AU2021106679A4

AU2021106679A4 - Enzyme loaded navigated nanomatrix systems to the inflamed synovial locus for the treatment of rheumatoid arthritis.

Info

Publication number: AU2021106679A4
Application number: AU2021106679A
Authority: AU
Inventors: K. K. Sahu; Shikha Shrivastava; Deependra Singh; Manju Rawat Singh
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-08-23
Filing date: 2021-08-23
Publication date: 2021-12-02
Anticipated expiration: 2029-08-23

Abstract

: Enzyme loaded navigated nanomatrix systems to the inflamed synovial locus for the treatment of rheumatoid arthritis. This invention describes a method of synthesizing and testing a nanocarriers containing potent antioxidant enzymes superoxide dismutase (SOD) adsorbed for controlled delivery of enzymes at the site of severe inflammation to meet the therapeutic needs of rheumatoid arthritis patients. The Nanomatrix core (NM) of Polypropylene Sulphide is formulated by a modified emulsion ring-opening polymerization method.The prepared systems were then sugar-coated by cellobiose and lyophilized (CNM). Folate was conjugated to SOD (Fol-SOD) through linking agent 1-ethyl-3-(3-dimethyl aminopropyl) (FE) carbodiimide adsorbed over cellobiose coated system. Signatory Manju Rawat Singh Deependra Singh K.K.Sahu Shikha Slirivastava 1

Description

TITLE Enzyme loaded navigated nanomatrix systems to the inflamed synovial locus for the treatment of rheumatoid arthritis

FIELD OF INVENTION

[0001] This invention relates to the pharmaceutical and medical sciences more particularly a nanocarriers containing potent antioxidant enzymes superoxide dismutase (SOD) adsorbed for controlled delivery of enzymes at the site of severe inflammation to meet the therapeutic needs of rheumatoid arthritis patients.

PRIOR ART AND PROBLEM TO BE SOLVED

[0002] Worldwide, Rheumatoid arthritis (RA) is becoming one of the major health problems of society affecting all age groups majorly elderly ones, and women. The actual triggering factor for the cause of this particular autoimmune disease is still unknown. An imbalance between mitochondrial oxidative stress and total antioxidant potential majorly accelerates the cause of RA. The vitality of macrophages, leucocytes directly correlates with the severity of RA. During the progression of RA, macrophages are maximally reported at the site of inflammation and cartilage pannus junction. Reactive oxygen species (ROS) produced from inflammatory macrophages causing oxidative stress is becoming one of the major causative factors stimulating the breakdown of biological components, provoking an autoimmune reaction and inflammatory disorders. The accrual of free radicals provokes inflammatory interleukins, generates tumor necrosis factors, and mediates signal transduction and transcription pathways causing autoimmune disorders. Thus, the virulence of free radicals can be effectively attenuated by the use of potential antioxidant enzymes i.e. Superoxide Dismutase (SOD) and Catalase (CAT). SOD as a potent antioxidant enzyme easily converts free radicals to peroxides and CAT potentially converts peroxide to non-toxic molecules. Though being effective and potential, the delivery of these enzymes has been always being question marked

[0003] To resolve the above problem here propylene sulphide nanomatrix mimicking as catalase coated with cellobiose over which folate linked-SOD (FECNM) are adsorbed for a macrophage-targeting. The folic acid conjugated enzyme potentiates the activity of antioxidant enzymes SOD and CAT to scavenge ROS and navigates the active moiety to the targeted site at activated macrophages. The drug is targeted to the site for the delivery of the enzyme for the treatment of rheumatoid arthritis.

THE OBJECTIVES OF THE INVENTION:

[0004] Rheumatoid arthritis (RA) is a chronic diseases that is very costly to society, both in terms of reduced productivity and quality of life of the afflicted individuals, and in terms of the increased health care costs. The exact causes of RA and diabetes are unknown at present but appear to be multifactorial with both genetics (polygenic) and environmental factors playing important roles.

[0005] It has already been proposed wherein pharmaceuticals of various types are used with the object of controlling the inflammatory process and also to remedy the damage that the inflammatory process itself caused to the osteocartilaginous structures. Among these we have gold salts, antimalarials, penicillamine, and furthermore all the analgesic and steroid and nonsteroid anti-inflammatory pharmaceuticals. In particular, the symptomatic therapy is carried out by resorting to antiphlogistic drugs (nonsteroidal anti inflammatory agents and cortisone compounds) and the base therapy using synthesized antimalarials, penicillamine, azathioprine, cyclophosphamide, gold salts, etc All these therapies, however, along with undoubtedly beneficial effects, create, to different degrees, undesired side effects. Such effects become more evident in all those forms which, like rheumatoid arthritis, require continuous therapy and especially targeted and controlled delivery of medication to the site of inflammation.

[0006] The principal objective of the invention is the composition of Enzyme loaded navigated nanomatrix systems for treating rheumatoid arthritis.

[0007] Another objective of the invention is the preparation of propylene sulphide nanomatrix mimicking as catalase coated with cellobiose over which folate linked-SOD (FECNM) are adsorbed for a macrophage-targeting.

[0008] The further objective of the invention is a mechanism of controlled delivery of enzymes at the site of severe inflammation to meet the therapeutic needs of rheumatoid arthritis patients.

[0009] The further objective of the invention is the formultation of Nanomatrix core (NM) of polypropylene sulphide by a modified emulsion ring-opening polymerization.

SUMMARY OF THE INVENTION

[0010] There are a number of rheumatologic and neurologic disorders as well as clinical musculo-skeletal syndromes where free radicals play a primary or secondary role in the clinical signs and symptoms of these distinct entities. Exercise, whether as calisthenics, weight lifting, swimming, running or jogging, generates free radical species. Exercise may be followed by muscle strain and aches or sprains, or result in painful sport injuries. The most common diseases affecting joints are rheumatoid arthritis and osteoarthritis. The former is an autoimmune disease where the articular inflammation in part leads to the generation of free radicals causing further inflammation and damage to the lining (synovium) of the affected joints. Free radicals also arise in rheumatoid arthritis and the other autoimmune related diseases, as periarteritis, lupus and scleroderma, through the mechanism of ischemia reperfusion, similar to that in myocardial damage from coronary artery disease. The common syndromes of low back pain, fibrositis, and other neuro-muscular entities cause chronic pain from local inflammation. Thus, locally administered synergistic antioxidants play a role as adjuvant therapy alone or in combination with anti-inflammatory and analgesic medications, including topical capsaicin.So here for targeted deliver of enzymes, nanocarriers containing potent antioxidant enzymes superoxide dismutase (SOD) adsorbed for controlled delivery of enzymes at the site of severe inflammation to meet the therapeutic needs of rheumatoid arthritis patients.

DETAILED DESCRIPTION OF THE INVENTION

[0011] While the present invention is described herein by way of example, using various embodiments and illustrative drawings, those skilled in the art will recognize that the invention is neither intended to be limited to the embodiment of drawing nor drawings described nor designed to represent the scale of the various components. Further, some components that may form a part of the invention may not be illustrated with specific figures, for ease of illustration, and such omissions do not limit the embodiment outlined in any way. The drawings and a detailed description of it are not intended to restrict the invention to the form disclosed, but on the contrary, the invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention as defined by the appended claims. The headings are used for organizational purposes only and are not meant to limit the scope of the description or the claims. As used throughout this specification, the word "may" be used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning, must).

[0012] Further, the words "an" or "a" means "at least one" and the word "plurality" means one or more unless otherwise mentioned. Furthermore, the terminology and phraseology used herein are solely used for descriptive purposes and should not be construed as limiting in scope. Language such as "including," "comprising," "having," "containing," or "involving," and variations thereof, is intended to be broad and encompass the subject matter listed thereafter, equivalents and any additional subject matter not recited, and is not supposed to exclude any other additives, components, integers, or steps. Likewise, the term "comprising" is considered synonymous with the terms "including" or "containing" for applicable legal purposes. Any discussion of documents acts, materials, devices, articles, and the like are included in the specification solely to provide a context for the present invention.

[0013] In this disclosure, whenever an element or a group of elements is preceded with the transitional phrase "comprising", it is also understood that it contemplates the same element or group of elements with transitional phrases "consisting essentially of, "consisting", "selected from the group comprising", "including", or "is" preceding the recitation of the element or group of elements and vice versa.

[0014] Before explaining at least one embodiment of the invention in detail, it is to be understood that the present invention is not limited in its application to the details outlined in the following description or exemplified by the examples.

The invention is capable of other embodiments or of being practised or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for description and should not be regarded as limiting.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Besides, the descriptions, materials, methods, and examples are illustrative only and not intended to be limiting. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0016] The present invention provides a method of preparation and testing nanocarriers containing potent antioxidant enzymes superoxide dismutase (SOD) adsorbed for controlled delivery of enzymes at the site of severe inflammation to meet the therapeutic needs of rheumatoid arthritis patients.

[0017] In this embodiment the material required consists of Bovine collagen Type-II (Condrex), Complete Freund's adjuvant (Sigma), Incomplete Freund's adjuvant (Sigma), ELISA kits of IL-6, IL- and TNF-a, BSA (Himedia), Primary and secondary antibody (Invitrogen), Acetic acid and HCl from Loba Chemie, protease inhibitor cocktail tablet (Sigma).

[0018] Nanomatrix core (NM) of polypropylene sulphide was formulated by a modified emulsion ring-opening polymerization method as followed by Hu et al. Prepared systems were then sugar-coated by cellobiose and lyophilized (CNM). Folate was conjugated to SOD (Fol-SOD) through linking agent 1 ethyl-3-(3-dimethyl aminopropyl) (FE) carbodiimide adsorbed over cellobiose coated system.

[0019] For the testing of the nanomatrix, initially rats were induced with Arthritis. At 4°C collagen was overnight solubilized in 0.05 M acetic acid at a concentration of 2 mg/ml. The prepared solution was cleared by centrifugation, diluted when necessary with 0.1 M acetic acid, and emulsified with an equal volume of complete Freund's adjuvant (CFA) in ratio 1:1 for 1 hr at low speed using an Eppendorf shaker. A total volume of 400 pg- 200 pg of the cold emulsion was injected intradermally into each rat at 4-5 sites on the back, paw, and tail. Similarly, a booster dose was given on the 9 th day of 1:1 ratio of collagen II to incomplete Freund's adjuvant (ICFA) and rats were kept under observation for the occurrence of arthritis and scored for severity

[0020] For the threatment of the arthritis induces rats and to understand the effects, the experimental animals were divided into seven groups containing six rats in each group. Group I received normal saline and served as control. Group II served as collagen control (positive control), Group III received the doses of standard drug Indomethacin 5 mg/kg b. w. Group IV received the doses of 30 pg of Fol-SOD (TI), Group V received the doses of 50 pg of Fol-SOD (T2) and dose equivalent to 30 pg of FECNM (TI1) to Group VI and 50 pg of FECNM (T22) to Group VII.

[0021] Rats were observed every alternate day for the progression and severity of arthritis. To assess the disease progression, the volume of hind paws was measured using a digital plethysmometer by dipping paw in 0.9 % NaCl solution. The volume was adjusted each time before dipping the paw. The measurement was carried out prior to the onset of arthritis at an interval of 3 days, for up to 35 days. The arthritis index was calculated as given by AI =

[Hind paw volume on day X-Hind paw volume on day 0]/ Hind paw volume on day 0] X 100.

[0022] A decrease in paw volume or arthritic index was calculated as percent CIA inhibition and calculated using the formula, CIA inhibition(%)= 100-[(AIt

AIc/Aica) X 100] where these are the arthritic indices of the treated, healthy control and arthritic group respectively. The level of CIA inhibition by treatment was evaluated by a decrease in the Al of the treated compared to the Al of the untreated CIA group.

[0023] On day 42 right-hand limbs were removed and fixed in 10% buffered formalin for radiological evaluation. All radiographs were taken with x-ray film using MBR 1505R. Settings for radiography were 5 Ma, 40 Kv, and 1 min exposure. Films were placed 60 cm below the X-ray source. An investigator blinded for the treatment regime performed the score. The following radiographed criteria were considered: score 0: no damage; score 1: tissue swelling and edema; score 2: joint erosion; score 3: bone erosion and osteophyte formation.

[0024] Blood samples were drawn from rats after euthanization using cardiac puncture and were collected in vacutainers having anticoagulant. Blood plasma was separated by centrifugation at 2000 rpm for 10 min at room temperature and stored in aliquots at -80°C until further analysis. To confirm the inflammation ELISA (Enzyme-Linked Immunosorbent Assay) was carried out for quantitative estimation of inflammatory cytokines IL6, IL-1, and TNF-a. Plasma samples were diluted (2[1/200tl) in a coating buffer of pH 9.6, and coated into 96 well micro-titer plates, and kept overnight at 4°C. The plates were washed using 100[ 1of washing buffer [0.1% tween-20 in a phosphate buffer saline (PBS)]. To reduce non-specificity, the wells were incubated with 100[ 1of 1% bovine serum albumin in PBS each well for 1h at RT. Afterward, the wells were washed and reacted with commercially available diluted (1:2000) anti-IL6 and anti-TNFa antibody for 2 h at RT and incubated with 100[ diluted (1:1000) HRP-conjugated secondary anti-mouse antibody, at RT for 1 h after washing. Wells were developed using substrate ortho-phenylene diamine (1mg/ml) [Dissolved into 0.05M citrate-phosphate buffer and 5 l/ml 18 H 2 0 2 ]. The reaction was terminated by adding 50[ 1of

2N H 2 SO4 stop solution in each well and the optical density was observed with absorbance at 492nm in an ELISA reader in contrast to the blank demonstrated previously (Rioja, 2005; Sahu, 2012). For joint cytokine measurement, whole joints were stored within liquid nitrogen and were homogenized using a mortar and pestle filled with liquid nitrogen. Powdered tissue was weighed and homogenized by taking 1 mL phosphate buffer per 200 mg of protein-containing protease inhibitor cocktail tablet per 25 ml buffer. Homogenate was first centrifuged at 4000 rpm, 4°C for 15 min to remove the debris and bony portions. The supernatant was centrifuged at 16,000 rpm for 15 min and the resultant clear supernatant was collected and stored at -80°C for estimation [12]. Further, these supernatants were used for the analysis of cytokines IL6, IL-I, TNF-a, and lipid peroxidation assay.

[0025] The lipid peroxidation assay was carried out by taking joint tissue homogenate and plasma. To the homogenate added 100 pl of sodium dodecyl sulphate (4%), followed by the addition of 750 pl of 2M HCl (1 % acetic acid) and 0.8% of thiobarbituric acid. The complete sample was kept at 95°C for an hour in a water bath. After1-hour samples were cooled and centrifuged at 2000 rpm for 10 min. The absorbance of the color developed was taken at 532 nm using UV Spectrophotometer. The amount of thiobarbituric acid reactive substances was estimated as malondialdehyde/mg protein in nanomoles by taking tetra methoxy propane as standard.

[0026] The plasma protein equivalent to 50 g after performing Bradford assay from control, CIA, and treated groups were run in 12% SDS-PAGE. The gel was then transferred to the nitrocellulose (NC) membrane using a blot transfer unit. The proteins were thereby transferred to the NC membrane. The further procedure was followed by blocking with 5% bovine serum albumin consisting of 0.5% Tween-20 for 1 h at room temperature (RT) with mild shaking. After washing the membrane with sterile phosphate buffer saline (PBS) and 0.05% Tween-20 (thrice) to reduce non-specificity, the membrane was incubated with primary anti-TTR antibody in antibody dilution (1:2000 dilutions) buffer (1X-PBS+0.05% Tween-20) and incubated overnight at 4C on a shaker. The membrane was then incubated with an HRP-conjugated anti mouse secondary antibody, with (1:5000 dilutions), at RT for 1 h on gentle shaking condition. Enhanced chemiluminescence (ECL), a highly sensitive substrate detector, was used to develop the membrane and by using ChemiDoc Imagers

[0027] It was observed that the treated groups reported remarked improved locomotors activity compared to CIA rats as reported in table 1. As shown in the graph of figure 1 treatment with an antioxidant enzyme, FECNM and indomethacin observed to be favorable then CIA-associated weight loss, when compared to vehicle, treated CIA rats. Thus, confirms antioxidant enzyme is not toxic at a given dose and positively contributes to a weight gain of collagen-II immunized rats in a dose-dependent manner. The control rats show a gradual increase in weight gain with time

[0028] CIA response during the duration of 1 5th day to 2 7th day with the mean arthritic index of 140.32.9 on the 1 8 th day and 141.67 %on the 21" day, followed by a slow decrease in arthritic index [Figure 2 (i)]. Treatment of CIA rats at both the doses 30 pg and 50 pg lead to a significant decrease in CIA severity as measured by an Al soon after the treatment for 6 days. The percentage CIA inhibitions for different treatment at 33rd day were calculated to be 32.67+5.4 (IND); 45.343.2 (T1); 43.3+4.7 (T11); 42.12+2.6 (T2) and 32.13.2 (T22) respectively. Treatment efficacies of both the doses were also observed by a reduction in arthritic index calculated from paw volume. The arthritic score during the duration of the 1 5 th day to the 2 7th day [Figure 2 (ii)]. Treatment efficacies of formulations were observed by a reduction in arthritic score compared to the untreated one.

[0029] The blood of rats was analyzed for WBC, RBC, hemoglobulin, erythrocytes sedimentation rate, and C-reactive protein. The results of hematological studies obtained are shown in table 2. The inflamed condition appeared in CIA rats compared to control. The normal range of Hg (g/dl) in female Wistar rat's is [13-17]; WBC count (x10 3 ) [1.13-7.49]; RBC (x106) [7.07 9.03]; ESR [3-5 mm/hr]; CRP pg/mL [150-230]. In the case of CIA range of WBC, CRP and ESR were found to be higher compared to control, and treated groups demonstrating inflammation in the CIA group. Similarly, a reduced level of RBC and Hb demonstrated in CIA compared to control and treated one. The inflamed condition remarkably ameliorated on treatment with T22 and Ti1 as compared to others..

[0030] The extent and changes in the level of lipid peroxidation are addressed in figure 3(i). Oxidative stress causes excessive lipid peroxidation. In the case of the CIA, lipid peroxidation was found to be higher in both plasma and joints compared to control. The lowest level was obtained in the case of TI1 and T22 i.e nearly to the level of control. Thus, from the study, it is demonstrated that treatment helps in the reduction of MDA levels in joints and plasma

[0031] Proinflammatory cytokines released from macrophages are a major cause of pathogenesis in RA. Probably the ELISA study demonstrates an increase in the level of cytokines IL-1, IL-6, and TNF-a in plasma and joint tissues both of CIA rats compared to healthy control. The level of these inflammatory cytokines was found to be significantly reduced in rats treated with FECNM at both the doses (p< 0.05) and indomethacin (p < 0.05). The quantification of cytokines in plasma of TNF-a, IL-6, and IL- Ihas been shown in figure 3 (ii), (iii), and (iv), respectively. The quantification of cytokines in joints of TNF-a, IL-6, and IL-i has been reported in figure 4(i), (ii) and (iii), respectively.

[0032] The delivery system appears to be a promising carrier for controlled delivery of enzymes at the site of severe inflammation to meet the therapeutic needs of RA patients.

[0033] While there has been illustrated and described embodiments of the present invention, those of ordinary skill in the art, to be understood that various changes may be made to these embodiments without departing from the principles and spirit of the present invention, modifications, substitutions and modifications, the scope of the invention being indicated by the appended claims and their equivalents.

FIGURE DESCRIPTION

[0034] The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate an exemplary embodiment and, together with the description, explain the disclosed embodiment. In the figures, the left and rightmost digit(s) of a reference number identify the figure in which the reference number first appears. The same numbers are used throughout the figures to reference features and components. Some embodiments of the system and methods of an embodiment of the present subject matter are now described, by way of example only, and concerning the accompanying figures, in which:

[0035] Figure 1 shows the Effect of treatment over changes in body weight in arthritis induced animal model.

[0036] Figure 2 has the illustration of various anatomical and physiological changes occurs during the treatment on arthritis induced animal model: (i) Arthritic Index (AI); (ii) Arthritic score; (iii) X-ray report of a) Control, b) CIA, c)

IND treated, d) T1, e) T11, f) T2, g) T22; (iv) Radiographic scoring of X ray report to compare efficacy of treatment.

[0037] Figure 3 shows the estimation of various biochemical molecules as markers of pathogenesis and progression of RA: (i) Lipid peroxidation assay; (ii) Expression of TNF-a in plasma; (iii) Expression of IL-6 in plasma; (iv) Expression of IL- IPin plasma.

[0038] Figure 4 shows the estimation of various biochemical molecules as markers of pathogenesis and progression of RA: (i) Expression of TNF-a in joints; (ii) Expression of IL-6 in joints; (iii) Expression of IL- Iin joints;

[0039] Table 1: Locomotors test of all the experimental rats

[0040] Table 2: Hematological assay of experimental animals

Claims

CLAIMS: I/We claim that

1. A method of making and testing nanocarriers containing potent antioxidant enzymes superoxide dismutase (SOD) adsorbed for controlled delivery of enzymes at the site of severe inflammation to meet the therapeutic needs of rheumatoid arthritis patients consisting of; Nanomatrix core (NM) of polypropylene sulphide; cellobiose and lyophilized (CNM); Folate; superoxide dismutase (Fol-SOD); 1-ethyl-3-(3-dimethyl aminopropyl) (FE) carbodiimide.

2. The nanocarrier as claimed in claim - 1, has formulation of Nanomatrix core (NM) of polypropylene sulphide by a modified emulsion ring-opening polymerization.

3. The nanocarrier as claimed in claim - 1, has propylene sulphide nanomatrix mimicking as catalase coated with cellobiose over which folate linked-SOD (FECNM) are adsorbed for a macrophage-targeting.

4. The nanocarriers claimed in claim - 1, does controlled delivery of enzymes at the site of severe inflammation to meet the therapeutic needs of rheumatoid arthritis patients.

Signatory

Manju Rawat Singh

Deependra Singh

K.K.Sahu

Shikha Sfrivastava

Sheet 1 of 6

APPLICANT 23 Aug 2021

Manju Rawat Singh Deependra Singh K.K.Sahu Shikha Shrivastava 2021106679

Figure – 1

Signatory

Manju Rawat Singh

Deependra Singh

K.K.Sahu

Shikha Shrivastava

Sheet 2 of 6

APPLICANT 23 Aug 2021

Manju Rawat Singh Deependra Singh K.K.Sahu Shikha Shrivastava 2021106679

Figure – 2

Signatory

Manju Rawat Singh

Deependra Singh

K.K.Sahu

Shikha Shrivastava

Sheet 3 of 6

APPLICANT 23 Aug 2021

Manju Rawat Singh Deependra Singh K.K.Sahu Shikha Shrivastava 2021106679

Figure – 3

Signatory

Manju Rawat Singh

Deependra Singh

K.K.Sahu

Shikha Shrivastava

Sheet 4 of 6

APPLICANT 23 Aug 2021

Manju Rawat Singh Deependra Singh K.K.Sahu Shikha Shrivastava 2021106679

Figure – 4

Signatory

Manju Rawat Singh

Deependra Singh

K.K.Sahu

Shikha Shrivastava

Sheet 5 of 6

APPLICANT 23 Aug 2021

Manju Rawat Singh Deependra Singh K.K.Sahu Shikha Shrivastava 2021106679

Control CIA CIA+ CIA+T1 T11 CIA+T2 CIA+T22 IND Lines 122.34± 12.24±4.5 62.5±13.24 52.32±11.64 61.42±13.81 59.74±12.25 101.23±8.3 Crossed 28.7 (cm) Defecation 1.34±1.19 9±3.02 3.12±1.86 4.98±2.23 3.41±1.65 3.09±2.83 1.06±1.2 ()

Table 1

Signatory

Manju Rawat Singh

Deependra Singh

K.K.Sahu

Shikha Shrivastava

Sheet 6 of 6

APPLICANT 23 Aug 2021

Manju Rawat Singh Deependra Singh K.K.Sahu Shikha Shrivastava 2021106679

Con CIA IND T1 T11 T2 T22 Total WBC 4.9±1.2 14.5±0.9 7.2±0.7 8.6±1.1 6.4±0.61 6.4±0.98 5.2±1.4 (x 103) RBC 5.9±0.03 3.2±0.06 4.1±0.05 3.8±0.12 4.6±0.03 4.9±0.04 5.1± (x106) Hb (g/dL) 13.2±0.4 5.9±0.6 7.4±0.3 6.2±0.4 8.9±0.5 8.1±0.3 10.2±1.2 ESR 3.23±0.67 9.78±0.72 5.4±0.5 6.8±0.3 4.8±1.43 5.2±0.9 3.49±1.1 (mm/hr) CRP µg/mL 169.7±4.21 389.4±5.4 248.2±2.5 301.3±3.1 232.2±1.97 251.3±5.3 215±3.2

Table 2

Signatory

Manju Rawat Singh

Deependra Singh

K.K.Sahu

Shikha Shrivastava