PROTEIN FOR TREATMENT OF INFLAMMATORY DISEASES
20220047671 · 2022-02-17
Assignee
Inventors
Cpc classification
A61P29/00
HUMAN NECESSITIES
A61K38/16
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to a lectin for use in the treatment, reduction in progression and curing of inflammation in a subject in need thereof. Pharmaceutical compositions comprising said lectin are also described. The lectin may comprise an amino acid sequence having sequence of SEQ ID NO: 1 or at least 60% homology to SEQ ID NO: 1.
Claims
1. A lectin protein for use in the treatment or prevention of inflammation in a subject in need thereof.
2. The lectin protein for use as claimed in claim 1, wherein the lectin protein comprises an amino acid sequence having at least 60% homology to SEQ ID NO. 1.
3. The lectin protein for use as claim in claim 1 or 2, wherein the lectin protein comprises an amino acid sequence having at least 60% homology to SEQ ID NO: 2, 3, or 4.
4. The lectin protein for use as claimed in claim 2, wherein the lectin protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4.
5. The lectin protein for use as claimed in any preceding claim, wherein the lectin protein is recombinant.
6. The lectin protein for use as claimed in any one of claims 2 to 4, wherein the amino acid sequence has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO. 1.
7. The lectin protein for use as claimed in any preceding claim, wherein the lectin protein is capable of inhibiting the secretion of inflammatory cytokines.
8. The lectin protein for use as claimed in any preceding claim, wherein the lectin protein is capable of reducing C-reactive protein (CRP) levels in blood.
9. The lectin protein for use as claimed in any preceding claim, wherein the lectin protein is capable of stimulating ACP activity and/or collagen levels.
10. The lectin protein for use as claimed in any preceding claim, wherein the subject has a chronic inflammatory disease.
11. The lectin protein for use as claimed in claim 9, wherein the chronic inflammatory disease is psoriasis, psoriatic arthritis, rheumatoid arthritis (RA), hepatitis, asthma, inflammatory bowel disease (including crohn's disease, colitis and irritable bowel syndrome (IBS)), temporal arteritis, atopic dermatitis, systemic lupus, multiple sclerosis or sarcoidosis.
12. The lectin protein for use as claimed in claim 10, wherein the chronic inflammatory disease is rheumatoid arthritis (RA).
13. The lectin protein for use as claimed in one of claims 1 to 8, wherein the subject has acute inflammation.
14. The lectin protein for use as claimed in claim 12, wherein the acute inflammation is caused by an allergic reaction, an infection or trauma.
15. The lectin protein for use as claimed in any preceding claim, wherein said treatment comprises curing the inflammation or the chronic inflammatory disease, or reducing symptoms associated with the inflammation.
16. The lectin protein for use as claimed in any preceding claim, wherein said prevention comprises preventing the onset of the inflammation, or preventing or slowing the progression of the chronic inflammatory disease.
17. The lectin protein for use as claimed in any preceding claim, wherein said treatment or prevention comprises administering the lectin protein at a dose of 1 to 50 mg/kg.
18. A pharmaceutical composition comprising a lectin protein and a pharmaceutically acceptable diluent or excipient.
19. A method of treatment or prevention of inflammation in a subject, the method comprising administering a lectin protein or the pharmaceutical composition of claim 18 to the subject.
20. The method as claimed in claim 19, wherein the inflammation is an inflammatory disease.
21. The method as claimed in claim 20, wherein the inflammatory disease is arthritis.
22. The method as claimed in one of claims 19 to 21, wherein the lectin protein is a recombinant lectin.
23. The method as claimed in one of claims 19 to 22, wherein the method comprises administering the lectin protein at a dose of 1 to 50 mg/kg.
Description
DESCRIPTION OF DRAWINGS AND TABLES
[0111] The invention will now be described by reference to the following Figures and Tables, in which:
[0112]
[0113]
[0114]
[0115]
[0116] Table 1: Effect of a recombinant lectin on cytokine secretion in a human synovial cell line (SW982) after 24 hours;
[0117] Table 2. Effect of a recombinant lectin on cytokine secretion in murine splenocytes after 48 hours;
[0118] Table 3. Effect of SEQ ID NO:2 on cytokine secretion in a murine macrophage cell line (RAW264.7) after 24 hours; and
[0119] Table 4. Effect of SEQ ID NO:2 on ALP activity and collagen levels in an osteoblast cell line (MG-63) after 5 days.
[0120] Inflammatory diseases are diseases caused by the impairment of immune system. Inflammatory diseases can be chronic inflammatory disease or acute inflammatory disease. Inflammatory diseases include arthritis, psoriasis, temporal arteritis, dermatitis, inflammatory bowel disease (IBS), systemic lupus, hepatitis, asthma, allergic reactions, sarcoidosis and similar diseases as understood by the person skilled in the art.
[0121] Key signaling pathways which play an important role in the pathogenesis of inflammation are MAPK/EGFR/Ras/Raf, Cannabinoid receptor 2 (CNR2), IL-4/STAT, PI3K/AKT/FOXO3, TNF-α/JNK, NLRP3 and NLRP1, PTPN22, PADI4, STAT4, TRAF1-05 and TNFAIP3, MyD88, IRAKs, and TRAF6.
[0122] The present invention relates to the treatment of inflammatory diseases using a lectin, such as lectin having the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4. The invention also relates to a method for the prevention of, the onset of or the progression of inflammatory diseases in the subject using a lectin, such as a lectin having the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4. It further relates to a method to cure or reduce the effect of inflammatory diseases in the subject using a lectin, such as a lectin having the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4.
[0123] The present invention relates to the method of treatment of arthritis in a subject using a lectin having an amino acid sequence of SEQ ID NO:1 or its homologues. The invention further relates to a lectin having an amino acid sequence of SEQ ID NO: 1 or its homologues used in the treatment of arthritis in the subject. The invention also provides a method for the prevention of onset of or progression of arthritis in the subject using a recombinant lectin having the amino acid sequence of SEQ ID NO:1 or its homologues. Further the invention relates to a method to cure or reduce the effect of arthritis in a subject using a recombinant lectin having the amino acid sequence of SEQ ID NO:1 or its homologues.
[0124] The applicant's earlier patent application, 350/MUM/2009, discloses a recombinant lectin with the amino acid sequence of SEQ ID NO: 1 having high binding specificity toward the carbohydrate antigen (Galβ1-3GalNAc-α-O-Ser/Thr). Lectins are generally known to cause inflammation. However the present inventors, to their surprise, have found that the lectin with the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4 is anti-inflammatory. The detailed study confirmed the efficacy of recombinant lectin with the amino acid sequence of SEQ ID NO: 2 towards arthritis. The novel and inventive aspect of the present invention is the use of a lectin, such as the recombinant lectin with the amino acid sequence of SEQ ID NO: 2 in the prevention, cure or reduction of proliferation of inflammation, such as that causing arthritis.
[0125] The effect of a recombinant lectin having the sequence of SEQ ID NO:2 on the modulation of key signaling pathways which play an important role in the pathogenesis of inflammation was evaluated in cell based assays. A recombinant lectin having the amino acid sequence of SEQ ID NO: 2 showed inhibition of signaling pathways which are key pathways in the pathogenesis of inflammation related to arthritis. These results, detailed below, suggest the possible efficacious effect of lectins, such as the recombinant lectin having the amino acid sequence of SEQ ID NO: 2 in inflammation related to arthritis.
[0126] In vitro studies of a recombinant lectin having the amino acid sequence of SEQ ID NO: 2 demonstrated strong anti-inflammatory activity by inhibiting pro-inflammatory cytokines and inhibition of signaling pathways which play a key role in the pathogenesis of inflammation. It also stimulated ALP activity and collagen levels in human osteoblast cells supporting its anti-inflammatory potential.
[0127] Synovial fibroblasts produce excessive levels of inflammatory cytokines which destroy cartilage and bone in inflamed arthritic joint. A human synoviocyte cell line is characterized by expression of inflammatory cytokines. In-vitro cell based assays on human synoviocyte cell lines were performed using a recombinant lectin having the amino acid sequence of SEQ ID NO: 2 to assess its effect on the secretion of inflammatory cytokines such as interleukins, interferons, granulocyte-macrophage colony-stimulating factor, (GM-CSF), PI3Kδ/γ and TNF-α. against IFN-γ stimulation. The effect of using a recombinant lectin having the amino acid SEQ ID NO:2 in an in-vitro cell based LPS induction assay on the immune cells murine splenocytes and an in-vitro cell based IFN-γ stimulation assay on the murine macrophage cell line RAW264.7 were also assessed. A recombinant lectin having the amino acid sequence of SEQ ID NO: 2 significantly inhibited the secretion of inflammatory cytokines. The secretion of IL-8 and IL-6 was inhibited by 10% to 30% and 20% to 50%, respectively, in synovial cells against interferon gamma (IFN-γ) stimulation (Table 1). Similarly, a recombinant lectin having the amino acid sequence of SEQ ID NO: 2 inhibited the secretion of TNF-α and IL-1-β by 15 to 35% and 20 to 32% respectively in splenocytes stimulated with LPS (Table 2). The inhibition of the secretion of TNF-α in a murine macrophage (RAW264.7) cell line stimulated with IFN-γ was from 5 to 30% (Table 3). The results demonstrated that a recombinant lectin having the amino acid sequence of SEQ ID NO: 2 inhibits key inflammatory cytokines in a synovial cell line as well as immune cells.
[0128] A recombinant lectin having the amino acid sequence of SEQ ID NO: 2 was assessed for its effect on the stimulation of bone markers such as Alkaline Phosphatase (ALP) activity and collagen levels in a human osteoblast cell line. A recombinant lectin having the amino acid sequence of SEQ ID NO: 2 stimulated ALP activity in osteoblasts by 10% to 45% with respect to the control. A recombinant lectin having the amino acid sequence of SEQ ID NO. 2 also stimulated collagen levels by 3% to 50% as compared to the control (Table 4).
[0129] A recombinant lectin having the amino acid sequence of SEQ ID NO: 2 was also tested for its effect on the inhibition of inflammatory cytokines in an in vivo study using an LPS-induced cytokine release mouse model. The mice were treated with lipopolysaccharide (LPS) alone (50 μg/animal) and LPS (50 μg/animal) along with a recombinant lectin having the amino acid sequence of SEQ ID NO: 2(5 to 50 mg/kg body weight). The dosing of recombinant lectin having the sequence of SEQ ID NO: 2 was done 1 hour before the injection of LPS. Normal control animals received TBS (tris-buffered saline), at the dose volume of 5 mL/kg. All doses were administered intraperitoneally. Excessive secretion of TNF-α (675.7±76.6 pg/mL) and IL-6 (3256.0±203.1 pg/mL) was observed in the blood serum when LPS alone was injected. The recombinant lectin having the amino acid sequence of SEQ ID NO: 2 significantly inhibited the secretion of cytokines (
[0130] The effect of the recombinant lectin having the sequence of SEQ ID NO: 2 was further tested on an adjuvant induced arthritis (MA) model in rats. Animals were injected with a recombinant lectin having the amino acid sequence of SEQ ID NO: 2 intraperitoneally at a concentration of 20 mg/kg of body weight for 21 days (from day 8 to 28 after adjuvant injection). Treatment with the recombinant lectin having the amino acid sequence of SEQ ID NO: 2 significantly reduced paw and ankle thickness compared to vehicle-treated adjuvant-induced arthritis animals (
[0131] The method may comprise administration of a lectin having the amino acid sequence of SEQ ID NO:1 or its homologuesin the range of about 1 mg to 50 mg/kg body weight of the subject, wherein the recombinant lectin having the amino acid sequence of SEQ ID NO:1 is administered as such or in the pharmaceutically acceptable form.
[0132] A preferred range of a recombinant lectin having an amino acid sequence of SEQ ID NO: for its homologues is from about 5 mg to 30 mg/kg body weight of the subject. A most preferred range of a recombinant lectin having an amino acid sequence of SEQ ID NO: 1 or its homologues is from about 5 mg to 20 mg/kg body weight of the subject.
[0133] An anti-arthritic effect of a recombinant lectin having an amino acid sequence of SEQ ID 2 is at least 10% to up to 100%.
EXAMPLES
[0134] The following examples are given to demonstrate the best mode of performance of the invention. Examples do not limit the invention in any manner.
Example 1
[0135] A study was conducted to assess the effect of a recombinant lectin having the amino acid sequence of SEQ ID NO:2 on the secretion of inflammatory cytokines (IL-8 and IL-6) in a human synoviocyte cell line (SW982). The recombinant lectin showed inhibition of the secretion of the inflammatory cytokines from the cell line.
[0136] Human synoviocyte (SW982) cells were first cultured for 24 hours and sera starved for 6 hours, followed by treatment of the cells with the recombinant lectin having the amino acid sequence of SEQ ID NO: 2 at non-cytotoxic concentrations (1 ng/ml-1000 ng/ml) with the inflammatory stimulus (100 ng/ml IFN-γ) for another 24 hours. The levels of cytokines (IL-6 and IL-8) in supernatants were determined by ELISA. Inhibitory effect of recombinant lectin on secretion of cytokine was calculated as follows:
% inhibition of cytokines=[(A−B)/A]*100
Where,
[0137] A=concentration of cytokine in control cells (IFN-γ alone)
B=concentration of cytokine in treated cells (recombinant lectin+IFN-γ)
[0138] It was seen that the recombinant lectin having the amino acid sequence of SEQ ID NO: 2 inhibited IL-8 secretion from synovial cells (SW982) against IFN-γ stimulation (11.2%-27.9%) and inhibited IL-6 secretion from synovial cells (SW982) against IFN-γ stimulation (23.7%-49.4%).
Example 2
[0139] Another study was conducted to determine the effect of a recombinant lectin having the amino acid sequence of SEQ ID NO: 2 on the secretion of inflammatory cytokines (TNF-α and IL-1-β) from immune cells (murine splenocytes and the murine macrophage cell line (RAW264.7)).
[0140] Splenocytes were isolated from C57BL/6 mice and treated with a recombinant lectin having the amino acid sequence of SEQ ID NO: 2 at non-cytotoxic concentrations (1 ng/ml-1000 ng/ml) along with 25 ng/ml of LPS for 48 hours.
[0141] RAW264.7 cells were cultured for 24 hours and sera starved for another 24 hours followed by co-treatment of the cells with a recombinant lectin having the amino acid sequence of SEQ ID NO: 2 at non-cytotoxic concentrations (1 ng/ml-500 ng/ml) and the inflammatory stimulus IFN-γ (100 ng/ml) for 24 hours. Levels of cytokines (TNF-α and IL-1-β) in supernatants were determined by ELISA.
[0142] Inhibitory effect of recombinant lectin on secretion of cytokine was calculated as follows:
% inhibition of cytokines=[(A−B)/A]*100
Where,
[0143] A=concentration of cytokine in control cells (LPS or IFN-γ alone)
B=concentration of cytokine in treated cells (recombinant lectin+LPS or IFN-γ)
[0144] It was observed that in murine splenocytes, the recombinant lectin having the amino acid sequence of SEQ ID NO: 2 demonstrated the inhibition of the secretion of cytokines from splenocytes against LPS stimulation (TNF-α by 17.2%-32.9% and IL-1-β by 23.5%-30.4%).
[0145] In murine macrophages (RAW264.7), the recombinant lectin having the amino acid sequence of SEQ ID NO: 2 inhibited TNF-α secretion from macrophages (RAW264.7) against LPS stimulation (6.1%-26.5%)
Example 3
[0146] Another study was conducted to determine the effect of a recombinant lectin having the amino acid sequence of SEQ ID NO: 2 on the bone markers (ALP & Collagen) in a human osteoblasts cell line (MG-63). First, MG-63 cells were cultured for 24 hours and sera starved for another 24 hours followed by treatment of the cells with a recombinant lectin having the amino acid sequence of SEQ ID NO: 2 at non-cytotoxic concentrations (1 ng/ml-1000 ng/ml) for 5 days. ALP activity in cell lysates was determined by a PnPP based assay. Collagen levels in cell lysates were determined by a Sircoll dye based assay. The recombinant lectin having the amino acid sequence of SEQ ID NO: 2 increased ALP activity (11%-42.8%) and collagen levels (3.2%-45.3%) in the human osteoblasts cell line (MG-63).
Example 4
[0147] Another study was conducted to consider the effect of a recombinant lectin having the amino acid sequence of SEQ ID NO: 2 on key signaling pathways which play an important role in the pathogenesis of Rheumatoid Arthritis.
[0148] The effect of a recombinant lectin having the amino acid sequence of SEQ ID NO: 2 on the modulation of key signaling pathways was determined in cell based assays using Geneblazer and Tango technologies.
[0149] It was seen that the recombinant lectin having the amino acid sequence of SEQ ID NO: 2 inhibited the following pathways: MAPK/EGFR/Ras/Raf, Cannabinoid receptor 2 (CNR2), IL-4/STAT, PI3K/AKT/FOXO3 and TNF-α/JNK, further strengthening the suggested role of the lectin having the amino acid sequence of SEQ ID NO: 1 or its homologuesin its favor in the case of rheumatoid arthritis.
In Vivo Experiments
Example 5
[0150] Another study was conducted to check the effect of a recombinant lectin having the amino acid sequence of SEQ ID NO: 2 on inflammatory cytokines (TNF-α and IL6) in an LPS-induced cytokine release model in mice.
[0151] 30 female C56BL6 mice were randomized based on body weight and assigned to five groups (n=6). For groups receiving LPS (G2), a single dose of 50 μg/animal of LPS was injected intraperitoneally (IP). For groups receiving a recombinant lectin having the amino acid sequence of SEQ ID NO: 2 at 10 & 20 mg/kg+LPS, (G3 & G4) a single IP injection of 10 & 20 mg/kg of the recombinant lectin having the amino acid sequence of SEQ ID NO: 2 was administered 1 hr before the injection of LPS. For a group of Dexamethasone (G5), 1 mg/kg+LPS, a single IP injection of 1 mg/kg of dexamethasone was administered 1 hr min before injection of the LPS. Normal control animals (G1) received TBS intraperitoneally at the dose volume of 5 mL/kg.
[0152] Blood was collected 90 min after treatment with LPS and the blood was centrifuged for the collection of serum. The serum samples were stored at −80° C. Serum TNF-α and IL-6 were measured using an ELISA kit.
Serum Cytokine Levels
[0153] TNF-α was 102.3±9.2 pg/mL in the serum of TBS treated mice, while the IL-6 concentration in the serum of TBS-treated mice was 204.0±14.3 pg/mL.
[0154] A significant increase in the serum level of TNF-α was observed in the LPS group at 90 minutes to an average of 675.7±76.6 pg/mL. This was inhibited by the recombinant lectin having the amino acid sequence of SEQ ID NO: 2 to 566.2±52.3 and 471.8±48.3 pg/mL for 10 mg/kg and 20 mg/kg respectively, giving a percentage reduction of 16.2% and 30.2%, respectively.
[0155] A significant increase in the serum level of IL-6 was observed in the LPS group at 90 minutes to an average of 3256.0±203.1 pg/mL. This was inhibited by the recombinant lectin having the amino acid sequence of SEQ ID NO: 2 to 2601.3±268.2 and 1866.2±139.3 pg/mL for 10 mg/kg and 20 mg/kg, respectively, giving a percentage reduction of 20.1% and 42.7%, respectively.
[0156] In short, it is demonstrated that the lectin having the amino acid sequence of SEQ ID NO:1 or its homologueshas a promising anti-inflammatory activity due to its ability to suppress the pro-inflammatory cytokines, TNF-α and IL-6 in an LPS-induced mouse inflammation model.
Example 6
[0157] Another study was conducted to see the effect of a recombinant lectin having the amino acid sequence of SEQ ID NO:2 on an adjuvant induced-arthritis model in rats.
[0158] Adjuvant arthritis was induced by the injection of 100 μL (0.5 mg) of Mycobacterium butyricum desiccated—Complete Freund's Adjuvant-CFA (Chondrex) into the left hind paw (Day 0) of 34 rats. On day 0 of the experiment, 24 animals were selected, randomized and assigned to four groups of 6 animals each.
[0159] The groups were as follows:
G1: Normal Control
[0160] G2: Adjuvant induced arthritis control (MA)+TBS
G3: AIA+10 mg/Kg recombinant lectin
G4: AIA+20 mg/Kg recombinant lectin
G5: AIA+0.5 mg/kg dexamethasone
[0161] A recombinant lectin having the amino acid sequence of SEQ ID NO:2 compound was administered from days 8 to 28 to two groups (G3 and G4) after adjuvant injection, a total of 21 days treatment. Control groups were administered the following. Normal control animals (G1, n=6) were injected with 100 μL of paraffin oil into the left hind paw on day 0 and then dosed on days 8 to 28 with Tris Buffer Saline, 5 mL/kg, IP. Another control group received Complete Freund's Adjuvant (CFA) on day 0 followed by the intraperitoneal injection of Tris Buffer Saline on days 8 to 28 (G2). For the groups receiving the recombinant lectin having the amino acid sequence of SEQ ID NO:2, IP injection of 10 & 20 mg/kg of the drug (G3 & G4 respectively), was administered daily for 21 days (days 8 to 28). For the group of Dexamethasone (G5), an IP injection of 0.5 mg/kg of dexamethasone was administered daily for 21 days (days 8 to 28) following CFA on day 0.
[0162] Paw thickness was measured by a digimatic micrometer (Mitutoyo). The angle of the ankle joint by degree was also recorded. On day 28 after adjuvant injection and day 21 post treatment, whole blood was collected from the animals and centrifuged to separate serum. The serum samples were stored at −80° C. Serum Uric acid, C-Reactive Protein (CRP) and IL-6 was measured using kits mentioned below.
TABLE-US-00005 Name of Kit Manufacturer Catalogue no. Rat C-Reactive CUSABIO CSB-E07922r Protein, CRP ELISA Kit Rat Interleukin 6, IL-6 CUSABIO CSB-E04640r ELISA KIT Rat Uric Acid (UA) MYBIOSOURCE MBS9346935 ELISA Kit
Assessment of Joint Destruction (Radiographic Evaluation):
[0163] On the day of termination (day 28), the hind limb was sectioned below the hip joint. Radiographs were taken of the hind limb. The severity of bone damage on the radiographs was assessed by the grading of periostitis and bone destruction.
[0164] Radiography of the hind limb from the vehicle-treated adjuvant-induced arthritis group (G2), showed severe disorganization (periostitis and destruction) of bone in the distal tibia, tarsus, metatarsus, and calcaneus. 21 days of intraperitoneal injection of 20 mg/kg a recombinant lectin having the amino acid sequence of SEQ ID NO: 2, moderately ameliorated this periostitis and bone destruction (G4) whereas 10 mg/kg of a recombinant lectin having the amino acid sequence of SEQ ID NO: 2 showed a palpable effect (G3). Dexamethasone 0.5 mg/kg, IP showed a significant amelioration (G5).
Effect of a Recombinant Lectin Having the Amino Acid Sequence of SEQ ID NO: 2 on Ankle Bone Radiography in Adjuvant-Induced Rat Model of Arthritis
[0165] Ankle joint radiography from the vehicle-treated adjuvant-induced arthritis group (G2) showed the severe destruction of bone when compared to normal animals (G1). 21 days of an IP injection of 10 mg/kg and 20 mg/kg (G3 and G4) of a recombinant lectin having the amino acid sequence of SEQ ID NO: 2, mild to moderately ameliorated this bone destruction. Dexamethasone 0.5 mg/kg, IP showed significant amelioration (G5).
Effect of a Recombinant Lectin Having the Amino Acid Sequence of SEQ ID NO: 2 on Joint Angle (Joint Mobility) in an Adjuvant-Induced Rat Model of Arthritis
[0166] The vehicle-treated adjuvant-induced arthritis group (G2) showed a significant decrease in joint angle when compare to the normal control group (G1). 21 days of an intraperitoneal injection of 10 mg/kg and 20 mg/kg of a recombinant lectin having the amino acid sequence of SEQ ID NO: 2(G3 & G4), dose dependently increased the joint mobility with a percentage improvement of 7.9% and 34.2%, respectively. Dexamethasone 0.5 mg/kg, showed significant amelioration (G5) with 61.8%.
Inhibitory Effects of a Recombinant Lectin Having the Amino Acid Sequence of SEQ ID NO: 2 on Paw and Ankle Thickness in an Adjuvant-Induced Rat Model of Arthritis
[0167] The vehicle-treated adjuvant-induced arthritis group (G2) showed a significant increase in paw and ankle thickness. 21 days of intraperitoneal injection of 20 mg/kg of a recombinant lectin having the amino acid sequence of SEQ ID NO: 2, significantly reduced paw and ankle swelling (G4), whereas 10 mg/kg showed a poor effect (G3). Dexamethasone 0.5 mg/kg, IP, showed significant amelioration (G5).
Effect of a Recombinant Lectin Having the Amino Acid Sequence of SEQ ID NO: 2 on Serum Uric Acid Levels in an Adjuvant-Induced Rat Model of Arthritis
[0168] Dexamethasone decreased the level of serum uric acid significantly (G5) in adjuvant-induced rats with arthritis. 21 days of intraperitoneal injection of a recombinant lectin having the amino acid sequence of SEQ ID NO: 2 at 10 mg/kg and 20 mg/kg dose (G3 and G4) dependently decreased the uric acid level with a percentage reduction of 11.0% and 22.0%, respectively.
Effect of a Recombinant Lectin Having the Amino Acid Sequence of SEQ ID NO: 2 on Serum IL-6 and CRP Levels in an Adjuvant-Induced Rat Model of Arthritis
[0169] C-Reactive Protein (CRP) was 290.6±15.5 pg/mL in the serum of Tris Buffered Saline (TBS) treated rats (G1), while the IL-6 concentration in the serum of TBS treated rats was 265.8±22.7 pg/mL. A significant increase in the serum level of IL-6 was observed in the arthritis group (G2) to an average of 6989.2±475.2 pg/mL. This was inhibited by a recombinant lectin having the amino acid sequence of SEQ ID NO: 2 to 6219.8±359.8 and 5393.6±420.4 pg/mL for 10 mg/kg and 20 mg/kg dosages respectively (G3 and G4), giving a percentage reduction of 11.0% and 22.8%, respectively.
[0170] A significant increase in the serum level of CRP was observed in the Arthritis group (G2) to an average of 748.2±21.8 pg/mL. This was inhibited by a recombinant lectin having the amino acid sequence of SEQ ID NO: 2 to 726.4±36.6 and 523.3±110.5 pg/mL for 10 mg/kg and 20 mg/kg (G3 and G4), respectively, giving a percentage reduction of 2.9% and 30.1%, respectively.
Effect of a Recombinant Lectin Having the Amino Acid Sequence of SEQ ID NO: 2 and Dexamethasone on Thymus and Spleen Weight in an Adjuvant-Induced Rat Model of Arthritis
[0171] The vehicle-treated adjuvant-induced arthritis group (G2) showed a significant decrease in thymus weight when compared to the normal control group (G1). 21 days of intraperitoneal injection of 10 mg/kg and 20 mg/kg of a recombinant lectin having the amino acid sequence of SEQ ID NO: 2(G3 & G4), showed 14.48% gain and 3.98% loss, respectively compared to the G2 group. Dexamethasone 0.5 mg/kg, IP, showed a considerable reduction (i.e. 19.33%) in thymus weight compared to the G2 group.
[0172] The vehicle-treated adjuvant-induced arthritis group (G2) showed a marginal increase in spleen weight when compared to the normal control group (G1). 21 days of intraperitoneal injection of 10 mg/kg and 20 mg/kg of a recombinant lectin having the amino acid sequence of SEQ ID NO: 2(G3 & G4), showed a 40.55% and 12.17% increase, respectively compared to the G2 group. Dexamethasone 0.5 mg/kg, IP, showed a significant reduction (i.e. 51.57%) in spleen weight compared to the G2 group.
Effect of a Recombinant Lectin Having the Amino Acid Sequence of SEQ ID NO: 2 and Dexamethasone on Hematology in an Adjuvant-Induced Rat Model of Arthritis
[0173] The vehicle-treated adjuvant-induced arthritis group (G2) showed a moderate to significant alteration in all the tested hematological parameters when compared to the normal control group (G1). 21 days of intraperitoneal injection of 10 mg/kg and 20 mg/kg of a recombinant lectin having the amino acid sequence of SEQ ID NO: 2(G3 & G4), did not alter the change caused by arthritis. However, Dexamethasone 0.5 mg/kg, IP, showed moderate to significant changes (i.e. WBC-60.3% reduction, RBC-23.4% Increase, Hb-25.2% increase and HCT-22.4% increase) in hematology compared to the G2 group.
Effect of a Recombinant Lectin Having the Amino Acid Sequence of SEQ ID NO: 2 and Dexamethasone on Body Weight in an Adjuvant-Induced Rat Model of Arthritis
[0174] Recombinant lectin treated groups (G3 & G4) and the vehicle-treated adjuvant-induced arthritis group (G2), showed a similar trend to the normal control group (G1) in gaining body weight. However, the dexamethasone 0.5 mg/kg, IP group (G5) showed no gain in body weight throughout the treatment period.
TABLE-US-00006 TABLE 1 Inhibition of cytokines (%) Sample IL-8 IL-6 IFN-γ 100 ng/ml Control Control IFN-γ 100 ng/ml + SEQ ID NO: 2 (ng/ml) 1 22.4 46.3 10 27.9 49.4 250 14.9 31.9 500 11.2 23.7 1000 −40.8 −25.2
TABLE-US-00007 TABLE 2 Inhibition of cytokines (%) Sample TNF-α IL-1-β LPS 25 ng/ml Control Control LPS 25 ng/ml + SEQ ID NO: 2 (ng/ml) 1 24.8 30.4 10 18.6 24.4 25 29.2 23.5 100 32.9 27.6 500 26.1 26.7 1000 17.2 24.4
TABLE-US-00008 TABLE 3 Sample Inhibition of TNF-α (%) IFN-γ 100 ng/ml Control IFN-γ 100 ng/ml + SEQ ID NO: 2 (ng/ml) 1 26.5 10 6.1 100 11.7 500 11
TABLE-US-00009 TABLE 4 % increase in ALP activity wrt to % increase in Sample control collagen level Recombinant lectin 1 37.7 3.2 10 42.8 45.3 25 30.9 26.7 100 27.3 26.8 1000 11 13.5 Vitamin D3 (Positive control) 1 31.6 14 10 60.8 30.9