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Therapeutic Drug Monitoring Jun 2009Azathioprine and 6-mercaptopurine are often used in the treatment of patients with inflammatory bowel disease (IBD). They are prodrugs and undergo a complex metabolism...
Azathioprine and 6-mercaptopurine are often used in the treatment of patients with inflammatory bowel disease (IBD). They are prodrugs and undergo a complex metabolism to active and inactive metabolites. Thiopurine treatment is monitored in many laboratories by measuring metabolite concentrations in erythrocytes (red blood cells). The metabolites of interest are not measured directly but as hydrolysis products, which can be produced from several metabolites. The aim of this study was to examine which metabolites are actually measured during routine monitoring. Samples from 18 patients treated with a thiopurine were analyzed by a typical routine high-performance liquid chromatography method for therapeutic drug monitoring and by a newly developed specific method measuring thioguanosine monophosphate (TGMP), thioguanosine diphosphate (TGDP), and thioguanosine triphosphate (TGTP), as well as methylthioinosine monophosphate (meTIMP), and the results were compared. 6-Thioguanine nucleotide (TGN) values detected by the routine method were 69% (range 40%-90%) of the sum of TGMP, TGDP, and TGTP measured by the specific method. TGTP and TGDP contributed 85% (range 78%-90%) and 14% (range 10%-21%) of the TGN total, respectively. Thioguanosine was not found in any patient sample. The concentration of meTIMP obtained by the routine method was 548% of the value obtained by the specific method (range 340%-718%). The difference in TGN measurements between the routine and specific methods can be explained by low hydrolysis efficiency in the routine method, although the most likely explanation for the difference in meTIMP values is that not yet identified metabolites are codetermined in the routine high-performance liquid chromatography method. Concentrations reported as TGN during therapeutic drug monitoring of thiopurine metabolites consist of TGDP and TGTP with a minor contribution of the TGMP. Concentrations reported as meTIMP or methyl mercaptopurine consist in part of meTIMP, but other not yet identified metabolites are codetermined.
Topics: Adult; Aged; Aged, 80 and over; Azathioprine; Drug Monitoring; Female; Humans; Inflammatory Bowel Diseases; Male; Mercaptopurine; Middle Aged; Young Adult
PubMed: 19363461
DOI: 10.1097/FTD.0b013e3181a1ea58 -
Current Protocols in Nucleic Acid... Sep 2004The N(6)-alkyladenosines and 2-methylthio-N(6)-alkyladenosines are the most common modified adenosine nucleosides, and transfer ribonucleic acids (tRNA) are particularly... (Review)
Review
The N(6)-alkyladenosines and 2-methylthio-N(6)-alkyladenosines are the most common modified adenosine nucleosides, and transfer ribonucleic acids (tRNA) are particularly rich in these modified nucleosides. They are present at position 37 of the anticodon arm, and the contributions of these hypermodified nucleosides to codon-anticodon interactions as well as to translation are significant, although they are not fully understood. This unit describes a new chemical synthesis method for oligoribonucleotides containing N(6)-alkyladenosines and 2-methylthio-N(6)-alkyladenosines via postsynthetic modifications of precursor oligoribonucleotides. To obtain oligoribonucleotides containing N(6)-alkyladenosines, a precursor oligoribonucleotide carrying 6-methylthiopurine riboside residues was used, whereas for the synthesis of oligoribonucleotides containing 2-methylthio-N(6)-alkyladenosines, a precursor oligoribonucleotide carrying the 2-methylthio-6-chloropurine riboside was applied. This allowed synthesis of modified oligoribonucleotides containing naturally occurring modified nucleosides such as N(6)-isopentenyladenosine (i(6)A), N(6)-methyladenosine (m(6)A), 2-methylthio-N(6)-isopentenyladenosine (ms(2)i(6)A), and 2-methylthio-N(6)-methyladenosine (ms(2)m(6)A), as well as several unnaturally modified adenosine derivatives.
Topics: Adenosine; Alkylation; Inosine; Methylthioinosine; Models, Biological; Oligoribonucleotides; Organophosphorus Compounds
PubMed: 18428929
DOI: 10.1002/0471142700.nc0423s17 -
The Journal of General Virology Apr 2008The family Flaviviridae comprises positive-strand RNA viral pathogens of humans and livestock with few treatment options. We have previously shown that azathioprine...
The family Flaviviridae comprises positive-strand RNA viral pathogens of humans and livestock with few treatment options. We have previously shown that azathioprine (AZA) has in vitro activity against bovine viral diarrhea virus (BVDV). While the mechanism of inhibition is unknown, AZA and related thiopurine nucleoside analogues have been used as immunosuppressants for decades and both AZA metabolites and cellular genes involved in AZA metabolism have been extensively characterized. Here, we show that only certain riboside metabolites have antiviral activity and identify the most potent known antiviral AZA metabolite as 6-methylmercaptopurine riboside (6MMPr). The antiviral activity of 6MMPr is antagonized by adenosine, and is specific to BVDV and not to the related yellow fever virus. An essential step in the conversion of AZA to 6MMPr is the addition of a methyl group onto the sulfur atom attached to position six of the purine ring. Intracellularly, the methyl group is added by thiopurine methyltransferase (TPMT), an S-adenosyl methionine-dependent methyltransferase. Either chemically bypassing or inhibiting TPMT modulates antiviral activity of AZA metabolites. TPMT exists in several variants with varying levels of activity and since 6MMPr is a potent antiviral, the antiviral activity of AZA may be modulated by host genetics.
Topics: Adenosine; Animals; Antiviral Agents; Azathioprine; Bovine Virus Diarrhea-Mucosal Disease; Cattle; Cell Line; Diarrhea Viruses, Bovine Viral; Methylthioinosine; Methyltransferases; Virus Replication
PubMed: 18343842
DOI: 10.1099/vir.0.83381-0 -
Molecules (Basel, Switzerland) Aug 20056-Methylpurine-beta-D-riboside (beta-D-MPR) has been synthesized by coupling 6-methylpurine and 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribose using conditions that produce the...
6-Methylpurine-beta-D-riboside (beta-D-MPR) has been synthesized by coupling 6-methylpurine and 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribose using conditions that produce the beta-D-anomer exclusively. The in vitro antitumor effects of beta-D-MPR and 6-methyl-purine-alpha-D-riboside (alpha-D-MPR) in five human tumor cell lines showed that beta-D-MPR was highly active (IC(50) values ranging from 6 to 34 nM). alpha-D-MPR, although less active than beta-D-MPR, also exhibited significant antitumor effects (IC50 values ranging from 1.47 to 4.83 microM).
Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Humans; Methylthioinosine; Stereoisomerism
PubMed: 18007371
DOI: 10.3390/10081015 -
British Journal of Clinical Pharmacology Jan 2008Azathioprine and 6-mercaptopurine are steroid-sparing drugs used in inflammatory bowel disease (IBD). The polymorphic enzyme thiopurine S-methyltransferase (TPMT) is of...
AIMS
Azathioprine and 6-mercaptopurine are steroid-sparing drugs used in inflammatory bowel disease (IBD). The polymorphic enzyme thiopurine S-methyltransferase (TPMT) is of importance for thiopurine metabolism and occurrence of adverse events. The role of other thiopurine-metabolizing enzymes is less well known. This study investigated the role of inosine-5'-monophosphate dehydrogenase (IMPDH), which is a key enzyme in the de novo synthesis of guanine nucleotides and also strategically positioned in the metabolic pathway of thiopurines.
METHODS
IMPDH was measured in 100 healthy blood donors. IMPDH, TPMT and metabolite concentrations were studied in 50 patients with IBD on stable thiopurine therapy. IMPDH activity was measured in peripheral blood mononuclear cells. TPMT activity, 6-methylthioinosine 5'-monophosphate (meTIMP) and 6-thioguanine nucleotide (6-TGN) concentrations were measured in red blood cells, which is the current practice in clinical monitoring of thiopurines. Enzyme activities were related to metabolite concentrations and clinical characteristics.
RESULTS
A wide range of IMPDH activity was observed both in healthy blood donors (median 13.1, range 4.7-24.2 nmol mg(-1) protein h(-1)) and IBD patients (median 14.0, range 7.0-21.7). There was a negative correlation between IMPDH activity and dose-normalized meTIMP concentrations (r(s) = -0.31, P = 0.03), but no evident correlation to 6-TGN concentration or the meTIMP/6-TGN ratio. There were no significant correlations between TPMT activity and metabolite concentrations.
CONCLUSION
Even though the meTIMP concentrations correlated inversely to the IMPDH activity, the role of IMPDH in balancing the formation of methylated and phosphorylated metabolites was not evident. Taken together, the results give cause to question established opinions about thiopurine metabolism.
Topics: Adult; Aged; Aged, 80 and over; Azathioprine; Biomarkers; Female; Humans; IMP Dehydrogenase; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methyltransferases; Middle Aged; Polymorphism, Genetic
PubMed: 17662091
DOI: 10.1111/j.1365-2125.2007.02985.x -
Gastroenterologia Y Hepatologia Nov 2006Determination of the activity of thiopurine methyltransferase (TPMT) and of thiopurine metabolites (6-thioguanine and 6-methylmercaptopurine nucleotides) could be useful... (Review)
Review
Determination of the activity of thiopurine methyltransferase (TPMT) and of thiopurine metabolites (6-thioguanine and 6-methylmercaptopurine nucleotides) could be useful for individualized monitoring of azathioprine (AZA) and 6-mercaptopurine (6-MP) doses. TPMT activity in the general population follows a trimodal distribution, in which approximately 0.3% of the population is homozygotic for the low-activity allele. A notable correlation has been observed between the low TPMP activity genotype or phenotype and the risk of myelotoxicity. Patients with a high TPMT activity genotype or homozygous phenotype should receive immunosuppressive doses that have clearly been demonstrated to be effective. In contrast, in patients with a low TPMT activity genotype or homozygous phenotype, the use of AZA/6-MP should be contraindicated or only very small doses should be administered. Importantly, TPMP deficiency explains only some cases of myelotoxicity and consequently periodic laboratory testing should be performed in patients receiving AZA/6-MP, even though TPMP function may be normal. Currently, the utility of routine thiopurine metabolite determinations in patients undergoing AZA/6-MP therapy has not been established and this practice should be limited to specific situations such as lack of response to thiopurine therapy or the occurrence of thiopurine-related adverse effects. Randomized trials comparing the routine strategy of AZA/6-MP dosing (based exclusively on the patient's weight) versus individualized monitoring (based on quantification of TPMP activity and/or thiopurine metabolites) are required before definitive conclusions on the most effective alternative can be drawn.
Topics: Antimetabolites; Azathioprine; Drug Monitoring; Genotype; Humans; Immunosuppressive Agents; Mercaptopurine; Methylthioinosine; Methyltransferases; Phenotype; Thioguanine
PubMed: 17129552
DOI: 10.1157/13094355 -
The Journal of Biological Chemistry Sep 2006Purine nucleoside phosphorylase (PNP) is an important component of the nucleotide salvage pathway in apicomplexan parasites and a potential target for drug development....
Purine nucleoside phosphorylase (PNP) is an important component of the nucleotide salvage pathway in apicomplexan parasites and a potential target for drug development. The intracellular pathogen Toxoplasma gondii was therefore tested for sensitivity to immucillins, transition state analogs that exhibit high potency against PNP in the malaria parasite Plasmodium falciparum. Growth of wild-type T. gondii is unaffected by up to 10 microm immucillin-H (ImmH), but mutants lacking the (redundant) purine salvage pathway enzyme adenosine kinase are susceptible to the drug, with an IC50 of 23 nm. This effect is rescued by the reaction product hypoxanthine, but not the substrate inosine, indicating that ImmH acts via inhibition of T. gondii PNP. The primary amino acid sequence of TgPNP is >40% identical to PfPNP, and recombinant enzymes exhibit similar kinetic parameters for most substrates. Unlike the Plasmodium enzyme, however, TgPNP cannot utilize 5'-methylthio-inosine (MTI). Moreover, TgPNP is insensitive to methylthio-immucillin-H (MT-ImmH), which inhibits PfPNP with a Ki* of 2.7 nm. MTI arises through the deamination of methylthio-adenosine, a product of the polyamine biosynthetic pathway, and its further metabolism to hypoxanthine involves PfPNP in purine recycling (in addition to salvage). Remarkably, analysis of the recently completed T. gondii genome indicates that polyamine biosynthetic machinery is completely lacking in this species, obviating the need for TgPNP to metabolize MTI. Differences in purine and polyamine metabolic pathways among members of the phylum Apicomplexa and these parasites and their human hosts are likely to influence drug target selection strategies. Targeting T. gondii PNP alone is unlikely to be efficacious for treatment of toxoplasmosis.
Topics: Amino Acid Sequence; Animals; Escherichia coli; Humans; Inhibitory Concentration 50; Kinetics; Molecular Sequence Data; Nucleotides; Plasmodium falciparum; Purine-Nucleoside Phosphorylase; Sequence Homology, Amino Acid; Substrate Specificity; Toxoplasma
PubMed: 16829527
DOI: 10.1074/jbc.M602624200 -
Scandinavian Journal of Gastroenterology Oct 20056-thioguanine (6-TG) has emerged as a promising therapeutic alternative in patients with Crohn's disease intolerant or resistant to azathioprine (AZA) and/or... (Clinical Trial)
Clinical Trial Comparative Study
OBJECTIVE
6-thioguanine (6-TG) has emerged as a promising therapeutic alternative in patients with Crohn's disease intolerant or resistant to azathioprine (AZA) and/or 6-mercaptopurine (6-MP). The aim of the present study was to evaluate the safety and efficacy of 6-TG in patients with ulcerative colitis (UC) or indeterminate colitis (IC) intolerant or resistant to AZA/6-MP.
MATERIAL AND METHODS
Twenty patients with an acute flare, steroid-dependent or steroid-refractory disease attending our outpatient department were included in the study. Measurement of 6-TG nucleotide levels was done to check compliance. Complete, partial and non-response were defined by means of the clinical activity index and the daily steroid demand. Secondary outcome parameters included changes in cumulative steroid doses, C-reactive protein (CRP) levels, and an endoscopic score.
RESULTS
Out of 20 patients 4 were excluded owing to noncompliance; 2/16 compliant patients (13%) had to be prematurely withdrawn because of adverse events, which ceased upon drug discontinuation. By per-protocol analysis, 5/14 patients (36%) were complete, 6/14 (43%) partial and 3/14 (21%) non-responders. In addition to the reduction of the cumulative steroid dose over 3 months, CRP decreased in the study population and the endoscopic score decreased in treatment responders.
CONCLUSIONS
Treatment with 6-TG was effective in patients with UC or IC previously intolerant or resistant to AZA/6-MP. Future work is needed to define a subpopulation of patients at low risk for its potential hepatotoxicity, which we assume will benefit from 6-TG.
Topics: Adult; Aged; Anti-Inflammatory Agents; Azathioprine; Biomarkers; C-Reactive Protein; Colitis, Ulcerative; Dose-Response Relationship, Drug; Endoscopy, Gastrointestinal; Female; Genotype; Guanine Nucleotides; Humans; Male; Mercaptopurine; Methylthioinosine; Methyltransferases; Middle Aged; Orosomucoid; Patient Compliance; Prospective Studies; Quality of Life; Thioguanine; Thionucleotides; Treatment Outcome
PubMed: 16265777
DOI: 10.1080/00365520510023369 -
The Journal of Biological Chemistry Aug 2005Human purine nucleoside phosphorylase (huPNP) is essential for human T-cell division by removing deoxyguanosine and preventing dGTP imbalance. Plasmodium falciparum...
Human purine nucleoside phosphorylase (huPNP) is essential for human T-cell division by removing deoxyguanosine and preventing dGTP imbalance. Plasmodium falciparum expresses a distinct PNP (PfPNP) with a unique substrate specificity that includes 5'-methylthioinosine. The PfPNP functions both in purine salvage and in recycling purine groups from the polyamine synthetic pathway. Immucillin-H is an inhibitor of both huPNP and PfPNPs. It kills activated human T-cells and induces purine-less death in P. falciparum. Immucillin-H is a transition state analogue designed to mimic the early transition state of bovine PNP. The DADMe-Immucillins are second generation transition state analogues designed to match the fully dissociated transition states of huPNP and PfPNP. Immucillins, DADMe-Immucillins and related analogues are compared for their energetic interactions with human and P. falciparum PNPs. Immucillin-H and DADMe-Immucillin-H are 860 and 500 pM inhibitors against P. falciparum PNP but bind human PNP 15-35 times more tightly. This common pattern is a result of kcat for huPNP being 18-fold greater than kcat for PfPNP. This energetic binding difference between huPNP and PfPNP supports the k(chem)/kcat binding argument for transition state analogues. Preferential PfPNP inhibition is gained in the Immucillins by 5'-methylthio substitution which exploits the unique substrate specificity of PfPNP. Human PNP achieves part of its catalytic potential from 5'-OH neighboring group participation. When PfPNP acts on 5'-methylthioinosine, this interaction is not possible. Compensation for the 5'-OH effect in the P. falciparum enzyme is provided by improved leaving group interactions with Asp206 as a general acid compared with Asn at this position in huPNP. Specific atomic modifications in the transition state analogues cause disproportionate binding differences between huPNP and PfPNPs and pinpoint energetic binding differences despite similar transition states.
Topics: Animals; Aspartic Acid; Cattle; Humans; Hydrogen-Ion Concentration; Kinetics; Models, Chemical; Plasmodium falciparum; Polyamines; Protein Binding; Protein Structure, Tertiary; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Purines; Pyrimidinones; Pyrroles; Static Electricity; Substrate Specificity; T-Lymphocytes
PubMed: 15961383
DOI: 10.1074/jbc.M505033200 -
The Journal of Biological Chemistry Mar 2005Plasmodium falciparum is unable to synthesize purine bases and relies upon purine salvage and purine recycling to meet its purine needs. We report that purines formed as...
Plasmodium falciparum is unable to synthesize purine bases and relies upon purine salvage and purine recycling to meet its purine needs. We report that purines formed as products of polyamine synthesis are recycled in a novel pathway in which 5'-methylthioinosine is generated by adenosine deaminase. The action of P. falciparum purine nucleoside phosphorylase is a convergent step of purine salvage, converting both 5'-methylthioinosine and inosine to hypoxanthine. We used accelerator mass spectrometry to verify that 5'-methylthioinosine is an active nucleic acid precursor in P. falciparum. Prior studies have shown that inhibitors of purine salvage enzymes kill malaria, but potent malaria-specific inhibitors of these enzymes have not been described previously. 5'-Methylthio-immucillin-H, a transition state analogue inhibitor that is selective for malarial relative to human purine nucleoside phosphorylase, kills P. falciparum in culture. Immucillins are currently in clinical trials for other indications and may also have application as anti-malarials.
Topics: Adenine; Adenosine Deaminase; Amino Acid Sequence; Animals; Conserved Sequence; Escherichia coli; Humans; Hypoxanthine; Inosine; Methylthioinosine; Molecular Sequence Data; Plasmodium falciparum; Purine-Nucleoside Phosphorylase; Purines; Sequence Alignment; Sequence Homology, Amino Acid
PubMed: 15576366
DOI: 10.1074/jbc.M412693200