Stable Isotopes Labeling of Drugs in Pediatric Clinical Pharmacology (1999)

Stable isotope labeling (SIL) still has not been used very much in pediatric and perinatal clinical pharmacology. However, this method has numerous advantages. It allows one to determine the concentration of drugs with great sensitivity, thereby allowing quantification in small amounts of biologic samples. It also allows quantification with great specificity, eliminating interference by drug metabolites found in some radioactive tracer methods. SIL is safe because stable isotopes are not radioactive. The only possible toxicity that may be related to an isotope effect is a slowing of biochemical reactions because of the greater mass of the stable isotope. Because of the great difference between deuterium and hydrogen, a significant isotope effect occurs only with deuterium. However, toxicity related to the isotope effect of deuterium can only be produced by very high levels of deuteration (>15% of body water), far higher than the amount of deuterium in a typical tracer dose of drug.1 SIL also allows noninvasive in vivo studies such as the co2 breath test (CBT). All of these advantages explain the great potential interest in SIL in pediatric pharmacology. The costs of stable isotopes and limited facilities available for sample analysis are undoubtedly the greatest limitations to their increased use.SIL is useful in different types of studies,2 such as studies on the fate of drugs (drug metabolic pathways and pharmacokinetics), determination of compliance, and assessment of therapeutic and unwanted effects of drugs.The use of SIL of drugs in positron emission tomography investigations is not addressed in this article.In the periodic table, the elements are ranked according to their atomic number, ie, the number of protons contained in the nucleus. All elements located at the same place in the table are isotopes of the same family, for example, the family of carbon. All members of the same family are characterized by the same number of protons, but they differ by the number of neutrons.The structure of the nucleus of radioactive isotopes is unstable, and a disintegration occurs with emission of an ionizing radiation. The radioactive isotopes can be identified by the detection of this radiation. On the contrary, the structure of the nucleus of stable isotopes is stable and no disintegration occurs. Stable isotopes contain one or more additional neutrons and therefore are heavier than the normally abundant isotope of the considered atom. Because of this difference in atomic weight, the stable isotopes can be identified and differentiated by mass spectrometry. Some isotopes are naturally occurring, others are manmade. The natural occurrence varies very much from one isotope to another.Stable isotopes can be incorporated into a molecule, producing a molecule with a higher molecular weight than that of the unlabeled molecule.The natural SIL of molecules is proportional to the natural occurrence of the considered stable isotope and yields a low rate of labeling. Artificial SIL allows higher rates of labeling.When an atom in a chemical radical is replaced by a stable isotope (eg, hydrogen replaced by deuterium), the mass of the radical is modified and the physical molecular interactions may be modified with kinetic changes on chemical reactions that define the isotopic effect. For example, the replacement of the hydrogen atoms of a methyl group by atoms of deuterium may decrease the rate of demethylation of a chemical compound.Mass spectrometry is a technique that allows identification and quantification of compounds labeled with a stable isotope.Biologic samples to be assayed using mass spectrometry have to undergo an extraction and a separation that is performed most often by gas chromatography and, more recently, by high-pressure liquid chromatography (HPLC).Separation by gas chromatography is used for volatile compounds and compounds that can be derivatized, ie, that can be bound to a radical that increases the mass of the fragment. Separation by HPLC has to be used for thermolabile molecules, large molecules (eg, prostaglandins), and molecules difficult to obtain in a vapor state (eg, glycuronoconjugates). The separated fractions are injected in the vapor state into a chamber of ionization. The liquid fractions obtained during HPLC have to be nebulized to be in a vapor state while the sample is dried under a flow of nitrogen. The most common ionization process is achieved by bombardment of the molecules with electrons at 70 electron volts of energy (electron impact ionization) or of ions (chemical ionization). Most of the injected molecules are broken into fragments, and both fragments and unbroken molecules are ionized and accelerated in an electric field. They enter a magnetic field, and the deflection of their path is proportional to their electric charge and inversely proportional to the mass and speed. In the mass spectrum obtained for each fraction of the chromatogram, the ion clusters allow the identification of labeled fragments and unbroken molecules. Ion clusters are characteristic twin peaks of the mass spectrum corresponding to a molecular fragment or to the unbroken molecule (molecular ion) and its corresponding stable isotope-labeled homolog. A computer-reconstructed total ion chromatogram is obtained from the consecutive mass spectra obtained from the same sample.The isotope cluster technique is a powerful method for the detection and structural identification of drug metabolites. It also is a useful tool for tracing the origin and sequence of the addition of groups to a drug during biotransformation.The N7 methylation of theophylline into caffeine (CAF) has been demonstrated in premature neonates by Brazier and colleagues using SIL.3,4CAF had already been detected in plasma of premature neonates receiving theophylline for treatment and prevention of apnea.56–8Plasma concentrations of CAF correlated with those of theophylline, with variations occurring in parallel. Three hypotheses have been raised to explain how CAF was present in plasma: 1) transferred transplacentally; 2) transferred via human breast milk; or 3) theophylline was transformed, in vivo, into CAF. Indeed, CAF is a methylxanthine, like theophylline, but CAF has an additional methyl group on the N7 position.To verify the third hypothesis, Brazier and colleagues used theophylline labeled with two stable isotopes, nitrogen 15 on the 1 and 3 positions, carbon 13 on the 2 positions.3,4 Two premature twin infants received theophylline, 3 mg/kg every 8 hours, starting on day 2 for 8 days as a mixture of 46% of the labeled form and 54% of the unlabeled form. Using gas chromatographic mass spectrometry, the investigators showed that in plasma and urine, CAF molecules have the same labeling as the administered theophylline molecules.The mass spectrum of CAF extracted from the urine showed the molecular ion cluster of CAF, 194 and 197, that corresponds to unlabeled and trilabeled CAF, respectively. The other ion clusters produced by the fragmentation of CAF and theophylline demonstrate that the CAF molecules had the same labeling as the administered theophylline molecules.The presence of labeled CAF in plasma and urine of infants treated with labeled theophylline confirms the biotransformation of theophylline into CAF via N7 methylation. This metabolic pathway is virtually undetectable in adults.9The CBT allows one to study the demethylation and decarboxylation of molecules.The principle of the CBT is as follows. One or several carbon atoms of an administered molecule is metabolized via a demethylation or a decarboxylation into formaldehyde, formic acid, formyltetrahydrofolate, and co2. If the molecule is artificially enriched with 13C on the position to be metabolized, the expired gas is enriched with -co2proportionately to the drug metabolism. When the carbon atom belongs to a methyl group, the CBT allows a noninvasive measurement of the demethylation of the molecule, as long as the transformation of formaldehyde produced into co2 is not a limiting step of the production of co2. It has been shown in rodents that the CBT is an effective method for measuring the activity of the isoenzymes depending on CYP 1A2.10,11Because it is not easy to measure directly the activity of the enzyme in humans, a correlation between the rate of expired labeledco2 and an indirect measure of the enzyme activity, such as plasma clearance, has been sought. Such a correlation already has been evidence for aminopyrine12 and CAF13,14 demethylations. This is consistent with the understanding that demethylation explains most of the aminopyrine and CAF clearance.The CBT has been used to study the maturation ofN-demethylation of CAF in neonates and infants.15 The study was performed in 12 children, 4 premature neonates and 8 infants (1–19 months old). They received oral CAF citrate solution for treatment and prevention of apnea. The usual morning maintenance dose was substituted by 1,37–13C trimethylxanthine.The expired gas was collected over a 1-minute 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-evaluated glass flasks. Five breath samples were collected between two consecutive doses: the day before the administration of labeled CAF (day 1) for endogenous labeled co2 production and the day after (day 2). They were collected just before the dose and 2, 3, and 6 hours after this dose and before the next scheduled dose. Blood samples were drawn by heel prick after each breath sample collection on day 2. Urine samples were collected on day 2.The 13C-co2 enrichment arising from labeled CAF demethylation was determined by mass spectrometry and expressed as the % increase from baseline. The instantaneously labeled co2 excretion rate was calculated as percent of administered labeled CAF at each sampling time. The cumulative labeled co2 excretion rate was calculated from the area under the curve of the instantaneously labeledco2 excretion rate as a function of time at 2, 4, and 6 hours after labeled CAF administration. CAF plasma concentration and CAF metabolite concentrations in urine samples were determined using HPLC.16CAF plasma clearance has been calculated as the ratio of the administered dose, assuming 100% bioavailability, to the area under the curve of CAF 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 urine samples to the number of methyl groups contained in the molecules of the parent CAF.16The cumulative labeled co2 excretion rate correlated with the CAF plasma clearance. CAF plasma clearance has been used as an indirect method of validating the CBT as a monitor of CYP 1A2, assuming that CAF plasma clearance correlates withCYP 1 A2-dependent CAF N-demethylation. This assumption should be valid because N-demethylation is the major route of CAF metabolism and CAF plasma clearance is expected to be sensitive only to hepatic intrinsic clearance. Because CAFD is a drug with a low extraction ratio and low protein binding, CAF plasma clearance is not expected to be influenced either by hepatic blood flow or by protein binding. The correlation between cumulated13C-co2 excretion rate and CAF plasma clearance already has been found in smoking and nonsmoking adults.13This method allowed us to describe the maturational profile of CAFN-demethylation as a function of age. No detectable change in expired 13C-co2 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.16,17CAF plasma clearance increases with postnatal age according to a single exponential curve. The plateau is reached during the second trimester of life and accounts for ∼60% of all methyl groups. Demethylated metabolites are detectable as soon as 22 days' postnatal age and 34 weeks' postconceptional age.6The use of the CBT also has allowed study of the maturation ofN-demethylation of CAF during puberty. Lambert and colleagues measured the changes of -co2excretion rate after a 3 mg/kg administration of CAF labeled on the N3 methyl group in 62 subjects 3 to 20 years of age. 18The labeled co2 excretion rate was higher in children before puberty than in adults, and decreased with increasing age. Using the five pubertal stages of Tanner, Lambert and colleagues showed that the labeled co2 excretion rate decreased with increasing pubertal maturation. The maturational profile during puberty differed by gender. The excretion rate of adult patients was reached earlier in puberty in girls (early puberty) than in boys (midpuberty). These changes in co2 excretion rate suggest decreasing enzyme function during puberty and a different maturational profile according to gender.The use of the CBT allowed Levitsky and associates19 to study the effect of GH therapy in GH-deficient children on cytochrome P-450-dependent 3-N-demethylation of CAF. Six 4- to 15-year-old GH-deficient children received CAF labeled on the 3-N-methyl group as a single 3 mg/kg oral dose before and after a 4-week treatment with human GH, 0.1 IU/kg sc, three times a week. The 3-N-demethylation of CAF as measured by the CBT was significantly decreased after 1 month of GH therapy, suggesting a possible role of GH in the expression of CYP 1A2.There are no available data in the literature on the use of SIL 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 debrisoquine20 and the metabolism of other enantiomers. In children, changes related to enzymatic maturation may affect significantly the relative rate of biotransformation of enantiomers as a function of age.Using stable-isotope tracer methods, it is possible to perform pharmacokinetic studies that measure steady-state values and evaluate time-dependent and dose-dependent pharmacokinetic changes without exposing the subject to radiation, without creating radioactive waste, and without withholding necessary medication.Serial kinetic studies during maintenance therapy using SIL have been used in children to show 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 both the labeled and the unlabeled compound, using the same blood samples.Carbamazepine (CBZ) is used in children for the treatment of partial seizures. The decrease in steady-state plasma concentrations of CBZ during long-term treatment suggests that CBZ induces its own metabolism.Bertilsson and colleagues21 used tetradeuterium-labeled CBZ in three 10- to 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 subsequent 4 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 5 months of maintenance therapy, a portion of the CBZ dose was replaced by an equivalent amount of labeled CBZ. The plasma kinetics was studied after each of these doses. The steady-state CBZ total plasma concentrations during multiple dosing were lower after dose 3 (days and dose 4 than after dose 2 (day They were two times lower than the steady-state plasma concentrations using the kinetic obtained from the initial they were to those using the kinetic obtained after labeled CBZ during multiple was a increase to in plasma clearance between dose 1 and dose 3 without subsequent CBZ is via changes in kinetic may be to changes in metabolism. This study demonstrated the of CBZ metabolism and its time time-dependent kinetic changes may be studied in children using of of and the effect of age during long-term to study by SIL also for dose-dependent kinetic ie, stable isotope-labeled studies at different steady-state plasma and colleagues a using labeled theophylline in the to the of This method allows one to in the same with the labeled drug to the and with the unlabeled drug to the and to study the kinetics of both labeled and unlabeled drug in both and method is of more limited use in because of drug in the of the is but sampling of the blood is The stable isotope administration technique therefore may be of great potential interest to evaluate and to drug to the a of different stable isotope-labeled forms of the same drug at different times before a single collection of blood via a of the for or therapeutic one can obtain the same the as be obtained by a single dose of the drug and of This has several of a of stable isotopes of a drug is of several stable isotopes of a drug may by increasing with and interference by endogenous One have an of the time for the drug concentration to to times for administration of stable isotope-labeled and However, it also have to be for sample using The be to the potential of several of the drug stable isotope administration technique only a single biologic sample also be to the measurement of the rate of of drugs into or other than blood or as of a or kinetic studies of potential interest in pediatric pharmacology the study of the enantioselectivity of kinetics of and, the determination of and relative in isotope-labeled of a drug have been used to study of a in to not in is as the rate and of of a drug from its form into the is measured as the ratio of the of a of a drug to the of the same drug after administration. is measured as the ratio of the of two of the same drug. is as the equivalent of two or more of the isotopic is that the drug can be administered by either of two (eg, or or in two (eg, or a single of blood samples to describe the time of the or The administration in administration of the that each subject as its own time equivalent stable isotope can the number of subjects by at in The single for both forms The method both drug exposure and to the subject by the time and number of samples and is no need for the to undergo the more than this technique is to the kinetics of a single dose during multiple or dosing can be with also be used in children as tracer to determine The with the use of stable isotopes in studies is the of the a labeled with a stable isotope for the of such a using the enriched isotope that is deuterium. For drugs administered as (eg, be in the drug as a be used as a in Using this to with dose at the in adult is no available of such a use in is related to the presence both in children and in The of in children as varies between and for of that and therefore are and difficult to use in breath test is a noninvasive technique to the presence of activity in the The principle of the breath test is that in the presence of the enzyme administered is and If the carbon is labeled with either the stable isotope 13C or it can be detected in the breath as is the most common and therefore a breath test can be with the presence of a breath test with breath test The great of the breath test in children is the of radioactive the radioactive dose is total is of the test that be at the use of radioactive isotopes at a The breath test has to be very The of and and of the breath test as in children corresponds to that in of has was a need for noninvasive that and separate therapeutic according to their showed that breath test is the equal of of the in a clinical in the treatment was for its to use of to in children is of in a pediatric because of the of of the collection of samples is for is and analysis is to either from an of or or from mg/kg showed that children with a total of of the dose by 2 hours, whereas those with because of from of the dose in the same period of time. The increase of 13C in the breath the activity of the enzyme When were the children with and excretion increased that the treatment potential interest for the of is the breath test The a is produced by a common and the stable isotope carbon A production of in the the presence of has been used to study the effects of on protein metabolism and of in of was not was it a of an increase in protein a decrease in protein or an acid, during the the of only from the of endogenous steady state the rate of of the rate of both into and into protein and can be estimated by the measurement of flow in method was used to the effects of a treatment hours to a of in subjects on metabolism from a of during both the and increased the of from protein but not protein more in the state and to in the this study has not been performed in children, this of study is of great potential interest in children on drug potential interest are the metabolic studies on the of drugs on nitrogen and energy after administration of and labeled isotope have been to and studied three children with and These children demonstrated that decreased after treatment with adults, numerous for its therapy, and other such used in children, isotopic studies a to evaluate their pharmacokinetic the occurrence of isotope effect can to biologic and and therefore should be In isotope effects are used at their It is possible to drug metabolic pathway to a metabolite by labeling the drug with deuterium at metabolic and in animals the of the different of a of the drug in with the unlabeled drug suggests that the deuterium was at a metabolic for the of the metabolite and that of the metabolite was by kinetic isotope is in Two available the and the breath to study the of drugs on of the liquid and the