Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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

a technology of glucuronide derivatives and products, applied in the field of biotechnology, can solve the problems of corroding vulnerable equipment, stricter security conditions, and degradation of sensitive compounds

Inactive Publication Date: 2018-03-08
LA PIEDRA BIOTECHA SPA
View PDF1 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method to detect products derived from glucuronide metabolites in a sample using an enzyme with β-glucuronidase activity from genus Brachyspira or any variant or mutant thereof. The method can detect these products in various samples such as saliva, whole blood, plasma, urine, hair, skin, teeth, soft tissues, meconium, vitreous humor, water, and food. The enzyme with β-glucuronidase activity can be added to the sample and incubated for a determined period of time before detection of the product using any suitable analytical technique. The invention also provides a reagent containing the enzyme and an appropriate vehicle for detection of glucuronide metabolites in a sample.

Problems solved by technology

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.
Generally, preparations of enzymes obtained from molluscs tend to have more contaminant substances that may interfere with further analysis of glucuronide metabolites.
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).

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for detecting products derived from glucuronide metabolites with the enzyme b-glucuronidase, and a reagent comprising said enzyme
  • Method for detecting products derived from glucuronide metabolites with the enzyme b-glucuronidase, and a reagent comprising said enzyme
  • Method for detecting products derived from glucuronide metabolites with the enzyme b-glucuronidase, and a reagent comprising said enzyme

Examples

Experimental program
Comparison scheme
Effect test

examples of implementation

Example 1

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

[0047]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. ...

example 2

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

[0052]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 ti...

example 3

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

[0063]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 m...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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[0001]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[0002]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.[0003]Detection of these glucuronide metabolites in biological ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): G01N33/569C12Q1/34
CPCG01N33/56916C12Q1/34G01N30/72G01N33/948G01N33/9486G01N2333/924C12Y302/01031
Inventor ROZAS ANDREU, MANUELALMONACID CORONADO, DANIEL E.
Owner LA PIEDRA BIOTECHA SPA
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com