Our Products
PolyUGTs Kits for Glucuronides
PolyUGTs Metabolite Kits
Easy-to-use in-vitro enzyme system for generation of glucuronides of drugs, agrochemicals, and other small molecules
PolyUGTs® metabolite kits can be used to screen for, and scale-up production of, a variety of glucuronides of drugs and agrochemicals.
Hypha’s PolyUGT enzymes are mined from selected biotransforming strains in Hypha’s microbial collection, expressed in E.coli, and subsequently purified. The enzymes provided in the screening kit have been tested for biocatalytic activity against a number of substrates known to be subject to glucuronidation. This includes glucuronidation of alkyl and phenolic hydroxyls for O-glucuronides, carboxylic acids for acyl-glucuronides, as well as aromatic nitrogen-containing systems for N-glucuronides. Glucuronidation of some non-aromatic alkyl N-moieties is also observed.
Currently 11 PolyUGT isoforms are available in a screening kit and scale-up kit format for clients to use in their own labs. Hit reactions can be scaled up to obtain material for structure elucidation by NMR to pinpoint the position of attachment, biological activity assessment, validation of sample handling processes for bioanalysis, and for in vitro drug-drug interaction testing.
There is also the option to outsource screening, scale-up, purification and structure elucidation of glucuronide metabolites to Hypha using our wider variety of proprietary techniques.
PolyUGTs glucuronide kits are complementary to Hypha’s PolyCYPs kits used for producing CYP and other phase I metabolites of drugs and agrochemicals.
Content of PolyUGTs glucuronide kit
All reagents are provided as lyophilised powders in sealed vials together with a 24-well reaction plate and an adhesive plate seal. Everything is included in the kit that you will need to perform the reactions in your lab, just add your compound and water!
11 PolyUGT enzymes
Uridine diphosphate glucuronic acid (UDPGA)
Formulation reagent to aid the solubilisation of test compounds of poor aqueous solubility
Positive control substrate (4-methylumbelliferone)
Extra enzyme (PolyUGT 216) for use with control compound
Optional pH reduction buffer vial for use when an acyl-glucuronide is anticipated. This will reduce the pH to ~pH6.5 to help reduce the formation of acyl migration isomers, and potentially increase yields.
PolyUGTs glucuronide scale-up
PolyUGT enzyme reactions are scalable from mg to gram quantities through a number of routes, either by resupply of lyophilised enzymes for mg scale production in house, or larger scale production at Hypha, with optional purification and structure elucidation.
Options:
- Multiple 10ml scale-up vials, suitable for converting 1mg+ of parent per vial, can be ordered off-the-shelf for any isoform for in-house scale-up in a kit format.
- UDPGA and formulant are also included along with a 24-well block suitable for performing 4 x 2.5 ml reactions per 10 ml vial. Other incubation formats can also be used, as recommended in the instructions.
- Scale-up vials can also be used for initial hit optimisation, such as scoping higher doses of parent compound and incubation times ahead of scale-up.
- For accessing larger quantities of glucuronides, Hypha can scale-up reactions using bulk enzyme preparations or via streptomycete clones containing the UGT of interest. Our selected host strains synthesise the UDPGA cofactor for the reactions thereby negating the need to add expensive exogenous UDPGA.
We can also process and purify metabolites from the reaction extracts and undertake structure elucidation using cryoprobe NMR spectroscopy.
Contact us to find out more about our service options.
Case studies
Use of PolyUGTs metabolite kits for making acyl glucuronides of drug compounds
A range of drugs containing carboxylic acid groups known to form acyl glucuronides in human liver microsome incubations have been tested with PolyUGTs glucuronide kits. Since some acyl glucuronides may form acyl migration isomers at neutral / slightly alkaline pH, we include an acid buffer in the kit for use if formation of an acyl glucuronide is anticipated. Incubations can be run with and without the acid buffer to determine the best conditions for your specific acyl glucuronide.
In all instances of the evaluation with the drug compounds summarised in the bar chart below, the human liver microsome product matched those observed in PolyUGTs incubations by LC-MS analysis. All of the compounds are known to be metabolised to acyl glucuronides in humans, particularly by UGT1A3 and UGT2B7.
The majority of the reactions performed better with addition of the acid buffer for this set of compounds, with either increased yields or reduced formation of acyl migration isomers.
Use of PolyUGTs for synthesis of a major human glucuronide
In this case study PolyUGTs were used to screen for and subsequently generate a major human glucuronide of a drug for definitive structure elucidation by NMR spectroscopy.
O-glucuronides of the drug compound were produced by several isoforms in the glucuronide screening kit. The highest yielding enzyme, PolyUGT 179, was scaled up using a UDPGA-providing Streptomyces host into which the UGT had been cloned. Two O-glucuronides were purified and structures elucidated by NMR spectroscopy, reflecting what was observed in human liver microsomes and published in the literature.
The phenoxy glucuronide is unusual in that it is a pharmacologically active metabolite, and is more potent at inhibiting cholesterol absorption in the gut than the parent drug. Repeated enterohepatic circulation results in a long duration of action.
Formation of an N-carbamoyl glucuronide using PolyUGTs and bicarbonate buffer
The N-carbamoyl glucuronide of lorcaserin was not initially seen in PolyUGTs incubations. However Gunduz et al. investigated the mechanism of N-carbamoyl glucuronidation of another compound using stable labelled CO2, showed incorporation of CO2 from the bicarbonate buffer, in equilibrium with exogenous CO2 into the carbamoyl moiety of the subsequently formed N-carbamoyl glucuronide in liver microsome incubations.
Since bicarbonate buffer contains 200-fold higher dissolved CO2 than phosphate buffer at atmospheric pressure, a switch to use of a bicarbonate buffer during screening of lorcaserin vs PolyUGTs resulted in a 15% conversion to the N-carbamoyl glucuronide.
Ref: Gunduz, M. et al. (2010). Identification of a novel N-carbamoyl glucuronide: in vitro, in vivo, and mechanistic studies. Drug Metab Dispos. 38 (3): 361-7. DOI: 10.1124/dmd.109.030650
Why use PolyUGTs glucuronide kits?
Quick and easy access to a variety of glucuronides for MetID and biological testing, including drug-drug interaction studies
Applicable to a wide variety of drug, agrochemical and other small molecules
Capability to provide acyl-glucuronides to allow purification for acyl-migration lability testing
Kit comes with everything needed – reagents are provided as stable lyophilised powders (just add water) and includes a 24-well reaction block and plate seal
Predispensed UDPGA co-factor vials included - no need to buy additional expensive co-factor
Scalable - use scale-up vials or outsource to Hypha to a live cell system with good capacity for dose escalation to increase metabolite yields.
Resources
Explore our library of resources comprising brochures, case studies, posters and publications about the work we do.
In this case study at least 2 mg of a monohydroxylated metabolite (M4), originally observed in rat liver microsomes, was required by a US pharma company. Using PolyCYPs, the target metabolite was supplied to the client together with a Certificate of Analysis within 22 days from receipt of order, exemplifying the short timelines achievable using PolyCYPs to access CYP-derived metabolites.
Once a target metabolite or oxidised derivative has been synthesised by one or more PolyCYPs enzymes in the screening kit, a scale-up reaction with the best performing isoform is performed in order to access mg amounts of material for MetID and biological testing. The quickest and most cost-effective route for generating low mg amount of product is through the use of scale-up vials. Higher amounts of product can be generated using either a recombinant E.coli cell paste or through fermentation of a recombinant streptomyces clone expressing the isoform responsible for the biotransformation.
Hypha’s PolyCYPs kits are in routine use by pharma and agchem companies for producing human and other mammalian metabolites. One application involves use of PolyCYPs for creating radiolabelled metabolites for direct comparison with the radio profiles from mass balance and distribution study samples, necessary for regulatory filing. PolyCYPs provides a clean route for scalable access to more of the CYP-derived metabolites observed in these matrices, for definitive MetID and any tox studies deemed necessary. This is especially useful where low concentrations or unstable metabolites in the mass balance sample make structural identification difficult.
Related Services
We offer a number of solutions for synthesis of phase 1 and phase 2 metabolites using our one-stop metabolite shop approach
Chemical Synthesis
Late-stage chemical glucuronidation
Mammalian biotransformation
Panels of liver S9s / microsomes
Recombinant enzymes
PolyCYPs, hrCYPs, AOX, FMOs
Microbial biotransformation
Proven panels of bacteria and fungi
Gut metabolism
Human faecal incubations under anaerobic conditions
Purification & structure elucidation
COAs, acquisition / interpretation of NMR data
“I am thrilled to share my testimonial on the remarkable achievement of synthesizing the N-glucuronide and O-glucuronide of our development compound at Hypha Discovery. As a chemist experienced in both synthetic and analytical chemistry, I am pleased to provide my feedback on this significant accomplishment. This synthesis posed numerous formidable challenges due to the structural complexity, resulting in the formation of multiple glucuronide isomers and purification challenges, which were overcome with great success. I extend my heartfelt congratulations and sincere gratitude to the Hypha Team for their exceptional dedication and skill in completing both the synthesis and purification stages.”
Vijay Reddy PhD, Senior Director Pharmacokinetics/Pharmacodynamics and ADME
BioXcel Therapeutics, USA
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Hypha Discovery is a UK-based CRO supporting pharmaceutical and agrochemical companies worldwide through the production of metabolites and new derivatives of drugs and agrochemicals in discovery and development.