SBIR-STTR Award

The measurement of testosterone levels is central to the diagnosis of androgen disorders in men and women. Circulating testosterone is bound largely to sex hormone binding globulin (SHBG) and albumin; only the unbound or free fraction is biologically active. Therefore, in conditions that affect SHBG concentrations, such as aging, obesity, and diabetes, the determination of free testosterone is necessary to obtain an accurate assessment of androgen status. The current methods for the determination of free testosterone - equilibrium dialysis, ultrafiltration, tracer analog methods, and the use of algorithms based on the law-of-mass-action - suffer from problems of imprecision or inaccuracy. Equilibrium dialysis method, widely considered the reference method, is labor-intensive and cumbersome, and susceptible to errors due to tracer impurities, leading the Endocrine Society's Expert Panel to conclude that '...the calculation of free testosterone is the most useful estimate of free testosterone in plasma...' Therefore, algorithms for calculating free testosterone concentrations from total testosterone, SHBG and albumin concentrations using the law-of-mass-action equations have been used widely. Our preliminary data show that the current model of homogeneous testosterone binding sites on SHBG - the conceptual basis of the extant law-of-mass action equations - is erroneous, and that the free testosterone concentrations derived from the available law-of-mass action equations differ substantially from those measured by equilibrium dialysis.. Because of these limitations of the available methods, there is an enormous unmet need for an accurate method to determine free testosterone concentrations that can be applied conveniently in clinical practice. New evidence shows that testosterone's binding to SHBG conforms to an Ensemble Allostery Model (EAM). Based on this new EAM model of testosterone binding to SHBG, we have constructed a novel algorithm for calculating free testosterone levels that provides excellent conformity with values determined by equilibrium dialysis. This phase I SBIR application aims to characterize the technical performance of the novel algorithm by determining its precision, accuracy, functional sensitivity, intra- and inter-assay variation, and specificity, stability, and matrix effects (Aim 1). Aim 2 will incorporate SHB genotype in the algorithm and apply it to men with different SHBG genotypes. Aim 3 will characterize the performance of the assay in young and older men and women, and in obese men with alterations in SHBG concentrations. The product of the phase I studies will be a copyright-protected validated algorithm available as a stand-alone application that physicians can download on their personal computers or hand-held devices for use in their office or even on the bedside. An interdisciplinary team of investigators, strong preliminary data, and a validation plan that conforms to FDA guidance will assure the successful generation of a product for which there is considerable unmet need and which will advance our understanding of testosterone's binding to SHBG.

Thesaurus Terms:
Affect;Affinity;Aging;Albumins;Algorithms;Analog;Androgens;Base;Binding (Molecular Function);Binding Sites;Biological Assay;Clinical;Clinical Practice;Copyright;Cost Effective;Data;Devices;Diabetes Mellitus;Diagnosis;Diagnostic;Dialysis Procedure;Dimer;Disease;Endocrine;Equation;Equilibrium;Evaluation;Exhibits;Funding;Funding Agency;Generations;Genetic Polymorphism;Genotype;Guidelines;Hand;Hiv Infections;Hormones;Hyperthyroidism;Improved;Laws;Legal Patent;Liver Diseases;Measurement;Measures;Men;Menopause;Methods;Modeling;Novel;Obesity;Older Men;Outcome;Performance;Personal Computers;Persons;Phase;Phase 1 Study;Physicians;Plasma;Population;Public Health Relevance;Recombinants;Research Personnel;Sampling;Sex Hormone-Binding Globulin;Small Business Innovation Research Grant;Societies;Specificity;Steroid Hormone;Sweden;Testosterone;Tracer;Ultrafiltration;United States National Institutes Of Health;Validation;Variant;Woman;Young Man;

The measurement of testosterone (T) levels is central to the diagnosis of androgen disorders, such as hypogonadism in men and polycystic ovary syndrome (PCOS) in women. Circulating T is bound with high affinity to sex hormone binding globulin (SHBG) and with substantially lower affinity to albumin; only the free fraction is biologically active. Conditions that affect SHBG concentrations, such as aging and obesity, alter total but not free T concentrations; in these conditions, the determination of free T is necessary to obtain an accurate assessment of androgen status. Tracer analog method, the most widely used method for free T, has been shown to be inaccurate. Equilibrium dialysis method, considered the reference method, is technically difficult to implement and standardize, and is not available in most hospital laboratories, leading the Endocrine Society's Expert Panel to conclude that “…the calculation of free testosterone is the most useful estimate of free testosterone in plasma…” Therefore, there is an unmet need for algorithms that provide accurate estimates of free T that match those derived from equilibrium dialysis. We have designed a novel and accurate TruTTM algorithm for the determination of free T, based on the characterization of testosterone's binding to SHBG using modern biophysical techniques. We have discovered that testosterone's binding to SHBG is a dynamic multistep process that includes allosteric interaction between the two binding sites on an SHBG dimer. Our computational frame-work incorporates the correct binding parameters derived experimentally in these studies, the non-linear dynamics in T:SHBG association, and allsotery. In phase I studies, we demonstrated that TruTTM algorithm provides accurate free T values that match those obtained using the equilibrium dialysis in healthy and hypogoandal men. We have also shown that the binding parameters that have formed the basis of previous equations (e.g., Vermeulen) are incorrect, and that free T values derived using these equations deviate substantially from free T measured by equilibrium dialysis. The phase I studies have led to adoption of the TruTTM algorithm at several institutions. The phase II program will continue the development of the TruTTM algorithm by validating it in common conditions characterized by altered SHBG concentrations, such as obesity and aging (AIM 1), in healthy women across the menstrual cycle, and in women with PCOS (Aim 2). We will generate population-based reference ranges for free T (Aim 3). Phase II also includes plans for commercialization of the TruTTM algorithm using a HIPAA compliant infrastructure for its clinical adoption. The phase II program will provide validation of TruTTM algorithm in the two most common clinical indications for free T measurement – men suspected of hypogonadism and altered SHBG levels, and women with hyperandrogenic disorders. It will also enable the development of a HIPAA compliant platform that can be embedded into electronic medical record for wider clinical adoption and for improving clinical care.

Public Health Relevance Statement:
Measurements of free testosterone are clinically indicated for the accurate diagnosis of common androgen disorders such as hypogonadism in men and polycystic ovary syndrome (PCOS) in women. We have designed a novel and accurate TruTTM algorithm for the determination of free testosterone, based on the characterization of testosterone's binding to SHBG using modern biophysical techniques. Building upon the success of a productive phase I program, our phase II application will continue the development of the TruTTM algorithm by validating it in common conditions characterized by altered SHBG concentrations; in women across the menstrual cycle; and in women with PCOS, and by generating population-based reference ranges for free T. Phase II also includes plans for commercialization of the TruTTM algorithm using a HIPAA compliant application for integration into the electronic medical record for its clinical adoption.

Project Terms:
accurate diagnosis; Adoption; Affect; Affinity; Age; Aging; Albumins; Algorithms; analog; Androgens; base; Binding; Binding Proteins; Binding Sites; Biological; biophysical techniques; Calorimetry; Clinical; clinical care; Clinical Research; commercialization; Communities; computer framework; Computerized Medical Record; design; Development; Diagnosis; Diagnostic; Dialysis procedure; dimer; Dimerization; Disease; Endocrine; Equation; Equilibrium; Estradiol; Health Insurance Portability and Accountability Act; hospital laboratories; Hypogonadism; improved; Institutes; Institution; International; Legal patent; Measurement; Measures; Medical; medical schools; men; Menstrual cycle; Methods; Modernization; Nonlinear Dynamics; novel; Obesity; Performance; Persons; Phase; phase 1 study; Plasma; Polycystic Ovary Syndrome; population based; Process; programs; Radioimmunoassay; Reference Values; reproductive; Research; Research Infrastructure; Sex Hormone-Binding Globulin; Societies; Standardization; stoichiometry; success; Testosterone; Titrations; Tracer; Universities; uptake; Validation; Woman