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Chapter 19 Applications to pediatric pharmacology
347
CHAPTER 19
APPLICATIONS TO PEDIATRIC PHARMACOLOGY
GERARD PONS and ELISABETH REY Service de Pharmacologie P~rinatale et P~diatrique, Universit~ Ren~ Descartes, Paris 5 H5pital Saint-Vincent de Paul, 82 Av. Denfert Rochereau, 75674 Paris C~dex 14, France
1. I N T R O D U C T I O N
Stable isotope labeling has not been used extensively in pediatric and perinatal clinical pharmacology. This method has, however, numerous advantages. It allows one to determine the concentration of drugs with great sensitivity, therefore allowing quantification in small volumes of biological samples. The method also allows quantification with great specificity, eliminating interference by drug metabolites found in some radioactive tracer methods. Stable isotope labeling is safe, since stable isotopes are not radioactive. The only possible toxicity may be related to an isotope effect, a slowing of biochemical reactions because of the greater mass of the stable isotope. Due to the large mass difference between deuterium and hydrogen, a significant isotope effect occurs only with deuterium labeling. However, significant toxicity related to the isotope effect of deuterium can only be produced by very high levels of deuteration (greater than 15 percent of body water) far greater than the amount of deuterium in a typical tracer dose of drug (1). Stable isotope labeling also allows noninvasive in vivo studies like the CO2 breath test. All these advantages explain the great potential interest of stable isotope labeling in pediatric pharmacology. Stable isotope labeling is useful in four different types of study (2): (i) drug metabolic pathways; (ii) pharmacokinetic studies; (iii) determination of compliance; and (iv) assessment of the therapeutic or unwanted effects of drugs.
348 2. STUDIES ON DRUG METABOLIC PATHWAYS
The isotope cluster technique is a powerful method of detection and structural identification of drug metabolites and a useful tool to trace the origin and sequence of addition of groups to a drug during biotransformation.
2.1. N7 Methylation of Theophylline in Neonates The N7 methylation of theophylline into caffeine (CAF) has been demonstrated in premature neonates by Brazier and colleagues using stable isotope labeling (3-4). CAF had already been detected in plasma of premature neonates receiving theophylline for treatment and prevention of apneas (5-8). Plasma concentration of CAF correlated with those of theophylline, variations occurring in parallel. Three hypothesis were raised in order to explain that CAF was present in plasma: (i) CAF either was transferred transplacentally; (ii) or transferred via human breast milk; or (iii) theophylline was transformed, in vivo, into caffeine. Indeed, CAF is a methylxanthine, like theophylline, but CAF has an additional methyl group on the N7-position. In order to verify the third hypothesis, Brazier and colleagues used theophylline labeled with two stable isotopes: nitrogen 15 on the 1 and 3 positions, and carbon 13 on the 2 position (3-4). Two premature twin babies received theophylline, 3 mg/kg every 8 hr, starting on day 2, for 8 days, as a mixture of 46 percent of the labeled form, and 54 percent of the unlabeled one. Using gas chromatographic-mass spectrometry, the authors showed that in plasma and urine caffeine molecules have the same labeling as the administered theophylline molecules. The mass spectrum of caffeine extracted from the urine showed the molecular cluster of caffeine, 194 and 197, that corresponds to unlabeled and trilabeled CAF, respectively. The other ion clusters produced by the fragmentation of caffeine and theophylline demonstrate that the caffeine molecules had the same labeling as the administered theophylline ones. The presence of labeled caffeine in plasma and urine of babies treated by labeled theophylline confirms biotransformation of theophylline into caffeine via N7 methylation. This metabolic pathway is virtually not detectable in adults (9).
2.2. C02 Breath Test The C02 breath test (CBT) allows one to study the demethylation and the decarboxylation of molecules. The principle of the C02 breath test is as
349 follows. One or several carbon atoms of an administered molecule is metabolized via a demethylation or a decarboxylation into formaldehyde, formic acid, formyltetrahydrofolate and C02. If the molecule is artificially enriched with C13 on the position to be metabolized, the expired gas is enriched with ~3CC02 proportionally to the drug metabolism. When the carbon atom belongs to a methyl group, the C02 breath test allows a noninvasive measurement of the demethylation of the molecule, as long as the transformation of formaldehyde produced into C02 is not a limiting step of the production of C02. It has been shown in rodents that the C02 breath test is a good method for measuring the activity of the isoenzymes depending on CYP 1A2 (10-11). Since it is not easy to measure directly the activity of the enzyme in human, a correlation between the rate of expired labeled C02 and an indirect measure of the enzyme activity, such as plasma clearance, has been looked for. Such a correlation has already been evidenced for aminopyrine (12) and caffeine (13-14) demethylations. This is consistent with the knowledge that demethylation explains most of aminopyrine and caffeine clearance.
2.2.1. Maturation of N-demethylation of caffeine in neonates and infants The C02 breath test has been used to study the maturation of N-demethylation of caffeine in neonates and infants (15). The study was performed in 12 children, 4 premature neonates and 8 infants 1-10 months old. They received oral caffeine citrate solution for treatment and prevention of apnea. The usual morning maintenance dose was substituted by 1, 3, 7-~3C trimethyl-xanthine. The expired gas was collected over a 1-min period using a face mask and a small dead volume two-way valve into a latex bag. The volume of expired gas was measured with a pneumotachograph connected to a flow transducer. Aliquots were transferred to vacuum-evacuated glass flasks. Five breath samples were collected between two consecutive doses; samples also were collected the day before the administration of labeled CAF for endogenous labeled C02 production, and during the test day. On the test day, samples were collected just before the labeled dose, 2, 4 and 6 hr after this dose and before the next scheduled dose. Blood samples were drawn by heelprick after each breath sample collection on the test day. Urines were collected on the test day. The ~3C-C02 enrichment arising from labeled CAF demethylation was determined by mass spectrometry and expressed as the A ~176176 increase from baseline. This instantaneous labeled C02 excretion rate was calculated as a percentage of administered labeled CAF at each sampling time. The cumulative labeled C02 excretion rate was calculated from the area under the curve of the instantaneous labeled C02 excretion rate as a function of time at 2, 4
350 and 6 hr after labeled CAF administration. CAF plasma concentration and CAF metabolites concentrations in urines were determined using HPLC (16). Caffeine plasma clearance has been calculated as the ratio of the administered dose, assuming 100 percent bioavailability, to the area under the curve of caffeine plasma concentration as a function of time between two consecutive doses. The demethylation process was estimated by the ratio of the number of methyl groups absent in the metabolites recovered in urines to the number of methyl groups contained in the molecules of the parent CAF (16). The cumulative labeled C02 excretion rate correlated with the CAF plasma clearance. CAF plasma clearance has been used as an indirect method of validating the C02 breath test as a monitor of CYP 1A2, assuming that CAF plasma clearance correlates with CYP 1A2-dependent CAF N-demethylation. This assumption should be valid since N-demethylation is the major route of CAF metabolism, and CAF plasma clearance is expected to be sensitive only to hepatic intrinsic clearance. As CAF is a drug with low extraction ratio and low protein binding, CAF plasma clearance is not expected to be influenced either by hepatic blood flow or protein binding. The correlation between cumulated ~3C-C02 excretion rate and CAF plasma clearance has already been found in smoking and nonsmoking adults (13). This method allowed us to describe the maturational profile of CAF Ndemethylation as a function of age: - No detectable change in expired ~3C-C02 from basal values was observed in neonates and young infants, whereas, changes were measurable in all infants older than 33 days postnatal age, and older than 45 weeks postconceptional age. - These changes parallel those of CAF plasma clearance and of demethylation ratio calculated from the urine data (16-17). CAF plasma clearance increases with post-natal age according to a single exponential curve. The plateau is reached during the second trimester of life (17). The demethylation ratio increases with post-natal age as a monoexponential curve. The plateau is reached during the second trimester of life and accounts for about 60 percent of all methyl groups. Demethylated metabolites are detectable as soon as 22 days postnatal age and 34 weeks post-conceptional age (16).
2.2.2. Maturation of N-demethylation of caffeine during puberty The use of the C02 breath test also has allowed study of the maturation of N-demethylation of caffeine during puberty. Lambert et al. (18) measured the
351 changes of 13C-C02 excretion rate after a 3 mg/kg administration of caffeine labeled on the N3 methyl group in 62 subjects 3-20 years old. The labeled C02 excretion rate was higher in children before puberty than in adults and decreased with increasing age. Using the five pubertal stages of Tanner, the authors showed that the labeled C02 excretion rate decreased with increasing pubertal maturation. The maturational profile during puberty differed according t o the gender. The excretion rate of adult patients was reached earlier in puberty in girls (early puberty) than in boys (mid-puberty). These changes in C02 excretion rate suggest decreasing enzyme function during puberty and a different maturational profile according to gender. 2.2.3. Effect of growth hormone therapy in growth hormone-deficient children on demethylation of caffeine.
The use of the C02 breath test allowed Levistsky et al. (19) to study the effect of growth hormone therapy in growth hormone-deficient children on cytochrome P450-dependent 3-N-demethylation of CAF. Six 4-15 year old GH-deficient children received caffeine labeled on the 3-N-methyl group as a single 3-mg/kg oral dose before and after a 4-week treatment with human growth hormone 0.1 IU/kg s . c . - three times a week. The 3-N-demethylation of CAF, as measured by the CBT, was significantly decreased following 1 month of growth hormone therapy suggesting a possible role of growth hormone on the expression of CYP 1A2. 2.3. Enantioselectivity
There is no available data in the literature on the use of stable isotope labeling in pediatric pharmacology for the study of enantioselectivity of drug metabolism. Using stable isotopes in adults, a highly stereoselective metabolism has been demonstrated for 4-hydroxylation of debrisoquine (20) and the metabolism of other enantiomers (see Chapter 22). In children, changes related to enzymatic maturation may significantly affect the relative rate of biotransformation of enantiomers as a function of age.
3. PHARMACOKINETIC STUDIES
Using stable isotope tracer methods, it is possible to perform pharmacokinetic studies that measure true steady state values and evaluate time-dependent and dose-dependent pharmacokinetic changes without exposing the subject
352 to radiation, without creating radioactive waste and without withholding necessary medication.
3.1. Time-Dependent Pharmacokinetic Studies Serial kinetic studies during maintenance therapy using stable isotope labeling have been used in children to study time-dependent kinetic changes at steady state without need to interrupt the ongoing treatment and without exposure to radioactivity. A unit dose is replaced, totally or in part, by an equal amount of the labeled drug. Mass spectrometry allows one to study separately the kinetics of the labeled and unlabeled compound, using the same blood samples.
3.1.1. Auto-induction of drug metabolism Carbamazepine (CBZ) is used in children for the treatment of partial seizures. The decrease in steady state plasma concentration of CBZ during long-term treatment suggests that CBZ induces its own metabolism. Bertilsson and colleagues (21) used tetradeuterium-labeled CBZ in three 10-13 year old children. A preliminary study in animals ruled out an isotope effect (22). Patients were given equimolecular amounts of labeled and unlabeled CBZ as an initial oral single dose. The plasma concentrations of the two forms of CBZ were determined simultaneously by mass spectrometry during the four subsequent days. The single oral dose kinetics of CBZ and CBZ-D4 were almost identical, indicating no isotope effect of deuterium labeling in these patients. Maintenance therapy with unlabeled CBZ was started on day 5. On three separate occasions during the first five months of maintenance therapy, a portion of the CBZ dose was replaced by an equivalent amount of labeled CBZ. The plasma kinetics were studied after each of these doses. The steady state CBZ total plasma concentrations during multiple dosing were lower after dose III (days 21 to 36) and IV (about 5 months) than after dose II (day 6). They were two times lower than the theorical steady state plasma concentrations predicted using the kinetic parameters obtained from the initial single-dose experiment. But they were close to those predicted using the kinetic parameters obtained after labeled CBZ administrations during multiple dosing. There was a considerable increase (around 0.7 to 1.9 times) in plasma clearance between dose I and III without subsequent increase. Since CBZ is primarily eliminated via metabolism, changes in kinetic parameters may be attributed to changes in metabolism. This study demonstrated
353 the auto-induction of CBZ metabolism and described its time course: occurring rapidly, and maximum within 2 to 4 weeks.
3.1.2. Drug interactions Other time-dependent kinetic changes may be studied in children using stable isotopes labeling: (i) induction of biotransformation; (ii)inhibition of biotransformation (23); and (iii) the effect of age during long term therapy.
3.2. Dose-Dependent Pharmacokinetic Changes Stable isotope labeled tracer methods also have been successfully employed for describing the pharmacokinetic properties of drugs with dose-dependent (nonlinear) pharmacokinetics in adults by Browne's group (see Chapter 16).
3.3. Other Pharmacokinetic Studies 3.3.1. Placental transfer of drugs Brazier et al. (24) developed a model using labeled theophylline in a pregnant ewe in order to investigate the placental transfer of drugs. This method allows one to carry out simultaneously in the same animal, infusions with the labeled drug to the fetus and with the unlabeled drug to the mother, and to study the kinetics of both labeled and unlabeled drug in both fetus and mother. This method is of more limited use in human since injection of drug in the umbilical vein of the fetus is possible, but serial sampling of the umbilical blood is not possible. The staggered stable isotope administration technique, therefore, may be of great potential interest to evaluate placental transfer and to calculate drug entry half-life. By infusing intravenously to the mother, a series of different stable isotope-labeled forms of the same drug at different times before a single collection of fetal blood via a puncture of the umbilical vein for diagnostic or therapeutic purposes, one can obtain the same information about the drug's distribution as would be obtained by infusing a single dose of the drug and performing serial collections of umbilical venous blood (25). However, this technique has several drawbacks. Synthesis of a series of stable isotopes of a drug is expensive. Presence of several stable isotopes of a drug may create analytic difficulties by increasing problems with overlap and interference by endogenous substances. One must have an approximate idea of the time required for the umbilical drug concentration to attain equilibrium in order to select proper times for administration of stable isotope-
354
labeled analogues and specimen collection. It would, however, also have to be known for serial sample collections using alternative techniques. The mother would be exposed to the potential morbidity of several intravenous infusions of the drug being investigated.
3.3.2. Other studies Other kinetic studies are of potential interest in pediatric pharmacology: (i) the study of enantioselectivity of drugs; and (ii) particularly the determination of absolute and relative bioavailability in early infancy.
3.3.2.1. Study of the enantioselectivity of kinetics of drugs Stable isotope-labeled isomers of a drug have been used to study stereospecific aspects of a drug's disposition in adults (26-28) but, to our knowledge, not in children (see Chapter 16). 3.3.2.2. Studies of drug bioavailability and bioequivalence Bioavailability is defined as the rate and extent of absorption of a drug from its dosage form into the systemic circulation. Absolute bioavailability is measured as the ratio of the availability of a pharmaceutic formulation of a drug to the availability of the same drug after intravenous administration. Relative bioavailability is measured as the ratio of the availability of two nonintravenous pharmacokinetic alternatives of the same drug. Bioequivalence is defined as the equivalent bioavailability of two or more pharmaceutic alternatives. The primary advantage of the isotopic method is that the drug can be administered concomitantly with by two routes (e.g. parenteral and oral) or in two formulations (e.g. solution and solid dosage). Thus, a single set of blood samples serves to describe the time course of the routes or formulations being compared. The concomitant administration reduces intrasubject variability inherent in dual administration of the conventional cross-over designs, considering that each subject serves as its own "real time" control. To achieve equivalent statistical certainty, the stable isotope technique can reduce the number of subjects by at least 50 percent when compared with the cross-over technique, resulting in reduced cost (29-31). The single assay for both forms further reduces variation. The stable isotope method minimizes both drug exposure and discomfort to the subject by reducing the time and number of samples collected, and there is no need for the individual to undergo the procedure more than once. Furthermore this technique is well suited to "pulse" administration, wherein the kinetics of a single dose during
355
multiple or chronic dosing regimens can be compared with single-dose kinetics. See Chapter 13 for more information.
4. STUDY OF COMPLIANCE
Stable isotope labeling could also be used in children as tracer to determine compliance (32). The main problem with the use of stable isotopes in compliance studies is the cost of the tracer, a biologically acceptable substance labeled with a rare stable isotope (~3C for example). To minimize the cost of such a study, Schwartz (32) proposed utilizing the cheapest enriched light isotope that is available, namely, deuterium. For drugs administered as liquids (e.g. insulin), D20 could be included in the drug as a diluent. Deuterated glucose could be used as a "filler" in solid drugs. Using this method, Schwartz could detect failure to comply with prescribed dose at the 10 percent deviation level in adult subjects. There is no available example of such a use in children.
5. STUDY OF THE EFFECT OF DRUGS
5.1. Treatment of Helicobacter Pylori Infection Active chronic gastritis is clearly related to the presence of Helicobacter pylori, both in children and adults. The prevalence of H. pylori infection in children diagnosed as having primary gastritis varies between 70 and 84 percent. Present methods for detecting H. pylori comprise techniques (bacterial cultures, urease tests, histological examination of antral biopsy specimens) that need endoscopy and, therefore, are invasive and difficult to use in children. The urea breath test is a noninvasive technique designed to identify the presence of urease activity in the gastro-intestinal tract. The principle of the urea breath test is that, in the presence of the enzyme urease, orally administered urea is hydrolyzed to C02 and ammonia. If the urea carbon is labeled with either the stable isotope 13C, or radioactive 14C, it can be detected in the breath as labeled C02 (33). H. pylori is the most common urease containing gastric pathogen and, therefore, a positive urea breath test can be generally equated with the presence of an H. pylori infection. The ~3C-urea breath test results compare well with the ~4C-urea breath test results (34). The great advantage of the ~3C-urea breath test in children is the absence of radioactive risk. Although the radioactive dose with ~4C urea is low (35), total avoidance
356 is better. Furthermore, repetition of the test that will be required at intervals, theoretically places the use of radioactive isotopes at a distinct disadvantage. The urea breath test has proven to be very robust. The degree of sensitivity (96 percent), specificity (93 percent), positive (83 percent) and negative predictive value (99 percent) of the ~3C-urea breath test compared with cultures as "gold standard" (36) in children corresponds to that reported in adults (37). Eradication of H. pylori infections has proven difficult. There was a need for simple, noninvasive tests that will rapidly identify and separate therapeutic modalities according to their effectiveness. Graham et al. (38) showed that the ~3C-urea breath test is fully the equal of any of the alternative invasive procedures in monitoring a clinical trial in which the treatment was tested for its ability to eradicate H. pylori infection. 5.2. Effect of Exogenous Pancreatic Enzymes on Fat Malabsorption in Cystic Fibrosis Patients The use of ~3C-labeled lipids to detect fat malabsorption in children is of particular advantage in a pediatric population because of the simplicity of tests based on ~3C-C02 measurements of respiratory C02: the collection of fecal samples is inconvenient for outpatients, expensive if hospitalization is required; and stool analysis is unpleasant to laboratory technicians (39). Fat malabsorption results from either an absence of bile acids or pancreatic lipase or from inadequate intestinal mucosa. Watkins et al. (40) administering 10 mg/kg 1-(~3C)-trioctanoin showed that children with normal fat absorption excreted a total of 25 _+ 2.5 percent of the dose as ~3C-C02 by 2 hr, while those with steatorrhea due to pancreatic insufficiency resulting from cystic fibrosis excreted 3.5_+ 2.5 percent of the dose in the same period of time. When exogenous pancreatic enzymes were fed, the children with cystic fibrosis excreted less fat, and the ~3C-C02 excretion increased 4-fold, showing the treatment decreases fat malabsorption (40). 5.3. Effect of Drug on Protein Metabolism and Turnover of Amino-acids Stable isotope labeling has been used to study the effects of steroids on protein metabolism in man (41). The mechanism of steroid related myopathy was not known: increase in protein catabolism? decrease in protein synthesis? or both? Being an essential amino-acid, during the post absorptive state, the flux of leucine equals the rate of disappearance. Leucine undergoes both irreversible oxidation into exhaled CO= and recycling into protein synthesis.
357 Protein catabolism and synthesis can be estimated by the measurement of leucine flow in vivo. This method was used to assess the effects of a short-term high-dose prednisone treatment (40 mg/m2/24 hr - max 60 mg/24 hr) in eight normal subjects in leucine metabolism from a primed continuous infusion of L-(113C)-Ieucine during both the post-absorptive and fed state. Prednisone increased the flux of leucine coming from protein (protein catabolism) but did not modify protein synthesis. Protein balance (synthesis minus catabolism) became more negative in the post-absorptive state and failed to become positive in the fed state. Although this study has not been performed in children, this type of study is of great potential interest in children on drug therapy.
5.4. Teratogenicity In pharmacokinetic studies, the occurrence of isotope effect can lead to grossly misleading biologic and analytic results and, therefore, should be avoided. In mechanistic studies, isotope effects can be used to advantage. It is possible to elucidate which drug metabolic pathway leads to a teratogenic metabolite by labeling the drug with deuterium at various metabolic sites and determining in animals the teratogencity of the different deuterated forms (42). Reduced teratogenicity of a deuterium-labeled analogue of the drug, in comparison with the unlabeled drug, suggests that the deuterium label was placed at a metabolic site important for the formation of the teratogenic metabolite and that formation of the metabolite was inhibited by kinetic isotope effect.
REFERENCES
1. P.D. Klein and E.R. Klein, J. Clin. Pharmacol., 26 (1986) 378. 2. T.R. Browne, A. Van Langenhove, C.E. Costello, K. Bieman and D.J. Greenblatt, Ther. Drug Monit., 6 (1984) 3. 3. J.L. Brazier, B. Ribon, M. Desage and S. Salle, Biomed. Mass. Spectrom, 7 (1980) 189. 4. J.L. Brazier, B. Salle, B. Ribon, M. Desage and H. Renaud, Dev. Pharmcol. Ther., 2 (1981) 137. 5. H.S. Bada, N.N. Khanna, S.M. Somani and A.A. Tin, J. Pediatr., 94 (1979) 993. 6. C. Bory, P. Bltassat, M. Porthault, M. Bethenod, A. Frederich and J.V. Aranda, J. Pediatr., 94 (1979) 988. 7. M.J. Boutroy, P. Vert, R.J. Royer, P. Monin and M.J. Royer-Morrot, J. Pediatr., 94 (1970) 996. 8. J.L. Brazier, H. Renaud, B. Ribon and B.L. Salle, Arch. Dis. Child., 54 (1979) 194.
358 9. D.D.S. Tang-Liu and S. Riegelman, Res. Com. Chem. Pathol. Pharmacol., 34 (1981) 371. 10. K. Biagas and A.N. Kotake, Fed. Proc., 42 (1983) 2872A. 11. G.J. Lambert, H. Leitz, D. Pang and A.N. Kotake, Pediatr. Res., 17 (1983) 150A. 12. R.J. Shulman, C.S. Irving, T.W. Boutton, W.N. Wong, B.L. Nichols and P.D. Klein, Pediatr. Res., 19 (1985) 441. 13. A.N. Kotake, D.A. Scholler, G.H. Lambergt, A.L. Baker D.D. Schaffer and H. Josephs, Clin. Pharmcol. Ther., 32 (1982) 261. 14. E. Renner, H. Weitholtz, R. Hugenin M.J. Arnaud and R. Presig, Hepatology, 4 (1984) 38. 15. G. Pons, J.C. Blais, E. Rey, N. Plissonnier, M.O. Richard, O. Carrier, P. D'Athis, C. Moran, J. Badoual and G. Olive, Pediatr. Res., 23 (1988) 632. 16. O. Carrier, G. Pons, E. Rey, M.O. Richard, C. Moran, J. Badoual and G. Olive, Clin. Pharmacol. Ther., 44 (1988) 145. 17. G. Pons, O. Carrier, M.O. Richard, E. Rey, P. D'Athis, C. Moran, J. Badoual and G. Olive, Dev. Pharmacol. Ther., 11 (1988) 258. 18. G.H. Lambert, D.A. Schoeller, A.N. Kotake, C. Flores and D. Hay, Dev. Pharmacol. Ther., 9 (1986) 375. 19. L.L. Levitsky, D.A. Schoeller, G.H. Lambert and D.V. Edidin, Dev. Pharmacol. Ther., 12 (1989)90. 20. M. Eichelbaum, L. Bertilsson, A. Kupfer, E. Steiner and C.O. Messe, Br. J. Clin. Pharmacol., 25 (1988) 505. 21. L. Bertilsson, B. Hojer, G. Tybring, J. Osterloh and A. Rane, Clin. Pharmacol. Ther., 27 (1980) 83. 22. J. Osterloh and L. Bertilsson, Life Science, 23 (1978) 83. 23. I.M. Kapetanovic, H.J. Kupferberg, R.J. Porter, W. Theodore, E. Schulman and J.K. Penry, Clin. Pharmacol. Ther., 29 (981) 480. 24. J.L. Brazier, B. Ribon, M. Desage, F. Comet, M. Lievre, M. Berland and B. Salle, Dev. Pharmacol. Ther., 7 (1984) 52. 25. T.R. Browne, J.E. Evans, D.L. Kasdon, G.K. Szabo, B.A. Evans and D.J. Greenblatt, J. Clin, Pharmacol., 26 (1986) 425. 26. T.A. Baillie and A.W. Rettenmeier, J. Clin. Pharmacol., 26 (1986) 448. 27. T.A. Baillie, A.W. Rettenmeier, L.A. Peterson and N.J. Castagnoli, Ann. Rep. Med. Chem., 19 (184) 273. 28. W.F. Trager, J. Clin. Pharmacol., 26 (1986) 443. 29. M. Eichelbaum, G.E. Von Unruh and A. Somogyi, Clin. Pharmacokinet., 7 (1982) 490. 30. M. Eichelbaum, H.J. Dengler, A. Somogyi and G.E. Von Unruh. Eur. J. Clin. Pharmacol., 19 (1981) 127. 31. H. d'A.Heck, S.E. Buttrill, N.W. Flynn, R.L. Dyer, M. Anbar, T. Cairns, S. Dighe and B.E. Cabana, Pharmacokinet. Biopharm., 7 (1979) 233. 32. H.P. Schwartz, Contr. Clin. Trials, 5 (1984) 573. 33. D.Y. Graham and P.D. Klein, Am. J. Gastroenterol., 86 (1991) 1118. 34. S. Dill, J.J. Payne-James, J.J. Misiewicz, G.K. Grimble, D. McSwiggan, K. Pathak, A.J. Wood, C.M. Scrimgeour and M.J. Rennie, Gut., 31 (1990) 1237. 35. B.J. Marshall, M.W. Planke, S.R. Hoffman, C.L. Boyd, K.R. Dye, H.F. Frieirson, R.L. Guerrant and R.W. McCallum, Am. J. Gastroenterol., 86 (1991) 438. 36. Y. Vandenplas, U. Blecker, T. Devreker, E. Keppens, J. Nijs, S. Cadranel, M. Pipeleers-Marichal, A. Goossens and S. Lauwers, Pediatrics, 90 (1992) 608. 37. D.Y. Graham, P.G. Klein, A.R. Opekun and T.W. Boutton, J. Infect. Dis., 157 (1988) 777.
359 38. D.Y. Graham, P.D. Klein, D.G. Evans, D.J. Evans, L.C. Albert, A. Opekun, G.R. Jerdack and D.R. Morgan, Am. J. Gastroenterol., 86 (1991) 1158. 39. P.D. Klein and E.R. Klein, J. Pediatr. Gastroenterol. Nutr., 4 (1985) 9. 40. J.B. Watkins, D.A. Schoeller, P.D. Klein, D.G. Ott, A.D. Newcomer and A.F. Hofmann, J. Lab. Clin. Med., 90 (1977) 422. 41. B. Beaufrere and H.W. Haymond, Ann. Endocrinol., 46 (1985) 347. 42. H. Spielman and H. Nau, J.Clin. Pharmacol., 26 (1986) 474.
CHAPTER 19
APPLICATIONS TO PEDIATRIC PHARMACOLOGY
GERARD PONS and ELISABETH REY Service de Pharmacologie P~rinatale et P~diatrique, Universit~ Ren~ Descartes, Paris 5 H5pital Saint-Vincent de Paul, 82 Av. Denfert Rochereau, 75674 Paris C~dex 14, France
1. I N T R O D U C T I O N
Stable isotope labeling has not been used extensively in pediatric and perinatal clinical pharmacology. This method has, however, numerous advantages. It allows one to determine the concentration of drugs with great sensitivity, therefore allowing quantification in small volumes of biological samples. The method also allows quantification with great specificity, eliminating interference by drug metabolites found in some radioactive tracer methods. Stable isotope labeling is safe, since stable isotopes are not radioactive. The only possible toxicity may be related to an isotope effect, a slowing of biochemical reactions because of the greater mass of the stable isotope. Due to the large mass difference between deuterium and hydrogen, a significant isotope effect occurs only with deuterium labeling. However, significant toxicity related to the isotope effect of deuterium can only be produced by very high levels of deuteration (greater than 15 percent of body water) far greater than the amount of deuterium in a typical tracer dose of drug (1). Stable isotope labeling also allows noninvasive in vivo studies like the CO2 breath test. All these advantages explain the great potential interest of stable isotope labeling in pediatric pharmacology. Stable isotope labeling is useful in four different types of study (2): (i) drug metabolic pathways; (ii) pharmacokinetic studies; (iii) determination of compliance; and (iv) assessment of the therapeutic or unwanted effects of drugs.
348 2. STUDIES ON DRUG METABOLIC PATHWAYS
The isotope cluster technique is a powerful method of detection and structural identification of drug metabolites and a useful tool to trace the origin and sequence of addition of groups to a drug during biotransformation.
2.1. N7 Methylation of Theophylline in Neonates The N7 methylation of theophylline into caffeine (CAF) has been demonstrated in premature neonates by Brazier and colleagues using stable isotope labeling (3-4). CAF had already been detected in plasma of premature neonates receiving theophylline for treatment and prevention of apneas (5-8). Plasma concentration of CAF correlated with those of theophylline, variations occurring in parallel. Three hypothesis were raised in order to explain that CAF was present in plasma: (i) CAF either was transferred transplacentally; (ii) or transferred via human breast milk; or (iii) theophylline was transformed, in vivo, into caffeine. Indeed, CAF is a methylxanthine, like theophylline, but CAF has an additional methyl group on the N7-position. In order to verify the third hypothesis, Brazier and colleagues used theophylline labeled with two stable isotopes: nitrogen 15 on the 1 and 3 positions, and carbon 13 on the 2 position (3-4). Two premature twin babies received theophylline, 3 mg/kg every 8 hr, starting on day 2, for 8 days, as a mixture of 46 percent of the labeled form, and 54 percent of the unlabeled one. Using gas chromatographic-mass spectrometry, the authors showed that in plasma and urine caffeine molecules have the same labeling as the administered theophylline molecules. The mass spectrum of caffeine extracted from the urine showed the molecular cluster of caffeine, 194 and 197, that corresponds to unlabeled and trilabeled CAF, respectively. The other ion clusters produced by the fragmentation of caffeine and theophylline demonstrate that the caffeine molecules had the same labeling as the administered theophylline ones. The presence of labeled caffeine in plasma and urine of babies treated by labeled theophylline confirms biotransformation of theophylline into caffeine via N7 methylation. This metabolic pathway is virtually not detectable in adults (9).
2.2. C02 Breath Test The C02 breath test (CBT) allows one to study the demethylation and the decarboxylation of molecules. The principle of the C02 breath test is as
349 follows. One or several carbon atoms of an administered molecule is metabolized via a demethylation or a decarboxylation into formaldehyde, formic acid, formyltetrahydrofolate and C02. If the molecule is artificially enriched with C13 on the position to be metabolized, the expired gas is enriched with ~3CC02 proportionally to the drug metabolism. When the carbon atom belongs to a methyl group, the C02 breath test allows a noninvasive measurement of the demethylation of the molecule, as long as the transformation of formaldehyde produced into C02 is not a limiting step of the production of C02. It has been shown in rodents that the C02 breath test is a good method for measuring the activity of the isoenzymes depending on CYP 1A2 (10-11). Since it is not easy to measure directly the activity of the enzyme in human, a correlation between the rate of expired labeled C02 and an indirect measure of the enzyme activity, such as plasma clearance, has been looked for. Such a correlation has already been evidenced for aminopyrine (12) and caffeine (13-14) demethylations. This is consistent with the knowledge that demethylation explains most of aminopyrine and caffeine clearance.
2.2.1. Maturation of N-demethylation of caffeine in neonates and infants The C02 breath test has been used to study the maturation of N-demethylation of caffeine in neonates and infants (15). The study was performed in 12 children, 4 premature neonates and 8 infants 1-10 months old. They received oral caffeine citrate solution for treatment and prevention of apnea. The usual morning maintenance dose was substituted by 1, 3, 7-~3C trimethyl-xanthine. The expired gas was collected over a 1-min period using a face mask and a small dead volume two-way valve into a latex bag. The volume of expired gas was measured with a pneumotachograph connected to a flow transducer. Aliquots were transferred to vacuum-evacuated glass flasks. Five breath samples were collected between two consecutive doses; samples also were collected the day before the administration of labeled CAF for endogenous labeled C02 production, and during the test day. On the test day, samples were collected just before the labeled dose, 2, 4 and 6 hr after this dose and before the next scheduled dose. Blood samples were drawn by heelprick after each breath sample collection on the test day. Urines were collected on the test day. The ~3C-C02 enrichment arising from labeled CAF demethylation was determined by mass spectrometry and expressed as the A ~176176 increase from baseline. This instantaneous labeled C02 excretion rate was calculated as a percentage of administered labeled CAF at each sampling time. The cumulative labeled C02 excretion rate was calculated from the area under the curve of the instantaneous labeled C02 excretion rate as a function of time at 2, 4
350 and 6 hr after labeled CAF administration. CAF plasma concentration and CAF metabolites concentrations in urines were determined using HPLC (16). Caffeine plasma clearance has been calculated as the ratio of the administered dose, assuming 100 percent bioavailability, to the area under the curve of caffeine plasma concentration as a function of time between two consecutive doses. The demethylation process was estimated by the ratio of the number of methyl groups absent in the metabolites recovered in urines to the number of methyl groups contained in the molecules of the parent CAF (16). The cumulative labeled C02 excretion rate correlated with the CAF plasma clearance. CAF plasma clearance has been used as an indirect method of validating the C02 breath test as a monitor of CYP 1A2, assuming that CAF plasma clearance correlates with CYP 1A2-dependent CAF N-demethylation. This assumption should be valid since N-demethylation is the major route of CAF metabolism, and CAF plasma clearance is expected to be sensitive only to hepatic intrinsic clearance. As CAF is a drug with low extraction ratio and low protein binding, CAF plasma clearance is not expected to be influenced either by hepatic blood flow or protein binding. The correlation between cumulated ~3C-C02 excretion rate and CAF plasma clearance has already been found in smoking and nonsmoking adults (13). This method allowed us to describe the maturational profile of CAF Ndemethylation as a function of age: - No detectable change in expired ~3C-C02 from basal values was observed in neonates and young infants, whereas, changes were measurable in all infants older than 33 days postnatal age, and older than 45 weeks postconceptional age. - These changes parallel those of CAF plasma clearance and of demethylation ratio calculated from the urine data (16-17). CAF plasma clearance increases with post-natal age according to a single exponential curve. The plateau is reached during the second trimester of life (17). The demethylation ratio increases with post-natal age as a monoexponential curve. The plateau is reached during the second trimester of life and accounts for about 60 percent of all methyl groups. Demethylated metabolites are detectable as soon as 22 days postnatal age and 34 weeks post-conceptional age (16).
2.2.2. Maturation of N-demethylation of caffeine during puberty The use of the C02 breath test also has allowed study of the maturation of N-demethylation of caffeine during puberty. Lambert et al. (18) measured the
351 changes of 13C-C02 excretion rate after a 3 mg/kg administration of caffeine labeled on the N3 methyl group in 62 subjects 3-20 years old. The labeled C02 excretion rate was higher in children before puberty than in adults and decreased with increasing age. Using the five pubertal stages of Tanner, the authors showed that the labeled C02 excretion rate decreased with increasing pubertal maturation. The maturational profile during puberty differed according t o the gender. The excretion rate of adult patients was reached earlier in puberty in girls (early puberty) than in boys (mid-puberty). These changes in C02 excretion rate suggest decreasing enzyme function during puberty and a different maturational profile according to gender. 2.2.3. Effect of growth hormone therapy in growth hormone-deficient children on demethylation of caffeine.
The use of the C02 breath test allowed Levistsky et al. (19) to study the effect of growth hormone therapy in growth hormone-deficient children on cytochrome P450-dependent 3-N-demethylation of CAF. Six 4-15 year old GH-deficient children received caffeine labeled on the 3-N-methyl group as a single 3-mg/kg oral dose before and after a 4-week treatment with human growth hormone 0.1 IU/kg s . c . - three times a week. The 3-N-demethylation of CAF, as measured by the CBT, was significantly decreased following 1 month of growth hormone therapy suggesting a possible role of growth hormone on the expression of CYP 1A2. 2.3. Enantioselectivity
There is no available data in the literature on the use of stable isotope labeling in pediatric pharmacology for the study of enantioselectivity of drug metabolism. Using stable isotopes in adults, a highly stereoselective metabolism has been demonstrated for 4-hydroxylation of debrisoquine (20) and the metabolism of other enantiomers (see Chapter 22). In children, changes related to enzymatic maturation may significantly affect the relative rate of biotransformation of enantiomers as a function of age.
3. PHARMACOKINETIC STUDIES
Using stable isotope tracer methods, it is possible to perform pharmacokinetic studies that measure true steady state values and evaluate time-dependent and dose-dependent pharmacokinetic changes without exposing the subject
352 to radiation, without creating radioactive waste and without withholding necessary medication.
3.1. Time-Dependent Pharmacokinetic Studies Serial kinetic studies during maintenance therapy using stable isotope labeling have been used in children to study time-dependent kinetic changes at steady state without need to interrupt the ongoing treatment and without exposure to radioactivity. A unit dose is replaced, totally or in part, by an equal amount of the labeled drug. Mass spectrometry allows one to study separately the kinetics of the labeled and unlabeled compound, using the same blood samples.
3.1.1. Auto-induction of drug metabolism Carbamazepine (CBZ) is used in children for the treatment of partial seizures. The decrease in steady state plasma concentration of CBZ during long-term treatment suggests that CBZ induces its own metabolism. Bertilsson and colleagues (21) used tetradeuterium-labeled CBZ in three 10-13 year old children. A preliminary study in animals ruled out an isotope effect (22). Patients were given equimolecular amounts of labeled and unlabeled CBZ as an initial oral single dose. The plasma concentrations of the two forms of CBZ were determined simultaneously by mass spectrometry during the four subsequent days. The single oral dose kinetics of CBZ and CBZ-D4 were almost identical, indicating no isotope effect of deuterium labeling in these patients. Maintenance therapy with unlabeled CBZ was started on day 5. On three separate occasions during the first five months of maintenance therapy, a portion of the CBZ dose was replaced by an equivalent amount of labeled CBZ. The plasma kinetics were studied after each of these doses. The steady state CBZ total plasma concentrations during multiple dosing were lower after dose III (days 21 to 36) and IV (about 5 months) than after dose II (day 6). They were two times lower than the theorical steady state plasma concentrations predicted using the kinetic parameters obtained from the initial single-dose experiment. But they were close to those predicted using the kinetic parameters obtained after labeled CBZ administrations during multiple dosing. There was a considerable increase (around 0.7 to 1.9 times) in plasma clearance between dose I and III without subsequent increase. Since CBZ is primarily eliminated via metabolism, changes in kinetic parameters may be attributed to changes in metabolism. This study demonstrated
353 the auto-induction of CBZ metabolism and described its time course: occurring rapidly, and maximum within 2 to 4 weeks.
3.1.2. Drug interactions Other time-dependent kinetic changes may be studied in children using stable isotopes labeling: (i) induction of biotransformation; (ii)inhibition of biotransformation (23); and (iii) the effect of age during long term therapy.
3.2. Dose-Dependent Pharmacokinetic Changes Stable isotope labeled tracer methods also have been successfully employed for describing the pharmacokinetic properties of drugs with dose-dependent (nonlinear) pharmacokinetics in adults by Browne's group (see Chapter 16).
3.3. Other Pharmacokinetic Studies 3.3.1. Placental transfer of drugs Brazier et al. (24) developed a model using labeled theophylline in a pregnant ewe in order to investigate the placental transfer of drugs. This method allows one to carry out simultaneously in the same animal, infusions with the labeled drug to the fetus and with the unlabeled drug to the mother, and to study the kinetics of both labeled and unlabeled drug in both fetus and mother. This method is of more limited use in human since injection of drug in the umbilical vein of the fetus is possible, but serial sampling of the umbilical blood is not possible. The staggered stable isotope administration technique, therefore, may be of great potential interest to evaluate placental transfer and to calculate drug entry half-life. By infusing intravenously to the mother, a series of different stable isotope-labeled forms of the same drug at different times before a single collection of fetal blood via a puncture of the umbilical vein for diagnostic or therapeutic purposes, one can obtain the same information about the drug's distribution as would be obtained by infusing a single dose of the drug and performing serial collections of umbilical venous blood (25). However, this technique has several drawbacks. Synthesis of a series of stable isotopes of a drug is expensive. Presence of several stable isotopes of a drug may create analytic difficulties by increasing problems with overlap and interference by endogenous substances. One must have an approximate idea of the time required for the umbilical drug concentration to attain equilibrium in order to select proper times for administration of stable isotope-
354
labeled analogues and specimen collection. It would, however, also have to be known for serial sample collections using alternative techniques. The mother would be exposed to the potential morbidity of several intravenous infusions of the drug being investigated.
3.3.2. Other studies Other kinetic studies are of potential interest in pediatric pharmacology: (i) the study of enantioselectivity of drugs; and (ii) particularly the determination of absolute and relative bioavailability in early infancy.
3.3.2.1. Study of the enantioselectivity of kinetics of drugs Stable isotope-labeled isomers of a drug have been used to study stereospecific aspects of a drug's disposition in adults (26-28) but, to our knowledge, not in children (see Chapter 16). 3.3.2.2. Studies of drug bioavailability and bioequivalence Bioavailability is defined as the rate and extent of absorption of a drug from its dosage form into the systemic circulation. Absolute bioavailability is measured as the ratio of the availability of a pharmaceutic formulation of a drug to the availability of the same drug after intravenous administration. Relative bioavailability is measured as the ratio of the availability of two nonintravenous pharmacokinetic alternatives of the same drug. Bioequivalence is defined as the equivalent bioavailability of two or more pharmaceutic alternatives. The primary advantage of the isotopic method is that the drug can be administered concomitantly with by two routes (e.g. parenteral and oral) or in two formulations (e.g. solution and solid dosage). Thus, a single set of blood samples serves to describe the time course of the routes or formulations being compared. The concomitant administration reduces intrasubject variability inherent in dual administration of the conventional cross-over designs, considering that each subject serves as its own "real time" control. To achieve equivalent statistical certainty, the stable isotope technique can reduce the number of subjects by at least 50 percent when compared with the cross-over technique, resulting in reduced cost (29-31). The single assay for both forms further reduces variation. The stable isotope method minimizes both drug exposure and discomfort to the subject by reducing the time and number of samples collected, and there is no need for the individual to undergo the procedure more than once. Furthermore this technique is well suited to "pulse" administration, wherein the kinetics of a single dose during
355
multiple or chronic dosing regimens can be compared with single-dose kinetics. See Chapter 13 for more information.
4. STUDY OF COMPLIANCE
Stable isotope labeling could also be used in children as tracer to determine compliance (32). The main problem with the use of stable isotopes in compliance studies is the cost of the tracer, a biologically acceptable substance labeled with a rare stable isotope (~3C for example). To minimize the cost of such a study, Schwartz (32) proposed utilizing the cheapest enriched light isotope that is available, namely, deuterium. For drugs administered as liquids (e.g. insulin), D20 could be included in the drug as a diluent. Deuterated glucose could be used as a "filler" in solid drugs. Using this method, Schwartz could detect failure to comply with prescribed dose at the 10 percent deviation level in adult subjects. There is no available example of such a use in children.
5. STUDY OF THE EFFECT OF DRUGS
5.1. Treatment of Helicobacter Pylori Infection Active chronic gastritis is clearly related to the presence of Helicobacter pylori, both in children and adults. The prevalence of H. pylori infection in children diagnosed as having primary gastritis varies between 70 and 84 percent. Present methods for detecting H. pylori comprise techniques (bacterial cultures, urease tests, histological examination of antral biopsy specimens) that need endoscopy and, therefore, are invasive and difficult to use in children. The urea breath test is a noninvasive technique designed to identify the presence of urease activity in the gastro-intestinal tract. The principle of the urea breath test is that, in the presence of the enzyme urease, orally administered urea is hydrolyzed to C02 and ammonia. If the urea carbon is labeled with either the stable isotope 13C, or radioactive 14C, it can be detected in the breath as labeled C02 (33). H. pylori is the most common urease containing gastric pathogen and, therefore, a positive urea breath test can be generally equated with the presence of an H. pylori infection. The ~3C-urea breath test results compare well with the ~4C-urea breath test results (34). The great advantage of the ~3C-urea breath test in children is the absence of radioactive risk. Although the radioactive dose with ~4C urea is low (35), total avoidance
356 is better. Furthermore, repetition of the test that will be required at intervals, theoretically places the use of radioactive isotopes at a distinct disadvantage. The urea breath test has proven to be very robust. The degree of sensitivity (96 percent), specificity (93 percent), positive (83 percent) and negative predictive value (99 percent) of the ~3C-urea breath test compared with cultures as "gold standard" (36) in children corresponds to that reported in adults (37). Eradication of H. pylori infections has proven difficult. There was a need for simple, noninvasive tests that will rapidly identify and separate therapeutic modalities according to their effectiveness. Graham et al. (38) showed that the ~3C-urea breath test is fully the equal of any of the alternative invasive procedures in monitoring a clinical trial in which the treatment was tested for its ability to eradicate H. pylori infection. 5.2. Effect of Exogenous Pancreatic Enzymes on Fat Malabsorption in Cystic Fibrosis Patients The use of ~3C-labeled lipids to detect fat malabsorption in children is of particular advantage in a pediatric population because of the simplicity of tests based on ~3C-C02 measurements of respiratory C02: the collection of fecal samples is inconvenient for outpatients, expensive if hospitalization is required; and stool analysis is unpleasant to laboratory technicians (39). Fat malabsorption results from either an absence of bile acids or pancreatic lipase or from inadequate intestinal mucosa. Watkins et al. (40) administering 10 mg/kg 1-(~3C)-trioctanoin showed that children with normal fat absorption excreted a total of 25 _+ 2.5 percent of the dose as ~3C-C02 by 2 hr, while those with steatorrhea due to pancreatic insufficiency resulting from cystic fibrosis excreted 3.5_+ 2.5 percent of the dose in the same period of time. When exogenous pancreatic enzymes were fed, the children with cystic fibrosis excreted less fat, and the ~3C-C02 excretion increased 4-fold, showing the treatment decreases fat malabsorption (40). 5.3. Effect of Drug on Protein Metabolism and Turnover of Amino-acids Stable isotope labeling has been used to study the effects of steroids on protein metabolism in man (41). The mechanism of steroid related myopathy was not known: increase in protein catabolism? decrease in protein synthesis? or both? Being an essential amino-acid, during the post absorptive state, the flux of leucine equals the rate of disappearance. Leucine undergoes both irreversible oxidation into exhaled CO= and recycling into protein synthesis.
357 Protein catabolism and synthesis can be estimated by the measurement of leucine flow in vivo. This method was used to assess the effects of a short-term high-dose prednisone treatment (40 mg/m2/24 hr - max 60 mg/24 hr) in eight normal subjects in leucine metabolism from a primed continuous infusion of L-(113C)-Ieucine during both the post-absorptive and fed state. Prednisone increased the flux of leucine coming from protein (protein catabolism) but did not modify protein synthesis. Protein balance (synthesis minus catabolism) became more negative in the post-absorptive state and failed to become positive in the fed state. Although this study has not been performed in children, this type of study is of great potential interest in children on drug therapy.
5.4. Teratogenicity In pharmacokinetic studies, the occurrence of isotope effect can lead to grossly misleading biologic and analytic results and, therefore, should be avoided. In mechanistic studies, isotope effects can be used to advantage. It is possible to elucidate which drug metabolic pathway leads to a teratogenic metabolite by labeling the drug with deuterium at various metabolic sites and determining in animals the teratogencity of the different deuterated forms (42). Reduced teratogenicity of a deuterium-labeled analogue of the drug, in comparison with the unlabeled drug, suggests that the deuterium label was placed at a metabolic site important for the formation of the teratogenic metabolite and that formation of the metabolite was inhibited by kinetic isotope effect.
REFERENCES
1. P.D. Klein and E.R. Klein, J. Clin. Pharmacol., 26 (1986) 378. 2. T.R. Browne, A. Van Langenhove, C.E. Costello, K. Bieman and D.J. Greenblatt, Ther. Drug Monit., 6 (1984) 3. 3. J.L. Brazier, B. Ribon, M. Desage and S. Salle, Biomed. Mass. Spectrom, 7 (1980) 189. 4. J.L. Brazier, B. Salle, B. Ribon, M. Desage and H. Renaud, Dev. Pharmcol. Ther., 2 (1981) 137. 5. H.S. Bada, N.N. Khanna, S.M. Somani and A.A. Tin, J. Pediatr., 94 (1979) 993. 6. C. Bory, P. Bltassat, M. Porthault, M. Bethenod, A. Frederich and J.V. Aranda, J. Pediatr., 94 (1979) 988. 7. M.J. Boutroy, P. Vert, R.J. Royer, P. Monin and M.J. Royer-Morrot, J. Pediatr., 94 (1970) 996. 8. J.L. Brazier, H. Renaud, B. Ribon and B.L. Salle, Arch. Dis. Child., 54 (1979) 194.
358 9. D.D.S. Tang-Liu and S. Riegelman, Res. Com. Chem. Pathol. Pharmacol., 34 (1981) 371. 10. K. Biagas and A.N. Kotake, Fed. Proc., 42 (1983) 2872A. 11. G.J. Lambert, H. Leitz, D. Pang and A.N. Kotake, Pediatr. Res., 17 (1983) 150A. 12. R.J. Shulman, C.S. Irving, T.W. Boutton, W.N. Wong, B.L. Nichols and P.D. Klein, Pediatr. Res., 19 (1985) 441. 13. A.N. Kotake, D.A. Scholler, G.H. Lambergt, A.L. Baker D.D. Schaffer and H. Josephs, Clin. Pharmcol. Ther., 32 (1982) 261. 14. E. Renner, H. Weitholtz, R. Hugenin M.J. Arnaud and R. Presig, Hepatology, 4 (1984) 38. 15. G. Pons, J.C. Blais, E. Rey, N. Plissonnier, M.O. Richard, O. Carrier, P. D'Athis, C. Moran, J. Badoual and G. Olive, Pediatr. Res., 23 (1988) 632. 16. O. Carrier, G. Pons, E. Rey, M.O. Richard, C. Moran, J. Badoual and G. Olive, Clin. Pharmacol. Ther., 44 (1988) 145. 17. G. Pons, O. Carrier, M.O. Richard, E. Rey, P. D'Athis, C. Moran, J. Badoual and G. Olive, Dev. Pharmacol. Ther., 11 (1988) 258. 18. G.H. Lambert, D.A. Schoeller, A.N. Kotake, C. Flores and D. Hay, Dev. Pharmacol. Ther., 9 (1986) 375. 19. L.L. Levitsky, D.A. Schoeller, G.H. Lambert and D.V. Edidin, Dev. Pharmacol. Ther., 12 (1989)90. 20. M. Eichelbaum, L. Bertilsson, A. Kupfer, E. Steiner and C.O. Messe, Br. J. Clin. Pharmacol., 25 (1988) 505. 21. L. Bertilsson, B. Hojer, G. Tybring, J. Osterloh and A. Rane, Clin. Pharmacol. Ther., 27 (1980) 83. 22. J. Osterloh and L. Bertilsson, Life Science, 23 (1978) 83. 23. I.M. Kapetanovic, H.J. Kupferberg, R.J. Porter, W. Theodore, E. Schulman and J.K. Penry, Clin. Pharmacol. Ther., 29 (981) 480. 24. J.L. Brazier, B. Ribon, M. Desage, F. Comet, M. Lievre, M. Berland and B. Salle, Dev. Pharmacol. Ther., 7 (1984) 52. 25. T.R. Browne, J.E. Evans, D.L. Kasdon, G.K. Szabo, B.A. Evans and D.J. Greenblatt, J. Clin, Pharmacol., 26 (1986) 425. 26. T.A. Baillie and A.W. Rettenmeier, J. Clin. Pharmacol., 26 (1986) 448. 27. T.A. Baillie, A.W. Rettenmeier, L.A. Peterson and N.J. Castagnoli, Ann. Rep. Med. Chem., 19 (184) 273. 28. W.F. Trager, J. Clin. Pharmacol., 26 (1986) 443. 29. M. Eichelbaum, G.E. Von Unruh and A. Somogyi, Clin. Pharmacokinet., 7 (1982) 490. 30. M. Eichelbaum, H.J. Dengler, A. Somogyi and G.E. Von Unruh. Eur. J. Clin. Pharmacol., 19 (1981) 127. 31. H. d'A.Heck, S.E. Buttrill, N.W. Flynn, R.L. Dyer, M. Anbar, T. Cairns, S. Dighe and B.E. Cabana, Pharmacokinet. Biopharm., 7 (1979) 233. 32. H.P. Schwartz, Contr. Clin. Trials, 5 (1984) 573. 33. D.Y. Graham and P.D. Klein, Am. J. Gastroenterol., 86 (1991) 1118. 34. S. Dill, J.J. Payne-James, J.J. Misiewicz, G.K. Grimble, D. McSwiggan, K. Pathak, A.J. Wood, C.M. Scrimgeour and M.J. Rennie, Gut., 31 (1990) 1237. 35. B.J. Marshall, M.W. Planke, S.R. Hoffman, C.L. Boyd, K.R. Dye, H.F. Frieirson, R.L. Guerrant and R.W. McCallum, Am. J. Gastroenterol., 86 (1991) 438. 36. Y. Vandenplas, U. Blecker, T. Devreker, E. Keppens, J. Nijs, S. Cadranel, M. Pipeleers-Marichal, A. Goossens and S. Lauwers, Pediatrics, 90 (1992) 608. 37. D.Y. Graham, P.G. Klein, A.R. Opekun and T.W. Boutton, J. Infect. Dis., 157 (1988) 777.
359 38. D.Y. Graham, P.D. Klein, D.G. Evans, D.J. Evans, L.C. Albert, A. Opekun, G.R. Jerdack and D.R. Morgan, Am. J. Gastroenterol., 86 (1991) 1158. 39. P.D. Klein and E.R. Klein, J. Pediatr. Gastroenterol. Nutr., 4 (1985) 9. 40. J.B. Watkins, D.A. Schoeller, P.D. Klein, D.G. Ott, A.D. Newcomer and A.F. Hofmann, J. Lab. Clin. Med., 90 (1977) 422. 41. B. Beaufrere and H.W. Haymond, Ann. Endocrinol., 46 (1985) 347. 42. H. Spielman and H. Nau, J.Clin. Pharmacol., 26 (1986) 474.