Means and methods for treating calcium crystal deposition diseases
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- MAASTRICHT UNIVERSITY
- Filing Date
- 2022-06-27
- Publication Date
- 2026-07-30
Smart Images
Figure 0007897920000005 
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of drug therapy, particularly for the treatment of humans, and more particularly for the treatment of humans with calcium crystal deposition diseases. In one aspect, the present invention provides polypeptides that prevent or reduce the precipitation of calcium-containing crystals (also known as ectopic calcification) in the human body.
Background Art
[0002] Abnormal deposition of calcium crystals, also known as soft tissue calcification, is a major problem in many systemic and chronic diseases such as atherosclerosis (1), renal failure (2), osteoarthritis (3). In atherosclerosis and osteoarthritis, the importance of calcium deposition was not initially recognized (4, 5), but it is well recognized in kidney diseases (6). Calcium-containing crystals are often difficult to detect in the form of fine particles, but it has gradually become clear that they can actually cause or at least exacerbate diseases (7, 8). Pathological calcium deposition mainly consists of basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPP).
[0003] Controlling calcium crystal deposition is crucial in multicellular organisms where high concentrations of metal ions and anions, such as inorganic phosphate (π), are present in the intracellular and extracellular environments. Extracellular matrix components, such as collagen, can promote calcification by acting as nucleation centers (9). On the other hand, mineralization is extremely important for bone development and homeostasis (10). Therefore, calcium ion concentration is maintained within a strict limit in the bloodstream (1.15-1.33 mmol / L) by PTH and calcitonin, hormones important for muscle contraction and nerve impulse generation (11). Total calcium concentration is 2.2-2.7 mM, with approximately 45% ionic, 45% protein-bound, and 10% complex (12). Phosphate concentration is known to fluctuate depending on dietary intake (0.8-1.5 mM), and protein-bound phosphate accounts for only a small portion (approximately 10-15%) (13). Intrinsic calcification inhibitors include inorganic pyrophosphate (ppi), which has been shown to be stimulated by ectonucleotide pyrophosphatase / phosphodiesterase 1 (Enpp1;(14)), ankyrin 1 (Ank1;(15)), and ATP-binding cassette subfamily C member 6 (Abcc6;(16)) in mouse knockout animal experiments. Other important calcification inhibitors include matrix Gla protein (MGP), Gla-rich protein (GRP), klotho (KL), and α-2-HS-glycoprotein (AHSG, also known as fetuin A). [Overview of the project]
[0004] Despite the existence of several endogenous calcification inhibitors, there are no compositions that are effective in treating or preventing calcium crystal deposition disease. [Brief explanation of the drawing]
[0005] [Figure 1]This figure shows the calcium precipitation inhibitory effect of the peptide shown in Sequence ID No. 1 and β-fetuin over a wide concentration range in vitro. It shows a 10-step dilution series of the peptide (total 10¹⁵) in a calcium precipitation assay (2 hours). 100% precipitation is achieved even without the addition of β-fetuin or peptide 1 (dashed line). [Figure 2] These are electron microscope images obtained from precipitates formed after 2 hours in the absence (left panel) or presence (right panel) of peptide (20 μM). The precipitate formed in the presence of β-fetuin is shown in the center panel. Absorption measurement before sample processing (dialysis) confirmed a precipitate suppression rate of ~50%. The scanning electron microscope magnification was 35,000x. The bars represent 5 micrometers. Center panel: 0.5 mg / ml β-fetuin added; Right panel: 20 μM peptide 1 added. [Figure 3] This figure shows the inhibition of cellular calcification in human articular chondrocytes, vascular smooth muscle cells, and bone marrow stromal cells by the peptide shown in Sequence ID No. 1. Representative phase-contrast microscopy images from in vivo cell assays are shown. A) Human articular chondrocytes (HACs) were stimulated for 7 days with or without 1 mM ATP and 20 μM peptide. Results were obtained from three chondrocyte donors, each with 3 biological replicas. B) Vascular smooth muscle cells (VSMCs) were stimulated for 7 days with 4.5 mM ionic calcium in or without 20 μM peptide. Results were obtained from 3 biological replicas from each of three VSMC donors. C) BMSCs were differentiated for 21 days in osteogenic differentiation medium containing β-glycerophosphate (BGP), in or without 2.1 μM peptide. BMSCs: 4 biological replicas from one donor were used. Left panel: Phase-contrast images were obtained on the final day of each experiment. Calcification can be seen as black dots. One representative image is shown for each condition. The scale bar represents 200 μm. Right panel: Total calcium precipitate per well, measured using a Randox total calcium assay, was quantified and normalized by total protein. Positive controls for each donor were normalized to 100%. Statistical comparisons were performed using the Student's t-observer test. *=p value < 0.05. [Figure 4]This figure shows that intra-articular injection of peptides improved cartilage histopathology and motor function in a rat model of osteoarthritis. Osteoarthritis was induced by surgical tearing of the medial meniscus and transection of the collateral ligament (N=40). 50 μl of 20 μM peptide or 50 μl of vehicle (0.9% NaCl) were injected intra-articularly every other week (N=20 in each group). Necropsy was performed on day 28, and histological analysis was performed according to the OARSI scoring system. A) Representative toluidine blue stained tissue sections from the best, mean, and worst histological appearances. Cartilage tissue is stained dark blue. B) OARSI scores were calculated by multiplying the grade and stage of cartilage damage per animal by two blinded observers. Statistical comparisons were performed using a two-sided t-test. *=p value<0.05. C) Gait analysis score on day 20 of the experiment. Four measurements were taken per animal, and each point represents the mean for one animal in the graph. Statistical significance between groups was determined by the Mann-Whitney U test. *=p-value<0.05. [Modes for carrying out the invention]
[0006] The present invention relates to a cyclic polypeptide having the amino acid sequence shown in Sequence ID No. 1, wherein the amino acids are D-amino acids. The present invention also relates to a pharmaceutical composition comprising such a polypeptide and a pharmaceutically acceptable carrier, and its use in the treatment of diseases. More specifically, the present invention relates to the polypeptide described herein for the treatment of diseases selected from the group consisting of osteoarthritis, periarthritis of the shoulder, heterotopic ossification, vascular calcification, kidney stones, and calcification.
[0007] (Detailed description of the invention) The inventors chemically synthesized a cyclic polypeptide consisting of 30 D-amino acids. Its sequence is LIYRQPNCDDPETEEAALVAIDYIAPHGPG (hereinafter also referred to as peptide 1, peptide, or peptide shown in Sequence ID No. 1), in which the amino acid D-leucine (L) at position 1 is chemically bonded to the amino acid D-glycine (G) at position 30 to form a cyclic polypeptide. In this specification, "polypeptide" and "peptide" are used interchangeably.
[0008] To evaluate the inhibitory ability of the peptide shown in Sequence ID No. 1, an established in vitro calcium phosphate precipitation assay (17-19) was used, as described in Example 2. As a positive control, bovine fetuin (β-fetuin) at a concentration of 1 mg / ml (20.6 μM), which was found to completely inhibit precipitation, was used.
[0009] Attempts were made to dilute the peptide until it lost its effectiveness. The peptide shown in SEQ ID NO: 1 showed a positive dose-response effect as the peptide concentration increased. Bovine fetuin (β-fetuin) lost its inhibitory effect at concentrations of 1–10 μg / ml (20.6–200.6 nM), but the peptide shown in SEQ ID NO: 1 remained active up to the atomolecular concentration range (Figure 1).
[0010] It was found that precipitation could be statistically significantly suppressed at a concentration of 20.6 aM, not just 2.6 aM, compared to the control (Figure 1). This 20.6 aM concentration was estimated to correspond to 1,250 peptide molecules in a reaction volume of 100 μl.
[0011] In summary, the peptide shown in SEQ ID NO: 1 inhibits the in vitro precipitation of calcium phosphate more efficiently than β-fetuin. Therefore, it can be concluded that the peptide shown in SEQ ID NO: 1 is more effective than β-fetuin in preventing calcium crystal deposition in in vitro models of calcium crystal deposition disease.
[0012] Next, the deposits formed after 2 hours in in vitro assays in or without β-fetuin or the peptide shown in SEQ ID NO: 1 were analyzed by scanning electron microscopy (Example 3). In the control, relatively large particles (~1 μM) and small particles (50~200 nM) were observed (Figure 2; left panel). The larger particles had distinct crystalline edges, which is typical of BCP crystal formation (20). The particles obtained after contact with β-fetuin had a completely different morphology, lacking these sharp edges (Figure 2; center panel). The particles were also somewhat smaller (in the range of approximately 0.5 μM). The particles obtained with the peptide shown in SEQ ID NO: 1 appeared even smaller and more compact. They were triangular, square, and rarely pentagonal or hexagonal in shape (Figure 2; right panel). The smallest discernible particles obtained from the peptide-treated samples were approximately 67 nm ± 7 (mean ± SD, n=4) in size and resembled primary CPP (<100 nM). Particles obtained using the peptide were susceptible to electron beam decomposition at high magnification (>35,000x), whereas particles from the control sample did not. β-fetuin is known to inhibit CPP growth by coating the outer surface. Without being constrained by theory, we hypothesized that this peptide also interacts with calcium phosphate particles.
[0013] To test this, particles were allowed to form for 2 hours or 24 hours in the presence or absence (control) of β-fetuin or the peptide shown in SEQ ID NO: 1. These particles were then collected by centrifugation, washed, dissolved in acid, and subsequently measured for total calcium, phosphate, and protein in the pellet and supernatant.
[0014] The calcium content in the control pellet remained similar between the 2-hour and 24-hour reaction periods.
[0015] In pellets obtained with β-fetuin and peptide, the amount of precipitated calcium was less than 1% after 2 hours of incubation, but increased to over 2% after 24 hours of incubation, showing a function of time. This was still significantly lower than the control without peptide or β-fetuin (26% after 2 hours, 28% after 24 hours).
[0016] Phosphate content in the pellets increased from 2% to 14% of the control pellets over a period of 2 to 24 hours. Addition of β-fetuin or peptide significantly reduced the phosphate content to 9% after 24 hours.
[0017] Finally, when the total protein content was measured, it was confirmed that both β-fetuin and the peptide were incorporated into the pellet. Up to 4% β-fetuin and 22% peptide were incorporated into the pellet. Taken together, these analyses demonstrate that both β-fetuin and the peptide shown in SEQ ID NO: 1 are incorporated into the calcium phosphate particles, altering their morphology and composition.
[0018] To investigate whether this peptide exhibits anti-calcification activity in vivo, its effects were tested in three different well-established cellular calcification models: the ATP-induced human articular chondrocyte calcification model (HAC), the calcium-induced vascular smooth muscle cell (VSMC) calcification model, and the β-glycerophosphate-induced bone marrow-derived mesenchymal stem cell (BMSC) osteogenic differentiation model (Examples 6, (21-23)).
[0019] After stimulating chondrocytes with ATP for 7 days, the inhibition of calcification by peptides was 22-33% compared to the control group (defined here as the maximum precipitate obtained in the presence of ATP) (Figure 3A).
[0020] In a 7-day VSMC calcification model, the peptide inhibited calcification by 58-61% (Figure 3B).
[0021] Finally, in the 3-week BMSC differentiation model, the peptide showed a strong inhibition of calcium precipitation (81%) (Figure 3C). The effect of peptide treatment was clearly confirmed in phase-contrast microscopy images (Figure 3C).
[0022] In conclusion, the peptide inhibits in vivo calcification in three different cell models.
[0023] Also, in a rat osteoarthritis model, the effect of injecting the peptide intra-articularly every other week was evaluated (Figure 4). Osteoarthritis was induced by surgical transection of the medial meniscus and transection of the collateral ligament (N = 40). 50 μl of a 20 μM peptide or 50 μl of vehicle (0.9% NaCl) was injected intra-articularly every other week (N = 20 per group). Necropsy was performed on day 28, and histological analysis was performed according to the OARSI scoring system (24).
[0024] On day 28 after surgery, in the knee joints of rats treated with the peptide, the histopathological OA score was significantly reduced by 38% compared to the group treated with the vehicle (Figure 4B). Most notably, there was a large and significant improvement in the walking score after peptide administration, with 16 / 20 showing normal walking, compared to only 5 / 20 in the vehicle control administration group (Figure 4C).
[0025] In summary, in a rat osteoarthritis model, it was found that intra-articular injection of the peptide improved the degree of the osteoarthritis condition and enhanced the locomotor ability of the animals.
Example
[0026] Example 1: Peptide synthesis The lyophilized peptide was synthesized by PepScan (NL). Batches were produced with a purity of over 90%. Purification was performed by pHPLC, and the mass and UV profiles were evaluated by MS-UPLC analysis (C18 RP-HPLC column).
[0027] Example 2: In vitro calcium phosphate precipitation assay The calcium precipitation assay was performed essentially the same as before, with some modifications (19). 2.4 mM ionized calcium (0.1 M stock) was added to 50 mM Tris / HCl buffer (pH 7.4) in a 1.5 ml Eppendorf tube. Peptide or β-fetuin was added and incubated at room temperature for 15 minutes. Then, 1.6 mM phosphate buffer (0.1 M stock) was added and the mixture was incubated at 37°C for 120 minutes. To ensure that timing did not affect the results, a positive control without peptide or β-fetuin was prepared twice, at the start and end of the series.
[0028] Data were normalized in each experiment against the first positive control (normalized to 100%). Bovine fetuin (β-fetuin, Sigma-Aldrich, #F2379) at concentrations of 1 mg / ml, 0.5 mg / ml, and 0.25 mg / ml was used as an internal reference for precipitation inhibition in each experiment. After 2 hours, samples were transferred to cuvettes (1.0 ml reaction solution), and absorbance was measured using a plate reader (Biorad) with an A620. Each condition consisted of three biological replicates.
[0029] Example 3: Scanning Electron Microscope Two hours after extracorporeal calcium phosphate precipitation, the sample was transferred to a dialysis membrane to stop the reaction. After overnight dialysis, the sample was freeze-dried, mounted on a stub, and gold-coated to enhance contrast before SEM examination (25). Images were obtained at 10.0 kV, 0.40 nA.
[0030] Example 4: Calcium assay The deposited calcium was quantified by hydrolyzing the deposited minerals with 0.1 M HCl, followed by the use of a calcium quantification kit (Randox, London, UK) according to the manufacturer's instructions. The calcium values were normalized to protein content using the micro DC Protein Assay (Termo Scientifc, Braiswijk, Netherlands). To enable the measurement of protein content, the acid was neutralized with an equal amount of 0.1 M NaOH, and cells were lysed using a cell calcification assay with 0.1% SDS (final concentration).
[0031] Example 5: Phosphate assay The deposited phosphate was quantified by solubilizing the deposit with 0.1 M HCl, followed by a phosphate colorimetric assay (Sigma, MK030) according to the manufacturer's instructions.
[0032] Example 6: Cellular calcification model Human primary smooth muscle cells (VSMCs) were obtained from tissue excisions of patients who underwent surgery. The tissue was cleaved into ±5 mm² sections and isolated using the previous method (26). Briefly, the cells were cultured in M199 with 20% FCS and 1% Pen / Strep (Gibco). Successful isolation was determined by immunofluorescence staining for positive expression of α-smooth muscle actin (αSMA), smooth muscle protein 22α (SM22α), phosphorylated myosin light chain 2 (pMLC), and the absence of S100 C calcium-binding protein 4 (S100A4). Cells from passages 5–8 were used in the experiment, and 1 cm² was used. 2 10,000 cells were seeded per cell. After 24 hours, the culture medium was changed to calcification medium (growth medium with 5.4 mM calcium, or growth medium with peptide and 5.4 mM calcium). The medium was refreshed every 2 or 3 days until calcification was visually confirmed on day 7.
[0033] Human primary chondrocytes (HACs) were isolated as residual material from the articular cartilage of patients undergoing total knee arthroplasty in end-stage osteoarthritis patients, after written informed consent (METC2017-0183). Cells were cultured in DMEM / F12 supplemented with 10% FCS, 1% Pen / Strep (Gibco), and 1% non-essential amino acids (Gibco). Cells from passages 3-5 were used in experiments, and 1 cm³ was used. 2 Approximately 30,000 cells were seeded for the experiment. After 24 hours, the culture medium was changed to calcification medium (growth medium supplemented with 1 mM ATP, or growth medium supplemented with the indicated concentration of peptide and 1 mM ATP). The medium was refreshed every 2 or 3 days until calcification was visually confirmed on day 7.
[0034] Bone marrow-derived stromal cells (BMSCs) were isolated from human bone marrow aspirates collected from the iliac crest of young individuals (METC08-4-056). Cells were cultured in DMEM high glucose (Thermo Fisher) supplemented with 10% FCS and 1% Pen / strep (Gibco). Osteogenesis was performed in a medium supplemented with 50 μg / ml ascorbic acid, 100 nM dexamethasone, and 10 mM β-glycerophosphate. The medium was refreshed every 2 or 3 days until calcification was visually confirmed on day 21.
[0035] Example 7: Rat model of osteoarthritis Lewis rats (n=40) underwent surgery for medial meniscus tear and collateral ligament transection of the right knee joint (27). 50 μl of vehicle (0.9% NaCl) (n=20) or the peptide shown in Sequence ID No. 1 (20 μM; n=20) was injected intra-articularly on postoperative days 7, 10, 14, 17, 21, and 24. Gait analysis was performed on day 20, and autopsy was performed on postoperative day 28. Tissue sections of the knee joint were decalcified and paraffin-embedded, stained with toluidine blue (28), and histologically scored (24). Experiments were conducted at Bolder Biopath (Bolder, USA), and tissue sections were stained. Example 8: Statistical Analysis
[0036] A two-sided Student's t-test was used to compare two groups. Data from the precipitation assay were assumed to be normally distributed. For comparisons of multiple groups, one-way ANOVA with Dunnett's post-hoc test or Bonferroni post-hoc test was used. For the rat OA model, a two-sided Student's t-test (D'Agostino-Pearson normality test) was used for normally distributed data, and the Mann-Whitney U test was used for non-normally distributed data to compare between groups. Statistical analysis was performed using Graphpad Prism 8.
[0037] [Table 1-1] [Table 1-2] [Table 1-3]
[0038] [Table 2]
Claims
1. A cyclic polypeptide consisting of the amino acid sequence shown in Sequence ID No. 1, where the amino acids are D-amino acids.
2. A pharmaceutical composition comprising a cyclic polypeptide as described in claim 1 and a pharmaceutically acceptable carrier.
3. A polypeptide according to claim 1 or a pharmaceutical composition according to claim 2 for the treatment of a disease selected from the group consisting of osteoarthritis, periarthritis of the shoulder, heterotopic ossification, vascular calcification, kidney stones, and calcification.