous proteins. In this study, we used protease overexpression and deletion strategies to examine the effect of the Clp protease system in Corynebacterium glutamicum on the recombinant protein and to produce a highly efficient heterologous protein expression host. In this study, we identified seven genes in the Clp protease family in Corynebacterium glutamicum ATCC 13032 through bioinformatics analysis, and studied their effects on the enhanced green fluorescent protein (EGFP) reporter protein. The fluorescence intensity of the knockout strain was significantly higher, and the effect of the clpS deletion strain was the most obvious. To verify the universal effect of the lack of clpS, the excellent industrial strain C. glutamicum 1.15647 was transformed to form recombinant 15647-ΔclpS. Based on the results, 15647-ΔclpS had a more significant effect on improving protein expression. Furthermore, recombinant human teriparatide (rhPTH) and variable domain of heavy chain of heavy-chain antibody (VHH) were selected to verify the universal applicability of the knockout strain for expressing heterologous proteins. Accordingly, we found that protease deficiency could increase the production of heterologous proteins. Finally, through a large-scale fermentation, the 15647-ΔclpS strain was used to produce VHH. Its yield was approximately 530 mg/L, which was 65% higher than that of WT-15647. In this study, a host that could effectively increase heterologous protein expression was successfully obtained.

Parathyroid hormone and its related peptides in bone metabolism.

Parathyroid hormone (PTH) is an 84-amino-acid peptide hormone that is secreted by the parathyroid gland. It has different administration modes in bone tissue through which it promotes bone formation (intermittent administration) and bone resorption (continuous administration) and has great potential for application in sbone defect repair. PTH regulates bone metabolism by binding to PTH1R. PTH plays an osteogenic role by acting directly on mesenchymal stem cells, cells with an osteoblastic lineage, osteocytes, and T cells. It also participates as an osteoclast by indirectly acting on osteoclast precursor cells and osteoclasts and directly acting on T cells. In these cells, PTH activates the Wnt signaling, cAMP/PKA, cAMP/PKC, and RANKL/RANK/OPG pathways and other signaling pathways. Although PTH(1-34), also known as teriparatide, has been used clinically, it still has some disadvantages. Developing improved PTH-related peptides is a potential solution to teriparatide's shortcomings. The action mechanism of these PTH-related peptides is not exactly the same as that of PTH. Thus, the mechanisms of PTH and PTH-related peptides in bone metabolism were reviewed in this paper.

Non-chromatographic purification of Teriparatide with a pH-responsive CspB tag.

Cell surface protein B (CspB) from Corynebacterium glutamicum is used as a pH-responsive peptide tag to enable a simple solid-liquid separation method for isolating a CspB fusion protein. Here we demonstrate the first application of a CspB tag for the purification of Teriparatide, which is a biologic drug that is prescribed for osteoporosis. The Teriparatide was constructed as CspB50TEV-Teriparatide, comprising 50 amino acid residues of CspB, the cleavage site of TEV protease, and Teriparatide. CspB50TEV-Teriparatide was expressed in a culture supernatant by C. glutamicum secretion system at 3.0 g/L (equivalent to approximately 1.2 g/L Teriparatide). The CspB50TEV-Teriparatide was precipitated by reducing the pH of the culture supernatant, and the precipitate was then dissolved in a neutral buffer. A TEV protease treatment was applied to cleave the Teriparatide from the CspB50TEV-Teriparatide. Then, the remaining digested CspB50TEV, undigested CspB50TEV-Teriparatide, and TEV protease were precipitated in an acidic pH, whereas the soluble Teriparatide remained in the supernatant. The process had a yield of 96.5% and resulted in Teriparatide with a purity of 98.0% and productivity of 1.1 g/L of C. glutamicum culture. Thus, tag-free Teriparatide was successfully purified from the CspB fusion protein using only pH changes, centrifugation, and protease digestion without the need for chromatography. This versatile purification protocol is expected to be applicable to various proteins from laboratory to industrial scales.

[Pharmacological treatment for pregnancy and lactation associated osteoporosis.]

Pregnancy and lactation associated osteoporosis(PLO)is a rare disorder for women during pregnancy, the post-partum period, or while breastfeeding. It still remains unknown factors in its pathogenesis. That makes it there is no evident strategy for PLO now. In most cases, bone mineral density(BMD)of PLO patients spontaneously recovers after giving lactation up. On the other hand, patients with severe cases sometimes need bone specific therapy. There are some reports that show bisphosphonate, teriparatide and/or denosumab are effective on PLO cases. When the patients have wishes for having babies, we have to pay attention if the prescription effect on next pregnancy.

[ASBMR topics from clinical research(osteoporosis and sarcopenia).]

There are a lot of progressive topics about osteoporosis and sarcopenia in 2018 ASBMR Annual Meeting, involving an association between diabetes and bone microarchitecture, associations between atypical femoral fractures and bisphosphonate drug holidays as well as pre-treatment bone mineral density, an effect of combined denosumab and high-dose teriparatide on bone parameters, and relationships between muscle and deuterated creatine, a selective androgen receptor modulator, and high-dose vitamin D supplementation.

Time-resolved fluorescence ligand binding for G protein-coupled receptors.

G protein-coupled receptors (GPCRs) and their ligands are traditionally characterized by radioligand-binding experiments. These experiments yield excellent quantitative data, but have low temporal and spatial resolution. In addition, the use of radioligands presents safety concerns. Here we provide a general procedure for an alternative approach with high temporal and spatial resolution, based on Tb(+)-labeled fluorescent receptor ligands and time-resolved fluorescence resonance energy transfer (TR-FRET). This protocol and its design are detailed here for the parathyroid hormone receptor, a class B GPCR, and its fluorescently labeled 34-amino acid peptide ligand, but it can be easily modified for other receptors and their appropriately labeled ligands. We discuss three protocol options that use Tb(+)-labeled fluorescent ligands: a time-resolved fluorescence separation option that works on native receptors but requires separation of bound and unbound ligand; a TR-FRET option using SNAP-tag-labeled receptors for high-throughput screening; and a TR-FRET option that uses fluorescently labeled antibodies directed against an epitope engineered into the Flag-labeled receptors' N terminus. These protocol options can be used as standard procedures with very high signal-to-background ratios in order to characterize ligands and their receptors in living cells and in cell membranes via straightforward plate-reader measurements.

ß-arrestin-selective G protein-coupled receptor agonists engender unique biological efficacy in vivo.

Biased G protein-coupled receptor agonists are orthosteric ligands that possess pathway-selective efficacy, activating or inhibiting only a subset of the signaling repertoire of their cognate receptors. In vitro, D-Trp(12),Tyr(34)-bPTH(7-34) [bPTH(7-34)], a biased agonist for the type 1 PTH receptor, antagonizes receptor-G protein coupling but activates arrestin-dependent signaling. In vivo, both bPTH(7-34) and the conventional agonist hPTH(1-34) stimulate anabolic bone formation. To understand how two PTH receptor ligands with markedly different in vitro efficacy could elicit similar in vivo responses, we analyzed transcriptional profiles from calvarial bone of mice treated for 8 wk with vehicle, bPTH(7-34) or hPTH(1-34). Treatment of wild-type mice with bPTH(7-34) primarily affected pathways that promote expansion of the osteoblast pool, notably cell cycle regulation, cell survival, and migration. These responses were absent in ß-arrestin2-null mice, identifying them as downstream targets of ß-arrestin2-mediated signaling. In contrast, hPTH(1-34) primarily affected pathways classically associated with enhanced bone formation, including collagen synthesis and matrix mineralization. hPTH(1-34) actions were less dependent on ß-arrestin2, as might be expected of a ligand capable of G protein activation. In vitro, bPTH(7-34) slowed the rate of preosteoblast proliferation, enhanced osteoblast survival when exposed to an apoptotic stimulus, and stimulated cell migration in wild-type, but not ß-arrestin2-null, calvarial osteoblasts. These results suggest that bPTH(7-34) and hPTH(1-34) affect bone mass in vivo through predominantly separate genomic mechanisms created by largely distinct receptor-signaling networks and demonstrate that functional selectivity can be exploited to change the quality of G protein-coupled receptor efficacy.

A claudin-4 modulator enhances the mucosal absorption of a biologically active peptide.

Biologics, such as peptides, proteins and nucleic acids, are emerging pharmaceuticals. Passage across the epithelium is the first step in the absorption of biologics. Tight junctions (TJ) function as seals between adjacent epithelial cells, preventing free movement of solutes across the epithelium. We previously found that modulation of a key TJ component, claudin-4, is a potent method to enhance jejunal absorption when we used dextran as a model drug and the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) as a claudin-4 modulator. Here, we investigated whether the claudin-4 modulator enhances jejunal, nasal and pulmonary absorption of a biologics human parathyroid hormone derivative, hPTH(1-34). The claudin-4 modulator enhanced nasal but not jejunal and pulmonary absorption of hPTH(1-34). C-CPE is hydrophobic with low solubility of less than 0.3mg/ml, but deletion of 10 amino acids at the N-terminal of C-CPE increased its solubility by 30-fold. Moreover, the N-terminal truncated C-CPE bound to claudin-4, modulated the TJ-barrier and enhanced jejunal absorption of dextran. The N-terminal-truncated C-CPE also enhanced jejunal and pulmonary absorption of hPTH(1-34). This report is the first to indicate that a claudin-4 modulator may be a promising enhancer of the jejunal, pulmonary and nasal absorption of a peptide drug.

Nmp4/CIZ: road block at the intersection of PTH and load.

Teriparatide (parathyroid hormone, [PTH]) is the only FDA-approved drug that replaces bone lost to osteoporosis. Enhancing PTH efficacy will improve cost-effectiveness and ameliorate contraindications. Combining this hormone with load-bearing exercise may enhance therapeutic potential consistent with a growing body of evidence that these agonists are synergistic and share common signaling pathways. Additionally, neutralizing molecules that naturally suppress the anabolic response to PTH may also improve the efficacy of treatment with this hormone. Nmp4/CIZ (nuclear matrix protein 4/cas interacting zinc finger)-null mice have enhanced responses to intermittent PTH with respect to increasing trabecular bone mass and are also immune to disuse-induced bone loss likely by the removal of Nmp4/CIZ suppressive action on osteoblast function. Nmp4/CIZ activity may be sensitive to changes in the mechanical environment of the bone cell brought about by hormone- or mechanical load-induced changes in cell shape and adhesion. Nmp4 was identified in a screen for PTH-responsive nuclear matrix architectural transcription factors (ATFs) that we proposed translate hormone-induced changes in cell shape and adhesion into changes in target gene DNA conformation. CIZ was independently identified as a nucleocytoplasmic shuttling transcription factor associating with the mechano-sensitive focal adhesion proteins p130Cas and zxyin. The p130Cas/zyxin/Nmp4/CIZ pathway resembles the beta-catenin/TCF/LEF1 mechanotransduction response limb and both share features with the HMGB1 (high mobility group box 1)/RAGE (receptor for advanced glycation end products) signaling axis. Here we describe Nmp4/CIZ within the context of the PTH-induced anabolic response and consider the place of this molecule in the hierarchy of the PTH-load response network.

Prediction of parathyroid hormone signalling potency using SVMs.

Parathyroid hormone is the most important endocrine regulator of calcium concentration. Its N-terminal fragment (1-34) has sufficient activity for biological function. Recently, site-directed mutagenesis studies demonstrated that substitutions at several positions within shorter analogues (1-14) can enhance the bioactivity to greater than that of PTH (1-34). However, designing the optimal sequence combination is not simple due to complex combinatorial problems. In this study, support vector machines were introduced to predict the biological activity of modified PTH (1-14) analogues using mono-substituted experimental data and to analyze the key physicochemical properties at each position that correlated with bioactivity. This systematic approach can reduce the time and effort needed to obtain desirable molecules by bench experiments and provide useful information in the design of simpler activating molecules.

Pulsatile release of parathyroid hormone from an implantable delivery system.

Intermittent (pulsatile) administration of parathyroid hormone (PTH) is known to improve bone micro-architecture, mineral density and strength. Therefore, daily injection of PTH has been clinically used for the treatment of osteoporosis. However, this regimen of administration is not convenient and is not a favorable choice of patients. In this study, an implantable delivery system has been developed to achieve pulsatile release of PTH. A well-defined cylindrical device was first fabricated with a biodegradable polymer, poly(l-lactic acid) (PLLA), using a reverse solid-free form fabrication technique. Three-component polyanhydrides composed of sebacic acid, 1,3-bis(p-carboxyphenoxy) propane and poly(ethylene glycol) were synthesized and used as isolation layers. The polyanhydride isolation layers and PTH-loaded alginate layers were then stacked alternately within the delivery device. The gap between the stacked PTH-releasing core and the device frame was filled with PLLA to seal. Multi-pulse PTH release was achieved using the implantable device. The lag time between two adjacent pulses were modulated by the composition and the film thickness of the polyanhydride. The released PTH was demonstrated to be biologically active using an in vitro assay. Timed sequential release of multiple drugs has also been demonstrated. The implantable device holds promise for both systemic and local therapies.

Parathyroid hormone/parathyroid hormone-related peptide modulates growth of avian sternal cartilage via chondrocytic proliferation.

Parathyroid hormone (PTH; 10(-7) to 10(-15) M) decreased terminal chondrogenesis in the avian sterna. During the first half of an 8-day culture, 100 nM PTH (1-34) significantly increased sternal length and downregulated the deposition of type X collagen and its mRNA expression. However, it remains unclear how PTH increased cartilaginous growth. In this study, we examined growth by both cell proliferation and analysis of cyclin d1 and collagen mRNA. Types II, IX, and X collagens and cyclin d1 mRNA were quantified through real-time RT-PCR, while Ki-67 was used as an immunohistochemical proliferation marker. Extracellular matrix content was measured through mRNA quantification of types II, IX, and X collagen and observing deposition of the same collagens. PTH significantly increased the proliferation marker Ki-67 in the sternal cephalic region. There was less type II and X collagen in PTH-treated sterna with concomitant decreases in mRNA production, suggesting that proliferation was the major contributor to cartilage growth in the presence of PTH/PTH-related peptide receptor activation. In conclusion, these experiments demonstrated that PTH increased cartilage growth by upregulating cell proliferation or other extracellular matrix components.

In vitro studies on Itm2a reveal its involvement in early stages of the chondrogenic differentiation pathway.

The Integral membrane protein 2A (Itm2A) is a transmembrane protein belonging to a family composed of at least two other members, Itm2B and Itm2C, all of them having a different expression pattern. The Itm2a gene serves as a marker for early stages in endochondral ossification. In order to understand the role of Itm2A in this process, expression of the gene was investigated in different cell systems. In C3H10T1/2 cells, the gene was upregulated early on when the cells were induced to the chondrogenic lineage but less to the osteogenic lineage. In MCT cells, expression was upregulated at permissive temperatures but not at non-permissive temperatures. When induced with insulin, ATDC5 cells expressed Itm2a in early stages but not at late stages. Furthermore, PTH treatment seems to upregulate Itm2a transcription. In order to understand the role of Itm2a in the chondrogenic differentiation process in more detail, we constitutively overexpressed exogenous Itm2A in mouse ATDC5 cells. Two clones expressing high levels of Itm2a were isolated and characterized. Gene expression analysis of the overexpresser clones demonstrated that expression of collagen type X was delayed. These results demonstrate that overexpression of Itm2a in mouse ATDC5 cells impede the transition to hypertrophic cells. Taken together, our observation supports the involvement of Itm2a in the early stages of chondrogenesis in vitro.

Regulation of PTH1 receptor expression by uremic ultrafiltrate in UMR 106-01 osteoblast-like cells.

BACKGROUND: Homologous down-regulation/desensitization of the parathyroid hormone receptor (PTH1R)/adenylate cyclase system has been demonstrated in uremia, and may contribute to parathyroid hormone (PTH) resistance; however, additional studies have shown that parathyroidectomy fails to normalize the down-regulation of the PTH1R. The present studies were designed to test directly, in vitro, the hypothesis that factors circulating in the uremic environment, other than PTH, decrease the response of osteoblastic cells to PTH. METHODS: Studies were conducted in confluent cultures of UMR 106-01 osteoblast-like cells. Uremic ultrafiltrate (UUF) was obtained from patients on hemodialysis. Cells were exposed to media containing 50% uremic ultrafiltrate for periods of up to 72 hours. Control cultures were exposed to a buffered salt solution containing a comparable ionic composition to that of the UUF. PTH-stimulated cyclic adenosine monophosphate (cAMP) generation was determined by radioimmunoassay (RIA), PTH binding and PTH1R mRNA levels were determined by radioligand binding and Northern analysis, respectively. RESULTS: PTH-stimulated cAMP generation from cultures treated with uremic ultrafiltrate for 48 hours was 1385.8 +/- 183.2 pmol/culture/5 minutes, whereas control cultures generated 2389.5 +/- 271 pmol cAMP/culture/5 minutes (P < 0.05). PTH binding was decreased by 30% in cultures incubated with UUF as compared to controls. The decrease in binding induced by UUF was accompanied by a decrease in PTH1R mRNA levels. CONCLUSION: These findings demonstrate that factors present in UUF decrease PTH-stimulated cAMP generation by a mechanism that involves a decrease in the levels of PTH1R mRNA levels. Thus, the skeletal resistance to PTH in the setting of chronic kidney disease, may be explained, at least in part, by circulating factors other than PTH.