Association of cytochrome P450 CYP2C9 (rs1057910) gene polymorphism and ibuprofen response in preterm neonates diagnosed with patent ductus arteriosus

Abstract

Background

Patent ductus arteriosus (PDA) closure is one of the most significant changes necessary to transition to extrauterine life. The failure of closure in preterm infants has been associated with a variety of complications.

Aim and objectives

This study aim to investigate the correlation between cytochrome P450 CYP2C9 gene polymorphism and the response to ibuprofen treatment in preterm neonates with PDA.

Subjects and methods

This prospective study was conducted on 64 preterm neonates with patent ductus arteriosus (hsPDA). The neonates were treated with ibuprofen and diagnosed using clinical and echocardiographic examinations. The study was carried out at the Neonatal Intensive Care Unit (NICU), Pediatric Department, Kasr Alainy and ElMounira Pediatric Hospitals, Cairo University, in June 2018.

Results

A statistically significant difference in respiratory rate was observed between both groups (p = 0.047). Additionally, there was a significant difference in the duration of treatment with ibuprofen (p = 0.021). Treatment with ibuprofen had no impact on renal function parameters. The platelet count decreased after treatment with no statistical difference.

Conclusion

Use of Oral ibuprofen is a highly effective treatment for HsPDA in preterm neonates, demonstrating a remarkable success rate of 92.2% and fewer adverse effects. Whilst no correlation between the CYP2C9 (rs1057910) gene polymorphism and the efficacy of oral ibuprofen response, Other factors affecting the response of oral ibuprofen and subsequent PDA closure include gestational age, birth weight, Apgar score at 5 min, ductal diameter, RDS, and sepsis.

Keywords

CYP2C9 ibuprofen PDA preterm neonates

Introduction

The ductus arteriosus (DA) is the vascular connection between the aortic arch and the pulmonary artery during fetal circulation. DA closure is a crucial step in the transition from intrauterine to extrauterine life.¹ In healthy full-term newborns, DA generally undergoes functional closure between 24 and 72 h of life.² The incidence of PDA in Egypt is 57.2%.

Currently, the most rational approach is to limit treatment to HsPDA. The administration of non-steroidal anti-inflammatory Drugs (NSAIDs) for the treatment of PDA varies significantly in practice among NICUs. Ibuprofen is the first-choice drug for HsPDA treatment.³

Ibuprofen has demonstrated efficacy in PDA closure while maintaining cerebral and mesenteric blood flow, and it has a minor effect on renal perfusion compared to indomethacin. The decision to administer ibuprofen for PDA closure was determined based on the individual’s clinical condition (e.g., increased respiratory support or hypotension) and echocardiographic findings (e.g., large ductus >2 mm or low peak systolic ductal flow). Infants with right-to-left or bidirectional shunting PDA, intraventricular hemorrhage grade ≥3, or poor renal function (serum creatinine >1.8 mg/dl or oliguria <1 mL/kg/h) were not eligible for ibuprofen treatment, as per unit policy. The attending neonatologist also decided to administer treatment to the patient (using either oral ibuprofen suspension or intravenous ibuprofen). A single course of ibuprofen treatment involved the administration of three consecutive doses of 10, 5, and 5 mg/kg/dose, with each dose administered orally and intravenously, with a 24-hour interval between each treatment. Responder to ibuprofen treatment was defined as the absence of ductal flow in echocardiography within 24 h after completion of treatment.⁴

Individuals can exhibit varying drug responses, as evidenced by pharmacogenomics. Currently, it is common to give newborns of varying gestational ages and postnatal ages the same amount of ibuprofen. Genetic polymorphisms may influence various clinical and side effects, categorizing treated patients into extensive and poor metabolizers.⁵

In addition, the study assessed the impact of CYP450 polymorphism on the pharmacokinetics of S-ibuprofen and R-ibuprofen in healthy volunteers treated with ibuprofen. Specifically, the influence of genetic variants CYP2C9*2 and CYP2C9*3 on drug metabolism showed a gender predisposition.⁶

Infants who possess the variant allele would experience reduced clearance of ibuprofen, higher drug exposure, and potentially more profound clinical effects. The impact of genetic variability on the incidence and treatment of PDA is significant but has not been thoroughly studied.⁷

The objective of this study was to assess the impact of pharmacogenetic factors (CYP2C9 genotypes) on the efficacy of ibuprofen response. Additionally, the study investigated the potential relevance of genotype-based dosing of ibuprofen, which is expected to increase the rate of PDA closure and subsequently reduce neonatal morbidity.

Patients and methods

This prospective study was performed on 64 preterm neonates with hsPDA who were treated with ibuprofen. The diagnosis of HsPDA was made based on clinical evaluation and echocardiographic examination. This study was carried out at the Neonatal Intensive Care Unit (NICU), Pediatric Department, Kasr Alainy and ElMounira Pediatric Hospitals, Cairo University in June 2018. The study protocol was approved by the Local Ethical Committee of the Pediatric Hospital at Cairo University, and parental consent was obtained.

Sample size calculation

Sample size calculation was done using SPSS power, sample calculation software, version 3.0.11 for MS Windows (William D. Dupont and Walton D Vanderbilt, USA).

The Fischer exact test was calculated by comparing two proportions from independent samples in a prospective study. The α-error level was fixed at 0.05, the power was set at 80%, and the intervention group (cases and control) ratio was set at 1.

Durrmeyer et al.⁸ showed that the ibuprofen response rate among polymorphic preterm neonates was 52%, while it was 73% in non-polymorphic matched neonates. Accordingly, the minimum optimum sample size was 64 studied preterm neonates.

Patients

Preterm newborn babies with gestational age equal to or less than 35 weeks, aged 1–3 days of life, diagnosed to have PDA (hemodynamically significant) clinically and by echocardiography.

After admission to the NICU, all preterm infants with hemodynamically significant PDA (HsPDA) received medical treatment. Three daily doses of ibuprofen were given orally on three consecutive days at a 24-hour interval. Each course of therapy was composed of a standard dose of 10 mg/kg/dose/day on the first day of treatment, followed by two subsequent doses of 5 mg/kg/dose/day on the second and third days.

Ibuprofen administration started between 12 h and 72 h of life. In most cases, patients were evaluated by color Doppler ultrasonography using 5–7 MHz probe ultrasound. Patients with partially closed PDA after the first course of treatment received the second course 38 h after the first course.

Inclusion criteria of the patients

Preterm newborns ≤35 weeks, preterm newborns aged 1–3 days of life, and clinically and echocardiography diagnosed with HsPDA.

Exclusion criteria of the patients

Full-term neonates, defined as newborn babies with gestational age more than 37 weeks of age. Preterm neonates with gross congenital anomalies, and preterm neonates with congenital heart disease except for patent foramen oval, mild mitral and tricuspid regurgitation (Tables 1 and 2).

Table 1.

Clinical characteristics and demographic data of the study population according to CYP2C9 genotype.

Demographic data	CYP2C9		Chi-square test (p-value)
	AA (n = 48)	AC (n = 16)
	Mean ± SD	Mean ± SD
GA (weeks)	31.25 ± 1.862	31.81 ± 1.424	0.274 (NS)
Birth weight (g)	1620.83 ± 261.101	1693.75 ± 203.204	0.313 (NS)
APGAR at 1 min	4.33 ± 0.834	4.56 ± 0.727	0.33 (NS)
APGAR at 5 min	6.77 ± 0.951	6.88 ± 0.719	0.69 (NS)
HR (minute)	176.1 ± 2.97	175.13 ± 2.604	0.244 (NS)
RR (minute)	72.98 ± 4.76	70.25 ± 4.328	0.047 (S)
Ductal diameter (mm) detected by Echo	2.892 ± 0.6881	2.863 ± 0.8172	0.889 (NS)
Duration of treatment with ibuprofen	3.67 ± 1.326	3.13 ± 0.5	0.021 (S)

Table 2.

The outcomes in preterm neonates with HsPDA after the course of oral ibuprofen treatment in responders and non-responders groups.

Outcomes		Response to ibuprofen						Chi-square test (p-value)
		Responders (n = 59)		Non-responders (n = 5)		Total (n = 64)
		No	%	No	%	No	%
Surgical ligation	Yes	0	0	3	60	3	4.7	<0.001 (S)
Surgical ligation	No	59	100	2	40	61	95.3	<0.001 (S)
ROP	Yes	1	1.7	0	0	1	1.6	1 (NS)
ROP	No	58	98.3	5	100	63	98.4	1 (NS)
NEC	Yes	0	0	1	20	1	1.6	0.001 (S)
NEC	No	59	100	4	80	63	98.4	0.001 (S)
GIT bleeding	Yes	0	0	3	60	3	4.7	<0.001 (S)
GIT bleeding	No	59	100	2	40	61	95.3	<0.001 (S)
IVH	Yes	0	0	1	20	1	1.6	0.001 (S)
IVH	No	59	100	4	80	63	98.4	0.001 (S)
Oliguria	Yes	0	0	3	60	3	4.7	0.001 (S)
Oliguria	No	59	100	2	40	61	95.3	0.001 (S)
Sepsis	Yes	13	23.7	4	80	18	28.6	0.021 (S)
Sepsis	No	45	76.3	4	20	46	71.4	0.021 (S)
Hyperbilirubinemia	Yes	31	52.5	0	0	31	48.4	0.024 (S)
Hyperbilirubinemia	No	28	47.5	5	100	33	51.6	0.024 (S)
Death	Yes	0	0	2	40	2	3.1	0.005 (S)
Death	No	59	100	3	60	62	96.9	0.005 (S)

All patients were treated and evaluated similarly during the study period.

Methods

All studied patients were subjected to the following: History taking (maternal, antenatal, and natal history), Ballard score system,⁹ Clinical examination, and Echocardiographic examination and Laboratory investigations.

Blood sample collection

Two mL of blood were collected from each participant in a sterile violet topped ethylene diamine tetra acetate (EDTA) and stored −20°C in a microcentrifuge tube for genotyping of rs1057910 variant by Real Time-PCR.

Genomic DNA extraction from EDTA blood samples using Gene JET Whole Blood Genomic DNA Purification Mini Kit) provided by (Thermofisher Scientific, Lot No. 00616093)**Thermofisher scientific company Watltham, Massachusetts USA.

DNA amplification and real-time PCR allelic discrimination assays

An optimized protocol of real-time PCR allelic discrimination assay was done for genotyping of CYP2C9 gene rs1057910 (probe ID C__27104892_10 (Applied biosystems CA94404, Foster city, USA) and a Step-one Real-Time Polymerase Chain Reaction System.

Data management and analysis

The collected data was revised, coded, tabulated, and fed into the computer using Microsoft Excel 2013 for data entry and the Statistical Package for Social Science (SPSS), version 22 (SPSS, Armonk, Newyork: International Business Machine Corporation) was used for data analysis. Bivariate relationship was displayed in cross-tabulations and comparison of proportions was performed using chi-square test or Fischer exact test whenever appropriate. T test was used to compare normally distributed quantitative data. Level of significance (p-value): p > 0.05: Non-significant (NS), p < 0.05: Significant (S), and p < 0.01: Highly significant (HS).

Results

There was no significant difference between the two groups as regards gestational age (weeks) (p = 0.274), birth weight (gm) (p = 0.313), APGAR at 1 min (p = 0.33), APGAR at 5 min (p = 0.69), HR (minute) (p = 0.244) and ductal diameter (detected by echocardiography) (p = 0.889).

There was a significant difference in the respiratory rate between both groups (p = 0.047). The duration of treatment with ibuprofen differed significantly between the homozygous (wild type) group (3.7 ± 1.3) and the heterozygous type group (mean duration: 3.1 ± 0.5), with a statistically significant difference (p = 0.021). As regards surgical ligation and GIT bleeding, there were statistically highly significant differences between responder and non-responder groups (p < 0.001).

For necrotizing enterocolitis (NEC), interventricular hemorrhage (IVH), and oliguria, there were statistically significant differences (p = 0.001) between the two groups. Regarding retinopathy of prematurity (ROP), there was no statistically significant (p = 1) between the two groups. Nevertheless, regarding sepsis, there was a statistically significant difference (p = 0.021) between the two groups.

Concerning hyperbilirubinemia, there was a significant difference (p = 0.024) between the two groups. In addition, the two groups had a statistically significant difference in mortality rates (p = 0.005).

There were no statistically significant differences regarding outcomes between the two groups in both homozygous (wild type) and heterozygous type groups. As regards surgical ligation, ROP, NEC, GIT bleeding, IVH, oliguria, and mortality, they were present in the homozygous type group and absent in the heterozygous group. However, sepsis was more prevalent in the homozygous type (33.3%) compared to the heterozygous type (12.5%). By comparison, hyperbilirubinemia was more common in the heterozygous type (50%) than in the wild type (47.9%).

There was no statistical significance between response to ibuprofen and CYP2C9 genotype (p = 0.319) (Tables 3 and 4).

Table 3.

The outcomes in preterm neonates with HsPDA after the course of oral ibuprofen treatment in homozygous (AA) and heterozygous (AC) groups according to CYP2C9 genotypes.

Outcomes		CYP2C9						Chi-square test (p-value)
		AA (n = 48)		AC (n = 16)		Total (n = 64)
		No	%	No	%	No	%
Surgical ligation	Yes	3	6.3	0	0	3	4.7	0.567 (NS)
Surgical ligation	No	45	91.7	16	100	61	95.3	0.567 (NS)
ROP	Yes	1	2.1	0	0	1	1.6	1 (NS)
ROP	No	47	97.9	16	100	63	98.4	1 (NS)
NEC	Yes	1	2.1	0	0	1	1.6	1 (NS)
NEC	No	47	97.9	16	100	63	98.4	1 (NS)
GIT bleeding	Yes	3	6.3	0	0	3	4.7	0.567 (NS)
GIT bleeding	No	45	91.7	16	100	61	95.3	0.567 (NS)
IVH	Yes	1	2.1	0	0	1	1.6	1 (NS)
IVH	No	47	97.9	16	100	63	98.4	1 (NS)
Oliguria	Yes	1	2.1	0	0	1	1.6	1 (NS)
Oliguria	No	47	91.7	16	100	63	98.4	1 (NS)
Sepsis	Yes	16	33.3	2	12.5	18	28.1	0.195 (NS)
Sepsis	No	32	66.7	14	87.5	46	71.9	0.195 (NS)
Hyperbilirubinemia	Yes	23	47.9	8	50	31	48.4	0.885 (NS)
Hyperbilirubinemia	No	25	52.1	8	50	33	51.6	0.885 (NS)
Death	Yes	2	4.2	0	0	2	3.1	0.407 (NS)
Death	No	46	95.8	16	100	62	96.9	0.407 (NS)

Table 4.

Relation between response to ibuprofen and CYP2C9 (rs1057910) genotype.

CYP₂C₉ (rs1057910) genotype	Responders (n = 59)		Non-responders (n = 5)		Total		Chi-square test
CYP₂C₉ (rs1057910) genotype	No	%	No	%	No	%	Chi-square test
AA	43	72.9	5	100	48	75	p = 0.319 (NS)
AC	16	27.1	0	0	16	25	p = 0.319 (NS)

Discussion

The current study results revealed a significant difference between the responder and the non-responder groups regarding retinopathy of prematurity. However, there was no significant difference between the homozygous and heterozygous groups.

Similarly, Ye et al.¹⁰ found no significant difference in retinopathy of prematurity between the homozygous and the heterozygous groups. Consistent with these results, El-Mashad et al.¹¹ showed no significant difference between the two groups.

Our results showed a significant difference in gastrointestinal bleeding between the responders and the non-responders. This aligns with Shahmirzadi et al.,¹² who showed a significant difference between the ibuprofen and paracetamol groups, indicating that ibuprofen is a preferred treatment option in their study.

There was a significant difference between the responders and the non-responders regarding oliguria (urine output <1 cc/kg/hour). This finding is consistent with Ohlssson et al.,¹³ who found a significant difference between the ibuprofen group and the placebo group in the incidence of oliguria, with a higher incidence in the ibuprofen group. Likewise, Ohlsson et al.¹³ found a statistically significant reduction in the proportion of infants with oliguria in the ibuprofen group than the indomethacin group (Figures 1–5).

Figure 1.

Study flow chart.

Figure 2.

Bar chart showing respiratory support (nasal O₂, NCPAP, and SIMV).

Figure 3.

The total numbers and percentages for each CYP₂C₉ genotype (AA, AC) in responders and non-responders groups.

Figure 4.

Efficacy of ibuprofen response for the treatment of HsPDA, with a response rate of 92.2% in preterm neonates.

Figure 5.

In-hospital outcomes of preterm infants with HsPDA after the course of OIBU treatment.

Our study also found no significant difference between the homozygous type and heterozygous type groups in the incidence of oliguria. This result agrees with Matsumura et al.,¹⁴ who showed no significant difference between the ibuprofen and indomethacin groups in the incidence of oliguria. Furthermore, Constance et al.¹⁵ found no statistical significance between the ibuprofen and control groups in the incidence of oliguria.

There was a significant difference between the responders and non-responders regarding hospital mortality. Consistent with our results, Aldana-Aguirre et al.¹⁶ found a significant difference in mortality rates between the AG and AGA groups of infants to treat symptomatic PDA).

When studying the mortality rate in treated preterm infants with symptomatic PDA, Ohlsson et al.¹³ discovered that there was no significant statistical difference in rates between the groups of preterm infants treated with oral and IV ibuprofen versus oral or IV indomethacin for symptomatic PDA.

Conclusion

Oral ibuprofen is a highly effective treatment and confirmed a place for HsPDA in preterm neonates, with minimal side effects, and has a success rate of 92.2%. There is no relationship between the CYP2C9 (rs1057910) gene polymorphism and the effectiveness of the response to oral ibuprofen. As currently performed, other factors affecting the response of oral ibuprofen and subsequent PDA closure include gestational age, birth weight, Apgar score at 5 min, ductal diameter, RDS, and sepsis.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethical approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Cairo University (Date 15-08-2018 /No MD08-2018).”

ORCID iD

Radwa Marawan Abdel Halim

Data availability statement

The authors declare that the data supporting the findings of this study are available within the paper.*

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