Particular Metabolic Profile of Patient with Total

International Journal of Pharmaceutical Science and Health Care Issue 5, Vol. 1 (Jan-Feb. 2015) Available online on http://www.rspublication.com/ijphc/index.html ISSN 2249 – 5738

PARTICULAR METABOLIC PROFILE OF PATIENT WITH TOTAL DIHYDROPYRIMIDINE DEHYDROGENASE DEFICIENCY (DPD) Saif Eddine GAMAOUNa,b, Nissem ABDEJALLILa, Mhamed Ali HAMZAb, Saad SAGUEMa aLaboratory of Metabolic Biophysics, Professional and Applied Environmental Toxicology, Medicine faculty of Sousse, Mohamed Karoui street, Sousse 4002, Tunisia bDepartment of chemistry, sciences faculty of Monastir, Environment street, Monastir 5019, Tunisia *Author for correspondence at present address: Saif Eddine GAMAOUN Higher Institute of Technological Studies (ISET) Ksar Hellal-Hadj Ali Soua street-5070-Ksar Hellal,Tunisia  Fonction : Engineer of analytical chemistry and instrumentation - Assistant teacher in ISET Ksar Hellal - Tunisia Tel.: (+216) 73 475 900 / (+216) 98 685 958 Fax: (+216) 73 475 163 E-mail: saifeddineg @ yahoo.fr

ABSTRACT

5-Fluorouracil (5-FU) remains one of the most used chemotherapeutics agents in the treatment of several types of cancers. Its use could nevertheless, raise a problem of pharmacovigilance. It may engender a very grave toxicity which can be fatal at certain patient's presenting an enzymatic deficit with dihydropyrimidine dehydrogenase (DPD). The identification of the subjects at risk through the detection of a partial or total deficit before starting the treatment with 5-FU, could allow to adapt individually the prescribed dose, to optimize the therapeutic efficacy and reduce potential toxicities at expanding patients of a partial deficit to DPD. Such a screening would especially become more important when it is about a complete deficit in DPD. Such metabolic abnormality whose frequency is of the order of one per thousand, would indeed constitute a contraindication absolved from any chemotherapy with 5-FU (mortal toxicities). In the present study, we optimized and validated a chromatographic method of analysis allowing to separate and to identify the structural analogues of 5-FU and studied a particular metabolic profile at a total overdrawn patient in DPD compared to a normal profile‘s patient.

KEY-WORDS: 5-Fluorouracil, Dihydropyrimidine dehydrogenase, Deficiency, Dihydrouracil/uracil ratio, Hypoxanthine, Xanthine.

CORRESPONDING AUTHOR: Saifeddine GAMAOUN

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International Journal of Pharmaceutical Science and Health Care Issue 5, Vol. 1 (Jan-Feb. 2015) Available online on http://www.rspublication.com/ijphc/index.html ISSN 2249 – 5738

INTRODUCTION

The anticancer agent 5-Fluorouracil (5-FU) remains one of the most prescribed chemotherapeutic drugs for the curative and palliative treatment of patients with cancers of the gastrointestinal tract, breast, head and neck [1-8]. Usually, only a small proportion of the administered dose (by intravenous way or orally) is amended by anabolism in fluoronucleotides which are responsible for the cytotoxic potency and the therapeutic of the 5-FU. More than 85% of the administered medicinal dose is excreted under forms of catabolites stemming essentially from the metabolic pathway catalyzed by the dihydropyrimidine dehydrogenase ( DPD), the hepatic enzyme responsible for the catabolism of the 5-FU but also for pyrimidics bases (Uracil and Thymine) [7-11]. A wide interpersonal variability of the enzymatic activity of the DPD has been reported. Therefore, the therapeutic efficiency and the toxic effects of the 5-FU would be indirectly controlled by this enzyme which, by catalysing the detoxifying way competing with that of the anabolism, controls the quantity of fluoronucleotides formed by the anabolic way. The partial or complete deficiency of DPD activity could lead to an overproduction of anabolites assets and engender serious toxicity which can be mortal in case of a total deficit of the enzymatic activity of the DPD [ 12 ]. In this study, we have conducted plasmatic analyzes of the 5-FU used in chemotherapy and its endogenous structural analogues in particular pyrimidine bases. We were able to develop the analytical method concerning the choices of the liquid-liquid extraction of plasma samples and of the chromatographic technique by HPLC used in reversed phase. Two of the structural analogues of the 5-FU were identified (Uracil and endogenous Thymine) as structures accumulated at the expanding patients with a complete deficit in DPD. We were also interested in the identification of two other endogenous metabolites having the particularity to accumulate in the body (blood plasma) to a subject which presents a complete DPD deficiency. We were able to identify these both endogenous metabolites (Hypoxanthine and Xanthine) by using the appropriate techniques of analyses in particular the mass spectrometry coupled with the chromatography (LC-MS). We were afterward able to confirm these chemical structures and it is true from the standard products thanks to their chromatographic behavior. So besides the accumulation of both Pyrimidine bases which reflects the complete deficiency of DPD, the accumulation of the endogenous hypoxanthine and the Xanthine may also reflect such a deficit although it could also establish an evolution of the disease as a marker of precocity or a metastatic degree of this disease. MATERIALS AND METHODS

Sample preparation A 5 mL sample of whole blood was collected in an EDTA tube. Centrifugation at 3500 rpm for 15 min was performed and plasma was isolated. Ammonium sulfate (600 mg) was then added and vortex-mixed for 2 min. addition of 3 mL of the extraction solution (propanol - ethyl acetate (15:85, v/v)) was then performed. The sample was vortex-mixed for 3 min and

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International Journal of Pharmaceutical Science and Health Care Issue 5, Vol. 1 (Jan-Feb. 2015) Available online on http://www.rspublication.com/ijphc/index.html ISSN 2249 – 5738 centrifuged for 20 min (4000 rpm). Two milliliters of the organic phase were collected and evaporated to dryness at 55 °C under a flow of nitrogen gas. The dried residue was dissolved in 50 µL of mobile phase (potassium phosphate buffer KH2PO4 0.05 M, pH = 3.2). A 5 µL of this sample extract were injected into the HPLC. HPLC apparatus and chromatographic conditions The chromatographic analysis was developed using a HPLC system which consisted of an Agilent series 1200 LC system (Agilent Technologies, Waldbronn Germany) equipped with a multiwavelength UV detector (diode array detector). Data acquisition and processing were accomplished automatically by computing integrator. Chromatographic separation was achieved by the use of two Hypersil ODS (5 µm, 250 mm x 4 mm and 5 µm, 150 mm x 4 mm respectively) columns maintained at 33 °C and protected by a guard column containing the same packing material. Elution was carried out isocratically with a mobile phase consisting of 0.05 M potassium phosphate buffer (pH = 3.2) at a flow rate of 0.5 mL/min. For each analysis the UV detection of the Dihydrouracil (UH2) , Uracil (U), Thymine, Hypoxanthine and Xanthine was monitored at 205-210 and 268 nm.

LC-MS conditions The LC–MS system consisted of an Agilent 1100 system coupled to a tandem quadrupole 4000 Qtrap system. Chromatographic separations were performed on a Kromasil ODS column (5 µm, 250 mm × 4.6 mm). The column temperature was held at 40 °C. The optimum separation for these analytes was achieved using the mixture of water, acetonitrile and formic acid (85:14:1, v/v/v) as a mobile phase. A simple and rapid isocratic LC/MS coupled with electrospray ionization (ESI) method for simultaneous separation and determination of Hypoxanthine and Xanthine in human plasma was developed.

RESULTS AND DISCUSSION

The dihydropyrimidine dehydrogenase (DPD) is an enzyme involved in the catabolism of pyrimidine bases (Uracil and Thymine), but also of 5-fluorouracil (5-FU) [13,14] a drug widely used in chemotherapy for the treatment of several types of cancers and in particular tumors of the digestive system, breast and brain. The indirect role of this enzyme appears as a factor modulating the therapeutic and toxic effects of 5-FU by controlling the proportion of anabolised medicine [15]. A decrease in the activity of this enzyme decreases the clearance of 5-FU and increases its toxicity. This deficit favors the occurrence of severe toxicity in patients treated with 5-FU. This toxicity is usually fatal in the case of complete DPD deficiency despite of available intensive care reanimation [16, 17]. Looking for a DPD deficiency should therefore be performed before therapy with 5-FU to anticipate severe toxicities. And it should lead to a dose adjustment with individual monitoring of the pharmacokinetics of 5-FU in the case of a partial deficiency of DPD activity. However a complete DPD deficiency should be an absolute contraindication against the use of chemotherapy based on 5-FU in order to avoid the occurrence of fatal toxicities [18]. Several DPD deficits screening methods were suggested and were based on genetic or metabolic approaches. However they were not suitable in clinical routine for relatively heavy and expensive technology. In this work, we performed chromatographic analysis of plasma

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International Journal of Pharmaceutical Science and Health Care Issue 5, Vol. 1 (Jan-Feb. 2015) Available online on http://www.rspublication.com/ijphc/index.html ISSN 2249 – 5738 extracted from a blood sample from a normal patient with no DPD deficiency (Figure 1) using a simultaneous detection at different wavelength and a plasma sample from a blood sample from a patient having a total DPD deficiency (Figure 2). Metabolic index assessing the activity of DPD is obtained by calculating the ratio UH2 / U [19, 20]. The complete deficiency results in no relation characterized by the total absence of catabolite UH2 and an abnormal accumulation of plasmatic Uracil.

Full scale : 6 mAU

UH2 unknown 1

U unknown 2

Fig 1: Chromatogram of a plasma sample obtained from a normal subject who is not deficient in DPD, recorded at three wavelengths: 205, 210 and 268 nm (UH2 is not detected to 268 nm).

Full scale : 350 mAU

absence of UH2

U unknown 1

unknown 2 Thymine

Fig 2: Chromatogram of a plasma sample from a subject with a total DPD deficiency recorded at three wavelengths: 205, 210 and 268 nm (UH2 is not detected to 268 nm).

A ratio greater than 1 indicates a normal DPD activity. Greater than zero but less than unity value indicates the presence of a partial deficiency of the enzyme activity and requires an adjustment in the dose of 5-FU required to optimize therapeutic efficacy and reduce drug toxicity [19]. By comparing the two chromatograms represented in figures 1 and 2, relating to a plasma sample taken from a normal subject with no DPD deficiency and a plasma sample taken from an individual having a total DPD deficiency, there is a plasmatic accumulation of endogenous Uracil and thymine and also two other unknown metabolites 1 and 2, unknown products that we have identified by liquid chromatography-mass spectrometry (LC-MS). These chemical

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Fig 3: Chromatogram after the injection of 5 .mu.l of an aqueous mixture containing 0.025 mg/mL water of Hypoxanthine, Xanthine and Thymine, recorded at 205 nm. The elution times are respectively 22.21min, 26.5 min and 36.64 min. The absorption spectra of endogenous metabolites shown in figures 4 and 5 also confirm the results that we achieved.

220 240 260 280 300 320 340 360 380 nm

Fig 4: Hypoxanthine absorption spectrum.

220 240 260 280 300 320 340 360 380 nm

Fig 5: Xanthine absorption spectrum.

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International Journal of Pharmaceutical Science and Health Care Issue 5, Vol. 1 (Jan-Feb. 2015) Available online on http://www.rspublication.com/ijphc/index.html ISSN 2249 – 5738

Structural identification of these two metabolites (Hypoxanthine and Xanthine), Uracil and Thymine, would improve the understanding of the molecular and enzymatic mechanisms in complete DPD deficiency and would lead to markers for screening cancer patients who may develop severe toxicities before any treatment with 5-FU. CONCLUSION

As part of this work, we have optimized and have validated a chromatographic method of analysis, allowing to separate and to identify the structural analogues of 5-FU. We were able to develop the analytical method based on liquid-liquid extraction of plasma samples and the technique chromatographic by HPLC used in reversed phase. Two of the structural analogues of the 5-FU were already identified (Uracil and endogenous Thymine) as structures accumulated at the expanding patients of a complete deficit in DPD. We also identified two other endogenous metabolites having the feature to accumulate in the body (blood plasma) to a subject which presents a complete DPD deficiency. It is about, Hypoxanthine and Xanthine. So in addition to the accumulation of two Pyrimidine bases reflecting the complete deficiency of DPD, the accumulation of the endogenous Hypoxanthine and the Xanthine could also reflect such a deficit and may establish an evolution of the disease as a marker of precocity or a metastatic illness degree.

ACKNOWLEDGEMENT

This study was supported by the “Laboratory of Metabolic Biophysics, Professional and Applied Environmental Toxicology (LR 12ES02).

REFERENCES

[1] B.Robert, Diasio, E.Barry, and Harris, Clinical pharmacology of 5-fluorouracil, Clinical pharmacokinetics, Vol.16, pp.215-237, 1989.

[2] M.David, Acomprehensive Review of 5-Fluorouracil and Leucovorin in Patients With Metastatic Colorectal Carcinoma, Vol.80, 1997.

[3] H.Anderson and M.K. Palmer, Measuring quality of life: impact of chemotherapy for advanced colorectal cancer. Experience from two recent large phase III trials, British journal of cancer, Vol.77, pp.9-14, 1998.

[4] T.Andre, P.Colin, C.Louvet, E.Gamelin, O.Bouche, and E. Achille, Semimonthly versus monthly regimen

R S. Publication, [email protected] Page 35

International Journal of Pharmaceutical Science and Health Care Issue 5, Vol. 1 (Jan-Feb. 2015) Available online on http://www.rspublication.com/ijphc/index.html ISSN 2249 – 5738

of fluorouracile and leucovorin administered for 24 or 36 weeks as adjuvant therapy in stage II and III colon cancer: results of a randomized trial, J Clin Oncol, Vol.21, pp.2896-2903, 2003.

[5] L.Chaisemartin, and M.A.Loriot, Pharmacogénétique des medicaments anticancéreux, Pathologie Biologie, Vol.53, pp.116-124, 2005.

[6] F.Harmozian, J.Guido Shmitt , E.Sagulenko, M.Schwab, and L.Savelyeva, FRA1E common fragile site breaks map within a 370 kilobase pair region and disrupt the dihydropyrimidine dehydrogenase gene (DPYD), Cancer Letters, Vol.246, pp.82-91, 2007.

[7] K.Ogura, T.Ohnuma, Y.Minamide, A.Mizuno, T.Nishiyama, S.Nagashima, M.Kanamaru, A.Hiratsuka, T.Watabe, and T.Uematsu, Dihydropyrimidine dehydrogenase activity in 150 healthy japanese vollunteers and identification of novel mutations, Clin Cancer Res, Vol.11, pp.5104-5111,2005.

[8] M.Schwab, U.M.Zanger, C.Marx, E.Shaeffeler, K.Klein, J.Dippon, R.Kerb, J.Blievernicht, J.Fisher, U.Hofmann, C.Bokomeyer , and M.Eichelbauem, Role of genetic and no genetic factors for fluorouracil treatment related severe toxicity : A prospective clinical trial by the German 5-FU toxicity study group, J Clin Onco, Vol.26, pp.2131-2138, 2008.

[9] R.Coriat, and S.Chaussade, Pour ou contre le phénotypage/génotypage des patients traits par le 5-fluorouracile pour prévenir les effets indésirables, Thérapie, Vol.62, pp.1-5, 2007.

[10] J.Brockmöller, and M.V.Tzvetkov, Pharmacogenetics: data, concepts and tools to improve drug discovery and drug treatment, Eur J Clin Pharmacol, Vol.64, pp.133-157, 2008.

[11] M.R.Johnson, W.Kangsheng, and R.B.Diasio, Profound Dihydropyrimidine dehydrogenase deficiency resulting from a novel compound heterozygote genotype, Clin Cancer Res, Vol.8, pp.768-774, 2002.

[12] A.B.P.Van kuilenburg, H.J.Klumpen, A.M.Westermann, L.Zoetekouw, H.Van Lenthe, P.J.M.Bakker, D.J.Richel, and H.J.Guchelaar, Increased Dihydropyrimidine dehydrogenase activity associated with mild toxicity in patients treated with 5-Fluorouracil and leucovorin, Eur J Cancer, Vol.43, pp.459-465, 2007.

[13] T.Aparicio, M.Ducreux, and S.Chaussade S, 5-Fluorouracil : metabolism and current indications in

R S. Publication, [email protected] Page 36

International Journal of Pharmaceutical Science and Health Care Issue 5, Vol. 1 (Jan-Feb. 2015) Available online on http://www.rspublication.com/ijphc/index.html ISSN 2249 – 5738

digestive cancer treatment, Gastroenterol Clin Biol, Vol.26, pp.38-47, 2002.

[14] D.J.Sweeny, S.Barnes , and R.B.Diasio, Production of cholestasis by 2 fluoro--alanine- chenodeoxycholic acid: possible role of this metabolite in the cholestasis associated with hepatic arterial infusion of fluoropyrimidines, Proceedings of the American association for cancer research, Vol.29, pp.486, 1988.

[15] C.Foa, Bondiau, R.Largillier, G.Milano, and N.Magné, Déficit en Dihydropyrimidine déshydrogénase (DPD) et toxicités au 5-fluoro-uracile, Rev Med Interne, Vol.21, pp.1133-1134, 2000.

[16] T.Lecomte, P.Laurent-Puig, and M.A.Loriot, Pharmacogénétique en hépato- gastroentérologie, Hépato Gastro, Vol.13, pp.275-287, 2006.

[17] G.Milano, M.C.Etienne, N.Renee, A.Thyss, M.Schneider, and A.Ramioli, Relation ship between fluorouracil systemic exposure and tumor response and patient survival, Journal of clinical oncology, Vol.12, pp.1291-1295,1994.

[18] G.Milano, M.C.Etienne, and V.Pierrefite, Dihydropyrimidine dehydrogénase deficiency and fluorouracil- related toxicity, Br J Cancer , Vol.79, pp.627-630, 1999.

[19] E.Gamelin, M.Boisdron-Cell, V.Guerin-Meyer, R.Delva, A.Lortholary, F.Genevieve, F.Larra, N.Ifrah, and J.Robert, Correlation between uracil and dihydrouracil plasma ratio, fluorouracil (5-FU) pharmacokinetic parameters, and tolerance in patients with advanced colorectal cancer: a potential interest for predicting 5-FU toxicity and determining optimal 5-FU dosage, J Clin Oncol, Vol.17, pp.1105- 1110, 1999.

[20] E.Gamelin, M.Boisdron-Celle, A.Turcant, A.Larra, and P. Allain P, Rapid and sensitive high-performance liquid chromatographic analysis of halogenopyrimidines in plasma, Journal of chromatography B, Vol.695, pp.409-416, 1997.

R S. Publication, [email protected] Page 37