Systems and methods for predicting fracture risk using circulating f2-isoprostanes

Abstract

Blood plasma samples are obtained from type 2 diabetes (T2D) patients and a concentration of F2-isoprostanes (F2I) in the sample is quantified. Plasma F2I levels assess oxidative stress levels in the patient and indicate elevated risk of T2D-associated complications including incident clinical fracture, cardiovascular complications, renal disease, etc. in the T2D cohort, independently of other risk factors. Fracture risk in the T2D population is heightened for a given bone mineral density (BMD) and is underestimated by evaluation tools such as Fracture Risk Assessment Tool (FRAX). The patient's BMD T-score can be adjusted based on F2I concentrations, capturing reductions in bone quality as well as quantity that lead to the elevated fracture risk in T2D patients. These diagnostic procedures can be effectively deployed in a patient's course of treatment merely by supplementing an existing blood panel, rather than with a separate blood draw.

Description

SYSTEMS AND METHODS FOR PREDICTING FRACTURE RISK USING CIRCULATING F2-ISOPROSTANESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional PatentApplication No. 63 / 604,287, filed November 30, 2023, which is incorporated by reference as if disclosed herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with U.S. Government support under Grant Number AG075654 awarded by the National Institutes of Health. The United States Government has certain rights in the invention.

[0003] This invention was made with government support Grant Number C210154 awarded by Empire State Development's Division of Science, Technology and Innovation (NYSTAR). The government may have certain rights in the invention.BACKGROUND

[0004] Globally, there are approximately 529 million people currently living with diabetes, a great majority of cases being type 2 diabetes (T2D). Almost 98% of T2D individuals have at least one diabetic complication, such as bone fragility, cardiovascular complications, end-stage renal disease, neuropathy, retinopathy, etc. Diabetic complications are responsible for the major individual health and society economic burdens associated with diabetes, leading to increased morbidity, impaired quality of life, and profound demand on health care services.

[0005] One of the comorbidities that is gaining more awareness is bone fragility. Individuals with T2D have increased fracture risk, despite having normal or increased bone mineral density (BMD). The prediction of skeletal outcomes from the fall events remains challenging and is particularly relevant for T2D patients. The underprediction of fracture risk among T2D individuals leads to improper preventive treatment strategies, increasing morbidity and mortality, which further results in significant increase in healthcare cost. In addition, individuals with T2D have a higher tendency of experiencing fall events, due to other diabetic complications such as neuropathy, retinopathy, and complications from medication use.

[0006] Current standard of care tools evaluating fracture risk, including Fracture Risk Assessment Tool (FRAX) and other BMD-based assessments, fail to account for the pathogeneses of diabetes-induced bone fragility, and therefore underestimate the fracture risk in T2D. Therefore, other aspects of bone quality beyond BMD that can be measured clinically should be used to account for T2D-related bone fragility. For example, elevated oxidative stress in T2D plays a role in developing diabetic comorbidities, both microvascular and macrovascular. Oxidative stress is caused by the mitochondrial overproduction of reactive oxygen species, including free oxygen radicals, whose accumulation in cells and tissues cannot be detoxified by the antioxidants in the biologic systems. There is a growing interest in developing surrogate markers that can be used to evaluate the efficacy of treatments of diabetic and fragility fractures.

[0007] There is a need to identify other clinically measurable biomarkers independent of BMD to improve T2D fracture prediction and inform strategies to better manage T2D fractures.SUMMARY

[0008] Aspects of the present disclosure are directed to methods of treating diabetes- related complications in a patient. In some embodiments, the methods include obtaining a sample from a patient suspected of having or known to have type 2 diabetes (T2D); quantifying a concentration of F2-isoprostanes (F2I) in the sample; diagnosing an elevated risk of diabetes- related complications in the patient in response to elevated concentrations of F2I; and administering a treatment to the patient corresponding to the elevated risk of diabetes-related complications.

[0009] In some embodiments, diagnosing the elevated risk of diabetes-related complications in the patient includes comparing the concentration of F2I with a threshold F2I concentration value and confirming an elevated fracture risk for bones in the patient when the concentration of F2I is greater than or equal to the threshold F2I concentration value. In some embodiments, the threshold F2I concentration value is about 41 pg / mL.

[0010] In some embodiments, administering the treatment to the patient corresponding to the elevated risk of diabetes-related complications in the patient includes determining a bone mineral density (BMD) T-score for the patient and reducing the BMD T-score of the patient by a predetermined standard deviation (SD). In some embodiments, the predetermined SD is between 0.5 and about 0.8 when the concentration of F2I in the sample is between about 41 pg / mL and about 54 pg / mL. In some embodiments, the predetermined SD is between about 0.8and about 1.2 when the concentration of F2I in the sample is between about 54 pg / mL and about 68 pg / mL. In some embodiments, the predetermined SD is at least 1.2 when the F2I concentration in the sample is at least 68 pg / mL.

[0011] In some embodiments, the diabetes-related complications include bone fracture, cardiovascular complications, renal disease, or combinations thereof. In some embodiments, the sample includes blood plasma. In some embodiments, the treatment includes an effective amount of a therapeutic compound. In some embodiments, the treatment includes prophylactic treatment of the patient for decreased BMD.

[0012] Aspects of the present disclosure are directed to methods of monitoring treatment of diabetes-related complications including obtaining a first sample from a T2D patient at a first instance; quantifying a concentration of F2I in the first sample; diagnosing an elevated risk of diabetes-related complications in the patient in response to elevated concentrations of F2I; administering a first treatment to the patient corresponding to the elevated risk of diabetes- related complications; obtaining a second sample from the T2D patient at a second instance; quantifying a concentration of F2I in the second sample; and administering a second treatment to the patient corresponding to a change in concentration of F2I from the first sample to the second sample. In some embodiments, the first sample, the second sample, or combinations thereof, includes blood plasma.

[0013] In some embodiments, diagnosing the elevated risk of diabetes-related complications in the patient includes comparing the concentration of F2I in the first sample with a threshold F2I concentration value and confirming an elevated fracture risk for bones in the patient when the concentration of F2I in the first sample is greater than or equal to the threshold F2I concentration value. In some embodiments, administering the first treatment to the patient corresponding to the elevated risk of diabetes-related complications includes determining a BMD T-score for the patient and reducing the BMD T-score of the patient by a predetermined SD when the concentration of F2I in the first sample is above the threshold F2I concentration value, wherein the predetermined SD is at least 0.5. In some embodiments, administering the second treatment to the patient corresponding to the change in concentration of F2I from the first sample to the second sample includes reducing the BMD T-score of the patient by at least 0.8 SD when the concentration of F2I in the second sample is above 54 pg / mL. In some embodiments, administering the second treatment to the patient corresponding to the change in concentration of F2I from the first sample to the second sample includes reducingthe BMD T-score of the patient by at least 1.2 SD when the concentration of F2I in the second sample is above 68 pg / mL.

[0014] Aspects of the present disclosure are directed to methods of treating diabetes- related complications in a patient. In some embodiments, the method includes obtaining a blood plasma sample from a T2D patient; quantifying a concentration of F2I in the blood plasma sample; determining a BMD T-score for the patient; reducing the BMD T-score of the patient by a predetermined SD; administering a treatment to the patient corresponding to an elevated risk of bone fracture; monitoring for an increased F2I concentration in the patient over time; and modifying the treatment for increased risk of diabetes complications in the patient corresponding to the increased F2I concentration.

[0015] In some embodiments, the BMD T-score of the patient is reduced by about 0.5 when the concentration of F2I in the sample is greater 41 pg / mL. In some embodiments, the BMD T-score of the patient is reduced by about 0.8 when the concentration of F2I in the sample is greater than 54 pg / mL. In some embodiments, the BMD T-score of the patient is reduced by about 1.2 when the concentration of F2I in the sample is greater than 68 pg / mL.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The drawings show embodiments of the disclosed subject matter for the purpose of illustrating the invention. However, it should be understood that the present application is not limited to the precise arrangements and instrumentalities shown in the drawings, wherein:

[0017] FIG. l is a chart of a method of treating diabetes-related complications in a patient according to some embodiments of the present disclosure;

[0018] FIG. 2 is a chart of a method of treating diabetes-related complications in a patient according to some embodiments of the present disclosure;

[0019] FIG. 3 is a chart of a method of treating diabetes-related complications in a patient according to some embodiments of the present disclosure;

[0020] FIG. 4 is a chart of a method of monitoring treatment of diabetes-related complications according to some embodiments of the present disclosure;

[0021] FIG. 5 is a graph portraying 10-year absolute incident clinical fracture risk plotted against femoral neck bone mineral density (BMD) T-score among type 2 diabetes (T2D) andnon-diabetes patients, demonstrating that a patient’s F2-isoprostanes (F2I) levels according to some embodiments of the present disclosure captures the elevated fracture risk in T2D patients;

[0022] FIG. 6 is a graph portraying Kaplan-Meier survival analysis demonstrating significantly increasing rates of fracture with increasing F2I levels in T2D patients but not in non-diabetes patients; and

[0023] FIG. 7 is a graph showing negative correlations between increased F2I concentrations and baseline total hip BMD in both T2D patients and non-diabetes patients, as well as the negative correlation between increased F2I concentrations and 4-year percent change in total hip BMD in T2D patients but not in non-diabetes patients.DETAILED DESCRIPTION

[0024] Referring now to FIG. 1, some embodiments of the present disclosure are directed to a method 100 of treating diabetes-related complications in a patient. In some embodiments, at 102, a sample is obtained from the patient. In some embodiments, the patient has type 2 diabetes (T2D). In some embodiments, the patient is at least suspected of having T2D. In some embodiments, the sample is any suitable sample from the patient, e.g., tissue sample, blood sample, etc., or combinations thereof. In some embodiments, the sample is a blood plasma sample. In some embodiments, the sample is obtained via any suitable means, e.g., traditional blood draw, etc. At 104, a concentration of F2-isoprostanes (F2I) in the sample is quantified. In some embodiments, quantifying 104 occurs through any suitable process or combination of processes. In some embodiments, quantifying 104 occurs via a blood panel. In some embodiments, quantifying 104 is performed on a blood plasma sample from a previous blood draw from the patient.

[0025] Still referring to FIG. 1, at 106, an elevated risk of diabetes-related complications is diagnosed in the patient in response to elevated concentrations of F2I. In some embodiments, the diabetes-related complications include bone fracture, cardiovascular complications, renal disease, etc., or combinations thereof. In some embodiments, diagnosing 106 includes comparing the concentration of F2I quantified in the sample at 104 with a threshold F2I concentration value, and then confirming the elevated risk of diabetes-related complications, e.g., elevated fracture risk for bones in the patient, when the concentration of F2I is greater than or equal to the threshold F2I concentration value. In some embodiments, the threshold F2I concentration value is greater than about 40 pg / mL. In some embodiments, the threshold F2I concentration value is about 41 pg / mL. In some embodiments, the threshold F2I concentrationvalue is greater than about 50 pg / mL. In some embodiments, the threshold F2I concentration value is about 54 pg / mL. In some embodiments, the threshold F2I concentration value is greater than about 60 pg / mL. In some embodiments, the threshold F2I concentration value is about 68 pg / mL.

[0026] Referring again to FIG. 1, at 108, a treatment is administered to the patient corresponding to the elevated risk of diabetes-related complications. In some embodiments, the treatment is any intervention or combination of interventions directed to treating the diabetes- related complication. Additionally, the particular treatment is tailored to account for the particular risk profile for diabetes-related complications in the patient as determined by the concentration of F2I, e.g., more aggressive treatments can be administered to patients with high or rapidly increasing concentrations of F2I and thus particularly elevated risk profiles. In some embodiments, treatment can be recommended early including prophylactic treatment of the patient for decreased BMD and / or increased fracture risk, for example, bisphosphonates, denosumab, romosozumab, etc. In some embodiments, patients with at least a low risk of elevated diabetes-related complications are administered treatment at 108. In some embodiments, patients with a low elevated risk of diabetes-related complications have a concentration of F2I between about 41 pg / mL and about 54 pg / mL. In some embodiments, patients with a medium elevated risk of diabetes-related complications have a concentration of F2I between about 54 pg / mL and about 68 pg / mL. In some embodiments, patients with a high elevated risk of diabetes-related complications have a concentration of F2I above about 68 pg / mL.

[0027] Referring now to FIG. 2, in some embodiments, administering 108 includes determining 202 a bone mineral density (BMD) T-score for the patient. The patient’s BMD T- score compares the BMD of the patient to that of the average young healthy human adult, with T-scores between -1 standard deviations (SD) and 1 SD generally indicating normal bone density. In some embodiments, at 204, the BMD T-score of the patient is reduced by a predetermined SD. This adjusted BMD T-score, based on patient’s F2I concentration, captures the pathology where individuals with T2D can exhibit significant increases in bone fracture risk, despite having the same or better BMD compared with an otherwise comparable individual without T2D. The adjusted T-score better reflects the degradation of bone quality in T2D patients, which is not captured in the traditional T-score or bone fracture risk evaluation tools such as Fracture Risk Assessment Tool (FRAX), as will be discussed in greater detail below. In some embodiments, the predetermined SD is between 0.5 and about 0.8 when the concentration of F2I in the sample is between about 41 pg / mL and about 54 pg / mL. In some embodiments, thepredetermined SD is between about 0.8 and about 1.2 when the concentration of F2I in the sample is between about 54 pg / mL and about 68 pg / mL. In some embodiments, the predetermined SD at least 1.2 when the F2I concentration in the sample is at least 68 pg / mL. For example, a T2D individual having a T-score of -1.3 but having a concentration of F2I above 68 pg / mL would have a corresponding adjusted T-score of at least -2.5. As discussed in greater detail below, this is the equivalent of the difference in fracture risk between a patient with mild osteopenia and a patient with osteoporosis. Clinicians can then utilize the adjusted T-score to treat the T2D patient with interventions commensurate with that corresponding elevated level of risk.

[0028] 8-epi-prostaglandin F2a, known as F2-isoprostanes, are a group of prostaglandinlike compounds formed due to arachidonic acid's free radical oxidation. F2-isoprostanes are a reliable indicator of lipid peroxidation and can be used to assess patient systemic oxidative stress levels. F2-isoprostanes has been implicated as a risk marker for multiple disease conditions. For example, a case-control study demonstrated an independent 30.8-fold increase in the risk of developing coronary heart disease in individuals with high F2-isoprostanes values. As a direct measurement for lipid peroxidation, F2-isoprostanes cause inflammation and induce vasoconstriction in multiple tissues and organs.

[0029] One of the overlooked potential contributors to bone fragility during T2D is elevated oxidative stress. Increased oxidative stress levels in T2D play a role in multiple diabetic complications. In bone, oxidative stress negatively impacts osteoblasts, osteocytes, and osteoclasts, compromising bone mineralization and accelerating bone loss. For example, reduced bone formation in T2D coincided with the significant elevation of 8- hydroxydeoxyguanosine, an oxidative stress marker, in a non-obese T2D rat model. Further, in the postmenopausal osteoporosis population, the levels of oxidative stress have been associated with a reduction of BMD and an increased hip fracture risk. In addition, oxidative stress also increases accumulation of advanced glycoxidation end-products (AGOEs) in bone, which negatively impact bone fragility during T2D, and are associated with T2D fracture risk. Notably, the association between baseline concentration of F2I and incident clinical fracture risk in T2D is independent of baseline BMD, indicating that oxidative stress in T2D results in the decline of bone quality and not just bone quantity, as will be discussed in greater detail below.

[0030] The extent of oxidative stress in T2D, reflected in circulating levels of F2- isoprostanes, dramatically impacts fracture outcomes within the T2D population. Specifically, for each SD increase in plasma F2-isoprostanes, fracture risk can increase by 93%. Thus, theembodiments of the present disclosure determine and monitor F2I concentrations in T2D patients, enabling clinicians to diagnosis and predict diabetic complications including bone fragility early, and tailor treatment strategies on the basis of personalized medicine.

[0031] Referring now to FIG. 3, some embodiments of the present disclosure are directed to a method 300 of treating diabetes-related complications in a patient. In some embodiments, at 302, a blood plasma sample is obtained from a T2D patient. As discussed above, in some embodiments, the blood plasma sample can be a fresh blood draw, a sample drawn during a previous procedure, etc. At 304, a concentration of F2I is quantified in the blood plasma sample. At 306, a bone BMD T-score for the patient is determined. At 308, the BMD T-score of the patient is reduced by a predetermined SD to arrive at the patient’s adjusted T-score. In some embodiments, the BMD T-score is reduced 308 by about 0.5 when the concentration of F2I in the sample is greater 41 pg / mL. In some embodiments, the BMD T-score is reduced 308 by about 0.8 when the concentration of F2I in the sample is greater than 54 pg / mL. In some embodiments, the BMD T-score is reduced 308 by about 1.2 when the concentration of F2I in the sample is greater than 68 pg / mL. At 310, a treatment is administered to the patient corresponding to an elevated risk of bone fracture. In some embodiments, treatment can be recommended early including prophylactic treatment of the patient for decreased BMD and increased fracture risk, for example, bisphosphonates, denosumab, romosozumab, etc. In some embodiments, the treatment administered at 310 further targets increased risk of cardiovascular complications, renal disease, or combinations thereof. At 312, the concentration of F2I in the patient is monitored over time. In the event that the concentration of F2I is unaffected by the treatments at 310, or otherwise maintains its level or increases, in some embodiments, at 314, the treatment for increased risk of diabetes complications is modified, e.g., corresponding to an increased F2I concentration.

[0032] Referring now to FIG. 4, some embodiments of the present disclosure are directed to a method 400 of monitoring treatment of diabetes-related complications. In some embodiments, at 402, a first sample is obtained from a T2D patient at a first instance. As discussed above, in some embodiments, the first sample includes blood plasma. At 404, a concentration of F2I is quantified in the first sample. At 406, an elevated risk of diabetes-related complications in the patient is diagnosed in response to elevated concentrations of F2I. At 408, a first treatment is administered to the patient corresponding to the elevated risk of diabetes-related complications.

[0033] As discussed above, in some embodiments, the diabetes-related complications include bone fracture, cardiovascular complications, renal disease, or combinations thereof. In some embodiments, diagnosing 406 the elevated risk of diabetes-related complications in the patient includes comparing the concentration of F2I in the first sample with a threshold F2I concentration value and confirming an elevated fracture risk for bones in the patient when the concentration of F2I in the first sample is greater than or equal to the threshold F2I concentration value. In some embodiments, the threshold F2I concentration value is greater than about 40 pg / mL. In some embodiments, the threshold F2I concentration value is about 41 pg / mL. In some embodiments, the threshold F2I concentration value is greater than about 50 pg / mL. In some embodiments, the threshold F2I concentration value is about 54 pg / mL. In some embodiments, the threshold F2I concentration value is greater than about 60 pg / mL. In some embodiments, the threshold F2I concentration value is about 68 pg / mL. In some embodiments, administering 406 the first treatment to the patient corresponding to the elevated risk of diabetes-related complications includes determining a BMD T-score for the patient and reducing the BMD T-score of the patient by a predetermined SD when the concentration of F2I in the first sample is above the threshold F2I concentration value. In some embodiments, the predetermined SD is at least 0.5.

[0034] Still referring to FIG. 4, at 410, a second sample is obtained from the T2D patient at a second instance. In some embodiments, the second sample includes blood plasma. At 412, a concentration of F2I is quantified in the second sample. At 414, a second treatment is administered to the patient corresponding to a change in concentration of F2I from the first sample to the second sample. In some embodiments, administering 414 the second treatment to the patient corresponding to the change in concentration of F2I from the first sample to the second sample includes reducing the BMD T-score of the patient by at least 0.8 SD when the concentration of F2I in the second sample is above 54 pg / mL. In some embodiments, administering 414 the second treatment to the patient corresponding to the change in concentration of F2I from the first sample to the second sample includes reducing the BMD T- score of the patient by at least 1.2 SD when the concentration of F2I in the second sample is above 68 pg / mL.

[0035] Referring now to FIG. 5, a longitudinal study population was selected from the Health, Aging, and Body Composition (Health ABC) study. The study was designed to evaluate body composition alterations with the aging process and investigate whether the changes explain the decline in function for the healthier older population. The cohort was recruited at two medical centers (University of Pittsburgh, Pittsburgh, PA, and University of Memphis,Memphis, TN) between 1997 and 1998. The study enrolled 3,075 black and white individuals with a baseline age of 70-79 years. Individuals with difficulty walking % mile or climbing up ten steps were excluded.

[0036] A subset of participants previously chosen for the random sub-cohort of a casecohort study who had plasma F2-isoprostane levels measured in stored baseline serum was isolated for further analysis. The subset was a random sample of those enrolled at baseline (N = 743), stratified by sex, race and baseline diabetes status. Baseline diabetes was defined by meeting any of the following criteria: (1) self-reported diagnosis of diabetes; (2) use of hypoglycemic medications; (3) fasting blood glucose test > 126 mg / dL; (4) oral glucose tolerance test (2 -hour plasma glucose) > 200 mg / dL. 14 participants were excluded due to lack of F2-isoprostane results, 15 due to lack of baseline data on glycated hemoglobin (HbAlc), and 11 due to lack of baseline BMD, leaving a study cohort population of 703, among which 132 individuals had baseline diabetes. In the study cohort, 521 participants had four-year followup BMD data available (T2D: N = 92; non-diabetes: N = 429).

[0037] From the collected venous blood, plasma was separated via centrifugation and stored at -80°C until analysis. F2-isoprostanes from plasma were analyzed using a gas chromatography-mass spectrometry-based method (Agilent 6890 Series GC and an Agilent 5973N Mass Selective Detector) at the Molecular Epidemiology and Biomarker Research Laboratory (MEBRL, University of Minnesota, Minneapolis, MN). The analytical variance of the assay was within 10%, and the coefficient of variation from the blind duplicate measurements among 5% of the subjects was 6.96%. Prior studies have shown that F2- isoprostanes are stable under these collection and storage conditions.

[0038] Every six months during the study period, the study participants were asked by phone call or during a clinical visit regarding the occurrence of a fracture for a follow-up period of up to 17.3 years. Reported fractures were verified through radiology reports. Fractures that occurred at the ribs, chest / sternum, skull / face, fingers, toes, and cervical vertebra, as well as pathologic fractures were excluded. The follow-up period was defined as the time between the baseline visit and the first recorded fracture for those who fractured, and overall study time for those who did not fracture.

[0039] All demographic information was self-reported. During the baseline visit, the participants’ height and weight were measured, and body mass index (BMI) was calculated as weight by height squared (kg / m2). Participants were asked to bring over-the-counter and prescription medications taken during the previous week. These medications were classified andcoded according to the Iowa Drug Information System. Dual energy x-ray absorptiometry (DXA) (QDR 4500A, software version 9.03; Hologic, Inc., Bedford, MA) was used to quantify bone mineral density (BMD) at the proximal femur. DXA quality assurance measurements, including use of daily and cross-calibration phantoms, were performed at both study sites to ensure scanner reliability. The CV for total hip BMD was 0.44% at Memphis and 0.41% at Pittsburgh, after applying a couple longitudinal corrections. For the study cohort, total hip BMD was recorded both at baseline and four-year follow-up.

[0040] Baseline HbAlc was measured from a blood sample utilizing the principles of HPLC (Biorad Variant, Bio-Rad Laboratories, Hercules, CA), performed at Fletcher Allen Health Care MCHV campus at Burlington, Vermont. Serum carboxymethyl-lysine (CML) was measured by a competitive enzyme-linked immunosorbent assay (ELISA) specific to CML (AGE-CML ELISA; Microcoat, Penzberg, Germany); Pentosidine (PEN) from urinary hydrolysate samples was quantified using high-performance liquid chromatography (HPLC). PEN data was available on a subset (N=95 for T2D, N=118 for non-diabetes). Both CML and PEN are stable during collection and storage for reliable measurements.

[0041] Bone turnover markers, including procollagen type 1 N-terminal propeptide (P1NP) and osteocalcin (OC) as markers of bone formation, as well as C-terminal telopeptide of type I collagen (CTX) as the marker of bone resorption, were measured in baseline serum in a subset of participants. A competitive radioimmunoassay (Orion Diagnostica UniQ, Espoo, Finland) was used to quantify serum intact P1NP. Serum CTX was measured using a sandwich ELISA (Serum CrossLaps; Nordic Biosciences, Herlev, Denmark). Serum intact OC was assessed through an equilibrium radioimmunoassay. The following bone turnover markers were available at baseline for a subset: serum P1NP: N=95 for T2D, N=148 for non-diabetes; serum CTX: N=95 for T2D, N=136 for non-diabetes; serum OC: N=49 for T2D, N=154 for non- diabetes.

[0042] For evaluation of renal functions, baseline serum cystatin-C was quantified by a particle-enhanced immunonephelometric assay (N Latex Cystatin C; Dade Behring, Inc., Deerfield, IL, USA) on a BNII nephelometer (Dade Behring, Inc.). Serum creatinine was measured using a colorimetric technique and analyzed via the enzymatic method. Estimated glomerular filtration rates (eGFRs) based on cystatin-C (eGFRCys) and based on creatinine (eGFRCr) were calculated respectively using the Chronic Kidney Disease Epidemiology Collaboration equations. All the assays above have been validated with small intra- and inter-assay coefficients of variation.

[0043] The characteristics of participants were reported separately in the T2D and the non-diabetes group. The categorical variables were reported as numbers and percentages, and the continuous variables were reported as mean ± SD. Because F2-isoprostanes values were not normally distributed, log-transformed values were used in the analyses.

[0044] To evaluate the relationship between baseline plasma F2-isoprostanes and subsequent incident fracture risk within T2D and non-diabetes, Kaplan-Meier survival analysis was used to test the assumption of linearity of the relationship between F2-isoprostanes (grouped by tertiles or quartiles) and fracture risk separately for T2D and non-diabetes. Statistical differences across the tertiles and quartiles were determined by the log-rank test. Cox proportional hazard models were used to estimate the association of F2-isoprostanes with fracture risk adjusted with multiple risk factors, with results presented as hazard ratios (HRs) and 95% confidence intervals (Cis) per SD increase in log F2-isoprostanes. All models included age, sex, race, and clinic site. Multivariate models also included current smoking status, total hip BMD, BMI, HbAlc, and medication use (vitamin D supplements, calcium supplements, and osteoporosis drugs). Multivariate models for the T2D group also included oral hypoglycemic medications use, insulin use, thiazolidinediones use, and diabetes duration. A causal directed acyclic graph was used to identify confounders and excluded any potential mediators to avoid overadjustment. In all Cox proportional hazard models, weights were applied to the 8 substrata (defined by race, sex, diabetes status) to reduce possible selection bias. Time-varying coefficients were tested using Schoenfeld residuals for each covariate to ensure that the proportionality assumption was met. Models’ goodness-of-fit were evaluated by the concordance-index.

[0045] Sensitivity analyses for the incident fracture outcome was conducted. First, five individuals from the non-diabetes cohort who had a later diagnosis of T2D during the study were identified and then tested if the exclusion of these five individuals impacted the relationship between plasma F2-isoprostanes level and incident clinical fracture risk. Next, eGFRCys and then eGFRCr were added as variables to the multivariate model. Finally, the competing risk of mortality was incorporated into the multivariate analysis to determine if mortality affected the association between F2-isoprostanes and fracture risk using the Fine-Gray sub distribution hazard model. The variables included in the competing risk models were the same as above.

[0046] To understand the contribution of plasma F2-isoprostanes on incident fracture risk, for a given T-score in the T2D and the non-diabetes group, the 10-year absolute incident fracture risk at the 25th and 75th percentiles of plasma F2-isoprostanes level were predictedusing Cox regression models adjusted only for T-score with age fixed at 75 years. The baseline survival functions were modeled as restricted cubic splines. Here, T-scores were evaluated based on femoral neck BMD, calculated using sex- and race-specific reference values at the age of 20-29 years from the third National Health and Nutrition Examination Survey (NHANES III)

[0047] Furthermore, to establish potential mechanistic pathways explaining the association between F2-isoprostanes and fracture risk, the Pearson (or Spearman) correlation of log plasma F2-isoprostanes was tested with BMD (baseline total hip BMD, four-year change in total hip BMD) and the following circulating baseline parameters: HbAlc, CML (log- transformed), PEN (log-transformed), P1NP, CTX, OC, eGFRCys, and eGFRCr, separately in T2D and the non-diabetes group. Percentage change in BMD was calculated by subtracting the baseline value from the Year 4 value, then dividing the difference by the baseline value, and finally multiplying the result by 100. The association between log plasma F2-isoprostanes and four-year change in BMD was further examined in a linear regression model, adjusting for the covariates and confounders included in the Cox proportional hazard models above. For all statistical tests, a p-value < 0.05 was considered statistically significant.

[0048] Table 1 below presents the baseline characteristics of the participants included in this analysis, stratified by diabetes status. Of the 703 participants, 132 (18.8%) were diabetic at baseline. The participants with T2D and without diabetes were frequency matched by sex and race. The average baseline age was similar in the two groups, at approximately 74 years old. Mean baseline plasma F2-isoprostanes was 59.3 ± 27.7 pg / mL for T2D and 62.7 ± 32.0 pg / mL for the non-diabetes group. BMI, mean baseline total hip BMD, and femoral neck BMD were higher in the diabetic group than in the non-diabetic group.Table 1: Cohort characteristics. Data are expressed as mean ± SD or number (percentage), except F2-Isoprostanes are expressed as geometric mean ± SD. BMI = body mass index; HbAlc = glycated hemoglobin.

[0049] Referring now to FIG. 6, in the T2D group, 34 (25.8%) participants experienced an incident clinical fracture during a mean follow-up of 6.2 ± 3.9 years; while among the nondiabetes group, incident clinical fracture occurred in 134 (23.5%) participants over a mean follow-up period of 8.0 ± 4.1 years. The univariate model confirms the linear relationship between F2-isoprostanes and T2D fracture risk, where the rate of incident clinical fracture increased with increasing F2-isoprostanes tertile (p=0.006) and quartile (p=0.004) among T2D but not among the non-diabetes group. Among individuals with T2D, the risk of incident clinical fracture increased by 51% (HR=1.51, 95% CI: 1.17-1.95, p=0.001, concordance-index=0.76) per SD increase in the log plasma F2-isoprostane levels in the minimally-adjusted model (see Table 2 below). In the multivariate-adjusted model, the incident clinical fracture risk increased by 93% (HR=1.93, 95% CI: 1.26-2.95, p=0.002, concordance-index=0.84) per SD increase in log plasma F2-isoprostanes in the T2D group. In contrast, there was no evidence of a relationship between plasma F2-isoprostanes and incident clinical fracture risk in the nondiabetes group for either model (minimally-adjusted model: HR=0.91, 95% CI: 0.75-1.11, p=0.37; multivariate-adjusted model: HR=0.98, 95% CI: 0.81-1.18, p=0.79; p for interaction <0.001 in both models). Excluding five individuals with a diabetes diagnosis during follow-up from the non-diabetes group had little effect on the associations (minimally-adjusted model: HR=0.92, 95% CI: 0.76-1.12, p = 0.40; multivariate-adjusted model: HR=0.98, 95% CI: 0.81-1.19, p = 0.81).Table 2: Risk of Incident Clinical Fracture per SD Increase in log plasma F2-isoprostanes. For minimal adjustments, the covariates include age, sex, race, and clinic site. For multivariate adjustments, covariates include age, race, sex, clinic site, current smoking status, total hip BMD, BMI, HbAlc, and medication use (vitamin D supplements, calcium supplements, osteoporosis drugs). In the T2D models, the following variables were also included: oral hypoglycemic medications use, insulin use, thiazolidinediones use, and diabetes duration.

[0050] To test whether the relationship between plasma F2-isoprostanes levels and incident clinical fracture risk may be mediated by renal function, the eGFRs based on cystatin C or creatinine were added as an additional covariate in the multivariate adjusted model. In the T2D group, the hazard ratio of plasma F2-isoprostanes levels for incident clinical fracture was slightly attenuated with either eGFRCys or eGFRCr added, however, it remained statistically significant (eGFRCys: HR=1.78, 95% CI: 1.09, 2.92, p=0.02; eGFRCr: HR=1.84, 95% CI: 1.14, 2.97, p=0.01). No association was identified between plasma F2- isoprostanes levels and incident clinical fracture risk in the non-diabetes group with or without adjustment of eGFRs (see Table 3 below).Table 3: Risk of Incident Clinical Fracture per SD Increase in log plasma F2-isoprostanes with eGFR added as an additional variable. eGFR = estimated glomerular filtration rate (calculated using Chronic Kidney Disease Epidemiology Collaboration equations, eGFRCys estimated based on cystatin C, eGFRCr estimated based on creatine).

[0051] The competing risk analysis using a Fine-Gray sub distribution hazard model showed that the inclusion of the competing risk of mortality did not affect the significant association between plasma F2-isoprostanes and incident clinical fracture risk in the T2D group. The minimally adjusted model in the non-diabetes group showed that higher levels of plasma F2-isoprostanes were associated with a lower risk of incident clinical fracture. However, the relationship became non-significant with additional adjustments (see Table 4 below).Table 4: Risk of Incident Clinical Fracture per SD Increase in log plasma F2-isoprostanes with Mortality as Competing Risk. For minimal adjustments, the covariates include age, sex, race, and clinic site. For multivariate adjustments, covariates include age, race, sex, clinic site, current smoking status, HbAlc, total hip BMD, BMI, and medication use (vitamin D supplements, calcium supplements, osteoporosis drugs). In the T2D models, the following variables were also included: oral hypoglycemic medications use, insulin use, thiazolidinediones use, and diabetes duration.

[0052] Referring again to FIG. 5, modeling the baseline survival functions as restricted cubic splines, the 10-year absolute incident fracture risk was evaluated separately in the T2D and the non-diabetes group in a model adjusted for T-score with age fixed at 75 years. FIG. 5 displays the 10-year incident fracture risk, stratified by diabetes status and for plasma F2- isoprostane levels at the 25th and 75th percentiles, plotted against the femoral neck BMD T- score. Here, in the T2D group, the 10-year incident fracture risk was higher in individuals with high plasma F2-isoprostane levels than in those with low plasma F2-isoprostane levels, whereas the difference in fracture risk caused by difference in plasma F2-isoprostane levels in the nondiabetes group was absent. At the T-score threshold of -2.5 for osteoporosis diagnosis for the non-diabetes group, the fracture risk was equivalent to those in the T2D group with high plasma F2-isoprostane level at a T-score of approximately -1.25.

[0053] Referring now to FIG. 7, to understand the association between F2-isoprostane levels and incident clinical fracture risk, the correlation of F2-isoprostanes (log-transformed) with other markers which potentially mediate fracture risk separately in T2D and non-diabetes was determined. Baseline total hip BMD was negatively correlated with the level of plasma F2- isoprostanes, regardless of diabetes status. 521 participants had a follow-up hip BMD available (N = 92 in the T2D group; N = 429 in the non-diabetes group). The characteristics of participants missing in Year 4 measurements were not distinguishably different from theindividuals who participated in Year 4 measurements (see Table 5 below). There was no association between plasma F2-isoprostanes and 4-year total hip bone loss in the non-diabetes group. Contrarily, in the T2D group, the elevated level of plasma F2-isoprostanes was significantly linked to a decrease in total hip BMD (r=-0.28; p=0.008) at four years. Furthermore, following adjustments of covariates and confounders in the linear regression model, the association between plasma F2-isoprostanes and four-year decrease in total hip BMD remained significant (p=0.01) in the T2D group with minimal adjustment, whereas in the nondiabetes group the relationship is not significant (p=0.31). However, with multivariate adjustments, the relationship was not significant in either group (p=0.09 for T2D, p=0.11 for non-diabetes). The correlations between plasma F2I and other circulating glycoxidation markers, bone turnover markers, and kidney function markers were also measured (see Table 6 below).Table 5: Baseline characteristics for individuals participating and not participating at Year 4.Data are expressed as mean ± SD or number (percentage), except F2-Isoprostanes are expressed as geometric mean ± SD.

[0054] No correlation was identified between baseline log F2-isoprostanes and HbAlc or circulating AGOEs in the T2D group. In the non-diabetes group, only a weak correlation approaching statistical significance was discovered between log F2-isoprostanes and log PEN. Log F2-isoprostanes levels were also not correlated with bone turnover markers in either group. In contrast, a significant negative correlation between log F2-isoprostanes and eGFRs was identified in both groups, where the correlation coefficient is higher in T2D than in nondiabetes (see Table 6 below).Table 6: Correlations between plasma F2-isoprostanes and other circulating glycoxidation markers, bone turnover markers, and kidney function markers. HbAlc = glycated hemoglobin; CML = carboxymethyl -lysine; PEN = pentosidine; P1NP = procollagen type 1 N-terminal propeptide; CTX = carboxy-terminal collagen crosslinks; OC = osteocalcin; eGFR = estimated glomerular filtration rate (calculated using Chronic Kidney Disease Epidemiology Collaboration equations, eGFRcysestimated based on cystatin C, eGFRcrestimated based on creatine).

[0055] In T2D patients, enhanced bone fragility is also attributable to a decline in renal functions, where diabetic nephropathy can lead to abnormal bone microarchitecture and defects in mineralization and collagen structures. Oxidative stress has been shown to play a role in the progression of chronic kidney disease, and the levels of esterified F2-isoprostanes were found to be significantly higher in diabetic end-stage renal disease patients than controls. Withoutwishing to be bound by theory, plasma F2-isoprostanes levels can mediate bone fracture outcomes via alteration of renal functions. As described above, plasma F2-isoprostanes significantly correlated with eGFR in T2D and non-diabetes groups. With the incorporation of eGFR measures in the multivariate model, the hazard ratio of F2-isoprostanes for incident clinical fracture risk was slightly attenuated in the T2D group, suggesting that higher F2- isoprostanes levels indeed enhance bone fragility partially through the association with impaired renal functions.

[0056] Methods and systems of the present disclosure are advantageous to identify a patient’s risk of complications based on F2I and develop personalized treatment of T2D patients by measuring F2I concentrations in newly drawn or existing blood samples. The levels of plasma F2-isoprostanes precisely capture global oxidative stress levels, which contributes to advanced glycation end-products accumulations in tissues and organs. As discussed above, bone fracture risk in the T2D population is heightened for a given BMD level and is underestimated by the standard of care evaluation tools such as FRAX. As demonstrated above, to be comparable with the fracture risk at the -2.5 T-score threshold for individuals without diabetes, lowering the T-score by 0.5 SD is insufficient in the T2D group above certain plasma F2- isoprostane threshold levels, e.g., at the 25th percentile. Consequently, the fracture risk would still be drastically underestimated in the T2D group with high plasma F2-isoprostane levels. In embodiments of the present disclosure, individuals with T2D with quantified high plasma F2- Isoprostane levels, e.g., at the 50th and 75th percentiles, have the T-score reduced an additional 0.8 SD and 1.2 SD, respectively.

[0057] Embodiments of the present disclosure advantageously recontextualize the treatment of increased bone fracture risk in T2D patients as a monitoring of the patient’s bone quality, rather than quantity. In terms of bone quality, microvascular impairment in the skeletal system has been implicated as a contributor to T2D-induced bone fragility. For example, reduced bone blood flow and oxygen supply can contribute to increased cortical porosity and degraded bone quality in T2D. These changes in bone quality, including microstructural alterations cannot be detected by BMD, derived from dual-energy x-ray absorptiometry. In comparison, the embodiments of the present disclosure enable bone quality monitoring via a simple blood panel.

[0058] Plasma F2I levels, which assess oxidative stress levels measured in a patient through a simple blood test, can be advantageously used to identify elevated incident clinical fracture risk in the T2D cohort, independently of baseline BMD, medication use, and other riskfactors. Further, the levels of F2-isoprostanes are significantly higher in diabetic end-stage renal disease patients than controls. Plasma F2-isoprostanes also significantly correlated with eGFR in T2D and non-diabetes groups, which is the marker for renal function. Embodiments of the present disclosure can be effectively deployed in a physician’s workflow and / or a patient’s course of treatment merely by supplementing an existing blood panel, rather than with a separate draw. In the case of orthopedic implants, plasma F2-isoprostanes level can serve as an indicator of bone implant quality, informing clinicians whether to use chemical treatments to refunctionalize the bone implants.

[0059] Although the invention has been described and illustrated with respect to exemplary embodiments thereof, it should be understood by those skilled in the art that the foregoing and various other changes, omissions and additions may be made therein and thereto, without parting from the spirit and scope of the present invention.

Claims

CLAIMSWhat is claimed is:

1. A method of treating diabetes-related complications in a patient, comprising: obtaining a sample from a patient suspected of having or known to have type 2 diabetes (T2D); quantifying a concentration of F2-isoprostanes (F2I) in the sample; diagnosing an elevated risk of diabetes-related complications in the patient in response to elevated concentrations of F2I; and administering a treatment to the patient corresponding to the elevated risk of diabetes-related complications.

2. The method according to claim 1, wherein the diabetes-related complications include bone fracture, cardiovascular complications, renal disease, or combinations thereof.

3. The method according to claim 1, wherein the sample includes blood plasma.

4. The method according to claim 1, wherein diagnosing the elevated risk of diabetes- related complications in the patient includes: comparing the concentration of F2I with a threshold F2I concentration value, and confirming an elevated fracture risk for bones in the patient when the concentration of F2I is greater than or equal to the threshold F2I concentration value.

5. The method according to claim 4, wherein the threshold F2I concentration value is about 41 pg / mL.

6. The method according to claim 4, wherein administering the treatment to the patient corresponding to the elevated risk of diabetes-related complications in the patient includes: determining a bone mineral density (BMD) T-score for the patient, andreducing the BMD T-score of the patient by a predetermined standard deviation (SD).

7. The method according to claim 6, wherein the predetermined SD is: between 0.5 and about 0.8 when the concentration of F2I in the sample is between about 41 pg / mL and about 54 pg / mL; between about 0.8 and about 1.2 when the concentration of F2I in the sample is between about 54 pg / mL and about 68 pg / mL; and at least 1.2 when the F2I concentration in the sample is at least 68 pg / mL.

8. The method according to claim 1, wherein the treatment includes an effective amount of a therapeutic compound.

9. The method according to claim 8, wherein the treatment includes prophylactic treatment of the patient for decreased BMD.

10. A method of monitoring treatment of diabetes-related complications, including: obtaining a first sample from a type 2 diabetes (T2D) patient at a first instance; quantifying a concentration of F2-isoprostanes (F2I) in the first sample; diagnosing an elevated risk of diabetes-related complications in the patient in response to elevated concentrations of F2I; administering a first treatment to the patient corresponding to the elevated risk of diabetes-related complications; obtaining a second sample from the T2D patient at a second instance; quantifying a concentration of F2I in the second sample; and administering a second treatment to the patient corresponding to a change in concentration of F2I from the first sample to the second sample.

11. The method according to claim 10, wherein the diabetes-related complications include bone fracture, cardiovascular complications, renal disease, or combinations thereof.

12. The method according to claim 10, wherein the first sample, the second sample, or combinations thereof, includes blood plasma.

13. The method according to claim 10, wherein diagnosing the elevated risk of diabetes- related complications in the patient includes: comparing the concentration of F2I in the first sample with a threshold F2I concentration value, and confirming an elevated fracture risk for bones in the patient when the concentration of F2I in the first sample is greater than or equal to the threshold F2I concentration value.

14. The method according to claim 13, wherein the threshold F2I concentration value is about 41 pg / mL.

15. The method according to claim 14, wherein administering the first treatment to the patient corresponding to the elevated risk of diabetes-related complications includes: determining a bone mineral density (BMD) T-score for the patient, and reducing the BMD T-score of the patient by a predetermined standard deviation (SD) when the concentration of F2I in the first sample is above the threshold F2I concentration value, wherein the predetermined SD is at least 0.5.

16. The method according to claim 15, wherein administering the second treatment to the patient corresponding to the change in concentration of F2I from the first sample to the second sample includes: reducing the BMD T-score of the patient by at least 0.8 SD when the concentration of F2I in the second sample is above 54 pg / mL; and reducing the BMD T-score of the patient by at least 1.2 SD when the concentration of F2I in the second sample is above 68 pg / mL.

17. A method of treating diabetes-related complications in a patient, comprising: obtaining a blood plasma sample from a type 2 diabetes (T2D) patient; quantifying a concentration of F2-isoprostanes (F2I) in the blood plasma sample;determining a bone mineral density (BMD) T-score for the patient; reducing the BMD T-score of the patient by a predetermined standard deviation (SD) by: about 0.5 when the concentration of F2I in the sample is greater 41 pg / mL; about 0.8 when the concentration of F2I in the sample is greater than 54 pg / mL; and about 1.2 when the concentration of F2I in the sample is greater than 68 pg / mL, and administering a treatment to the patient corresponding to an elevated risk of bone fracture.

18. The method according to claim 17, wherein the treatment includes an effective amount of a therapeutic compound.

19. The method according to claim 18, wherein the treatment further targets increased risk of cardiovascular complications, renal disease, or combinations thereof.

20. The method according to claim 17, further comprising: monitoring for an increased F2I concentration in the patient over time; and modifying the treatment for increased risk of diabetes complications in the patient corresponding to the increased F2I concentration.

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