2000-2003 Masters of Science: Graduate Biochemistry and Organic Research at The University of Northern Colorado

Research Advisor: Richard M. Hyslop


Investigations into Synthesis of 6-Thiopurine (6TP) Analogs, Development of HPLC & LC/MS Methods for the Assessment of UDP-Glucose Dehydrogenase (UDPGDH) in vivo & in vitro, and Inhibition Studies of 6-TP and its Metabolites towards UDPGDH.


The research conducted at UNC (Greeley) is threefold:  HPLC & LC/MS Methodologies for UDPGDH Assessment, Synthesis of 6-Thiopurine Analogs and Inhibition Studies of 6-TP and Metabolites toward UDPGDH.

Background:  The drug 6-thiopurine (6TP) is used as a standard treatment for childhood, non-B, acute lymphocytic leukemia (ALL) and non-Hogkins lymphoblastic leukemia, and has been, for the past 50 years (Elion et al., 1952; Burchenal et al., 1953).  The administration of this drug does allow beneficial therapeutic effects, however, several side effects are also associated with its use.

In 1951 Gertrude Elion first synthesized 6TP as part of a program that was initiated by G.H. Hitchings of the Wellcome Research Laboratories.  The goal of the research was to synthesize compounds that were structurally analogous to naturally occurring nucleic acid bases (Elion, 1986).  Upon its synthesis, 6TP was evaluated utilizing the microorganism Lactobacillus casei.  This microorganism requires a source of nitrogen, normally supplied from media containing purines and thymine or folic acid for the nitrogen source (Hitching et al., 1945).  Results of the evaluation showed that 6TP was a potent inhibitor of L. casei growth (Elion, 1953).  Further testing was performed on S-180 Cocker mouse sarcoma systems at the Sloan-Kettering Institute.  Testing showed that 6TP inhibited growth, which was unusual compared to other purine antagonists. 

Clarke et al. (1952) conducted experiments demonstrating that tumor size decreased with the administration of 6TP.  Also, along with decreased tumor size, tumors became non-viable.  Burchenal et al. (1953) confirmed these findings.  Observations in mice tumors specifically showed that 6TP was active.  Burchenal and colleagues also reported that 6TP caused the remission of acute lymphocytic leukemia (ALL) in children.  In 1953, 6TP was approved as an antileukemic agent. 

There are three major routes of metabolism for 6TP, which are thought to be associated with its cytotoxicity.  Firstly, 6TP can be metabolized via several intermediates to 6-thioguanosine-S-mono (di or tri) phosphate.  This compound can then be incorporated into DNA (Elion, 1986).  A second route is the metabolism of 6TP to S-methyl-6-thioinosine-5’-monophosphate (MeTIMP).  MeTIMP has been shown to be a potent inhibitor of purine synthesis (Zhang et al., 1998).  Thirdly, 6TP can be oxidized via cytochrome P450 to purine-6-sulfenic acid.  This compound can bind to proteins that possess a thiol group (SH), which may produce a conformational change in the protein structure.  This change in structure can potentially lead to cell destruction (Hyslop and Jardine, 1981). 

6TP can be degraded metabolically to 6-thiouric acid (6TU) by the enzyme xanthine oxidase.  6TU is far more polar than 6TP and hence more excretable.  Due to its polarity, 6TU is found in urine as a major metabolite of 6TP in humans (Hamiliton and Elion, 1954; 1967) and in animals, such as rats and rabbits (Elion et al., 1954; 1961).  The drugs allopurinal and methotrexate have been demonstrated to inhibit xanthine oxidase (Zimm et al., 1983).  Allopurinal was shown to increase the amount of 6TP available in the body (Zimm et al., 1983), while methotrexate was shown to only marginally increase the amount (Balis et al., 1987).  The conversion of 6TP to 6TU was completely inhibited when allopurinal was administered in conjunction with 6TP (Coffey, 1972).  The use of these inhibitors should allow for a lower dosage of 6TP required, hence decreasing side effects associated with the administration of the drug.  

Toxicity studies of 6TP have shown that side effects are associated with its therapeutic effects against ALL.  Reticulocytopenia, leukopenia, and depletion of cellularity in bone marrow was observed after the administration of 6TP in mice, rats, and dogs (Clarke et al., 1953).  Similar studies have shown that 6TP did in fact allow remission of ALL, but in those cases the patients did exhibit signs of some degree of hepatic toxicity (Burchenal et al., 1953; Gaffney & Cooper, 1954; Frei et al., 1958).  Toxicity of 6TP in humans was observed as side effects including jaundice, hepatic necrosis, anorexia, and diarrhea (Clarke et al., 1953; Philips et al., 1954).  Shorey and colleagues (1968) observed that 5-40% of patients treated with 6TP developed jaundice conditions.  The symptoms of jaundice disappeared once 6TP treatment was terminated (Bach, 1972).  Symptoms reoccurred, at a faster rate than the initial, when treatment was resumed (Einhorn et al., 1964). 

Jaundice, characterized by skin and the white of the eyes turning yellowish in color, is the result of an increased concentration of bilirubin in the blood.  When an erythrocyte is degraded, the heme contained within must be degraded and excreted, a process known as the bilirubin pathway.  Once the heme is released, it is converted to bilirubin; which is a large non-polar molecular.  In order for bilirubin to be excreted, it must be conjugated or made more polar using UDP-glucuronic acid via the microsomal enzyme UDP-glucuronosyl transferase.  Once conjugated, the bilirubin-glucuronic acid complex can be excreted.  UDP-glucuronic acid is produced from UDP-glucose by the enzyme UDP-glucose dehydrogenase (UDPGDH). 

It has been postulated that jaundice is caused by the inhibition of the enzyme UDPGDH when 6TP is administered.  Eliott and Hyslop (1979) reported that both 6TP and 6TU could inhibit UDPGDH, 6TU was observed to be a more potent inhibitor.  Young and Hyslop (1997) reported that both 6TP and 6TU are noncompetitive inhibitors of UDPGH.  In addition, they reported that 6TU inhibits approximately 10 times more potently than 6TP at comparable concentrations. 


HPLC and LC/MS Methodologies for the Assessment of UDP-Glucose Dehydrogenase
 
Previous Work: Two fluorescence procedures have been developed to assay the activity of UDPGDH (Burrows and Cintron, 1983; Singh et al., 1980).  The enzyme incubation was performed identically, following the protocols of Singh et al. for their enzymatic production of UDPGA.  Tissue extracts were incubated at 35 °C for 10 min in 0.1 mL of 5 mM glycine and 20 mM sodium phosphate, pH 8.7, with various concentrations of substrates in a total volume of 3 mL.  Once 10 min elapsed from the addition of UDPGDH, 0.1 mL of 0.1 M Tris-HCl, pH 7.3, containing 20 mM MgCl2, 0.1% Triton X-100, 0.2 mM UDP-Xylose, 0.05 mM 3-hydroxybenzo[a]pyrene, and 0.5 mg/mL guinea pig liver microsomal protein was added to each incubation medium and allowed to incubated for an additional 30 minutes.  Both studies require the derivization of UPDGA, which is performed by 3-hydroxybenzo-[a]pyrene in methanol to yield the corresponding glucuronylbenzo[a]-pyrene.  Unreacted 3-hydroxybenzo[a]pyrene was extracted by the addition of 0.8 mL of 0.4 M NaCl and 6 mL of 2:1 chloroform:methanol.  The aqueous phase was removed and analyzed via fluorometry.  Burrows and Clintron (1983) reported the optimal wavelengths for the derivatived glucuronic acid to be 382 nm and 424 nm for excitation and emission, respectively.  Singh et al. reported optimal wavelengths for the glucuronic acid derivative to be 378 nm and 425 nm for excitation and emission, respectively.  The only difference that might address the excitation wavelength difference is that Burrows and Clintron performed a second chloroform/methanol extraction. 

Gainey and Phelps (1975) performed kinetic studies upon UDPGDH via UV detection.  Enzymatic activity was assessed in a 1 cm cuvette at 31 °C containing 100 μmol glycine-NaOH buffer, pH 8.7, 1 μmol of NAD+, 1 μmol of UDPG and variable amounts of UDPGDH to a final volume of 1 mL.  The cuvette was preincubated at the desired temperature for 2.5 minutes prior to the addition of UPDG, which initiated the reaction.  Kinetics determination was performed by monitoring the change in absorbance at 340 nm, the optimal wavelength for NADH.

Grubb et al. (1993) developed an HPLC method for the separation and detection of UDPGA in 45 minutes.  The method, unlike its predecessors, did not require the derivatization of UDPGA, nor allow for possible purine interferences in monitoring the production of NADH due to is chromatographic separation.  Incubation protocols required the addition of homogenate equivalent to 5x105 cells to be placed in a 2 mL glass vial that contained 0.1 M glycine buffer, pH 8.7, and 1.25 mM NAD+.  The reaction was initiated by the addition of UDPGA to give a final concentration of 250 μM and a final volume of 275 μL.  The vial was capped and vortexed for 10 s and placed in a shaking water bath at 37 °C for 1 hour.  Termination of the reaction was done by heat treatment at 100 °C for 2 min with the vial cap still in place.  A 40 μL aliquot of the incubation sample was injected onto an Alltech Hypersil ODS (4.5 x 150 mm) C-18 analytical column for separation of the substrates and products from the enzymatic reaction.  Chromatographic separation was achieved by gradient elution, which used two mobile phase systems.  System A comprised of 40 mM ammonium phosphate and 5 mM tetrabutyl ammonium dihydrogen sulfate, pH 7.16 and system B was comprised of system A containing 50% acetonitrile (v/v).  Separation of substrates and products was performed with a gradient program at a flow rate of 0.5 mL/min.  Mobile phase composition was 95% system A and 5% system B for the first 25 minutes.  After which system B was increased to 35% over a ten minute period and returned to 5% over ten minutes.  Kinetics were determined based upon the production of UDPGA, eluted at 24 minutes, with monitoring the wavelength 262 nm with a UV detector. 

Higley and Hyslop (1998) performed kinetics studies upon UDPGDH using the method developed by Grubb (1993) with modifications.  System A was comprised of    80 mM ammonium phosphate and 10 mM tetrabutyl ammonium dihydrogen sulfate, pH 7.16.  System B was prepared by adding 500 mL of acetonitrile to 500 mL of system A, thus having this system at 40 mM ammonium phosphate and 5 mM tetrabutyl ammonium dihydrogen sulfate.  Therefore, system B was comprised of 40 and 5 mM for the ammonium phosphate and ion-pairing agent, respectively, which is double the concentration as the protocol outlined by Grubb (1993).  The remaining 500 mL of system A was diluted to 1 liter with nanopure water.  Chromatography was preformed on a Supleco LC-18-DB (4.6 x 150 mm) analytical column rather than an Alltech Hypersil ODS C-18 analytical column, at a flow rate of 1.0 mL/min in comparison to the flow rate of 0.5 mL/min.  An isocratic program, 80% system A and 20% system B, was used to separate analytes.  Detection of UDPGA was done with a UV-detector set at 262 nm.  UDPGA eluted from the column within a range of 5.38 to 5.66 minutes.

Current Work:  The reverse-phase HPLC methodology developed employed a Discovery C18® analytical column, and allowed base line separation of all substrates and products as well as column regeneration within 14 minutes.  In addition to a faster and more enviromentally friendly separation method (mobile phase consisting of imidazole rather than acetonitrile), detection limits were increased by nearly 70% compared to previously developed methodologies. 


LC/MS Methodologies were also investigated and allowed separation of substrates and products with quantification and dection based upon UDPGA fragmentation in negative ESI mode of acquition.  Detection limits were further increased into fmol compared to the rHPLC methodologies also developed.  The only set back in method development was the inability of detecting the UDPGA fragment at in high (umol) and low concentrations (pmol or fmol).  Thus, LC/MS methodology for the assessment of UDPGDH activity based upon UDPGA detection requires two standard curves.  Futher investigation in currently underway to correct this issue.






Enzymatic methodologies for the assessment of UDPGDH activity, utilizing HPLC, have also been development.  The methodologies for assessment are currently being written for publication.  The method development allows a for a 4 minute assay window of UDPGDH activity towards 6TP, it's metabolites and analogs.

Synthesis of 6-Thiopurine Analogs

Current being written up for publication. 


Inhibition Studies of Various 6-TP analogs and Metabolites towards UDP-Glucose Dehydrogenase
 
To assess synthesized analogs of 6TP ability to reduce jaundice onset, a UV spectrophotometric method was developed to determine inhibitor constants for 6TP, its metabilities and analogs.  The method development utlitizies a glycylgylcine medium at UDPGDH's optimium pH to assess activity (specific methodology is currently being written for publication).  It was found that 6-thiouric acid (6TU) inhibits UDPGDH 10 times more potently than 6TP.  6-Thioxanthine (6TX) and 6-thioguanine (6TG) have also been assessed for inhibitor activity towards UDPGDH.  Results are currently being drafted for publication concerning 6TX adn 6TG.  Inhibitor data suggested that position 8 of the purine ring is key to inhibition, whereas position 2 barely effects UDGPDH activity.

















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