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Testosterone Replacement, Low T, HCG, & Beyond
Blood Test Discussion
Calculate Free Testosterone with TruT by FPT
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<blockquote data-quote="madman" data-source="post: 145380" data-attributes="member: 13851"><p>Three heavy weights in the field behind the invention of <strong><span style="color: rgb(184, 49, 47)">TruT.</span></strong></p><p></p><p>Ravi Jasuja, Shalender Bhasin and Mikhail N Zakharov.</p><p></p><p>Hoping people understand the significance/importance of this testing method and the huge impact it will have.</p><p></p><p>Very long read but deeply interesting to say the least <a href="https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2014138026&tab=PCTDESCRIPTION&maxRec=1000" target="_blank">WO2014138026 METHODS AND SYSTEMS FOR THE DIAGNOSIS AND TREATMENT OF ANDROGEN DISORDERS</a> </p><p></p><p></p><p></p><p></p><p><strong><span style="color: rgb(184, 49, 47)">1. (WO2014138026) METHODS AND SYSTEMS FOR THE DIAGNOSIS AND TREATMENT OF ANDROGEN DISORDERS </span></strong></p><table class='post-table ' style='width: 100%'><tr><td ><p><strong>Pub. No.:</strong></p></td><td ><p>WO/2014/138026</p></td><td ><p><strong>International Application No.:</strong></p></td><td ><p>PCT/US2014/020223</p></td></tr><tr><td ><p><strong>Publication Date:</strong></p></td><td ><p>12.09.2014</p></td><td ><p><strong>International Filing Date:</strong></p></td><td ><p><span style="color: rgb(184, 49, 47)"><strong>04.03.2014</strong></span></p></td></tr></table><strong>IPC:</strong></p><table class='post-table ' style='width: 100%'><tr><td ><p><em><strong>G01N 33/566</strong></em> (2006.01)</p></td><td></td></tr></table><strong>Applicants:</strong></p><table class='post-table ' style='width: 100%'><tr><td ><p><strong>FUNCTION PROMOTING THERAPIES, LLC</strong> [US/US]; 32 Highland Meadows Lane Weston, Massachusetts 02493, US</p></td></tr></table><strong><span style="color: rgb(0, 0, 0)">Inventors:</span></strong></p><table class='post-table ' style='width: 100%'><tr><td ><p><span style="color: rgb(184, 49, 47)"><strong>JASUJA, Ravi; </strong></span><span style="color: rgb(0, 0, 0)"><strong>US</strong></span></p></td></tr><tr><td ><p><span style="color: rgb(184, 49, 47)"><strong>BHASIN, Shalender; </strong></span><span style="color: rgb(0, 0, 0)"><strong>US</strong></span></p></td></tr><tr><td ><p><strong><span style="color: rgb(184, 49, 47)">ZAKHAROV, Mikhail N.; </span><span style="color: rgb(0, 0, 0)">US</span></strong></p></td></tr></table><strong>Agent:</strong></p><table class='post-table ' style='width: 100%'><tr><td ><p><strong>CHEN, Yahua</strong>; Wolf, Greenfield and Sacks, P.C. 600 Atlantic Avenue Boston, MA 02210, US</p></td></tr></table><strong>Priority Data:</strong></p><table class='post-table ' style='width: 100%'><tr><td ><p>61/772,054</p></td><td></td><td ><p>04.03.2013</p></td><td></td><td ><p>US</p></td></tr></table><strong>Title(EN)<span style="color: rgb(41, 105, 176)">METHODS AND SYSTEMS FOR THE DIAGNOSIS AND TREATMENT OF ANDROGEN DISORDERS</span></strong></p><p></p><p><strong>(FR)</strong>PROCÉDÉS ET SYSTÈMES POUR LE DIAGNOSTIC ET LE TRAITEMENT DE TROUBLES ANDROGÉNIQUES</p><p><strong>Abstract:</strong></p><table class='post-table ' style='width: 100%'><tr><td ><p><strong>(EN)</strong> The technology described herein is directed to the diagnosis and treatment of androgen disorders and/or deficiencies, e.g, low testosterone.<br /> <strong>(FR)</strong> La présente invention concerne une technologie pour le diagnostic et le traitement de troubles et/ou carences androgéniques, par exemple un faible taux de testostérone.</p></td></tr></table> </p><p></p><p></p><p></p><p></p><p></p><p></p><p></p><p></p><p></p><p><strong>SUMMARY</strong></p><p></p><p><strong><span style="color: rgb(184, 49, 47)">[0005] </span>Described herein is the <span style="color: rgb(184, 49, 47)">inventors' </span>demonstration that the <span style="color: rgb(184, 49, 47)">prevailing model</span> of <span style="color: rgb(184, 49, 47)">testosterone's binding to SHBG</span>, used in the <span style="color: rgb(184, 49, 47)">current methods of testing and diagnosis is flawed</span>,</strong> <strong>and their further <span style="color: rgb(184, 49, 47)">discovery</span> of an<span style="color: rgb(184, 49, 47)"> improved model </span>that permits methods, assays, and systems with greater <span style="color: rgb(41, 105, 176)">accuracy</span> and <span style="color: rgb(41, 105, 176)">reliability </span>in <span style="color: rgb(184, 49, 47)">measuring testosterone levels.</span></strong> <strong>The <span style="color: rgb(41, 105, 176)">multi-step dynamic binding model </span>with <span style="color: rgb(41, 105, 176)">complex allostery </span>described herein is a <span style="color: rgb(184, 49, 47)">new model for calculation of free testosterone.</span> The <span style="color: rgb(41, 105, 176)">multi-step dynamic binding model</span> with <span style="color: rgb(41, 105, 176)">complex allostery model</span> is a <span style="color: rgb(41, 105, 176)">modified ensemble allostery model </span>that takes into consideration the <span style="color: rgb(41, 105, 176)">specific SHBG-Testosterone binding interaction</span> described herein.</strong></p><p></p><p><strong><span style="color: rgb(184, 49, 47)">[0006]</span></strong> <strong>In one aspect, described herein is a</strong> <strong>computer implemented method for an assay, comprising: on a device having one or more processors and a memory storing one or more programs for execution by the one or more processors, the one or more programs including instructions for:</strong> <span style="color: rgb(184, 49, 47)"><strong>a) receiving data from </strong></span><strong><span style="color: rgb(184, 49, 47)">measuring </span></strong><span style="color: rgb(44, 130, 201)"><strong>i) a total SHBG concentration, ii) a total testosterone concentration, and iii) a total albumin concentration</strong> </span><strong>in a biological sample obtained from an individual, to determine free testosterone concentration from the individual; <span style="color: rgb(184, 49, 47)">b) attributing at least </span><span style="color: rgb(41, 105, 176)">two distinct interconverting microstates</span><span style="color: rgb(184, 49, 47)"> of an </span><span style="color: rgb(41, 105, 176)">unliganded SHBG dimer</span><span style="color: rgb(184, 49, 47)"> having a </span><span style="color: rgb(41, 105, 176)">first monomer</span><span style="color: rgb(184, 49, 47)"> and a </span><span style="color: rgb(41, 105, 176)">second monomer;</span></strong> <strong><span style="color: rgb(184, 49, 47)">and c) calculating the free testosterone concentration</span> in the individual using the <span style="color: rgb(41, 105, 176)">New Multi-Step Dynamic Binding Model with Complex Allostery</span> encompassing <span style="color: rgb(41, 105, 176)">readjustment of a first equilibria</span> between the <span style="color: rgb(41, 105, 176)">microstates upon binding</span> of a <span style="color: rgb(41, 105, 176)">first testosterone molecule </span>to the<span style="color: rgb(41, 105, 176)"> first monomer</span> and an <span style="color: rgb(41, 105, 176)">allosteric interaction </span>between <span style="color: rgb(41, 105, 176)">two binding sites </span>of the <span style="color: rgb(41, 105, 176)">SHBG dimer.</span></strong></p><p></p><p></p><p></p><p>*Refer to article to read [0007] - [0017]</p><p></p><p></p><p></p><p><strong><span style="color: rgb(184, 49, 47)">[0018] </span>In one aspect, described herein is a <span style="color: rgb(184, 49, 47)">method for determining</span> a need for <span style="color: rgb(184, 49, 47)">adjustment of a dose</span> of <span style="color: rgb(184, 49, 47)">testosterone administered</span> to an individual comprising</strong> <strong><span style="color: rgb(184, 49, 47)">a) determining the concentration of free testosterone in an individual receiving testosterone therapy at a first dose, wherein the concentration of free testosterone is determined by b) measuring </span><span style="color: rgb(41, 105, 176)">i) a total SHBG concentration, ii) a total testosterone concentration, and iii) a total albumin concentration in a biological sample obtained from an individual</span>, to determine<span style="color: rgb(184, 49, 47)"> free testosterone concentration </span>from the individual; <span style="color: rgb(184, 49, 47)">c) attributing at least </span><span style="color: rgb(41, 105, 176)">two distinct interconverting microstates </span><span style="color: rgb(184, 49, 47)">of an </span><span style="color: rgb(41, 105, 176)">unliganded SHBG dimer</span><span style="color: rgb(184, 49, 47)"> having a</span><span style="color: rgb(41, 105, 176)"> first monomer </span><span style="color: rgb(184, 49, 47)">and a </span><span style="color: rgb(41, 105, 176)">second monomer</span><span style="color: rgb(184, 49, 47)"> by applying the </span><span style="color: rgb(41, 105, 176)">New Multi-Step Dynamic Binding Model with Complex Allostery</span><span style="color: rgb(184, 49, 47)"> to the data of steps a) and b)</span>; <span style="color: rgb(184, 49, 47)">d) calculating the free testosterone concentration in the individual </span>using the <span style="color: rgb(44, 130, 201)">New Multi-step Dynamic Binding Model with Complex Allostery </span>encompassing <span style="color: rgb(41, 105, 176)">readjustment</span> of a <span style="color: rgb(41, 105, 176)">first equilibria</span> between the <span style="color: rgb(41, 105, 176)">microstates</span> upon<span style="color: rgb(41, 105, 176)"> binding </span>of a <span style="color: rgb(41, 105, 176)">first testosterone molecule </span>to the <span style="color: rgb(41, 105, 176)">first monomer</span> and an <span style="color: rgb(41, 105, 176)">allosteric interaction</span> between <span style="color: rgb(41, 105, 176)">two binding sites</span> of the <span style="color: rgb(41, 105, 176)">SHBG dimer</span>; <span style="color: rgb(184, 49, 47)">e) providing a second dose of testosterone</span> that is higher than the <span style="color: rgb(184, 49, 47)">first dose </span>when the <span style="color: rgb(184, 49, 47)">free testosterone concentration </span>is <span style="color: rgb(184, 49, 47)">below</span> the <span style="color: rgb(184, 49, 47)">lower end</span> of the <span style="color: rgb(184, 49, 47)">target therapeutic range</span> (<span style="color: rgb(184, 49, 47)">e.g.164 pg/ml</span>); and <span style="color: rgb(184, 49, 47)">f) providing a second dose of testosterone </span>that is lower than the <span style="color: rgb(184, 49, 47)">first dose</span> when the<span style="color: rgb(184, 49, 47)"> free testosterone concentration</span> is <span style="color: rgb(184, 49, 47)">above </span>the <span style="color: rgb(184, 49, 47)">upper end</span> of the <span style="color: rgb(184, 49, 47)">target therapeutic range </span><span style="color: rgb(0, 0, 0)">(</span><span style="color: rgb(184, 49, 47)">314 pg/ml</span></strong><span style="color: rgb(0, 0, 0)"><strong>)</strong></span><strong><span style="color: rgb(184, 49, 47)">.</span></strong></p><p><strong> </strong></p><p></p><p></p><p>[0019] In some embodiments of any of the foregoing aspects, the step of attributing can be performed according to Figures 2, 3, 5, and 7. In some embodiments, of any of the foregoing aspects, the step of calculating can be performed according to Figure 7 or Example 5. In some embodiments of any of the foregoing aspects, the individual is a male over the age of 35. In some embodiments of any of the foregoing aspects, the androgen disorder is selected from the group consisting of a testosterone deficiency, an androgen deficiency, a hyperandrogenic disorder, an androgen expressing tumor, and a hypogonadism disorder. In some embodiments of any of the foregoing aspects, the androgen disorder is a hyperandrogenic disorder selected from the group consisting of an acne disorder, a hirsutism disorder, and an androgenic alopecia disorder. In some embodiments of any of the foregoing aspects, the individual has been diagnosed with a disease selected from the group consisting of: diabetes, human immunodeficiency virus (HIV), hepatitis B, hepatitis C, hypothyroidism or hyperthyroidism, androgen insensitivity, acromegaly, anorexia, muscular dystrophy, liver disease, cancer cachexia, malnutrition, nephrotic syndrome, and obesity, and other conditions in which SHBG or albumin concentrations are altered. In some embodiments of any of the foregoing aspects, the assay, method, system, or medium can further comprise the step of classifying the individual into categories based on additional clinical symptoms. In some embodiments of any of the foregoing aspects, the assay, method, system, or medium can further comprise the step of using the free testosterone concentration determined using the new Multistep Dynamic Binding Model with Complex Allostery to determine the dose or to individually adjust the dose of a formulation of testosterone for the treatment of a medical disease, taking into account patient's age, body weight and body mass index, medical conditions, including any co-morbid conditions, albumin and SHBG, and/or LH and FSH concentrations, and other patient-specific factors. In some embodiments of any of the foregoing aspects, instead of steps a-c, the data received is a previously calculated concentration of free testostosterone.</p><p></p><p></p><p></p><p><strong>BRIEF DESCRIPTION OF THE DRAWINGS</strong></p><p></p><p></p><p></p><p>[ATTACH=full]7251[/ATTACH]</p><p><strong><span style="color: rgb(184, 49, 47)">[0020] Figs. 1A-1B</span> demonstrate <span style="color: rgb(184, 49, 47)">that</span> <span style="color: rgb(184, 49, 47)">free T values calculated</span> <span style="color: rgb(184, 49, 47)">using the Vermeulen/ Sodergard/ Mazer model </span>- the commonly used method for determining free T concentrations - <span style="color: rgb(184, 49, 47)">differ</span> from those measured using <span style="color: rgb(184, 49, 47)">equilibrium dialysis.</span> Fig. 1A depicts a graph of <span style="color: rgb(41, 105, 176)">free testosterone concentrations </span>in samples derived from a <span style="color: rgb(41, 105, 176)">randomized testosterone trial (The TED study)</span> were measured using the <span style="color: rgb(41, 105, 176)">equilibrium dialysis</span> and plotted against those <span style="color: rgb(41, 105, 176)">calculated using the Vermeulen/ Sodergard/ Mazer equation.</span> <span style="color: rgb(184, 49, 47)">Calculated free T concentrations differed systematically </span>from the <span style="color: rgb(184, 49, 47)">measured values. </span>Fig. 1B depicts a <span style="color: rgb(41, 105, 176)">Bland Altman plot</span> revealing <span style="color: rgb(41, 105, 176)">substantial discrepancy </span>between the <span style="color: rgb(41, 105, 176)">calculated and measured free T concentrations.</span></strong></p><p></p><p></p><p></p><p></p><p>[ATTACH=full]7252[/ATTACH]</p><p><strong><span style="color: rgb(184, 49, 47)">[0021] Figs. 2A-2C</span></strong> <strong>demonstrate <span style="color: rgb(184, 49, 47)">that binding of testosterone to SHBG</span> displays <span style="color: rgb(184, 49, 47)">complex behavior. </span>Fig. 2A depicts a graph demonstrating that <span style="color: rgb(41, 105, 176)">binding isotherms</span> display <span style="color: rgb(41, 105, 176)">complex behavior.</span> <span style="color: rgb(184, 49, 47)">Graded concentrations of testosterone</span> were incubated overnight with <span style="color: rgb(184, 49, 47)">various SHBG concentrations</span> and the amount of <span style="color: rgb(184, 49, 47)">bound testosterone </span>was plotted against <span style="color: rgb(184, 49, 47)">total T (added) concentrations</span> (squares) 20nM, (triangles) 10 nM, (circles) 5nM SHBG. </strong><span style="color: rgb(184, 49, 47)"><strong>The </strong></span><span style="color: rgb(41, 105, 176)"><strong>fit curves </strong></span><span style="color: rgb(184, 49, 47)"><strong>represent the result of the linked fit of data to the </strong></span><span style="color: rgb(41, 105, 176)"><strong>new Multi-step Dynamic Binding Model with Complex Allostery </strong></span><span style="color: rgb(184, 49, 47)"><strong>(Fig. 3).</strong></span> <strong>Fig. 2B depicts a graph of the depletion of <span style="color: rgb(41, 105, 176)">free testosterone </span>by <span style="color: rgb(41, 105, 176)">varying SHBG concentration</span> which is best described by the <span style="color: rgb(41, 105, 176)">new Multi-step Dynamic Binding Model with Complex Allostery.</span></strong> <strong>Shown is concentration of free testosterone in the equilibrium dialysis experiment. </strong><span style="color: rgb(184, 49, 47)"><strong>One side of the equilibrium dialysis membrane has </strong></span><span style="color: rgb(41, 105, 176)"><strong>varying concentration of SHBG in buffer</strong></span><span style="color: rgb(184, 49, 47)"><strong>, the other one has </strong></span><span style="color: rgb(41, 105, 176)"><strong>plain buffer. </strong></span><strong>Constant concentration of testosterone is added to each well of multi-well dialyzer: (triangles) 8.7nM.</strong> <span style="color: rgb(184, 49, 47)"><strong>Curves are the result of the linked fit of data in Panel B to the new Multi-step Dynamic Binding Model with Complex Allostery (Fig 3). </strong></span></p><p></p><p></p><p></p><p></p><p></p><p></p><p>[ATTACH=full]7253[/ATTACH]</p><p><strong>Fig. 2C depicts <span style="color: rgb(41, 105, 176)">a graph of the</span> <span style="color: rgb(41, 105, 176)">heat of T:SHBG association </span>measured by i<span style="color: rgb(41, 105, 176)">sothermal calorimetry (ITC). </span>Presented is the integrated ITC curve with buffer heats subtracted.</strong><span style="color: rgb(184, 49, 47)"> <strong>SHBG starting concentration is 5μΜ. Experimental points are shown by squares, fit to the model in Fig.3 is shown by a solid line.</strong></span></p><p></p><p></p><p></p><p></p><p></p><p>[ATTACH=full]7254[/ATTACH]</p><p><strong><span style="color: rgb(184, 49, 47)">[0022] Fig.</span></strong> <strong>3 depicts <span style="color: rgb(184, 49, 47)">a</span> <span style="color: rgb(184, 49, 47)">schematic representation </span>of the<span style="color: rgb(184, 49, 47)"> new Multi-step Dynamic Binding Model with Complex Allostery of testosterone's binding to SHBG and albumin</span>, developed in this study.</strong> <strong><span style="color: rgb(41, 105, 176)">Unliganded SHBG dimers (S2, S2')</span> exist in <span style="color: rgb(41, 105, 176)">conformational equilibrium. </span>Upon <span style="color: rgb(41, 105, 176)">binding of the first testosterone molecule to microstates S2 and S2' </span>can result in <span style="color: rgb(41, 105, 176)">conformationally heterogeneous intermediate states S2T and S2'T</span> respectively. These <span style="color: rgb(41, 105, 176)">singly-occupied microstates </span>then <span style="color: rgb(41, 105, 176)">converge to S2T2 </span>upon <span style="color: rgb(41, 105, 176)">binding of the second testosterone molecule. </span></strong></p><p></p><p></p><p></p><p></p><p>[ATTACH=full]7255[/ATTACH]</p><p><strong><span style="color: rgb(184, 49, 47)">[0023] Figs. 4A-4B </span>demonstrate a comparison of <span style="color: rgb(184, 49, 47)">the Free Testosterone Concentrations Derived Using the Vermeulen Equation</span> <span style="color: rgb(184, 49, 47)">(23) </span>as implemented by Mazer (24) or the <span style="color: rgb(184, 49, 47)">New Algorithm Based on the new Multi-step Dynamic Binding Model with Complex Allostery </span>with those measured using the <span style="color: rgb(184, 49, 47)">equilibrium dialysis </span>in samples from the <span style="color: rgb(184, 49, 47)">5alpha reductase trial.</span> Fig. 4A depicts a graph of a comparison of the <span style="color: rgb(41, 105, 176)">free testosterone concentration calculated by the Vermeulen equation </span>and the <span style="color: rgb(41, 105, 176)">new algorithm based on the new Multi-step Dynamic Binding Model with Complex Allostery </span>to that measured by <span style="color: rgb(41, 105, 176)">equilibrium dialysis</span> in samples from a randomized testosterone trial in men. <span style="color: rgb(184, 49, 47)">(■) free testosterone concentrations</span> derived using an <span style="color: rgb(184, 49, 47)">algorithm based on new Multi-step Dynamic Binding Model with Complex Allostery</span>; (•) free testosterone concentrations derived using the Vermeulen model (23) as implemented by Mazer (24).</strong> <strong><span style="color: rgb(184, 49, 47)">Solid lines are lines of best linear fit.</span></strong> <strong><span style="color: rgb(0, 0, 0)">Regression lines fit new Multi-step Dynamic Binding Model with Complex Allostery calculation (slope = 1.01±0.01, regression line fitting the squares)</span>, and the <span style="color: rgb(184, 49, 47)">Vermeulen model (slope 0.77±0.02, lower line fitting the dots).</span></strong> <strong>Magenta dashed line is the line of prefect correlation. <span style="color: rgb(184, 49, 47)">Fig. 4B </span>depicts <span style="color: rgb(41, 105, 176)">Bland Altman plots </span>of the relative frequency distribution of % difference of<span style="color: rgb(41, 105, 176)"> calculated and measured free testosterone</span> using either the <span style="color: rgb(41, 105, 176)">Vermeulen equation (squares)</span> or the <span style="color: rgb(41, 105, 176)">new algorithm based on the new Multi-step Dynamic Binding Model with Complex Allostery (black dots)</span> The relative deviations from the measured value are distributed around <span style="color: rgb(0, 0, 0)">0</span> <span style="color: rgb(0, 0, 0)">for </span><span style="color: rgb(41, 105, 176)">new Multi-step Dynamic Binding Model with Complex Allostery model </span>and are different from zero for the <span style="color: rgb(41, 105, 176)">Vermeulen model.</span> </strong></p><p></p><p></p><p></p><p></p><p></p><p>[ATTACH=full]7256[/ATTACH]</p><p><strong><span style="color: rgb(184, 49, 47)">Figs. 4C-4D </span>depict <span style="color: rgb(184, 49, 47)">a</span> <span style="color: rgb(184, 49, 47)">Comparison of the Free Testosterone Concentrations Derived Using the Vermeulen Equation </span>or the <span style="color: rgb(184, 49, 47)">New Algorithm Based on the new Multi-step Dynamic Binding Model with Complex Allostery</span> with those measured using the equilibrium dialysis. <span style="color: rgb(184, 49, 47)">Figs. 4C-4D </span>demonstrate a <span style="color: rgb(41, 105, 176)">Comparison of the Free Testosterone Concentrations Derived Using the Vermeulen Equation</span> or the <span style="color: rgb(41, 105, 176)">New Algorithm Based on the new Multi-step Dynamic Binding Model with Complex Allostery </span>with those measured using the <span style="color: rgb(41, 105, 176)">equilibrium dialysis</span> in samples from a randomized testosterone trial in men with ED.</strong> <strong><span style="color: rgb(184, 49, 47)">Fig. 4C</span></strong> <strong>depicts a graph of a comparison of the <span style="color: rgb(184, 49, 47)">free testosterone concentration calculated by the Vermeulen equation</span> and the <span style="color: rgb(184, 49, 47)">new algorithm based on the new Multi-step Dynamic Binding Model with Complex Allostery</span> to that measured by <span style="color: rgb(184, 49, 47)">equilibrium dialysis</span> in samples from a randomized testosterone trial in men. </strong><span style="color: rgb(184, 49, 47)"><strong>(squares) free testosterone concentrations derived using an algorithm based on new Multi-step Dynamic Binding Model with Complex Allostery</strong>; </span><span style="color: rgb(0, 0, 0)"><strong>(circles) free testosterone concentrations derived using the Vermeulen model (23) as implemented by Mazer (24).</strong></span><strong> Black regression line fits <span style="color: rgb(41, 105, 176)">new Multi-step Dynamic Binding Model with Complex Allostery model calculation (slope = 1.01±0.01 is shown)</span> Fig. 4D depicts Bland Altman plots of the relative frequency of % difference of <span style="color: rgb(41, 105, 176)">calculated</span> and <span style="color: rgb(41, 105, 176)">measured free testosterone </span>using either the <span style="color: rgb(41, 105, 176)">Vermeulen equation </span>or the <span style="color: rgb(41, 105, 176)">new algorithm based on the new Multi-step Dynamic Binding Model with Complex Allostery. </span>The relative deviations from the measured value are distributed around 0 for the<span style="color: rgb(184, 49, 47)"> new Multi-step Dynamic Binding Model with Complex Allostery model</span> and are different from zero for the <span style="color: rgb(184, 49, 47)">Vermeulen model.</span></strong></p><p></p><p></p><p></p><p></p><p>[ATTACH=full]7257[/ATTACH]</p><p><strong><span style="color: rgb(184, 49, 47)">[0024] Fig. 5A </span>depicts a graph of <span style="color: rgb(184, 49, 47)">the</span> <span style="color: rgb(184, 49, 47)">binding isotherm.</span> Graded concentrations of testosterone were incubated overnight with<span style="color: rgb(184, 49, 47)"> 20 nM SHBG</span> and the amount of <span style="color: rgb(184, 49, 47)">bound testosterone</span> was plotted against <span style="color: rgb(184, 49, 47)">total testosterone concentration.</span></strong> <strong>The fit curve represents the fit of data to the <span style="color: rgb(184, 49, 47)">new Multi-step Dynamic Binding Model with Complex Allostery. </span>Fig. 5B depicts a graph of the <span style="color: rgb(41, 105, 176)">depletion curve.</span></strong> <strong>A constant concentration of testosterone <span style="color: rgb(41, 105, 176)">(8.7 nM)</span> was incubated with <span style="color: rgb(41, 105, 176)">increasing SHBG concentrations.</span> <span style="color: rgb(184, 49, 47)">Free testosterone concentration</span> is plotted against <span style="color: rgb(184, 49, 47)">SHBG concentration. </span>Solid line represents the fit of data to new <span style="color: rgb(184, 49, 47)">Multi-step Dynamic Binding Model with Complex Allostery.</span> The depletion of <span style="color: rgb(41, 105, 176)">free testosterone by increasing SHBG concentrations</span> is best described by the <span style="color: rgb(41, 105, 176)">new Multi-step Dynamic Binding Model with Complex Allostery.</span></strong> <strong>Fig. 5C depicts a graph of the <span style="color: rgb(184, 49, 47)">heat of testosterone and SHBG association </span>measured by <span style="color: rgb(184, 49, 47)">isothermal calorimetry.</span> The integrated ITC curve was generated after subtracting the buffer heats. <span style="color: rgb(184, 49, 47)">SHBG starting concentration is 5 μΜ.</span> Experimental points are shown by (■), and fit of the data to the <span style="color: rgb(184, 49, 47)">new Multi-step Dynamic Binding Model with Complex Allostery model </span>is shown by a solid line. </strong></p><p></p><p></p><p></p><p></p><p>[ATTACH=full]7258[/ATTACH]</p><p><strong><span style="color: rgb(184, 49, 47)">[0025] Fig. 6</span></strong> <strong>depicts <span style="color: rgb(184, 49, 47)">schematic representations </span>of the <span style="color: rgb(184, 49, 47)">various models tested</span> in this study to <span style="color: rgb(184, 49, 47)">examine SHBG:T interaction.</span></strong> <strong>Model A. Vermeulen's model, <span style="color: rgb(41, 105, 176)">homogenous interaction of testosterone molecule with equal affinity for each monomer in SHBG dimer (Kd=lnM)</span>; Model B. <span style="color: rgb(184, 49, 47)">monomers within the SHBG dimer exhibit distinct affinity constants with Kdi=lnM and Kd2 allowed to vary for data fits</span></strong>; <strong>Model C. <span style="color: rgb(41, 105, 176)">Inter-subunit allostery with positive cooperativity for binding of two ligands such that Kd2=1nM, Kd1 allowed to vary and Kd2<Kd1</span>; Model D. <span style="color: rgb(184, 49, 47)">allostery with negative cooperativity for binding of two ligands such that Kd1=1nM, Kd2allowed to vary and Kd1<Kd2;</span> and, Model E. <span style="color: rgb(41, 105, 176)">The new Multi-step Dynamic Binding Model with Complex Allostery</span> which encompasses <span style="color: rgb(41, 105, 176)">two distinct SHBG microstates in equilibrium </span>such that the<span style="color: rgb(41, 105, 176)"> equilibria between the unliganded and mono-liganded states readjust</span> as testosterone concentration is increased. </strong></p></blockquote><p></p>
[QUOTE="madman, post: 145380, member: 13851"] Three heavy weights in the field behind the invention of [B][COLOR=rgb(184, 49, 47)]TruT.[/COLOR][/B] Ravi Jasuja, Shalender Bhasin and Mikhail N Zakharov. Hoping people understand the significance/importance of this testing method and the huge impact it will have. Very long read but deeply interesting to say the least [URL="https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2014138026&tab=PCTDESCRIPTION&maxRec=1000"]WO2014138026 METHODS AND SYSTEMS FOR THE DIAGNOSIS AND TREATMENT OF ANDROGEN DISORDERS[/URL] [B][COLOR=rgb(184, 49, 47)]1. (WO2014138026) METHODS AND SYSTEMS FOR THE DIAGNOSIS AND TREATMENT OF ANDROGEN DISORDERS [/COLOR][/B] [TABLE][TR][TD] [B]Pub. No.:[/B] [/TD] [TD] WO/2014/138026 [/TD] [TD] [B]International Application No.:[/B] [/TD] [TD] PCT/US2014/020223 [/TD][/TR] [TR][TD] [B]Publication Date:[/B] [/TD] [TD] 12.09.2014 [/TD] [TD] [B]International Filing Date:[/B] [/TD] [TD] [COLOR=rgb(184, 49, 47)][B]04.03.2014[/B][/COLOR] [/TD][/TR][/TABLE] [B]IPC:[/B] [TABLE][TR][TD] [I][B]G01N 33/566[/B][/I] (2006.01) [/TD] [TD] [/TD][/TR][/TABLE] [B]Applicants:[/B] [TABLE][TR][TD] [B]FUNCTION PROMOTING THERAPIES, LLC[/B] [US/US]; 32 Highland Meadows Lane Weston, Massachusetts 02493, US [/TD][/TR][/TABLE] [B][COLOR=rgb(0, 0, 0)]Inventors:[/COLOR][/B] [TABLE][TR][TD] [COLOR=rgb(184, 49, 47)][B]JASUJA, Ravi; [/B][/COLOR][COLOR=rgb(0, 0, 0)][B]US[/B][/COLOR] [/TD][/TR] [TR][TD] [COLOR=rgb(184, 49, 47)][B]BHASIN, Shalender; [/B][/COLOR][COLOR=rgb(0, 0, 0)][B]US[/B][/COLOR] [/TD][/TR] [TR][TD] [B][COLOR=rgb(184, 49, 47)]ZAKHAROV, Mikhail N.; [/COLOR][COLOR=rgb(0, 0, 0)]US[/COLOR][/B] [/TD][/TR][/TABLE] [B]Agent:[/B] [TABLE][TR][TD] [B]CHEN, Yahua[/B]; Wolf, Greenfield and Sacks, P.C. 600 Atlantic Avenue Boston, MA 02210, US [/TD][/TR][/TABLE] [B]Priority Data:[/B] [TABLE][TR][TD] 61/772,054 [/TD] [TD] [/TD] [TD] 04.03.2013 [/TD] [TD] [/TD] [TD] US [/TD][/TR][/TABLE] [B]Title(EN)[COLOR=rgb(41, 105, 176)]METHODS AND SYSTEMS FOR THE DIAGNOSIS AND TREATMENT OF ANDROGEN DISORDERS[/COLOR][/B] [B](FR)[/B]PROCÉDÉS ET SYSTÈMES POUR LE DIAGNOSTIC ET LE TRAITEMENT DE TROUBLES ANDROGÉNIQUES [B]Abstract:[/B] [TABLE][TR][TD] [B](EN)[/B] The technology described herein is directed to the diagnosis and treatment of androgen disorders and/or deficiencies, e.g, low testosterone. [B](FR)[/B] La présente invention concerne une technologie pour le diagnostic et le traitement de troubles et/ou carences androgéniques, par exemple un faible taux de testostérone. [/TD][/TR][/TABLE] [B]SUMMARY[/B] [B][COLOR=rgb(184, 49, 47)][0005] [/COLOR]Described herein is the [COLOR=rgb(184, 49, 47)]inventors' [/COLOR]demonstration that the [COLOR=rgb(184, 49, 47)]prevailing model[/COLOR] of [COLOR=rgb(184, 49, 47)]testosterone's binding to SHBG[/COLOR], used in the [COLOR=rgb(184, 49, 47)]current methods of testing and diagnosis is flawed[/COLOR],[/B] [B]and their further [COLOR=rgb(184, 49, 47)]discovery[/COLOR] of an[COLOR=rgb(184, 49, 47)] improved model [/COLOR]that permits methods, assays, and systems with greater [COLOR=rgb(41, 105, 176)]accuracy[/COLOR] and [COLOR=rgb(41, 105, 176)]reliability [/COLOR]in [COLOR=rgb(184, 49, 47)]measuring testosterone levels.[/COLOR][/B] [B]The [COLOR=rgb(41, 105, 176)]multi-step dynamic binding model [/COLOR]with [COLOR=rgb(41, 105, 176)]complex allostery [/COLOR]described herein is a [COLOR=rgb(184, 49, 47)]new model for calculation of free testosterone.[/COLOR] The [COLOR=rgb(41, 105, 176)]multi-step dynamic binding model[/COLOR] with [COLOR=rgb(41, 105, 176)]complex allostery model[/COLOR] is a [COLOR=rgb(41, 105, 176)]modified ensemble allostery model [/COLOR]that takes into consideration the [COLOR=rgb(41, 105, 176)]specific SHBG-Testosterone binding interaction[/COLOR] described herein.[/B] [B][COLOR=rgb(184, 49, 47)][0006][/COLOR][/B] [B]In one aspect, described herein is a[/B] [B]computer implemented method for an assay, comprising: on a device having one or more processors and a memory storing one or more programs for execution by the one or more processors, the one or more programs including instructions for:[/B] [COLOR=rgb(184, 49, 47)][B]a) receiving data from [/B][/COLOR][B][COLOR=rgb(184, 49, 47)]measuring [/COLOR][/B][COLOR=rgb(44, 130, 201)][B]i) a total SHBG concentration, ii) a total testosterone concentration, and iii) a total albumin concentration[/B] [/COLOR][B]in a biological sample obtained from an individual, to determine free testosterone concentration from the individual; [COLOR=rgb(184, 49, 47)]b) attributing at least [/COLOR][COLOR=rgb(41, 105, 176)]two distinct interconverting microstates[/COLOR][COLOR=rgb(184, 49, 47)] of an [/COLOR][COLOR=rgb(41, 105, 176)]unliganded SHBG dimer[/COLOR][COLOR=rgb(184, 49, 47)] having a [/COLOR][COLOR=rgb(41, 105, 176)]first monomer[/COLOR][COLOR=rgb(184, 49, 47)] and a [/COLOR][COLOR=rgb(41, 105, 176)]second monomer;[/COLOR][/B] [B][COLOR=rgb(184, 49, 47)]and c) calculating the free testosterone concentration[/COLOR] in the individual using the [COLOR=rgb(41, 105, 176)]New Multi-Step Dynamic Binding Model with Complex Allostery[/COLOR] encompassing [COLOR=rgb(41, 105, 176)]readjustment of a first equilibria[/COLOR] between the [COLOR=rgb(41, 105, 176)]microstates upon binding[/COLOR] of a [COLOR=rgb(41, 105, 176)]first testosterone molecule [/COLOR]to the[COLOR=rgb(41, 105, 176)] first monomer[/COLOR] and an [COLOR=rgb(41, 105, 176)]allosteric interaction [/COLOR]between [COLOR=rgb(41, 105, 176)]two binding sites [/COLOR]of the [COLOR=rgb(41, 105, 176)]SHBG dimer.[/COLOR][/B] *Refer to article to read [0007] - [0017] [B][COLOR=rgb(184, 49, 47)][0018] [/COLOR]In one aspect, described herein is a [COLOR=rgb(184, 49, 47)]method for determining[/COLOR] a need for [COLOR=rgb(184, 49, 47)]adjustment of a dose[/COLOR] of [COLOR=rgb(184, 49, 47)]testosterone administered[/COLOR] to an individual comprising[/B] [B][COLOR=rgb(184, 49, 47)]a) determining the concentration of free testosterone in an individual receiving testosterone therapy at a first dose, wherein the concentration of free testosterone is determined by b) measuring [/COLOR][COLOR=rgb(41, 105, 176)]i) a total SHBG concentration, ii) a total testosterone concentration, and iii) a total albumin concentration in a biological sample obtained from an individual[/COLOR], to determine[COLOR=rgb(184, 49, 47)] free testosterone concentration [/COLOR]from the individual; [COLOR=rgb(184, 49, 47)]c) attributing at least [/COLOR][COLOR=rgb(41, 105, 176)]two distinct interconverting microstates [/COLOR][COLOR=rgb(184, 49, 47)]of an [/COLOR][COLOR=rgb(41, 105, 176)]unliganded SHBG dimer[/COLOR][COLOR=rgb(184, 49, 47)] having a[/COLOR][COLOR=rgb(41, 105, 176)] first monomer [/COLOR][COLOR=rgb(184, 49, 47)]and a [/COLOR][COLOR=rgb(41, 105, 176)]second monomer[/COLOR][COLOR=rgb(184, 49, 47)] by applying the [/COLOR][COLOR=rgb(41, 105, 176)]New Multi-Step Dynamic Binding Model with Complex Allostery[/COLOR][COLOR=rgb(184, 49, 47)] to the data of steps a) and b)[/COLOR]; [COLOR=rgb(184, 49, 47)]d) calculating the free testosterone concentration in the individual [/COLOR]using the [COLOR=rgb(44, 130, 201)]New Multi-step Dynamic Binding Model with Complex Allostery [/COLOR]encompassing [COLOR=rgb(41, 105, 176)]readjustment[/COLOR] of a [COLOR=rgb(41, 105, 176)]first equilibria[/COLOR] between the [COLOR=rgb(41, 105, 176)]microstates[/COLOR] upon[COLOR=rgb(41, 105, 176)] binding [/COLOR]of a [COLOR=rgb(41, 105, 176)]first testosterone molecule [/COLOR]to the [COLOR=rgb(41, 105, 176)]first monomer[/COLOR] and an [COLOR=rgb(41, 105, 176)]allosteric interaction[/COLOR] between [COLOR=rgb(41, 105, 176)]two binding sites[/COLOR] of the [COLOR=rgb(41, 105, 176)]SHBG dimer[/COLOR]; [COLOR=rgb(184, 49, 47)]e) providing a second dose of testosterone[/COLOR] that is higher than the [COLOR=rgb(184, 49, 47)]first dose [/COLOR]when the [COLOR=rgb(184, 49, 47)]free testosterone concentration [/COLOR]is [COLOR=rgb(184, 49, 47)]below[/COLOR] the [COLOR=rgb(184, 49, 47)]lower end[/COLOR] of the [COLOR=rgb(184, 49, 47)]target therapeutic range[/COLOR] ([COLOR=rgb(184, 49, 47)]e.g.164 pg/ml[/COLOR]); and [COLOR=rgb(184, 49, 47)]f) providing a second dose of testosterone [/COLOR]that is lower than the [COLOR=rgb(184, 49, 47)]first dose[/COLOR] when the[COLOR=rgb(184, 49, 47)] free testosterone concentration[/COLOR] is [COLOR=rgb(184, 49, 47)]above [/COLOR]the [COLOR=rgb(184, 49, 47)]upper end[/COLOR] of the [COLOR=rgb(184, 49, 47)]target therapeutic range [/COLOR][COLOR=rgb(0, 0, 0)]([/COLOR][COLOR=rgb(184, 49, 47)]314 pg/ml[/COLOR][/B][COLOR=rgb(0, 0, 0)][B])[/B][/COLOR][B][COLOR=rgb(184, 49, 47)].[/COLOR] [/B] [0019] In some embodiments of any of the foregoing aspects, the step of attributing can be performed according to Figures 2, 3, 5, and 7. In some embodiments, of any of the foregoing aspects, the step of calculating can be performed according to Figure 7 or Example 5. In some embodiments of any of the foregoing aspects, the individual is a male over the age of 35. In some embodiments of any of the foregoing aspects, the androgen disorder is selected from the group consisting of a testosterone deficiency, an androgen deficiency, a hyperandrogenic disorder, an androgen expressing tumor, and a hypogonadism disorder. In some embodiments of any of the foregoing aspects, the androgen disorder is a hyperandrogenic disorder selected from the group consisting of an acne disorder, a hirsutism disorder, and an androgenic alopecia disorder. In some embodiments of any of the foregoing aspects, the individual has been diagnosed with a disease selected from the group consisting of: diabetes, human immunodeficiency virus (HIV), hepatitis B, hepatitis C, hypothyroidism or hyperthyroidism, androgen insensitivity, acromegaly, anorexia, muscular dystrophy, liver disease, cancer cachexia, malnutrition, nephrotic syndrome, and obesity, and other conditions in which SHBG or albumin concentrations are altered. In some embodiments of any of the foregoing aspects, the assay, method, system, or medium can further comprise the step of classifying the individual into categories based on additional clinical symptoms. In some embodiments of any of the foregoing aspects, the assay, method, system, or medium can further comprise the step of using the free testosterone concentration determined using the new Multistep Dynamic Binding Model with Complex Allostery to determine the dose or to individually adjust the dose of a formulation of testosterone for the treatment of a medical disease, taking into account patient's age, body weight and body mass index, medical conditions, including any co-morbid conditions, albumin and SHBG, and/or LH and FSH concentrations, and other patient-specific factors. In some embodiments of any of the foregoing aspects, instead of steps a-c, the data received is a previously calculated concentration of free testostosterone. [B]BRIEF DESCRIPTION OF THE DRAWINGS[/B] [ATTACH=full]7251[/ATTACH] [B][COLOR=rgb(184, 49, 47)][0020] Figs. 1A-1B[/COLOR] demonstrate [COLOR=rgb(184, 49, 47)]that[/COLOR] [COLOR=rgb(184, 49, 47)]free T values calculated[/COLOR] [COLOR=rgb(184, 49, 47)]using the Vermeulen/ Sodergard/ Mazer model [/COLOR]- the commonly used method for determining free T concentrations - [COLOR=rgb(184, 49, 47)]differ[/COLOR] from those measured using [COLOR=rgb(184, 49, 47)]equilibrium dialysis.[/COLOR] Fig. 1A depicts a graph of [COLOR=rgb(41, 105, 176)]free testosterone concentrations [/COLOR]in samples derived from a [COLOR=rgb(41, 105, 176)]randomized testosterone trial (The TED study)[/COLOR] were measured using the [COLOR=rgb(41, 105, 176)]equilibrium dialysis[/COLOR] and plotted against those [COLOR=rgb(41, 105, 176)]calculated using the Vermeulen/ Sodergard/ Mazer equation.[/COLOR] [COLOR=rgb(184, 49, 47)]Calculated free T concentrations differed systematically [/COLOR]from the [COLOR=rgb(184, 49, 47)]measured values. [/COLOR]Fig. 1B depicts a [COLOR=rgb(41, 105, 176)]Bland Altman plot[/COLOR] revealing [COLOR=rgb(41, 105, 176)]substantial discrepancy [/COLOR]between the [COLOR=rgb(41, 105, 176)]calculated and measured free T concentrations.[/COLOR][/B] [ATTACH=full]7252[/ATTACH] [B][COLOR=rgb(184, 49, 47)][0021] Figs. 2A-2C[/COLOR][/B] [B]demonstrate [COLOR=rgb(184, 49, 47)]that binding of testosterone to SHBG[/COLOR] displays [COLOR=rgb(184, 49, 47)]complex behavior. [/COLOR]Fig. 2A depicts a graph demonstrating that [COLOR=rgb(41, 105, 176)]binding isotherms[/COLOR] display [COLOR=rgb(41, 105, 176)]complex behavior.[/COLOR] [COLOR=rgb(184, 49, 47)]Graded concentrations of testosterone[/COLOR] were incubated overnight with [COLOR=rgb(184, 49, 47)]various SHBG concentrations[/COLOR] and the amount of [COLOR=rgb(184, 49, 47)]bound testosterone [/COLOR]was plotted against [COLOR=rgb(184, 49, 47)]total T (added) concentrations[/COLOR] (squares) 20nM, (triangles) 10 nM, (circles) 5nM SHBG. [/B][COLOR=rgb(184, 49, 47)][B]The [/B][/COLOR][COLOR=rgb(41, 105, 176)][B]fit curves [/B][/COLOR][COLOR=rgb(184, 49, 47)][B]represent the result of the linked fit of data to the [/B][/COLOR][COLOR=rgb(41, 105, 176)][B]new Multi-step Dynamic Binding Model with Complex Allostery [/B][/COLOR][COLOR=rgb(184, 49, 47)][B](Fig. 3).[/B][/COLOR] [B]Fig. 2B depicts a graph of the depletion of [COLOR=rgb(41, 105, 176)]free testosterone [/COLOR]by [COLOR=rgb(41, 105, 176)]varying SHBG concentration[/COLOR] which is best described by the [COLOR=rgb(41, 105, 176)]new Multi-step Dynamic Binding Model with Complex Allostery.[/COLOR][/B] [B]Shown is concentration of free testosterone in the equilibrium dialysis experiment. [/B][COLOR=rgb(184, 49, 47)][B]One side of the equilibrium dialysis membrane has [/B][/COLOR][COLOR=rgb(41, 105, 176)][B]varying concentration of SHBG in buffer[/B][/COLOR][COLOR=rgb(184, 49, 47)][B], the other one has [/B][/COLOR][COLOR=rgb(41, 105, 176)][B]plain buffer. [/B][/COLOR][B]Constant concentration of testosterone is added to each well of multi-well dialyzer: (triangles) 8.7nM.[/B] [COLOR=rgb(184, 49, 47)][B]Curves are the result of the linked fit of data in Panel B to the new Multi-step Dynamic Binding Model with Complex Allostery (Fig 3). [/B][/COLOR] [ATTACH=full]7253[/ATTACH] [B]Fig. 2C depicts [COLOR=rgb(41, 105, 176)]a graph of the[/COLOR] [COLOR=rgb(41, 105, 176)]heat of T:SHBG association [/COLOR]measured by i[COLOR=rgb(41, 105, 176)]sothermal calorimetry (ITC). [/COLOR]Presented is the integrated ITC curve with buffer heats subtracted.[/B][COLOR=rgb(184, 49, 47)] [B]SHBG starting concentration is 5μΜ. Experimental points are shown by squares, fit to the model in Fig.3 is shown by a solid line.[/B][/COLOR] [ATTACH=full]7254[/ATTACH] [B][COLOR=rgb(184, 49, 47)][0022] Fig.[/COLOR][/B] [B]3 depicts [COLOR=rgb(184, 49, 47)]a[/COLOR] [COLOR=rgb(184, 49, 47)]schematic representation [/COLOR]of the[COLOR=rgb(184, 49, 47)] new Multi-step Dynamic Binding Model with Complex Allostery of testosterone's binding to SHBG and albumin[/COLOR], developed in this study.[/B] [B][COLOR=rgb(41, 105, 176)]Unliganded SHBG dimers (S2, S2')[/COLOR] exist in [COLOR=rgb(41, 105, 176)]conformational equilibrium. [/COLOR]Upon [COLOR=rgb(41, 105, 176)]binding of the first testosterone molecule to microstates S2 and S2' [/COLOR]can result in [COLOR=rgb(41, 105, 176)]conformationally heterogeneous intermediate states S2T and S2'T[/COLOR] respectively. These [COLOR=rgb(41, 105, 176)]singly-occupied microstates [/COLOR]then [COLOR=rgb(41, 105, 176)]converge to S2T2 [/COLOR]upon [COLOR=rgb(41, 105, 176)]binding of the second testosterone molecule. [/COLOR][/B] [ATTACH=full]7255[/ATTACH] [B][COLOR=rgb(184, 49, 47)][0023] Figs. 4A-4B [/COLOR]demonstrate a comparison of [COLOR=rgb(184, 49, 47)]the Free Testosterone Concentrations Derived Using the Vermeulen Equation[/COLOR] [COLOR=rgb(184, 49, 47)](23) [/COLOR]as implemented by Mazer (24) or the [COLOR=rgb(184, 49, 47)]New Algorithm Based on the new Multi-step Dynamic Binding Model with Complex Allostery [/COLOR]with those measured using the [COLOR=rgb(184, 49, 47)]equilibrium dialysis [/COLOR]in samples from the [COLOR=rgb(184, 49, 47)]5alpha reductase trial.[/COLOR] Fig. 4A depicts a graph of a comparison of the [COLOR=rgb(41, 105, 176)]free testosterone concentration calculated by the Vermeulen equation [/COLOR]and the [COLOR=rgb(41, 105, 176)]new algorithm based on the new Multi-step Dynamic Binding Model with Complex Allostery [/COLOR]to that measured by [COLOR=rgb(41, 105, 176)]equilibrium dialysis[/COLOR] in samples from a randomized testosterone trial in men. [COLOR=rgb(184, 49, 47)](■) free testosterone concentrations[/COLOR] derived using an [COLOR=rgb(184, 49, 47)]algorithm based on new Multi-step Dynamic Binding Model with Complex Allostery[/COLOR]; (•) free testosterone concentrations derived using the Vermeulen model (23) as implemented by Mazer (24).[/B] [B][COLOR=rgb(184, 49, 47)]Solid lines are lines of best linear fit.[/COLOR][/B] [B][COLOR=rgb(0, 0, 0)]Regression lines fit new Multi-step Dynamic Binding Model with Complex Allostery calculation (slope = 1.01±0.01, regression line fitting the squares)[/COLOR], and the [COLOR=rgb(184, 49, 47)]Vermeulen model (slope 0.77±0.02, lower line fitting the dots).[/COLOR][/B] [B]Magenta dashed line is the line of prefect correlation. [COLOR=rgb(184, 49, 47)]Fig. 4B [/COLOR]depicts [COLOR=rgb(41, 105, 176)]Bland Altman plots [/COLOR]of the relative frequency distribution of % difference of[COLOR=rgb(41, 105, 176)] calculated and measured free testosterone[/COLOR] using either the [COLOR=rgb(41, 105, 176)]Vermeulen equation (squares)[/COLOR] or the [COLOR=rgb(41, 105, 176)]new algorithm based on the new Multi-step Dynamic Binding Model with Complex Allostery (black dots)[/COLOR] The relative deviations from the measured value are distributed around [COLOR=rgb(0, 0, 0)]0[/COLOR] [COLOR=rgb(0, 0, 0)]for [/COLOR][COLOR=rgb(41, 105, 176)]new Multi-step Dynamic Binding Model with Complex Allostery model [/COLOR]and are different from zero for the [COLOR=rgb(41, 105, 176)]Vermeulen model.[/COLOR] [/B] [ATTACH=full]7256[/ATTACH] [B][COLOR=rgb(184, 49, 47)]Figs. 4C-4D [/COLOR]depict [COLOR=rgb(184, 49, 47)]a[/COLOR] [COLOR=rgb(184, 49, 47)]Comparison of the Free Testosterone Concentrations Derived Using the Vermeulen Equation [/COLOR]or the [COLOR=rgb(184, 49, 47)]New Algorithm Based on the new Multi-step Dynamic Binding Model with Complex Allostery[/COLOR] with those measured using the equilibrium dialysis. [COLOR=rgb(184, 49, 47)]Figs. 4C-4D [/COLOR]demonstrate a [COLOR=rgb(41, 105, 176)]Comparison of the Free Testosterone Concentrations Derived Using the Vermeulen Equation[/COLOR] or the [COLOR=rgb(41, 105, 176)]New Algorithm Based on the new Multi-step Dynamic Binding Model with Complex Allostery [/COLOR]with those measured using the [COLOR=rgb(41, 105, 176)]equilibrium dialysis[/COLOR] in samples from a randomized testosterone trial in men with ED.[/B] [B][COLOR=rgb(184, 49, 47)]Fig. 4C[/COLOR][/B] [B]depicts a graph of a comparison of the [COLOR=rgb(184, 49, 47)]free testosterone concentration calculated by the Vermeulen equation[/COLOR] and the [COLOR=rgb(184, 49, 47)]new algorithm based on the new Multi-step Dynamic Binding Model with Complex Allostery[/COLOR] to that measured by [COLOR=rgb(184, 49, 47)]equilibrium dialysis[/COLOR] in samples from a randomized testosterone trial in men. [/B][COLOR=rgb(184, 49, 47)][B](squares) free testosterone concentrations derived using an algorithm based on new Multi-step Dynamic Binding Model with Complex Allostery[/B]; [/COLOR][COLOR=rgb(0, 0, 0)][B](circles) free testosterone concentrations derived using the Vermeulen model (23) as implemented by Mazer (24).[/B][/COLOR][B] Black regression line fits [COLOR=rgb(41, 105, 176)]new Multi-step Dynamic Binding Model with Complex Allostery model calculation (slope = 1.01±0.01 is shown)[/COLOR] Fig. 4D depicts Bland Altman plots of the relative frequency of % difference of [COLOR=rgb(41, 105, 176)]calculated[/COLOR] and [COLOR=rgb(41, 105, 176)]measured free testosterone [/COLOR]using either the [COLOR=rgb(41, 105, 176)]Vermeulen equation [/COLOR]or the [COLOR=rgb(41, 105, 176)]new algorithm based on the new Multi-step Dynamic Binding Model with Complex Allostery. [/COLOR]The relative deviations from the measured value are distributed around 0 for the[COLOR=rgb(184, 49, 47)] new Multi-step Dynamic Binding Model with Complex Allostery model[/COLOR] and are different from zero for the [COLOR=rgb(184, 49, 47)]Vermeulen model.[/COLOR][/B] [ATTACH=full]7257[/ATTACH] [B][COLOR=rgb(184, 49, 47)][0024] Fig. 5A [/COLOR]depicts a graph of [COLOR=rgb(184, 49, 47)]the[/COLOR] [COLOR=rgb(184, 49, 47)]binding isotherm.[/COLOR] Graded concentrations of testosterone were incubated overnight with[COLOR=rgb(184, 49, 47)] 20 nM SHBG[/COLOR] and the amount of [COLOR=rgb(184, 49, 47)]bound testosterone[/COLOR] was plotted against [COLOR=rgb(184, 49, 47)]total testosterone concentration.[/COLOR][/B] [B]The fit curve represents the fit of data to the [COLOR=rgb(184, 49, 47)]new Multi-step Dynamic Binding Model with Complex Allostery. [/COLOR]Fig. 5B depicts a graph of the [COLOR=rgb(41, 105, 176)]depletion curve.[/COLOR][/B] [B]A constant concentration of testosterone [COLOR=rgb(41, 105, 176)](8.7 nM)[/COLOR] was incubated with [COLOR=rgb(41, 105, 176)]increasing SHBG concentrations.[/COLOR] [COLOR=rgb(184, 49, 47)]Free testosterone concentration[/COLOR] is plotted against [COLOR=rgb(184, 49, 47)]SHBG concentration. [/COLOR]Solid line represents the fit of data to new [COLOR=rgb(184, 49, 47)]Multi-step Dynamic Binding Model with Complex Allostery.[/COLOR] The depletion of [COLOR=rgb(41, 105, 176)]free testosterone by increasing SHBG concentrations[/COLOR] is best described by the [COLOR=rgb(41, 105, 176)]new Multi-step Dynamic Binding Model with Complex Allostery.[/COLOR][/B] [B]Fig. 5C depicts a graph of the [COLOR=rgb(184, 49, 47)]heat of testosterone and SHBG association [/COLOR]measured by [COLOR=rgb(184, 49, 47)]isothermal calorimetry.[/COLOR] The integrated ITC curve was generated after subtracting the buffer heats. [COLOR=rgb(184, 49, 47)]SHBG starting concentration is 5 μΜ.[/COLOR] Experimental points are shown by (■), and fit of the data to the [COLOR=rgb(184, 49, 47)]new Multi-step Dynamic Binding Model with Complex Allostery model [/COLOR]is shown by a solid line. [/B] [ATTACH=full]7258[/ATTACH] [B][COLOR=rgb(184, 49, 47)][0025] Fig. 6[/COLOR][/B] [B]depicts [COLOR=rgb(184, 49, 47)]schematic representations [/COLOR]of the [COLOR=rgb(184, 49, 47)]various models tested[/COLOR] in this study to [COLOR=rgb(184, 49, 47)]examine SHBG:T interaction.[/COLOR][/B] [B]Model A. Vermeulen's model, [COLOR=rgb(41, 105, 176)]homogenous interaction of testosterone molecule with equal affinity for each monomer in SHBG dimer (Kd=lnM)[/COLOR]; Model B. [COLOR=rgb(184, 49, 47)]monomers within the SHBG dimer exhibit distinct affinity constants with Kdi=lnM and Kd2 allowed to vary for data fits[/COLOR][/B]; [B]Model C. [COLOR=rgb(41, 105, 176)]Inter-subunit allostery with positive cooperativity for binding of two ligands such that Kd2=1nM, Kd1 allowed to vary and Kd2<Kd1[/COLOR]; Model D. [COLOR=rgb(184, 49, 47)]allostery with negative cooperativity for binding of two ligands such that Kd1=1nM, Kd2allowed to vary and Kd1<Kd2;[/COLOR] and, Model E. [COLOR=rgb(41, 105, 176)]The new Multi-step Dynamic Binding Model with Complex Allostery[/COLOR] which encompasses [COLOR=rgb(41, 105, 176)]two distinct SHBG microstates in equilibrium [/COLOR]such that the[COLOR=rgb(41, 105, 176)] equilibria between the unliganded and mono-liganded states readjust[/COLOR] as testosterone concentration is increased. [/B] [/QUOTE]
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