TO A MEDICAL CONDITION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Serial No.

61/699,054, filed September 10, 2012. The disclosure of this provisional application is incorporated by reference herein in its entirety.

BACKGROUND

The following relates to the medical care arts, and related arts and more specifically concerns a method for diagnosing and/or monitoring a medical condition (e.g., rheumatoid arthritis) by detecting citrullination in saliva.

Rheumatoid arthritis is an inflammatory disorder that chronically and systemically affects many tissues and organs, particularly synovial joints. The inflammatory activity causes tethering of tendons and erosion and/or destruction of the joint surfaces which limits range of motion while causing pain, disability, comorbidity, and increased mortality.

Rheumatoid arthritis affects about 0.5 to 1% of the global population, is more common in women than in men and can occur at any age (See, Feldmann, et al., Cell 1996, 85, 307-10). Most frequently, onset occurs between the ages of 40 and 50. The onset of rheumatoid arthritis can occur over a period of time (e.g., over weeks or months) or the condition can present itself rapidly in an acute manner. Although the cause of rheumatoid arthritis is not well known, autoimmunity plays a key role. Accordingly, rheumatoid arthritis is considered to be an autoimmune disease (i.e., the body's immune system mistakenly attacks healthy tissue).

Citrullination is the enzymatic conversion of protein bound arginine to protein bound citrulline. Arginine and citrulline are amino acids and are illustrated below in Formulas (I) and (II), respectively.

Formula (I)

I ·

Formula (II)

This post translational modification is catalyzed by a family of calcium-dependent enzymes called peptidylarginine deiminases (PADs). PADs have a physiological role in gene regulation via citrullination of histones, which form the protein backbone of condensed deoxyribonucleic acid (DNA). For DNA to be transcribed into messenger ribonucleic acid (mRNA) and then translated into the proteins of life, local citrullination has to occur to unmask the relevant coding DNA. However, wholesale citrullination of the entire nuclear DNA has recently been described in the production of neutrophil extracellular traps (NETs), putatively powerful bacterial traps triggered by reactive oxygen radicals (e.g., HOC1). Other cell types also produce PADs for different physiological roles, such as epithelial cells, which produce a peptidylarginine deiminase 2 (PAD2) enzyme capable of citrullinating keratins within skin. The physiological role of citrullination can become pathological when citrullination becomes excessive and various environmental factors such as cigarette smoking or hormonal influences such as elevated oestradiol can active PAD2. The pathogen P. gingivalis may also give rise to excess citrullination. P. gingivalis is the only known bacterium found to express porphyromonas gingivalis peptidylarginine deiminase (PPAD).

Without wishing to be bound by theory, it is believed that proteins citrullinated in the oral cavity, either by PPAD or endogenous human PADs released during innate immunity, may form a point source for the break in immune tolerance that leads to anti- citrullinated protein antibody (ACPA) formation which can then undergo epitope spreading and lead, in time, to rheumatoid arthritis in some patients (Lundberg, et al, Nat. Rev. Rheum. 2010, 6, 727-30). ACPAs are also known as anti-cyclic citrullinated peptide antibodies (anti-CCP antibodies). Patients do not produce ACPAs against only one type of protein but instead can express a range of different citrullinated proteins (Wegner, et al, Immunol. Rev. 2010, 233, 34-54). The alteration in the charge of the amino acid side chain is likely to cause changes in protein structure, function, and antigenicity of the proteins in which they are found.

Approximately 60 to 70% of patients with rheumatoid arthritis have ACPAs. ACPAs appear before the onset of joint pain and swelling caused by rheumatoid arthritis. In fact, patients can test positive for ACPAs years before it develops into a clinical condition (Rantapaa-Dahlqvist, Arthritis Rheum. 2003, 48, 2741-9), implying that the initial loss of immune tolerance to citrullinated proteins may arise from inflammatory events outside the joint. Some ACPA-positive patients do not go on to develop rheumatoid arthritis. ACPA-positive rheumatoid arthritis may become a specific and well characterized subgenus of rheumatoid arthritis genus. The identification of citrullinated antigens themselves may precede antibody production and act as an early diagnostic marker, or risk predictor, of rheumatoid arthritis.

Some diagnostic tests for rheumatoid arthritis rely on subjective measures (e.g., signs of joint swelling, stiffness and pain on a squeeze test). Other diagnostic tests include measuring ACPAs in blood. However, such tests are invasive and non-specific. Identification of high levels of citrullinated proteins can provide a very early indicator of risk or susceptibility to rheumatoid arthritis and other chronic inflammatory diseases where citrullination is believed to underpin later clinical manifestation of disease (e.g. multiple sclerosis, glaucoma, myositis, and Alzheimer's disease).

Early diagnosis of rheumatoid arthritis and other medical conditions allows early aggressive treatment and more effective therapy. However, known diagnostic tests are invasive and somewhat costly.

Methods and diagnostic tests are disclosed which can overcome some of the problems with existing tests. BRIEF DESCRIPTION

The present disclosure relates, in various embodiments, to methods for estimating the susceptibility to a medical condition such as rheumatoid arthritis. The methods use citrullination of proteins in saliva as a biomarker.

Disclosed in some embodiments is a method for estimating susceptibility to a medical condition. The method includes determining the concentration of citrullinated proteins in a saliva sample, comparing the determined concentration to at least one threshold indicative of a risk level, and based on the comparison, reporting an estimate of susceptibility to the medical condition.

The determining may include performing mass spectroscopy on the saliva sample.

In some embodiments, the mass spectroscopy is tandem mass spectroscopy. In some embodiments, the mass spectroscopy includes a neutral loss scan. In some embodiments, the mass spectroscopy includes both collision induced dissociation and electron transfer dissociation. The collision induced dissociation may be performed on a first portion of the saliva sample and the electron transfer dissociation may be performed on a second portion of the saliva sample.

In some embodiments, the medical condition is rheumatoid arthritis, multiple sclerosis, glaucoma, polymyositis, Alzheimer's disease, or cancer.

The method may further include obtaining the saliva sample from a patient, such as a human or animal subject.

In some embodiments, the obtaining and determining are performed with a handheld device.

The citrullinated proteins may include alpha-enolase, fibrinogen, vimentin, and/or collagen type II.

In some embodiments, the plurality of ranges is two ranges. In other embodiments, the plurality of ranges includes at least four ranges.

The reporting may include providing a visual printout or display.

Optionally, the method further includes purifying the saliva sample prior to the determining.

Disclosed in other embodiments is a method for diagnosing rheumatoid arthritis.

The method includes determining the concentration of citrullinated proteins in the saliva sample, comparing the determined concentration to a plurality of ranges indicative of different risk levels for rheumatoid arthritis, and reporting an estimated risk level based on the comparison.

The reporting may include printing the estimated risk level. In some embodiments, the method further includes purifying the saliva sample prior to determining the concentration of citrullinated proteins in the saliva sample.

Further disclosed in embodiments is a device for diagnosing rheumatoid arthritis. The device includes a saliva sample input, a saliva purification chamber, an analysis chamber, and a reporter element. The analysis chamber is configured to measure a concentration of citrullinated proteins. The reporter element is for reporting the concentration of citrullinated proteins. In some embodiments, the device is a handheld device.

These and other non-limiting aspects and/or objects of the disclosure are more particularly described below. BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawings, which are presented for the purposes of illustrating the exemplary embodiments disclosed herein and not for the purposes of limiting the same.

FIGURE 1 is a flow chart illustrating a method for estimating susceptibility to a medical condition.

FIGURE 2 is a flow chart illustrating a method for diagnosing rheumatoid arthritis.

FIGURE 3 is a schematic illustration of a device for diagnosing rheumatoid arthritis.

DETAILED DESCRIPTION

Aspects disclosed herein relate to a method and a device for estimating susceptibility to a medical condition.

One advantage of the exemplary method and device, in embodiments disclosed herein, is that they enable an early, non- invasive and objective diagnosis of rheumatoid arthritis. With reference to FIGURE 1, a method 100 of estimating susceptibility to a medical condition is shown. The method 100 includes obtaining a bodily fluid, such as a saliva sample 110, optionally, purifying the saliva sample 120, determining the concentration of citrullinated proteins in the saliva sample 130, comparing the determined concentration to at least one threshold indicative of a risk level for the medical condition 140, and reporting a level of risk based on the comparison 150.

The comparing 140 can include comparing the determined concentration to a plurality of ranges indicative of different risk levels for the medical condition.

The reporting 150 can include reporting which range of the plurality of ranges the determined concentration is within.

The saliva sample may be obtained 110 from a subject or patient by a medical professional (e.g., via a swab). In other embodiments, the saliva sample is provided directly by the subject. Utilizing saliva samples is less invasive than using other bodily fluids, such as blood. However, in some embodiments, such fluids are also contemplated.

Optionally, the saliva sample may be purified 120 to remove contaminants. Such contaminants may interfere with subsequent analysis of the sample. Purification 120 may be performed inside a device used to perform other elements of the method or may be conducted external to such a device.

A standard proteomics workflow can be used for this purpose. In one embodiment, the samples are prepared for determination of citrullinated proteins by lysing the cells, e.g., with sonication. The cell lysate can be digested, e.g., using a protease enzyme, such as trypsin, to generate a mixture of peptides.

The determination of citrullinated protein concentration 130 may be performed on the mixture of peptides via mass spectroscopy. Mass spectroscopy is an analytical technique that measures the mass-to-charge ratio of charged particles. Mass spectroscopy works by ionizing chemical compounds (e.g., peptides) to generate charged molecules or molecule fragments and then measuring their mass-to-charge ratios. Ionization may be performed by electron impact, chemical ionization, atmospheric pressure ionization, electrospray ionization, fast atom bombardment, field desorption/field ionization, matrix assisted laser desorption/ionization, thermospray ionization, or any other suitable form of ionization. In some embodiments, tandem mass spectroscopy is utilized. Tandem mass spectroscopy allows for more powerful structure elucidation, selective detection of a target compound, greatly reduces interferences, and is useful for the study of ion-molecule reactions. Tandem mass spectroscopy employs two stages of mass analysis in order to examine selectively the fragmentation of particular ions. Instruments that can be used for tandem mass spectroscopy include those based on separation of the mass-analysis events in time and those based on measurements in physically separate analyzers.

There are two main categories of instruments that can be used in mass spectrometry applications. The first category includes instruments which comprise two different mass spectrometers assembled in tandem, e.g., two mass-analyzing quadrupoles; two magnetic analyzer instruments; and one mass-analyzing quadrupole and one magnetic analyzer instrument (see, for example, Creese, et al, Anal. Methods, 2011, 3, 259-266). The second category includes devices capable of storing ions (e.g., in an ion trap). Such devices allow the selection of particular ions by ejection of all others. The selected ion can be excited and fragmented during a selected time period and the fragment ions can be observed in a mass spectrum. The process may be repeated to observe fragments of fragments, over several generations. The first category may be used for tandem in space mass spectrometry wherein a sequence of mass spectrometers in space is utilized. The second category may be used for tandem in time mass spectrometry wherein one spectrometer with ion storage capability exploits a sequence of events in time.

In some embodiments, the scan mode in tandem mass spectrometry is a product ion scan, a precursor ion scan, or a neutral loss scan. In product ion scans, ions of a given mass-to-charge ratio are selected with a first mass spectrometer. The selected ions are passed into a collision cell. Typically, the collision cell is filled with an inert gas, such as helium, argon, or xenon gas. The ions are activated by collision and thereby induced to fragment. The product ions are then analyzed with a second mass spectrometer which is set to scan over a predetermined mass range. A product ion spectrum is obtained and allows fragments arising from a molecular ion of a specific compound to be recorded.

In precursor ion scans, the second mass spectrometer is set to pass only ions with a predetermined mass-to-charge ratio. The first mass spectrometer is scanned over a chosen mass range with a collision gas present in the instrument. Ions which pass through the first mass spectrometer may be detected if, after fragmentation in the collision cell, the predetermined product ion is produced. This product ion is the only ion that the second mass analyzer can transmit to the detector.

Neutral loss scans are performed with tandem in space mass spectrometry. Neutral loss scans are a form of functional group-selective scans but are more complex than precursor ion scans because both analyzers are scanned together but with a constant mass- to-charge ratio difference between the spectrometers. Neutral loss scans allow the selective recognition of all ions which, by fragmentation, lead to the loss of a given neutral fragment. See, for example, Louris, John N.; Wright, Larry G.; Cooks, R. Graham.; Schoen, Alan E. (1985). "New scan modes accessed with a hybrid mass spectrometer". Analytical Chemistry 57 (14): 2918, for a discussion of the neutral loss method.

The mass spectroscopy may include fragmenting citrullinated peptides via collision induced dissociation (CID). CID is a mechanism by which molecular ions are fragmented in the gas phase. In CID, the ions are typically excited to high kinetic energy. The ions then collide with neutral molecules (e.g., helium, nitrogen, and/or argon). At least a portion of the kinetic energy is converted into internal energy which results in bond breakage and fragmentation of the ion. The fragments are suitable for analysis via a mass spectrometer. In CID of citrullinated peptides, an intense signal is observed as the result of the loss of 43 Da, representing isocyanic acid (HNCO). The mass spectrometer may be programmed to trigger repeat analysis of the peptide with a different type of fragmentation: electron transfer dissociation (ETD). ETD is a method which induces fragmentation of cations (e.g., peptides and/or proteins) by transferring electrons to them. ETD retains the HNCO on the peptide backbone and allows for more accurate assignment of citrulline residues.

In one embodiment, the peptide mixture can be analyzed using a decision tree method. As an example, the instrument fragments with either CID or ETD (low resolution) depending on the mass to charge ratio, e.g., for a mass to charge ratio of 2+, CID only is used, whereas at higher mass to charge ratios, ETD may be used. This improves the proteome coverage. Peptides can then be identified as citrullinated from a database search. The method can be repeated to improve accuracy and/or a combination of CID and ETD used. In some embodiments, the determination of the concentration of citrullinated proteins may be performed without conducting an analysis of ACPAs. Since the citrullinated proteins themselves may precede antibody production, analyzing the citrullinated proteins themselves allows earlier diagnosis and risk prediction.

The method 100 also includes comparing the determined concentration to a threshold 140, e.g., a plurality of ranges indicative of different risk levels. The ranges are predetermined for the medical condition being tested for. The plurality of ranges may include two ranges (e.g., risk and no risk) or may include at least four ranges (e.g., no risk, mild risk, moderate risk, and severe risk).

Reporting the results 150 may include providing a visual or auditory signal to a subject and/or a medical professional. In some embodiments, the results are displayed on a display screen or a printout is provided. The reporting 150 may describe the concentration of citrullinated proteins, the risk level, and or a combination thereof.

With reference to FIGURE 2, a method 200 for diagnosing rheumatoid arthritis is shown. The method 200 includes obtaining a saliva sample 210, optionally, purifying the saliva sample 220, determining the concentration of citrullinated proteins in the saliva sample 230, comparing the determined concentration to a threshold, such as a plurality of ranges indicative of different risk levels for rheumatoid arthritis 240, and reporting based on the comparison, such as outputting which range of the plurality of ranges the determined concentration is within 250. The method of FIGURE 2 can be performed as for the method of FIGURE 1, except in that the threshold/ranges is/are defined based on a predetermined risk level for rheumatoid arthritis.

By way of example, the conversion of arginine to citrulline by peptidyl arginine deiminases results in a mass increase of 0.984 Da (the same increase as deamidation of asparagine and glutamine). The exemplary method can use an intense signal observed when citrullinated peptides are fragmented by collision induced dissociation (CID), this signal is the result of the loss of 43 Da representing FINCO. The mass spectrometer can be programmed to trigger repeat analysis of the peptide with a different type of fragmentation: electron transfer dissociation (ETD) which retains the FINCO on the peptide backbone and allows for more accurate assignment of the citrulline. An example method employs both the low resolution and high scanning speed of the ion trap mass analyzer with the high resolution to distinguish between ions of similar mass. Test results results show that with only CID analysis of potentially citrullinated protein samples, several peptides can be falsely reported as citrullinated. By triggering ETD of the potentially citrullinated peptide the false positive rate reduces significantly and more citrullinated peptides are identified with confidence. The exemplary method may be configured for detecting one, or two or more different citrullinated proteins, such as four, or more.

With reference to FIGURE 3, a device 300 for diagnosing rheumatoid arthritis is shown. The device 300 includes a saliva sample input 310, such as a collection vehicle for catchment of saliva, an optional purification chamber 320 for pre-analytical purification of saliva to obtain a sample freed from saliva interfering elements that could hinder subsequent analyses, a micro fluidic analysis chamber 330 configured for routing of the sample to the reading analysis and scanning element of the spectroscopic device in real time to measure a concentration of citrullinated proteins (or to perform the measurement within the handheld device). A reporter element 350 is provided for reporting information, such as a risk level, based on the measured concentration of citrullinated proteins. The device may be a handheld device.

The analysis chamber can include or be temporarily linked to a detection device such as a mass spectroscopy device as described above. Alternatively, detection hardware for protein biomarkers in saliva can be provided in a lab-on-a-chip device that is geared to detecting salivary proteins with various unique conjugates. Such hardware that can detect citrullinated proteins in particular in saliva may depend on the attached conjugate molecules that enable biochemical or biophysical detection. Conjugates may be enzymatic based and may rely on principles requiring real time absorbance, luminescence, chemiluminescence or fluorescence. Analytical thresholds for detection that take into account the biochemical nature of citrullination may entail experimental analysis to determine where quantitative limits should be set in a working assay to define optimal detection.

The reporter element 350 can include non-transitory memory which stores software instructions which compute, based on the measured concentration of citrullinated proteins, a decision, such as a risk level or the like. The memory may store a threshold value for one or more risk levels and the instructions may compare the concentration of citrullinated proteins with the threshold(s). The reporter element may also include a computer processor, such as a CPU, in communication with the memory, which executes the instructions, and which may also control other operations of the handheld device, including transferring the saliva sample to the analysis chamber. The reporter element may further include a display device, such as an LCD screen, an alarm, a light, or the like which outputs the result in a human-understandable form.

In practice, salivary citrullinated proteins can be detected in such a handheld device. As opposed to collection from blood or other tissues, collective of salivary proteins is non-invasive and thus preferable to patients. A user may simply apply a small volume of saliva to a chamber connected to a main body of the device. The device may analyze the sample and provide a real-time indicator regarding the levels of citrullinated proteins in the saliva sample relative to previously defined clinical thresholds indicative of risk susceptibility and/or early diagnosis of rheumatoid arthritis. An example device which could be adapted to use for detection of citrullinated proteins is the Concateno-Philips handheld product which is designed for detection of drugs in saliva.

The reading could be provided digitally or a printout of the results could allow a hardcopy data record to be available to the user. Interpretation of the reading could be binary (i.e., risk or no risk). Alternatively, more clinical thresholds for risk could be used (e.g., mild, moderate, or severe risk). Each risk level could be split into a plurality of benchmarked sublevels. These benchmarks could be incorporated and described in an easily understood, intuitive format for the benefit of the user. They could be incorporated in the directions for use (DFU) accompanying such a device.

Hospitals and medical practices can use this technology to screen for rheumatoid arthritis and other chronic inflammatory diseases, where excessive citrullination is part of the causal pathway. Patients may also be able to perform a "home test" if they suspect they are at risk, and would be encouraged to visit their doctor in the event of an indicative outcome.

The detection of citrullinated proteins in saliva samples can be used to predict risk and susceptibility in the diagnosis of rheumatoid arthritis. The exemplary hand held device allows for immediate chair- side detection. As an over the counter consumer product, the device can provide diagnostic guidance to a consumer at risk by informing them of their susceptibility to rheumatoid arthritis. As a preliminary self-assessment tool, the application of such a device would validate to the consumer the risks associated with disease development. Contingent on the outcome, the consumer could then seek further validation from the medical professional of recommended next steps to address/counteract the disease risks.

Although the present disclosure emphasizes diagnosis of rheumatoid arthritis, it is contemplated that the susceptibility to medical conditions other than rheumatoid arthritis may be estimated with the systems and methods of the present disclosure. Tables 1 and 2 (below) list the pathological roles for different medical conditions and physiological roles, respectively, of citrullination.

Table 1: Pathological Roles of Citrullination

Table 2: Physiological Roles of Citrullination

The following examples are for purposes of further illustrating the present disclosure. The examples are merely illustrative and are not intended to limit processes or devices made in accordance with the disclosure to the materials, conditions, or process parameters set forth therein.

EXAMPLES

Saliva samples were collected from several systemically healthy dentate and edentulous (i.e., toothless) patients without knowledge of whether the patients suffered from rheumatoid arthritis. The samples were tested for citrullination. Substantial citrullination was found in one edentulous patient's saliva. The patient was diagnosed with rheumatoid arthritis shortly thereafter.

Saliva samples were obtained from one healthy subject (Subject 1) and one edentulous subject (Subject 2). Cell debris from the samples was analyzed using a standard proteomics workflow to assess the number of proteins identified compared to saliva in order to compare healthy and edentulous subjects. The samples were sonicated for five (5) minutes to lyse the cells and the cell lysate was digested using trypsin. The resulting peptide mixtures were analyzed using a decision tree method (the instrument fragments with either CID or ETD (low resolution) depending on the mass to charge ratio: 2+ all CID, 3+ >m/z 650 ETD, 4+ >m/z 900 ETD, 5+ >m/z 950 ETD) which improves the proteome coverage. Several peptides were identified as citrullinated from a database search, though without both CID and high resolution CID spectra the presence of citrulline was ambiguous.

Two additional saliva samples from different subjects (Subjects 3 and 4) were worked up for analysis. Subject 3 was a healthy, dentate subject. Subject 4 was an edentulous subject. These samples were analyzed using the neutral loss method to improve confidence of identification.

The initial analysis of the sample of Subject 3 identified three (3) citrullinated peptides by ETD: VArKSAPATGGVK:Histone H3. lt, rGAFSSVSMSGGAGR:Keratin, and type II cytoskeletal 4.

The initial analysis of the sample of Subject 4 identified four (4) citrullinated peptides: LPGVSrSGFSSVSVSR:Keratin, type II cytoskeletal 6A, VArKSAPATGGVK:Histone H3.lt, GGPrGF S cGSAIVGGGK: Keratin, type II cytoskeletal 4, and rGAFSSVSMSGGAGR:Keratin, type II cytoskeletal 4. Repeat analysis of the samples of Subjects 2 and 4 was performed using the neutral loss method. The analysis found no citrullinated peptides in Subject 2's sample and five (5) citrullinated peptides in Subject 4's sample: DWYPHSrLFDQAFGLPR:Heat shock protein beta-1, TAENFrALSTGEK: Peptidylprolyl cis-trans isomerase A- like 4G, LPGVSrSGFSSVSVSR:Keratin, type II cytoskeletal 6A,

SQTVSHGGArEQGQTQTQPGSGQR:Cornulin, and GGPrGFS cG S AI VGGGK :Keratin, type II cytoskeletal 4.

In the examples, four (4) synthetic citrullinated peptides were identified which were spiked into a complex saliva tryptic digest. In the CID data, only one of the peptides was positively identified with high confidence. Twenty-two (22) additional peptides were identified as citrullinated. However, manual analysis proved that these were false positives. The ETD data identified three (3) of the peptides with high confidence. No additional citrullinated peptides were observed from the ETD spectra.

Both the low resolution and high scanning speed of an ion trap mass analyzer with high resolution to distinguish between ions of similar mass were employed. With only CID analysis, some peptides may be falsely reported as being citrullinated. However, using ETD in combination with CID may eliminate false positives and allows citrullinated peptides to be identified with confidence.

The difference in results for the two analyses of Subject 4's sample may have resulted from the complexity of the sample. Generally, when a sample is run through a mass spectrometer in duplicate, approximately 75% of the peptides are observed in both samples with 25% of the peptides unique to one sample or the other. This 25% is usually the less abundant peptides. This issue can be addressed by either running the samples more than twice (thus reducing the number of peptides which only appear once) or fractioning the samples to reduce the complexity of each run.

Thus it can be concluded that identification of citrullinated proteins in cell debris from saliva samples may function as a test to identify people at risk from developing rheumatoid arthritis, or other diseases, or those with pre-clinical symptoms. Such a noninvasive measure could be employed as a near-patient screening test, or even for patient self-assessment. The present disclosure has been described with reference to the preferred embodiments. Obviously, modifications and alterations will occur to others upon reading and understanding the preceding detailed description. It is intended that the present disclosure be construed as including all such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.