Non-invasive determination of progestogens in animals

Abstract

This disclosure relates to 5α-pregnane-3,6,20-triol isomers or glucuronidated forms of 5α-pregnane-3,6,20-triol in the determination of progestogens or multiple progestogens in non-human animal samples. This disclosure also relates to an in vitro method for determining progestogens or multiple progestogens in non-human animal samples.

Description

[Technical Field]

[0001] This disclosure relates to compounds that are 5α-pregnane-3,6,20-triol isomers or 5α-pregnane-3,6,20-triol isomers in a glucuronidated form. This disclosure also relates to the use of 5α-pregnane-3,6,20-triol isomers or 5α-pregnane-3,6,20-triol isomers in a glucuronidated form in the determination of progestogens or multiple progestogens in in vitro urine samples from non-human animals. This disclosure also relates to an in vitro method for determining progestogens or multiple progestogens in urine samples from non-human animals. [Background technology]

[0002] The optimal breeding time for dogs is primarily determined by progesterone analysis of serum samples. In this analysis, a rise in serum progesterone levels to a certain level indicates that ovulation has occurred, and as a result, conception is possible.

[0003] During the proestrus period, which typically lasts 3 to 21 days, the external genitalia and perineum of female dogs become swollen. Vaginal discharge, which may be creamy or bloody, is usually visible (Concannon, 2011). The length and volume of vaginal discharge vary considerably between individuals, and in some female dogs, the discharge may be detectable from the proestrus period to the early stages of teresus.

[0004] The onset of behavioral estrus is determined by the first day the dog is receptive to mating. When dogs are allowed to breed naturally, female dogs are usually mated during their fertile period. However, fertilization may require mating over several consecutive days. In practice and research, this is usually an extremely laborious task and, in some cases, impossible. Furthermore, because mating behavior is highly individual and sometimes difficult to recognize, monitoring estrus behavior is not a specific tool for predicting fertile timing.

[0005] In canids, the stage of estrus can be estimated by vaginal cytology. During proestrus, vaginal epithelial cells begin to divide, followed by morphological changes and keratinization at different stages of estrus. However, without additional methods, cytological analysis is not always applicable to determining the optimal breeding time (Moxon et al., 2010). In addition, vaginal secretions crystallize immediately after peripheral estrogen levels reach their peak. Crystallization detection can be used in conjunction with cytological analysis, but it is not accurate on its own for monitoring the stage of estrus (England and Allen, 1989).

[0006] Measuring the electrical resistance of vaginal secretions is a common method for detecting the optimal breeding and / or insemination time in domesticated foxes. The conductivity of vaginal secretions changes as electrical resistance increases, reaching its maximum level immediately after the LH peak, which is thought to be the first day of estrus. In dogs, this method is usable but is not common due to a slower and less significant increase in resistance (Gunzel et al., 1986).

[0007] The size and shape of the ovaries change with the growth of follicular cells during proestrus and estrus, as well as increased ovarian blood flow during the LH peak. These changes can be detected by ultrasound. While ultrasound provides the most accurate method for detecting ovulation, it is a laborious and time-consuming method. The increase in follicular size, pre-ovulatory corpus luteum formation, and distinctive features during ovulation are very subtle and require experience in interpreting ultrasound images. Furthermore, locating the ovaries can be difficult or even impossible, especially in larger breeds, if the dog is uncomfortable during the procedure. Experts can detect ovulation in progress in 50–92% of cases, but this may require daily observation or even multiple observations per day (Fontbonne and Malandain, 2006).

[0008] The onset of estrus (day 0) is often determined by the day when luteinizing hormone (LH) reaches its peak value, known as the LH peak. Similar to LH, the follicle-stimulating hormone (FSH) peak can be detected early in estrus. These hormones are gonadotropins that regulate gonadal functions such as ovulation. At the end of the anestrus period, FSH levels rise, stimulating follicles to increase in size and number. In proestrus, FSH levels decrease and then suddenly peak. Luteinizing hormone levels increase at the end of the anestrus period, stimulating follicular maturation. LH peaks simultaneously with FSH, but its peak duration is only about one-third that of the FSH peak. The LH peak lasts approximately 36 hours. The gonadotropin peak triggers ovulation within 1-2 days (Concannon, 2009). Due to the pulsatile secretion of gonadotropins, detecting peak values ​​requires daily blood samples or even multiple samples per day. However, determining the LH peak is often considered the most accurate method for predicting the timing of ovulation. In addition to enzyme immunoassays, LH can be detected from serum samples by a rapid test (WITNESS LH rapid test).

[0009] The follicles and corpus luteum secrete estrogens (estrone (E1), estradiol (E2), and estriol (E3)). In proestrus, mature follicles produce estrogen, which lowers FSH levels (Concannon, 2009). The rise in estrogen levels triggers external signs of estrus and pheromone excretion. After reaching its peak, estrogen levels suddenly decline. Gonadotropins peak a few days after the decline in estrogen levels. Estrogen levels are usually measured by enzyme immunoassays from blood samples, but the usefulness of these assays in determining optimal breeding times is questionable, and estrogen levels fluctuate significantly during the estrous cycle, not only between individuals but also within a single animal (Frank et al., 2010).

[0010] Progesterone is produced in follicular membrane cells, granulosa cells, the corpus luteum, and the adrenal cortex. In dogs, luteinization begins immediately after the peak of estrogen secretion, even before the LH peak. Pre-ovulatory luteinization can be detected by an increase in serum progesterone levels (approximately 0.35 to 0.8 ng / ml). During the LH peak, progesterone concentration is 1-2 ng / ml, and excretion accelerates immediately afterward. During ovulation, approximately two days after the LH peak, progesterone levels rise to a concentration of approximately 6 ng / ml. Progesterone secretion increases in the early stages of late estrus until the peak concentration (14-80 ng / ml) is reached within approximately 10 days after the LH peak, or at the latest within 3-5 weeks. Progesterone excretion rates are similar in non-pregnant and pregnant female dogs. However, in pregnant female dogs, a sudden drop in progesterone to basal levels is usually detected 24 hours before delivery, and, as in non-pregnant female dogs, the decline in progesterone can be slower and take longer (Concannon et al., 2009). Due to pre-ovulatory corpus luteum formation, serum progesterone analysis has been the most widely used method to detect the optimal breeding time in female dogs. However, blood sampling is invasive and unpleasant for dogs. Furthermore, it often requires taking blood samples multiple times, and the cost of having a specialist collect and analyze the blood is substantial.

[0011] Despite advances in technology for monitoring animal estrus cycles, improved non-invasive methods and products are still needed. [Overview of the project]

[0012] The purpose of this disclosure is to provide compounds that overcome the above-mentioned problems associated with currently used methods, as well as their use and methods.

[0013] This disclosure relates to compounds comprising a 5α-pregnane-3,6,20-triol isomer or a glucuronidated form of a 5α-pregnane-3,6,20-triol isomer.

[0014] The disclosure also relates to the use of 5α-pregnane-3,6,20-triol isomers or glucuronidated forms of 5α-pregnane-3,6,20-triol isomers in determining a progestogen or multiple progestogens in an in vitro sample of non-human animal origin, wherein the compound comprises 5α-pregnane-3,6,20-triol isomers or glucuronidated forms of 5α-pregnane-3,6,20-triol isomers.

[0015] The disclosure also relates to an in vitro method for determining a progestogen or multiple progestogens in a sample derived from a non-human animal, the method comprising the steps of: providing an in vitro urine sample derived from a non-human animal; measuring the amount of a 5α-pregnane-3,6,20-triol isomer or a 5α-pregnane-3,6,20-triol glucuronide isomer in the sample; and determining the presence of luteinized follicle theca cells and / or granulosa cells based on the amount of a 5α-pregnane-3,6,20-triol isomer or a 5α-pregnane-3,6,20-triol glucuronide isomer in the sample, wherein the progestogen level in the non-human animal sample is indicated by the presence of luteinized follicle theca cells and / or granulosa cells.

[0016] Specifically, this disclosure relates to 5α-pregnane-3,6,20-triol isomers or 5α-pregnane-3,6,20-triol glucuronide isomers, and their use for determining the estrous cycle phase in animals such as canids. [Brief explanation of the drawing]

[0017] [Figure 1]Figure 1 shows extracted ion chromatograms (EIC, m / z 495) from LC-MS data of commercially available PdG (pregnanediol-3-glucuronide, 5β-pregnane-3α,20α-diol glucuronide), enzymatically glucuronidated 5β-pregnane-3α,20α / β-diol, and two late-estrus urine samples. A) Commercially available PdG, B) Enzymatically glucuronidated 5β-pregnane-3α,20α-diol, C) Enzymatically glucuronidated 5β-pregnane-3α,20β-diol, D) Late-estrus urine sample from female dog A, and E) Late-estrus urine sample from female dog B. Retention times for PdG are marked with solid boxes, and retention times for 20β-type PdG are marked with dotted boxes. In dogs, the 20β type of PdG is found at higher concentrations than the 20α type. [Figure 2] Figure 2 shows a comparison of LC-MS data between urinary PdGα ​​(PdG 20α) and PdGβ (PdG 20β) levels in eight female dogs (EIC area m / z 495 mean ± SD). β reduction is favored at the C20 carbonyl group of PdG. [Figure 3]Figure 3 shows extracted ion chromatograms (EIC, m / z 511) from LC-MS data of enzymatically glucuronidated 5β-pregnane-3α,17α,20α / β-triol and two late-estrus urine samples. A) Enzymatically glucuronidated 5β-pregnane-3α,17α,20α-triol (17α-PtGα,20α type), B) Enzymatically glucuronidated 5β-pregnane-3α,17α,20β-triol (17α-PtGβ,20β type), C) Late-estrus urine sample from female dog A, D) Late-estrus urine sample from female dog B, E) Zoomed-in EIC (female dog A) and F) Zoomed-in EIC (female dog B). The retention time for 17α-PtGα is marked with a solid box, and the retention time for 17α-PtGβ is marked with a dotted box. In dogs, 20β reduction is more common than 20α reduction at the C20 carbonyl group of 17α-PtG, as is the case with PdG. In dogs, 17α-PtGα / β is excreted in small amounts in the urine, but a more abundant metabolite with the same m / z 511 can be seen with a retention time of 6.4 minutes. [Figure 4] Figure 4 shows the urinary 5β-pregnane-3α,17α,20α / β-triol-glucuronide (mean area ± SD from extracted ion chromatogram m / z 511 at a retention time of 8.7 minutes) including serum progesterone (P4, a~b; ng / ml ± SD) and serum 17α-hydroxyprogesterone (17α-OHP, cd; pg / ml ± SD) in eight female dogs measured by LC-MS. Day 0 was considered the day when serum estradiol (E2) reached its maximum concentration. In the figure, 17α-PtGα means 5β-pregnane-3α,17α,20α-triol-glucuronide (20α type), and 17α-PtGβ means 5β-pregnane-3α,17α,20β-triol-glucuronide (20β type). Since the 20α and 20β forms of 17α-PtG elute in close proximity, the area is calculated by manually integrating the split double peaks. [Figure 5]Figure 5 shows a comparison of serum progesterone (mean P4 ng / ml ± SD) and unidentified pregnantriol glucuronide (PtGX, retention time 6.4 min, mean peak area / Cr ± SD) in eight female dogs measured by LC-MS. The PtGX profile follows the serum P4 profile. [Figure 6] Figure 6 shows LC-MS data for two closely eluted isomers of PtG (pregnanetriol glucuronide) in the urine of two female dogs (A and B). The first PtG to elute (PtG1) was the most abundant urinary PtG in female dog A, and its retention time is shown by the dashed line. The second PtG to elute (PtG2) was the most abundant urinary PtG in female dog B, and is shown by the dotted line. [Figure 7] Figure 7 shows the extracted ions corresponding to pregnanediol glucuronide (PdG) in LC-MS data (EIC m / z 495). The retention time of PdG (5β-pregnane-3α,20α-diol-3-glucuronide), the major metabolite of progesterone in humans, is shown by the dashed line (retention time 8.7 minutes). In dogs, PdG is found only in trace amounts. [Figure 8] Figure 8 shows the extracted ions corresponding to pregnanetriol glucuronide (PtG) in LC-MS data (EIC m / z 511). The retention time of 5β-pregnane-3α,17α,20α-triol glucuronide, the major metabolite of 17α-hydroxyprogesterone in humans, is shown by the dashed line (retention time 8.46 mins). In dogs, 17α-PtG is excreted as 5β-pregnane-3α,17α,20β-triol glucuronide (the 20β form of 17α-PtG), with a retention time of only 0.05 mins (retention time 8.51 mins). Pregnanetriol glucuronide, the most abundant in dogs, is found with a retention time of 6.38 mins. [Figure 9] Figure 9 shows the extracted ions corresponding to pregnanediol-onglucuronide in LC-MS data (EIC m / z 509). In canine and human samples, only faint peaks (shown by dashed lines) are observed at the same retention time. [Figure 10] Figure 10 shows the extracted ions corresponding to pregnan-ol-dion glucuronide in the LC-MS data (EIC m / z 507). In dogs, only one peak can be seen (shown as a dashed line). The same peak is also seen in human samples. [Figure 11] Figure 11 shows the production of hydroxyprogesterone (OHP) from progesterone by ovarian and liver enzymes in the S9 fraction based on LC-MS data. Lines A - D indicate the retention times of reference hydroxyprogesterone: A) 6α-OHP, B) 16α-OHP, C) 6β-OHP, and D) 17α-OHP. The ovarian S9 enzyme has high 17α-hydroxylase activity. The liver S9 enzyme has high 6α-, 6β-, and 16α-hydroxylase activity. [Figure 12] Figure 12 shows the extracted ion chromatogram from the LC-MS data of pregnanetriol glucuronide (PtGs, m / z 511) formed by the reduction and glucuronidation of 16α-hydroxyprogesterone (A), and urine samples from two late estrus times from two female dogs (B and C). PtG1 is marked as a dashed line and PtG2 is marked as a dotted line. The most abundant synthetic PtG is shown as a solid line. [Figure 13] Figure 13 shows the extracted ion chromatogram from the LC-MS data of pregnanetriol glucuronide (PtGs, m / z 511) formed by the reduction and glucuronidation of 6β-hydroxyprogesterone (A), and urine samples from two late estrus times from two female dogs (B and C). PtG1 is marked as a dashed line and PtG2 is marked as a dotted line. The most abundant synthetic PtG is shown as a solid line. [Figure 14]Figure 14 shows the extracted ion chromatogram from the LC-MS data of pregnanetriol glucuronide (PtGs, m / z 511), in which A) a reference steroid formed by the reduction and glucuronidation of 6α-hydroxyprogesterone, B) a urine sample in the late estrus phase, C) a urine sample in the late estrus phase spiked with 0.5 μM reference steroid, and D) a urine sample in the late estrus phase spiked with 1.5 μM reference steroid are shown. PtG1 is marked as a dashed line and PtG2 is marked as a dotted line. The most abundant synthetic PtG is shown as a solid line. [Figure 15] Figure 15 shows the extracted ion chromatogram from the LC-MS data of pregnanediol-one (m / z 509), where A and B show the m / z 509 ions in urine samples at two late estrus times of two female dogs, and C shows a reference steroid of glucuronidated 5β-pregnane-3α,6α-diol-20-one. The reference glucuronide is shown as a solid line and the endogenous pregnanediol-one is shown as a dashed-dotted line. [Figure 16] Figure 16 shows the extracted ion chromatogram from the LC-MS data of pregnanetriol glucuronide (PtGs, m / z 511) formed by the reduction and glucuronidation of 5β-pregnane-3α,6α-diol-20-one (A), and urine samples (B and C) at two late estrus times from two female dogs. PtG1 is marked as a dashed line and PtG2 is marked as a dotted line. The most abundant synthetic PtG is shown as a solid line. [Figure 17] Figure 17 shows the extracted ion chromatogram from the LC-MS data of pregnanetriol glucuronide (PtGs, m / z 511) formed by A) deacetylation and glucuronidation of 5β-pregnane-3α,6α,20α-triol-20-acetate, B) deacetylation and glucuronidation of 5β-pregnane-3α,6α,20β-triol-20-acetate, C) and D) urine samples at two late estrus times from two female dogs. PtG1 is marked as a dashed line and PtG2 is marked as a dotted line. The synthetic PtG is shown as a solid line. [Figure 18]Figure 18 shows extracted ion chromatograms from LC-MS data of pregnanetriol glucuronides (PtGs, m / z 511) formed from A) reduction and glucuronidation of 5α-pregnane-3β,6α-diol-20-one, and B) and C) two late-estrus urine samples from two female dogs. The most abundant synthetic PtGs are marked as solid lines, and those with the same retention time as PtG1 (marked as dashed lines) are excluded. PtG2 is shown as a dotted line. [Figure 19] Figure 19 shows extracted ion chromatograms from LC-MS data of pregnanetriol glucuronides (PtGs, m / z 511) formed from A) reduction and glucuronidation of 5α-pregnane-3β,20β-diol-6-one, and B) and C) urine samples from two late-estrus stages of two female dogs. The most abundant synthetic PtGs are marked as solid lines, and those with the same retention time as PtG1 (marked as dashed lines) are excluded. PtG2 is shown as a dotted line. [Figure 20] Figure 20 shows extracted ion chromatograms from LC-MS data of pregnantriol glucuronide (PtGs, m / z 511) formed from A) reduction and glucuronidation of pregnenolone, and B) and C) urine samples from two late-estrus stages of two female dogs. A reference steroid was extracted by solid-phase extraction (SPE) and eluted in 50% methanol. [Figure 21] Figure 21 shows extracted ion chromatograms from LC-MS data of pregnanetriol glucuronide (PtGs, m / z 511) formed from urine samples of two female dogs at two late estrus stages: A) glucuronidation of 5α-pregnane-3β,6α,20α-triol (solid line), B) glucuronidation of 5α-pregnane-3β,6α,20β-triol (dashed line), C) and D) pregnanetriol glucuronide (PtGs, m / z 511) formed from urine samples from two female dogs at two late estrus stages. PtG1 is marked as a dashed line and PtG2 is marked as a dotted line. [Figure 22]Figure 22 shows extracted ion chromatograms from LC-MS data of pregnanetriol glucuronide (PtGs, m / z 511) formed from: A) synthetic 5α-PtG (5α-pregnane-3,6,20-triol glucuronide), B) late-estrus urine samples spiked with 1 μM 5α-PtG, C) late-estrus urine samples spiked with 0.5 μM 5α-PtG, and D) late-estrus urine samples without 5α-PtG addition. PtG1 is marked as a dashed line and PtG2 is marked as a dotted line. The increase in the area of ​​the PtG1 peak in urine was linear with the addition of 5α-PtG. [Figure 23] Figure 23 shows extracted ion chromatograms from LC-MS data of pregnanetriolglucuronide (m / z 511) formed by A) 5α-PtG (5α-pregnane-3,6,20-triolglucuronide), B) late-estrus urine samples spiked with 1 μM 5α-PtG, C) late-estrus urine samples spiked with 0.5 μM 5α-PtG, and D) late-estrus urine samples without 5α-PtG. PtG1 is marked as a dashed line, and PtG2 is marked as a dotted line. The increase in the area of ​​the PtG1 peak in urine was linear with the addition of the reference. The urine samples were from different individuals than those shown in Figure 20. [Figure 24] Figure 24 shows urinary 5α-PtG concentrations (sum of PtG1 and PtG2; nM ± SEM) and serum progesterone concentrations (ng / ml ± SEM) in eight female dogs measured by LC-MS. Urinary PtG concentrations correspond to serum progesterone concentrations. A) Serum and urinary concentrations were measured as results from the same day, and B) urinary results are considered to be the result of progesterone from the previous day. Ovulation was estimated to occur 2 days after serum progesterone levels of 5-6 ng / ml or 2 ng / ml (estimated LH peak). [Figure 25]Figure 25 shows the mean (±SEM) peak areas of urinary 5α-PtG (areas of co-elution peaks PtG1 and PtG2) and urinary PdGβ (PdG 20β type) in eight female dogs. Peaks were integrated from ion chromatograms extracted from LC-MS data. A) Mean areas shown on the same area axis, B) Mean areas shown on separate axes, with PdGβ similar to the 5α-PtG profile. Ovulation was estimated to occur 2 days after serum progesterone levels of 5-6 ng / ml or 2 ng / ml (estimated LH peak). [Figure 26] Figure 26 shows the average concentration (nM ± SEM) of urinary 5α-PtG from a series of 60 urine samples collected from 52 female dogs, measured by EIA. [Figure 27] Figure 27 shows the mean urinary 5α-PtG concentration (nM ± SEM) and serum progesterone concentration (ng / ml ± SEM) in 27 female dogs. The LH peak was estimated to occur when serum progesterone was approximately 2 ng / ml, or two days before ovulation (serum progesterone was 5-6 ng / ml). [Figure 28] Figure 28 shows a lateral flow immunochromatographic test strip containing a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. [Modes for carrying out the invention]

[0018] As mentioned above, determining the stage of estrus is important in order to detect the optimal time for reproduction and / or insemination in animals.

[0019] Therefore, it would be beneficial to provide a non-invasive, cost-effective, and rapid method for monitoring the estrous cycle.

[0020] Accordingly, this disclosure provides a novel method for monitoring the estrous cycle in animals, particularly non-human animals, such as canids. Monitoring the estrous cycle from excretory samples such as urine, saliva, and / or fecal samples is painless and cost-effective. Preferably, the sample is a urine sample. Rapid tests, such as rapid urine tests, provide an inexpensive tool for animal keepers, such as dog owners and breeders, to detect the optimal breeding time at home without stressful procedures such as blood or vaginal smear sampling. Samples can be collected using common household items at no additional cost.

[0021] Serum progesterone analysis is a commonly used method to determine the optimal breeding time in dogs. It can be used at any estrous cycle phase to estimate the estrous cycle phase in female dogs. However, the metabolism of progestogens in canids is unknown, and existing non-invasive methods for measuring steroid metabolites are not applicable to canids.

[0022] The inventors have noticed that progesterone (preg-4-ene-3,20-dione) hormone metabolism is unique among mammals in canids. Progestogens are usually reduced in mammals as 5β-pregnane-structured metabolites such as 5β-pregnane-3,20-diol glucuronide (PdG) and 5β-pregnane-3,17,20-triol glucuronide (17α-PtG) (Niemuller et al., 1993; O'Connor et al., 2003), but in canids such as dogs, they are reduced as 5α-pregnane derivatives. In humans, an increase in the amount of 5α-reducing metabolites can be a sign of various diseases such as breast cancer or pregnancy-related intrahepatic cholestasis (Meng et al., 1997; Wiebe, 2005).

[0023] In dogs, only trace amounts of PdG are excreted in the urine. On the other hand, the concentrations of 5β-pregnane-3α,17α,20α-triol glucuronide (17α-PtG) (both 20α and 20β isomers) fluctuate throughout the proestrus and estrus phases. Urinary pregnane-3,17,20-triol glucuronide does not follow serum progesterone or 17α-hydroxyprogesterone and is not a usable biomarker for progestogens. Interestingly, in dogs, 20β reduction of progesterone metabolites is far more common than 20α reduction, and in canine urine, the metabolites corresponding to PdG and 17α-PtG are 5β-pregnane-3α,20β-diol and 5β-pregnane-3α,17α,20β-triol glucuronide.

[0024] In dogs, instead of 17α-hydroxylation, progestogens are primarily excreted as the pregnane-3,6,20-triol glucuronide isomer. In humans, 5α-pregnane-3,6,20-triol is a cancer biomarker (Fennessey et al., 1986; Suzuki et al., 2002). The results indicate that, due to the unique metabolism in dogs, existing estrus / menstrual cycle monitoring or progestogen measurement methods used in humans or other mammals cannot be used in canids. These 5α-reduced pregnane-3,6,20-triols and their glucuronidated forms presented herein have never been used as progestogen biomarkers in estrus monitoring to date. This disclosure provides a novel tool for non-invasive progestogen detection in canids.

[0025] In particular, this disclosure relates to various methods for determining the estrous cycle phase of female dogs (Canis familiaris) by measuring progestogen metabolites in urine. This disclosure provides a method for detecting whether the canine ovary is secreting progestogens due to pre-ovulatory luteinization or luteinized follicles. This method can be used to detect the optimal breeding time (mating or insemination) in female dogs and is readily applicable to other canids.

[0026] Since circulating steroid hormones are excreted as metabolites in urine, saliva, and feces, the concentrations of these metabolites can be determined by preferred methods known to those skilled in the art. PdG and 17α-PtG, the most common metabolites of progesterone and 17α-hydroxyprogesterone, are not useful biomarkers for monitoring estrus in dogs.

[0027] This disclosure relates to 5α-pregnane-3,6,20-triol or its stereoisomers.

[0028] The molecule disclosed herein comprises a main structure (5α-pregnane-3,6,20-triol) and a side structure (glucuronide).

[0029] The molecular formula for 5α-pregnane-3,6,20-triol is C 21 H 36 It is O3, and the molecular weight of 5α-pregnane-3,6,20-triol is 336.51 g / mol.

[0030] The molecular formula for 5α-pregnane-3,6,20-triol glucuronide is C 27 H 44 It is O9, and the molecular weight of 5α-pregnane-3,6,20-triol glucuronide is 512.63 g / mol.

[0031] The general formula of the molecule is 5α-pregnane-3z,6z,20z-triol. The letter z represents the α or β configuration of the hydroxyl group on each chiral carbon. The C20 configuration can also be represented in the form of R and S.

[0032] The molecule can exist as a free form of 5α-pregnane-3z,6z,20z-triol, or as a glucuronidated form of 5α-pregnane-3z,6z,20z-triol glucuronide, in which one of the three hydroxyl groups is conjugated.

[0033] Due to both the chirality of the molecule and the alternative position of the glucuronide, the molecule has several different possible structures. Formula (I) below reflects the structure of the molecule when glucuronic acid is conjugated at the C3 position. Thus, the molecule of formula (I) is 5α-pregnane-3z,6z,20z-triol-3-glucuronide. The hydroxylated chiral carbon is highlighted with a dashed rectangle. [ka]

[0034] The chiral carbons (C3, C6 ja C20) in the dashed rectangle can be in an α or β configuration (C20 can alternatively be in an S or R configuration). Glucuronic acid can be conjugated from C3, C6, and C20.

[0035] The molecule 5α-pregnane-3z,6z,20z-triol and its C3 glucuronide form have several alternative spellings, and some examples of synonyms are listed below.

[0036] Pregnane-3,6,20-triol; 5α-Pregnane-3,6,20-triol; Pregnane-3,6,20-triol glucuronide; Pregnane-3,6,20-triol-3-glucuronide; 5α-Pregnane-3,6,20-triol Glucuronide; 5α-pregnane-3,6,20-triol-3-glucuronide; 5α-pregnane-3z,6z,20z-triol; 5α-pregnane-3z,6z,20z-triol-3-OzD-glucuronide; (3z,5alpha,6z,20z)-3,6,20-trihydroxypregnane; (3z,5alpha,6z,20z)-3,6,20-trihydroxypregnane-3-yl-zD-glucopyranosideuronic acid; (3z,5alpha,20z)-6,20-dihydroxypregnane; (3z,5 Alpha,20z)-6,20-dihydroxypregnane-3-yl-zD-glucopyranosideuronic acid; 6z,20z-dihydroxy-5α-pregnane; 6z,20z-dihydroxy-5α-pregnane-3z-yl-zD-glucopyranosideuronic acid

[0037] The molecule 5α-pregnane-3,6,20-triol has 8 stereoisomers, and its glucuronide has 48 stereoisomers. All stereoisomers represented by a single spelling are shown in Tables 1 and 2.

[0038] [Table 1]

[0039] [Table 2-1] [Table 2-2]

[0040] This disclosure describes compounds comprising a 5α-pregnane-3,6,20-triol isomer or a glucuronidated form of a 5α-pregnane-3,6,20-triol isomer.

[0041] In one embodiment, the compound comprises a 5α-pregnane-3,6,20-triol isomer.

[0042] In one embodiment, the compound is selected from the group consisting of 5α-pregnane-3α,6α,20α-triol, 5α-pregnane-3β,6α,20α-triol, 5α-pregnane-3α,6β,20α-triol, 5α-pregnane-3α,6α,20β-triol, 5α-pregnane-3β,6α,20β-triol, 5α-pregnane-3α,6β,20β-triol, and 5α-pregnane-3β,6β,20β-triol.

[0043] In one embodiment, the compound comprises a 5α-pregnane-3,6,20-triol glucuronide isomer.

[0044] In one embodiment, the compound is selected from the group consisting of 5α-pregnane-3α,6α,20α-triol glucuronide, 5α-pregnane-3β,6α,20α-triol glucuronide, 5α-pregnane-3α,6β,20α-triol glucuronide, 5α-pregnane-3α,6α,20β-triol glucuronide, 5α-pregnane-3β,6α,20β-triol glucuronide, 5α-pregnane-3α,6β,20β-triol glucuronide, and 5α-pregnane-3β,6β,20β-triol glucuronide.

[0045] In one embodiment, the compound is selected from the group consisting of 5α-pregnane-3,6,20-triol-3-O-α-D-glucuronide, 5α-pregnane-3,6,20-triol-6-O-α-D-glucuronide, 5α-pregnane-3,6,20-triol-20-O-α-D-glucuronide, 5α-pregnane-3,6,20-triol-3-O-β-D-glucuronide, 5α-pregnane-3,6,20-triol-6-O-β-D-glucuronide, or 5α-pregnane-3,6,20-triol-20-O-β-D-glucuronide.

[0046] In one embodiment, a combination or mixture of 5α-pregnane-3,6,20-triol isomers and / or glucuronidated forms of 5α-pregnane-3,6,20-triol isomers may be used.

[0047] This disclosure also describes the use of a compound in determining a progestogen or multiple progestogens in a sample derived from a non-human animal, wherein the compound comprises a 5α-pregnane-3,6,20-triol isomer or a glucuronidated form of the 5α-pregnane-3,6,20-triol isomer.

[0048] In one embodiment, the disclosure describes the use of a compound in the determination of a progestogen or a plurality of progestogens in an in vitro sample of non-human animal origin, wherein the compound comprises a 5α-pregnane-3,6,20-triol isomer or a glucuronidated form of the 5α-pregnane-3,6,20-triol isomer.

[0049] In particular, this disclosure describes the use of a compound in determining a progestogen or multiple progestogens in an in vitro urine sample derived from a non-human animal, wherein the compound comprises a 5α-pregnane-3,6,20-triol isomer or a glucuronidated form of the 5α-pregnane-3,6,20-triol isomer.

[0050] In one embodiment, the compound is a 5α-pregnane-3,6,20-triol isomer.

[0051] In one embodiment, the compound is a 5α-pregnane-3,6,20-triol glucuronide isomer.

[0052] In one embodiment, the disclosure describes the use of compounds selected from the group consisting of 5α-pregnane-3α,6α,20α-triol, 5α-pregnane-3β,6α,20α-triol, 5α-pregnane-3α,6β,20α-triol, 5α-pregnane-3β,6β,20α-triol, 5α-pregnane-3α,6α,20β-triol, 5α-pregnane-3β,6α,20β-triol, and 5α-pregnane-3β,6β,20β-triol in the determination of a progestogen or a plurality of progestogens in an in vitro sample of non-human animal origin.

[0053] In one embodiment, the disclosure describes the use of compounds selected from the group consisting of 5α-pregnane-3α,6α,20α-triolglucuronide, 5α-pregnane-3β,6α,20α-triolglucuronide, 5α-pregnane-3α,6β,20α-triolglucuronide, 5α-pregnane-3β,6β,20α-triolglucuronide, 5α-pregnane-3α,6α,20β-triolglucuronide, and 5α-pregnane-3β,6β,20β-triolglucuronide in determining a progestogen or a plurality of progestogens in an in vitro sample of non-human animal origin.

[0054] In one embodiment, the disclosure describes the use of a compound selected from the group consisting of 5α-pregnane-3,6,20-triol-3-O-α-D-glucuronide, 5α-pregnane-3,6,20-triol-6-O-α-D-glucuronide, 5α-pregnane-3,6,20-triol-20-O-α-D-glucuronide, 5α-pregnane-3,6,20-triol-3-O-β-D-glucuronide, 5α-pregnane-3,6,20-triol-6-O-β-D-glucuronide, or 5α-pregnane-3,6,20-triol-20-O-β-D-glucuronide in determining a progestogen or a plurality of progestogens in an in vitro sample of non-human animal origin.

[0055] In one embodiment, the sample is excrement. In a preferred embodiment, the excrement is selected from the group consisting of urine, saliva, and feces. In a more preferred embodiment, the sample is a urine sample.

[0056] In one embodiment, the Disclosure describes the use of the compounds according to the Disclosure in determining a progestogen or a group of progestogens in an in vitro urine sample derived from a non-human animal.

[0057] In one embodiment, the disclosure describes the use of compounds selected from the group consisting of 5α-pregnane-3α,6α,20α-triol, 5α-pregnane-3β,6α,20α-triol, 5α-pregnane-3α,6β,20α-triol, 5α-pregnane-3β,6β,20α-triol, 5α-pregnane-3α,6α,20β-triol, 5α-pregnane-3β,6α,20β-triol, and 5α-pregnane-3β,6β,20β-triol in the determination of a progestogen or a plurality of progestogens in an in vitro urine sample of non-human animal origin.

[0058] In one embodiment, the disclosure describes the use of compounds selected from the group consisting of 5α-pregnane-3α,6α,20α-triolglucuronide, 5α-pregnane-3β,6α,20α-triolglucuronide, 5α-pregnane-3α,6β,20α-triolglucuronide, 5α-pregnane-3β,6β,20α-triolglucuronide, 5α-pregnane-3α,6α,20β-triolglucuronide, and 5α-pregnane-3β,6β,20β-triolglucuronide in determining the presence of one or more progestogens in an in vitro urine sample of non-human animal origin.

[0059] In one embodiment, the disclosure describes the use of a compound selected from the group consisting of 5α-pregnane-3,6,20-triol-3-O-α-D-glucuronide, 5α-pregnane-3,6,20-triol-6-O-α-D-glucuronide, 5α-pregnane-3,6,20-triol-20-O-α-D-glucuronide, 5α-pregnane-3,6,20-triol-3-O-β-D-glucuronide, 5α-pregnane-3,6,20-triol-6-O-β-D-glucuronide, or 5α-pregnane-3,6,20-triol-20-O-β-D-glucuronide in determining the presence of one or more progestogens in an in vitro urine sample of non-human animal origin.

[0060] In one embodiment, the animal is selected from a group of canids consisting of domestic dogs, wild dogs, wolves, coyotes, foxes, bush dogs, short-eared dogs, jackals, and raccoons.

[0061] In one embodiment, the sample is an in vitro sample.

[0062] This disclosure provides an in vitro method for determining a progestogen or a plurality of progestogens in a sample derived from a non-human animal, the method comprising the steps of: providing an in vitro sample derived from a non-human animal; measuring the amount of a 5α-pregnane-3,6,20-triol isomer or a 5α-pregnane-3,6,20-triol glucuronide isomer in the sample; and determining the presence of luteinized follicle theca cells and / or granulosa cells based on the amount of a 5α-pregnane-3,6,20-triol isomer or a 5α-pregnane-3,6,20-triol glucuronide isomer in the sample, wherein the progestogen level in the non-human animal sample is indicated by the presence of luteinized follicle theca cells and / or granulosa cells.

[0063] In one embodiment, the method includes an in vitro method for determining a progestogen or a plurality of progestogens in a urine sample derived from a non-human animal, the method comprising the steps of: providing an in vitro urine sample derived from a non-human animal; measuring the amount of 5α-pregnane-3,6,20-triol isomer or 5α-pregnane-3,6,20-triol glucuronide isomer in the sample; and determining the presence of luteinized follicle theca cells and / or granulosa cells based on the amount of 5α-pregnane-3,6,20-triol isomer or 5α-pregnane-3,6,20-triol glucuronide isomer in the sample, wherein the progestogen level in the non-human animal sample is indicated by the presence of luteinized follicle theca cells and / or granulosa cells.

[0064] In one embodiment, the method includes an in vitro method for determining a progestogen or a plurality of progestogens in a urine sample derived from a non-human animal, the method comprising the steps of: providing an in vitro urine sample derived from a non-human animal; measuring the amount of 5α-pregnane-3,6,20-triol isomers in the sample; and determining the presence of luteinized follicle theca cells and / or granulosa cells based on the amount of 5α-pregnane-3,6,20-triol isomers in the sample, wherein the progestogen level in the non-human animal sample is indicated by the presence of luteinized follicle theca cells and / or granulosa cells.

[0065] In one embodiment, the method includes an in vitro method for determining a progestogen or a plurality of progestogens in a urine sample derived from a non-human animal, the method comprising the steps of: providing an in vitro urine sample derived from a non-human animal; measuring the amount of 5α-pregnane-3,6,20-triol glucuronide isomers in the sample; and determining the presence of luteinized follicle theca cells and / or granulosa cells based on the amount of 5α-pregnane-3,6,20-triol glucuronide isomers in the sample, wherein the progestogen level in the non-human animal sample is indicated by the presence of luteinized follicle theca cells and / or granulosa cells.

[0066] The stage of estrus can be determined based on the amount of 5α-pregnane-3,6,20-triol isomer or 5α-pregnane-3,6,20-triol glucuronide isomer in the sample. The presence of luteinized follicle theca cells and / or granulosa cells in the sample can be determined based on the amount of 5α-pregnane-3,6,20-triol or its glucuronidated form in the sample.

[0067] The optimal breeding time for animals such as canids can be determined by measuring the amount of 5α-pregnane-3,6,20-triol or its glucuronidated form in an in vitro sample.

[0068] In particular, the optimal breeding time for animals such as canids can be determined by measuring the amount of 5α-pregnane-3,6,20-triol or its glucuronidated form in an in vitro urine sample.

[0069] In one embodiment, a combination or mixture of 5α-pregnane-3,6,20-triol isomers and / or glucuronidated forms of 5α-pregnane-3,6,20-triol isomers may be used in this method.

[0070] This method can be used to determine the estrous cycle phase in animals such as canids. A rise in serum progesterone hormone concentration to a certain level indicates ovulation has occurred, making conception possible. Progestogen levels in non-human animal samples are indicated by the presence of theca cells and / or granulosa cells of luteinized follicles.

[0071] This method for determining a progestogen or multiple progestogens is performed by measuring the amount of 5α-pregnane-3,6,20-triol isomer or 5α-pregnane-3,6,20-triol glucuronide isomer in an in vitro urine sample from a non-human animal.

[0072] In this method, the quantification of a progestogen or multiple progestogens is performed by measuring the amount of 5α-pregnane-3,6,20-triol isomer or 5α-pregnane-3,6,20-triol glucuronide isomer in an in vitro sample of a non-human animal.

[0073] In one embodiment, the animal is preferably a canid. More preferably, the canid is selected from the group consisting of domestic dogs, wild dogs, wolves, coyotes, foxes, bush dogs, short-haired dogs, jackals, and raccoon dogs.

[0074] In one embodiment, the sample is excrement. Preferably, the excrement is selected from the group consisting of urine, saliva, and feces. More preferably, the sample is a urine sample.

[0075] In this method, the quantification of a progestogen or multiple progestogens is performed by measuring the amount of 5α-pregnane-3,6,20-triol isomer or 5α-pregnane-3,6,20-triol glucuronide isomer in an in vitro urine sample from a non-human animal.

[0076] Quantitative analysis may be performed using any suitable method known to those skilled in the art. In one embodiment, the quantitative analysis method is selected from the group consisting of immunoassay, binding assay, mass spectrometry, nuclear magnetic resonance (NMR), and chromatography.

[0077] In one embodiment of the method of this disclosure, an immunoassay may be used. An immunoassay is an immunochemical method based on the specific interaction between an antigen and the antibody to which it binds. The antigen-binding agent may also be only a portion of the antibody (e.g., immunoglobulin Fab domain) or a molecule other than the antibody, such as an aptamer, affimer, protein A (and others), or a receptor. Any molecule known to bind to a particular antibody can act as an antigen. In a basic immunoassay, one member of an antigen-antibody pair is immobilized on a solid support as a capture agent and is then probed by the other member of the pair. The captured molecule or complex is then detected using a labeled molecule, usually an antibody.

[0078] Numerous immunoassay-based methods have already been developed, and more are constantly being created. These methods can be applied almost indefinitely to any suitable environment. Suitable immunoassay-based methods include, for example, radioimmunoassays (RIA), enzyme immunoassays (EIA, ELISA), and fluorescence immunoassays (IFA, FIA, etc.), as well as immunoelectrophoresis and lateral flow immunoassay (LFIA). Those skilled in the art can select the appropriate method to use.

[0079] Immunoassay formats can be classified into two types, normal-phase and reverse-phase, depending on which target molecule (capture or probe) is involved in the assay. Furthermore, assay formats can be divided into competitive and non-competitive categories depending on the assay used. Competitive assays are mainly used to evaluate the expression levels of small molecules such as steroids or drugs, while non-competitive assays are generally used to measure (qualitatively or quantitatively) or analyze larger biomolecules such as proteins and antibodies.

[0080] Normal-phase assays use well-characterized molecules with known target specificity, immobilized on a solid support as a capture agent. The capture agent is probed with a sample solution, such as saliva, urine, or fecal supernatant, to measure the presence, absence, or concentration of the target molecule. In reverse-phase assays, antibodies are used to probe molecules immobilized on a solid phase. Analysis of the resulting binding profiles can help identify novel biomarker candidates for the development of diagnostic methods or can be used, for example, for antibody specificity screening.

[0081] Immunoassays are based on the adhesion of antigens / antibodies to a solid phase. Most solid surfaces to which desired molecules can adhere can serve as the basis for an immunoassay. The most common solid phase used in immunoassays is the conventional 96-well plate, but other substrates such as microscope slides, test tubes, membranes, rapid test platforms, and various micro / nanoparticle and gel matrices are also used.

[0082] The most commonly used solid support materials are plastics (e.g., polystyrene, polycarbonate, poly(methyl methacrylate), polyethylene terephthalate), glass (mainly silicon dioxide), and metals (gold, silver, platinum, aluminum, iron, cobalt, copper, titanium, etc.). Other materials such as silicon, carbon, lipids, and (nitro)cellulose are also used. The materials used to manufacture solid support materials are virtually limitless.

[0083] While some solid support materials can bind to proteins without pre-activation, assays are typically performed on functionalized surfaces. Surface modification can be divided into two types: two-dimensional (2-D), which includes surface activation by plasma treatment, irradiation, or chemical treatment, and three-dimensional (3-D), in which a chemically activated porous layer is fabricated on the solid support by physical or chemical methods. 3-D surface layers are typically polymers that can form membranes, hydrogels, layers, brushes, monoliths, or dendrimers. 3-D surfaces have increased binding capacity compared to 2-D surfaces due to their porosity and therefore their higher binding area.

[0084] Antigen / antibody attachment to a solid phase can be achieved by physical or chemical methods, or by using a support protein. Physical binding processes are based on the direct adsorption or capture of the protein, and can also be passively attached to a surface by nonspecific interactions, for example. Immobilization by direct adsorption is mainly based on hydrophobic, hydrophilic, and electrostatic bonding interactions between the protein and the solid surface. Chemical bonding involves the formation of new molecular bonds between the protein and the surface. Covalent bonding can occur between the chemically active portion of the protein (usually a primary amine or thiol group) and the surface of a solid support patterned with chemically active groups such as primary amines, carboxylic acids, aldehydes, or epoxides.

[0085] Proteins are widely used as carriers to enhance binding affinity and enable site-specific immobilization of molecules with only one antigen site, otherwise these molecules would be impossible to immobilize on solid supports. The use of protein-mediated immobilization requires the formation of a bond between the carrier protein and the capture agent. This can be achieved either by direct conjugation of the capture agent to the carrier protein via covalent bonding or by affinity binding via non-covalent specific recognition.

[0086] Examples of carrier proteins (for covalent bonding) in immunoassays include serum albumin obtained from either bovine (BSA) or human (HSA) sources, and ovalbumin protein derived from avian egg white, although other proteins are also used. Protein carriers are widely used for immobilizing low molecular weight chemical compounds such as steroids, toxins, and drugs that cannot be bound to solid supports without losing their antigenic properties. Furthermore, the need to use carrier proteins is often related to the need to induce an immune response, for example, in antibody and vaccine development, and therefore requires hapten analysis. Widely used immunogenic carrier proteins include BSA, ovalbumin, keyhole limpet hemocyanin (KLH), and blue carrier protein (mollusk-derived hemocyanin).

[0087] In one embodiment of the method disclosed herein, a binding assay may be used.

[0088] Affinity conjugation refers to binding based on non-covalent specific recognition between two different molecules. The most widely used affinity conjugation methods utilize the specific binding properties of antibodies and biotin-avidin (or streptavidin / neutraavidin) interactions. Antibody-mediated affinity conjugation is primarily used for immobilizing oriented antibodies. Oriented immobilization exposes antigen-binding sites and therefore significantly improves target binding efficiency and detection sensitivity. Antibodies can also be used as capture agents in unoriented immobilized form, particularly monoclonal antibodies with specific types of binding sites. When using antiserum or polyclonal antibodies that bind to multiple epitopes as capture agents, affinity conjugation via a secondary antibody system can be applied to increase the binding affinity of the specific antibody to the target antigen. Antibodies can also bind to other capture agents, such as glycoproteins.

[0089] Many different methods are used to detect formed antigen-antibody complexes in immunoassays, and more methods are constantly being developed. Traditionally, a desired antigen or antibody conjugated with some kind of label (either directly or using affinity-binding tags) is used for detection. In immunological assays, the most widely used labels are enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase, which function by producing a detectable color change (colorimetric) or precipitate, or by producing light or chemiluminescence when exposed to certain reagents. Widely used labels are also radioisotopes and fluorescent chemical compounds, but other methods such as DNA reporters and various detectable nanoparticles / microparticles (e.g., precipitates or turbidity) have also been described. Furthermore, detection by immunoassay can also be performed without labeling, using surface plasmon resonance techniques or by measuring the change in resistance at an electrode when the antigen binds to the electrode.

[0090] Progestogen metabolites can be measured by various immunological assays. In this method, either a polyclonal or monoclonal antibody produced against the 5α-pregnane-3,6,20-triol isomer or the 5α-pregnane-3,6,20-triol glucuronide isomer can be used as the primary antibody.

[0091] In one embodiment of the present disclosure, the immunoassay is selected from the group consisting of enzyme immunoassays (ELISA, EIA), radioimmunoassays (RIA), fluorescence immunoassays, immunoelectrophoresis, lateral flow immunoassays, electrochemiluminescence assays, and nanoparticle-based assays. Those skilled in the art can select an appropriate immunoassay method for use in detection.

[0092] In one embodiment of this disclosure, a rapid test is used for determination. In a typical semi-quantitative or quantitative lateral flow immunochromatography assay, the concentration of urinary progestogen metabolites is measured. In an example of such assay, urinary progestogen metabolites compete with progestogen metabolites conjugated to gold nanoparticles. Those skilled in the art can select a format suitable for a rapid test.

[0093] In one embodiment of this disclosure, the difference in urinary 5α-PtG concentration between estrous cycle phases in dogs is tested by side-flow chromatography. This rapid test provides a quick and cost-effective method for detecting urinary progestogen levels in dogs without requiring special equipment or laboratory facilities.

[0094] In one embodiment of the present disclosure, the rapid test is a lateral flow immunochromatography test strip comprising a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorption pad. Those skilled in the art can select appropriate materials to include in the rapid test. An example of a test strip is shown in Figure 29. A buffering agent is absorbed into the sample pad to smooth out variations between samples, for example, due to variations in pH and salt concentration. Antibodies, such as 5α-PtG antibody conjugated to gold particles, are dried on the conjugate pad. Conjugates, such as 5α-PtG conjugates, are immobilized on the nitrocellulose membrane as test lines, and anti-mouse IgG antibody is immobilized on the nitrocellulose membrane as a control line. Excess sample is finally absorbed into the absorption pad, which also contributes to capillary action on the test strip.

[0095] In one embodiment of the present disclosure, in a rapid test, the sample is placed on a sample pad and flows through a test strip by capillary force. On the conjugate pad, sample molecules, such as 5α-PtG molecules, contained in the sample bind to antibodies, such as 5α-PtG molecules conjugated to gold nanoparticles, and the formed complex moves to the membrane. If only a small amount of analyte, such as 5α-PtG, is present in the sample, the free antibody-gold nanoparticles flow through the membrane and bind to analytes, such as 5α-PtG-BSA, in the test line, generating a visible signal. When analyte molecules, such as 5α-PtG, in the sample bind to the flowing antibody-gold nanoparticles, the antibodies do not interact with analyte conjugates such as 5α-PtG-BSA conjugates, and no visible test line is formed. The remaining antibody-gold particles bind to anti-mouse IgG in the control line, generating a visible control line that confirms that the fluid has successfully passed through the sample pad and test line to reach the control line. Since the intensity of the test line depends on the amount of antigen present in the sample, the estimation of, for example, the estrous cycle phase can be achieved by simple visual evaluation.

[0096] In another embodiment, a method for measuring the amount of 5α-pregnane-3,6,20-triol isomer or 5α-pregnane-3,6,20-triol glucuronide isomer in an in vitro sample derived from a non-human animal is bringing the sample into contact with a capture portion and a detection portion, wherein the capture portion is 5α-pregnane-3,6,20-triol or 5α-pregnane-3,6,20-triol glucuronide isomer attached to a solid support conjugated with a carrier protein, and the capture portion is 5α-pregnane-3,6,20-triol glucuronide isomer. The detection portion can form a complex with an antibody against a gunan-3,6,20-triol or a 5α-pregnan-3,6,20-triol glucuronide isomer, the detection portion can bind to the antibody of the antibody-capture portion complex, and the formation of the complex includes contacting the sample with the capture portion and the detection portion, and detecting a signal from the detection portion, wherein the amount of the detected signal is proportional to the estrous cycle phase. In a preferred embodiment, the sample is urine. In a preferred embodiment, the non-human animal is selected from the group of Canidae animals consisting of domestic dogs, wild dogs, wolves, coyotes, foxes, bush dogs, short-haired dogs, jackals, and raccoon dogs.

[0097] In one embodiment, the detection portion includes a binding partner and a label, the label being selected from the group consisting of electrochemiluminescent labels or compounds, chemiluminescent compounds, enzyme labels, fluorophores, chromogenic compounds, radioactive labels, catalysts, latex particles, magnetic particles, radioactive elements, fluorescent dyes, phosphorescent dyes, dye microcrystals, gold colloid particles, silver colloid particles, selenium colloid particles, metal chelates, coenzymes, electroactive groups, oligonucleotides, and stable radicals.

[0098] In one embodiment of the present disclosure, the solid support includes beads, superparamagnetic beads, paramagnetic beads, plates, glass surfaces, plastic surfaces, metal surfaces, polystyrene surfaces, nitrocellulose surfaces, Sepharose, agar, fine particle surfaces, nanoparticle surfaces, channels in a lateral flow assay apparatus, or wells in a microtiter plate.

[0099] In one embodiment of the method of the present disclosure, chromatography may be used. In chromatography, an analyte of interest is separated from a mixture of compounds, for example, a body fluid. Chromatography is based on the interaction between a stationary phase and a mobile phase, in which the mixture is dissolved in the mobile phase (Beesley and Buglio, 2000).

[0100] Chromatography can be used for preparative (separation, purification) or analytical purposes (quantitative and qualitative analysis). Generally, separation is based on the structure and physicochemical properties of the analyte, including size (size exclusion chromatography, SEC), polarity, respective charges (ionic chromatography), and adsorption (expanded bed chromatography adsorption, EBA). Chromatographic techniques are usually named according to the bed shape (column chromatography and planar chromatography), the characteristics of the mobile phase (gas chromatography and liquid chromatography), or the separation mechanism (e.g., affinity, ion exchange, size exclusion, EBA). Chromatography is generally used in conjunction with measuring instruments such as mass spectrometers or UV detectors.

[0101] For analytical measurement, samples are prepared for analysis. Efficient sample preparation is repeatable and results in high analyte recovery. Sample preparation may be necessary before chromatography to minimize matrix effects in the sample, but various chromatographic methods can also be used for sample preparation. Body fluids such as blood, saliva, tissue fluid, and excretion must be homogenized and purified from solid particles. Steroid extraction removes undesirable compounds from the sample that may interfere with the analysis. Ultracentrifugation, precipitation, and dialysis are cost-effective methods for removing or isolating high molecular weight compounds such as proteins from a sample. In exclusion chromatography, large compounds are removed from the sample matrix by passing the sample through a stationary phase, and the pore size is suitable for the size of the target analyte. Solid-phase extraction (SPE) is a rapid method for sample preparation and purification. In SPE, complex matrices such as urine or blood can be easily purified by normal-phase, reverse-phase, or ion-exchange extraction, depending on the stationary phase packing. SPE provides an efficient technique for preparing samples before chromatographic analysis.

[0102] Sample preparation depends on the chromatography method. For example, in liquid chromatography, the analyte of interest is usually diluted with a suitable solvent such as acetonitrile, or the steroid is extracted by liquid-liquid extraction before chromatography. In gas chromatography (GC), the volatility of naturally occurring non-volatile steroids is increased by derivatization. The advantages of gas chromatography are the high sensitivity and efficiency of the GC detector, which is why GC provides accurate and precise results. Furthermore, the spectral library of derivatized steroids is comprehensive. In liquid chromatography (LC), steroids can be detected in their natural form, and derivatization is not necessary. Like GC, LC is considered an accurate and precise method for analysis. Those skilled in the art can select the appropriate chromatography method.

[0103] In one embodiment, the chromatography is selected from the group consisting of gas chromatography, gas chromatography-mass spectrometry, liquid chromatography, and liquid chromatography-mass spectrometry.

[0104] In one embodiment, progesterone-derived metabolites can be identified and measured by liquid chromatography-high-resolution mass spectrometry (LC-HRMS). The levels of these metabolites can be used as biomarkers for circulating progestogens.

[0105] In one embodiment of the method of this disclosure, mass spectrometry may be used. Mass spectrometry (MS) is a tool for many fields of research. MS is a highly sensitive and versatile analytical technique used to identify and quantify molecules in various sample types, including biological samples such as urine, saliva, and blood. It works by ionizing the sample, separating the ions based on their mass-to-charge ratio (m / z), and detecting them to determine their molecular weight and elemental composition.

[0106] In ionization, the analyte is converted into ions. Negatively or positively charged ions are accelerated in an electric field, extracted, and guided to a mass spectrometer and detector to provide information about the molecular composition of the sample. Ionization can be achieved by various methods, including electron ionization (EI), electrospray ionization (ESI), chemical ionization (CI), matrix-assisted laser desorption ionization (MALDI), and atmospheric pressure chemical ionization (APCI). EI involves removing electrons from sample molecules to produce positive ions, while CI involves reacting the sample with a reagent gas to produce ions. MALDI uses a laser to ionize the sample. In ionization, the analyte is fragmented, and the degree of fragmentation depends on the energy in ionization. Hard ionization (like EI) results in a high degree of fragmentation, while soft ionization (ESI, CI, MALDI, APCI, etc.) results in only slight fragmentation. Soft ionization provides an m / z close to the neutral mass of the analyte.

[0107] Mass spectrometers are used to separate ions based on their m / z values. Mass spectrometers use either static or dynamic fields and either magnetic or electric fields for separation. For example, time-of-flight (TOF) analyzers and quadrupole mass spectrometers use electric fields to accelerate ions. Tandem mass spectrometry (MS / MS) uses two or more analyzers to provide more accurate information about ions and fragmentation.

[0108] A time-of-flight (TOF) analyzer is a mass spectrometer that separates ions based on the time it takes for them to travel a set distance. It works by accelerating ions and measuring the time it takes for them to reach a detector at the end of a flight tube. Since the time it takes for ions to reach the detector is proportional to their mass-to-charge ratio, a TOF analyzer can separate and identify ions based on their mass.

[0109] A quadrupole mass spectrometer is a type of mass spectrometer that uses four parallel rods to focus and analyze ions based on their mass-to-charge ratio. The rods generate an alternating current potential that acts as a mass filter. Ions with a desired mass-to-charge ratio can pass through the quadrupole and be detected, while ions with other mass-to-charge ratios are deflected and do not reach the detector.

[0110] Orbitrap is a mass spectrometer that uses a combination of electric and magnetic fields to analyze ions based on their mass-to-charge ratio. Ions are trapped in a toroidal electric field, and their motion is analyzed as they orbit around the center of the electric field. The frequency of an ion's motion is proportional to its mass-to-charge ratio, and this information is used to determine the ion's mass.

[0111] A mass detector is an instrument used in mass spectrometry to measure the mass of ions. It works by measuring the response of ions to an electric or magnetic field, and the collected information is used to determine the ion's mass-to-charge ratio. The most commonly used mass detectors in mass spectrometry include quadrupole mass filters, time-of-flight (TOF) analyzers, and Orbitrap. Each type of mass detector has its own advantages and disadvantages, and the choice of detector depends on the specific application and requirements of the analysis. Mass detectors are essential components of mass spectrometers and play a crucial role in the analysis and identification of compounds in a sample.

[0112] Mass spectrometry is widely used in fields such as chemistry, biology, and health sciences for applications including the analysis of proteins and metabolites, the analysis of trace contaminants, and drug development. It offers many advantages, including high sensitivity, accuracy, and the ability to handle complex samples. Mass spectrometry provides a tool for the quantification and identification of unknown compounds.

[0113] In one embodiment of the method of this disclosure, immunoaffinity chromatography (IAC) may be used in combination with liquid / gas chromatography-mass spectrometry. The combinations of IAC-LCMS and IAC-GCMS offer the advantages of structure-specific antibodies and high-precision analysis. These methods are effective for extracting and concentrating compounds of interest from complex sample matrices, such as urine samples, and analyzing them with high-precision instruments. This method can be used for low-concentration samples, for example, when results cannot be achieved without an efficient concentration method, or when subsequent analysis / preparation methods require an efficient purification process.

[0114] In one embodiment of the method of this disclosure, nuclear magnetic resonance (NMR) spectroscopy may be used. NMR is an analytical technique for determining the structure of chemical and biological compounds. It relies on the interaction between the nucleus of a particular atomic isotope and a static magnetic field, detecting the energy absorbed by changes in the nuclear spin state. The most commonly used nuclei are 1 H and 13 C, 19 F and 31Isotopes of other elements, such as phosphorus (P), can also be studied. There are one-dimensional and two-dimensional methods; the latter is sometimes necessary when determining the structures of more complex molecules. NMR experiments are typically performed in solution.

[0115] Atomic nuclei have intrinsic spin properties, and different atoms within a molecule have different resonance frequencies in the same magnetic field; this phenomenon is known as chemical shift (δ). This means that in an NMR spectrum, different atoms will produce different peaks according to their specific chemical environment and interatomic bonding. Furthermore, resonance frequencies are perturbed by neighboring NMR-active nuclei, depending on the bonding electrons that link the nuclei, meaning that interatomic bonding on a molecule can be identified. The observable NMR signal is recorded by a spectrometer as free-induced decay (FID), then Fourier transformed to obtain a spectrum consisting of a set of peaks, each corresponding to a distinct chemical environment. The number of nuclei in a particular chemical environment is directly related to the area under the peak. Chemical shift is expressed in parts per million (ppm).

[0116] To avoid solvent interference, stabilize the magnetic field strength, and accurately define 0 ppm, NMR experiments should be performed in a deuterated solvent. Since spectral peaks can be solvent-dependent, comparisons with reference spectra should be performed using the same solvent. The most common solvents are deuterated chloroform, acetone, dimethyl sulfoxide, acetonitrile, methanol, and water.

[0117] In one embodiment of the present disclosure, the in vitro method includes the steps of: providing an in vitro urine sample from a canid; measuring the amount of 5α-pregnane-3,6,20-triol isomer or 5α-pregnane-3,6,20-triol glucuronide isomer in the sample; and determining the presence of luteinized follicle theca cells and / or granulosa cells based on the amount of 5α-pregnane-3,6,20-triol isomer or 5α-pregnane-3,6,20-triol glucuronide isomer in the sample, wherein the level of progestogen in the sample is indicated by the presence of luteinized follicle theca cells and / or granulosa cells.

[0118] The estrous cycle phase in canids is determined by urine testing for progestogen metabolites, specifically the 5α-pregnane-3,6,20-triol isomer or 5α-pregnane-3,6,20-triol glucuronide isomer. This allows for the detection of the optimal breeding time (mating or insemination) in female dogs.

[0119] This disclosure describes a kit for determining a progestogen or multiple progestogens in a urine sample from a non-human animal, the kit comprising an immunoassay means for measuring the amount of a 5α-pregnane-3,6,20-triol isomer or a 5α-pregnane-3,6,20-triol glucuronide isomer in an in vitro urine sample from a non-human animal, and instructions for determining a progestogen or multiple progestogens according to this method, the method is described as follows: a) A step of providing an in vitro urine sample derived from a non-human animal, b) A step of measuring the amount of 5α-pregnane-3,6,20-triol isomer or 5α-pregnane-3,6,20-triol glucuronide isomer in the sample, c) A step of determining the presence of theca cells and / or granulosa cells of a luteinized follicle based on the amount of 5α-pregnane-3,6,20-triol isomer or 5α-pregnane-3,6,20-triol glucuronide isomer in the sample, The progestogen level in the sample is indicated by the presence of theca cells and / or granulosa cells of luteinized follicles.

[0120] As technology advances, it will be apparent to those skilled in the art that the basic concepts of the present invention can be implemented in a variety of ways. Therefore, the present invention and its embodiments are not limited to the examples described above and may vary within the scope of the claims. [Examples]

[0121] Example 1 Discovery of novel biomarkers for estrus monitoring Progesterone-derived metabolites were discovered by liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Metabolites with a neutral mass of 512 can be used as biomarkers for circulating progestogens.

[0122] Collection and preparation of serum samples Serum samples (177 samples from 22 female dogs) were collected from proestrus to estrus and provided by Vetcare Ltd. The serum samples were collected during a study in South Africa (ClinVet International (Pty) Ltd) in 2015. Study plan CW 14 / 122 was approved by the ClinVet Committee for Animal Ethics and Welfare (CCAEW). Serum samples were stored frozen (below -20°C) until analysis. A total of 95 serum samples from 8 randomly selected female dogs were prepared and analyzed, excluding sample dilution, as described in Hakkinen et al. (2018). Prior to analysis, serum samples were slowly thawed to room temperature and diluted 1:5 with 0.9% NaCl solution (9 mg / ml injectable NaCl solution, Braun Medical Ltd, Finland).

[0123] Collection and preparation of urine samples Urine samples from female dogs were collected in clean containers from spontaneous urination by volunteer dog owners. The samples were transferred to sample tubes and kept frozen until analysis. Immediately before use, the urine samples were slowly thawed to room temperature, thoroughly mixed, and centrifuged (2-5 minutes, 13400 rpm). Urine samples were collected in parallel with serum samples using ClinVet International (Pty) Ltd. Urine samples were kept frozen until analysis. One urine sample was collected from a volunteer woman in the second trimester of pregnancy to compare differences between dogs and humans. Written informed consent was obtained from participants.

[0124] Measurement of urinary creatinine using the Jaffe reaction Urinary creatinine concentration was measured by a colorimetric method based on the Jaffe reaction. Creatinine (Merck, Darmstadt, Germany) was dissolved in ultrapure water at a concentration of 0.5 mg / ml on the day of the assay. The standard dilution series was prepared by diluting the stock solution a total of seven times in a 1:2 ratio (0.5–0.008 mg / ml). Water was used as a blank control. Urine samples centrifuged (13400 rpm, 1.5 min) were diluted 1:40 with ultrapure water. The standard and diluted urine samples were added to 96-well plates (655101; Greiner Bio-one GmbH, Flickenhausen, Germany) in two sets of 10 μl / well, followed by the addition of 100 μl of picric acid solution (10 parts of 4.5 mM picric acid, 1 part of 1.4 M sodium hydroxide (Reagena Ltd, Sirinjärvi, Finland)). The absorbance at 490 nm was measured after 30 minutes.

[0125] Urinary creatinine concentration was calculated using linear regression analysis (y=kx+b), and the results were multiplied by a dilution factor (40). Steroid hormone concentrations were normalized using creatinine concentration (ng / Crmg, pmol / μmol Cr, or EIC area / Cr).

number

[0126] Sample preparation by solid-phase extraction (SPE) Glucuronidated steroid hormones were extracted from urine samples by solid-phase extraction. A silica-based C18 solid-phase reversed-phase extraction column (Waters Sep-Pak C18 3 cc Vac cartridge, 500 mg adsorbent per cartridge, 55-105 μm (WAT020805)) was conditioned by discharging 4 ml of methanol followed by 6 ml of 10% methanol into ultrapure water. The sample (1-3 ml) was diluted in water or 10% methanol in a 1:3 ratio and loaded onto the column. The column was rinsed with 3 ml of 10% methanol in ultrapure water. The flow-through solution was discarded. Steroid glucuronides were eluted into glass tubes using 6 ml of 50% methanol in ultrapure water. The sample tubes were placed in a heating block (+50°C) and the solvent was evaporated in a nitrogen evaporator. The samples were dissolved in methanol or acetonitrile. The sample was diluted to the desired dilution with 30% methanol (total volume 100 μl) in ultrapure water and placed in a glass vial insert.

[0127] Simple sample preparation A portion of the supernatant was reconstituted with two parts ultrapure water, followed by the addition of three parts of a suitable solvent, such as HPLC ultragradient grade methanol or acetonitrile. The mixture was filtered to a 96-deep well plate using a filtration plate (Captiva 96-well filter plate, 0.2 μm pore size, polypropylene filter, Agilent Technologies) or to an HPLC vial using a syringe filter (20 μm pore size). The well plate was covered with a silicone well cap (Thermo Nunc 276002), and the vial was sealed with a red PTFE / silicone screw cap. The vial was stored at +4°C until analysis.

[0128] Urine samples from several female dogs during estrus or post-estrus were thawed at room temperature, vigorously vortexed, and centrifuged at 13400 rpm for 2–5 minutes. The supernatant was diluted 1:3 with ultrapure water and mixed with acetonitrile 1:1 (diluted sample: ACN). The urine sample solution was filtered through a 96-well plate to a 1 ml deep-well plate by centrifugation (2300 rpm, 5 minutes) and sealed in a glass vial using a silicone well cap or syringe filter (0.2 μm). 30% ACN was used as a blank control. Freshly prepared samples were stored in the refrigerator until analysis.

[0129] Glucuronidation of reference steroids Steroids were dissolved in ethanol or methanol at a concentration of 1–5 mg / ml, and 5 μg of steroid (1.9–2 nmol) was used in the reaction. The reaction was carried out under the following conditions: 0.1 M Tris-HCl, 4 mM MgCl, 1 mM UDPGA (uridine-5'-diphosphate-glucuronate ammonium salt, U5625, Sigma-Aldrich, Missouri, USA) and 100 μg of protein (untreated female beagle liver microsomes, "CLM", 20 mg / ml, D1500, Xenotech, Kansas, USA). The reaction solution was kept on ice until use. Canid liver microsomes (CLM) were slowly thawed on ice. The reaction was initiated by adding the reagents to the reaction tube and then adding the canid microsomes. The reaction tube was incubated in a plate shaker at +37°C for 1 hour, and the reaction was stopped by adding 300 μl of acetonitrile (ACN, high-purity HPLC grade, VWR Chemicals). The reaction solution was centrifuged at 13400 rpm for 10 minutes (Eppendorf Centrifuge 5415 D, Hamburg, Germany), and the supernatant was collected and stored at -20°C until analysis.

[0130] Non-targeted and semi-quantitative analysis of pregnanediols and pregnanetriols by LC-HRMS For the semi-quantitative analysis of progesterone metabolites, various steroid glucuronides were used as reference steroids. Steroids were diluted to concentrations of 1–5 μM in 30% acetonitrile. Thawed urine samples were prepared as described above. 30% acetonitrile was used as a blank control. Freshly prepared samples were stored in the refrigerator until analysis.

[0131] Semi-quantitative analysis of progestogen glucuronide was performed using a UHPLC-QTOF-MS system (Agilent Technologies 1290 LC, 6540 MS, Agilent Technologies, Santa Clara, California, USA) as described by Pekkinen et al. (2013). Prepared samples were analyzed by reverse-phase chromatography using negative and positive mode ESI on a Zorbax Eclipse XDB-C18 column (100 mm × 2.1 mm, 1.8 μm; Agilent Technologies, column temperature +50°C, flow rate 0.4 ml / min). The analysis was performed by gradient elution using ultrapure water (eluent A) and methanol containing 0.1% (V / V) formic acid (eluent B): 0-10 min: 2 → 100% B, 10-14.5 min: 100% B, 14.5-16.5 min: 2% B. The sample injection volume was 2 μl, and the sample was maintained at +4°C. The MS ion source conditions and MS2 analysis are shown in Table 3. Similar methods were also used with the Thermo Scientific Q Exactive Quadrupole-Orbitrap high-resolution MS / MS system and the Thermo Scientific Vanquish Binary Flex Binary 1000 bar UHPLC system.

[0132] [Table 3]

[0133] result Data analysis was performed using MassHunter Acquisition B.06.00 or later versions (Agilent Technologies, Santa Clara, California, USA). Pregnanediol and pregnanetriol glucuronides (PdG and PtG) were analyzed from extracted ion chromatograms (EIC). PdG was found at 495 [MH]-, 514 [M+NH4]+, and 519 [M+Na]+. PtG was found at m / z 511 [MH]-, m / z 530 [M+NH4]+, and m / z 535 [M+Na]+.

[0134] Two synthetic pregnanediols (5β-pregnane-3α,20α-diol and 5β-pregnane-3α,20β-diol) were conjugated with glucuronic acid by CLM, and the products were compared with commercially available PdG (5β-pregnane-3α,20α-diol-3-glucuronide, later PdGα) and urine samples from dogs in the late estrus period. Mass spectrometry showed that the progestogen was excreted in the urine as several glucuronidated pregnanediols, triols, and tetrol. While many of these metabolites remain unidentified, the results were interesting, suggesting that β-reduction of the carbonyl group in C20 is highly favorable than α-reduction.

[0135] These results indicated that PdG (m / z 495 [MH]-) was detected only in trace amounts. Semi-quantitative analysis revealed that only small amounts of PdGα ​​and 5β-pregnane-3α,20β-diol glucuronide (later PdGβ) could be detected in urine samples (Figure 1). PdG is mainly excreted in the 20β form. The average ratio of PdGβ to PdGα ​​was 5.0 (1.0~14.7; Figure 2).

[0136] Glucuronide-conjugated pregnantriol was compared to pregnantriol glucuronide in urine samples collected from two female dogs in the late estrus period (m / z 511 [MH]-, m / z 530 [M+NH4]+, m / z 535 [M+Na]+). According to the literature, the common metabolite of progesterone and / or 17α-hydroxyprogesterone is 5β-pregnane-3α,17α,20α-triol-3-glucuronide (later 17α-PtGα). In dogs, the carbonyl in C20 is preferably reduced to the 20β type rather than the 20α type (Figure 3). Urinary 17α-PtG was compared to serum progesterone and 17α-OHP concentrations. None of the metabolites followed the serum progesterone (Figure 4a-b) or serum 17α-hydroxyprogesterone (Figure 4c-d) profiles, and therefore could not be used as biomarkers for estrus timing. However, a significant amount of previously unidentified pregnantriol glucuronide (later PtGX) was present at retention times of 6.4–6.7 minutes (Figure 3). Retention times varied slightly between run and instrument.

[0137] The urinary PtGX profile was similar to the serum progesterone profile, suggesting that this metabolite can be used as a biomarker for estrus timing in a similar manner to serum progesterone (Figure 5). Mass spectrometry using a similar method with Thermo Scientific Q Exactive Quadrupole-Orbitrap high-resolution MS / MS revealed that PtGX is indeed two closely eluted isomers with retention times of 6.4–6.7 minutes (Figure 6). In female dog A, the most abundant PtG (later PtG1) was found 0.05 minutes earlier than the most abundant isomer (later PtG2) in female dog B. However, both metabolites were found in both female dogs, and both metabolites can be used as PtGX for estrus monitoring using a QTOF instrument.

[0138] Example 2 Comparison of progestogen-derived metabolites in dogs and humans Progestogen-derived metabolites were investigated and compared using liquid chromatography-high-resolution mass spectrometry (LC-HRMS).

[0139] Collection and preparation of urine samples The collection of canine urine samples is described in Example 1. To compare differences between dogs and humans, one urine sample was collected from a female volunteer in the second trimester of pregnancy. Written informed consent was obtained from the participant. The urine sample was prepared as described in Example 1 (simple sample preparation).

[0140] Non-targeted and semi-quantitative analysis of progestogen-derived metabolites by LC-HRMS Semi-quantitative analysis of progestogen glucuronide was performed using the UHPLC-QTOF-MS system described in Example 1 (Agilent Technologies 1290 LC, 6540 MS, Agilent Technologies, Santa Clara, California, USA; negative ionization). The ion chromatograms (m / z 511, 509, 507, 505, and 495) of each extracted sample were compared.

[0141] result Data analysis was performed using MassHunter Acquisition B.06.00 or later versions (Agilent Technologies, Santa Clara, California, USA). An ion equivalent to PdG (m / z 495) was found between retention times of 6.9 and 9.0 minutes (Figure 7). As expected, PdG was found to be the most abundant progesterone metabolite in human urine, although only trace amounts of PdG were found in dogs. An ion corresponding to pregnanetriol glucuronide (m / z 511) was found between RT 5.9 and 8.5 minutes. Human urine samples contained large amounts of 17α-PtG (Figure 8). Both urine samples analyzed contained several unidentified pregnanetriol glucuronides (m / z 511). According to Eriksson and Gustafsson (1970), urine from pregnant women contains at least 5β-pregnane-3α,17α,20α-triol, pregnane-3,16α,20-triol, and 5α-pregnane-3,20α,21-triol. Metabolites with m / z 509 (as pregnanediol-one) and 507 (as pregnane-ol-dione) are shown in Figures 9 and 10. Progesterone metabolism between dogs and humans is clearly significantly different from that of humans.

[0142] Example 3 Identification of the progestogen metabolite 5α-pregnane-3,6,20-triol Progesterone metabolites were identified by examining the production of hydroxyprogesterone metabolism in the ovaries and liver in vitro, and by synthesizing novel reference steroids for biomarker comparison.

[0143] In vitro progesterone metabolism in the ovaries and liver The ovaries of one female dog in the anestrus period and one female dog in the postestrus period were surgically removed during routine sterilization procedures at a veterinary clinic. Written permission for the scientific use of the ovaries was obtained from the owners, and no further harm was inflicted on the dogs. After removal, the ovaries were detached from the sac and frozen at -20°C (transferred to -80°C in 24 hours) or liquid nitrogen (transferred to -80°C in several weeks), depending on the resources of the veterinary clinic. The frozen dog ovaries were weighed, cut into smaller pieces on ice, and the pieces were placed in a centrifuge tube containing ice-bold homogenization buffer (0.1 M Tris-HCl, 1 mM K2EDTA, pH 7.4) and kept on ice during homogenization. Small fragments were coarsely homogenized in short bursts in a disperser (Ultra Turrax®, Janke & Kunkel IKA-Werke TP 18 / 10, 20000 rpm). The volume of homogenization buffer was four times the mass of the ovary (4 ml per gram of tissue). The crude homogenate was transferred in 5 ml batches to homogenization tubes, kept on ice, and thoroughly homogenized (Heidolph overhead stirrer RZR 2, Germany). The homogenate was divided into Beckman Coulter tubes and centrifuged at 10000 g for 15 minutes. The supernatant and S9 fraction were transferred to Eppendorf tubes and kept frozen (below -70°C) until use. After use, residual tissue was disposed of according to the University of Eastern Finland's waste disposal protocol.

[0144] In vitro progesterone hormone metabolism reactions were performed in 100 μl volumes. Progesterone was dissolved in ethanol at a concentration of 5 mg / ml, and 5 μg of steroid was used in the reaction. The reaction conditions were similar to those for glucuronidation with some modifications. In the Phase I reaction, 20 μl of ovarian S9 was used with or without the addition of cofactors (NAD+, NADH, NADP+, NADPH). The reaction time was 2 hours. The Phase II reaction was performed using liver S9 from canid animals, with or without the addition of cofactors and UDPGA. In the Phase I + II reaction, after a 1-hour reaction time with ovarian S9, liver S9 was added with or without UDPGA, and the reaction was stopped after 1 hour. Samples without steroids and / or S9 were used as control samples. Pregnenolone, progesterone, and 6α-, 6β-, 16α, and 17α-hydroxyprogesterone were used as steroid standards in mass spectrometry (LC-qTOF-MS). Furthermore, urine samples were measured during two late estrus periods, and the results were compared with urinary metabolites.

[0145] Synthesis of reference steroids To identify abundant urinary pregnanetriols, several steroids were chemically reduced, enzymatically glucuronidated, and analyzed by mass spectrometry. Unless otherwise specified, all reference steroids were purchased from Steraloids. 5α-pregnane-3β,6α,20α-triol, 5α-pregnane-3β,6α,20β-triol, and 5α-pregnane-3β,6α,20β-triol-6-O-β-glucuronide were ordered by custom synthesis from Toronto Research Chemicals.

[0146] reduction For reference steroid synthesis, 16α-,6α-, and 6β-hydroxyprogesterone (OHP) were reduced by method: 10% Pd / C in THF, 100 psi, stirred overnight. 5β-pregnane-3α,6α-diol-20-one, 5α-pregnane-3β,6α-diol-20-one, and 5α-pregnane-3β,20β-diol-6-one were reduced by the method described in Bournot et al. (1989), except that methanol was used instead of ethanol. Furthermore, 5α-pregnane-3β,20β-diol-6-one was selectively reduced using method: 10% Pd / C in THF, 50 psi, stirred overnight. Pregnenolone (P5,5-pregnen-3β-ol-20-one) was purchased from Sigma and reduced by the method described in Stappenbeck et al. (2012).

[0147] Glucuronidation Reducing steroids, along with 5β-pregnane-3α,6α-diol-20-one, 5α-pregnane-3β,6α,20α-triol, and 5α-pregnane-3β,6α,20β-triol, were enzymatically glucuronidated. 5β-pregnane-3α,6α,20α-triol-20-acetate and 5β-pregnane-3α,6α,20β-triol-20-acetate were glucuronidated using two different methods before and after deacetylation. The steroids were deacylated by adding 10M NaOH and vigorously vortexing (pH 10-11).

[0148] For analytical purposes, glucuronidation was performed in 100 μl volumes as described in Example 1. Reduced pregnenolone was glucuronidated in a larger volume (3 μmol). Glucuronidation and purification were carried out in the same manner as Gufford et al. (2015), with modifications. The steroid was dissolved in DMSO and added to a 50 ml reaction tube. The reaction conditions were similar to those for small-scale glucuronidation, but 0.5% BSA (w / V) and 25 μM aramethicine were added. The reaction was initiated by adding 1.25 mg of CLM, and the reaction tube was incubated for 20-24 hours. Ice-cold methanol (3 times the reaction volume) was used as a stop solution. The glucuronidated steroid hormone was extracted from the reaction solution by solid-phase extraction. Methanol was removed from the reaction solution by evaporating the solution to at least one-tenth of its original volume by a nitrogen gas stream at +50°C. A silica-based C18 solid reversed-phase extraction column (Waters Sep-Pak C18 3 cc Vac cartridge, 500 mg adsorbent per cartridge, 55-105 μm (WAT020805)) was conditioned by discharging 4 ml of methanol, followed by 3 ml of 10% methanol, into ultrapure water. A concentrated glucuronide solution (1-2 ml) was diluted in 1:3 ultrapure water with 10% methanol and loaded onto the column. The column was rinsed with 3 ml of 10% methanol in ultrapure water. The flow-through solution was discarded. Steroid glucuronides were eluted into a glass tube with 50% methanol in ultrapure water. Free steroids and low-polarity glucuronides were eluted with 100% methanol. The eluate was placed in a heating block (+50°C) and the solvent was evaporated in a nitrogen evaporator. The sample was dissolved in methanol to a concentration of approximately 1 mg / ml.

[0149] Analysis of reference steroids by mass spectrometry Commercially available reference steroids were analyzed by mass spectrometry before any treatment was performed on them. After chemical reduction, the steroids were analyzed by mass spectrometry both before and after glucuronidation. The steroids were diluted to 1–5 μM concentrations in 30% acetonitrile and compared with urine samples. Freshly prepared samples were stored in a refrigerator until analysis. Mass spectrometry was performed using either Agilent Qualitative Analysis B.07.00 (or a newer version) or Thermo Xcalibur Qual Browser 4.0 (or a newer version). Steroid analysis was performed by negative and / or positive ionization, and MS / MS spectra were also measured for several steroid molecules.

[0150] result As described in Example 1, glucuronidated synthetic pregnantriol (PtGs, m / z 511) was compared with urine samples collected from two female dogs during the late estrus period. When these urine samples were compared with each other, pregnantriol glucuronides were found to elute in proximity within 6.6–6.7 minutes (PtG1 and PtG2).

[0151] Progesterone in vitro metabolism In vitro studies using ovaries and liver S9 in canids demonstrated that the ovaries and liver possess high 6α-, 6β-, 16α-, and 17α-hydroxylase activity. In the presence of NADPH with progesterone, the ovaries synthesized large amounts of 17α-OHP and small amounts of 6α-, 6β-, and 16α-OHP. 17α-OHP levels significantly decreased upon the addition of liver S9, indicating that liver enzymes effectively metabolize 17α-OHP (Figure 11). Since 5β-pregnane and 3α,17α,20α-triol, common urinary metabolites of 17α-hydroxyprogesterone in many mammals, do not follow the 17α-OHP concentration in canine serum (Example 1), it is highly probable that 17α-OHP metabolized in the liver is excreted in the urine as pregnanediol compounds or pregnanetetrols, rather than as pregnanetriols. Other hydroxyprogesterones did not decrease significantly after the addition of liver S9, likely due to lower concentrations. Unlike ovarian S9, liver enzymes produced only small amounts of 17α-OHP or were directly further metabolized. Instead, larger amounts of 6α-, 6β-, and 16α-hydroxyprogesterones were detected. Furthermore, several unidentified hydroxyprogesterones were produced, although one of them (retention time 8.0 min), primarily produced in the liver, was later identified as 21-OHP. High 6-hydroxylase activity in the ovaries and liver reinforces the recognition that the major urinary progesterone hormone metabolite (e.g., 1) is 5α-pregnane-3,6,20-triol.

[0152] Identification of 5α-PtG using reference steroids 16α-hydroxyprogesterone (16α-OHP) was modified by chemical reduction and glucuronidated with CLM. Theoretically, all stereoisomers can be formed by reduction, yielding any 5-pregnane-3,16,20-triol-glucuronide. When chemically modified 16α-OHP was compared with urine samples from the late estrus period, it was clearly observed that most 16α-OH-based pregnanetriols were less polar than urinary PtG (Figure 12). Urinary PtG is not formed during the reduction and glucuronidation of 16α-hydroxyprogesterone.

[0153] Reduction and glucuronidation of 6β-hydroxyprogesterone (6β-OHP) yielded several PtGs (Figure 13). The corresponding molecules of the synthesized 6β-pregnanetriol glucuronide were not found in dog urine. Reduction and glucuronidation of 6α-hydroxyprogesterone (6α-OHP) yielded very few PtGs, with the most abundant being detected at 6.56 min, which was not found in the urine sample (Figure 14). However, the left-hand peak at 6.56 min and the shoulder of the peak detected at 6.67 min indicate that the urinary PtG is a 6α-pregnanetriol isomer.

[0154] Urine samples were compared to glucuronidated 5β-pregnane-3α,6α-diol-20-one (m / z 509). 5β-pregnane-3α,6α-diol-20-one is a known progesterone metabolite in sows (Jones and Erb, 1968). The urine samples showed two abundant pregnanediol-one structured metabolites that were not consistent with the reference steroid (Figure 15).

[0155] 5β-pregnane-3α,6α,20α / β-triol was synthesized by chemical reduction. Reduction of 5β-pregnane-3α,6α-diol-20-one yielded two reference steroids: 5β-pregnane-3α,6α,20α-triol and 5β-pregnane-3α,6α,20β-triol. The synthesized products were glucuronidated and compared with canine late-estrus urine samples. Furthermore, 5β-pregnane-3α,6α,20α-triol-20-acetate and 5β-pregnane-3α,6α,20β-triol-20-acetate were glucuronidated before and after deacetylation.

[0156] Reduction and glucuronidation of 5β-pregnane-3α,6α-diol-20-one yielded two abundant steroid glucuronides with retention times of 6.55 min and 7.39 min, respectively (Figure 16). The difference between the synthetic pregnanetriol glucuronide and the abundant metabolite was 0.05 min and 0.11 min, respectively. Only trace amounts of 5β-pregnane-3α,6α,20α / β-triol-glucuronide were found in dog urine. The area of ​​the corresponding peak from the abundant urinary PtG metabolite was 0.9–1.1%. 5β-pregnane-3α,6α,20α-triol was measured in the urine of Asian elephants, rabbits, and human neonates (Anderson et al., 1974; Niemuller et al., 1993; Senciall et al., 1990). When 5β-pregnane-3α,6α,20α / β-triol-20-acetate was deacetylated and glucuronidated, CLM formed only one glucuronide, despite the pregnanetriol having three available OH groups (Figure 17). The results indicate that glucuronidation of progesterone metabolites is highly dependent on the arrangement of hydroxyl groups, or that CLM cannot accurately mimic the in vitro natural reaction.

[0157] Reduction of 5α-pregnane-3β,6α-diol-20-one can produce 5α-pregnane-3β,6α,20α / β-triol, which can be glucuronidated from the hydroxyl groups at C3, C6, and C20, providing a total of six steroid criteria. Analysis of the reduced and glucuronidated steroids revealed 5-6 steroids, one of which had the same retention time as PtG1 (Figure 18). The left side of the split peak coincided with urinary PtG1 (RT 6.59 min), while the right side was detected slightly earlier than PtG2 (6.63 min vs. 6.66 min).

[0158] Reduction and glucuronidation of 5α-pregnane-3β,20β-diol-6-one produced three major products (Figure 19). One peak coincided with PtG1, indicating that PtG1 is 5α-pregnane-3β,6α / β,20β-triol. Considering previous results for reduced and glucuronidated 6α-OHP and 5α-pregnane-3β,6α-diol-20-one (see Figures 14 and 18), PtG1 is likely 5α-pregnane-3β,6α,20β-triol-glucuronide.

[0159] 5α-pregnane-3,6,20-triol was synthesized from pregnenolone (P5,5-pregnen-3β-ol-20-one) by reduction and glucuronidated by CLM. The mixture of glucuronidated and free steroids was purified by solid-phase extraction (SPE) with 10%, 50%, and 100% methanol. Two major PtGs were formed during steroid synthesis, which could be separated by SPE. Mass spectrometry showed that the retention times of PtGs in the 50% methanol fraction and in the abundant urinary metabolites were identical (Figure 20). MS2 spectra showed that the synthesized PtGs and urinary metabolites were similar (Table 4). According to the synthetic pathway (Stappenbeck et al., 2012) and all results achieved, the PtG1 metabolite was identified as 5α-pregnane-3β,6α,20β-triol glucuronide.

[0160] [Table 4]

[0161] In PtG identification, different PtG molecules obtained by reduction or present in urine could not be separated for NMR analysis. 6α-OHP, 6β-OHP, 5β-pregnane-3,6,20-trione, and 5α-pregnane-3β,20β-diol-6-one were chemically reduced, and the resulting pregnanetriol mixture and hydrolyzed urine samples were fractionated by preparative HPLC combined with an MS detector. Ionization was performed using positive electrospray ionization. Separation was achieved using a fixed-composition mobile phase system consisting of methanol-ammonium formate (1 mM)-formic acid (50:40:0.1, V / V / V) at a flow rate of 1 ml / min on a Phenomenex Gemini C18 column (150 mm × 21.2 mm, 5 μm), and fractions were collected at 1-minute time intervals.

[0162] The fractions were placed in a heating block (+50°C), and the solvent was evaporated in a nitrogen evaporator. The fractions were dissolved in 30% acetonitrile and analyzed by mass spectrometer (LC-QTOF-MS, m / z 354 [M+NH4]+ and m / z 301 [M-2H2O+H]+) in parallel with hydrolyzed urine samples (from untreated female dogs in the late estrus period) and the original pregnanetriol mixture (from the reduced starting molecules). The results indicated which of the reduced molecules formed the same pregnanetriol molecules found in dog urine.

[0163] Next, the fractions were enzymatically glucuronidated as described above, and subjected to mass spectrometry (LC-QTOF-MS, m / z 511 [MH]-) in parallel with the original glucuronidated pregnantriol mixture (from the reduced starting molecules) and untreated urine samples (from female dogs in the late estrus period). The results show the fractions containing PtG1 and PtG2, and the starting molecules from which they were obtained.

[0164] Based on the results, 5α-pregnane-3β,20β-diol-6-one was selected as the starting molecule, and the reduction product was fractionated into several batches. The fractions were analyzed by LC-MS, and fractions containing pregnanetriols with the same retention time were pooled. The pregnanetriol pools were glucuronidated and analyzed by LC-MS before and after glucuronidation. None of the pools contained PtG2. NMR analysis of the pool containing PtG1 molecules showed that the glucuronide was attached to the 6th carbon of the pregnanetriol.

[0165] The glucuronidated custom molecule 5α-pregnane-3β,6α,20α-triol and 5α-pregnane-3β,6α,20β-triol were compared with urine samples from two female dogs. The results showed that the major peaks of 5α-pregnane-3β,6α,20β-triol glucuronide and PtG1 were identical (Figure 21). NMR analysis of 5α-pregnane-3β,6α,20β-triol glucuronide confirmed that the glucuronide was attached to the 6 carbon atoms of the pregnane triol.

[0166] A custom-made 5α-pregnane-3β,6α,20β-triol-6-O-β-glucuronide was compared to urine samples from two female dogs, one with and one without a reference steroid (Figures 22-23). ​​The results showed that both the reference and PtG1 were detected with the same retention time. The spiked samples showed a linear increase in peak area. PtG1 was confirmed to be 5α-pregnane-3β,6α,20β-triol-6-O-β-D-glucuronide. Based on a similar excretion profile to PtG1, PtG2 is likely a stereoisomer of PtG1.

[0167] Example 4 Quantitative measurement of 5α-pregnane-3,6,20-triol (5α-PtG) by LC-HRMS A novel urinary biomarker for circulating progesterone was identified as 5α-pregnane-3β,6α,20β-triol-6-O-β-D-glucuronide. LC-HRMS and enzyme immunoassay methods were developed to measure the amount of urinary progestogen metabolites in dogs.

[0168] Dispensed urine sample series from 8 female dogs, and individual samples from 89 female dogs in estrus or post-estrus, were thawed at room temperature, vigorously vortexed, and centrifuged at 13400 rpm for 5 minutes. The samples were diluted 1:3 with ultrapure water and mixed with 200 nM diclofenac in acetonitrile 1:1 (dilution water: ACN). The urine sample solutions were filtered into 96-well plates by centrifugation (2300 rpm, 5 minutes) into 1 ml deep-well plates and sealed with silicone well caps. A standard dilution series (1 to 1000 nM) of 5α-pregnane-3β,6α,20β-triol-6-O-β-D-glucuronide was prepared in 30% ACN containing 100 nM diclofenac. Standard urine samples were spiked with 5α-pregnane-3β,6α,20β-triol-6-O-β-D-glucuronide. 30% ACN was used as a blank control, and pooled urine samples were used as a quality control. Urine samples were analyzed using a Thermo Scientific Q Exactive Quadrupole-Orbitrap high-resolution MS / MS system with a Thermo Scientific Vanquish Binary Flex Binary 1000 bar UHPLC system (using a method similar to that used for non-targeted analysis). Two closely eluted isomers (m / z 511 [MH]-) were found in the extracted ion chromatograms at a retention time of 6.7 minutes. Both metabolites were used for analysis and estrus monitoring. Semi-quantitative analysis of PdG was performed by comparing the peak areas in the extracted ion chromatograms of PdG (m / z 495) and 5α-PtG (m / z 511).

[0169] Quantitative data analysis was performed using the TraceFinder 5.0 application. The results were normalized by dividing the integrated area of 5α-PtG by the area of diclofenac and calculated using the equation of the standard curve (nM).

[0170] Results In mass spectrometry by Thermo Scientific Q Exactive Quadrupole-Orbitrap high-resolution MS / MS, progesterone was revealed to be metabolized as several different metabolites including two overlapping and proximate elution isomers of pregnanetriol glucuronide, and the first elution peak (PtG1) corresponded to 5α-PtG. When 5α-PtG was added to urine samples, the increase in PtG1 was linear, confirming that the steroids were identical. Due to proximate elution and similar profiles, the metabolites were assumed to be stereoisomers of each other and are collectively referred to as PtG in these results.

[0171] The excretion rates of these metabolites vary among individuals, but all female dogs excrete both PtG1 (5α-PtG) and PtG2, and both PtGs follow the profile of serum progesterone. These metabolites can be used for estrus monitoring either as individual metabolites or by summing the metabolites. Mass spectrometry measurements at lower resolution showed only one peak for both PtGs. Thus, these results are presented as the sum of PtG1 and PtG2.

[0172] According to quantitative measurements, the average concentration of 5α-PtG in urine (total of PtG1 and PtG2) increased to a concentration of 2.5 μM on day 4 after ovulation and showed a profile similar to serum progesterone (serum progesterone analysis was measured and reported by the dog owners). The results suggest that progesterone changes can be detected from urine samples at least the next day (r 2=0.9628) (Figure 24). Comparing the mean concentration before the estimated LH peak (2 days - from the estimated start of ovulation) with the increase in PtG (%), 5α-PtG concentration increased by more than 100% from the start of the LH peak to the estimated start of ovulation. Over a 6-day period (from day -2 to day +4), the concentration increased by 800%.

[0173] The superiority of this new biomarker, 5α-PtG, was demonstrated by semi-quantitatively comparing it to the 20β-stereoisomer of PdG, the most common progesterone metabolite (Figure 25A). In dogs, PdG is primarily metabolized as the 20β form (PdGβ). These results suggest that PdG 20β excretion is similar to PtG excretion (Figure 25B). However, due to its low concentration, it is not a suitable biomarker for progesterone excretion. On average, in eight female dogs, the area of ​​PdGβ (extracted ion chromatogram) was only 4.3% of the area of ​​5α-PtG. The amount of PdGβ varied among individuals. The average percentage (area of ​​PdG / 5α-PtG × 100%) in individuals ranged from 0.3 to 17.3.

[0174] Example 5 Quantitative measurement of 5α-PtG by enzyme immunoassay Imject® cultured keyhole limpet hemocyanin (mcKLH) in PBS, Imject® bovine serum albumin (BSA) in PBS, and Imject® cBSA immunomodulator (cBSA) in PBS were used as carrier proteins in immunization, antibody screening, and urinalysis. Hapten-protein conjugation was performed using EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodimide hydrochloride) and sulfo-NHS (N-hydroxysulfosuccinimide sodium salt). Both EDC and sulfo-NHS were dissolved in MES buffer (0.1M 2-(N-morpholino)-ethanesulfonic acid (0.9% NaCl, pH 4.6)) immediately before use. The antigen (5α-PtG, 5α-pregnane-3β,6α,20β-triol-6-O-β-D-glucuronide) was dissolved in DMSO to a concentration of 10 mg / ml (19.5 mM).

[0175] For conjugation, 500 mol excess and 50 mol excess 5α-PtG were used for KLH and (c)BSA conjugations, respectively. The molar concentration of EDC was 1.5 times that of 5α-PtG, and the sulfo-NHS concentration was 1.2 times that of 5α-PtG. Freshly dissolved sulfo-NHS was added to a glass vial containing 5α-PtG and vortexed thoroughly. Steroid activation was initiated by adding fresh EDC solution and vigorous vortexing. A reaction solution consisting of DMSO and MES buffer in a 1:1 ratio was incubated in a plate shaker (300 rpm) at room temperature for 1 hour. After activation, the reaction solution was slowly added to the protein solution, gently vortexed, and incubated in a plate shaker (300 rpm) for 2 hours. The protein conjugates were purified by centrifugal filtration (30k MWCO for KLH and 10k MWCO for BSA conjugates). The unconjugated steroid, conjugation buffer, and DMSO were removed by centrifugation (4000 rpm, 10-20 minutes), and the protein concentrate was washed three times with 5 ml of PBS (pH 7.4). The conjugate was diluted with PBS to a concentration of 1-5 mg / ml and stored in the refrigerator until use.

[0176] Monoclonal mouse 5α-PtG antibodies were prepared against 5α-PtG-KLH by Thermo Fisher Scientific (Rockford, Illinois, USA). The antibodies were screened against 5α-PtG-BSA. The cross-reactivity of the antibodies was as follows: 100% for 5α-pregnane-3β,6α,20β-triol glucuronide, 25.4% for 5α-pregnane-3β,6α,20β-triol, 0.5% for 5β-pregnane-3α,20β-diol, and 0.5% for 5α-pregnane-3β,6α-diol-20-one. No cross-reactivity was found with 5α-pregnane-3β,6α,20α-triol, 5β-pregnane-3α,20α-diol-3-glucuronide, 5β-pregnane-3α,20α-diol, 5β-pregnane-3α,17α,20α-triol, 5β-pregnane-3α,17α,20β-triol, 5α-pregnane-3β,17α,20β-triol, or progesterone.

[0177] 96-well polystyrene, MaxiSorp® plates were coated with antigen (5α-PtG-cBSA) by adding 100 μl of 0.25 μg / ml antigen to coating buffer (0.01 M sodium bicarbonate, pH 9.5) in each well. The plates were covered with plastic seals to prevent evaporation, protected from light, and incubated overnight at room temperature or at +4°C for several nights. The wells were emptied, washed once with 250 μl of general washing buffer (e.g., PBS (PBST) containing 0.05% Tween 20), filled with 200 μl of blocking buffer (5% sucrose and 0.5% BSA in PBST (w / V)), covered, and incubated at +37°C for 30 minutes. The contents of the plates were drained and emptied, and dried at +37°C for 3 hours. The dried plates were covered with plastic seals, packed in a resealable plastic bag containing silica beads, and stored at +4°C until use.

[0178] Hormone assays were performed on individual samples collected at different estrous cycle phases and on urine sample series from 60 female dogs. 5α-PtG-cBSA coated plates were washed once with 250 μl of PBST. Urine samples were diluted 1:20 with assay buffer (0.2% BSA (w / V) in PBS containing 0.01% Triton-X) and added to sample wells (50 μl). A standard dilution series was prepared by diluting 5α-PtG in assay buffer and added to standard wells (50 μl; 1200~1.6 nM). Maximum binding capacity (B0) was determined by adding additive-free assay buffer to two wells. A 1900 nM 5α-PtG control was diluted 1:20 with assay buffer and used as a recovery control in all assays. All samples and standards were measured twice in a repeated manner. A stock dilution of monoclonal mouse 5α-PtG antibody (1:100) was further diluted 1:100 with assay buffer, and 50 μl was added to each well. The plate was covered and incubated at +37°C for 30 minutes, after which the plate was washed three times. Horseradish peroxidase-labeled secondary antibody (goat anti-mouse IgG HRP) was diluted 1:20000 in 0.5% BSA (w / V) in PBST, and 100 μl was added per well, and incubated at +37°C for 30 minutes. After washing the plate three times with PBST, TMB (0.5% 3,3',5,5'-tetramethylbenzidine (w / V; TCIAT1023 Tokyo Chemical Industry)) in DMSO solution was diluted 1:50 with substrate buffer (0.1M sodium acetate trihydrate, 1.5mM citrate monohydrate, 0.005% hydrogen peroxide (30%)) and added to each well (100 μl), and incubated at room temperature for 30 minutes. The reaction was stopped by adding 50 μl of 1M sulfuric acid to each well. Absorbance was measured at 450 nm using a microplate reader (Hidex Sense).

[0179] The results were calculated using a 4-parameter logistic curve of the standard dilution series. 4PL curve fitting was performed using AssayFit v 1.4.1, and the results were calculated using the following formula:

number

[0180] result Progestogen metabolite concentrations were quantified by enzyme immunoassay (EIA). A total of 60 urine samples were measured by 5α-PtG EIA. The results showed that 5α-PtG concentrations were low during the anestrus and proestrus periods, and the average concentration began to increase approximately one week after the onset of vaginal discharge, reaching a maximum concentration over a period of three weeks (Figure 26). The correlation coefficient between urinary 5α-PtG concentrations and serum progesterone profiles (n=27 female dogs, Figure 27) was 0.988.

[0181] The results of 5α-PtG measurement are remarkable. This novel, previously unknown metabolite can be used as a biomarker for serum progesterone, which is actually used in studies to monitor the phases of the estrous cycle in dogs. The novel urinary biomarker provides a non-invasive method for monitoring the phases of the estrous cycle in female dogs. The results are promising, and the method can be easily adapted into a practical form.

[0182] Example 6 Quantitative measurement of 5α-PtG by capture enzyme immunoassay Urinary 5α-PtG was measured by an enzyme immunoassay using a capture antibody. Unless otherwise noted, the reagents, buffers, and analytical methods were the same as in Example 5. This assay provides an alternative to antigen-based EIA. For horseradish peroxidase (HRP) conjugation, 5α-PtG was conjugated to HRP (Thermo Scientific, 31490) using a process similar to that of 5α-PtG-BSA conjugation (Example 5), but the molar concentration of 5α-PtG was 8 times that of HRP. 96-well polystyrene plates (Thermo Scientific) were coated by adding 100 μl of 1 μg / ml anti-mouse IgG (GtxMu-003-D, goat anti-mouse IgG (H&L), ImmunoReagents) in coating buffer in each well. The plates were covered with plastic seals to prevent evaporation, protected from light, and incubated overnight at room temperature or for several nights at +4°C. The wells were emptied and washed three times with 250 μl of general washing buffer immediately before use.

[0183] A stock dilution of monoclonal mouse 5α-PtG antibody was diluted 1:20000 with assay buffer, and 100 μl was added to each well. The plate was covered and incubated at +37°C for 30 minutes, after which the plate was washed three times. A standard dilution series was prepared by diluting urine samples 1:20 with assay buffer and diluting 5α-PtG with assay buffer.

[0184] Horseradish peroxidase-labeled 5α-PtG was diluted 1:10000 in 0.5% BSA (w / V) in PBST and added to diluted urine samples and standards (1:1). 100 μl of the mixture of urine sample / or standard and HRP-labeled 5α-PtG was added to a well plate, and the plate was incubated at +37°C for 30 minutes. After washing the plate three times with PBST, TMB in DMSO solution was diluted 1:50 with substrate buffer and added to each well (100 μl), and incubated at room temperature for 30 minutes. The reaction was stopped by adding 50 μl of 1 M sulfuric acid to each well. Absorbance was measured at 450 nm using a microplate reader.

[0185] Example 7 Quantitative measurement of 5α-PtG by surrogate scavenging enzyme immunoassay This immunoassay is based on the method used in the capture EIA in Example 6. Unless otherwise noted, the reagents, buffers, and analytical methods were the same as in Examples 5 and 6. This assay is a simplified version of the capture EIA.

[0186] A 96-well polystyrene plate (Thermo Scientific) was coated by adding 100 μl of 0.2 μg / ml monoclonal 5α-PtG antibody IgG to the coating buffer in each well. The plate was covered with a plastic seal to prevent evaporation, protected from light, and incubated overnight at room temperature or at +4°C for several nights. The wells were emptied and washed three times with 250 μl of general washing buffer immediately before use.

[0187] A standard dilution series was prepared by diluting urine samples 1:20 with assay buffer and 5α-PtG with assay buffer. Horseradish peroxidase-labeled 5α-PtG was diluted 1:10000 in 0.5% BSA (w / V) in PBST and added to the diluted urine samples and standards (1:1). 100 μl of a mixture of urine sample / or standard and HRP-labeled 5α-PtG was added to a well plate, and the plate was incubated at room temperature for 1 hour. After washing the plate three times with PBST, TMB in DMSO solution was diluted 1:50 with substrate buffer and added to each well (100 μl), and incubated at room temperature for 30 minutes. The reaction was stopped by adding 50 μl of 1 M sulfuric acid to each well. Absorbance was measured at 450 nm using a microplate reader. Example 8 Qualitative analysis of 5α-pregnanetriol glucuronide by immunoaffinity chromatography and LC-HRMS.

[0188] Antibody cross-reactive steroids were analyzed by immunoaffinity chromatography. Cyanogen bromide (CNBr)-activated Sepharose® 4B (1 g, Cytiva 17-0430-01, Uppsala, Sweden) was suspended and washed multiple times with 1 mM HCl (200 ml, Sigma-Aldrich 30721). The Sepharose medium was transferred to a PD10 column and rinsed five times with coupling buffer (0.1 M NaHCO3, 0.5 M NaCl, pH 8.4). 3 mg of 5α-PtG antibody was diluted 1:5 with coupling buffer and added to the column. The sealed column was incubated at room temperature for 4 hours with gentle shaking. The column was rinsed twice with 5 ml of coupling buffer, blocked by adding 10 ml of blocking buffer (0.1 M Tris-HCl, pH 8) and incubated at room temperature for 2 hours. The column was washed with alternating pH cycles for 3 cycles. First, the column was rinsed with 5 ml of acid buffer (0.1 M sodium acetate, 0.5 M NaCl, pH 4), followed by 5 ml of alkaline buffer (0.1 M Tris-HCl, 0.5 M NaCl, pH 8). The prepared column was rinsed five times with 5 ml of PBS, followed by 10 ml of 20% ethanol. After draining several milliliters of 20% ethanol, the column was sealed and stored at +4°C.

[0189] Before use, the a-5α-PtG column was rinsed three times with 5 ml of PBS. A urine sample (3 ml) was diluted with 2 ml of PBS and added to the sealed column. The column was incubated at room temperature for 30 minutes with gentle shaking. After incubation, the sample was drained and the eluate was collected for the 5α-PtG EIA test. The column was rinsed four times with 5 ml of PBS, and the cross-reactive steroids were eluted with 80% methanol to separate the fractions (12 fractions in total, 1 ml each). After elution, the column was rinsed three times with 5 ml of PBS and stored in 20% ethanol as before.

[0190] The fraction was dried in a centrifugal evaporator (50°C) and suspended in 5% methanol (100 μl). The fraction was diluted 1:50 with sample buffer and tested by 5α-PtG EIA as described in Example 5. The fraction that showed the most inhibition by EIA was transferred to a glass vial and analyzed by liquid chromatography-mass spectrometry. Non-targeting analysis of pregnanetriol glucuronide was performed using a Thermo Scientific Vanquish Binary Flex Binary 1000 bar UHPLC system as described in Example 1.

[0191] As a result, 5α-PtG in urine samples can be extracted by immunoaffinity chromatography and detected by LC-HRMS (EIC m / z 511). No other significant cross-reactive pregnanetriol glucuronides were detected.

[0192] Example 9 Analysis of hydrolyzed urinary steroids Urinary steroid glucuronides were hydrolyzed before analysis. The centrifuged urine samples were diluted 1:3 with 75 mM KH2PO4 (pH 6.8), and 250-1250 units of IX-A type β-glucuronidase enzyme (Sigma G7396) were added. Hydrolysis was carried out at +37°C for 20 hours, and unconjugated steroids were extracted by SPE. The SPE method was the same as in Example 1 (sample preparation by solid-phase extraction), but unconjugated steroids were eluted with 100% methanol. The hydrolyzed urine samples could be used for estrus detection by LC-MS analysis, as described in Examples 1 and 2. Unconjugated pregnantriols were detected by positive ionization at m / z 301[M-2H2O+H]+ and m / z 354[M+NH4]+. Unconjugated pregnantriols could be measured by 5α-PtG EIA, as in Example 5.

[0193] Example 10 Rapid urine test for 5α-PtG detection Lateral flow chromatography was used to test the difference in urinary 5α-PtG concentration during different phases of the estrous cycle in dogs. This rapid test provides a quick and cost-effective method for detecting progestogen levels in dogs without requiring specialized equipment or laboratory facilities.

[0194] The lateral flow immunochromatography test (Figure 28) includes a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorption pad attached to a backing card and cut into 4 mm wide test strips. 5α-PtG antibody conjugated to gold particles is dried on the conjugate pad. The 5α-PtG-BSA conjugate is immobilized on the nitrocellulose membrane as the test line, and anti-mouse IgG antibody is immobilized on the nitrocellulose membrane as the control line.

[0195] In the rapid test, the sample is placed on a sample pad and flows through the test strip by capillary force. On the conjugate pad, 5α-PtG molecules in the sample bind to 5α-PtG antibodies conjugated to gold nanoparticles, and the formed complex moves to the membrane. If only a small amount of 5α-PtG is present in the sample, the free antibody-gold nanoparticles flow through the membrane and bind to 5α-PtG-BSA in the test line, generating a visible signal. If 5α-PtG molecules in the sample bind to the flowing antibody-gold nanoparticles, the antibody does not interact with the 5α-PtG-BSA conjugate, and no visible test line is formed. The remaining antibody-gold particles bind to anti-mouse IgG in the control line, generating a visible control line that confirms the fluid has successfully passed through the sample pad and test line to reach the control line. Since the intensity of the test line depends on the amount of antigen present in the sample, estimation of the estrous cycle phase can be achieved by simple visual assessment. Excess sample is eventually absorbed into the absorption pad, which also contributes to the capillary force on the test strip.

[0196] Urine samples were tested without pre-preparation. In the pilot study, 80 μl of urine sample was applied to the sample well. The intensity of the test line was estimated, and the presence of the control line was confirmed 20 minutes after sample application. Urine samples from the anestrus period containing only a small amount of 5α-PtG provided a test line of similar intensity to the control line. Urine from the postestrus period with a high concentration of 5α-PtG provided a test line that was barely visible. If the intensity of the test line in the anestrus sample was judged as 100%, the intensity of the test line in the proestrus period was approximately 90%, in the estrus period 50%, and in the postestrus period 10%.

[0197] References JPEG2026510817000009.jpg48158 JPEG2026510817000010.jpg253157

Claims

1. A compound characterized by containing a 5α-pregnane-3,6,20-triol isomer or a glucuronidated form of the 5α-pregnane-3,6,20-triol isomer.

2. The compound according to claim 1, characterized in that the compound comprises a 5α-pregnane-3,6,20-triol isomer.

3. The compound according to claim 1 or 2, characterized in that the compound is selected from the group consisting of 5α-pregnane-3α,6α,20α-triol, 5α-pregnane-3β,6α,20α-triol, 5α-pregnane-3α,6β,20α-triol, 5α-pregnane-3α,6α,20β-triol, 5α-pregnane-3β,6α,20β-triol, 5α-pregnane-3α,6β,20β-triol, and 5α-pregnane-3β,6β,20β-triol.

4. The compound according to any one of claims 1 to 3, characterized in that the compound comprises a 5α-pregnane-3,6,20-triol glucuronide isomer.

5. The compound according to any one of claims 1 to 4, characterized in that the compound is selected from the group consisting of 5α-pregnane-3α,6α,20α-triol glucuronide, 5α-pregnane-3β,6α,20α-triol glucuronide, 5α-pregnane-3α,6β,20α-triol glucuronide, 5α-pregnane-3α,6α,20β-triol glucuronide, 5α-pregnane-3β,6α,20β-triol glucuronide, and 5α-pregnane-3β,6β,20β-triol glucuronide.

6. The compound according to any one of claims 1 to 5, characterized in that the compound is selected from the group consisting of 5α-pregnane-3,6,20-triol-3-O-α-D-glucuronide, 5α-pregnane-3,6,20-triol-6-O-α-D-glucuronide, 5α-pregnane-3,6,20-triol-20-O-α-D-glucuronide, 5α-pregnane-3,6,20-triol-3-O-β-D-glucuronide, 5α-pregnane-3,6,20-triol-6-O-β-D-glucuronide, or 5α-pregnane-3,6,20-triol-20-O-β-D-glucuronide.

7. Use of the compound according to any one of claims 1 to 6 in determining a progestogen or multiple progestogens in an in vitro urine sample derived from a non-human animal.

8. The use according to claim 7, characterized in that the compound is a 5α-pregnane-3,6,20-triol isomer.

9. The use according to claim 7, characterized in that the compound is a 5α-pregnane-3,6,20-triol glucuronide isomer.

10. The use according to any one of claims 7 to 9, characterized in that the non-human animal is selected from a group of canids consisting of domestic dogs, wild dogs, wolves, coyotes, foxes, bush dogs, short-eared dogs, jackals, and raccoons.

11. An in vitro method for determining a progestogen or multiple progestogens in a urine sample derived from a non-human animal, wherein the method is A process for providing in vitro urine samples derived from non-human animals, A step of measuring the amount of 5α-pregnane-3,6,20-triol isomer or 5α-pregnane-3,6,20-triol glucuronide isomer in the sample, The step includes determining the presence of theca cells and / or granulosa cells of a luteinized follicle based on the amount of 5α-pregnane-3,6,20-triol isomer or 5α-pregnane-3,6,20-triol glucuronide isomer in the sample, A method characterized in that the progestogen level in the sample is indicated by the presence of theca cells and / or granulosa cells of a luteinized follicle.

12. The method according to claim 11, characterized in that the animal is selected from a group of canids consisting of domestic dogs, wild dogs, wolves, coyotes, foxes, bush dogs, short-haired dogs, jackals, and raccoons.

13. The method according to claim 11 or 12, characterized in that the measurement is performed using a method selected from the group consisting of binding assays, immunoassays, mass spectrometry, nuclear magnetic resonance, and chromatography.

14. A kit for determining progestogens or multiple progestogens in urine samples derived from non-human animals, wherein the kit is An immunoassay method for measuring the amount of 5α-pregnane-3,6,20-triol isomer or 5α-pregnane-3,6,20-triol glucuronide isomer in an in vitro urine sample derived from a non-human animal, A manual for determining a progestogen or multiple progestogens according to the above method, A process for providing in vitro urine samples derived from non-human animals, A step of measuring the amount of 5α-pregnane-3,6,20-triol isomer or 5α-pregnane-3,6,20-triol glucuronide isomer in the sample, The instructions include a step of determining the presence of theca cells and / or granulosa cells of a luteinized follicle based on the amount of 5α-pregnane-3,6,20-triol isomer or 5α-pregnane-3,6,20-triol glucuronide isomer in the sample, A kit characterized in that the progestogen level in the sample is indicated by the presence of theca cells and / or granulosa cells of a luteinized follicle.

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