WO2011143292A1 - Biomarkers for detecting erythropoietin use in human subjects - Google Patents

Abstract

Erythropoietin (Epo) is produced primarily in the kidneys upon low blood oxygen tension and stimulates erythropoiesis in the bone marrow. Recombinant human Epo (rHuEpo), a drug developed to increase arterial oxygen content in patients, is expensive and optimal dosing is difficult to determine and which can lead to waste. RHuEpo is also illicitly used by athletes to improve endurance performance. Described herein are methods for detecting exogenous Epo presence in a human subject by detecting an aberrant level of a biomarker such as isoforms of haptoglobin, transferrin, and/or hemopexin/albumin in serum samples.

Description

BIOMARKERS FOR DETECTING ERYTHROPOIETIN USE IN HUMAN SUBJECTS

Cross Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Application Serial No.

61/333,411, filed on May 11, 2010, the disclosure of which is expressly incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grant Nos. AG19899, AG031736, DK075436 awarded by the National Institutes of Health.

FIELD

[0003] The present invention relates generally to biomarkers for exogenous

erythropoietin (Epo) use in a subject and more specifically to the use of blood serum proteins to detect the presence of exogenous Epo in a subject.

BACKGROUND

[0004] Erythropoietin ("Epo") is known to affect the oxygen carrying capacity of the blood by increasing the number of erythrocytes, resulting in an increase in blood hemoglobin content and hematocrit. Epo is secreted primarily from the kidneys when blood oxygen availability decreases. Epo interacts with Epo receptors (Epo-R) on predominantly bone marrow colony-forming erythroid precursor-cells and has anti-apoptotic and proliferative effects.

Increased erythropoesis will eventually lead to decreased serum iron levels, since developing erythrocytes take up iron that is incorporated into hemoglobin. In the blood, iron is stored bound to ferritin, and the amount of ferritin in plasma reflects whether a shortage or excess of iron exists (i.e. high ferritin levels correlate with an excess of iron, while low ferritin reflects a need to store iron).

[0005] The gene for Epo was first cloned in 1985 and recombinant human Epo (rHuEpo) was approved for clinical use in 1989 for treatment of chronic anemia in patients with end-stage renal disease (ESRD). Additional FDA-approved clinical indications include patients with HIV, cancer, myelodysplastic syndromes, bone marrow transplantation, hepatitis C, and for the prevention of anemia in premature newborns. Clinically, rHuEpo is an effective but very expensive drug. As such, the medical community tries to carefully manage dosing, which in turn helps manage costs. However, since each patient will respond differently to the drug, standard dosing regimens can lead to ineffective dosing or inefficiencies that lead to waste where higher doses than need for efficacy are administered. Presently, hospitals utilize labor intensive and expensive disease management programs that require teams of nurses to establish higher levels of control to help identify effective yet efficient dosing regimens. These non- biomarker based clinical algorithms are typically nursing protocols based on clinical experience and routine testing. A less labor intensive method of identifying optimal dosing regimens for individual patients is needed.

[0006] In addition to clinical uses, Epo has been abused by athletes due to its ability to stimulate red blood cell production and when injected, to increase sub-maximal exercise performance by more than 50%. Use of rHuEpo in sports has been banned by the American Medical Association and the International Olympic Committee (IOC). However, the abuse continues and a sensitive and robust detection test for Epo is needed.

[0007] The current method directly measures urinary rHuEpo, and is based on differences in glycosylation between rHuEpo and endogenous Epo. However, this method is expensive and has low sensitivity. Also, whether or not this test will be effective against new Epo derivates, some of which are produced in human cell lines and thereby express the same glycosylation pattern as endogenous Epo, is unknown. Related to the reproducibility of the current detection test, two accredited (World Anti Doping Agency) WADA laboratories tested human samples from subjects injected with rHuEpo and found less than half to be positive for the drug. In fact, one laboratory did not detect any positive samples. This emphasizes the importance of the future development of new and more sensitive Epo-specific assays.

[0008] In addition to the abuse of rHuEpo injection, gene therapy is emerging as a futuristic doping practice. This procedure involves transfection of the Epo gene into selected tissues of the athletes' body. Stable long-term expression of Epo genes in experimental animals has been reported. Therefore, IOC and WADA in 2003 included "gene doping" on their list of "prohibited substances and methods in sport."

[0009] Moreover, compounds have been devised that exert epo-like activity by decreasing the clearance of endogenous Epo.

[0010] Thus, future doping tests should focus not only on detecting rHuEpo and related protein analogues, but also on biological markers that are altered by Epo exposure. Such markers are presently being investigated and include the assessment of total hemoglobin mass and reticulocyte percentage (OFF_hr-_scor_e) and the so-called Blood Passport. Unfortunately, both are not able to reliably identify Epo use. The cut-off limits for the OFF_hr__score are based on average population levels of total hemoglobin mass and reticulocyte percentage. Given the large inter-personal variability, the cut-off values (upper and lower levels) for this model is very wide in order to prevent false -positive results. This ultimately leads to low detection levels. More recently, the Blood Passport was implemented, making it possible to compare an athlete's current blood sample values with the same athletes' previous obtained blood values. However, it has been shown, that the Blood Passport does not guarantee a doping free sport.

SUMMARY

[0011] Certain exemplary aspects of the invention are set forth below. It should be understood that these aspects are presented merely to provide the reader with a brief summary of certain forms the invention might take and that these aspects are not intended to limit the scope of the invention. Indeed, the invention may encompass a variety of aspects that may not be explicitly set forth below.

[0012] As described above, there presently is no reliable and efficient method to detect if a subject is using exogenous Epo. One aspect of the invention provides a method of detecting the presence of exogenous Epo in a subject by detecting an aberrant level of a biomarker in a sample from the subject. This method of detecting the presence of exogenous Epo may be used to optimize the therapeutic efficacy of exogenous Epo in a subject such as in a clinical setting or the abuse of exogenous Epo such as by an athlete. The sample may be a blood sample, and in one exemplary embodiment may be the serum fraction. In one embodiment, the biomarker may be a protein, such as a serum protein. The biomarker may include isoforms of the serum proteins haptoglobin, transferrin, hemopexin, and albumin. In one embodiment, the method may include comparing the level of the biomarker in samples collected from the subject at different times. In another embodiment, the method may include comparing the biomarker with a reference biomarker from the same subject or a different subject.

[0013] Another aspect of the invention provides a kit for performing the method described above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the general description of the invention given above and the detailed description of the embodiments given below, serve to explain the principles of the present invention. [0015] Figure 1 is a representative 2D-gel of human serum showing protein spots that changed significantly (p<0.05) after 16 days of treatment with rHuEpo.

[0016] Figure 2A illustrates serum protein changes for haptoglobin isoforms a representative 3D image of protein spots in accordance with embodiments of the invention.

[0017] Figure 2B is a graph illustrating the mean changes in intensity for each of the spots for haptoglobin isoforms from the representative 3D image of protein spots shown in Figure 2A in accordance with embodiments of the invention.

[0018] Figure 2C is a graph illustrating spot intensity changes between baseline and day

16 in individual subjects from the representative 3D image of protein spots shown in Figure 2A.

[0019] Figure 3A illustrates serum protein changes for transferrin isoforms a representative 3D image of protein spots in accordance with embodiments of the invention.

[0020] Figure 3B is a graph illustrating the mean changes in intensity for each of the spots for transferrin isoforms from the representative 3D image of protein spots shown in Figure 3A in accordance with embodiments of the invention.

[0021] Figure 3C is a graph illustrating spot intensity changes between baseline and day

16 in individual subjects from the representative 3D image of protein spots shown in Figure 3 A in accordance with embodiments of the invention.

[0022] Figure 4A illustrates serum protein changes for hemopexin/albumin isoforms isoforms a representative 3D image of protein spots in accordance with embodiments of the invention.

[0023] Figure 4B is a graph illustrating the mean changes in intensity for each of the spots for hemopexin/albumin isoforms from the representative 3D image of protein spots shown in Figure 4A in accordance with embodiments of the invention.

[0024] Figure 4C is a graph illustrating spot intensity changes between baseline and day

16 in individual subjects from the representative 3D image of protein spots shown in Figure 4A in accordance with embodiments of the invention.

DETAILED DESCRIPTION

[0025] One or more specific embodiments of the present invention will be described below. In an effort to provide a concise description of these embodiments, all features of an actual implementation may not be described in the specification. It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation- specific decisions must be made to achieve the developers' specific goals, such as compliance with system-related and business-related constraints, which may vary from one implementation to another. Moreover, it should be appreciated that such a

development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

[0026] Certain aspects of the invention include methods of detecting the presence of exogenous Epo in a subject by detecting an aberrant level of at least one biomarker in a sample from the subject. Exemplary uses of the method include optimizing the therapeutic efficacy of exogenous Epo, such as in a clinical setting, or to detect exogenous Epo abuse, such as the abuse of exogenous Epo by athletes. For example, when the method is used for optimizing the therapeutic efficacy of exogenous Epo, if the level of the biomarker is below a lower threshold level, it would indicate a need to increase the dose of exogenous Epo. If the level is above another higher threshold, it would indicate a need to lower the dose of exogenous Epo. If the level is between a lower threshold and a higher threshold, it would indicate an optimal dose.

[0027] As used herein, the phrase "exogenous Epo" is understood to mean any Epo other than Epo naturally produced by the subject being tested, which includes, without limitation, Epo expressed as the result of gene doping and recombinant or other forms of Epo administered to the subject as well as Epo mimetics, and Epo that results from increases in Epo production and/or decreases in Epo clearance. In addition, the phrase "presence of exogenous Epo" is understood to include the presence of exogenous Epo from any source, such as recombinant human Epo, compositions that exert Epo-like activity by decreasing the clearance of endogenous Epo or increasing endogenous Epo production, and Epo mimetics. The phrase "presence of exogenous Epo" is also understood to include exogenous Epo that is the result of gene doping that increases Epo production or decreases Epo clearance, such as the transfection of an Epo gene into one or more cells in a subject that results in the cell producing the Epo protein using the transfected gene. The phrase "presence of exogenous Epo" is also understood to include the situations wherein the exogenous Epo has been cleared from the subject, but that had been present at an earlier time.

[0028] Also, as used herein, the phrase "aberrant level" is understood to mean levels of a biomarker that occur as a result of the presence of Epo in a subject that are different from levels of the biomarker in the absence of exogenous Epo in the subject. For example, an aberrant level of a biomarker may be detected as an increase or a decrease in the expression of the biomarker or the presence or absence of the biomarker. Additional exemplary methods of detecting aberrant levels of a biomarker are described in greater detail below.

[0029] Exemplary biomarkers include a protein, such as a serum protein, a carbohydrate, a lipid, a glycoprotein, a glycolipid, a protein fragment, a hormone, a steroid, a cytokine, a lymphokine, a chemokine, an immune modulator, a cell, a hematologic parameter, genomic expression from a cell in the sample, and combinations thereof. In one embodiment, the biomarker is an isoform of a serum protein, such as haptoglobin, transferrin, hemopexin, albumin, and combinations thereof. In some embodiments, the biomarker is an isoform of a protein. Isoform is understood to mean proteins that have undergone post-translational modifications. For example, a cell will generally transcribe and translate one gene into many related proteins. The differences in the proteins, called isoforms, are a result of alterations in the originally translated protein. There are over 800 known post-translational modifications which include, but are not limited to, protein cleavage fragments, phosphorylation, glycosylation, lipidation, crosslinking, protein folding, hydrogen bond interactions with other molecules, Van der Waals force interactions with other molecules, etc.

[0030] Examples of samples from a subjected that may be used for detecting biomarkers include whole blood, fractions of blood, such as the serum fraction, the plasma fraction, or the cellular fraction, such as white blood cells or red blood cells, other fluids (i.e., saliva, urine, cerebral spinal fluid, bile, extracellular fluid, cytosolic fluid, etc.), cells, tissues (such as skin, bone, muscle, hair, etc.), and combinations thereof.

[0031] Biomarker levels may be detected by analyzing a chemical parameter such as the size, charge, structure, and/or sequence of the biomarker, in addition to the association of the biomarker with other components. The aberrant level of the biomarker may also be detected with a compound that selectively binds the biomarker. The selective compound can include a polyclonal antibody, a monoclonal antibody, an antibody fragment, a receptor, a phage display, a peptide, a protein fragment, a nucleic acid, a sequence of nucleic acids, a ribonucleic acid, a deoxyribonucleic acid, a small molecule, and combinations thereof. For example, antibodies have been developed that selectively bind to specific isoforms of proteins, such as

phosphorylated protein with greater specificity than non-phosphorylated proteins are known. The selective compound may be coupled to or associated with a detection system to indicate the level of the detected biomarker. Exemplary detection systems may have elements that include, but are not limited to a test strip, chip, slide, microarray, titer plate, membrane, electrode, probe, bead, column, matrix, gel, and liquids. [0032] Various methods for analyzing biomarker levels can be used in combination. For example, the aberrant levels of the biomarker may be detected using gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, radio immune assay, protein blotting, a test strip, chromatography, liquid chromatography, gas chromatography, binding assays, mass spectrometry, microarray, genomic microarray, polymerase chain reaction (PCR), Reverse transcription PCR (RT-PCR), real time RT-PCR, and combinations thereof. In one embodiment, the size and charge of the biomarker is analyzed with two-dimensional gel electrophoresis that segregates the biomarkers, for example, by molecular weight and charge.

[0033] In one embodiment, the aberrant level of the biomarker is detected by comparing the level of the biomarker in at least two samples collected from the subject. The two samples can be collected at different times or from different tissues. The level of the biomarker from the first sample is compared with the level of the biomarker in the second sample. A difference in the level of the biomarker between the two samples indicates an aberrant level of the biomarker and the presence of exogenous Epo in the subject.

[0034] In one embodiment, the levels of the biomarker between the two samples is used to create a ratio. The ratio could indicate the normal or aberrant levels of the biomarker and thus the presence or absence of exogenous Epo.

[0035] In another embodiment, the aberrant level of the biomarker is detected by comparing the level of the biomarker with the level of a reference biomarker. Comparing the level of biomarker with the level of the reference biomarker can include determining the biomarker to reference biomarker ratio. This ratio could indicate the normal or aberrant levels of the biomarker and thus the presence or absence of exogenous Epo.

[0036] The reference biomarker can come from the same sample as the biomarker, or from a different sample. For example, the levels of both the biomarker and the reference biomarker may be measured in a sample of blood from the subject. Or the level of the biomarker may be measured in one sample and the reference biomarker measure in another sample from the subject. The two samples may come from the same tissue, such as the blood, that are collected at different time points, or come from different tissues, such as a blood sample and a hair sample, or combinations thereof. The reference biomarker may be a protein, a carbohydrate, a lipid, a glycoprotein, a glycolipid, a protein fragment, a nucleic acid, a sequence of nucleic acids, a ribonucleic acid, a deoxyribonucleic acid, a hormone, a steroid, a cytokine, a lymphokine, a chemokine, an immune modulator, a cell, a hematologic parameter, and combinations thereof. The hematologic parameter can be the concentration of hemoglobin, erythrocytes, iron, transferrin, ferritin, reticulocytes, creatinine, sodium, potassium, total albumin and haptoglobin, and combinations thereof. In one embodiment, the reference biomarker is an isoform of the biomarker. The difference in the ratio could also be used to optimize the therapeutic efficacy of exogenous Epo, such as when used or administered in a clinical setting.

[0037] In one embodiment, the reference biomarker is from another subject. "Another subject" in this context is understood to mean one or more other subjects and may represent the levels of reference biomarkers found in a population of subjects that are found to be indicative of either normal or aberrant levels of the biomarker in the population, and thus represent the presence or absence of exogenous Epo in the population.

[0038] In one embodiment, the ratio of biomarker to reference biomarker from a subject is compared with the ratio of biomarker to reference biomarker from another subject. Another subject in this context is understood to mean one or more other subjects and can represent the levels of ratios of biomarker to reference biomarkers found in a population of subjects that are found to be indicative of either normal levels of the biomarker, or represent the presence or absence of exogenous Epo in the population.

[0039] The reference biomarker can also come from another source, such as a virus, a microorganism, a non-human animal, a plant, and combinations thereof. The reference biomarker from another source could be produced or synthesized using standard procedures by chemical or biochemical techniques as are commonly used by both chemical and pharmaceutical companies such as by peptide synthesis, growing in bioreactors, and harvesting from organisms. The reference biomarker from another source can be used, for example, to standardize the level of biomarker in a sample. For example, the reference biomarker from another source could be used to establish a concentration curve for comparison with the level of biomarker in the sample so as to establish the actual levels of biomarker in the sample.

[0040] The invention also includes a kit for use in the methods described above for detecting the presence of exogenous Epo in a subject. Exemplary uses for the kit includes optimizing the therapeutic efficacy of exogenous Epo, such as when present in a clinical setting, or to detect Epo abuse, such as by athletes. The kit includes devices and reagents configured to detect normal or aberrant levels of a biomarker in a sample from the subject.

[0041] Various aspects of the present invention are further illustrated by the following non-limiting Example. EXAMPLE

[0042] Protein profiling provides a powerful method for characterizing the complete set of proteins expressed in a biological system. In this example, we found that the levels of several human serum proteins change following rHuEpo treatment. The changes in serum protein levels after treatment are an example of a method of detecting aberrant levels of a biomarker to detect the presence of exogenous Epo in a subject, which can be used, for example, to optimize the therapeutic efficacy of exogenous Epo, such as when present in a clinical setting, or to detect exogenous Epo abuse, such as by athletes.

Methods for the Example

Subjects

[0043] Eight healthy male volunteers (25 ± 4 yr, 183 ± 6 cm, 79 ± 7 kg, mean + SEM) were included in the present study. They did not take any medication and did not participate in competitive sports or training during the study period. All subjects gave a written informed consent before participating in the study, which was approved by the local human ethical committee of Copenhagen and Frederiksberg, Denmark (KF 01 269 637) in adherence to the declaration of Helsinki.

Study design

[0044] rHuEpo (epoetin β; NeoRecormon, Roche, Mannheim, Germany) was injected every second day (day 0, 2, 4, 6, 8, 10, 12, 14) subcutaneously at a dose of 5000 IU. Serum was collected approximately 1 week before the first Epo injection, and at days 8 (before the injection occurred on day 8) and 16 during the injection period. After clotting for 15 min at room temperature, serum was stored at -80°C for subsequent analysis. Also, blood pressure was monitored every second day throughout the study, and showed no indication of hypertension. Measurements of basic hematological parameters

[0045] Venous blood samples were analyzed for hemoglobin, erythrocytes, iron, transferrin, ferritin, reticulocytes, creatininum, sodium, and potassium using a Sysmex R-3000 (Sysmex Europe, Norderstedt, Germany). Furthermore, total albumin and haptoglobin was measured by Cobas c-systems (Roche Diagnostics, Mannheim, Germany).

Proteomics analysis

[0046] The procedures used for the proteomics analysis have been described previously and are briefly reviewed below.

Sample preparation

[0047] Before 2D electrophoresis, the serum samples were depleted of albumin and IgG using the Proteo Prep Blue Albumin & IgG Depletion kit (Sigma, St. Louis, MO). Serum samples containing 300μg of protein were diluted in sample buffer (7M urea, 2M thiourea, 1% w/v SB 3-10, 3% w/v CHAPS, 0.25% v/v Bio-Lyte 3/10 ampholytes (Bio-Rad Laboratories Inc., Hercules, CA)) containing 1.5% v/v protease inhibitor cocktail (Sigma, St. Louis, MO).

Disulfide bonds were reduced by addition of tributylphosphine and sulfhydryl groups were alkylated with iodoacetamide.

Two-dimensional gel electrophoresis ( 2DE)

[0048] For the first dimension, diluted and treated samples were loaded onto IPG strips

(isoelectric point (pi) 3-10 linear, Bio-Rad) and passively rehydrated for two hours. Then, strips were placed into a PROTEAN IEF cell (Bio-Rad) for isoelectric focusing consisting of 12 h of active rehydration at 250 V followed by separation at 4000 V for 60000 V h. The strips were then equilibrated for 45 min in equilibration buffer (0.375 M Tris-HCl pH 8.8, 6 M urea, 2% w/v SDS, and 20% v/v glycerol) and loaded on 15 % polyacrylamide gels. SDS-PAGE was run in a PROTEAN II XL cell (Bio-Rad) at 25 mA/gel and 270 V x h. Gels were fixed (40% ethanol, 2% acetic acid, 0.005% w/v SDS), washed three times (2% acetic acid, 0.005% w/v SDS), stained using SYPRO Orange (Molecular Probes, Inc., Eugene, OR), scanned and images captured using a PharosFX Plus Molecular Imager (Bio-Rad) with an excitation wavelength of 488 nm and emission detected at 605 nm.

Image analysis

[0049] Protein spots in the gels were matched using the image analysis software

PDQuest Advanced v. 8.0 (Bio-Rad) and all matches were confirmed manually. Protein spot intensities were normalized to the total image density in each gel, which depended on the total protein content of the sample. Protein spots displaying significant (p<0.05) intensity changes at the time points studied were manually excised from the gels and sent to Protea Biosciences Inc. Morgantown, WV for analysis by mass spectrometry (MS) and tandem-MS (MS/MS) using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) and MALDI-TOF-TOF.

Statistical analysis

[0050] The level of significance was set at p<0.05. Data were tested using a parametric repeated measure One-way ANOVA if data passed a normality test (Shapiro-Wilk). If non- normally distributed, a repeated measure ANOVA on ranks was used. Normally distributed data were also tested for sphericity (Mauchly's test), and Greenhouse-Geisser's correction factor was applied to non-spherical data. Tukey's HSD test was used for post-hoc comparisons. All spot intensity data were log transformed before analysis. SPSS v. 14.0 and SigmaPlot v. 11.0 were used for statistical analysis and graphical presentation. Results for the example

Hematological measurements

[0051] At baseline, all measured hematological parameters of the individuals were found to be within normal ranges (Table 1). At day 8 both the total number of reticulocytes and the number of reticulocytes per 1000 erythrocytes were up-regulated compared to baseline. The number did not change between day 8 and 16, and remained elevated at day 16. However, erythrocyte counts did not change at day 8 but increased significantly at day 16, resulting in a significantly increased hematocrit. Furthermore, hemoglobin also increased at day 16. In terms of iron related parameters, it was found that the levels of iron decreased significantly after Epo treatment (both at day 8 and 16). Transferrin levels were significantly increased at day 16, but the percentage of iron bound to transferrin was decreased at day 8 and 16. Also ferritin levels were significantly decreased at day 8 and 16. Haptoglobin levels were significantly decreased at day 8 and 16 compared to baseline. Total albumin did not change throughout the study

(Table 1).

Table 1; Clinical hematological values

Baseline Day 8 Day 16 P- value

B-hemoglobin (fe), mmol/1 9.24 ± 0.17 9.29 ± 0.16 9.68 ± 0.18 * 0.008, # 0.017

B-erythrocytes, billion/1 4.94 ± 0.08 4.97 ± 0.08 5.17 ± 0.06 * 0.003, # 0.008

B-erythrocytes, vol.fr. % 0.44 ± 0.01 0.45 ± 0.01 0.46 ± 0.01 * 0.01

Erythrocyte, MCV, fl 89.1 ± 0.8 89.6 + 1.1 89.0 ± 1.2 NS

Erythrocyte, MCHC, mmol/1 21.0 ± 0.1 20.9 ± 0.1 21.0 + 0.1 NS

P-iron, μπιοΐ/ΐ 19.9 ± 2.5 10.6 ± 1.6 9.5 ± 1.6 * <0.001, n <0.001

P-transferrin, μπιοΐ/ΐ 32.8 ± 1.0 34.8 ± 0.8 37.4 ± 1.1 * <0.01

P-transferrin, % iron binding 0.30 ± 0.04 0.15 ± 0.02 0.13 ± 0.02 * <0.001, n <0.001

P-ferritin, μg/l 114.6 ± 29.0 38.6 ± 10.7 24.2 ± 6.0 * <0.001, Π 0.002

Reticulocytes, 1/1000 erythrocytes 9.3 ± 0.6 20.6 ± 2.0 18.0 + 2.3 * <0.001, n <0.001

Reticulocytes, billion/1 46.1 ± 3.6 102.0 ± 10.0 91.9 ± 13.2 * <0.001, n <0.001

S-haptoglobin g/1 1.12 + 0.18 0.96 ± 0.15 0.85 ± 0.14 <0.001, n 0.022

S-albumin g/1 47.8 ± 1.0 48.3 ± 0.5 48.6 ± 1.2 NS

P-creatininum, mmol/1 0.088 ± 0.002 0.089 ± 0.002 0.090 ± 0.003 NS

P-sodium, mmol/1 144.6 ± 1.1 142.3 ± 0.4 141.8 ± 0.6 * 0.03 P-potassium, mmoM 3.98 ± 0.07 3.8 ± 0.08 4.0 ± 0.08 NS

S-basic phosphatase U/l 62.0 + 9.5 § 52.6 ± 3.9 56.3 + 2.8 NS

S-bilirubin μπιοΐ/ΐ 5.0 ± 0.6 § 5.6 ± 0.7 6.6 + 1.2 NS

S-LDH U/1 77.3 + 16.7 § 98.3 + 8.4 115.7 + 9.3 NS

Values shown as mean + SE. B = blood, P = plasma, S = serum MCV = mean corpuscular volume, MCHC = mean corpuscular hemoglobin, LDH = lactate dehydrogenase. * Baseline vs. day 16, it Baseline vs. day 8, # Day 8 vs. day 16, § (n=3)

Serum proteome patterns

[0052] The serum protein patterns observed for each subject were homogeneous.

Furthermore, the protein patterns were consistent in each subject before and after treatment with rHuEpo. Figure 1 shows a representative 2D gel. The high molecular weight region of the gel (>80 kDa) revealed low resolution of proteins, therefore, changes in proteins in this region were not analyzed.

Serum proteome changes after treatment with rHuEpo

[0053] A total of 97 proteins were analyzed in the serum 2D gels; among them, 92 were observed in all subjects. No protein intensities were altered after 8 days of rHuEpo treatment when compared to baseline. But the intensities of seven of the 92 spots were significantly down-regulated at day 16 (Figures 1, 2A-2C, 3A-3C, and 4A-4C). Six of these proteins were located in two different protein spot "trains", i.e. proteins with nearly the same molecular mass and varying iso-electric points (pis). Protein identities were determined by MS and MS/MS. Identity matches, scores, and sequence coverage values are shown in Table 2. The four proteins located in the ~75kDa "train" (A (p=0.014), B (p=0.006), C (p=0.013), and D (p=0.047)) were identified as haptoglobin, and displayed varying pis (A: -5.8, B: -6.0, C: -6.1, and D: -6.3). Additionally, the two proteins in the ~45kDa "train" (E (p=0.018) and F (p=0.047)), were identified as transferrin. The pis for spot E and F where -6.9 and -7.2, respectively. We have termed different versions of the same protein as "protein isoforms." Thus, we have detected four haptoglobin isoforms and two transferrin isoforms. Another protein that changed (G (p=0.050)) upon rHuEpo treatment was found to be a mixture of hemopexin and serum albumin (MW - 70 kDa and pi -6.3) (Table 2). Table 2: Mass spectrometry identity matches (significant spots)

Spot # Protein Uniprot # MS results MS/MS results

Max

Matched sequence Score Matched Max sequence Score fragments coverage (%) (Mascot) fragments coverage (%) (Mascot)

A Haptoglobin P00738 7/24 31 91 7/39 25 418

B Haptoglobin P00738 8/32 35 93 5/45 15 269

C Haptoglobin P00738 8/33 35 88 5/46 17 217

D Haptoglobin P00738 7/35 30 67 3/47 7 154

E Hemopexin P02790 12/39 36 120 7/53 25 651

Serum albumin P02768 10/39 22 68 3/53 7 50

F Transferrin P02787 17/41 31 186 10/56 21 707

G Transferrin P02787 19/38 36 202 10/53 21 643

Discussion of the results for the Example

[0054] The current study investigated the changes in serum protein profiles from healthy young men after rHuEpo treatment. Several isoforms of three different proteins were found to change significantly after 16 days of treatment.

Basic hematological results

[0055] The current study on the effects of rHuEpo administration in healthy young men resulted in significantly increased circulating levels of reticulocytes (immature erythrocytes) at 8 days. Our data show that iron levels, the levels of iron bound to transferrin, and ferritin levels were significantly decreased at day 8. Because iron is incorporated into maturing erythrocytes, when erythropoiesis is increased upon Epo stimulation, the need for iron also increases. This eventually leads to decreased iron binding to transferrin, a protein that transports iron from sites of absorption/heme degradation to those of storage and utilization. In addition, a decrease in ferritin indicates that less iron is stored in a soluble form in the blood. Furthermore, haptoglobin levels were decreased and total albumin levels unchanged at day 8.

[0056] The effects of Epo on blood parameters were more pronounced 16 days after rHuEpo treatment compared to day 8. The levels of reticulocytes, erythrocytes, hemoglobin, and hematocrit were significantly increased at day 16. The levels of transferrin were also

increased, but the percentage of iron bound to this protein was significantly decreased. These data are consistent with the decreased levels of iron found at day 8 and 16. Moreover, ferritin levels were still decreased at day 16, indicating less iron storage. Furthermore, haptoglobin levels stayed decreased at day 16 and total albumin levels were unchanged. Thus, as expected and in agreement with previous studies, our results show that 16 days of treatment with rHuEpo leads to a robust stimulation and production of red blood cells and utilization of iron.

Proteomic results

[0057] The proteomic data obtained in the current study showed no significant changes in serum protein intensities at day 8. However, at day 16, the levels of four isoforms of haptoglobin and one isoform of hemopexin were significantly decreased. Thus, the decrease in total haptoglobin is further supported by the decrease in four of its isoforms. Haptoglobin binds free plasma hemoglobin, thereby making hemoglobin accessible to degradative enzymes and preventing iron loss through the kidneys. Hemopexin binds heme and transports it to the liver for breakdown/recovery and prevents heme-mediated oxidative damage and heme-bound iron loss.

[0058] Both hemopexin and haptoglobin are class I acute-phase proteins which are produced primarily in the liver in relation to inflammation and hypoxia. Furthermore, interleukin (IL)-6 and IL-1 also induces these proteins. Recently, increased levels of Epo were shown to repress the expression of IL-Ιβ that accompanies traumatic brain injury, and repress IL-6 levels in an experimental autoimmune encephalomyelitis rat model. Hypoxia, leading to increased Epo secretion, has also been shown to induce the expression on a number of acute phase proteins, among them haptoglobin. Hypoxia and stimulation with IL-6 also lead to increased levels of both haptoglobin and hemopexin in a hapatocyte cell line. However, the increase in these acute phase proteins in response to hypoxia could not simply be attributed to autocrine IL-6 activation. Furthermore, studies on hemopexin and haptoglobin gene-disrupted mice suggest interactions between the hemopexin and haptoglobin in the clearance of hemoglobin and its oxidative product heme from the blood, but the mechanism responsible for this is currently unknown. Thus, there may be a link between the levels of hemopexin/haptoglobin with those of Epo that are mediated by IL-1 and IL-6. In the current study, IL-Ι β and IL-6 levels were found to be very low and not affected by the rHuEpo treatment. In agreement with our data, it has previously been shown that an acute bolus of rHuEpo does not lead to changed IL-6 levels. This agrees with the decrease in haptoglobin and hemopexin spot intensities measured in our subjects. Thus, rHuEpo might only lower cytokine levels when IL-Ιβ and IL-6 are elevated above normal levels.

[0059] Other possible explanations for a decrease in haptoglobin and hemopexin could be a decreased production in the liver due to decreased liver function or intravascular hemolysis. Liver function was evaluated by measurements of alanine-aminotransferase (ALAT) and basic phosphatase, both of which would be increased upon liver dysfunction. In the current study, almost all samples had ALAT concentrations below the detection level and the remaining in the lower part of the reference interval. Basic phosphatase levels were within normal range and did not change throughout the study. Thus, the decrease in haptoglobin and hemopexin are not due to altered liver function. LDH and bilirubin were measured in order to evaluate intravascular hemolysis. Both markers were within normal range and did not change throughout the study, thus, intravascular hemolysis is not the reason for the decreased levels of haptoglobin and hemopexin either. Together, these results suggest that the decrease in haptoglobin and hemopexin is most likely a result of the treatment with rHuEpo, however the underlying mechanism is unknown and merits further investigation.

In contradiction to the total levels of transferrin measured in the blood, we found that two isoforms of transferrin were significantly decreased at day 16. As stated above, transferrin is the major transporter of iron from its storage sites to the bone marrow.

[0060] According to our proteomics results, several isoforms of haptoglobin, transferrin and hemopexin, all proteins involved in transport of the degradation products of erythrocytes, are decreased. Erythrocytes normally circulate in the blood for 120 days before being degraded. Thus, although two weeks were sufficient to observe increased erythropoiesis; this time-point might be relatively early to detect the action of rHuEpo on degradation of these new erythrocytes.

[0061] The protein spot that was identified as hemopexin also contained a small amount of albumin. Although the serum samples were albumin-depleted before resolution by 2D gel electrophoresis, the procedure used for albumin-depletion is only -85% effective, thus, some albumin may still be present in the samples. Unfortunately, it is not possible to determine which of the two proteins (hemopexin and albumin) is responsible for the decrease in the intensity of this spot. Given that circulating albumin levels are positively correlated to total hemoglobin and that hemoglobin increased in the current study, it would have been expected that total albumin levels would increase. However, albumin levels were found to be stable throughout the study. As clearly shown for two transferrin isoforms, even though the total levels of a protein may vary in one direction, some of its isoforms might change in the opposite direction (or remain constant). Therefore, our conclusion is that levels of one or both of hemopexin and albumin in this particular spot have changed.

[0062] The change in the total amounts of these proteins, does not necessarily reflect the changes in individual isoforms of the particular protein, as was seen in the current study for transferrin. Different isoforms of a protein usually display mass and/or charge shifts generated primarily by post translational modifications such as protein cleavage, side-chain residue modifications (e.g. phosphorylation, sulfonation, oxidation), glycosylation and many others, that may induce a change in activity in the modified protein.

New biomarkers for a future doping test

[0063] The seven protein spots identified in our study show clear intensity variations in response to rHuEpo treatment. As such, these protein isoforms may be used as biomarkers to detect Epo doping.

CONCLUSION

[0064] A total of seven protein spots were found to be significantly decreased after 16 days of treatment with rHuEpo. Their identities were determined by MS analysis, and found to correspond to four isoforms of haptoglobin, two isoforms of transferrin and one isoform of hemopexin/albumin. These proteins are closely related to the transport of degradation products of erythrocytes after hemolysis. These proteins are new targets for detecting the presence of Epo in a subject such as would be useful in optimizing clinical efficacy or detecting Epo doping since there was a significant change of these proteins among all the subjects tested.

[0065] While the present invention has been disclosed by reference to the details of preferred embodiments of the invention, it is to be understood that the disclosure is intended as an illustrative rather than in a limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the invention and the scope of the amended claims.

WHAT IS CLAIMED IS:

Claims

1. A method for detecting the presence of exogenous erythropoietin in a subject comprising detecting an aberrant level of a biomarker in a sample from the subject wherein the aberrant level of the biomarker is indicative of the presence of exogenous erythropoietin.

2. The method of claim 1 wherein the biomarker is a protein, a carbohydrate, a lipid, a glycoprotein, a glycolipid, a protein fragment, a hormone, a steroid, a cytokine, a

lymphokine, a chemokine, an immune modulator, a cell, a hematologic parameter, a nucleic acid, a sequence of nucleic acids, a ribonucleic acid, a deoxyribonucleic acid, and

combinations thereof.

3. The method of claim 1 wherein the biomarker is a serum protein.

4. The method of claim 1 wherein the biomarker is a plasma protein.

5. The method of claim 1 wherein the biomarker is a blood protein.

6. The method of claim 1 wherein the biomarker is at least one of haptoglobin, transferrin, hemopexin, and albumin.

7. The method of claim 1 wherein the sample is whole blood, a fraction of blood, a serum fraction of blood, a plasma fraction blood, a cellular fraction of blood, saliva, urine, cerebral spinal fluid, bile, extracellular fluid, cytosolic fluid, cells, tissues, skin, bone, muscle, hair, and combinations thereof.

8. The method of claim 7 wherein the blood fraction is at least one of serum or plasma.

9. The method of claim 1 wherein the aberrant level of the biomarker is detected by analyzing a chemical parameters of the biomarker including its size, charge, structure, sequence, and combinations thereof.

10. The method of claim 1 wherein the aberrant level of the biomarker is detected with a compound that selectively binds or interacts with the biomarker.

11. The method claim 10 wherein the selective compound includes a polyclonal antibody, a monoclonal antibody, an antibody fragment, a receptor, a phage display, a peptide, a protein fragment, a small molecule, a nucleic acid, a sequence of nucleic acids, a ribonucleic acid, a deoxyribonucleic acid, and combinations thereof.

12. The method of claim 10 wherein the selective compound is coupled to or associated with a detection system.

13. The method of claim 12 wherein the detection system includes at least one of a test strip, a chip, a slide, a microarray, a titer plate, a membrane, an electrode, a probe, a bead, a column, a matrix, a gel, liquid reagent, and combinations thereof.

14. The method of claim 1 wherein the aberrant level of the biomarker is detected by gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, protein blot, a test strip, chromatography, liquid chromatography, gas chromatography, binding assay, radio immune assay, a probe, a chip, mass spectrometry, a microarray, genomic microarray, polymerase chain reaction (PCR), Reverse transcription PCR (RT-PCR), real time RT-PCR, and combinations thereof.

15. The method of claim 14 wherein the gel electrophoresis is two dimensional gel electrophoresis.

16. The method of claim 1 wherein the sample is a first sample and the aberrant level of the biomarker is detected by comparing the level of the biomarker in the first sample with the level of the same biomarker in a second sample from the subject, wherein the first and second samples are collected at different times.

17. The method of claim 1 wherein the aberrant level of the biomarker is detected by comparing the biomarker to the level of a reference biomarker.

18. The method of claim 17 wherein the reference biomarker is from at least one of the first sample or a second sample.

19. The method of claim 1 wherein the sample is a first sample and the aberrant level of the biomarker in the first sample is detected by determining the ratio between the level of the biomarker and the level of a reference biomarker.

20. The method of claim 19 further comprising determining the biomarker to reference biomarker ratio for at least a second sample and comparing the biomarker to reference biomarker ratios from the first sample and the second sample.

21. The method of claim 20 wherein the second sample is from a second subject.

22. The method of claim 17 wherein the reference biomarker includes a protein, a carbohydrate, a lipid, a glycoprotein, a glycolipid, a protein fragment, a hormone, a steroid, a cytokine, a lymphokine, a chemokine, an immune modulator, a cell, a hematologic parameter, a nucleic acid, a sequence of nucleic acids, a ribonucleic acid, a deoxyribonucleic acid, and combinations thereof.

23. The method of claim 22 wherein the hematologic parameter is the concentration of protein, hemoglobin, erythrocytes, iron, transferrin, ferritin, reticulocytes, creatinine, sodium, potassium, and total levels of albumin, hemopexin, and/or haptoglobin, and combinations thereof.

24. The method of claim 17 wherein the reference biomarker is an isoform of the biomarker.

25. The method of claim 1 wherein the aberrant level of the biomarker is detected by comparing the level of the biomarker to the level of at least one of a biomarker or a reference biomarker from a second subject.

26. The method of claim 1 wherein the aberrant level of the biomarker is detected by comparing the level of the biomarker to the level of a reference biomarker from another source.

27. The method of claim 26 wherein the another source is from a virus, a microorganism, a non-human animal, a plant, and combinations thereof.

28. A kit for detecting the presence of erythropoietin in a subject comprising a device configured to detect an aberrant level of a biomarker in a sample from the subject.

29. The kit of claim 28 wherein the biomarker is a protein, a carbohydrate, a lipid, a glycoprotein, a glycolipid, a protein fragment, a nucleic acid, a sequence of nucleic acids, a ribonucleic acid, a deoxyribonucleic acid, a hormone, a steroid, a cytokine, a cell, a hematologic parameter, and combinations thereof.

30. The kit of claim 28 wherein the biomarker is a serum protein or a plasma protein.

31. The kit of claim 28 wherein the serum protein is at least one of haptoglobin, transferrin, hemopexin, and albumin.

32. The kit of claim 28 wherein the serum protein is at least one isofom of haptoglobin, transferring, hemopexin, and albumin.

33. The kit of claim 28 wherein the sample is whole blood, a fraction of blood, a serum fraction of blood, a plasma fraction blood, a cellular fraction of blood, saliva, urine, cerebral spinal fluid, bile, extracellular fluid, cytosolic fluid, cells, tissues, skin, bone, muscle, hair, and combinations thereof.

34. The kit of claim 28 wherein the blood fraction is at least one of serum or plasma.

35. The kit of claim 28 wherein the device analyze at least one chemical parameter of the biomarker including the size, charge, structure, and/or sequence of the biomarker.

36. The kit of claim 28 wherein the device includes a compound that selectively binds or interacts with the biomarker.

37. The kit of claim 36 wherein the selective compound includes at least one of a polyclonal antibody, a monoclonal antibody, an antibody fragment, receptor, a phage display, a peptide, a protein fragment a nucleic acid, a sequence of nucleic acids, a ribonucleic acid, a deoxyribonucleic acid, a small molecule, and combinations thereof.

38. The kit of claim 28 further including at least one of a test strip, a chip, a slide, a microarray, a titer plate, a membrane, an electrode, a probe, a bead, a column, a matrix, a gel, liquid reagent, and combinations thereof.

39. The kit of claim 28 wherein the device includes at least one of a test strip, a gel electrophoresis apparatus, a ELISA apparatus, an immunoprecipitation apparatus, a chromatography apparatus, a liquid chromatography apparatus, a gas chromatography apparatus, a binding assay apparatus, radio immune assay, a probe, a chip, a mass spectrometer, a microarray, a genomic microarray, a polymerase chain reaction (PCR) apparatus, Reverse transcription PCR (RT-PCR) apparatus, real time RT-PCR apparatus, and combinations thereof.

40. The kit of claim 28 wherein the device is configured to detect the level of the biomarker in a first sample and a second sample.

41. The kit of claim 28 wherein device is configured to compare the level of the biomarker with the level of a reference biomarker.

42. The kit of claim 28 wherein device is configured to compare a ratio between the level of the biomarker and the level of a reference biomarker from a first sample with the ratio between the level of the biomarker and the level of a reference biomarker from a second sample.