By Al-Saleh, Eyad Al-Harmi, Jehad; Al-Rashdan, Ibrahim; Al-Shammari, Majed; Nandakumaran, Moorkath
Abstract Objective. Folate antagonists are widely used in the treatment of diverse cancerous states. A paucity of data on transport characteristics of one such widely used drug, methotrexate, in the human placenta, prompted us to study its permeation characteristics in vitro.
Methods. Placentas from normal pregnancies were collected post- partum. Methotrexate, along with antipyrine as reference marker were injected as a single bolus (100 [mu]L) into the maternal arterial circulation of isolated perfused placental lobules; perfusate samples were collected from both maternal and fetal circulations over a study period of five minutes. National Culture and Tissue Collection medium, diluted with Earle’s buffered salt solution was used as the perfusate. The concentration of methotrexate in various samples was determined by high performance liquid chromatography, while antipyrine concentration was assayed by spectrophotometry. Transport and pharmacokinetic data of study and reference substances were computed using standard parameters.
Results. Differential transport rate of methotrexate for 10, 25, 50, 75 and 90% efflux fractions in fetal venous effluent averaged 0.52, 1.30, 2.37, 3.57 and 4.43 minutes in 12 perfusions, representing 1.01+0.08, 1.03 + 0.06, 0.95 + 0.03, 0.93 + 0.03, 0.93 + 0.03 respectively times antipyrine reference value. Student’s r- test showed varying differences between the control and study group data. Transport Fraction (TF) of methotrexate, expressed as fraction of the drug appearing in fetal vein, during study period of 5 minutes averaged 24.00 + 2.50% of bolus dose while antipyrine TF averaged 68.73 + 2.01% of injected bolus dose, representing 24.00 percent of reference marker value. Student’s ss-test showed methotrexate and reference marker TF values to be significantly different (p
Conclusions. We report for the first time that the transport of methotrexate from maternal to fetal circulation is not negligible in human placenta at term. It is reasonable to assume that a direct risk for the fetus from methotrexate use in pregnancy cannot be excluded, and caution is warranted when it is used in emergency clinical situations.
Keywords: Methotrexate, placental transport, in vitro perfusion, antipyrine, anti-cancer drugs
Folie acid antagonists are widely used in the clinical treatment of various neoplastic as well as inflammatory states. One such agent, methotrexate ((2S)-2-(4(2,4-diaminopteridin-6- yl)methyl)methylaminobenzoylpentanedioic acid) is known to exert its action by impairing the enzyme dehydrofolate reductase and by interfering with production of purine nucleotides [1,2]. Folic acid is essential for methylation of DNA, which in turn is essential for normal embryogenesis.
The use of methotrexate (MTX) has been reported in the treatment of diverse clinical conditions such as breast cancer, lymphoma and lymphadenoma, gastric ulcer, urinary bladder cancer, psoriasis, rheumatoid arthritis, etc. [3-11]. A study from our research group has shown the efficacy of MTX in the treatment of ectopic pregnancies , and similar success has also been reported by other research groups [13,14].
Considering the reported teratogenicity of methotrexate in experimental animals , it is customary to avoid the use of this class of drug in pregnant women , so as to avoid possible harm to the fetus and neonate. Several studies have attributed congenital abnormalities in newborns of mothers who received MTX treatment [16- 19] in pregnancy to its use. Studies from other research groups, however, have found no malformations or abnormalities in children exposed to MTX in utero [20,21]. Nevertheless, when the mother’s life is in danger, the use of these life-saving drugs in pregnancy cannot be excluded. The transplacental passage of methotrexate to the fetus has been assumed in humans  by noting chromosomal aberrations in a newborn who received MTX treatment in pregnancy. However, there have been no detailed reports to-date assessing the maternal-fetal transport characteristics of this drug in humans. Hence, we thought it interesting to study the maternal-fetal transport characteristics of MTX in human placenta in late gestation, using in vitro perfusion of isolated placental lobules. This method has been previously used by our research group to investigate maternal-fetal transport characteristics of a wide variety of drugs and nutrients both in normal as well as in disease states [22-24], and has the added advantage of assessing placental transport parameters, independent of maternal and fetal hemodynamic and metabolic influences. Antipyrine, a freely diffusing substance, was used as the internal standard.
Materials and methods
Human placentas were collected postpartum after uncomplicated, normal pregnancies. Perfusion of suitable placental lobules was performed as previously described [22-24]. National Culture and Tissue Collection (NCTC-13 5) medium (Sigma Chemicals, USA) diluted with buffered, oxygenated Earle’s salt solution was used as the perfusate [23,24], and circulation of the perfusate through the maternal and fetal circuits was done using a Harvard digital pump. Advantages and suitability of using NCTC medium as the perfusate have been detailed elsewhere . Perfusion flow rates in fetal and maternal circuits were measured by Brooks R215 flow meters, and perfusion pressure in both the circuits was monitored by mercury manometers.
After an initial wash-out period of 10 minutes, MTX at twice the therapeutic concentration of 1 mg/mL  and antipyrine as reference marker (concentration: 100 /ig/L) were injected as a 100 pL bolus into the maternal circulation, at a site close to the insertion of the microcannulas in the maternal basal plate. All chemicals used were analytically ultra pure grade (Merck, Germany). After a period of 1 min, serial perfusate samples were collected from the fetal venous outflow every 30 s for a period of 5 min. The lag time of 1 min before the start of sample collection was based on our preliminary study . The study period of 5 min was based on the time required for 90% of injected substance in the bolus to appear in the combined fetal and maternal venous efflux in control experiments. The fetal perfusion flow rate averaged 5.8 + 0.5 mL/ min, while the maternal flow rate averaged 12.5 +- 0.8 mL/min in 12 successful perfusions. Cotyledon weights averaged 29.4 + 1.6 g. Viability of placental perfusions was assessed by determination of the oxygen consumption of the perfused tissue during the period of study and by evaluating absence or negligible presence of intracellular enzyme, lactate dehydrogenase (LDH) in perfusate effluents. Those perfusions with greater than 5% leak of LDH in venous effluents compared to the enzyme level at the beginning of the experiment and with greater than 5% fetal arterio-venous flow rate mismatch were judged as unsuitable and data collected from the experiments were discarded.
Assay of study and reference substances
The concentration of MTX in perfusate samples was determined by high performance liquid chromatography  using a reverse phase column (Novapak CIS, Waters Associates, Milford, MA, USA) and using 4-aminoacetophenone as internal standard. The mobile phase consisted of phosphate buffer, methanol, and acetonitrile (84:11:5). MTX and internal standard were detected at 313 nm, using a UV detector, and the concentration of study substance was quantified using standard criteria. Antipyrine concentrations in perfusate samples were determined using a colorimetric technique .
The maternal-fetal transport of substances studied was determined as differential transport rate (TR), expressed as time in min for a given fraction to he transported across to the fetal vein [23,24].
Efflux fractions (EF) of methotrexate and antipyrine were calculated as per the following formula: EF = EFSTTEFVS, where EFS = concentration of the element studied in the fetal venous sample and TEFVS = total inorganic element concentration of the element studied in fetal venous outflow for the period of 5 min. From the curve obtained by plotting various cumulative efflux fractions, time in min for efflux of 10, 25, 50, 75 and 90% of total venous outflow were then computed.
Transport fractions (TF) of different substances were calculated [23,24] using the following equation: TF = TEFV5/TEMb, where TEFV5 = total content of the substance studied in fetal venous efflux for a period of 5 min and TEMb = total content of the substance studied in the injected maternal bolus.
Area under the curve (AUC)
Other kinetic transport parameters such as clearance, Kel (elimination constant), Tmax (time of maximum response), absorption rate and elimination rate were determined using a computer program based on IMSL Fortran Subroutine software specially adapted for statistical applications.
Data were analyzed using Stat-View 402 statistics package, (SPSS 11, USA). Student’s ss-test or analysis of var (ANOVA) or analysis of covariance (ANCOVA) tests were used, where appropriate. Results were considered significant if p
Clinical characteristics of mothers from whom placental samples were collected and details of their newborns are shown in Table I. Twenty-five percent of the women were primiparous and 33% had a history of previous abortions. No woman had diabetes or hepatic or renal disease complicating pregnancy and the placental weights and weights of newborns at delivery were within normal limits. The differential TR of MTX and antipyrine for 10, 25, 50, 75 and 90% efflux into fetal vein in 12 perfusions are summarized in Table II. TR of MTX for 10, 25, 50, 75 and 90% efflux fractions averaged 0.52, 1.30, 2.37, 3.57 and 4.43 minutes, respectively, while the values for corresponding efflux fractions of reference marker averaged 0.51, 1.26, 2.52, 3.78 and 4.52 minutes, respectively (Figure 1). Student’s f-test did not show any significant difference (p > 0.05) between study and reference substance values for the 10, 25 and 50 percent efflux fractions. However, MTX TF values for 75 and 90% efflux were significantly lower (p
Table I. Clinical characteristics of mothers and newborns (N = 12).
Pharmacokinetic parameters of MTX and antipyrine in the 12 perfusions are summarized in Table III. Area under the curve (AUC), clearance, elimination constant (Ke;), time for maximum response (Tmax), absorption rate, and elimination rate of MTX averaged 305 429.2 [mu]g/h, 17.4 mL/min, 655.5, 181.7 s, 377.9 [mu]g/min and – 291.9 [mu]g/min, respectively, while corresponding values of reference marker averaged 375 974.2 [mu]g/h, 0.05 mL/min, 5893.2, 191.8s, 432.1 [mu]g/min and -289.9 [mu]g/min. Excepting Tmax, absorption rate and elimination rate, all the other parameters were significantly different (Student’s t-test; p 0.05) between the above- mentioned ratios of study and reference substances.
When maternal-fetal transport rate values of antipyrine and MTX were plotted (Figure 4) as a fraction of the injected maternal load, the slope of the MTX curve was found to be significantly lower (ANCOVA, p
Table II. Differential transport rates (TR) of methotrexate and antipyrine.
Figure 1. TR50 values of methotrexate and antipyrine in fetal venous efflux normal placentas. TR50, transport rate for 50% of efflux fraction. Statistical significance (p) was assessed by unpaired Student’s t-test.
We report for the first time data on maternal-fetal transport characteristics of a widely used anti-cancer agent, MTX, in human placenta in vitro conditions. The possibility of placental transfer of this drug across the human placenta in vivo has been assumed by noting the presence of chromosomal aberrations in the neonate and noting the presence of the drug in the umbilical blood of a woman who received the drug for the treatment of cancer in pregnancy . But no attempt was made by the investigators to quantify the amount of drug thus transferred from the maternal to fetal circulation. Our results conclusively prove that transport of MTX across the human placenta is not negligible in vitro, compared to the freely difiusible reference marker, representing about 24% of injected drug load and about 69% of antipyrine transport value. Since the placental membrane is able to discriminate between transport behavior of the study and reference substances in our in vitro experimental conditions, we have reason to believe that the same results can be anticipated in vivo as well.
Results on maternal-fetal transport of the reference marker, antipyrine, are in accord with previous reports from our research group [22,30] as well as with those reported by others in the perfused human placental lobule in vitro [31,32]. Free diffusibility of antipyrine across the placental membrane has been established in humans in vivo as well [33,34]. Use of this reference substance as an internal marker has permitted us to minimize errors from experimental artifacts such as size of lobule perfused, membrane surface area, shunts, etc. [21,32] and to reduce the variability between experiments.
Figure 2. Transport fraction (TF) of methotrexate and antipyrine, expressed as % of injected maternal bolus dose in normal placentas. Statistical significance (p) was assessed by unpaired Student’s r- test.
Table III. Pharmacokinetic parameters of methotrexate and antipyrine.
Although it is generally believed that drugs below the molecular weight of 600 Daltons pass freely through the placenta! membrane , the present results on MTX transport from maternal to fetal circulation are consistent with our earlier observation of water- soluble substances crossing the placental membrane, as a function of their molecular weights and depending on their ionized nature [36,37].
The relatively low transport of MTX compared to antipyrine can be attributed to the differences in physicochemical characteristics of the two drugs. While antipyrine has a smaller molecular weight (MW=180) and is highly lipid-soluble [30,38], MTX is mainly water- soluble and minimally lipidsoluble [1,2]. Furthermore, while antipyrine is mainly unionized at physiological pH and binds negligibly to plasma or tissue proteins [38,39], MTX is reported to bind moderately to plasma proteins [1,2] and with pKas of 4.8 and 5.5 [1,2] has higher ionized fraction, as per Henderson-Hasselbach equation, at physiological pH . The low lipid solubility and higher ionization of MTX at physiological pH, coupled with the greater degree of protein binding and higher molecular weight compared to the reference marker, could account for the reduced transfer of this drug from the maternal to fetal circulation. Such a possibility of water-soluble drugs being transported in human placenta in vitro as an inverse function of molecular weight has been demonstrated in the case of certain sympathomimetic drugs , as well as certain neuroleptics . Interestingly, the transport fraction index of MTX compared to reference marker is apparently higher than that expected from a substance of similar molecular weight as per the standard curve outlined by us earlier for water- soluble substances . The possibility of a carrier-mediated transport of the drug cannot be discounted in the placenta, considering the reported carrier-mediated active transport of the drug, particularly at lower concentrations in the gastrointestinal tract  in humans.
Figure 3. Absorption rate:elimination rate index (TF) of methotrexate and antipyrine in normal placentas. Statistical significance (p) was assessed by unpaired Student’s t-test.
Figure 4. Cumulative concentration curves of methotrexate and antipyrine in fetal vein, expressed as cumulative percentage of injected bolus dose. *TF = Transport Fraction. The regression curve for methotrexate was drawn using the following parameters: b – 0.0545454545; b 0.0826666667; r^sup 2^ = 0.9990471309; while that for antipyrine was drawn using the following parameters: b – 0.0322727273; b 0.2283545455; r^sup 2^ = 0.999790324. Statistical significance (p) was assessed by analysis of co-variance (ANCOVA) test (p = 0.036).
It is unclear whether the relatively moderate protein binding of MTX of about 50% [1,2], as mentioned earlier, to plasma or tissue proteins compared to negligible binding of the reference marker [38,39] has played a role in determining their contrasting transfer behaviors in our short-duration experiments. The lower absorption:elimination rate ratio of the drug compared to antipyrine observed in our experiments is indicative of possible placental tissue accumulation of the drug when used in pregnancy.
There have been reports of successful pregnancies after chemotherapy with MTX, with few side effects for fetus and offspring [20,21], although many others implicate maternal MTX use with varying teratogenic disorders [16-19]. Most of the teratogenic side effects of MTX use in pregnancy in animals and humans have involved the central nervous system, cranial ossification and the palate [15,19]. The difference in teratogenic response to MTX treatment may be due to the difference in developmental stage at which the fetus was exposed to the drug or to the difference in fraction of drug that crossed over to the fetus from the maternal circulation. Since MTX transport towards the fetus is not negligible, as noted in this study, it will be advantageous for use in pregnancy, especially in second and third trimesters when the danger of teratogenic effects to the fetus are minimal. Alternatively, if MTX use in the first trimester is imperative, patients may be advised to start or continue MTX use only after safe contraception has been established  and after educating the patient about the possible danger or harm to the fetus from the use of the drug in the first trimester.
Further, since neurotoxicity [41,42], thrombocytopenia, stomatitis, liver function changes, bone maral dysfunction [45,46], and chromosomal aberrations  have been reported in patients receiving MTX therapy, the possibility, albeit small, of such undesirable effects on the fetus of the mother receiving such treatment in pregnancy cannot be discounted as well. The treatment regimen needs to assess the benefit to risk ratio for the mother and the baby and the long-term sequelae of potentially undesirable postnatal and post-partum effects of anti-folate drug therapy. Acknowledgements
The authors wish to thank Ms Teena Sadan for her excellent technical assistance.
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EYAD AL-SALEH1, JEHAD AL-HARMI1, IBRAHIM AL-RASHDAN2, MAJED AL- SHAMMARI1, & MOORKATH NANDAKUMARAN1
1 Department of Obstetrics and Gynecology, Faculty of Medicine, University of Kuwait, Kuwait and 2 Department of Medicine, Faculty of Medicine, University of Kuwait, Kuwait
(Received 22 December 2006; revised 27 December 2006; accepted 28 December 2006)
Correspondence: Dr Eyad Al-Saleh, Vice Dean, Research, Faculty of Medicine, University of Kuwait, PO Box 24923, Safat 13110, Kuwait. Tel: +965 4986155/+965 5319601. Fax: +965 5318454. E-mail: [email protected]
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