Evidence of impaired cisapride metabolism in neonates

Abstract

Aims

Cisapride has been shown to cause QTc prolongation in neonates in the absence of any of the known risk factors ascribed to children or adults (excessive dosage, drug–drug interactions). Our hypothesis was that the early neonatal liver may show defective elimination of cisapride resulting in its accumulation in the immature child. Owing to the difficulties associated with in vivo pharmacokinetic studies in a paediatric population, we explored the in vitro metabolism of cisapride by human cytochrome P450.

Methods

Experiments were conducted with recombinant CYPs stably expressed in mammalian cells and with liver microsomes obtained from human foetuses, neonates, infants and adults. Cisapride metabolites were measured by high performance liquid chromatography.

Results

The rate of biotransformation of cisapride was greater by recombinant CYP3A4 than by CYP3A7 (0.77 ± 0.5 and 0.01 ± 0.01 nmol metabolites formed in 24 h, respectively), whereas CYP1A1, 1A2, 2C8, 2C9 and 3A5 showed no activity. Norcisapride formation was significantly correlated with testosterone 6β-hydroxylation, a CYP3A4 catalysed reaction (r = 0.71, P = 0.03) but not with the 16-hydroxylation of dehydroepiandrosterone, catalysed by CYP3A7 (r = 0.30, P = 0.29) by microsomes from a panel of livers from foetuses, neonates and infants. No or negligible cisapride metabolic activity was observed in microsomes from either foetuses or neonates aged less than 7 days, which contained mostly CYP3A7 and no CYP3A4. The metabolism of cisapride steadily increased after the first week of life in parallel with CYP3A4 activity to reach levels exceeding adult values.

Conclusions

The low content of CYP3A4 in the human neonatal liver appears to be responsible for its inability to oxidize cisapride and could explain its accumulation in plasma leading to the cases of QTc prolongation reported in this paediatric population.

Introduction

Cisapride, a substituted piperidinyl benzamide, is one of the most widely used agents to treat gastroesophageal reflux in neonates and infants [1–5]. There is growing evidence of an association between a lengthening of the electrocardiographic QT interval and the use of cisapride. Serious cardiac dysrhythmias including ventricular tachycardia, ventricular fibrillation and torsades de pointes have been reported in patients taking this drug [6, 7]. Cisapride prolongs cardiac repolarization without slowing conduction by a selective blockage of the rapid component of the delayed rectifying K⁺ current (Ikr) leading to a lengthening of the action potential [8]. Complications are detected when cisapride is administered in excessive dosage [6, 9] or in combination with other drugs that might reduce its hepatic metabolism [10–13].

From July 1993 to December 1999, 341 cases of cisapride related QT prolongation were reported spontaneously, including 80 deaths. QT prolongation was associated with the known risk factors in adults (excessive dosage, drug–drug interactions) in approximately 85% of cases [14]. The manufacturer, in consultation with the FDA, decided to discontinue the sale of cisapride and recommended its use only through a limited access programme including the three following treatment protocols: adults – gastroesophageal reflux disease (GERD), gastroparesis, pseudo-obstruction, and severe chronic constipation; children – refractory GERD (associated with failure to thrive, asthma, bradycardia, apnea, or other serious conditions) or pseudo-obstruction; and neonates – feeding intolerance.

A prospective survey of the effects of cisapride in 49 neonates given cisapride reported a significant increase in QT interval [15]. These neonates had none of the known risk factors described for children or adults, implicating age per se as a risk factor for cisapride cardiac toxicity. Janssen Pharmaceutica issued a warning to all health care professionals, cautioning against the use of cisapride in premature newborns [16]. However, the cause and mechanism of cisapride toxicity in newborns remains unknown.

Based on reported clinical [11–13, 17, 18] and in vitro studies [19], there is growing evidence that cisapride is a substrate of cytochrome P450 3A (CYP3A) leading to the formation of biologically inactive metabolites (Figure 1). The CYP3A subfamily is composed of three isoforms: CYP3A4, 3A5 and 3A7 [20, 21]. CYP3A4 is the major CYP isoform present in adult livers [22, 23], but is absent in the foetal liver. CYP3A4 expression rises during the first weeks of life to reach 30–40% of the adult level as early as one month of age [24]. CYP3A5 is similar to CYP3A4 in terms of substrate specificity but is expressed in only 25% of the adult population [25]. Conversely, CYP3A7 is the major isoform expressed in foetal liver. Its liver content and activity are maximal during the first weeks of life and then progressively decline to reach very low levels in adult livers [24]. Our hypothesis was that CYP3A7 may have no or little affinity for cisapride resulting in the inability of neonatal liver to eliminate the drug. In vivo, this may lead to its accumulation in plasma during the first weeks of life and cardiotoxicity.

Figure 1.

Structures of cisapride and its metabolites.

Because of the difficulty of performing pharmacokinetic studies in this paediatric population, we decided to study the in vitro metabolism of cisapride by liver microsomes from foetuses, neonates, infants and adults, and to compare the catalytic activity of CYP isoforms using stably transfected cells.

Methods

Materials

Cisapride and its major metabolites: M1 (norcisapride, R049469), M2 (R063908), M5 (R062864), M7 (R062869) were kindly provided by Janssen, Pharmaceutica. Cisapride was dissolved in 0.5 m lactic acid to obtain a 1 mm stock solution. All other reagents and solvents were of the highest analytical grade. Testosterone and DHEA were from the Sigma, Chemical Co. The preparation of antibodies against CYP3A has been described previously [24].

Expression of recombinant CYP

Ad 293 cells transfected with CYP1A1, 1A2, 2C8, 2C9 3A4, 3A5 and 3A7 full length cDNAs in the expression vector pMT2 were selected and plated as previously detailed [24, 26, 27]. Activities of recombinant P450s were confirmed with the model substrates ethoxyresorufin deethylase (CYP1A1), methoxyresosufin demethylase (CYP1A2), taxol 6β-hydroxylation (CYP2C8), tolbutamide hydroxylation (CYP2C9), and steroid hydroxylations (CYP3A). Cells were grown under 5% CO₂ at 37 °C in Dulbecco's modified Eagle medium supplemented with 4500 mg l⁻¹ glucose, 10% fetal calf serum, 100 IU ml⁻¹ penicillin and 100 µg streptomycin. Stable transfectants were selected for the capacity to express roughly similar levels of CYP proteins when tested by immunoblotting with the appropriate antibody and to display efficient catalytic activities against reference compounds as reported earlier [24, 26, 27]. Cells transfected for CYP3A proteins were cotransfected with the human NADPH-cytochrome P-450 reductase cDNA provided by Dr Urban (Gif, France) inserted into pMT2 to increase the catalytic activity of CYP3A proteins. For other CYPs, the addition of extra copies of the NADPH-P450 reductase gene has no stimulatory effect on catalytic activities. Reductase activity in native Ad293 cells and in cells transfected with CYP but not with reductase (2C8, 2C9, 1A1, 1A2) was about 20 nmol min⁻¹mg⁻¹ microsomal protein and rose to 190–350 nmol min⁻¹mg⁻¹ microsomal protein in cells transfected with 3A4, 3A5 or 3A7 plus reductase.

Microsome preparation

Liver samples from stillborn or aborted fetuses, neonates, infants and adults were collected according to the recommendations of the Ethics Committee of Institut National de la Santé et de la Recherche Médicale and obtained surgically within the hour following death [28]. Clinical data on the patients are shown in Table 1. The adult livers were from renal transplant donors with no known liver disease. Microsomal preparations were characterized for their total cytochrome P450 content by the Greim procedure [29] and for the presence of CYP3A by immunoblotting after SDS-gel electrophoresis, as reported elsewhere [24]. Protein concentrations were determined with the bicinchoninic acid reagent (BioRad, Hercules, CA). The 6β-hydroxylation of testosterone and 16-hydroxylation of dehydroepiandrosterone were measured as described previously [24].

Table 1.

Clinical data on paediatric patients included in the in vitro cisapride metabolic study.

Patient	Postnatal age (days)	Gestational age (weeks)	Pathology
Fœtus 1		20	Spontaneous abortion
Fœtus 2		31	Therapeutic abortion for Down syndrome
Fœtus 3		33	Therapeutic abortion for urinary tract malformations
Fœtus 4		33	Therapeutic abortion for myelomeningocoele
Neonate 1	1	28	Down syndrome sepsis
Neonate 2	2	33	Respiratory distress
Neonate 3	5	28	Hyaline membrane disease
Neonate 4	7	29	Hyaline membrane disease
Neonate 5	35	N.A.	Sudden infant death
Neonate 6	47	40	Sudden infant death
Neonate 7	90	N.A.	Sudden infant death
Neonate 8	90	N.A.	Sudden infant death
Neonate 9	183	38	Sudden infant death

N.A. not available.

Metabolism of cisapride

Cisapride was incubated for 24 h with CYP-expressing cells at near confluence (approximately 5 × 10⁵ cells) at a final concentration of 5 µm in 5 ml DMEM culture medium without fetal calf serum. Mock-transfected cells (transfected with pMT2 alone) were used as a control. After incubation, the culture medium was removed and processed for analysis by h.p.l.c. Human liver microsomes containing to 0.3 nmol P450 were incubated in 0.1 m phosphate buffer, pH 7.45, in the presence of a NADPH-generating system consisting of 10 mm glucose 6-phosphate and 0.5 mm NADP⁺, and 20 µm cisapride in a final volume of 1 ml at 37 °C. The reaction was initiated by the addition of 1 IU of glucose 6-phosphate dehydrogenase and stopped after 30 min by immersing the tube in an ice-cold water bath. Incubations were performed in duplicate. Blank samples were incubated under identical conditions but without the presence of the NADPH-generating system.

Analytical procedure

After centrifugation at 1500 g for 5 min, a fixed volume of internal standard (RIV 2093: benzamide analogue) and 0.1 ml 1 m sodium hydroxide was added successively to 1 ml culture medium or 0.5 ml microsomal incubation mixture. The sample was vortex mixed and extracted with 5 ml ethylacetate. The organic phase was removed and evaporated to dryness under a stream of nitrogen. The residue was dissolved in 100 µl of mobile phase (70% acetonitrile/30% ammonium acetate 0.01 m with 0.5‰ triethylamine pH 9). Separation was achieved on a Satisfaction C8-Plus column (250 × 3 mm, 5 µm, Cluzeau, Sainte Foy la Grande, France) using a gradient as follows: the acetonitrile concentration was increased from 30 to 60% in 30 min and after 5 min at 100%, the column was reequilibrated at 30% acetonitrile before the next injection. The flow was maintained at 0.6 ml min⁻¹ and column eluant was monitored at 230 nm. Under these conditions, retention times were: M2, 5.7 min; M1, 6.6 min; M5, 16.6 min; M7, 24.0 min; cisapride, 26.7 min; internal standard, 11.1 min. Standard curves were linear in the range 5–1250 ng ml⁻¹ (approximately 0.01–2.5 µm) for cisapride metabolites. Reproducibility, assessed as the coefficient of variation, was lower than 10% for all the analytes and their limit of detection was 0.01 µm.

Statistical analysis

Relationships between variables were assessed by the Spearman-rank test.

Results

Metabolism of cisapride by recombinant CYP

No appreciable metabolism of cisapride was observed in either mock-transfected (native Ad 293 cells) or in cells expressing CYP1A1, 1A2, 2C8, 2C9 and 3A5. In contrast, cisapride was metabolized by Ad 293 cells expressing CYP3A4. The main metabolite resulted from the loss of the fluorophenoxypropyl side-chain (norcisapride or M1), its formation exceeding the production of M2 (loss of the fluorophenyl moiety) and that of the oxidation products M5 and M7. CYP3A7 exhibited very limited activity towards cisapride and only M1 was produced at a detectable level (Table 2).

Table 2.

Cisapride metabolism by transfected cells expressing CYP1A1, CYP1A2, CYP2C8, CYP2C9, CYP3A4, CYPA3A5, CYP3A7 and controls. Results are expressed as nmol metabolites formed in 24 h and are the mean±s.e. mean for four determinations, except for CYP3A4 and CYP3A7 (six determinations).

		Metabolites (nmol formed in 24 h)
Transfected cells	M1	M2	M5	M7
Ad293	N.D.	N.D.	N.D.	N.D.
CYP1A1	N.D.	N.D.	N.D.	N.D.
CYP1A2	N.D.	N.D.	N.D.	N.D.
CYP2C8	N.D.	N.D.	N.D.	N.D.
CYP2C9	N.D.	N.D.	N.D.	N.D.
CYP3A4	0.77 ± 0.5	0.025 ± 0.02	0.02 ± 0.03	0.02 ± 0.03
CYP3A5	N.D.	N.D.	N.D.	N.D.
CYP3A7	0.01 ± 0.03	N.D.	N.D.	N.D.

N.D. not detectable (< 0.005 nmol 24 h⁻¹).

Metabolism of cisapride by liver microsomes

The major metabolite produced by adult liver microsomes was M1 while M2, M5 and M7 were formed to a lesser but detectable extent (Table 3). The conversion of cisapride was low in foetal preparations (Table 3). Liver microsomes from three neonates had no detectable or an extremely low activity. Their respective gestational and postnatal ages were 28, 33, 29 weeks, and 1, 2, 7 days, respectively. In contrast, liver microsomes from two other neonates were very active in the biotransformation of cisapride. The first was a 7 day old boy who had died from hyaline membrane disease. He had received three doses of exogenous surfactant, dobutamine, vancomycin, amikacin, ampicillin, fentanyl and frusemide but was not given drugs known to be drug-metabolizing enzyme inducers. The second child who was 3 months old, had received no drugs. The cause of death was attributed to sudden infant death syndrome.

Table 3.

CYP3A content and activity from foetal, neonate and adult livers (values are the mean of duplicate determinations).

				Metabolite formation (nmol min−1 mg−1 protein) cisapride
	Immunoreactive CYP3A (unit mg−1 protein)	Test 6OH (CYP3A4)	DHEA 16α (CYP3A7)	M1	M2	M5	M7	Total
Fœtus 1	1.38	N.D.	0.823	0.11	0	0	0	0.11
Fœtus 2	1.07	0.002	1.12	0	0	0	0	0
Fœtus 3	0.51	N.D.	0.039	0.06	0	0	0	0.06
Fœtus 4	1.32	0.004	0.176	0.25	0.01	0	0	0.26
Neonate 1	0.06	0.005	0.32	0	0	0	0	0
Neonate 2	0.19	0.007	0.411	0	0	0	0	0
Neonate 3	0.92	0.045	2.77	0.68	0.01	0	0	0.69
Neonate 4	0.93	0.029	2.93	40.14	1.30	5.43	3.62	50.49
Neonate 5	0.63	N.D.	0.225	0.40	0.01	0.04	0	0.45
Neonate 6	0.57	N.D.	0.035	0.04	0	0.03	0	0.07
Neonate 7	1.12	0.096	0.716	1.62	0.05	0.17	0.14	1.98
Neonate 8	2.1	N.D.	0.643	9.24	0.46	1.38	0.51	11.59
Neonate 9	1.57	0.105	0.274	1.76	0.06	0.22	0.16	2.2
Adult 1	1.1	0.102	0.088	1.55	0.09	0.23	0.2	2.07
Adult 2	N.D.	N.D.	N.D.	0.52	0.05	0.06	0.04	0.67

N.D. not determined.

Combining the data from liver microsomes of fetuses, neonates and infants, norcisapride formation was significantly correlated with testosterone 6β-hydroxylase, a CYP3A4 associated activity (r_s = 0.71, n = 9, P = 0.03) (Figure 2), but not with the 16-hydroxylation of dehydroepiandrosterone (r_s = 0.30; n = 14, P = 0.29), a reliable index of CYP3A7 activity [24]. No significant relationship was evident between immunodetected CYP3A (the sum of CYP3A4, CYP3A5 and CYP3A7) and the formation of cisapride metabolites (Table 3). Furthermore, the formation of the four cisapride metabolites seems to depend on the same CYP isoform since their rates of production were highly correlated to each other, with r_s values ranging from 0.82 to 0.98 (P < 0.001) (Figure 2).

Figure 2.

(a) The relationship between testosterone 6β-hydroxylation and the rate of cisapride M1 metabolite formation and (b) the relationship between the rate of formation of the M1 and M5 metabolites of cisapride in microsomes from fœtal (•), neonate (▴) and adult (▪) human livers.

Discussion

To gain insight into the basis of cisapride toxicity in neonates we investigated the in vitro hepatic metabolism of cisapride in children. Lacroix et al. [24] have shown that CYP3A4 is not present or barely detectable during the early neonatal period, irrespective of the gestational age at birth. CYP3A7 is present during this period, but does not catalyse the same reactions as CYP3A4. Thus, we studied cisapride metabolism in CYP isoforms expressed in stably transfected cells. Only CYP3A4 was shown to metabolize cisapride leading to the formation of all four derivatives by either cleavage of the fluorophenoxypropyl side-chain or hydroxylation of the parent molecule. Although expressed to the same extent as CYP3A4 in transfected cells, CYP3A7 activity toward cisapride was low and the other CYP isoforms investigated showed no activity. These findings suggest that CYP3A4 is the dominant isoform involved in cisapride biotransformation, which is in agreement with a recent report [19]. Furthermore CYP3A4-transfected cells and adult liver microsomes displayed the same profile of metabolites, and a positive correlation was observed between the conversion of cisapride into its four metabolites and the CYP3A4 content of hepatic microsomes. Collectively, these data strongly support earlier work, that CYP3A4 is responsible for cisapride metabolism in adult liver.

A previous report on the ontogenic profile of CYP3A subfamily members has demonstrated that CYP3A4 is very low at birth and its expression begins to rise after only 1 week of age [24], which would suggest that cisapride cannot be metabolized very well by young neonates. This was tentatively confirmed by our findings. Thus, liver microsomes from foetuses and neonates aged less than 1 week lacked CYP3A4 and showed no detectable metabolism of cisapride. After 1 week of age, the CYP3A4 activity and the metabolism of cisapride by neonatal liver began to increase. It is recognised that these findings are based on a relatively small numer of liver samples. However studies of this nature are disadvantaged by the limited availability of paediatric liver tissue.

Clinical studies have been performed in neonates and infants at doses of cisapride between 0.15 and 0.3 mg kg⁻¹ given 3–4 times daily, but no definitive pharmacokinetic data have been obtained in relation to the postnatal age of children [30–32]. Recently Benatar [33] compared the QT interval for two groups of children aged 2–24 weeks receiving cisapride therapy for gastroesophagol reflux with those receiving no therapy. Patients and controls were further divided into three age groups: < 3 months, 3–6 months, > 6 months. It was clearly demonstrated that the QTc interval significantly increases on cisapride therapy only in infants aged less than 3 months [34]. This clinical observation is consistent with our in vitro results and suggests that cisapride should be used with caution in neonates during the first weeks after birth.