METHOD FOR PREDICTING THE COURSE OF A VIRAL DISEASE

Abstract

The invention relates to a method for predicting the course of a viral disease in a male subject infected with an influenza virus or coronavirus which is based on measuring testosterone and/or estradiol levels in said subject. The invention further relates to a method for monitoring the course of a viral disease in a male subject infected with an influenza vims or coronavirus which comprises predicting the course of the disease in said subject and assigning the subject to preventive or therapeutic measures if a severe course of said viral disease is to be expected. The invention further relates to an aromatase inhibitor for use in a method of treating or preventing a severe course of a viral disease in a male subject infected with an influenza virus or coronavirus, wherein said subject has decreased testosterone levels and/or increased estradiol levels as compared to reference values. Finally, the invention also relates to a kit for carrying out one of the aforementioned methods.

Claims

1. Method for predicting the course of a viral disease in a male subject infected with an influenza virus or coronavirus, said method comprising: (a) providing a body fluid sample from the subject that is infected with said influenza virus or coronavirus; (b) determining in said sample the concentration of testosterone and/or estradiol; and (c) comparing the concentration obtained in step (b) with at least one testosterone and/or estradiol reference value; wherein the comparison of the concentration obtained in step (b) with said at least one reference value indicates whether a severe course of said viral disease is to be expected in said subject.

2. Method according to claim 1, wherein said body fluid sample is a blood, plasma or serum sample.

3. Method according to claim 1, wherein said method comprises the comparison of the concentration obtained in step (b) with a testosterone reference value, wherein a severe course of said viral disease is to be expected if the concentration obtained in step (b) falls below the reference value.

4. Method according to claim 1, wherein said testosterone reference value is 8.69 nMol/l blood serum in males between 18-50 years or 6.68 nMol/l blood serum in males older than 51 years.

5. Method according to claim 1, wherein said method comprises the comparison of the concentration obtained in step (b) with an estradiol reference value, wherein a severe course of said viral disease is to be expected if the concentration obtained in step (b) exceeds the reference value.

6. Method according to claim 1, wherein said estradiol reference value is 52.2 pg/ml blood serum, more preferably 60 pg/ml blood serum.

7. Method according to claim 1, wherein said method comprises: (i) the comparison of the testosterone concentration obtained in step (b) with a testosterone reference value, wherein said reference value preferably is 8.69 nMol/l blood serum in males between 18-50 years or 6.68 nMol/l blood serum in males older than 51 years, and (ii) the comparison of the estradiol concentration obtained in step (b) with an estradiol reference value, wherein said reference value preferably is 52.2 pg/ml blood serum, wherein a severe course of said viral disease is to be expected if the testosterone concentration obtained in step (b) falls below the testosterone reference value and the estradiol concentration obtained in step (b) exceeds the estradiol reference value.

8. Method according to claim 1, wherein said influenza virus is H7N9 or said coronavirus is SARS-CoV-2.

9. A method for monitoring the course of a viral disease in a subject infected with an influenza virus or coronavirus, said method comprising: (a) repeatedly conducting a method as defined in claim 1 in predefined time intervals, and (b) assigning the subject to preventive or therapeutic measures if based on the results obtained in step (a) a severe course of said viral disease is to be expected.

10. Method according to claim 1, wherein said severe course of the viral disease includes the development of the acute respiratory distress syndrome (ARDS).

11. A method of treating or preventing a severe course of a viral disease in a subject infected with an influenza virus or coronavirus, the method comprising administering to the subject an aromatase inhibitor, wherein said subject has (a) decreased testosterone levels, and/or (b) increased estradiol levels as compared to reference values prior to said administering step.

12. The method of claim 11, wherein said inhibitor is selected from the group consisting of aminoglutethimide, testolactone, anastrozole, letrozole, exemestane, vorozole, formestane, and fadrozole.

13. The method of claim 11, wherein said severe course of the viral disease includes the development of the acute respiratory distress syndrome (ARDS).

14. The method of claim 11, wherein said method further comprises administering testosterone or a testosterone derivative to said subject.

15. A method of inhibiting virus dissemination in a subject infected with an influenza virus or coronavirus, said method comprising administering an aromatase inhibitor to said subject.

16. The method of claim 15, wherein said subject has (a) decreased testosterone levels, and/or (b) increased estradiol levels as compared to reference values.

17. The method of claim 15, wherein said inhibitor is selected from the group consisting of aminoglutethimide, testolactone, anastrozole, letrozole, exemestane, vorozole, formestane, and fadrozole.

18. The method of claim 15, wherein said subject is a male subject.

19. A method of treating or preventing a severe course of a viral disease in a male subject infected with an influenza virus or coronavirus, said method comprising administering testosterone or a testosterone derivative to said male subject, wherein said male subject has (a) decreased testosterone levels compared to the normal reference levels and/or (b) increased estradiol levels compared to normal reference levels prior to said administering step.

20. Kit for carrying out the method of claim 1, comprising: (a) means for determining whether a subject is infected with an influenza virus or coronavirus; (b) means for determining the concentration of testosterone and/or estradiol; and (c) optionally, buffers and diluents.

Description

DESCRIPTION OF THE FIGURES

[0048] FIG. 1 shows the testosterone and estradiol levels determined in a number of COVID-19 patients. (A) Table depicting the testosterone and estradiol levels measured in male and female COVID-19 patients. (B) Graphic depiction of the testosterone (a, b) and estradiol (c, d) levels were measured in the sera or plasma from COVID-19 patients and aged-matched (≥40 y) healthy controls. Male COVID-19 patients (a, c) were subdivided into patients requiring connection to an ECMO (+ECMO) and patients not being placed on ECMO (−ECMO). Bargraphs (a, c) represent males (COVID-19+ECMO, n=5; COVID-19 −ECMO, n=34; healthy controls, n=30) and bargraphs (b, d) represent females (COVID-19, n=11, healthy controls, n=20). Statistical significance was assessed by Student's t-test (* P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001).

[0049] FIG. 2 shows the results from total testosterone expression level measurements in H7N9 male infected with H7N9 influenza A virus.

[0050] FIG. 3 shows the results of measuring CYP19A1 expression in the Syrian golden hamster. (a) CYP19A1 mRNA expression levels in lungs of SARS-CoV-2 infected male and female Syrian golden hamsters at 3 d p.i. (n=9-10). Relative CYP19A1 mRNA expression values in PBS treated hamsters for each sex were set to 1 after normalization against HPRT (Hypoxanthine Phosphoribosyltransferase 1). Values are shown as means and error bars are shown as SD. Statistical significance was assessed by Kruskal-Wallis one-way ANOVA followed by Dunn's multiple comparisons test (*p<0.05, ****p<0.0001). (b) CYP19A1 protein expression in lungs of SARS-CoV-2 infected male (upper panel) and female (lower panel) Syrian golden hamsters at 3 d p.i. Representative pictures for each sex (n=5) are shown. The arrowheads indicate positive signal for CYP19A1 expression.

[0051] FIG. 4 shows the results of measuring virus titer and MIP-1a/MIP-1b expression levels in different organs of hamsters treated with placebo or letrozole. (a-c) Virus titer in lungs (a), brain (b) and testis (c) of PBS and SARS-CoV-2 infected male Syrian golden hamsters treated with placebo or letrozole at 3 d p.i. (each n=6). (d-e) Protein expression levels of MIP-1a (d) and MIP-1b (e) in lungs of PBS and SARS-CoV-2 infected male Syrian golden hamsters treated with placebo or letrozole at 6 d p.i. (each n=6). (f-h) Virus titer in lungs (f), brain (g) and plasma (h) of PBS and SARS-CoV-2 infected female Syrian golden hamsters treated with placebo or letrozole at 3 d p.i. (each n=6). (i-j) Protein expression levels of MIP-1a (i) and MIP-1b (j) in lungs of PBS and SARS-CoV-2 infected female Syrian golden hamsters treated with placebo or letrozole at 6 d p.i. (each n=6). Values are shown as means and error bars are shown as SD. Statistical significance was assessed by Kruskal-Wallis one-way ANOVA followed by Dunn's multiple comparisons test (*p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001). n.d. not detectable, n.s. not significant.

[0052] FIG. 5 shows the results of measuring CYP19A1 expression in the human lung of fatal Covid-19 cases. (a) CYP19A1 mRNA expression levels in lungs from fatal male Covid-19 cases and controls (non-Covid-19) who died for other reasons (non-Covid-19: n=5, Covid-19: n=9). Values are shown as means; error bars are shown as SD. Statistical significance was assessed by Kruskal-Wallis one-way ANOVA by Dunn's multiple comparisons test (*p<0.05). (b) CYP19A1 protein expression in lungs of fatal male (upper panel) and female (lower panel) Covid-19 cases or controls who died of other reasons (non-Covid-19). Detection of SARS-CoV-2 RNA by in situ hybridization. Representative pictures are shown (males: n=8, females: n=3). The squares indicate macrophages.

EXAMPLES

[0053] The invention is described in the following on the basis of examples, for the purpose of illustration, without limiting the invention. It will be evident to a person skilled in the art that modifications and variations of the examples described are possible without deviating from the idea of the invention.

Example 1: Determination of Hormone Status in COVID-19 Patients

[0054] 45 COVID-19 patients at the University Hospital Hamburg Eppendorf requiring intensive care were examined. Among these patients, 35 were males and 10 were females. The median age within males and females was comparable with 62 and 67.5, respectively. Majority of the patients presented an elevated body mass index (BMI) (31.4% of males and 30% of females with a BMI ≥30). All patients presented comorbidities in males and females, such as adipositas (males 69%; females 50%), followed by diabetes type II (males 22.9%; females 20%), hypertension (males 45.7%, females 33.3%) and cancer (males 22.9%, females 33.3%). Acute respiratory distress (ARDS) detected was classified as moderate or severe in most male (37% or 26%) and female (33% or 33%) patients. Sequential organ failure assessment (SOFA) scores were evaluated in males and females presenting high (4-7) or very high (8-11) scores in males (35% or 25%) and females (40% or 60%). Due to the strong sex bias of males-to-females with a ratio of 3.5:1, sex hormones known to play a key role not only in fertility but also in innate and adaptive immunity were measured.

[0055] Results: The results are shown in Table 1. Total testosterone levels were reduced in 69% of males. Herein, 26% of males showed very low and 43% of males extremely low testosterone levels. In 60% of females, testosterone levels were increased to high (50%) or very high (10%) levels. Estradiol levels were elevated in male COVID-19 patients (46%), either to high (30%) or very high (16%) levels. Comparably, 60% of females also showed elevated estradiol concentration to high (40%) or very high (20%) levels. Thus, the vast majority of male COVID-19 patients have very low testosterone levels and very high estradiol levels. In contrast, female COVID-19 patients tend to have high testosterone and estradiol levels. A shift in sex hormones, as seen here in male patients, hints towards increased aromatase (CYP19A1) activity, i.e. the enzyme that converts testosterone to estradiol.

Example 2: Determination of Hormone Status in H7N9 Influenza Patients

[0056] A total of n=44 avian H7N9 influenza positive cases of reproductive age (18-49 years) were enrolled with a median age of 42 years. A total of n=54 avian H7N9 influenza positive cases were included in those 50 year olds with the median age of 61 years. The male H7N9 cases accounted for 75% in the younger and 70% in the older age groups, which is consistent with previous epidemiological studies based on larger laboratory-confirmed H7N9 cohorts. Blood samples of H7N9 patients were collected within acute phases after illness onset.

[0057] In order to assess the role of testosterone for the outcome of H7N9 infections, the testosterone concentrations were measured in all cohorts. Testosterone levels were strongly reduced in H7N9 infected men of both age groups assessed compared to virus negative H7N9 controls. Low testosterone levels strongly correlated with lethal outcome in H7N9 infected men in the age group of 18-49 year olds (P<0.001) (FIG. 2). These data show that low testosterone levels in H7N9 infected men of 18-49 years of age correlate with an enhanced risk for lethal outcome.

Example 3: Virus Isolation and Animal Infection

[0058] The SARS-CoV-2 isolate (SARS-CoV-2/Germany/Hamburg/01/2020) was isolated by inoculation of VeroE6 cells with 200 μl of a human nasopharyngeal swab sample of a confirmed male COVID-19 patient in Hamburg, Germany and propagated for three serial passages in VeroE6 cells. VeroE6 were cultivated in DMEM (Sigma-Aldrich GmbH) with 2% fetal bovine serum, 1% penicillin-streptomycin and 1% L-glutamine at 37° C. for virus propagation and were tested negative for Mycoplasma sp. by PCR. All infection experiments with SARS-CoV-2 were performed in a biosafety level 3 (BSL-3) laboratory.

[0059] All animal experiments were performed in strict accordance with the guidelines of German animal protection law and were approved by the relevant German authority (Behörde für Gesundheit and Verbraucherschutz; protocols N 32/2020). Male and female Syrian golden hamsters (8-10 weeks old) were purchased from Janvier and were kept under standard housing conditions (21±2° C., 40-50% humidity, food and water ad libitum) with a 12:12 light-dark cycle. For infection, hamsters were anaesthetized with 150 mg/kg ketamine and 10 mg/kg xylazine by intraperitoneal injection. The animals were intranasally inoculated with 10.sup.5 plaque forming units (pfu) SARS-CoV-2, mock infected with PBS or were administered with 1 mg kg-1 Poly(I:C). On day 3 p.i., five animals per group were euthanized by intraperitoneal injection of an overdosis of pentobarbital, and blood was drawn by cardiac puncture.

[0060] For RNA isolation, the lungs were stored in RNAprotect Tissue Reagent (QIAGEN). For histopathological examinations the collected lungs were fixed by immersion in 10% neutral-buffered formalin and embedded in paraffin.

Example 4: Determination of CYP19A1 mRNA Expression

[0061] For the determination of CYP19A1 mRNA expression levels by real-time quantitative PCR (RT-qPCR), RNAprotect-fixed lungs from hamsters were homogenized in 700 μl lysis buffer RL with 5 sterile, stainless steel beads (diameter 2 mm, Retsch) at 30 Hz and 4° C. for 10 min in the mixer mill MM400 (Retsch). Total RNA was isolated from homogenized lung supernatants using the innuPREP RNA Mini Kit 2.0 (Analytik Jena) according to the manufacturer's instructions with an additional on column DNase I treatment using the RNase-free DNase Set (QIAGEN). The RNA was eluted in RNase-free water and mixed with 1 U μl-1 RiboLock RNase inhibitor (Thermo Fisher Scientific). For cDNA synthesis random nonamer primers (Gene Link, pd(N)9, final concentration: 5 μM) and the SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) were used according to the manufacturer's instructions, using 2 pg total RNA.

[0062] The cDNA was generated using the GeneAmp PCR System 9700 (Applied Biosystems; cycle: 25° C. for 5 min, 50° C. for 60 min, 70° C. for 15 min, 4° C. hold). Reactions were set up with PCR grade water (Roche Life Science) in LightCycler® 480 Multi-well Plate 96 Reaction Plate (Roche Life Science). Briefly, 2 μl of cDNA template were added to 10 μl FastStart Essential DNA Green Master (Roche Life Science) and 300 nM of forward and reverse primer. RT-qPCR runs were conducted on the LightCycler® 96 Real-Time PCR System (Roche Life Science) with endpoint fluorescence detection: 10 min at 95° C. and 45 amplification cycles (15s at 95° C., 10s at 65° C. and 20s at 72° C.) Analysis was performed in duplicate for CYP19A1 and reference gene (hamster: HPRT, human: RPL32) in each sample. Negative controls and samples without reverse transcriptase were included to detect contaminations.

[0063] Relative expression values were determined using a modified E.sup.−ΔΔCt method. Rn-values were exported from the LightCycler® 96 Software v1.1.0.1320 (Roche) to Microsoft Office Excel 2016 and N.sub.0-value for the starting concentration of the transcript in the original sample were obtained with LinReg PCR Software v2018.0 (Ruijter et al. 2009). The averaged N.sub.0-value of the CYP19A1 gene was then normalized with the average N.sub.0-value for HPRT (N.sub.0(HPRT)) or RPL32 (N.sub.0(RPL32)) of the respective sample. The relative N.sub.0(CYP19A1)/N.sub.0(HPRT)- or N.sub.0(CYP19A1)/N.sub.0(RPL32)-expression values of the biological replicates are presented.

[0064] The following primer sequences were used for qRT-PCR of HPRT1 (hypoxanthin-guanin-phosphoribosyltransferase 1) and CYP19A1 in the hamster lung:

TABLE-US-00001 HPRT1 forward (SEQ ID NO: 1) 5′-TCCCAGCGTCGTGATTAGTG-3′ HPRT1 reverse (SEQ ID NO: 2) 5′-GTGATGGCCTCCCATCTCTT-3′ CYP19A1 forward (SEQ ID NO: 3) 5′- ATGCGGCACATCATGCTGAA-3′ CYP19A1 reverse (SEQ ID NO: 4) 5′- TCTTTCAAGTCCTTGGCGGAT-3′

[0065] Results: The results are shown in FIG. 3(a). It can be seen that the aromatase expression as determined by RT-qPCR is significantly higher in animals infected with SARS-CoV-2 compared to non-infected animals.

Example 5: Immunohistochemistry

[0066] For immunohistochemical detection of aromatase, the EnVision+ System (Dako Agilent Pathology Solutions) was used. Serial sections of tissue were dewaxed and rehydrated in isopropanol and 96% ethanol followed by blockage of endogenous peroxidase by incubation in 85% ethanol with 0.5% H.sub.2O.sub.2 for 30 min at room temperature. Antigen retrieval was performed by incubation in citrate buffer (10 mM citric acid, 0.05% Tween 20) for 20 min in a microwave at 800 W, followed by 20 min at room temperature. Sections were afterwards transferred to Shandon Coverplates™ (Thermo Electron GmbH) and stained with a polyclonal antibody directed against aromatase (Abcam, ab18995) diluted 1:500 in PBS containing 1% BSA, 0.3% Triton X-100 over night at 4° C. Sections were subsequently rinsed, and the peroxidase-labeled polymer was applied as secondary antibody for 30 minutes. Visualization of the reaction was accomplished by incubation in chromogen 3,3-diaminobenzidine tetrahydrochloride (DAB, 0.05%) and 0.03% H.sub.2O.sub.2 in PBS for 5 min and afterwards counterstained with Mayer's hematoxylin for 1 min. For negative controls, the primary antibody was replaced by rabbit normal serum (1:3,000).

[0067] Results: The results are shown in FIG. 3(b). It can be seen that aromatase protein expression can be detected in lungs of SARS-CoV-2-infected male (upper panel) and female (lower panel) hamsters. No aromatase protein expression can be detected in the lungs of control animals.

Example 6: Letrozole Treatment

[0068] All animal experiments were performed in strict accordance with the guidelines of German animal protection law and were approved by the relevant German authority (Behörde für Gesundheit and Verbraucherschutz; protocols N 103/2020). Male and female Syrian golden hamsters (8-12 weeks old) were purchased from Janvier or bread at the Heinrich Pette Institute (Leibniz Institute for Experimental Virology, Hamburg, Germany) and were kept under standard housing conditions (21±2° C., 40-50% humidity, food and water ad libitum) with a 16:8 light-dark cycle. For infection, hamsters were anaesthetized with 150 mg/kg ketamine and 10 mg/kg xylazine by intraperitoneal injection. The animals were intranasally inoculated with 10.sup.5 plaque forming units (p.f.u.) SARS-CoV-2 or mock infected with PBS. At 3 hours and each following day p.i., animals were treated with 0.18 mg kg-1 letrozole or placebo by intraperitoneal injection. On day 3 and 6 p.i., six animals per group were euthanized by intraperitoneal injection of an overdose of pentobarbital and blood was drawn by cardiac puncture. For virus titer determination and cytokine measurements, lungs, brains and testis were collected, homogenized in 1 ml 1×PBS and stored at −80° C.

[0069] Homogenization of organs was performed in 1 ml 1×PBS with 5 sterile, stainless steel beads (Ø 2 mm, Retsch) at 30 Hz for 10 min in the mixer mill MM400 (Retsch). The plaque assays were performed on VeroE6 cell monolayers and stained with crystal violet after 72 hours. The tissue homogenisates were titrated on VeroE6 cells in 10-fold serial dilutions for 30 min at 37° C. and overlaid with MEM (Sigma-Aldrich) supplemented with 0.2% BSA, 1% L-glutamine, 1% penicillin-streptomycin, 1 μg ml-1 L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) treated trypsin (Sigma-Aldrich) and 1.25% Avicel. After 72 hours p.i., cells were fixed with 4% paraformaldehyde and the plaques were visualized by crystal violet staining.

[0070] Protein expression levels of macrophage inflammatory protein 1α and 1β (MIP-1α, MIP-1β) were measured in homogenized lungs using a custom-made Bio-Plex Prom Mouse Cytokine multiplex (Bio-Rad) in a Bio-Plex 200 System with high-throughput fluidics (HTF; Bio-Rad) according to the instructions provided by the manufacturer.

[0071] All data were analysed with Prism software (GraphPad, 9.0.1) using Kruskal-Wallis one-way analysis of variance (ANOVA) followed by Dunn's multiple comparisons test. Statistical significance was defined as p<0.05 (*p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).

[0072] Results: The results are shown in FIG. 4. It can be taken from FIGS. 4 (a)-(c) that treatment with the aromatase inhibitor letrozole results in lower virus titers in the lungs, brain and testis in SARS-CoV-2-infected male animals compared to placebo. Similarly, FIGS. 4 (f)-(h) demonstrate that treatment with the aromatase inhibitor letrozole results in lower virus titers in the lungs, brain and plasma in SARS-CoV-2-infected female animals compared to placebo. These data suggest that the aromatase inhibitor may inhibit virus dissemination. FIG. 4 further shows that the expression levels MIP-1α and MIP-1β are lower in the lungs of animals treated with letrozole both in male (d)-(e) and female (i)-(j) animals.

Example 7: CYP19A1 Expression in Lung Autopsies of Men with Fatal Covid-19

[0073] It was analyzed whether the data derived from preclinical animal model are also reflected in humans. Therefore, autopsy material from the lungs of men and women who died of Covid-19 (n=54) was analyzed. As controls, lung material obtained from men and women who died for other reasons (non-Covid-19 control group) was analyzed as well. Pathological assessment was performed at three independent study sites: in Hamburg (n=26 males, n=8 females), in Tübingen (n=8 males, n=3 females) and in Rotterdam (n=12 males, n=1 female).

[0074] Total RNA from formalin-fixed, paraffin-embedded human lung tissue sections was purified using the RNeasy® FFPE Kit (Qiagen) according to the manufacturer's instructions. To detect SARS-CoV-2 RNA in lung tissue, in situ hybridization (ISH) was performed by hybridizing lung tissue sections using specific probes for SARS-CoV-2 (ACD, Newark, Calif., USA) followed by the RNAscope 2.5 HD Detection Kit Red from ACD (Newark, Calif., USA) according to the manufacturer's protocol.

qRT-PCR was performed as described in Example 4 above, wherein The following primer sequences were used for qRT-PCR of RPL32 (Ribosomal Protein L32) and CYP19A1 in the human lung:

TABLE-US-00002 RPL32 forward (SEQ ID NO: 5) 5′- GAAGTTCCTGGTCCACAACG-3′ RPL32 reverse (SEQ ID NO: 6) 5′ -GCGATCTCGGCACAGTAAG-3′ CYP19A1 forward (SEQ ID NO: 7) 5′ -CGGCCTTGTTCGTATGGTCA-3′ CYP19A1 reverse (SEQ ID NO: 8) 5′- CAGAAGGGTCAACACGTCCA-3′

[0075] Results: At all sites, CYP19A1 was abundantly expressed in the lungs of Covid-19 males compared to non-Covid-19 male controls. In general, CYP19A was expressed in epithelial cells, in endothelial cells but most profoundly in macrophages at all three study sites independently. Noteworthy, SARS-CoV-2 NP protein or RNA was still detectable in the lungs of most deceased females, while viral antigen or RNA was expressed at low levels or was already cleared at the time point of death in males. Quantification of CYP19A1 mRNA levels revealed a transcriptional increase up to ˜10-times in the lungs of Covid-19 males compared to non-Covid-19 males. These findings show that CYP19A1 is also abundantly expressed at the time point of death in the lungs of men with Covid-19. The result are depicted in FIG. 5.

LITERATURE

[0076] 1. Ankarberg-Lindgren et al. (2018), J Steroid Biochem Mol Biol, 183: 116-124. [0077] 2. Siqueira Ferreira et al. (2017), Journal of Chromatography B 1064, 109-114. [0078] 3. Star-Weinstock & Dey (2019), Clinical Mass Spectrometry, 13, 27-35. [0079] 4. Wooding et al (2015), Steroids, 96:89-94, [0080] 5. Van Nuland et al. (2019), J Pharm Biomed Anal., 170: 161-168.