US20180067116A1

US20180067116A1 - Method for detecting products derived from glucuronide metabolites with the enzyme b-glucuronidase, and a reagent comprising said enzyme

Info

Publication number: US20180067116A1
Application number: US15/442,607
Authority: US
Inventors: Manuel ROZAS ANDREU; Daniel E. ALMONACID CORONADO
Original assignee: La Piedra Biotecnologia SpA
Current assignee: La Piedra Biotecnologia SpA
Priority date: 2016-08-26
Filing date: 2017-02-24
Publication date: 2018-03-08
Also published as: US20200116719A1; GB2553142A; GB201614546D0; GB2553142B; US20230140113A1

Abstract

The present invention discloses a method and a highly efficient reagent to detect products derived from glucuronide metabolites presents in a sample, comprising the steps of adding to said sample an enzyme with β-glucuronidase activity originated from genus Brachyspira or any variant or mutant derived thereof; incubating the sample with the enzyme during a determined period of time; and detecting the product derived from glucuronide metabolite by means of a suitable technique. The reagent of the present invention comprises an enzyme with β-glucuronidase activity originated from genus Brachyspira or any variant or mutant derived thereof; and a suitable vehicle.

Description

TECHNICAL FIELD

The present invention is related with the technical field of biotechnology, and particularly provides a method for detecting glucuronide derivatives present in a sample, using for this purpose a β-glucuronidase enzyme from bacterial origin, as well as a reagent including said enzyme.

BACKGROUND OF THE INVENTION

Conjugation of endogenous and exogenous compounds with D-glucuronic acid is a metabolic pathway commonly known in animals and humans, which is normally considered as a process for detoxification of organisms. Through this process, glucuronide conjugates or metabolites are produced, which are excreted through urine or bile (Boppana V et al, J. Pharm. Sci. 1989. Vol 78(2):127-131). Compounds that are excreted in these ways are mainly lipophilic, such as bilirubin, androgens, glucocorticoids, among others, and xenobiotic substances such as opioids, cannabinoids, benzodiazepines, among others.
Detection of these glucuronide metabolites in biological fluids allows identification and quantitation of endogenous or exogenous compounds present in the organism. Said detection can be performed by means of chemical hydrolysis (acid hydrolysis) or by means of enzymatic hydrolysis of said glucuronide metabolites. Chemical hydrolysis is performed with an acid which hydrolyses most of conjugates in a relative reduced period of time (from 60 to 90 minutes) and at a low cost. However, the acid conditions and temperature required in this process degradate sensitive compounds, for example, benzodiazepines. Moreover, working with acids in the laboratory requires stricter security conditions, producing corrosion of vulnerable equipment. For this reason, testing laboratories for these purposes mainly employ enzymatic hydrolysis, consisting in the use of β-glucuronidases for hydrolyzation of glucuronide metabolites in conditions more manageable than acid hydrolysis.
Currently there exist diverse β-glucuronidase enzymes available from different origins, each one with optimum conditions for hydrolysis of metabolites. Thus, parameters such as concentration, pH, incubation period and temperature of reaction vary depending on the enzyme used.
For example, there exist alternatives of enzymes obtained from molluscs such as a β-glucuronidase enzyme originated from Patella vulgata described in document U.S. Pat. No. 4,473,640 of Combie et al, which requires a temperature between 60 and 70 degrees Celsius, pH 5, and at least one hour to obtain only 60% of hydrolysis of the glucuronide metabolite, and approximately three hours as the optimum time to reach complete quantification of the hydrolyzed metabolite. There also exist β-glucuronidase enzymes described from species Helix pomatia, Helix aspersa, Haliotis rufescens, among others (WO 95/16050, Scheinmann et al; Kemp et al, FAA. 2015. Report No. DOT/FAA/AM-15/6). Generally, preparations of enzymes obtained from molluscs tend to have more contaminant substances that may interfere with further analysis of glucuronide metabolites.
On the other hand, there also exist alternatives to enzymes from bacterial origin, such as β-glucuronidase from Escherichia coli. Comparative studies show that at an optimum pH and depending on the type of glucuronide metabolite/substrate, it is required an hour of incubation to obtain 40-80% of hydrolysis, and between three to five hours to reach a complete hydrolysis (Wakabashi et al, J Biol. Chem. 1961. Vol 236(4):996-1001). These detection times are excessive for testing laboratories which analyze drugs in biological samples, and the situation is even more dramatic in cases such as analysis of natural opioids (codeine and morphine) and semi-synthetic opioids (hydromorphone, oxycodone and oxymorphone). The hydrolysis percentage of conjugated metabolites of these opioids widely varies depending on the origin of the enzyme used: after an incubation period of two hours of the biological sample with β-glucuronidase from Patella vulgata, only a 21° A of codeine and a 64% of morphine are recovered; an 11° A of codeine and a 35% of morphine are obtained when β-glucuronidase from Helix pomatia is used; and only a 9% of codeine and morphine are recovered when β-glucuronidase from Escherichia coli is used (Wang et al., J Anal. Toxicol. 2006. Vol 30:570-575). For this reason, diverse researchers seek enzymes capable of reduce the time needed to hydrolyze glucuronide metabolites. The recently published patent application US 2016/0090582 discloses a recombinant β-glucuronidase enzyme from E. coli (IMCSzyme™), which, according to the applicant, detects the presence of drugs in biological samples in 30 minutes or less, whose specific activity is three times higher than wild type enzyme. However, in the examples section it is shown that 60 minutes are required to incubate the mutant enzyme with a sample containing codeine-6-glucuronide at 55 degrees Celsius to recover 93.5% of codeine.
Therefore, new β-glucuronidase enzymes are needed to achieve quick hydrolysis of glucuronide metabolites and to obtain high recovery percentages of their derivatives, in order to implement a highly efficient method for detection and quantification of the same.

SUMMARY OF INVENTION

The present invention discloses a method to detect products derived from glucuronide metabolites in a sample, comprising the steps of:

- a) providing the sample in which said product is to be detected,
- b) adding to said sample an enzyme with β-glucuronidase activity from genus Brachyspira or any variant or mutant derived thereof,
- c) incubating the sample with the enzyme for a determined period of time; and
- d) detecting the product derived from said glucuronide metabolite through any suitable technique.

This method allows to detect any product derived from glucuronide metabolites, whether from natural, semi-synthetic or synthetic origin. In a preferred embodiment, said derived products are selected from the group consisting of an opioid, an opiate, a cannabinoid, a benzodiazepine or any derivative thereof.
In a preferred embodiment, the sample from which these products derived from glucuronide metabolites are detected, is selected from the group consisting of saliva, whole blood, plasma, urine, hair, skin, teeth, soft tissues, meconium, vitreous humor, water and food.
The enzyme with β-glucuronidase activity of the present invention, whose amino acid sequence is shown in SEQ. ID. NO.1, preferably originates from species Brachyspira pilosicoli. The invention also includes any derived enzyme or mutant thereof, provided that it maintains its β-glucuronidase activity. Preferably, the mutant enzyme with β-glucuronidase activity shares at least an 80% of identity with the sequence defined in SEQ. ID. NO.1.
In another preferred modality of the method of the present invention, incubation period of sample with the enzyme is of at least one minute, preferably between 2 and 120 minutes, and even more preferably between 3 and 30 minutes. Incubation is performed in a range of temperature between 20 and 60 degrees Celsius, preferably between 50 and 55 degrees Celsius, and in a pH range between 4.0 and 9.0, preferably between 6.5 and 7.5.
Detection of products derived from glucuronide metabolites is performed through any suitable analytical technique, preferably selected from the group consisting of liquid chromatography (LC) or gas chromatography (GC), high performance liquid chromatography (HPLC), followed by mass spectrometry (LC-MS, or GC-MS), or high resolution mass spectrometry (LC-HRMS, HPLC-HRMS), or tandem mass spectrometry (LC-MS-MS, GC-MS-MS, HPLC-MS-MS) or any other suitable technique or combination of detection methods.
A second object of the present invention is a reagent for detection of products derived from glucuronide metabolites in a sample, which comprises an enzyme with β-glucuronidase activity originated from genus Brachyspira or any derived enzyme or mutant thereof; and an appropriate vehicle. Preferably, the reagent contains an enzyme with β-glucuronidase activity originated from species Brachyspira pilosicoli, whose amino acid sequence is shown in SEQ. ID. NO.1, or any enzyme derived from said sequence maintaining β-glucuronidase activity. Preferably, the derived or mutant enzyme with β-glucuronidase activity shares at least an 80% of identity with the sequence defined in SEQ. ID. NO.1.
In a preferred embodiment, the reagent contains the enzyme in a suitable vehicle which is selected from the group consisting of water, salts, buffer solutions, Tris, HEPES, DTT, citric acid, EDTA, glycerol, sugars, amino acids or a combination thereof. Salts are preferably selected from the group consisting of sodium phosphate, potassium phosphate, sodium carbonate, sodium acetate, sodium citrate, sodium chloride and potassium chloride. At the same time, sugars are selected from the group consisting of sorbitol, trehalose, sucrose, glucose, lactose, mannitol and raffinose.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows vector pET-28a with the optimized nucleotide sequence encoding β-glucuronidase enzyme originated from B. pilosicoli to be expressed in E. coli.

FIG. 2 shows an SDS-PAGE gel of β-glucuronidase enzyme purified with column Ni-NTA.

FIG. 3 shows recovery of codeine and morphine versus time, using β-glucuronidase from B. pilosicoli.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for detection of products derived from glucuronide metabolites present in a sample, comprising the use of a new β-glucuronidase enzyme originated from Brachyspira genus. Hydrolysis efficiency of said enzyme is widely superior than all β-glucuronidases currently known, with a specific activity 6.6 times higher than mean specific activity of a β-glucuronidase obtained from E. coli. This allows to detect products derived from glucuronide metabolites in less than 15 minutes with a high recovery rate.
All the technical and scientific terms used to describe the present invention have the same meaning understood by any person with basic knowledge in the field under review. Nevertheless, to define more clearly the scope of the invention, a list of terminology used in this description is given below.
It should be understood that as used herein, the term “glucuronide metabolite” refers to any compound conjugated with glucuronic acid through a glycosidic bond. It is to be understood that a glucuronide metabolite can be natural or synthetic, and produced by chemical or enzymatic methods. The terms “conjugated compound”, “conjugated metabolite”, “glucuronide compound”, and “glucuronide substrate” are indistinctly used to describe the technical characteristics of the present invention.
The term “products derived from glucuronide metabolites” or “recovered analytes” should be understood as the molecule or original compound that is produced after hydrolysis or glycosidic bond cleavage of a glucuronide metabolite. These derived products or original compounds can be natural or synthetic, and produced by chemical or enzymatic methods.
The term “enzyme” should be understood as a sequence of amino acids which form a functional protein of interest for the present invention. It is to be understood that the present invention is not related with enzymes in their natural state, but it relates to proteins in an isolated state, purified or partially purified, or recombinant, obtained by any method of genetic engineering known in the state of the art. Likewise, it is to be understood that the term “mutant” or “mutant enzyme” refers to a modified protein derived from the original amino acid sequence maintaining its β-glucuronidase enzymatic activity; and sharing at least 70%, preferably 80% and more preferably 90% of identity with the original sequence.
It is to be understood that the term “identity” among amino acid sequences refers to the percent of identical amino acids that the compared sequences share among them, in a particular sequence alignment window. The percentage of identity can be calculated using a sequence comparison algorithm or by manual alignment together with visual inspection. For example, sequences and percentages of identity can be obtained using computer resources available on internet such as BLAST (http://blast.ncbi.nlm.nih.gov/) or FastDB computer programs.
The term “Unit (U)” should be understood as the unit used to measure the specific activity of an enzyme. One unit will produce 1.0 μg of phenolphthalein from phenolphthalein glucuronide (PPG) metabolite per hour at 37 degrees Celsius at pH 6.8.
The term “vehicle” should be understood as any substance that is added to an active principle, in this case an enzyme, which is useful as a support and allows to dilute or improve stability or durability of the main compound.
As described before, an object of the present invention relates to a method for detection of glucuronide metabolites in a sample, which comprises the steps of adding an enzyme with β-glucuronidase activity from genus Brachyspira or any variant or mutant derived thereof to the sample in which a product derived from a glucuronide metabolite is to be detected, incubating the sample with the enzyme for a determined period of time; and detecting the product derived from said glucuronide metabolite through any suitable technique.
This method is superior than others described in the state of the art, since it includes the use of the new enzyme with β-glucuronidase activity originated from genus Brachyspira, which has an improved affinity for glucuronide metabolites, a specific activity 6.6 times higher than β-glucuronidase originated from E. coli, and therefore it is capable of hydrolyse glucuronide metabolites to be detected in a period of time of at least one minute, preferably between three and 30 minutes. This enzyme is preferably originated from species Brachyspira pilosicoli, whose amino acid sequence is shown in SEQ. ID. NO.1, but it can be obtained from any species of genus Brachyspira. Additionally, in the method of the present invention, any derivative or mutant of this protein can be used, provided that it maintains its β-glucuronidase activity. Preferably, the derived or mutant enzyme with β-glucuronidase activity shares at least a 70% of sequence identity, more preferably an 80% of identity, and even more preferably a 90% of identity with the sequence defined in SEQ. ID. NO.1.
The β-glucuronidase enzyme originated from Brachyspira can hydrolyse any glucuronide metabolite, whether from natural, semi-synthetic or synthetic origin. Some examples of these metabolites, without limitation, are: morphine-3-glucuronide, morphine-6-glucuronide, codeine-6-glucuronide, oximorphone-glucuronide, hydromorphone-glucuronide, norbuprenorphine-glucuronide, buprenorphine-glucuronide, oxazepam-glucuronide, temazepam-glucuronide, lorazepam-glucuronide, alprazolam-glucuronide, midazolam-glucuronide, nordiazepam-glucuronide, psilocin-glucuronide, carboxy-tetrahydrocannabinol-glucuronide, tetrahydrocannabinol-glucuronide, tetrahydrocannabinolic acid-glucuronide, naloxone-3-glucuronide, tapentadol-glucuronide, tricyclic antidepressant-glucuronide, cannabidiol-glucuronide, among others. It is to be understood for the scope of the present invention that the β-glucuronidase enzyme originated from Brachyspira can hydrolise any glucuronide metabolite and it is not limited to the examples previously mentioned.
Therefore, this method allows to detect any product derived from glucuronide metabolites, whether from natural, semi-synthetic or synthetic origin. For example, the method can be used to analyze drugs, toxic compounds derived from fungi, androgens, estrogens, mineralocorticoids, glucocorticoids, fatty acid derivatives, retinoids, among others.
In a preferred embodiment, products derived from glucuronide metabolites are selected from the group consisting of opioids, opiates, cannabinoids, benzodiazepines or any other derived thereof. Preferably, the products derived from glucuronide metabolites are selected from the group consisting of morphine, codeine, oximorphone, hydromorphone, norbuprenorphine, buprenorphine, oxazepam, temazepam, lorazepam, alprazolam, midazolam, nordiazepam, psilocin, 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH), tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCa), naloxone, tapentadol, tricyclic antidepressant, cannabidiol, among others, without being limited to the examples described herein.
In a preferred embodiment of the present invention, the method allows to detect any product derived from glucuronide metabolites in any type of sample, which is preferably selected from the group consisting of saliva, whole blood, plasma, urine, hair, skin, teeth, soft tissues, water, food, meconium and vitreous humor, without restriction to these types of samples. The following biological samples can also be used: bile, earwax, phlegm, vomit, aqueous humour, tears, amniotic fluid, vaginal secretion, semen, pre-ejaculate fluid, mucus, sebum, sweat, excrement, or any other suitable sample without limitation to the animal fluids previously mentioned. Other suitable samples originate from a non-animal source, such as food, water or chemical solutions from laboratories.
In the method of the present invention, β-glucuronidase enzyme originated from genus Brachyspira is added to the sample and the mixture is incubated for a determined period of time, in suitable conditions. In a preferred embodiment, incubation period of sample with the enzyme is of at least one minute, that is to say, only one minute of reaction is enough to detect the presence of a product derived of a glucuronide metabolite in a sample. However, incubation period can be extended to 16 hours or even more, provided that time is not an impediment to the technician performing the method.
More preferably, incubation period is between 2 and 120 minutes, and even more preferably, incubation period is between 3 and 30 minutes, since obtaining the results efficiently and in the shortest time is commonly desired. Additionally, adequate incubation conditions refer to ideal conditions of temperature and pH in order to allow the reaction to be produced in the time periods mentioned. Regarding temperature, incubation is performed preferably in a range between 20 and 60 degrees Celsius, and even more preferably in a range of 50 to 55 degrees Celsius. Regarding pH, incubation is performed in a range between 4.0 and 9.0 and preferably between 6.5 and 7.5. As part of the elements routinely used in this assays, incubation is performed in presence of buffer solutions, standards of products derived from glucuronide metabolites, polar or apolar solvents, among others.
After incubating the β-glucuronidase enzyme with the sample and before separating the products derived from glucuronide metabolites through separation columns, an extraction step of said metabolites can be optionally included to increase sensitivity of analytical techniques. This extraction step can be performed by means of liquid-liquid extraction (LLE), solid-liquid extraction (SLE), extraction in solid phase (SPE), or any other suitable technique. However, the step of extracting the products derived from glucuronide metabolites adds a laborious step to the analytical method and increases operational costs due to the use of solvents and other materials.
To the method of the present invention, the latter step is optional due to the availability of highly selective analytical techniques that allow direct analysis by means of the technique named “dilute and shoot”, which consist of diluting the sample with an adequate solution and injecting it directly into a separation column. Moreover, the method does not require elimination of the enzyme since the β-glucuronidase of the present invention is highly purified and has a high reactivity; and therefore a small volume of the enzyme is required to react with glucuronide metabolites. In a preferred embodiment the volume range of enzyme: liquid sample used, for example, urine, is from 1:1 to 1:14. Preferably, optimum concentration is between 0.02 and 0.25 mg of enzyme per mL of total reaction. In this manner, the volume of enzyme used does not affect or damage the separation column in case it is directly injected to it. The adequate solvent can be for example, water or a mixture of water with methanol, or any mixture of solvents that can be used to dilute the sample.
Finally, detection of products derived from glucuronide metabolites is performed through the use of separation columns and analytical techniques which are selected from the group consisting of: gas chromatography (GC), liquid chromatography (LC) or high performance liquid chromatography (HPLC), solid phase microextraction (SPME), with mass spectrometry detectors (GC-MS; LC-MS; HPLC-MS; SPME-MS), high resolution mass spectrometry (GC-HRMS; LC-HRMS; HPLC-HRMS), tandem mass spectrometry (GC-MS/MS; LC-MS/MS; SPME-GC-MS/MS), time of flight mass spectrometry (LC-TOF-MS), ultraviolet-visible spectrophotometry (GC-UV/Vis; LC-UV/Vis; SPME-LC-UV/Vis), flame ionization detection (GC-FID; LC-FID; SPME-GC-FID), photodiode array detection, (LC-DAD), or any other suitable detection method, without limiting to the examples previously mentioned.
A second object of the present invention is a reagent for detection of products derived from glucuronide metabolites in a sample, which comprises an enzyme with β-glucuronidase activity originated from genus Brachyspira or any derived enzyme or mutant thereof; which is included in a suitable vehicle.
Preferably, the reagent contains an enzyme with β-glucuronidase activity originated from species Brachyspira pilosicoli, or any derived enzyme or mutant thereof. Said enzyme has an amino acid sequence shown in SEQ. ID. NO.1, but it can be any enzyme mutant from said sequence and maintaining β-glucuronidase activity. Preferably, the derived or mutant enzyme with β-glucuronidase activity shares at least a 70% or 80% of identity with the sequence defined in SEQ. ID. NO.1, more preferably a 90% of identity with the sequence defined in SEQ. ID. NO.1.
In a preferred embodiment, the reagent contains between 0.1 and 2 mg/mL of enzyme, in which the same has a β-glucuronidase activity from 200,000 U/mL to 800.000 U/mL. In addition to β-glucuronidase enzyme, the reagent includes an appropriate vehicle that varies depending on the product format and on the function that the same performs. For example, the reagent including the enzyme can be in a liquid format, which could require only water as a vehicle.
To improve the stability of the enzyme, salts can be added (at concentrations μM or mM), or ethylenediamine tetraacetic acid (EDTA), Tris, dithiothreitol (DTT), hydroxyethylpiperazin-ylethanesulfonic acid (HEPES), citric acid, among others, or a combination thereof. Preferably, salts are selected from the group consisting of sodium phosphate, potassium phosphate, sodium carbonate, sodium acetate, sodium citrate, sodium chloride and potassium chloride, without limitation to the salts previously described. In case that the reagent is frozen, the vehicle to be used can include glycerol, preferably at a concentration of 50%. On the other hand, if the reagent format is powder, which can be produced by means of lyophilisation, spray drying or any other pulverization method; vehicles able to protect the enzyme from drying process are required such as sugars, amino acids and some salts, which are present at a concentration range between 0.001 to 2% based on liquid previous to the drying process. In this case, sugars are preferably selected from the group consisting of sorbitol, trehalose, sucrose, glucose, lactose, mannitol and raffinose, without limitation to these described examples. Therefore, the vehicle of the present invention fulfils the purpose of being a diluting, preservating or stabilizing medium for the enzyme.
The following examples are considered to illustrate the invention and its preferred embodiments, but they must not be considered under any circumstance as a restriction to the scope of the invention, which is defined by the claims attached hereto.

EXAMPLES OF IMPLEMENTATION

Example 1

Synthesis and Expression of β-glucuronidase Enzyme from B. pilosicoli

To express the β-glucuronidase enzyme, nucleotide sequence from said protein was used, originated from Brachyspira pilosicoli strain B2904 described in Genbank (access code CP003490), which is shown in SEC. ID. NO.2, and codon optimization was performed using the algorithm OptimumGene™ (GenScript) to efficiently express said sequence in E. coli. To do this, codon adaptation index (CAI), understood as distribution of frequency of codon usage through the sequence, was adjusted from 0.66 to 0.88, in which a CAI value of 1.0 is considered to be perfect in the desired expression organism; and a CAI value greater than 0.8 is regarded as good in terms of high gene expression levels. Guanine-cytosine content was also optimized to extend mRNA half-life and stem-loop structures were eliminated, since they disrupt ribosome binding and mRNA stability. Optimized nucleotide sequence obtained is shown in SEC. ID. NO.3. Amino acid sequence of the enzyme is shown in SEC. ID. NO.1.
The optimized nucleotide sequence of the enzyme was cloned into vector pET-28a, thus vector pET-28a-glucuronidase was obtained, which is shown in FIG. 1. To express β-glucuronidase enzyme from B. pilosicoli in E. coli, the following protocol was carried out: 5 μL of competent E. coli strain BL21 cells were incubated in ice with 10 ng of pET-28a-glucuronidase vector during 20 minutes. Then, bacterial cells were submitted to heatshock at 37 degrees Celsius during 3 minutes. Bacterial cells were then put in ice for 30 seconds and later cells were recovered with 250 μL of LB io medium at 37 degrees Celsius and plated in LB-agar with 50 mg/L of kanamycin, and colonies were grown. Finally, a β-glucuronidase activity test was carried out on plates with IPTG (isopropyl-β-thiogalactoside) and X-Gal (5-bromo-4-chloro-3 indolyl-beta-D-thiogalactopyranoside).
In order to purify the enzyme, E. coli BL21 transformed cells were grown in 500 mL of LB medium supplemented with kanamycin (50 mg/L), until obtention of an optical density of 0.3, measured using a spectrophotometer at 600 nm (DO₆₀₀).
Then, IPTG at a concentration of 1 mM was added to the culture and then incubated at 37 degrees Celsius during 6 hours. Cells were subjected to sonication during 3 minutes with an amplitude of 60 (cycles of 15 seconds in ice). Later, the enzyme was purified by means of a system for protein purification in columns with Ni-NTA (Nickel-Nitriloacetic acid) resins with elution of 2 mL of Tris-HCl 50 mM, pH 5.6 with imidazole 100 mM pH 5.9 or with imidazole 500 mM pH 4.5.
FIG. 2 shows an SDS-PAGE gel of purified proteins in column Ni-NTA. Each well contained proteins at a concentration of 15 pg/mL, except in crude extract (E. coli pET28a-glucuronidase without purification in the column), in which 5 μL were loaded. The acronym Std corresponds to molecular weight standard in kilodaltons (kD), Crude ex corresponds to crude extract, Glu E1 corresponds to elution with imidazole 100 mM pH 5.9 and Glu E2 corresponds to elution with imidazole 500 mM pH 4.5.

Example 2

Incubation Time of a Sample with β-glucuronidase from B. pilosicoli

The required time to hydrolyze morphine-3-glucuronide and codeine-6-glucuronide with the enzyme β-glucuronidase in excess (named BGTurbo™) from B. pilosicoli was evaluated with the following protocol: 10 μL of glucuronide metabolites at a concentration of 100 μg/mL, free standards (free codeine and morphine calibrators at concentrations of 312.5, 625, 1,000, 1,250 and 1,875 ng/mL), and internal standards (40 μL of deuterated free drug at a concentration of 1 μg/mL to each sample) were added to a sample of 0.4 mL of blank urine and then submitted to vortex agitation. Then, 360 μL of sodium phosphate (pH 7; 140 mM) solution was added to each sample. Then, 240 μL of β-glucuronidase (BGTurbo™) were added to each sample and then agitated by vortex. Samples were incubated at 55 degrees Celsius in an oven during 0, 10, 20, 30, 40, 50 and 60 minutes. A total of 21 samples were used: 7 points of measure at different times, each one in triplicate. Calibrators were incubated during 30 minutes. The samples incubated at different times were removed from oven and the reaction was stopped by adding 700 μL of sodium phosphate buffer solution 1, 5 M and agitated by vortex. Then, pH 8-9 was verified with pH paper, and 2 mL of chloroform: isopropanol 50:50 were added to samples, and they were agitated by vortex during 2 minutes. Aqueous phase was discarded and organic phase was transferred to 12×75 mm assay tubes and then evaporated under nitrogen gas (in a water bath at 50 degrees Celsius) until approximately 1 mL. Samples were then agitated by vortex previous to evaporation until dryness. Samples were reconstituted in 1 mL of HCl 0, 5 N in methanol for LC-MS/MS analysis.
Calculation of concentration of equivalents was performed as follows:

Morphine:

- Molecular weight of morphine: 285.34 g/mol
- Molecular weight of morphine-3-glucuronide: 461.462 g/mol
- Proportion of molecular weight of morphine/morphine-3-glucuronide:

Morphine equivalents:
$0.6183  2, 500 \frac{ng}{mL} Morphine - 3 - glucuronide = 1, 545.8 \frac{ng}{mL} Morphine$

Codeine:

- Molecular weight of codeine: 299.364 g/mol
- Molecular weight of codeine-6-glucuronide: 475.49 g/mol
- Proportion of molecular weight of codeine/codeine-6-glucuronide:

Codeine equivalents:
$0.62959  2, 500 \frac{ng}{mL} Codeine - 6 - glucuronide = 1, 574.0 \frac{ng}{mL} Codeine$
Table 1 and FIG. 3 show the results obtained. Hydrolysis of 100% of codeine-6-glucuronide was obtained in less than 10 minutes. In the case of morphin, approximately 65% of hydrolysis was obtained within 10 minutes, but these values can be improved by optimizing parameters of temperature, pH or the method to stop reaction. Due to the use of internal controls for recovery of products derived from glucuronide metabolites, values have a standard deviation up to 20%.

TABLE 1

Tabulated results of FIG. 3.

Time	Codeine	% Yield	Morphine	% Yield
(minutes)	(ng/mL)	Codeine	(ng/mL)	Morphine

0	30.0	1.9	195.8	12.7
10	1826.9	116.1	997.4	64.5
20	1867.5	118.6	969.2	62.7
30	1694.3	107.6	871.2	56.4
40	1821.0	115.7	811.5	52.5
50	1844.3	117.2	847.4	54.8
60	1819.5	115.6	823.4	53.3

Example 3

Calculation of Specific Activity of β-glucuronidase from B. pilosicoli

Specific activity of the β-glucuronidase enzyme from B. pilosicoli (BGTurbo™) was calculated and compared with a mean value of three calculations of the specific activity of β-glucuronidase from E. coli, using the following protocol: 350 μL of sodium phosphate buffer solution (NaH₂PO₄) 0.1 M, pH 6.8 were mixed with 350 μL of phenolphthalein β-D-glucuronide sodium salt (0.64 mg/mL) and the mixture was incubated at 37 degrees Celsius. Then, 50 μL of solution containing the enzyme were added to the mixture and incubated at 37 degrees Celsius during 30 minutes. After io the first 15 minutes the mixture was gently agitated and stopped at 30 minutes from the beginning of the reaction with 2.5 mL of a glycine solution 0.2 M, pH 10.4. Absorbance was read at 540 nm and the amount of enzyme used was calculated (U/mL) when adjusting the values in a calibration curve. Protein concentration was measured using Bradford method, as widely known in literature. As shown in Table 2, results indicate that BGTurbo™ enzyme has a specific activity 6.6 times higher than mean specific activity of β-glucuronidase from E. coli.

TABLE 2

Enzymatic activity of β-glucuronidase from B. pilosicoli.

	Activity	Proteins	Specific Activity
Enzyme	(U/mL)	(mg/mL)	(U/mg)

BGTurbo ™	427,805	2.08	205,676
E. coli 1	68,779	2.28	30,166
E. coli 2	102,710	3.17	32,401
E. coli 3	85,084	2.89	29,441

	E. coli mean value	30,669

Example 4

Comparison of β-glucuronidase from B. pilosicoli with β-glucuronidases from Different Sources

To test the efficiency of β-glucuronidase from B. pilosicoli (BGTurbo™), this enzyme was compared with two β-glucuronidases currently available in the market: DR2102 (Campbell Science) and BG100™ (KURA Biotec). To do this, 300 ng of each of the glucuronide metabolites of buprenorphine, codeine, hydromorphone, lorazepam, morphine, oxymorphone and 11-nor-9-carboxy-tetrahydrocannnabinol (THC-COON) were added to a sample of blank urine, therefore obtaining spiked urine. Each enzyme was used under the conditions proposed by each manufacturer. Hydrolysis of 50 μL of urine was performed, diluting it with 250 μL of buffer solution at pH 6.8, 20 μL of internal standards (ISTD) and 10 μL of DR2102 or BG100 enzymes, or 20 μL of BGTurbo™. Incubation conditions are shown in Table 3.

TABLE 3

Conditions used for each enzyme.

Conditions	DR2102	BG100 ™	BGTurbo ™

Urine (μL)	50	50	50
ISTD (μL)	20	20	20
Buffer (μL)	25	25	25
Enzyme (μL)	10	10	20
Temperature (° C.)	62	68	50
Time (minutes)	60	30	15

After hydrolysis, samples were centrifuged at 24,000 g, diluted with 450 μL of a solution comprising 95% water and 5% methanol, and charged to an HPLC column (Phenomenex). Protein amount in samples was measured before and after centrifugation at 24,000 g using a NanoDrop spectrophotometer (Thermo Scientific). As a result, the reactions performed were carried out with 1.787 mg/mL of DR2102, 0.554 mg/mL of BG100™ and 0.103 mg/mL of BGTurbo™.
Recovery yields of products derived from glucuronide metabolites are shown in Table 4. As shown, mean recovery of derived products was improved in a 15% when hydrolysis was performed with BGTurbo™ in only 15 minutes and using 70% and 94% less of protein amount in comparison to the other two enzymes. It is interesting to mention that β-glucuronidase enzyme from B. pilosicoli is more reactive with codeine-6-glucuronide than the other enzymes.

TABLE 4

Results of analites recovered from hydrolysis of
glucuronide metabolites with three β-glucuronidases
enzymes from different origins

	DR2102	BG100 ™	BGTurbo ™
	60 minutes	30 minutes	15 minutes
Recovered Analyte	(ng/mL)	(ng/mL)	(ng/mL)

Buprenorphine	223.1	271.1	224.4
Codeine	159.4	173.8	243.2
Hydromorphone	176.8	196.0	196.2
Lorazepam	214.9	202.5	185.9
Morphine	174.5	190.9	198.1
Oximorphone	155.8	189.0	169.4
THC-COOH	75.5	146.0	140.6
Protein in sample	1.787 (mg/mL)	0.554 (mg/mL)	0.103 (mg/mL)
pre-centrifugation
Protein in sample	0.725 (mg/mL)	0.280 (mg/mL)	0.085 (mg/mL)
post-centrifugation and
injected on HPLC
column

Example 5

Comparison of β-glucuronidase from B. pilosicoli with Other β-glucuronidases from Bacterial Origin

In order to test enzyme speed of β-glucuronidase from B. pilosicoli (BGTurbo™) in comparison with β-glucuronidases from bacterial origin, BGTurbo™ was compared with EBG enzyme (recombinant β-glucuronidase from E. coli, KURA Biotec) and with IMCSzyme™ (Integrated Micro-Chromatography Systems) at different incubation periods and concentration. The method previously described was used, but analyzing only hydrolysis of codeine-6-glucuronide and morphine-3-glucuronide with the conditions described in Table 5.

TABLE 5

Conditions used for each enzyme.

BGTurbo ™

EBG

IMCSzyme ™

Control

Conditions	A	B	C	D	F	—	E

Buffer (μL)	360	360	360	360	360	360	360
Water (μL)	0	120	180	210	180	180	120
Spiked urine (μL)	400	400	400	400	400	400	400
Enzyme (μL)	240	120	60	30	60	60	0
Internal Standard (μL)	40	40	40	40	40	40	40
Storage buffer control (μL)	0	0	0	0	0	0	120
Total Volume (μL)	1,040	1,040	1,040	1,040	1,040	1,040	1,040
Incubation Time (minutes)	10	10	10	10	0, 10, 20	0, 10, 20	10
pH	7.0	7.0	7.0	7.0	7.0	7.0	7.0
Temperature (° C.)	55	55	55	55	55	55	55

Each condition shown in Table 5 was assayed in duplicate, and each analysis was measured in triplicate. Calculation of concentration of equivalents was performed as described in Example 2.
Table 6 shows the results obtained from analysis of codeine, and Table 7 shows results obtained from analysis of morphine. As can be noticed, codeine-6-glucuronide was hydrolyzed in a mean value of 109,59% within 10 minutes, using 240 μL of BGTurbo™. The value mentioned is greater than 100% because standards of glucuronide metabolites used in the analysis are not completely pure, and present a fraction of the original compound that is added to the final result. On the other hand, when comparing hydrolysis yield of codeine-6-glucuronide and morphine-3-glucuronide using the same volume of the enzymes (60 μL), it is shown that BGTurbo™ is better than the other EBG and IMCSzyme™ enzymes within 10 minutes. Even after an incubation period of 20 minutes of the samples with EBG and IMCSzyme™, these enzymes could not produce the same hydrolysis yield that was obtained with BGTurbo™ within 10 minutes.

TABLE 6

Results of codeine analysis.

Condition/		Codeine	Codeine	Average of
Assay	Sample	(ng/mL)	% Yield	Assay Yield (%)

A/1	BGTurbo ™ (10 min, 240 μL)	1,740	110.55	109.59
A/2	BGTurbo ™ (10 min, 240 μL)	1,710	108.64
B/1	BGTurbo ™ (10 min, 120 μL)	1,270	80.69	78.14
B/2	BGTurbo ™ (10 min, 120 μL)	1,190	75.60
C/1	BGTurbo ™ (10 min, 60 μL)	879	55.84	54.19
C/2	BGTurbo ™ (10 min, 60 μL)	827	52.54
D/1	BGTurbo ™ (10 min, 30 μL)	230	14.61	22.20
D/2	BGTurbo ™ (10 min, 30 μL)	469	29.80
F/1	EBG (0 min, 60 μL)	No Peak	0.00	0.00
F/2	EBG (0 min, 60 μL)	No Peak	0.00
F/1	EBG (10 min, 60 μL)	154	9.78	10.48
F/2	EBG (10 min, 60 μL)	176	11.18
F/1	EBG (20 min, 60 μL)	396	25.16	27.10
F/2	EBG (20 min, 60 μL)	457	29.03
1	IMCSzyme ™ (0 min, 60 μL)	No Peak	0.00	0.00
2	IMCSzyme ™ (0 min, 60 μL)	No Peak	0.00
1	IMCSzyme ™ (10 min, 60 μL)	189	12.01	11.85
2	IMCSzyme ™ (10 min, 60 μL)	184	11.69
1	IMCSzyme ™ (20 min, 60 μL)	395	25.10	27.26
2	IMCSzyme ™ (20 min, 60 μL)	463	29.42
E/1	Control (10 min)	No Peak	0.00	0.00
E/2	Control (10 min)	No Peak	0.00

TABLE 7

Results of the morphine analysis.

Condition/		Morphine	Morphine	Average of
Assay	Sample	(ng/mL)	% Yield	Assay Yield (%)

A/1	BGTurbo ™ (10 min, 240 μL)	796	51.49	51.49
A/2	BGTurbo ™ (10 min, 240 μL)	796	51.49
B/1	BGTurbo ™ (10 min, 120 μL)	717	46.38	46.90
B/2	BGTurbo ™ (10 min, 120 μL)	733	47.42
C/1	BGTurbo ™ (10 min, 60 μL)	687	44.44	43.70
C/2	BGTurbo ™ (10 min, 60 μL)	664	42.96
D/1	BGTurbo ™ (10 min, 30 μL)	498	32.22	38.46
D/2	BGTurbo ™ (10 min, 30 μL)	691	44.70
F/1	EBG (0 min, 60 μL)	179	11.58	12.71
F/2	EBG (0 min, 60 μL)	214	13.84
F/1	EBG (10 min, 60 μL)	609	39.40	39.62
F/2	EBG (10 min, 60 μL)	616	39.85
F/1	EBG (20 min, 60 μL)	629	40.69	43.47
F/2	EBG (20 min, 60 μL)	715	46.25
1	IMCSzyme ™ (0 min, 60 μL)	142	9.19	10.19
2	IMCSzyme ™ (0 min, 60 μL)	173	11.19
1	IMCSzyme ™ (10 min, 60 μL)	603	39.01	39.20
2	IMCSzyme ™ (10 min, 60 μL)	609	39.40
1	IMCSzyme ™ (20 min, 60 μL)	662	42.83	43.41
2	IMCSzyme ™ (20 min, 60 μL)	680	43.99
E/1	Control (10 min)	No Peak	0.00	0.00
E/2	Control (10 min)	No Peak	0.00

Claims

1. A method to detect products derived from glucuronide metabolites in a sample, comprising the steps of:

a) providing the sample in which said product is to be detected,

b) adding to said sample an enzyme with β-glucuronidase activity from genus Brachyspira or any variant or mutant derived thereof,

c) incubating the sample with the enzyme for a determined period of time; and

d) detecting the product derived from said glucuronide metabolite through any suitable technique.

2. The method of claim 1, wherein products derived from glucuronide metabolites are from natural, semi-synthetic or synthetic origin.

3. The method of claim 2, wherein products derived from glucuronide metabolites are selected from the group consisting of an opioid, an opiate, a cannabinoid, a benzodiazepine or any derivative thereof.

4. The method of claim 1, wherein the sample is a biological sample.

5. The method of claim 4, wherein the biological sample is selected from the group consisting of saliva, whole blood, plasma, urine, hair, skin, teeth, soft tissues, meconium and vitreous humor.

6. The method of claim 1, wherein the enzyme with β-glucuronidase activity originates from species Brachyspira pilosicoh.

7. The method of claim 6, wherein the enzyme with β-glucuronidase activity is selected from the group consisting of:

a) an amino acid sequence comprising the amino acid sequence shown in SEQ. ID. NO.1;

b) an amino acid sequence sharing at least a 70% of identity with the amino acid sequence defined in a).

8. The method of claim 1, wherein the incubation period of the sample and the enzyme is between 1 and 30 minutes.

9. The method of claim 1, wherein incubation of sample and enzyme is performed at a temperature range between 20 and 60 degrees Celsius.

10. The method of claim 1, wherein incubation of sample and enzyme is performed at a range of pH between 4.0 and 9.0.

11. The method of claim 1, wherein the suitable technique to detect products derived from glucuronide metabolites is selected from the group consisting of LC-MS, GC-MS, HPLC-MS, LC-HRMS, HPLC-HRMS, LC-MS-MS, GC-MS-MS and HPLC-MS-MS.

12. A reagent to detect products derived from glucuronide metabolites in a sample, comprising the steps of:

a) an enzyme with β-glucuronidase activity originated from genus Brachyspira or any variant or mutant derived thereof; and

b) a suitable vehicle.

13. The reagent of claim 12, wherein the enzyme with β-glucuronidase activity originates from species Brachyspira pilosicoli.

14. The reagent of claim 13, wherein the enzyme with β-glucuronidase activity is selected from the group consisting of:

15. The reagent of claim 12, wherein the suitable vehicle is selected from the group consisting of: water, salts, buffer solutions, Tris, HEPES, DTT, citric acid, EDTA, glycerol, sugars, amino acids or a combination thereof.

16. The reagent of claim 15, wherein the salts are selected from the group consisting of: sodium phosphate, potassium phosphate, sodium carbonate, sodium acetate, sodium citrate, sodium chloride and potassium chloride.

17. The reagent of claim 15, wherein sugars are selected from the group consisting of sorbitol, trehalose, sucrose, glucose, lactose, mannitol and raffinose.