during the last decade , several new chemotherapeutic agents have been evaluated for their beneficial effect in patients with non - small - cell lung cancer ( nsclc ) .
so far , gemcitabine plus cisplatin has emerged as one of the standard regimens for the treatment of advanced nsclc .
gemcitabine ( 2,2-difluorodeoxycytidine ; dfdc ) is a fluoropyrimidine antimetabolite that is transported into cells by equilibrative nucleoside transporters . within the cell
, gemcitabine is phosphorylated into gemcitabine monophosphate ( dfdcmp ) by deoxycytidine kinase ( dck ) and thereafter into di- and triphosphates .
gemcitabine triphosphate ( dfdctp ) is incorporated in dna , and subsequently only one deoxynucleotide molecule more can be inserted , which leads to the halt of dna synthesis .
this process is amplified through inhibition of the enzyme ribonucleotide reductase ( rnr ) by gemcitabine diphosphate ( dfdcdp ) .
inhibition of rnr leads to intracellular depletion of dcdp and dctp , which favours the incorporation of dfdctp into dna [ 6 , 7 ] .
only a small part of the gemcitabine dose is responsible for the cytotoxic effects , since more than 90% of the dose is inactivated by the enzyme cytidine deaminase ( cda ) into 2,2-difluorodeoxyuridine ( dfdu ) .
cda is ubiquitous in the human body , catalyzing the hydrolytic deamination of ( deoxy-)cytidine to ( deoxy-)uridine .
the total enzyme capacity of cda in all organs and tissues determines the biotransformation rate of gemcitabine into dfdu and thereby indirectly the duration of exposure to gemcitabine .
interestingly , cloning of human cda has revealed a 79a  >  c nonsynonymous coding single nucleotide polymorphism ( csnp ) in exon 1 of the cda gene corresponding with two protein variants with more than twofold difference in vitro deamination rates [ 8 , 9 ] .
the a genotype corresponds to the wild type lys - carrying enzyme and the c genotype to a gln - carrying variant ( gln27 cda ) .
the wild type cda enzyme has been reported to exert a 1.3- to 3.3-fold higher deamination rate of cytarabine than gln27 cda .
the aim of the current study was to evaluate the impact of the common 79a  >  c csnp in the cda gene on the pharmacokinetics of gemcitabine and its metabolite dfdu in gemcitabine - treated nsclc patients .
this study was performed as a site study of a phase iii trial of gemcitabine plus epirubicin versus gemcitabine plus cisplatin in advanced nsclc patients .
radiotherapy should have been completed at least 4  weeks before inclusion , and patients should have recovered from any toxic side effect .
all patients had to have a performance status    2 according to the eastern cooperative oncology group ( ecog ) scale and a life expectancy of at least 12  weeks .
an adequate bone marrow reserve and normal renal ( creatinine clearance    60  ml / min ) and liver function were required .
the glomerular filtration rate ( gfr ) was calculated according to the formula of cockcroft and gault .
patients were excluded if they had active infections , second primary malignancies , uncorrected hypercalcemia , or an lvef    45% measured by multiple gated acquisition ( muga ) scan .
gemcitabine ( gemzar , lilly , nieuwegein , the netherlands ) in a dose of 1,125  mg / m in 250  ml 0.9% nacl solution was administered intravenously as a 30  min infusion .
patients co - treated with epirubicin received the epirubicin dose after completion of the gemcitabine infusion .
blood sampling blood sampling was carried out on the first day of the first chemotherapy cycle .
for pharmacokinetic sampling , a cannula was placed intravenously in the arm of the patient contralateral to the side of drug administration .
blood samples of 9  ml were collected in heparinized tubes containing 0.25  mg tetrahydrouridine ( thu ) in 50  l water , just before chemotherapy and at t  =  25 , 40 , 50 , 60 , 75 , 90 , 105 , 120 , 150 , 180 , 270 , and 360  min after the start of the gemcitabine infusion .
blood samples were immediately placed on ice and centrifuged at 2,500  g for 10  min within 1  h after collection .
genotyping material harvesting of oral mucosa cells was performed before chemotherapy by thorough mouth rinsing for 30  s with 5  ml 0.9 % nacl solution .
the cell suspension was collected in plastic cups and subsequently placed on ice . within 1  h after collection ,
the cell suspension was centrifuged for 10  min at 190  g at 4c .
the supernatant was discarded and cells were washed in 10  ml ice cold phosphate buffered saline , ph  =  7.40 ( pbs ) and centrifuged for 10  min at 190  g at 4c .
the remaining suspension was transferred to a microcentrifuge cup and centrifuged briefly ( 15  s ) at 10,000  g. the supernatant was discarded , and the pellet was kept frozen at 80c until further analysis . gemcitabine hydrochloride and dfdu
the analysis of gemcitabine and dfdu in plasma was carried out by high performance liquid chromatography as described by freeman et al . .
the cda exon 1 polymorphism ( c / a ) at codon position 27 was genotyped by direct sequencing , in both directions , of pcr - amplified genomic dna .
first , the cda region flanking the polymorphic site was amplified using pcr with forward primer 5- agtagcttccccttccagtagc and reverse primer 5-cctcttcctgtacatcttcctctg .
the 25  l reactions contained 2.5 units taq polymerase ( amersham biosciences , uppsala , sweden ) ; 0.5  mm dntp mix ( roche diagnostics , mannheim , germany ) ; 1x pcr buffer ( roche diagnostics ) , 0.05  mm mgcl2 ; 0.2  m of each primer ; and approximately 50  ng genomic dna . the amplification was performed on a ptc-225 thermal cycler ( mj research , waltham , ma ) , using a stepdown protocol .
the first five cycles were carried out at 94c , 65c , and 72c , each for 30  s. the next five cyles were carried out at 94c , 63c , and 72c , each for 30  s. the last 25 cyles were at 94c , 60c , and 72c , each for 30  s. following cycling the pcr products were purified with the qiagen qiaquick pcr purification kit ( westburg , leusden , the netherlands ) .
subsequently , 100  ng of the purified pcr product was cycle sequenced with a dyeterminator kit ( us81090 , amersham biosciences , roosendaal , the netherlands ) in a thermal cycler ( mj research ) , using 0.05  mm sequencing primer .
for the reverse reaction , the same primer was used as in the pcr , but for the forward reaction an internal primer was used ( 5-ggtaccaacatggcccagaag ) . after the cycle reaction the sequencing products were cleaned on a sephadex plate ( amersham biosciences ) by centrifugation for 5  min at 910  g. the eluted sample was vacuum dried for 45  min at 65c .
finally , 20  l of loading solution ( amersham biosciences ) was added to dissolve the sequencing products .
the samples were analyzed on a megabace 1000 capillary sequencer ( amersham biosciences ) by injecting the samples for 45  s at 3  kv and running them for 5  h at 4  kv .
the data were processed with sequence analyzer 3.0 ( amersham biosciences ) and seqman ii ( dnastar , madison , wi ) .
pharmacokinetic data were analyzed with the mw\pharm software package ( version 3.5 ; mediware , groningen , the netherlands ) using the kinfit module .
for gemcitabine and its metabolite dfdu , the auc ( using the trapezoid rule ) , clearance , distribution volume , and elimination half - lives were calculated by non - compartmental analysis .
differences in laboratory and demographic data between groups were tested using the mann - whitney test .
cda genotype - related differences in pharmacokinetic data were investigated by analyzing the wild type aa genotype patient group against the combined heterozygote ac and homozygote cc genotype patient group using the mann - whitney test .
we assumed an effect of 30% of the alternative genotype on the gemcitabine auc as clinically relevant . from the literature
, the allele frequency of the wild type genotype was estimated at 0.7 , and therefore an almost equal distribution of patients in aa wild type ( 49% ) and combined homozygote cc plus heterozygote ac ( 51% ) patient groups was expected .
we calculated that group sample sizes of 10 and 10 were needed to achieve 92% power to detect a difference of 30% between the null hypothesis that both group means are equal and the alternative hypothesis that the mean auc of the mutant group is 30% larger , with group standard deviations of 20% and with a significance level of 0.05 using a two - side mann - whitney test assuming that the actual distribution is logistic .
additionally , differences between uncombined cda genotype groups regarding gemcitabine auc , clearance , and half - life were also analyzed using the kruskall - wallis test .
statistical analyses were performed using the spss 14.0 statistical package ( spss 2005 , chicago , il ) .
power calculations were performed using ncss 2004 statistical & power analysis software ( ncss , kaysville , ut ) .
this study was performed as a site study of a phase iii trial of gemcitabine plus epirubicin versus gemcitabine plus cisplatin in advanced nsclc patients .
radiotherapy should have been completed at least 4  weeks before inclusion , and patients should have recovered from any toxic side effect .
all patients had to have a performance status    2 according to the eastern cooperative oncology group ( ecog ) scale and a life expectancy of at least 12  weeks .
an adequate bone marrow reserve and normal renal ( creatinine clearance    60  ml / min ) and liver function were required .
the glomerular filtration rate ( gfr ) was calculated according to the formula of cockcroft and gault .
patients were excluded if they had active infections , second primary malignancies , uncorrected hypercalcemia , or an lvef    45% measured by multiple gated acquisition ( muga ) scan .
gemcitabine ( gemzar , lilly , nieuwegein , the netherlands ) in a dose of 1,125  mg / m in 250  ml 0.9% nacl solution was administered intravenously as a 30  min infusion .
patients co - treated with epirubicin received the epirubicin dose after completion of the gemcitabine infusion .
blood sampling blood sampling was carried out on the first day of the first chemotherapy cycle .
for pharmacokinetic sampling , a cannula was placed intravenously in the arm of the patient contralateral to the side of drug administration .
blood samples of 9  ml were collected in heparinized tubes containing 0.25  mg tetrahydrouridine ( thu ) in 50  l water , just before chemotherapy and at t  =  25 , 40 , 50 , 60 , 75 , 90 , 105 , 120 , 150 , 180 , 270 , and 360  min after the start of the gemcitabine infusion .
blood samples were immediately placed on ice and centrifuged at 2,500  g for 10  min within 1  h after collection .
genotyping material harvesting of oral mucosa cells was performed before chemotherapy by thorough mouth rinsing for 30  s with 5  ml 0.9 % nacl solution .
the cell suspension was collected in plastic cups and subsequently placed on ice . within 1  h after collection ,
the cell suspension was centrifuged for 10  min at 190  g at 4c .
the supernatant was discarded and cells were washed in 10  ml ice cold phosphate buffered saline , ph  =  7.40 ( pbs ) and centrifuged for 10  min at 190  g at 4c .
the remaining suspension was transferred to a microcentrifuge cup and centrifuged briefly ( 15  s ) at 10,000  g. the supernatant was discarded , and the pellet was kept frozen at 80c until further analysis .
the analysis of gemcitabine and dfdu in plasma was carried out by high performance liquid chromatography as described by freeman et al . .
the cda exon 1 polymorphism ( c / a ) at codon position 27 was genotyped by direct sequencing , in both directions , of pcr - amplified genomic dna .
first , the cda region flanking the polymorphic site was amplified using pcr with forward primer 5- agtagcttccccttccagtagc and reverse primer 5-cctcttcctgtacatcttcctctg .
the 25  l reactions contained 2.5 units taq polymerase ( amersham biosciences , uppsala , sweden ) ; 0.5  mm dntp mix ( roche diagnostics , mannheim , germany ) ; 1x pcr buffer ( roche diagnostics ) , 0.05  mm mgcl2 ; 0.2  m of each primer ; and approximately 50  ng genomic dna .
the amplification was performed on a ptc-225 thermal cycler ( mj research , waltham , ma ) , using a stepdown protocol .
the first five cycles were carried out at 94c , 65c , and 72c , each for 30  s. the next five cyles were carried out at 94c , 63c , and 72c , each for 30  s. the last 25 cyles were at 94c , 60c , and 72c , each for 30  s. following cycling the pcr products were purified with the qiagen qiaquick pcr purification kit ( westburg , leusden , the netherlands ) .
subsequently , 100  ng of the purified pcr product was cycle sequenced with a dyeterminator kit ( us81090 , amersham biosciences , roosendaal , the netherlands ) in a thermal cycler ( mj research ) , using 0.05  mm sequencing primer . for the reverse reaction ,
the same primer was used as in the pcr , but for the forward reaction an internal primer was used ( 5-ggtaccaacatggcccagaag ) .
after the cycle reaction the sequencing products were cleaned on a sephadex plate ( amersham biosciences ) by centrifugation for 5  min at 910  g. the eluted sample was vacuum dried for 45  min at 65c .
finally , 20  l of loading solution ( amersham biosciences ) was added to dissolve the sequencing products .
the samples were analyzed on a megabace 1000 capillary sequencer ( amersham biosciences ) by injecting the samples for 45  s at 3  kv and running them for 5  h at 4  kv .
the data were processed with sequence analyzer 3.0 ( amersham biosciences ) and seqman ii ( dnastar , madison , wi ) .
pharmacokinetic data were analyzed with the mw\pharm software package ( version 3.5 ; mediware , groningen , the netherlands ) using the kinfit module .
for gemcitabine and its metabolite dfdu , the auc ( using the trapezoid rule ) , clearance , distribution volume , and elimination half - lives were calculated by non - compartmental analysis .
differences in laboratory and demographic data between groups were tested using the mann - whitney test .
cda genotype - related differences in pharmacokinetic data were investigated by analyzing the wild type aa genotype patient group against the combined heterozygote ac and homozygote cc genotype patient group using the mann - whitney test .
we assumed an effect of 30% of the alternative genotype on the gemcitabine auc as clinically relevant . from the literature ,
the allele frequency of the wild type genotype was estimated at 0.7 , and therefore an almost equal distribution of patients in aa wild type ( 49% ) and combined homozygote cc plus heterozygote ac ( 51% ) patient groups was expected .
we calculated that group sample sizes of 10 and 10 were needed to achieve 92% power to detect a difference of 30% between the null hypothesis that both group means are equal and the alternative hypothesis that the mean auc of the mutant group is 30% larger , with group standard deviations of 20% and with a significance level of 0.05 using a two - side mann - whitney test assuming that the actual distribution is logistic .
additionally , differences between uncombined cda genotype groups regarding gemcitabine auc , clearance , and half - life were also analyzed using the kruskall - wallis test .
statistical analyses were performed using the spss 14.0 statistical package ( spss 2005 , chicago , il ) .
power calculations were performed using ncss 2004 statistical & power analysis software ( ncss , kaysville , ut ) .
table  1patient characteristicsall patients ( n  =  20)cytidine deaminase genotype subgroupsa / a ( n  =  8)a / c ( n  =  8)c / c ( n  =  4)gender ( m / f)17/38/06/23/1age ( years)65 ( 4473)67 ( 5573)65 ( 5773)59 ( 4473)weight ( kg)77 ( 5897)81(6097)75 ( 5887)85 ( 6388)length ( cm)175 ( 161192)177 ( 168193)173 ( 161185)182 ( 169192)bsa ( m)1.93 ( 1.612.27)2.00 ( 1.692.27)1.90 ( 1.612.03)2.09 ( 1.722.13)creatinine ( mol / l)78 ( 49119)80 ( 60119)75 ( 4999)81 ( 6692)crcl ( ml / min ) ( normal : > 60)97 ( 51134)97 ( 51130)88 ( 64106)104 ( 59134)ast ( u / l ) ( normal : < 45)22 ( 1535)22 ( 1235)22 ( 1528)21 ( 1727)alt ( u / l ) ( normal : < 45)27 ( 747)23 ( 732)29 ( 1547)26 ( 1236)bilirubin ( mol / l ) ( normal : < 20)10 ( 617)10 ( 616)10 ( 617)10 ( 911)co - treatmentepirubicin10532cisplatin10352values are medians with the ranges in parentheses patient characteristics values are medians with the ranges in parentheses the groups were comparable with respect to demographic parameters , renal function , and liver enzymes .
all patients , except one , had ast levels below the upper limit of normal .
two patients had a slightly reduced creatinine clearance ( crcl ) , between 50 and 60  ml / min .
mean pharmacokinetic curves of gemcitabine and dfdu in plasma are presented in fig .  1 according to the different genotypes .
gemcitabine was rapidly cleared from plasma and undetectable after 3  h in all patients .
1pharmacokinetics of gemcitabine and its metabolite dfdu after a 30  min infusion of 1,125  mg / m gemcitabine grouped according to genotype of the cytidine deaminase 79a > c polymorphismtable  2gemcitabine pharmacokineticsall patients ( n  =  20)cytidine deaminase genotype subgroupsp - value ( mw)p - value ( kw)a / a ( n  =  8)a / c ( n  =  8)c / c ( n  =  4)a / c+c / c ( n  =  12)gemcitabinedose ( mg)2,098 ( 1,9352,800)2,199 ( 1,9702,400)2,024 ( 2,0002,200)2,359 ( 1,9352,800)2,093 ( 1,9352,800)auc03 h ( mgh / l)8.6 ( 6.022.2)8.7 ( 6.122.2)8.6 ( 7.117.3)9.7 ( 6.012.9)8.6 ( 6.017.3)0.9390.988vdistribution ( l)72 ( 37120)83 ( 37120)75 ( 59105)52 ( 3978)70 ( 39105)0.3160.137clmetabolic ( l / h)246 ( 106466)251 ( 106328)232 ( 118300)248 ( 164466)232 ( 118466)0.5370.736t1/2 elimination ( h)0.22 ( 0.080.48)0.26 ( 0.080.48)0.23 ( 0.170.31)0.16 ( 0.080.21)0.21 ( 0.080.31)0.3160.094dfduauc03 h ( mgh / l)60.5 ( 44.698.6)64.7 ( 46.898.6)55.8 ( 44.671.8)60.6 ( 53.981.1)56.1 ( 44.681.1)0.5890.648 t 1/2 elimination ( h)2.43 ( 1.625.32)2.49 ( 2.095.32)2.14 ( 1.624.25)2.21 ( 1.673.80)2.13 ( 1.624.25)0.1900.419values are medians with the ranges in parenthesesp - values resulting from the mann - whitney ( mw ) test on the difference between aa versus combined ac+cc genotypep - values resulting from the kruskal - wallis ( kw ) test on differences among aa , ac , and cc genotypes pharmacokinetics of gemcitabine and its metabolite dfdu after a 30  min infusion of 1,125  mg / m gemcitabine grouped according to genotype of the cytidine deaminase 79a > c
polymorphism gemcitabine pharmacokinetics values are medians with the ranges in parentheses p - values resulting from the mann - whitney ( mw ) test on the difference between aa versus combined ac+cc genotype p - values resulting from the kruskal - wallis ( kw ) test on differences among aa , ac , and cc genotypes the influence of the presence of epirubine on gemcitabine pharmacokinetics was checked by grouping the patients according to treatment schedule ( gemcitabine plus cisplatin or gemcitabine plus epirubicine ) .
no difference in gemcitabine pharmacokinetics was measured between the two groups using the mann - whitney u - test .
the gemcitabine pharmacokinetic data of all 20 patients were analyzed with respect to the impact of the 79a  >  c polymorphism in the cda gene .
the frequencies of the a / a , a / c , and c / c genotypes were 0.40 , 0.40 , and 0.20 respectively .
the allele frequency of the 79a  >  c polymorphism was 0.40 in our study .
the mean pharmacokinetic curves of gemcitabine and dfdu according to cda genotype are presented in fig .  1 .
no differences in pharmacokinetic parameters were observed between the wild type aa genotype group and the combined heterozygote ac and homozygote cc genotype group , using the mann - whitney test .
additionally , in the kruskal - wallis analysis , we found no significant differences in gemcitabine auc , clearance , or serum half - life among the different cda genotypes ( see table  2 ) .
table  1patient characteristicsall patients ( n  =  20)cytidine deaminase genotype subgroupsa / a ( n  =  8)a / c ( n  =  8)c / c ( n  =  4)gender ( m / f)17/38/06/23/1age ( years)65 ( 4473)67 ( 5573)65 ( 5773)59 ( 4473)weight ( kg)77 ( 5897)81(6097)75 ( 5887)85 ( 6388)length ( cm)175 ( 161192)177 ( 168193)173 ( 161185)182 ( 169192)bsa ( m)1.93 ( 1.612.27)2.00 ( 1.692.27)1.90 ( 1.612.03)2.09 ( 1.722.13)creatinine ( mol / l)78 ( 49119)80 ( 60119)75 ( 4999)81 ( 6692)crcl ( ml / min ) ( normal : > 60)97 ( 51134)97 ( 51130)88 ( 64106)104 ( 59134)ast ( u / l ) ( normal : < 45)22 ( 1535)22 ( 1235)22 ( 1528)21 ( 1727)alt ( u / l ) ( normal : < 45)27 ( 747)23 ( 732)29 ( 1547)26 ( 1236)bilirubin ( mol / l ) ( normal : < 20)10 ( 617)10 ( 616)10 ( 617)10 ( 911)co - treatmentepirubicin10532cisplatin10352values are medians with the ranges in parentheses patient characteristics values are medians with the ranges in parentheses the groups were comparable with respect to demographic parameters , renal function , and liver enzymes .
all patients , except one , had ast levels below the upper limit of normal .
two patients had a slightly reduced creatinine clearance ( crcl ) , between 50 and 60  ml / min .
mean pharmacokinetic curves of gemcitabine and dfdu in plasma are presented in fig .  1 according to the different genotypes .
gemcitabine was rapidly cleared from plasma and undetectable after 3  h in all patients .
1pharmacokinetics of gemcitabine and its metabolite dfdu after a 30  min infusion of 1,125  mg / m gemcitabine grouped according to genotype of the cytidine deaminase 79a > c polymorphismtable  2gemcitabine pharmacokineticsall patients ( n  =  20)cytidine deaminase genotype subgroupsp - value ( mw)p - value ( kw)a / a ( n  =  8)a / c ( n  =  8)c / c ( n  =  4)a / c+c / c ( n  =  12)gemcitabinedose ( mg)2,098 ( 1,9352,800)2,199 ( 1,9702,400)2,024 ( 2,0002,200)2,359 ( 1,9352,800)2,093 ( 1,9352,800)auc03 h ( mgh / l)8.6 ( 6.022.2)8.7 ( 6.122.2)8.6 ( 7.117.3)9.7 ( 6.012.9)8.6 ( 6.017.3)0.9390.988vdistribution ( l)72 ( 37120)83 ( 37120)75 ( 59105)52 ( 3978)70 ( 39105)0.3160.137clmetabolic ( l / h)246 ( 106466)251 ( 106328)232 ( 118300)248 ( 164466)232 ( 118466)0.5370.736t1/2 elimination ( h)0.22 ( 0.080.48)0.26 ( 0.080.48)0.23 ( 0.170.31)0.16 ( 0.080.21)0.21 ( 0.080.31)0.3160.094dfduauc03 h ( mgh / l)60.5 ( 44.698.6)64.7 ( 46.898.6)55.8 ( 44.671.8)60.6 ( 53.981.1)56.1 ( 44.681.1)0.5890.648 t 1/2 elimination ( h)2.43 ( 1.625.32)2.49 ( 2.095.32)2.14 ( 1.624.25)2.21 ( 1.673.80)2.13 ( 1.624.25)0.1900.419values are medians with the ranges in parenthesesp - values resulting from the mann - whitney ( mw ) test on the difference between aa versus combined ac+cc genotypep - values resulting from the kruskal - wallis ( kw ) test on differences among aa , ac , and cc genotypes pharmacokinetics of gemcitabine and its metabolite dfdu after a 30  min infusion of 1,125  mg / m gemcitabine grouped according to genotype of the cytidine deaminase 79a > c polymorphism gemcitabine pharmacokinetics values are medians with the ranges in parentheses p - values resulting from the mann - whitney ( mw ) test on the difference between aa versus combined ac+cc genotype p - values resulting from the kruskal - wallis ( kw ) test on differences among aa , ac , and cc genotypes the influence of the presence of epirubine on gemcitabine pharmacokinetics was checked by grouping the patients according to treatment schedule ( gemcitabine plus cisplatin or gemcitabine plus epirubicine ) . no difference in gemcitabine pharmacokinetics
the gemcitabine pharmacokinetic data of all 20 patients were analyzed with respect to the impact of the 79a  >  c polymorphism in the cda gene .
the frequencies of the a / a , a / c , and c / c genotypes were 0.40 , 0.40 , and 0.20 respectively .
the allele frequency of the 79a  >  c polymorphism was 0.40 in our study .
the mean pharmacokinetic curves of gemcitabine and dfdu according to cda genotype are presented in fig .  1 .
no differences in pharmacokinetic parameters were observed between the wild type aa genotype group and the combined heterozygote ac and homozygote cc genotype group , using the mann - whitney test .
additionally , in the kruskal - wallis analysis , we found no significant differences in gemcitabine auc , clearance , or serum half - life among the different cda genotypes ( see table  2 ) .
in this study , we investigated the impact of the common 79a  >  c nonsynonymous single nucleotide polymorphism in the cytidine deaminase gene on gemcitabine and dfdu pharmacokinetics in nsclc patients . in all patients gemcitabine
was rapidly cleared from plasma , as was expected from previous reports . the most important factor that determines the clearance rate of gemcitabine is the activity of cda .
the total enzyme capacity of cda in all organs and tissues determines the biotransformation rate of gemcitabine into dfdu and thus the duration of exposure to gemcitabine . despite increasing interest in dna polymorphisms related to gemcitabine metabolism ,
so far only a few reports have been published on the relation between cda polymorphisms and gemcitabine pharmacokinetics .
the 79a  >  c csnp is the most common cda polymorphism with previously reported allele frequencies of 0.20 in japanese , 0.11 in african americans , 0.04 in africans , 0.30 in caucasian americans , and 0.36 in european caucasians [ 8 , 1416 ] .
the polymorphism at nucleotide position 79 corresponds to codon27 , which is located within an n - terminal core domain and might be involved in binding pocket loops that cooperate with zinc ligands [ 15 , 17 ] .
the a genotype corresponds to the wild type lys - carrying enzyme and the c genotype to a gln - carrying variant ( gln27 cda ) .
kirch et al . found that the deamination rate of cytarabine by bacterial wild type cda enzyme was a 1.33.3 fold higher than by gln27 cda .
recently reported in a functional genomics study in mammalian cells that the activity of human recombinant gln27 cda was about 66% of the wild type cda activity for gemcitabine .
thus , assuming highest cda activity in a / a , intermediate activity in a / c , and lowest in c / c diplotypes , we studied the consequences of this polymorphism on gemcitabine auc , clearance , and serum half - life .
all patients in our study were caucasian europeans , and in this group we found an allele frequency of 0.40 , which is in line with the allele frequencies of 0.30 and 0.36 earlier reported for caucasians [ 8 , 16 ] .
obviously , our study was powered to detect differences of at least 30% in population means and smaller differences in pharmacokinetic parameters may have gone unnoticed . in the post - hoc statistical analysis , we found a much larger variation in gemcitabine auc values , ranging from 3050% , than the initially expected 20% . as a result we achieved only 25% power to detect a difference .
we reasoned however that , due to the fact that the data in both groups largely overlap , it seems very unlikely that dose adjustment according to cda genotype will become clinically feasible , if a difference might exist .  
interestingly , this matter has recently been elucidated , as , in parallel with our study , sugiyama et al .
investigated the 79a  >  c polymorphism in a population of 251 japanese patients . in line with our data
, they could not detect any effect of the 79a  >  c polymorphism on the gemcitabine pharmacokinetics after a 30  min 8001,000  mg / m infusion .
it is important to realize that the two studies differ with respect to administered gemcitabine dose and patient ethnicity .
the mean dose in our study was 38% higher as a result of both a higher standard dose ( 1,125  mg / m ) and generally higher body surface area ( bsa ) values in caucasians . obviously , these factors do not change or modulate the observed lack of impact on pharmacokinetic outcomes of the 79a  >  c polymorphism .
one explanation for our findings may be a far more complex regulation of cda gene transcription , leading to substantial interindividual differences in quantities of cda enzyme , unrelated to the 79a  >  c polymorphism .
demonstrated reduced enzyme activity with the 79a  >  c polymorphism using in vitro deamination of ara - c and dfdc , respectively , it is possible that additional polymorphisms within cda also influence enzyme activity or that variants in other enzymes in gemcitabine metabolism play a greater than expected role .
5 flanking region contains numerous potential transcription factor binding sites that can cause variations in transcription .
unfortunately , we did not perform direct measurement of cda activity in our patients and thus could not check the genotype - phenotype relation at the enzyme level .
moreover , we hypothesized that possibly the total capacity of cda in the body may far exceed the amount needed to cope with plasma levels resulting from 8001,125  mg / m gemcitabine doses . in that case , differences in pharmacokinetics may only become manifest at much higher plasma concentrations when michaelis - menten enzyme saturation is becoming relevant .
finally , the pharmacokinetic results might have been dispersed as a result of co - administration of other chemotherapeutic agents .
indeed , all patients in our study received combined chemotherapy , but cisplatin was administered on day 2 and therefore did not interact with our results .
we analyzed that co - administration of epirubicin did not influence the pharmacokinetics of gemcitabine , which is in line with previously reported findings regarding the pharmacokinetics of both drugs when combined in treatment schedules [ 1921 ] .
>  c polymorphism on gemcitabine pharmacokinetics , the polymorphism still may be relevant at the cellular level .
recently demonstrated that the post - induction treatment - related mortality in children treated for acute myeloid leukemia with high dose cytarabine was significantly higher in children bearing the cc compared to the ac or aa genotype .
furthermore , tibaldi et al . showed that the wild type cda lys27lys ( aa ) genotype was associated with better clinical outcome , longer time to progression , and overall survival .
interestingly , they also found an increased cda enzymatic activity in red blood cells associated with the cda gln / gln ( cc ) genotype . although these findings contradict previous results from in - vitro functional genomics experiments by kirch et al . and gilbert et al .
, this clinical study illustrates that the association between genotype and treatment efficacy may be quite complex . recently two new polymorphisms in the cda gene were reported . these concern
the 208g > a and 435t > c polymorphism with allele frequencies of 0.04 and 0.30 in japanese patients , respectively .
the 208g > a polymorphism produces an alanine - to - threonine substitution ( ala70thr ) within the conserved catalytic domain .
introduction of this gene in yeast null mutants resulted in a 20% reduction of the 50% inhibitory concentration value for cytarabine .
sugiyama et al . also studied both variants and found a decreased gemcitabine clearance in patients with the 208g > a polymorphism .
however , the 208g > a polymorphism has not been detected so far in caucasians and was therefore not included in our study .
the 435t > c polymorphism ( thr145thr ) does not encode for an amino acid change .
since an impact of this silent 425t > c polymorphism on cda enzyme activity was expected to be less probable , we did not include this polymorphism in our investigations .
we conclude that the common 79a > c cda polymorphism does not have a major consistent , significant effect on gemcitabine plasma clearance
. the full pharmacogenetic picture of cda in relation to gemcitabine clearance and gemcitabine pharmacodynamics may nevertheless be quite complex and can only be elucidated in large clinical trials .
 objectiveto study the impact of the 79a > c polymorphism in the cytidine deaminase ( cda ) gene on the pharmacokinetics of gemcitabine and its metabolite 2,2-difluorodeoxyuridine ( dfdu ) in non - small - cell lung cancer ( nsclc ) patients.patients and methodspatients ( n  =  20 ) received gemcitabine 1,125  mg / m2 as a 30  min i.v . 
 infusion as part of treatment for nsclc . 
 plasma samples were collected during 06  h after gemcitabine administration . 
 gemcitabine and dfdu were quantified by high performance liquid chromatography with ultraviolet detection . 
 the cda 79a > c genotype was determined with pcr and dna sequencing.resultsgemcitabine was rapidly cleared from plasma and undetectable after 3  h. the allele frequency of the 79a > c polymorphism was 0.40 . 
 diplotypes were distributed as a / a n  =  8 , a / c n  =  8 , and c / c n  =  4 . 
 no significant differences were found between the different cda genotypes and gemcitabine or dfdu auc , clearance , or half-life.conclusionthe 
 79a > c polymorphism in the cda gene does not have a major consistent and signficant impact on gemcitabine pharmacokinetics . 
Introduction
Patients and methods
Patient selection
Treatment and sample collection
Pharmacokinetic analysis
Cytidine deaminase genotyping
Pharmacokinetic modelling
Statistical analysis
Results
Patients
Pharmacokinetics
Pharmacogenetic analysis
Discussion
Conclusions