METHOD FOR DIAGNOSING RHEUMATOID ARTHRITIS

Abstract

This disclosure describes the use of plasma hydrogen sulfide metabolites as biomarkers for Rheumatoid arthritis, as well as the potential use of these metabolites as therapeutic efficacy targets.

Claims

1. A method for diagnosing Rheumatoid arthritis in a human patient comprising: obtaining a plasma sample from the patient; using an analytical instrument to determine a level of H.sub.2S in the sample; and diagnosing a patient with Rheumatoid arthritis when the level of H.sub.2S in the sample is above a control level of H.sub.2S.

2. The method of claim 1 wherein the level of H.sub.2S in the sample and level of H.sub.2S in the control refers to one of a level of acid labile sulfide, total sulfide, and both acid labile sulfide and total sulfide.

3. The method of claim 2 further comprising diagnosing a patient with Rheumatoid arthritis when the level of acid labile sulfide in the plasma is above one of 0.004, 0.005, 0.006, and 0.007 pmol sulfide/mg protein.

4. The method of claim 2 further comprising diagnosing a patient with Rheumatoid arthritis when the level of total sulfide in the plasma is above one of 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, and 0.020 pmol sulfide/mg protein.

5. The method of claim 2 further comprising diagnosing a patient with Rheumatoid arthritis when the level of acid labile sulfide in the plasma is above one of 0.004, 0.005, 0.006, and 0.007 pmol sulfide/mg protein and the level of total sulfide in the plasma is above one of 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, and 0.020 pmol sulfide/mg protein.

6. The method of claim 2 further comprising diagnosing a patient with Rheumatoid arthritis when the level of acid labile sulfide in the plasma is above one of 0.001, 0.002, 0.003, and 0.004 pmol sulfide/mg protein above the level of acid labile sulfide in the control.

7. The method of claim 2 further comprising diagnosing a patient with Rheumatoid arthritis when the level of total sulfide in the plasma is above one of 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, and 0.007 pmol sulfide/mg protein above the level of total sulfide in the control.

8. The method of claim 2 further comprising diagnosing a patient with Rheumatoid arthritis when the level of acid labile sulfide in the plasma is above one of 0.001, 0.002, 0.003, and 0.004 pmol sulfide/mg protein above the level of acid labile sulfide in the control and the level of total sulfide in the plasma is above one of 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, and 0.007 pmol sulfide/mg protein above the level of total sulfide in the control.

9. The method of claim 2 further comprising diagnosing a patient with Rheumatoid arthritis when the level of acid labile sulfide in the plasma one of 10%, 20% 40%, 80%, 100%, 150%, and 175% greater than the level of acid labile sulfide in the control.

10. The method of claim 2 further comprising diagnosing a patient with Rheumatoid arthritis when the level of total sulfide in the plasma one of 10%, 20% 30%, 40%, 50%, and 60% greater than the level of total sulfide in the control.

11. The method of claim 2 further comprising diagnosing a patient with Rheumatoid arthritis when the level of acid labile sulfide in the plasma one of 10%, 20% 40%, 80%, 100%, 150%, and 175% greater than the level of acid labile sulfide in the control and the level of total sulfide in the plasma one of 10%, 20% 30%, 40%, 50%, and 60% greater than the level of total sulfide in the control.

12. The method of claim 1 wherein the patient is previously suspected to have Rheumatoid arthritis based on diagnostic criteria in addition to the elevated level of H.sub.2S, including joint pain.

13. A method for diagnosing Rheumatoid arthritis in a human patient comprising: obtaining a plasma sample from the patient; using an analytical instrument to determine a level of H.sub.2S in the sample, wherein a level of H.sub.2S refers to a level of one or more of free sulfide, acid labile sulfide, bound sulfane, and total sulfide; and diagnosing a patient with Rheumatoid arthritis when the level of one or more of free sulfide, acid labile sulfide, bound sulfane, and total sulfide in the sample is above a respective control level of free sulfide, acid labile sulfide, bound sulfane, and total sulfide.

14. A method for diagnosing and treating Rheumatoid arthritis in a patient comprising: analyzing a patient plasma sample for an elevated level of H2S, wherein the patient is diagnosed with RA if an elevated level of H2S is detected; and administering pharmacologically effective dose of a therapeutic agent to the diagnosed patient.

15. The method of claim 14 wherein the therapeutic agent is one of a steroid, a corticosteroid, a disease-modifying antirheumatic drug, a biologic, a JAK inhibitor, and a nonsteroidal anti-inflammatory drug.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate various embodiments of the invention and together with the general description of the invention given above and the detailed description of the drawings given below, serve to explain the principles of the invention. It is to be appreciated that the accompanying drawings, as graphical representations of experimental data, are drawn to scale. The invention will now be described, by way of example, with reference to the accompanying drawings in which:

[0017] FIG. 1 is a graph of plasma H.sub.2S levels for individuals with RA and for individuals without RA (as Control) for free H.sub.2S, acid labile H.sub.2S, bound sulfane H.sub.2S, and total sulfide H.sub.2S according to a first example;

[0018] FIG. 2A is a graph of acid labile H.sub.2S for individuals with RA and for individuals without RA (as Control) according to a second example; and

[0019] FIG. 2B. is a graph of total sulfide H.sub.2S for individuals with RA and for individuals without RA (as Control) according to the second example.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention will be understood by reference to the following detailed description, which should be read in conjunction with the appended drawings. It is to be appreciated that the following detailed description of various embodiments is by way of example only and is not meant to limit, in any way, the scope of the present invention.

[0021] Turning now to FIG. 1, a brief description concerning the various components of the present invention will now be briefly discussed.

[0022] Materials & Methods: Patients were stratified into two main cohorts: first, eleven (11) patients without RA, who served as normal controls, and second, five (5) patients with RA were defined by American College of Rheumatology criteria. Arterial blood samples (10 mL) were collected via venipuncture and were prepared for detecting free H.sub.2S by centrifuging. The plasma was transferred to a PCR tube containing Tris-HCL and MBB solution. The solution was incubated in a hypoxic chamber at room temperature. The reaction was stopped by sulfosalicylic acid solution to precipitate the protein which was then vortexed and placed on ice. The tubes were centrifuged and the supernatant was transferred to an HPLC vial.

[0023] The supernatant was subsequently injected into the Reversed-phase high-performance liquid chromatography (RP-HLC) system with an Agilent Eclipse XDB-C18 column equilibrated with 15% CH.sub.3CN in water containing 0.1% (v/v) TFA. Monobromobimane and sulfide-dibimane were separated using the gradient of two mobile phases: (A) water containing 0.1% (v/v) TFA and (B) 99.9% CH3CN, 0.1% (v/v) TFA at a flow rate of 0.6 mL/min. The amount of H.sub.2S (from linear plots of the HPLC peak areas of sulfide-dibimane) versus known concentration of sulfide solution was measured. The values obtained were multiplied by 6.6 to account for dilutions prior to analysis.

[0024] Samples were prepared for detecting acid-labile sulfide and bound sulfane sulfur in plasma using two sets of BD vacutainer tubes. Phosphate buffer was added to one tube and phosphate buffer with TCEP was added to the other tube. The tubes were incubated on the nutator and the solution is then removed through the cap with a spinal needle. The sulfide gas is trapped by adding 500 microliters of 100 mM Tris-HCl buffer into the BD vacutainer tube, using a 30G needle and the sample is further incubated on the nutator. The solution is removed and the sulfide level in the vacutainer is measured by the MBB method. The remaining blood/plasma sample is frozen in liquid nitrogen and stored for future IRB approved research.

[0025] Discussion: As shown in the Figure, results of the RP-HLC analysis demonstrated that overall levels of total plasma H.sub.2S in RA patients were significantly higher than the levels of plasma H.sub.2S in the control population (p=0.02). Furthermore, the levels of bound H.sub.2S were also significantly higher in the RA patient population (p=0.01). While the level of acid labile plasma H.sub.2S appeared to vary greatly between the two populations, from this example, the degree of difference had a p=0.08. Furthermore, from this example, levels of free plasma H.sub.2S among the RA patients and control populations had a p=0.18.

[0026] Turning to FIGS. 2A and 2B a second example is shown with further investigation of the findings in the first example. In the second example, plasma samples were collected via venipuncture from 19 patients with RA and 8 healthy subjects. Sulfide metabolites were stabilized using methods previously published by our laboratory (Peter E A, Shen X, Shah S H, Pardue S, Glawe J D, Zhang W W, Reddy P, Akkus NI, Varma J, Kevil C G. Plasma free H2S levels are elevated in patients with cardiovascular disease, J Am Heart Assoc. 2013 Oct. 23; 2(5):e000387. doi:10.1161/JAHA.113.000387. PubMed PMID: 24152982; PubMed Central PMCID: PMC3835249), such method fully incorporated herein. Sulfide metabolite levels were measured using MBB HPLC analytical biochemical methods as we have previously reported (Shen X, Peter E A, Bir S, Wang R, Kevil C G. Analytical measurement of discrete hydrogen sulfide pools in biological specimens, Free Radic Biol Med. 2012 Jun. 1-15; 52(11-12):2276-83. doi: 10.1016/j.freeradbiomed.2012.04.007. Epub 2012 Apr. 19. PubMed PMID: 22561703; PubMed Central PMCID: PMC4413934) such method fully incorporated herein. Plasma acid labile sulfide levels were significantly higher in patients with RA compared to controls. Moreover, the overall total sulfide metabolite levels were significantly elevated in plasma of RA patients compared to controls.

[0027] This disclosure serves to describe a novel method for diagnosis of RA, including noting the variance in levels between the different forms of plasma H.sub.2S in patients with RA. By measuring levels of H.sub.2S in the plasma instead of the synovial fluid, the option of a less invasive technique is provided to patients to monitor disease activity who may are already be suffering from joint pain. Furthermore, the discovery of the RP-HPLC technique as a way to measure the three pools of H.sub.2S in plasma, allows a more precise, specific, and humane manner of measuring and diagnosing RA as compared to previous methods.

[0028] A further embodiment of the disclosed invention relates to the treatment of RA, as treatment of a condition is improved when the accurate diagnosis of the disease causing a symptom is achieved, and arcuate monitoring of the condition is improved..

[0029] Therapeutic agent: A substance that demonstrates some therapeutic effect by restoring or maintaining health, such as by alleviating the symptoms associated with a disease or physiological disorder, or delaying (including preventing) progression or onset of a disease. In some instances, the therapeutic agent is a chemical or pharmaceutical agent, or a prodrug. A therapeutic agent may be an agent that prevents or inhibits one or more signs or symptoms or laboratory findings associated with RA. Steroids and corticosteroids, including prednisone, prednisolone and methyprednisolone, are potent and quick-acting anti-inflammatory medications; DMARDs, an acronym for disease-modifying antirheumatic drugs, DMARDs are drugs that work to modify the course of the RA disease, include methotrexate, hydroxycholorquine, sulfasalazine, leflunomide, cyclophosphamide and azathioprine, which may be taken by mouth, self-injected or given as an infusion; Biologics, which are a subset of DMARDs, and are injected or given by infusion, and they target specific steps in the inflammatory process, they don't wipe out the entire immune response, and include abatacept (Orencia), adalimumab (Humira), anakinra (Kineret), certolizumab (Cimzia), etanercept (Enbrel), golimumab (Simponi), infliximab (Remicade), rituximab (Rituxan), tocilizumab (Actemra) and tofacitinib (Xeljanz); JAK inhibitors, a further subcategory of DMARDs that block the Janus kinase, or JAK, pathways, which are involved in the body's immune response, including Tofacitinib, and which can be taken by mouth; and NSAIDs, or nonsteroidal anti-inflammatory drugs, which can relieve pain and reduce inflammation and include ibuprofen (Advil, Motrin IB) and naproxen sodium.

[0030] A therapeutically effective amount or therapeutically effective dose is that amount or dose sufficient to inhibit or prevent onset or advancement, to treat outward symptoms, or to cause regression, of a disease. The therapeutically effective amount or dose also can be considered as that amount or dose capable of relieving symptoms caused by the disease. Thus, a therapeutically effective amount or dose of an anti-RA agent is that amount or dose sufficient to achieve a stated therapeutic effect.

[0031] While various embodiments of the present invention have been described in detail, it is apparent that various modifications and alterations of those embodiments will occur to and be readily apparent those skilled in the art. However, it is to be expressly understood that such modifications and alterations are within the scope and spirit of the present invention, as set forth in the appended claims. Further, the invention(s) described herein is capable of other embodiments and of being practiced or of being carried out in various other related ways. In addition, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of including, comprising, or having and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items while only the terms consisting of and consisting only of are to be construed in the limitative sense.