METHODS FOR TREATING TESTOSTERONE DEFICIENCY IN MEN AND METHODS FOR PRECISE DOSING OF UGT2B17 SUBSTRATE DRUGS
1. A method for treating testosterone deficiency in a subject, comprising the steps of:
- obtaining a sample from a subject suspected of having testosterone deficiency;
determining the presence in the sample of one or more biomarkers of UGT2B17 activity;
identifying an effective therapeutic amount of one or more agents for treating testosterone deficiency; and
administering the effective therapeutic amount of the agent to treat testosterone deficiency in the subject in need thereof.
Treatment of testosterone deficiency in men by a precision medicine approach using a biomarker of activity of UGT2B17 that is involved in testosterone urinary elimination. By inhibiting UGT2B17, alone or in combination with administration of testosterone, testosterone deficiency in men can be treatable. Further, a method of dose selection for precise dosing of UGT2B17 substrate drugs is provided. Additionally, methods for safe dosing of pharmaceutical agents that undergo UGT2B17-mediated acyl glucuronidation are provided.
- 1. A method for treating testosterone deficiency in a subject, comprising the steps of:
obtaining a sample from a subject suspected of having testosterone deficiency; determining the presence in the sample of one or more biomarkers of UGT2B17 activity; identifying an effective therapeutic amount of one or more agents for treating testosterone deficiency; and administering the effective therapeutic amount of the agent to treat testosterone deficiency in the subject in need thereof.
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
- 13. A method for treating a subject with a pharmaceutical agent that is metabolized by UGT2B17-dependent acyl glucuronidation, comprising the steps of:
obtaining a sample from a subject; determining the presence of one or more biomarkers of UGT2B17 activity in the sample; identifying an effective therapeutic amount of a pharmaceutical agent that is metabolized by UGT2B17-dependent acyl glucuronidation; and administering the effective therapeutic amount of the pharmaceutical agent to the subject in need thereof.
- View Dependent Claims (14, 15, 16, 17, 18, 19)
- 20. A method for testing an individual for testosterone doping, the method comprising:
(a) obtaining a biological sample from an individual subject to testosterone doping testing; (b) determining a ratio of testosterone glucuronide (TG) to aldosterone glucuronide (AG) in the biological sample; and (c) comparing the ratio of testosterone glucuronide (TG) to aldosterone glucuronide (AG) in the sample with that from a retention sample.
This application claims the benefit of U.S. Provisional Application No. 62/655,705, filed Apr. 10, 2018, the disclosure of which is expressly incorporated herein by reference in its entirety.
This invention was made with government support under Grant No. R01 HD081299 awarded by the National Institutes of Health. The government has certain rights in the invention.
Testosterone (T) is the key male sex-hormone. T plays an indispensable role in the development of male reproductive system during fetal life, and triggers the development of secondary sexual characteristics at puberty and maintains sexual function and bone and muscle health during adulthood. Low concentrations of T can occur for a variety of reasons. Men with low T experience fatigue, depression, diminished libido, loss of muscle strength, osteoporosis, visceral obesity, and insulin resistance. T-replacement therapy (TRT) is the first-line treatment for T deficiency and has been shown to improve bone density, alleviate depression and maintain sexual function. Currently available options for TRT in the United States (US) include intramuscular injections, transdermal patches and gels. The development of an oral TRT has been extremely challenging because of extensive first pass metabolism. As a result, the 2-4 million hypogonadal men in the US requiring TRT mainly rely on intra-muscular or topical T dosage forms, both of which are challenging in terms of compliance, and have sub-optimal pharmacokinetics (PK) or variable absorption. Clearly, the development of a safe and effective formulation of oral T is a major unmet need for these men.
Oral T is readily absorbed from small intestine by diffusion in humans, but the absorbed T dose is metabolized in both the intestine and the liver before reaching to the circulation, resulting in a very poor bioavailability of 2-8%. Exogenously administered T is metabolized to multiple unconjugated and conjugated metabolites, where the major urinary metabolites are glucuronide conjugates which are formed in the liver and intestine by various human UDP-glucuronosyl transferases (UGTs). In particular, UGT2B17 is the major T-glucuronidation enzyme. UGT2B17 is of interest as it is highly polymorphic and copy number variations (CNVs) in its gene are associated with multiple potentially T-related pathologies, e.g., obesity, prostate cancer, osteoporosis, ankylosing spondylitis, and endometrial cancer. UGT2B17 is highly expressed in the intestine and the liver and leads to formation of acyl glucuronide metabolites, which are often associated with hepatotoxicity or extra-hepatic toxicity of pharmaceutical agents.
Thus, because of UGT2B17 high variability, there is a need for a biomarker that can identify and stratify individuals who would experience greater levels of UGT2B17-mediated acyl glucuronides formation than others and allow precise, individualized dosing of oral testosterone and other pharmaceutical agents which are subject to UGT2B17-mediated metabolism.
This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
In one aspect, provided herein is a method for treating testosterone deficiency in a subject, comprising the steps of:
obtaining a sample from a subject suspected of having testosterone deficiency;
determining the presence in the sample of one or more biomarkers of UGT2B17 activity;
In some embodiments, the one or more biomarkers of UGT2B17 activity is a normalized testosterone glucuronide (normalized TG), for example, a ratio of concentration of testosterone glucuronide (TG) to concentration of aldosterone glucuronide (AG), a ratio of concentration of testosterone glucuronide (TG) to concentration of etiocholanolone glucuronide (EG), a ratio of concentration of testosterone glucuronide (TG) to concentration of testosterone sulfate (TS), a ratio of concentration of testosterone glucuronide (TG) to concentration of hydroxyl-testosterone, or a ratio of concentration of testosterone glucuronide TG to concentration of epitestosterone glucuronide (epiT). In some embodiments, the sample is blood, urine, plasma, or serum.
In some embodiments, the one or more agents for treating testosterone deficiency comprises orally administered testosterone, an inhibitor of UGT2B17, such as ibuprofen, diclofenac, or imatinib, or a combination of orally administered testosterone with one or more UGT2B17 inhibitors.
In another aspect, provided herein is a method for treating a subject with a pharmaceutical agent that is metabolized by UGT2B17-dependent acyl glucuronidation, comprising the steps of:
obtaining a sample from a subject;
quantifying one or more biomarkers of UGT2B17 activity in the sample, such as normalized TG;
In some embodiments, the effective therapeutic amount is an amount that does not result in hepatotoxicity caused by UGT2B17 mediated acyl-glucuronidation in the subject.
In yet another aspect, provided herein is a method for testing an individual for testosterone doping, the method comprising:
(a) obtaining a biological sample from an individual subject to testosterone doping testing;
The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings.
The disclosure addresses treating testosterone deficiency in men using a precision medicine approach. Based on the analysis of human samples, the inventors have identified that a critical enzyme UGT2B17, which is involved in testosterone elimination, is highly variable in men and have subsequently identified a specific UGT2B17 biomarker as described below.
Thus, in one aspect, the disclosure provides a method for treating testosterone deficiency in a subject, comprising the steps of:
obtaining a sample from a subject suspected of having testosterone deficiency;
determining the presence in the sample of one or more biomarkers of UGT2B17 activity;
In some embodiments, the one or more biomarkers of UGT2B17 activity is a phenotypical biomarker. In some embodiments, the one or more biomarkers of UGT2B17 activity is a normalized testosterone glucuronide (normalized TG). In some embodiments, normalized TG is a ratio of the concentration of TG to the concentration of another T metabolite, for example, aldosterone glucuronide (AG), in a sample. As discussed below, in some embodiments, the normalized TG can be a ratio of concentration of testosterone glucuronide (TG) to concentration of aldosterone glucuronide (AG), a ratio of concentration of testosterone glucuronide (TG) to concentration of etiocholanolone glucuronide (EG), a ratio of concentration of testosterone glucuronide (TG) to concentration of testosterone sulfate (TS), a ratio of concentration of testosterone glucuronide (TG) to concentration of hydroxyl-testosterone, or a ratio of concentration of testosterone glucuronide TG to concentration of epitestosterone glucuronide (epiT). In some embodiments, normalized TG is a ratio of concentrations of TG and aldosterone glucuronide (AG), in a sample. In certain embodiments, a molar ratio of testosterone glucuronide (TG) to aldosterone glucuronide (AG) is used as a biomarker of UGT2B17 activity.
Any suitable sample can be used in the methods disclosed herein. In some embodiments, the sample is a biological sample selected from blood, urine, plasma, or serum. The determining of the presence of one or more biomarkers of UGT2B17 activity in the sample can be done by any method that is sufficiently sensitive to detect the presence of one or more biomarkers of UGT2B17 activity. In some embodiments, the method includes liquid chromatography and mass spectrometry. In some embodiments, the method includes LC/MS or LC-MS/MS. In some embodiments, the method comprises extracting one or more testosterone metabolites, such as TG, AG, EpiT, and the like, from the biological sample, for example, by solid phase extraction. In some embodiments, the method comprises separation of one or more testosterone metabolites by liquid chromatography, such as gradient liquid chromatography. In some embodiments, the determining the presence in the sample of one or more biomarkers of UGT2B17 activity comprises reverse phase liquid chromatography. Detection and/or quantification of the concentration of one or more testosterone metabolites, such as TG, AG, EpiT can be done by any suitable method, for example, by tandem mass spectrometry or MS/MS. In some embodiments, determining the presence in the sample of one or more biomarkers of UGT2B17 activity comprises the use of one or more internal standards.
In some embodiments, the one or more agents for treating testosterone deficiency comprise testosterone. In some embodiments, testosterone is administered orally for instance, as a pharmaceutical formulation comprising one or more excipients. Any suitable oral formulation of testosterone can be used in the methods of the disclosure. For example, in some embodiments, the testosterone is formulated for gastroretentive dosage. In some embodiments, the methods use gastroretentive formulations of testosterone. As used herein, gastroretentive formulations include formulations designed to remain in the stomach for a prolonged and predictable period of time. Consequently, gastric residence time of drug substances, such as testosterone, is extended and their bioavailability improved. Non-limiting examples of gastroretentive drug delivery approaches include co-administration of drugs or pharmaceutical excipients influencing gastric motility pattern and thereby delaying gastric emptying process, magnetic systems, mucoadhesive systems, size-increasing systems due to swelling or unfolding, density-controlled systems that either float on gastric contents or sediment, and combination systems.
In some embodiments, the amount of normalized TG in an individual'"'"'s biological sample can be used to determine the therapeutically effective oral dose of testosterone for the subject. In some embodiments, the dose is proportional to the amount of the one or more biomarkers of UGT2B17 activity, for example, a ratio of TG/AG.
In some embodiments, the one or more agents for treating testosterone deficiency comprise an inhibitor of UGT2B17. Any suitable inhibitor of UGT2B17 can be used in the methods of the disclosure. For example, in some embodiments, the inhibitor of UGT2B17 activity comprises a nonsteroidal anti-inflammatory drug (NSAID) or a small molecule kinase inhibitor. Useful NSAIDs include ibuprofen, diclofenac, and combinations thereof. In some embodiments, the inhibitor of UGT2B17 is imatinib. In some embodiments, the inhibitor of UGT2B17 activity is co-administered with testosterone. Co-administration of one or more therapeutic agents, as used herein, includes administration of one or more pharmaceutical agents simultaneously, for example, in a single formulation, and sequentially.
In another aspect, provided herein are methods of reducing hepatotoxcicity and/or adverse effects of pharmaceutical agents that undergo UGT2B17-dependent acyl glucuronidation. Thus, the disclosure provides a method for treating a subject with a pharmaceutical agent that undergoes UGT2B17-dependent acyl glucuronidation, comprising the steps of:
obtaining a sample from a subject;
determining the presence of one or more biomarkers of UGT2B17 activity in the sample;
identifying an effective therapeutic amount of a pharmaceutical agent; and
UGT2B17-mediated metabolism of certain pharmaceutical agents generally leads to formation of acyl glucuronides which are often associated with hepatotoxicity or extra-hepatic toxicity of the parent pharmaceutical agents. For example, diclofenac forms acyl glucuronide which leads to its toxicity, and an individual with high UGT2B17 is expected to form higher diclofenac glucuronide that will eventually lead to greater toxicity of diclofenac. Because UGT2B17 is highly variable, a suitable biomarker, such as normalized TG, that identifies individuals who would experience greater levels of acyl glucuronide than others, can allow personalization of dosing of such pharmaceutical agents.
In some embodiments, the methods disclosed herein can be used in drug development, for example, to predict and/or pre-empt potential toxicity of a pharmaceutical agent in clinical trials. In some embodiments, the methods disclosed herein can be used to identify individuals with a greater risk of adverse drug-drug interactions. Acyl glucuronides are known to inactivate another drug metabolizing enzyme, Cytochrome P450 2C8 (CYP2C8), which is involved in metabolism of certain pharmaceutical agents. For example, clopidogrel is an antiplatelet drug that is metabolized to clopidogrel acid glucuronide (CAG) by UGT2B17. CAG is a potent inactivator of CYP2C8 and thus leads to increased plasma concentration of CYP2C8 substrates (e.g., repaglinide, pioglitazone, rosiglitazone, and cerivastatin), in clinic when co-administered. In particular, increased cerivastatin levels have been shown to be associated with rhabdomyolysis and even deaths, resulting withdrawal of this drug from market in early 2000, which was primarily observed in patients taking drugs such as gemfibrozil and clopidogrel, both of which form acyl glucuronides. Therefore, the UGT2B17 phenotypic biomarker, for example, normalized TG, can identify individuals with greater risks to such drug-interactions.
In some embodiments of the methods disclosed herein, the pharmaceutical agent is an agent that undergoes UGT2B17-dependent acyl glucuronidation. In certain embodiments, the pharmaceutical agent is selected from the group consisting of vorinostat, exemestane, and clopidogrel.
As described above, any suitable biological sample can be used, such as blood, urine, plasma, or serum. In some embodiments, the sample is urine.
In some embodiments, the determining the presence of one or more biomarkers of UGT2B17 activity in the sample is done by LC-MS/MS as described above.
In certain embodiments, the one or more biomarkers of UGT2B17 activity is a phenotypical biomarker. In some embodiments, the one or more biomarkers of UGT2B17 activity is normalized testosterone glucuronide (normalized TG), for example, a ratio of concentration of testosterone glucuronide (TG) to concentration of aldosterone glucuronide (AG), a ratio of concentration of testosterone glucuronide (TG) to concentration of etiocholanolone glucuronide (EG), a ratio of concentration of testosterone glucuronide (TG) to concentration of testosterone sulfate (TS), a ratio of concentration of testosterone glucuronide (TG) to concentration of hydroxyl-testosterone, or a ratio of concentration of testosterone glucuronide TG to concentration of epitestosterone glucuronide (epiT). In some embodiments, normalized TG is a ratio of concentrations of TG and aldosterone glucuronide (AG) in a sample. In certain embodiments, a molar ratio of testosterone glucuronide (TG) to aldosterone glucuronide (AG) is used as a biomarker of UGT2B17 activity.
As used herein, the effective therapeutic amount is an amount of a pharmaceutical agent that does not result in hepatotoxicity caused by UGT2B17 mediated acyl-glucuronidation of the pharmaceutical in the subject.
In yet another aspect, the disclosure provides a method for testing a biological sample for testosterone doping, comprising: obtaining a biological sample from a subject and determining the amount of normalized TG in the biological sample. In some embodiments, the methods further comparing the amount of normalized T with that from a control or a retention sample.
Testosterone doping is typically tested by measuring the total testosterone in urine, which is primarily (about 90%) testosterone glucuronide (TG). Because TG is formed via UGT2B17-mediated glucuronidation of T, variability in this enzyme leads to variable test outcomes. For example, UGT2B17 high expressers show higher total urinary TG levels, which can result in a false positive doping test. Similarly, low expression of UGT2B17 in an individual can lead to false negative test results. Because of these reasons, some testosterone doping tests rely on TG/EpiTG to address individual variability. However, TG/EpiTG has poor sensitivity, and a testosterone doping test that accounts for an individual UGT2B17 activity is highly desirable. Thus, in some embodiments, the testosterone doping tests disclosed herein comprise determining a molar ratio of testosterone glucuronide (TG) to aldosterone glucuronide (AG) in a sample, for example, a urine sample, which can be done by any suitable method as described above.
In another aspect, the disclosure provides a method for identification of individuals with greater risk to UGT2B17-associated diseases (e.g., chronic lymphocytic leukemia, osteoporosis, and prostate cancer) and tobacco carcinogenicity by determining normalized TG (e.g., TG/AG) in a biological sample obtained from the individuals suspected to have greater risk of UGT2B17-associated diseases. It has been shown that UGT2B17 gene deletion is associated with chronic lymphoid leukemia (CLL), prostate cancer, and osteoporosis. However, these results are controversial because of the high non-genetic variability in UGT2B17. Thus, using a phenotypic biomarker of UGT2B17 such as the normalized TG (e.g., TG/AG) as disclosed herein, susceptibility to CCL can be predicted.
While each of the elements of the present invention is described herein as containing multiple embodiments, it should be understood that, unless indicated otherwise, each of the embodiments of a given element of the present invention is capable of being used with each of the embodiments of the other elements of the present invention and each such use is intended to form a distinct embodiment of the present invention.
When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, denoting somewhat more or somewhat less than the stated value or range, to ±10% of the stated value.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. It is understood that any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
The following examples are presented for the purpose of illustrating, not limiting, the invention.
Development of an oral testosterone (T) therapy has proven extremely challenging because of extensive and variable first-pass metabolism. The in vivo metabolism of T with increasing oral doses of T, both alone and with the co-administration of dutasteride (5α-reductase inhibitor) by LC-MS/MS are described herein.
Seven healthy male volunteers with mean age 24.2±8.7 years were recruited. T emulsion in sesame oil, mixed with milk, was administered orally to human subjects with acyline-induced experimental hypogonadism. The blood samples were collected and the serum was isolated and stored in −80° C. Steroids and their metabolites were extracted from serum using protein precipitation followed by solid-phase extraction and analyzed by UPLC coupled with SCIEX 6500 MS/MS.
In eugonadal men prior to dosing, the circulating concentration of T, androstenedione (A), etiocholanolone-glucuronide (EtioG), and androsterone-glucuronide (AG) was 8.6, 20.9, 9.1, and 55.3%, respectively, of the total T-related species, whereas T-glucuronide (TG) was ˜1%. When T was dosed orally to men with experimental hypogonadism, a proportion of T-glucuronide increased to 13%. Dutasteride treatment significantly decreased levels of androsterone and its metabolites.
Extensive metabolism of orally dosed T to androsterone glucuronide via androstenedione was revealed, with T-glucuronide appearing to be the second most important metabolite. This information is of importance in the development of an effective oral T therapy and has implications for T doping research.
Oral T is readily absorbed from small intestine by diffusion in humans, but the absorbed T dose is metabolized in both the intestine and the liver before reaching to the circulation, resulting in a very poor bioavailability of 2-8%. Exogenously administered T has previously been reported to be metabolized to multiple unconjugated and conjugated metabolites (
Other than UGTs, 17β-HSD2, 5-AR, CYP3A4, and sulfotransferases (SULTs) can also metabolize T, however, the quantitative contribution of these enzymes in T first-pass metabolism is unknown. Dutasteride is a selective inhibitor of the type 1 and type 2 isoforms of the 5-AR enzyme (
Materials and Methods
LC-MS/MS grade acetonitrile, methanol, chloroform and formic acid were purchased from Fisher scientific (Fair Lawn, N.J., USA). Un-conjugated and conjugated steroid standards were purchased from Cerilliant corporation (Round Rock, Tex.) and Steraloid (Newport, R.I.). Recombinant human UGT2B7, UGT2B15, and UGT2B17 superosomes were purchased from Corning Life Science (Corning, N.Y.), and dutasteride for the in vitro experiments was from Sigma Aldrich (St. Louis, Mo.). Iodoacetamide (IAA), dithiothreitol (DTT), and pierce trypsin protease (MS grade) were purchased from Thermo Fisher Scientific (Rockford, Ill.). Ammonium bicarbonate buffer (ABC, 98% purity) was purchased from Acros Organics (Geel, Belgium). Human serum albumin (HSA) and bovine serum albumin (BSA) were obtained from Calbiochem (Billerica, Mass.) and Thermo Fisher Scientific (Rockford, Ill.), respectively. Surrogate peptides were produced by Thermo Fisher Scientific (Rockford, Ill.). UDPGA and MgCl2 were purchased from Sigma-Aldrich (St. Louis, Mo.). Hepatocyte were provided gratis by Lonza Inc (Walkersville, Md.), hepatocyte thawing media (UCRM™), hepatocyte plating media (UPCM™), hepatocyte induction media (HIM™) and hepatocyte quantification media (HQM™) were procured from IVAL LLC (Columbia, Md.).
For targeted T metabolomics study, previously collected and stored samples from seven subjects who received oral T, were used. Clinical study design is discussed below. All subjects provided written, informed consent prior to any study procedures. The study was approved by the University of Washington (UW) Investigational Review Board Committee. The oral T in sesame oil was manufactured by the compounding pharmacy at the UW, micronized T (U.S.P. grade, Spectrum Quality Projects, Gardena, Calif.) was suspended at 100 mg/ml in sesame oil (N.F. grade, Spectrum Quality Projects) and mixed thoroughly to create a homogenous T plus sesame oil emulsion. The emulsion was vigorously mixed by shaking with milk and administered to the subject. The drug exposure period lasted 11 days. On day 0, subjects received a single injection of the gonadotropin-releasing hormone (GnRH) antagonist acyline (300 μg/kg, subcutaneous), which has been shown to suppress T production in normal men for a minimum of 15 days. After one, two, and three days of acyline administration, subjects were given 200, 400, or 800 mg T-orally. Subjects self-administered dutasteride purchased by the University of Washington investigational pharmacy (0.5 mg, orally, once daily) on day 5-10 after acyline injection, and doses of T were repeated on days 8, 9, and 10, respectively. After each dose of T, subjects had blood drawn via a heparin-locked intravenous line at 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h for measurement of serum T and its unconjugated and conjugated metabolites. Serum was obtained from the blood by centrifugation and stored in −80° C. freezer until analysis by LC-MS/MS.
Serum was pooled from control subjects (before acyline dose, baseline) and from all time points after single oral dose of T at 200 mg (T200) and 800 mg (T800). While study was conducted with 200, 400 and 800 mg T dose, only T200 and T800 were quantified. T and its metabolites were extracted from the human serum sample using a protein precipitation with ice cold methanol (containing internal standard mix, Table 1) followed by solid-phase extraction (SPE) in C18 HLB cartridges (Waters). Calibration curve (CC), quality control (QC), or test samples (500 μL) were transferred to 5 mL micro-centrifuge tube. Protein precipitation was carried out using a 1:4 by volume methanol (2 ml) containing internal standard mix. Samples were mixed for 1 min by vortex mixing, and then centrifuged for 10 min at 3500×g at 4° C. The supernatant was transferred to glass tubes and dried under nitrogen evaporator. The resulting dried residue was re-dissolved in 2 mL of 5% methanol containing 0.2% formic acid and was used for SPE. The SPE cartridges were mounted on the positive pressure manifold and activated with 2 mL methanol and conditioned with 2 mL 0.2% formic acid. Samples (2 mL) were loaded onto the cartridges and flow through was discarded. Samples were washed with 1 mL of 5% methanol containing 0.2% formic acid to get rid of hydrophilic impurities. T metabolites were then eluted with 2 mL of methanol and collected in glass tubes. The eluate was then dried under nitrogen evaporator and reconstituted with 0.1 mL methanol:water (1:1, v/v) for LC-MS/MS analysis.
LC-MS/MS analyses were carried out on SCIEX 6500 triple quadrupole mass spectrometer coupled to a Waters Acquity UPLC system. Chromatographic separation of steroid metabolites was achieved using a reversed phase HSS T3 C18 column (2.1×100 mm, 1.8μ particle size). Mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) was used. The LC gradient system is reported in Table 2. All the MRM transitions and mass spectrometry parameters are reported in Table 2. Data were acquired by Analyst 1.6 software. LC-MS/MS method was validated for linearity, precision, accuracy, extraction efficiency, and auto-sampler stability.
HLM samples were isolated from 9 individual liver tissues from the University of Washington, School of Pharmacy liver bank and quantified for UGT2B7, UGT2B15 and UGT2B17 protein abundance using a validated trypsin digestion protocol (Vrana, M., Whittington, D., Nautiyal, V. & Prasad, B. Database of Optimized Proteomic Quantitative Methods for Human Drug Disposition-Related Proteins for Applications in Physiologically Based Pharmacokinetic Modeling. CPT Pharmacometrics Syst Pharmacol 6, 267-76 (2017), the disclosure of which is incorporated herein by reference). Glucuronidation activity assay was performed in HLM (0.1 mg/ml total protein) or recombinant human UGTs (10 μg/ml total protein) containing 5 mM MgCl2-100 mM phosphate buffer at pH 7.4, alamethicin (0.1 mg/ml), BSA (0.02%) and substrate, T/A/Etio (1 μM to 10 μM). The reaction mixture was incubated for 15 min on ice to allow alamethicin pore formation. Reaction was initiated by adding UDPGA (2.5 mM) and incubating for 30 minutes at 37° C. The reaction was quenched by acetonitrile containing internal standard and the sample were centrifuged at 2000×g, 4° C. for 5 min. The supernatant was analyzed by LC-MS/MS to quantify substrate depletion and glucuronide formation.
Fractional contribution of each UGTs in the glucuronidation of androgens in the intestinal and liver microsomes using the protein abundance data was also extrapolated, and the in vitro in vivo extrapolation (IVIVE) approach discussed elsewhere. Briefly, intestine and liver abundance of UGT2B7, UGT2B15 and UGT2B17 of 2.83, 0.0 and 8.87 vs. 36.6, 21.2, and 0.92 pmol/mg microsomal proteins, respectively were considered for IVIVE of recombinant data to liver and intestinal microsomes.
The relative concentrations of circulating T metabolites in eugonadal men (i.e., baseline levels prior to the induction of hypogonadism) is depicted in (
The average serum T AUC0-24 h was 81.6 nmol/L*h at T200 (
The pooled serum T levels were 1.4 and 2.4-fold higher after dutasteride co-administration at T200 and T800, respectively as compared to T alone. The average AUC0-24 h was 3277, 2583, and 373.4 nmol/L*h for EtioG, AG, and TG, respectively after 200 mg T with dutasteride (T200+D), which were 151, 21, and 43% with respect to T alone. The average AUC0-24 h at 800 mg T with dutasteride (T800+D) were 180, 22, and 60% as compared to T alone (
Consistent with the in vivo data, co-treatment of dutasteride with T resulted in complete inhibition of 5-AR type 1 and 2 mediated metabolism of T in human hepatocyte. This resulted in significant decreases in DHT, A, AG and 6β-0H-T formation (
In human liver microsomes (HLM), T is primarily glucuronidated by UGT2B17 with minor contribution from UGT2B15 (
Approximately 2-5% men are affected by hypogonadism, which requires TRT. Because of high-first pass metabolism, only non-oral T formulations are available in the US, and these formulations are frequently associated with poor patient compliance and variable response. In order to develop strategies to ensure optimum T bioavailability, it is important to characterize mechanisms that affect first-pass T metabolism. As described herein, it was observed that the metabolism of T differs between eugonadal men and those same men treated with oral T after induction of hypogonadism. Particularly, distinctly high AG, TG, and EtioG concentrations in T-treated vs. untreated samples indicate that glucuronidation is the major mechanism of T first-pass elimination. Of particular note, the inventors observed that, contrary to the prevailing thinking, glucuronidation of oral T to AG appeared to be more prevalent than formation of TG. While the clearance of TG and AG may differ (depending on differential renal clearance) and therefore individual contribution of each pathway (fm) cannot be calculated from the circulating metabolite concentration data, this finding suggests that “back-formation” of AED via 17β-HSD2 is the primary route of metabolism of oral T. Interestingly, relative proportions of AG and EtioG to all metabolites (
Although AED can also be biosynthesized from dehydroepiandrosterone (DHEA) by 3α-HSD, T to AED via 17β-HSD2 was observed as the major T metabolic pathway, which subsequently metabolized to the major metabolites, AG and EtioG. These data are corroborated by Bhasin et al., which showed the association of aldo-keto reductase 1C3 (AKR1C3/17β-HSD5) polymorphism (rs12529) with variable T circulating levels in men (Bhasin, S. et al. Contributors to the substantial variation in on-treatment testosterone levels in men receiving transdermal testosterone gels in randomized trials. Andrology 6, 151-7 (2018)). High levels of AED and its metabolites with oral T are presumably due to interaction of T with 17β-HSDs and AED with 5α/β-reductase (5-AR/5-BR) in the gastrointestinal tract and liver during the first-pass. High in vitro formation of AED (46%) from T using primary human hepatocyte was confirmed, but it was marginally higher than TG levels (42%). Because 17β-HSDs are also expressed in the intestine, T to AED formation can occur in the enterocyte. Thus inhibition of 17β-HSD2 in the intestine and the liver can significantly increase T circulating levels, and could be used as a potential therapeutic target for augmenting the effect of oral TRT. This is important as the development of 17β-HSDs inhibitors is in the early stage for treatment of osteoporosis (17β-HSD1/2) and prostate cancer (17β-HSD3/5).
Although relative to AED, TG formation was observed to be a minor pathway in overall T metabolism, it is noteworthy that T to TG conversion is mediated by a highly variable enzyme, UGT2B17. Because ˜50% population has a very low expression (non-detectable in the liver, T-glucuronidation will be important only in the high UGT2B17 expressers (e.g., in 10% population). This is supported with controversial association of UGT2B17 with T-related pathophysiological conditions, prostate cancer. That is, while some studies support association of UGT2B17 gene deletion with prostate cancer, a recent large study failed to confirm the same, perhaps due to high variability in UGT2B17. Particularly, that UGT2B17 is associated with single nucleotide polymorphism (rs7436962, rs9996186, rs28374627 and rs4860305), age and gender besides gene deletion has been demonstrated.
Here, a 4-fold increase in dose (200 to 800 mg) only resulted in 2-fold increase in circulating T concentrations. For the first time the inventors demonstrated that T metabolism to TG and AED increases more than proportionally with increasing dose. Higher TG formation after 800 mg dose could be because of the cumulative effect of a) the dose-limiting T solubility resulting in longer gastro intestinal (GI) retention of T and b) higher UGT2B17 abundance in lower part of GI tract (unpublished data). Interestingly, AG and EtioG formation seems to saturate at the higher dose (800 mg). Because metabolite ratios are used to create individual passports, these findings should be considered when analyzing doping test results.
Dutasteride co-administration resulted 2-fold higher serum T concentration after 800 mg oral T dose. This increase in serum T levels are due to dual inhibition of 5-AR type 1 and 2, and would likely not be evident with a specific inhibitor for Type-2 5-AR e.g., finasteride. Interestingly, there was no significant increase in serum T levels at 200 mg T dose with dutasteride perhaps due to low levels of AED in T200 as compared to T800 which seemed necessary to drive the back-conversion (
The above described study has several limitations, including, a small number of subjects, and the non-availability of urine samples and DNA for genotyping. The sulfate conjugates could not be detected in this study due to analytical challenges of these low level metabolites or perhaps due to rapid elimination of sulfates in urine. Nevertheless, the data presented herein are novel with respect to levels of circulating glucuronides, non-linear PK and effect of dutasteride on T metabolite profiles. In particular, the relative prominence of the AG vs. the TG pathway for the metabolism of oral T was unexpected and may be useful in the development of novel formulation strategies of oral T. In addition, the non-linear PK of oral T observed in this study should be taken into consideration while developing anti-doping strategies. Multiple peaking phenomenon in the PK profiles suggests a potential role of microbiome in deconjugation of glucuronides, indicating another potential reasons of inter-individual variability. This is consistent with data that revealed that transfer of cecal contents from male mice to female mice was associated with increased circulating T in female mice (Markle, J. G. et al. Sex differences in the gut microbiome drive hormone-dependent regulation of autoimmunity. Science 339, 1084-8 (2013)).
UGT2B17 is a highly variable enzyme, which is responsible for metabolism of drugs and androgens. Because UGT2B17 variability could not be explained by genetic polymorphism alone, it is important to identify a circulating biomarker of UGT2B17 activity for stratification of patients receiving UGT2B17 substrates such as vorinostat, exemestane and testosterone (T). The objective was to identify a potential serum biomarker of UGT2B17 by (i) accomplishing metabolomics analysis after oral T dosing, and (ii) performing an in vitro mechanistic study to compare correlation of steroid glucuronides vs. UGT2B17 protein abundance in T-treated human hepatocytes.
A targeted metabolomics study was performed on previously collected samples from an oral T pharmacokinetics (PK) study (800 mg). T and its primary and secondary metabolites in serum were quantified, and PK data of these steroids were analyzed using the non-compartmental method. UGT2B reaction phenotyping was also performed to identify whether UGT2B7, UGT2B15, and/or UGT2B17 metabolize T, androsterone (A) and etiocholanolone (Etio) in recombinant UGT2Bs and human liver microsomes characterized for UGT2B abundance. Additionally, T-metabolism study was also performed in human hepatocytes for 1 h. The media and cell pellets were collected for subsequent metabolomics and proteomics analyses, respectively. Effect of dutasteride treatment on androgen metabolome was also investigated, both in vivo and in vitro. A UGT2B17 biomarker was identified based on correlation of TG or TG/AG vs. UGT2B17 abundance in human hepatocytes and the approach was applied to predict in vivo UGT2B17 variability.
With 800 mg dose, AUC0-24 h of T-glucuronide (TG), androsterone glucuronide (AG), etiocholanolone glucuronide (EtioG) was 85, 389 and 87-fold higher in human serum as compared to T AUC0-24 h (nmol·h/L). Similarly, TG and AG were also detected in hepatocyte media, albeit TG levels were 3.7-fold lower than AG in the media. AG formation rate (i.e., AG/A) was constant in all the four hepatocyte donors, whereas TG/T ratio was highly variable, which strongly correlates with UGT2B17 protein abundance (r2=0.93). The correlation between TG and TG/AG remained strong when 5α-reductase was inhibited by dutasteride (r2=0.90). Consistent to these findings, the superosomes and microsomes reaction phenotyping data also confirmed UGT2B17 as a selective T-glucuronidation enzyme, with limited AG and EtioG formation activity. This approach when applied to the in vivo data, confirmed high variability in TG/AG ratio consistent with UGT2B17 in vivo expression, which was also reflected by strong correlation (r2=0.84) between serum TG and TG/AG was also observed.
TG/AG ratio can be used as a serum biomarker of UGT2B17 variability because TG and AG formation is mediated by different UGTs. The approach demonstrated herein can be used in precision therapy of vorinostat, exemestane, and T. Further, the serum biomarker of UGT2B17 variability is useful in T doping control.
Testosterone has poor and variable oral bioavailability, thus leading to unreliable predictions of its oral pharmacokinetics. Testosterone glucuronidation is the major route of testosterone elimination, and UGT2B15 and UGT2B17 are considered the main enzymes responsible.
In particular, UGT2B17 exhibits gene deletion which has been associated with false negative anti-doping test results, increased risk of prostate cancer, and drug development failure. The inventors have shown that high variability has been observed with UGT2B17 with ontogenic and gender effects (
In order to predict testosterone hepatic metabolism and its variability, the inventors estimated the fractional contributions of UGT2B15 and UGT2B17 in testosterone hepatic glucuronidation by performing in vitro activity and quantitative proteomic assays in human liver microsome (HLM) samples genotyped for UGT2B17 deletion.
The present disclosure addresses treating testosterone deficiency in men using a precision medicine approach. Based on analysis of human samples, the inventors have identified that a critical enzyme involved in testosterone urinary elimination is highly variable in men. The inventors then identified a specific testosterone metabolite as a biomarker in this special population of men using metabolomics, genomics, and proteomics approach. By inhibiting this enzyme, alone or in combination of testosterone, testosterone deficiency in men can be treated.
Human liver tissue samples were obtained from the University of Washington Human Liver Bank (Seattle, Wash., USA) (n=29). Protein abundance was measured by total protein quantification, trypsin digestion, and resulting surrogate peptides quantification using AB Sciex 6500 triple quadrupole MS/MS (
Calculation of individual fraction metabolized in UGT2B15 and UGT2B17 was done as follows:
- 1. Total individual microsomal activity (A)
- 2. Activity in UGT2B17 del/del samples=UGT2B15 activity (B)
- 3. Average UGT2B15 activity (C)=[Average B]/[Average UGT2B15 expression]
- 4. Individual UGT2B15 activity (D)=Individual UGT2B15 expression x (C)
- 5. Total individual microsomal activity−(D)=individual UGT2B17 activity (E)
- 6. Individual UGT2B15 fm=D/A
- 7. Individual UGT2B17 fm=E/A
Enzyme kinetic parameters (Vmax and Km) were determined for UGT2B17 using three human liver microsomes (HLMs) with high, mid, or null UGT2B17 abundance (9.7, 2.3, and 0.06 pmol/mg microsomal protein, respectively) with comparable UGT2B15 abundance (27, 20, and 34 pmol/mg microsomal protein, respectively). Assays were performed in triplicates with varying testosterone concentrations (0.05, 0.25, 0.5, 2.5, 5, 10, 15, 25 μM). Assays were performed in triplicates with 10 μg of protein in 5 mM MgCl2 and 100 mM potassium phosphate buffer (pH 7.4), 0.01% BSA, and 0.1 mg/mL of alamethicin in 100 μL final volume with testosterone. After 15 min of pretreatment on ice, reactions were initiated by addition of 2.5 mM UDP-glucuronic acid (UDPGA), incubated for 30 min (37° C.) at 300 rpm, and terminated using 200 μL of ice-cold acetonitrile containing deuterated TG as validated internal standards. Samples were centrifuged at 10,000 g for 5 min (4° C.). TG was quantified in supernatant by LC-MS/MS. The results are shown in
To validate in vivo utility of TG/AG, association of an exogenous marker of UGT2B17, i.e., dihydroexemestatne glucuronide (DHE-G) with TG/AG was performed in a genotype-guided urine samples from patients receiving exemestane (EXE). Genotyping was performed by real-time PCR using a CNV genotyping assay (Hs03185327_c, Life Technologies) with RNase P as a control (Cat #4403326, Life Technologies) using a Bio-Rad CFX384 real-time PCR machine. DHE-G, DHE, TG, and AG were by validated LC-MS/MS methods as described above. The results are shown in
UGT2B17 inhibition assays was performed with two HLMs expressing high and below limit of detection (LOD) UGT2B17 abundance (9.7 and 0.06 pmol/mg microsomal protein) with comparable UGT2B15 abundance (27 and 24 pmol/mg microsomal protein). IC50 of UGT2B17 inhibition by imatinib was determined by incubating HLM samples with varying inhibitor concentrations (0.01, 0.02, 0.05, 0.1, 0.5, 1, 10, 50, and 100 μM) followed by the testosterone glucuronidation assay in triplicates. UGT2B17 activity was measured with TG formation with 1 μM testosterone. Oxazepam was used as a probe substrate for UGT2B15 activity by measuring oxazepam glucuronide formation (m/z 463.3→287.1, 269.1; DP 70; CE 20) with 10 μM oxazepam. The results are shown in
Donor-matched intestinal tissue segments (n=6) were procured from Pomeranian Medical University, Szczecin, Poland, and approved by the local Bioethics Committee. Microsome isolation from donor-matched intestinal sections Donor-matched human intestinal microsomes (HIMs) were isolated from different sections of intestine, i.e., duodenum (D), jejunum (J1-J3, three sections), ileum (I), and colon (C), from six individual donors. Tissue samples were disrupted using cell crusher paddles in liquid-nitrogen and HIMs were isolated using protocols from Microsome isolation kit (Abcam, Cambridge, United Kingdom). Total protein was determined by bicinchoninic acid (BCA) assay using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, Ill.) per manufacturer'"'"'s protocol.
Membrane proteins were extracted from the cells using Mem-PER Plus Membrane Protein Extraction kit protocol (Rockford, Ill.). Briefly, previously washed and frozen hepatocytes (0.7-6 million) and enterocytes (6.7-37 million) were resuspended with permeabilization buffer (200 or 400 μL), gently mixed, and incubated on a Compact Digital Waving Rotator (Thermo Fisher Scientific, Rockford, Ill.) for 30 min (4° C.) at 300 rpm. Permeabilized cells were centrifuged at 16,000 g for 15 min (4° C.), and resulting supernatant containing cytosolic proteins was removed. Remaining pellet was resuspended with equivolume solubilization buffer, gently mixed, incubated for 60 min at 300 rpm (4° C.), and centrifuged at 16,000 g for 15 min (4° C.). Resulting supernatant containing membrane proteins was collected and total protein quantification was measured with BCA assay (Thermo Fisher Scientific, Rockford, Ill.). Remaining solubilized membrane fraction was stored at −80° C. for subsequent digestion and LC-MS/MS analysis.
UGT2B17 quantification in donor-matched individual HLMs was performed by digesting the membrane proteins using trypsin using an optimized protocol as described in M. Drozdzik et al, Protein abundance of clinically relevant drug-metabolizing enzymes in the human liver and intestine: a comparative analysis in paired tissue specimens. Clin. Pharmacol. Ther. (2017).
The inventors have demonstrated that UGT2B17 protein expression in human liver is highly variable due to effect of genotype, age, and gender, and that UGT2B17, when present, is the predominant enzyme responsible for testosterone glucuronidation. At the same time, UGT2B15 may be an important enzyme for testosterone glucuronidation in UGT2B17 deletion or low-expressing individuals. Because of the high variability in UGT2B17 protein expression, knowing fractional contribution (fm) for both isoforms in testosterone glucuronidation is critical in predicting testosterone drug interactions at individual levels. This approach allows precision dosing of UGT2B17/15 substrate drugs by calculating individualized doses that would eliminate or minimize adverse effects associated with UGT2B17/15 glucuronidation.
From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.