Use of Cholecystokinin-Octapeptide for the Prevention of Parenteral Nutrition-Associated Cholestasis
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《小儿科》
Department of Surgery
Department of Radiology, C.S. Mott Children's Hospital
School of Public Health, University of Michigan, Ann Arbor, Michigan
Hasbro Children's Hospital, Brown Medical School, Providence, Rhode Island
St Vincent's Mercy Children's Hospital, Toledo, Ohio
Strong Memorial Hospital, University of Rochester, Rochester, New York
Baylor College of Medicine, Houston, Texas
Johns Hopkins University School of Medicine, Baltimore, Maryland
Columbus Children's Hospital, Columbus, Ohio
ABSTRACT
Objective. To determine whether cholecystokinin-octapeptide (CCK-OP) would prevent or ameliorate parenteral nutrition-associated cholestasis (PNAC) among high-risk neonates treated with total parenteral nutrition.
Study Design. This was a multicenter, double-blind, randomized, controlled trial conducted between 1996 and 2001.
Patients. Neonates at risk for the development of PNAC included very low birth weight neonates and those with major surgical conditions involving the gastrointestinal tract.
Setting. Tertiary care hospitals.
Intervention. Patients were randomized to receive CCK-OP (0.04 μg/kg per dose, twice daily) or placebo. Eligible infants were all <30 days of age. Patients were enrolled within 2 weeks after birth or within 7 days after surgery.
Outcome Measures. The primary outcome measure was conjugated bilirubin (CB) levels, which were measured weekly. Secondary outcome measures included incidence of sepsis, times to achieve 50% and 100% of energy intake through the enteral route, number of ICU and hospital days, mortality rate, and incidences of biliary sludge and cholelithiasis.
Results. A total of 243 neonates were enrolled in the study. CCK-OP administration did not significantly affect CB levels (1.76 ± 3.14 and 1.93 ± 3.31 mg/dL for CCK-OP and placebo groups, respectively; mean ± SD). Secondary outcome measures also were not significantly affected by the study drug.
Conclusions. Use of CCK-OP failed to reduce significantly the incidence of PNAC or levels of CB. CCK-OP had no effect on other secondary measures and should not be recommended for the prevention of PNAC.
Key Words: cholecystokinin-octapeptide parenteral nutrition-associated cholestasis bilirubin
Abbreviations: PNAC, parenteral nutrition-associated cholestasis PN, parenteral nutrition CB, conjugated bilirubin CCK-OP, cholecystokinin-octapeptide NEC, necrotizing enterocolitis TPN, total parenteral nutrition CCK, cholecystokinin
Parenteral nutrition-associated cholestasis (PNAC) is a significant complication of prolonged parenteral nutrition (PN).1 The disease is particularly common among premature neonates and patients who undergo major gastrointestinal operations in the newborn period.2 PNAC is associated with a 50% risk of sepsis and mortality rates as high as 20% to 30%.3 Currently, there is no known treatment or means to prevent PNAC among patients who cannot tolerate enteral feedings. Attempts at addressing this problem have used a number of therapeutic interventions, including taurine-supplemented PN,4 tauroursodeoxycholic acid,5 and enteral antibiotics.6 The removal of several agents, including manganese, copper, and phytosterols, from intravenous lipids has also been suggested to be beneficial.7–9 Despite some encouraging reports, no definitive treatment or mode of prevention of PNAC has been proved.
One factor potentially contributing to PNAC is a lack of gastrointestinal hormonal stimulation, which facilitates bile flow. Cholecystokinin (CCK) is a gastrointestinal hormone that is released during enteral stimulation and can promote both intrahepatic and extrahepatic bile flow.10–12 We and others demonstrated previously that CCK-octapeptide (CCK-OP) could be used to partially correct hyperbilirubinemia associated with PNAC.13–15 CCK-OP was also shown in a retrospective study to prevent a rapid increase in conjugated bilirubin (CB) levels (used as a marker for PNAC), compared with untreated infants who were matched with respect to gestational age, primary disease, and duration of PN.16 That study showed that patients who received CCK-OP demonstrated a reduced incidence of severe cholestasis (defined as a direct bilirubin level of 5.0 mg/dL), compared with untreated patients (9.5% vs 38%, P = .015).
The study was conducted as a randomized, double-blind, controlled, multicenter trial over 8 weeks. We hypothesized that the use of CCK-OP could prevent the development of PNAC in an at-risk group of neonates. The primary outcome measure was CB levels, as a marker of the severity of PNAC. Secondary outcome measures were the incidence of sepsis, times to achieve 50% and 100% of energy intake through the enteral route, number of ICU and hospital days, mortality rate, and incidences of cholelithiasis and biliary sludge. The study examined severely premature neonates, as well as infants undergoing major surgical procedures in the newborn period (eg, necrotizing enterocolitis [NEC], gastroschisis, or severe intestinal atresia), who during their study course received >50% of total energy needs through the parenteral route.
METHODS
Trial Design
This study was a double-blind, randomized, multicenter, controlled trial to assess whether CCK-OP could prevent or reduce the severity of PNAC. In a small preliminary study,16 we observed rates of PNAC (defined as CB levels of >2.5 mg/dL) of 9% and 38% for CCK-OP-treated subjects and historical control subjects, respectively. Therefore, we designed this study to have 80% power to identify a difference of 15% (10% vs 25%) with a 2-tailed, 5% level of significance. To achieve this power, a total of 252 neonates at risk for PNAC would be needed (126 in each of the placebo and study drug groups). A neonatal population was selected because it was identified by several investigators as having the patients with the highest risk of developing PNAC.1 The primary outcome measure was CB levels, as a measure of the development and severity of PNAC. Secondary outcome measures included the incidence of sepsis, times to achieve 50% and 100% of energy intake through the enteral route, number of ICU and hospital days, mortality rate, and incidences of biliary sludge and cholelithiasis. The study was assigned IND 42898 and was funded by the Food and Drug Administration Orphan Products Grant Program.
Treatment Specifications
Drug Name and Source
Sincalide (Kinevac; Bracco Diagnostics, Princeton, NJ) is a synthetic form of CCK-OP that has been approved by the Food and Drug Administration. Dosing of CCK-OP was 0.04 μg/kg per dose, administered intravenously (in 10–15 minutes) every 12 hours, based on a mean of weekly body weights. Dose selection was based on previous use of the drug and because this was twice the dose recommended for stimulation of gallbladder contraction.16 Placebo consisted of intravenously administered 0.9% sodium chloride, given on the same schedule as the study drug. CCK-OP was diluted to the correct concentration in 0.9% sodium chloride. Appearance, size, and volume were identical to those of the study drug.
Total Duration
Patients remained on the CCK-OP or placebo therapy for no longer than 8 weeks, regardless of whether they achieved enteral feeding or not. CCK-OP or placebo administration was discontinued once the enteral component was >50% of the patient's nutritional requirements. If CCK-OP or placebo treatment was discontinued because >50% of energy needs were provided through the enteral route but enteral nutrition decreased to <50% of total required energy for any reason, then the patient was again treated with either CCK-OP or placebo, according to the previous assignment. All caretakers were masked with respect to group assignment; only the investigational drug service was aware of the results of the randomization.
Study Population
Study Centers
The 8 centers used in this proposal were chosen because of their homogeneity. Each is a tertiary care facility for neonates with a large regional referral pattern that exceeds 10000 live births per year. Therefore, it was anticipated that a fairly uniform population of patients would be examined among the sites.
Inclusion Criteria
Participating sites and the numbers of recruited patients in the study are shown in Table 1. All patients were receiving PN at the time of recruitment. Although the study was designed to include patients who would require prolonged periods of PN, after enrollment the patients remained in the study regardless of the duration of PN. Initially, the study population consisted of severely premature infants (<1000 g at birth and with an estimated [Dubowitz] gestational age of 28 weeks). However, because the majority of these patients were found to achieve >50% of nutritional intake through the enteral route by 7 to 14 days, the inclusion criteria were changed in 1999, to include surgical neonates (<30 days of age at the time of enrollment) who had a major gastrointestinal disorder preventing enteral intake. These criteria included 3 diagnoses, ie, (1) NEC, (2) gastroschisis, and (3) severe jejunal-ileal atresia. Diagnoses of NEC met Bell grade II or higher criteria.17 Severe jejunal-ileal atresia was defined as an intestinal loss of 50% of the anticipated small-bowel length for a given gestational age.18
A daily screening (based on discussions with the attending intensive care physicians) of all neonates in the each study center was performed to identify potential candidates for the study. All patients were recruited into the study within 7 days after the diagnosis of any one of the surgical disorders. Patients with hemodynamic instability (see below) were recruited but were not begun in the study until they were in hemodynamically stable condition (but still <30 days of age).
Exclusions Before Initiation of the Study
Patients in hemodynamically unstable condition were excluded. However, they could be enrolled if their condition stabilized within the first 2 weeks of life and before the initiation of feedings. Instability was defined as requiring fluid boluses of >40 mL/kg in the previous 24 hours in addition to maintenance intravenous fluids or requiring adrenergic support of >10 μg/kg per minute dopamine or dobutamine or 0.1 μg/kg per minute epinephrine. Also excluded were infants with a metabolic pathway defect (eg, hereditary fructose intolerance, galactosemia, or neonatal tyrosinemia), hepatic insufficiency (defined as a CB level of >1.0 mg/dL or a coagulation abnormality of an international normalized ratio >1.5 times normal), or progressive renal failure (defined as a creatinine level of >1.5 mg/dL). In addition, infants with primary or secondary liver disease (including hepatitis), regardless of liver function; use of extracorporeal life support; suspected congenital obstruction of the hepatobiliary tree (eg, biliary atresia or a choledochal cyst); or a diagnosis of HIV infection were excluded. No infants were enrolled if their CB levels were >1.0 mg/dL. Finally, infants were excluded if they received ursodeoxycholic acid before the study; no patients received this drug during the study.
After enrollment, all infant data were recorded and analyzed regardless of whether study drug/placebo treatment was stopped. Data were analyzed with an intent-to-treat approach. If neonates underwent a surgical procedure, then the study drug/placebo was withheld for a minimum of 12 hours and a maximum of 96 hours after surgery.
Randomization and Masking
All patients were enrolled after signed detailed informed consent was obtained from either a parent or guardian. The consent form and the entire study were approved fully by the institutional review boards of all participating hospitals. Infants were randomized to receive the study drug or placebo on the basis of an established randomization chart. Block randomization with a randomly chosen block size of 2 or 4 within center was used. Randomization tables and envelopes were generated by the Department of Biostatistics at the University of Michigan and were distributed to each site. Control of drug randomization and distribution of drug were performed by the investigational drug service at each study site. Patients, medical professionals, and investigators were masked from the results of the randomization. Enrollment was performed by either study coordinators or principal investigators at each study site. The flow of enrollment, study performance, and analysis is shown in Figure 1.
PN Guidelines
To ensure a uniform delivery of PN among patients and sites, the following standards were used. Total nonprotein energy delivery did not exceed 378 to 420 kJ/kg per day. Carbohydrate administration began at 4 to 8 mg/kg per minute on the first day of PN and was increased to 11 to 18 mg/kg per minute by day 3. Provision was made for patients who were glucose intolerant during this increase. Protein administration began at 1.0 g/kg per day on the first day of PN and was increased by 0.5 to 1 g/kg per day to a maximum of 2.5 to 3.0 g/kg per day. Lipid administration began at 1.0 g/kg per day on the first day of PN and was increased by 0.5 to 1 g/kg per day to a maximum of 3.0 g/kg per day. A pediatric amino acid formulation that contained taurine was used for PN; this was either Aminosyn-PF (Abbott, Abbott Park, IL; 10% solution containing 70 mg/100 mL in the bulk solution) for 76 patients or Trophamine (B. Braun, Bethlehem, PA; 10% solution containing 25 mg/100 mL in the bulk solution) for 127 patients. For the first 40 patients enrolled, taurine was not included in the formulation, and neonates received a standard adult formulation (Aminosyn; Abbott).
Primary Outcome Measure
CB levels were measured within 72 hours before or on the day of recruitment into the study and weekly thereafter. To ensure uniformity in laboratory determinations of the primary outcome measure, a Kodak Ektachem 750 system (Johnson and Johnson, New Brunswick, NJ) was used for all sample measurements.
Secondary Outcome Measures
Assessment of Patient Morbidity and Mortality Rates
Secondary outcome measures included the incidence of sepsis (both generalized and catheter-related), time in days to achieve 50% enteral feedings, time in days to achieve 100% enteral feedings, number of days in the ICU, number of hospital days, and mortality rates. Monitoring and recording of all in-hospital complications were performed on a weekly basis. Sepsis in the neonatal period used previously established definitions.19 Catheter sepsis was defined with a modified definition established by the Centers for Disease Control and Prevention.20 Percentage of energy intake was calculated by totaling enteral intake and dividing the result by the total number of joules delivered through the enteral and parenteral routes, including all protein sources. Infants had to tolerate a level of feeding (50% or 100%) for 3 consecutive days before reaching this definition. All patient deaths were recorded. Death was defined as a death occurring at any time during the child's admission to the hospital. The cause of death and autopsy findings were also recorded.
Ultrasonographic Assessment of the Hepatobiliary Tree
Formation of biliary sludge was assessed among children treated at 2 sites (University of Michigan and St Vincent's Mercy Hospital). The techniques at these sites were compared closely (because of their close geographic locations) and allowed accurate analysis of the data. An ultrasound study was performed within 48 hours after recruitment into the study, and a follow-up ultrasound study was performed 2 weeks after initiation of the study. Before the ultrasound studies, all infants had feedings withheld for at least 3 hours. Biliary ultrasonography was performed with 5- to 8-MHz sector transducers (Acuson 128XP/10; Acuson, Mountain View, CA; or ATL HDI 5000; ATL, Bothell, WA), depending on patient size. The gallbladder and biliary tree were assessed with longitudinal, transverse, and oblique scans, as indicated by the anatomic features. The following measurements were scored: presence of biliary sludge, cholelithiasis, choledocholithiasis, bile duct dilation (intra- or extrahepatic), and size of the common bile duct (transverse diameter at the liver hilum).
Monitoring
Monitoring Schedule
During the study period, neonates were monitored daily. During each infusion period, assessment for the development of adverse reactions was performed (see below). After completion of the 8-week study, infants were monitored daily during the NICU stay and every third day while out of the NICU but in the hospital. A final check for adverse reactions was performed, via telephone, 30 days after patient discharge.
Adverse Events
Adverse events were defined as any untoward events that occurred during the performance of the study. Each event was recorded. A determination of whether the event was associated with the administration of the study drug was then recorded as likely, possibly, unlikely, or not associated. On the basis of these criteria, if likely or possibly associated was selected, then the event was defined as an adverse drug reaction. In general, 3 major reactions were checked for routinely, namely, pain, feeding intolerance, and hemodynamic changes. The study coordinator at each center monitored for drug-related adverse reactions, including pain presumed to be attributable to gastrointestinal cramping, intolerance of oral feedings because of spasm of the pylorus, and hypotension. Nursing and physician staff members administering the drug were made aware of potential adverse reactions and were instructed to notify the investigators at each center if such reactions developed. All adverse reactions were reported to the principal investigator, the independent monitoring committee (see below), the study center's institutional review board, and the Food and Drug Administration. Significant adverse events included the following: death, sepsis, drug-related adverse events, prolongation of hospitalization, overdose, and disabling injury. If a significant adverse event was considered possibly associated with the study drug, then this was grounds for discontinuation of the study. One case of drug overdose occurred in the study; this did not result in any other adverse events. In that case, a 10-fold higher concentration of the study drug (CCK-OP) was given, which was recognized by the investigational drug service after the drug had been administered. Despite this larger amount of drug, no hemodynamic changes or feeding intolerance was noted. In addition, the pain score (see below) did not change, compared with predosing scores. The randomization code was broken for this patient; however, the patient remained in the study because of an intent to treat all recruited patients. A full report of this adverse event was made to all institutional review boards and the Food and Drug Administration.
Grading of pain among premature neonates used the Premature Infant Pain Profile system.21 Feeding intolerance had to be associated consistently (2 times) with the administration of CCK-OP, within a 1-hour period, to be considered related to administration of the study drug. To assess alterations in blood pressure, standard blood pressure curves were used and a 20% decrease from a previously normal mean, within 10 minutes after CCK-OP administration, was defined as CCK-OP-induced hypotension.
Independent Monitoring Committee
An independent monitoring committee was established to monitor the study for safety and to determine whether a statistical difference had been achieved between the 2 groups for the primary outcome measure. This committee examined data for adverse events after the first and second thirds of recruited patients had completed the study. The committee was charged with determining whether administration of study drug or placebo resulted in an increase in the number of adverse events. The study was not terminated early.
Statistical Methods
Data were summarized with descriptive measures, including means and SDs. Logarithmic transformations were also used for data that had skewed distributions with a long upper tail. Baseline values were compared between groups with a 2-sample t test for continuous measures and a 2 test for dichotomous measures. A comparison between study centers was also performed, to ensure that similar groups of patients were studied. A standardized scoring system, the Score for Neonatal Acute Physiology, was computed for each neonate.22,23 The scores were found to be similar among study sites.
The primary analysis used the intent-to-treat approach. The primary outcome measures were compared between treatment groups with a 2-sample t test for continuous measures and a 2 test for dichotomous measures.
To assess whether CCK-OP was effective over time, a repeated-measures analysis of variance was fitted to the postrandomization CB levels. A dummy variable for treatment group and a term for interaction between the treatment group and the week of treatment were included in the models. Also, multivariate regression analysis was performed for comparisons of the postrandomization CB levels between treatment groups, controlling for potential confounding variables that might influence the development of either PNAC or elevated CB levels. These latter variables included inclusion criteria at enrollment (prematurity, NEC, or surgical group), gestational age, and use of taurine. A total of 216 cases with complete sets of covariates were included in these models. Because several of the covariates were highly correlated, backward elimination was used to identify a minimal subset of covariates.
RESULTS
Baseline Characteristics
Primary Outcome Measure
CB results are shown in Table 2 and Figure 2. No significant difference was noted in maximal levels of CB between the placebo and CCK-OP groups. Because of the skew of the distribution in each group, logarithmic transformation of the data was performed. This analysis on the logarithmic scale also failed to demonstrate any significant differences between the 2 groups (Table 2). Levels were then plotted over weeks of the study (Fig 2). Although CB levels among infants in the CCK-OP group were consistently lower than those in the placebo group, these differences were not significant. Of the 3 cohorts of patients (premature, NEC, and surgical), patients who were stratified to the surgical group (operative NEC, gastroschisis, or atresia) were at significantly (P < .017) greater risk of developing PNAC (52% had CB levels of 5.0 mg/dL), compared with those stratified to the premature group (28%). To address stratification as a potential confounding factor, a general linear model that included as covariates the infants' diagnosis and degree of prematurity and whether they received a taurine-conjugated PN solution was fit to the CB data. This analysis also showed no significant impact on the use of CCK-OP to lower CB levels (P = .68). Because of the large number of preterm infants, compared with the other 2 cohorts (169 preterm infants, compared with 72 other patients), treatment effects were estimated separately for the premature group and the surgical and NEC groups. The estimates of the treatment effects trended toward CB reduction, and were –0.13 ± 0.18 for the premature group and –0.43 ± 0.98 for the NEC group. Both of these represented small effect sizes and were not significant. In addition, it would be highly unlikely that much larger numbers of patients would influence this result.
Because total PN (TPN) duration might have an influence on the efficacy of CCK-OP, reanalysis was performed for infants who received TPN for >14 days and >25 days. At 14 days, 126 patients remained on <50% enteral nutrition; 118 of these had sufficient data for complete analysis. The estimate of the treatment effect for infants who received TPN for >14 days was 0.1423 (95% confidence interval: –0.3223 to 0.6069), with a P value of .5450. At study day 25, 72 patients remained in the study, and 70 patients had sufficient data for complete analysis. For patients who received TPN for >25 days, the treatment effect for CCK-OP was 0.0813 (95% confidence interval: –0.9666 to 1.1293), with a P value of .8772.
Secondary Outcome Measures
The overall incidence of adverse events was quite high in our study. This did not appear to be attributable to the effect of the drug, however. In fact, we intentionally recruited high-risk patients to yield a greater number of patients who would develop PNAC. Sepsis occurred for 51% of all enrolled patients. The incidence of sepsis did not differ statistically between the study groups (51% in the CCK-OP group and 49% in the placebo group). Mortality rates were also high in this series but that was not surprising, because of the selected population of neonates who required long-term PN; again, no differences were noted between the CCK-OP and placebo groups (15% in each group).
Administration of CCK-OP did not show any immediate adverse side effects. Specifically, there was no change in the incidence of pain, intolerance of oral feeding, or hypotension that could be linked clearly with the period of time around drug administration. Lengths of ICU and hospital stays did not differ between the study groups (Table 3). In addition, the times to 50% enteral feeding and 100% enteral feeding did not differ between the groups (Table 3).
Ultrasonography was performed for a subgroup of patients, to assess the presence of sludge in the gallbladder. Evaluations were performed at baseline and at 2 weeks (Table 4) if the patient remained on either study drug or placebo. The presence of sludge in the gallbladder at 2 weeks showed no correlation for patients receiving study drug versus those receiving placebo. Biliary sludge was noted for 58% of patients in the CCK-OP group and 42% of those in the placebo group (P = .33). Interestingly, with stratification of patients between the premature group and other cohorts, a significantly (P < .0001) greater percentage of infants with NEC and surgical problems developed gallbladder sludge (64.7%), compared with premature infants (20.5%).
DISCUSSION
A number of studies have linked the development of PNAC to several risk factors. Perhaps the first association was the finding of an inverse relationship between the incidence of PNAC and both gestational age and birth weight.2,24–27 In a series reported by Beale et al,28 50% of infants who were <1000 g at birth developed PNAC, whereas the incidence decreased to <10% among infants weighing >1500 g. The longer PN is used for neonates, the greater risk they have of developing PNAC; virtually 100% of neonates developed PNAC if they received PN for >8 weeks.24 Another potential risk factor that may worsen the degree of PNAC is the occurrence of sepsis.29 The precise pathogenesis of PNAC is unknown, although a number of causes have been proposed.27,30,31 The process may well be multifactorial. Among these causes is a potential lack of essential factors in TPN, including choline.32 In addition, the administration of PN leads to mucosal atrophy, with a loss of epithelial barrier function.33 This can lead subsequently to endotoxins and proinflammatory cytokines entering the liver, with resultant hepatic injury.34 This may explain why the addition of enteral antibiotics has shown efficacy in the treatment of PNAC.35 In addition, a number of bile canalicular transport proteins have been shown to be disturbed with PN, and these may contribute significantly to the development of PNAC.36 Finally, a loss of enteral nutrition leads to a decline in the expression of a number of gastrointestinal hormones, including CCK, which may also account for the observed decline in bile flow and biliary sludge with TPN administration.16 In addition to this lack of understanding regarding the pathogenesis of PNAC, currently there are no known clearly effective pharmacologic modalities to treat or to prevent this condition.5
The clinical implications of PNAC include increased rates of sepsis, cirrhosis, and death. In one series, the incidence of sepsis was 56% among infants with PNAC, compared with 13% among neonates receiving PN without PNAC.3 Liver failure occurs with end-stage PNAC. In one review, infants with PNAC had a 31% mortality rate, compared with a 3% mortality rate for those receiving PN without cholestasis.3 Among a series of patients with short-bowel syndrome receiving long-term PN, 16 of 23 children (70%) had direct bilirubin levels of >2.5 mg/dL).37 If infants maintained a CB of 3.0 for >3 months, there was an associated 78% risk of mortality.
Current modalities used to treat cholestatic jaundice involve primarily early initiation of enteral feeding. Unfortunately, for many infants the intestinal tract either is too immature or, because of a variety of pathologic disorders, is without sufficient functional integrity to tolerate feedings. Other important care aspects include prevention of sepsis, confirmation that the patient is not being overfed (prevention of hepatic steatosis), and early cycling of PN. Medical options that have been suggested or used include the removal of certain trace elements, such as copper and manganese, from the PN.7 Both of these elements are excreted primarily in bile. Depositions of copper have been observed in cholestatic livers, and manganese deposits have been found in the brains of individuals receiving PN.1
Ursodeoxycholic acid has demonstrated usefulness in some cases of intrahepatic cholestasis, including primary biliary sclerosis, and for patients with cystic fibrosis.38 Its mechanism of action includes the exchange of hydrophobic bile acids for hydrophilic ones, leading to an improvement in bile flow. Although some studies suggest that it may be potentially effective in the care of neonates with PNAC,39 a prospective, randomized, controlled trial with tauroursodeoxycholic acid failed to demonstrate any benefit in preventing or reducing the severity of PNAC.5 Other modalities to treat PNAC include the use of enteral antibiotics. These antibiotics may cause a reduction in intraluminal bacteria, which may lead to the development of bacterial translocation and subsequent sepsis. Furthermore, bacteria produce endotoxins, which are associated with proinflammatory cytokines. Cytokines such as tumor necrosis factor- and interleukin-1 are associated with chronic liver injury. Clinical use of oral antibiotic therapy has shown some promise,35 but its use may lead to the development of resistant organisms.
CCK has the ability to induce gallbladder contraction and to increase intrahepatic bile flow.40,41 Because of these actions, administration of CCK has been thought to be efficacious in the prevention or treatment of PNAC. Studies with rodents receiving PN demonstrated that CCK was beneficial in reducing the degree of cholestasis.42 In another study, the use of CCK-OP was investigated with prepubescent rabbits maintained on PN. That study demonstrated that there was a reduction in periportal inflammation and fibrosis among CCK-OP-treated rabbits.43 In addition, significant increases in basal bile flow rates and clearance of sulfobromophthalein were noted in the CCK-OP-treated group.
Patients with short-bowel syndrome may also have low CCK-OP levels, compared with control patients, which suggests that a deficit in CCK-OP may be common among patients receiving long-term PN.44 This suggests that loss of intestinal length or function may affect CCK levels and may be one reason why patients in the NEC and surgical cohorts of the current study had significantly more biliary sludge. CCK-OP has been used to prevent the formation of biliary sludge among adult patients receiving long-term (>21 days) PN.12 In addition, 2 clinical reports showed that CCK-OP might be beneficial in reducing direct bilirubin levels among infants who have already developed PNAC, potentially contributing to resolution of the process.13,15 In those 2 reports, reductions of direct bilirubin levels were noted for several infants. However, the extent of the decreases was quite variable, and reductions did not occur among infants with evidence of cirrhosis.
The current study was prompted by the findings for a prospectively studied group of 21 neonates treated prophylactically with CCK-OP, compared with 21 historically treated neonates matched with respect to gestational age, diagnosis, and duration of PN.16 Two severities of PNAC were examined, low (direct bilirubin levels of >2.0 mg/dL but <5.0 mg/dL) and high (>5.0 mg/dL). Although no difference in the incidence of PNAC was seen for infants with low-severity PNAC, a significant reduction in the number of neonates with high-severity PNAC was observed in the CCK-OP-treated group, compared with the untreated group (9% and 38%, respectively). The study was preliminary but provided sufficient data to predict the needed size of the current prospective randomized trial.
In the current study, we attempted to use CCK-OP to prevent the development of PNAC. Despite a lack of statistical difference in the incidences of PNAC between the 2 groups, it was interesting to note that there was a persistent trend of lower levels of CB in the CCK-OP group. However, because of the large SDs in CB levels, no statistical difference could be noted between the study groups. It is conceivable that we did not achieve a statistical difference because of a type II error (insufficient number of patients). However, on the basis of our power analysis, we do not think this is the case. We certainly do not think that another study with larger numbers of patients would be warranted. We also considered the fact that an insufficient dose of CCK-OP might have been used in this study. The dose selected was 2 times higher than the standard dosage based on weight. In a previous study by our group, dosing in this range was effective in decreasing bilirubin levels among patients who had already developed PNAC.13 This dose was as efficacious as higher doses of CCK-OP and was associated with very few or no drug-related adverse events. Although CCK-OP might have had some effect in increasing bile flow (ie, the numerically lower bilirubin levels), other factors that likely contribute to PNAC (see above) may be more than CCK-OP treatment can overcome. The fact that biliary sludge did not decrease with CCK-OP treatment might be attributable to the fact that infants respond to CCK-OP differently than do adults. It might also suggest that other factors, aside from a lack of CCK, contribute to sludge. In fact, studies in mouse models suggested that PN might alter bile composition by downregulating bile canalicular transport proteins.45 Therefore, PNAC may continue to progress, despite the actions of CCK-OP. Clearly, this would be consistent with the general concept that PNAC is a multifactorial process.
CONCLUSIONS
CCK-OP was not effective in preventing the development of or reducing the severity of PNAC. Clearly, greater understanding of the pathogenesis and treatment of this devastating disease process is needed. It is hoped that additional studies can identify therapeutic strategies to treat or to prevent this process.
ACKNOWLEDGMENTS
This work was supported by the Food and Drug Administration (grant FD-R-001449-01).
We thank the following participants: independent study monitors: Carol Braunschweig, PhD, and Mary Fran Sowers, PhD; statistical design: Anthony Shork, PhD; study monitoring: Janet Lord, Jennifer Thibault, RN, Jill Knox, Stephanie Adams, RN, Virginia McCann, RN, Sarah Borror, and Jason Rytelewski.
We thank the following individuals for assistance in the performance of this study: Robert Schumacher, MD, James Padbury, MD, Arlet G. Kurkchubasche, MD, Lisa Judge, MD, and Judith Ivacko, MD. We thank the following individuals for investigational drug services: Roberta Tankanow, RPh, Helen Tamer, RPh, Chuck Bork, RPh, Bill Johnston, RPh, and Kathleen Truelove, RPh.
Additional support in Toledo, Ohio, was from the Frederick M. Douglas Foundation.
FOOTNOTES
Accepted Sep 28, 2004.
No conflict of interest declared.
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Iyer K, Spitz L, Clayton P. New insight into mechanisms of parenteral nutrition-associated cholestasis: role of plant sterols. J Pediatr Surg. 1998;33 :1 –6
Kaminski DL, Rose RC, Nahrwold DL. Effect of pentagastrin on cholecystokinin and secretin choleresis in the dog. J Surg Res. 1974;17 :26 –29
Dawes LG, Muldoon JP, Greiner MA, Bertolotti M. Cholecystokinin increases bile acid synthesis with total parenteral nutrition but does not prevent stone formation. J Surg Res. 1997;67 :84 –89
Sitzman J, Pitt H, Steinborn P, Pasha Z, Sanders R. Cholecystokinin prevents parenteral nutrition induced biliary sludge in humans. Surg Gynecol Obstet. 1990;170 :25 –31
Teitelbaum DH, Han-Markey T, Schumacher RE. Treatment of parenteral nutrition-associated cholestasis with cholecystokinin-octapeptide. J Pediatr Surg. 1995;30 :1082 –1085
Schwartz JB, Merritt RJ, Rosenthal P, Diament M, Sinatra FR, Ramos A. Ceruletide to treat neonatal cholestasis [letter]. Lancet. 1988;1 :1219 –1220
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Teitelbaum D, Han-Markey T, Drongowski R, et al. Use of cholecystokinin to prevent the development of parenteral nutrition-associated cholestasis. JPEN J Parenter Enteral Nutr. 1997;21 :100 –103
Bell M, Kosloske A, Benton C, Martin L. Neonatal necrotizing enterocolitis in infancy: prevention of perforation. J Pediatr Surg. 1973;8 :601 –605
Touloukian RJ, Walker-Smith GJ. Normal intestinal length in preterm infants. J Pediatr Surg. 1983;83 :720 –725
Garner J, Jarvis W, Emori T, Horan T, Hughes J. CDC definitions for nosocomial infections, 1988. Am J Infect Control. 1988;16 :128 –140
Garland J, Henrickson K, Maki D. The 2002 Hospital Infection Control Practices Advisory Committee Centers for Disease Control and Prevention guideline for prevention of intravascular device-related infection. Pediatrics. 2002;110 :1009 –1013
Stevens B, Johnston C, Petryshen P, Taddio A. Premature Infant Pain Profile: development and initial validation. Clin J Pain. 1996;12 :13 –22
Escobar G, Fischer A, Li D, Kremers R, Armstrong M. Score for neonatal acute physiology: validation in three Kaiser Permanente neonatal intensive care units. Pediatrics. 1995;96 :918 –922
Richardson D, Gray J, McCormick M, Workman K, Goldmann D. Score for neonatal acute physiology: a physiologic severity index for neonatal intensive care. Pediatrics. 1993;91 :617 –623
Drongowski RA, Coran AG. An analysis of factors contributing to the development of total parenteral nutrition-induced cholestasis. JPEN J Parenter Enteral Nutr. 1989;13 :586 –589
Merritt R. Cholestasis associated with total parenteral nutrition. J Pediatr Gastroenterol Nutr. 1986;5 :9 –22
Beath SV, Davies P, Papadopoulou A, et al. Parenteral nutrition-related cholestasis in postsurgical neonates: multivariate analysis of risk factors. J Pediatr Surg. 1996;31 :604 –606
Moss RL, Amii LA. New approaches to understanding the etiology and treatment of total parenteral nutrition-associated cholestasis. Semin Pediatr Surg. 1999;8 :140 –147
Beale EF, Nelson RM, Bucciarelli RL, Donnelly WH, Eitzman DV. Intrahepatic cholestasis associated with parenteral nutrition in premature infants. Pediatrics. 1979;64 :342 –347
Beath S, Davies P, Papadpoulou A, et al. Parenteral nutrition-related cholestasis in postsurgical neonates: multivariate analysis of risk factors. J Pediatr Surg. 1996;31 :604 –606
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Brown M, Thunberg B, Golub L, Maniscalco W, Cox C, Shapiro D. Decreased cholestasis with enteral instead of intravenous protein in the very low birth weight infant. J Pediatr Gastroenterol Nutr. 1989;9 :21 –27
Buchman AL, Ament M, Sohel M, et al. Choline deficiency causes reversible hepatic abnormalities in patients receiving parenteral nutrition: proof of a human choline requirement: a placebo-controlled trial. JPEN J Parenter Enteral Nutr. 2001;25 :260 –268
Khan J, Iiboshi Y, Nezu R, et al. Total parenteral nutrition increases uptake of latex beads by Peyer's patches. JPEN J Parenter Enteral Nutr. 1997;21 :31 –35
Haque SM, Chen K, Usui N, et al. Effects of endotoxin on intestinal hemodynamics, glutamine metabolism, and function. Surg Today. 1997;27 :500 –505
Kubota A, Okada A, Imura K, et al. The effect of metronidazole on TPN-associated liver dysfunction in neonates. J Pediatr Surg. 1990;25 :618 –621
Tazuke Y, Drongowski RA, Btaiche I, Coran AG, Teitelbaum DH. Effects of lipid administration on liver apoptotic signals in a mouse model of total parenteral nutrition (TPN). Pediatr Surg Int. 2004;20 :224 –228
Teitelbaum D, Drongowski R, Spivak D. Rapid development of hyperbilirubinemia in infants with the short bowel syndrome as a correlate to mortality: possible indication for early small bowel transplantation. Trans Proc. 1996;28 :2677 –2678
Trauner M, Graziadei IW. Review article: mechanisms of action and therapeutic applications of ursodeoxycholic acid in chronic liver diseases. Aliment Pharmacol Ther. 1999;13 :979 –996
Beau P, Labat-Labourdette J, Ingrand P, Beauchant M. Is ursodeoxycholic acid an effective therapy for total parenteral nutrition-related liver disease J Hepatol. 1994;20 :240 –244
Magnusson I, Thulin L, Einarsson K. Effects of substance P and somatostatin on taurocholate-stabilized and CCK- or secretin-induced choleresis in the anesthetized dog. Scand J Gastroenterol. 1984;19 :1007 –1014
Magnusson I, Thulin L. Effect of substance P on CCK- or VIP-induced choleresis in anesthetized dogs. Acta Physiol Scand. 1979;106 :293 –297
Innis S. Effect of cholecystokinin-octapeptide on total parenteral nutrition-induced changes in hepatic bile secretion and composition in the rat. J Pediatr Gastroenterol Nutr. 1986;5 :793 –798
Curran TJ, Uzoaru I, Das JB, Ansari G, Raffensperger JG. The effect of cholecystokinin-octapeptide on the hepatobiliary dysfunction caused by total parenteral nutrition. J Pediatr Surg. 1995;30 :242 –247
Ling PR, Sheikh M, Boyce P, et al. Cholecystokinin (CCK) secretion in patients with severe short bowel syndrome (SSBS). Dig Dis Sci. 2001;46 :859 –864
Tazuke Y, Kiristioglu I, Heidelberger KP, Eisenbraun MD, Teitelbaum DH. Hepatic P-glycoprotein changes with total parenteral nutrition administration. JPEN J Parenter Enteral Nutr. 2004;28 :1 –7(Daniel H. Teitelbaum, MD,)
Department of Radiology, C.S. Mott Children's Hospital
School of Public Health, University of Michigan, Ann Arbor, Michigan
Hasbro Children's Hospital, Brown Medical School, Providence, Rhode Island
St Vincent's Mercy Children's Hospital, Toledo, Ohio
Strong Memorial Hospital, University of Rochester, Rochester, New York
Baylor College of Medicine, Houston, Texas
Johns Hopkins University School of Medicine, Baltimore, Maryland
Columbus Children's Hospital, Columbus, Ohio
ABSTRACT
Objective. To determine whether cholecystokinin-octapeptide (CCK-OP) would prevent or ameliorate parenteral nutrition-associated cholestasis (PNAC) among high-risk neonates treated with total parenteral nutrition.
Study Design. This was a multicenter, double-blind, randomized, controlled trial conducted between 1996 and 2001.
Patients. Neonates at risk for the development of PNAC included very low birth weight neonates and those with major surgical conditions involving the gastrointestinal tract.
Setting. Tertiary care hospitals.
Intervention. Patients were randomized to receive CCK-OP (0.04 μg/kg per dose, twice daily) or placebo. Eligible infants were all <30 days of age. Patients were enrolled within 2 weeks after birth or within 7 days after surgery.
Outcome Measures. The primary outcome measure was conjugated bilirubin (CB) levels, which were measured weekly. Secondary outcome measures included incidence of sepsis, times to achieve 50% and 100% of energy intake through the enteral route, number of ICU and hospital days, mortality rate, and incidences of biliary sludge and cholelithiasis.
Results. A total of 243 neonates were enrolled in the study. CCK-OP administration did not significantly affect CB levels (1.76 ± 3.14 and 1.93 ± 3.31 mg/dL for CCK-OP and placebo groups, respectively; mean ± SD). Secondary outcome measures also were not significantly affected by the study drug.
Conclusions. Use of CCK-OP failed to reduce significantly the incidence of PNAC or levels of CB. CCK-OP had no effect on other secondary measures and should not be recommended for the prevention of PNAC.
Key Words: cholecystokinin-octapeptide parenteral nutrition-associated cholestasis bilirubin
Abbreviations: PNAC, parenteral nutrition-associated cholestasis PN, parenteral nutrition CB, conjugated bilirubin CCK-OP, cholecystokinin-octapeptide NEC, necrotizing enterocolitis TPN, total parenteral nutrition CCK, cholecystokinin
Parenteral nutrition-associated cholestasis (PNAC) is a significant complication of prolonged parenteral nutrition (PN).1 The disease is particularly common among premature neonates and patients who undergo major gastrointestinal operations in the newborn period.2 PNAC is associated with a 50% risk of sepsis and mortality rates as high as 20% to 30%.3 Currently, there is no known treatment or means to prevent PNAC among patients who cannot tolerate enteral feedings. Attempts at addressing this problem have used a number of therapeutic interventions, including taurine-supplemented PN,4 tauroursodeoxycholic acid,5 and enteral antibiotics.6 The removal of several agents, including manganese, copper, and phytosterols, from intravenous lipids has also been suggested to be beneficial.7–9 Despite some encouraging reports, no definitive treatment or mode of prevention of PNAC has been proved.
One factor potentially contributing to PNAC is a lack of gastrointestinal hormonal stimulation, which facilitates bile flow. Cholecystokinin (CCK) is a gastrointestinal hormone that is released during enteral stimulation and can promote both intrahepatic and extrahepatic bile flow.10–12 We and others demonstrated previously that CCK-octapeptide (CCK-OP) could be used to partially correct hyperbilirubinemia associated with PNAC.13–15 CCK-OP was also shown in a retrospective study to prevent a rapid increase in conjugated bilirubin (CB) levels (used as a marker for PNAC), compared with untreated infants who were matched with respect to gestational age, primary disease, and duration of PN.16 That study showed that patients who received CCK-OP demonstrated a reduced incidence of severe cholestasis (defined as a direct bilirubin level of 5.0 mg/dL), compared with untreated patients (9.5% vs 38%, P = .015).
The study was conducted as a randomized, double-blind, controlled, multicenter trial over 8 weeks. We hypothesized that the use of CCK-OP could prevent the development of PNAC in an at-risk group of neonates. The primary outcome measure was CB levels, as a marker of the severity of PNAC. Secondary outcome measures were the incidence of sepsis, times to achieve 50% and 100% of energy intake through the enteral route, number of ICU and hospital days, mortality rate, and incidences of cholelithiasis and biliary sludge. The study examined severely premature neonates, as well as infants undergoing major surgical procedures in the newborn period (eg, necrotizing enterocolitis [NEC], gastroschisis, or severe intestinal atresia), who during their study course received >50% of total energy needs through the parenteral route.
METHODS
Trial Design
This study was a double-blind, randomized, multicenter, controlled trial to assess whether CCK-OP could prevent or reduce the severity of PNAC. In a small preliminary study,16 we observed rates of PNAC (defined as CB levels of >2.5 mg/dL) of 9% and 38% for CCK-OP-treated subjects and historical control subjects, respectively. Therefore, we designed this study to have 80% power to identify a difference of 15% (10% vs 25%) with a 2-tailed, 5% level of significance. To achieve this power, a total of 252 neonates at risk for PNAC would be needed (126 in each of the placebo and study drug groups). A neonatal population was selected because it was identified by several investigators as having the patients with the highest risk of developing PNAC.1 The primary outcome measure was CB levels, as a measure of the development and severity of PNAC. Secondary outcome measures included the incidence of sepsis, times to achieve 50% and 100% of energy intake through the enteral route, number of ICU and hospital days, mortality rate, and incidences of biliary sludge and cholelithiasis. The study was assigned IND 42898 and was funded by the Food and Drug Administration Orphan Products Grant Program.
Treatment Specifications
Drug Name and Source
Sincalide (Kinevac; Bracco Diagnostics, Princeton, NJ) is a synthetic form of CCK-OP that has been approved by the Food and Drug Administration. Dosing of CCK-OP was 0.04 μg/kg per dose, administered intravenously (in 10–15 minutes) every 12 hours, based on a mean of weekly body weights. Dose selection was based on previous use of the drug and because this was twice the dose recommended for stimulation of gallbladder contraction.16 Placebo consisted of intravenously administered 0.9% sodium chloride, given on the same schedule as the study drug. CCK-OP was diluted to the correct concentration in 0.9% sodium chloride. Appearance, size, and volume were identical to those of the study drug.
Total Duration
Patients remained on the CCK-OP or placebo therapy for no longer than 8 weeks, regardless of whether they achieved enteral feeding or not. CCK-OP or placebo administration was discontinued once the enteral component was >50% of the patient's nutritional requirements. If CCK-OP or placebo treatment was discontinued because >50% of energy needs were provided through the enteral route but enteral nutrition decreased to <50% of total required energy for any reason, then the patient was again treated with either CCK-OP or placebo, according to the previous assignment. All caretakers were masked with respect to group assignment; only the investigational drug service was aware of the results of the randomization.
Study Population
Study Centers
The 8 centers used in this proposal were chosen because of their homogeneity. Each is a tertiary care facility for neonates with a large regional referral pattern that exceeds 10000 live births per year. Therefore, it was anticipated that a fairly uniform population of patients would be examined among the sites.
Inclusion Criteria
Participating sites and the numbers of recruited patients in the study are shown in Table 1. All patients were receiving PN at the time of recruitment. Although the study was designed to include patients who would require prolonged periods of PN, after enrollment the patients remained in the study regardless of the duration of PN. Initially, the study population consisted of severely premature infants (<1000 g at birth and with an estimated [Dubowitz] gestational age of 28 weeks). However, because the majority of these patients were found to achieve >50% of nutritional intake through the enteral route by 7 to 14 days, the inclusion criteria were changed in 1999, to include surgical neonates (<30 days of age at the time of enrollment) who had a major gastrointestinal disorder preventing enteral intake. These criteria included 3 diagnoses, ie, (1) NEC, (2) gastroschisis, and (3) severe jejunal-ileal atresia. Diagnoses of NEC met Bell grade II or higher criteria.17 Severe jejunal-ileal atresia was defined as an intestinal loss of 50% of the anticipated small-bowel length for a given gestational age.18
A daily screening (based on discussions with the attending intensive care physicians) of all neonates in the each study center was performed to identify potential candidates for the study. All patients were recruited into the study within 7 days after the diagnosis of any one of the surgical disorders. Patients with hemodynamic instability (see below) were recruited but were not begun in the study until they were in hemodynamically stable condition (but still <30 days of age).
Exclusions Before Initiation of the Study
Patients in hemodynamically unstable condition were excluded. However, they could be enrolled if their condition stabilized within the first 2 weeks of life and before the initiation of feedings. Instability was defined as requiring fluid boluses of >40 mL/kg in the previous 24 hours in addition to maintenance intravenous fluids or requiring adrenergic support of >10 μg/kg per minute dopamine or dobutamine or 0.1 μg/kg per minute epinephrine. Also excluded were infants with a metabolic pathway defect (eg, hereditary fructose intolerance, galactosemia, or neonatal tyrosinemia), hepatic insufficiency (defined as a CB level of >1.0 mg/dL or a coagulation abnormality of an international normalized ratio >1.5 times normal), or progressive renal failure (defined as a creatinine level of >1.5 mg/dL). In addition, infants with primary or secondary liver disease (including hepatitis), regardless of liver function; use of extracorporeal life support; suspected congenital obstruction of the hepatobiliary tree (eg, biliary atresia or a choledochal cyst); or a diagnosis of HIV infection were excluded. No infants were enrolled if their CB levels were >1.0 mg/dL. Finally, infants were excluded if they received ursodeoxycholic acid before the study; no patients received this drug during the study.
After enrollment, all infant data were recorded and analyzed regardless of whether study drug/placebo treatment was stopped. Data were analyzed with an intent-to-treat approach. If neonates underwent a surgical procedure, then the study drug/placebo was withheld for a minimum of 12 hours and a maximum of 96 hours after surgery.
Randomization and Masking
All patients were enrolled after signed detailed informed consent was obtained from either a parent or guardian. The consent form and the entire study were approved fully by the institutional review boards of all participating hospitals. Infants were randomized to receive the study drug or placebo on the basis of an established randomization chart. Block randomization with a randomly chosen block size of 2 or 4 within center was used. Randomization tables and envelopes were generated by the Department of Biostatistics at the University of Michigan and were distributed to each site. Control of drug randomization and distribution of drug were performed by the investigational drug service at each study site. Patients, medical professionals, and investigators were masked from the results of the randomization. Enrollment was performed by either study coordinators or principal investigators at each study site. The flow of enrollment, study performance, and analysis is shown in Figure 1.
PN Guidelines
To ensure a uniform delivery of PN among patients and sites, the following standards were used. Total nonprotein energy delivery did not exceed 378 to 420 kJ/kg per day. Carbohydrate administration began at 4 to 8 mg/kg per minute on the first day of PN and was increased to 11 to 18 mg/kg per minute by day 3. Provision was made for patients who were glucose intolerant during this increase. Protein administration began at 1.0 g/kg per day on the first day of PN and was increased by 0.5 to 1 g/kg per day to a maximum of 2.5 to 3.0 g/kg per day. Lipid administration began at 1.0 g/kg per day on the first day of PN and was increased by 0.5 to 1 g/kg per day to a maximum of 3.0 g/kg per day. A pediatric amino acid formulation that contained taurine was used for PN; this was either Aminosyn-PF (Abbott, Abbott Park, IL; 10% solution containing 70 mg/100 mL in the bulk solution) for 76 patients or Trophamine (B. Braun, Bethlehem, PA; 10% solution containing 25 mg/100 mL in the bulk solution) for 127 patients. For the first 40 patients enrolled, taurine was not included in the formulation, and neonates received a standard adult formulation (Aminosyn; Abbott).
Primary Outcome Measure
CB levels were measured within 72 hours before or on the day of recruitment into the study and weekly thereafter. To ensure uniformity in laboratory determinations of the primary outcome measure, a Kodak Ektachem 750 system (Johnson and Johnson, New Brunswick, NJ) was used for all sample measurements.
Secondary Outcome Measures
Assessment of Patient Morbidity and Mortality Rates
Secondary outcome measures included the incidence of sepsis (both generalized and catheter-related), time in days to achieve 50% enteral feedings, time in days to achieve 100% enteral feedings, number of days in the ICU, number of hospital days, and mortality rates. Monitoring and recording of all in-hospital complications were performed on a weekly basis. Sepsis in the neonatal period used previously established definitions.19 Catheter sepsis was defined with a modified definition established by the Centers for Disease Control and Prevention.20 Percentage of energy intake was calculated by totaling enteral intake and dividing the result by the total number of joules delivered through the enteral and parenteral routes, including all protein sources. Infants had to tolerate a level of feeding (50% or 100%) for 3 consecutive days before reaching this definition. All patient deaths were recorded. Death was defined as a death occurring at any time during the child's admission to the hospital. The cause of death and autopsy findings were also recorded.
Ultrasonographic Assessment of the Hepatobiliary Tree
Formation of biliary sludge was assessed among children treated at 2 sites (University of Michigan and St Vincent's Mercy Hospital). The techniques at these sites were compared closely (because of their close geographic locations) and allowed accurate analysis of the data. An ultrasound study was performed within 48 hours after recruitment into the study, and a follow-up ultrasound study was performed 2 weeks after initiation of the study. Before the ultrasound studies, all infants had feedings withheld for at least 3 hours. Biliary ultrasonography was performed with 5- to 8-MHz sector transducers (Acuson 128XP/10; Acuson, Mountain View, CA; or ATL HDI 5000; ATL, Bothell, WA), depending on patient size. The gallbladder and biliary tree were assessed with longitudinal, transverse, and oblique scans, as indicated by the anatomic features. The following measurements were scored: presence of biliary sludge, cholelithiasis, choledocholithiasis, bile duct dilation (intra- or extrahepatic), and size of the common bile duct (transverse diameter at the liver hilum).
Monitoring
Monitoring Schedule
During the study period, neonates were monitored daily. During each infusion period, assessment for the development of adverse reactions was performed (see below). After completion of the 8-week study, infants were monitored daily during the NICU stay and every third day while out of the NICU but in the hospital. A final check for adverse reactions was performed, via telephone, 30 days after patient discharge.
Adverse Events
Adverse events were defined as any untoward events that occurred during the performance of the study. Each event was recorded. A determination of whether the event was associated with the administration of the study drug was then recorded as likely, possibly, unlikely, or not associated. On the basis of these criteria, if likely or possibly associated was selected, then the event was defined as an adverse drug reaction. In general, 3 major reactions were checked for routinely, namely, pain, feeding intolerance, and hemodynamic changes. The study coordinator at each center monitored for drug-related adverse reactions, including pain presumed to be attributable to gastrointestinal cramping, intolerance of oral feedings because of spasm of the pylorus, and hypotension. Nursing and physician staff members administering the drug were made aware of potential adverse reactions and were instructed to notify the investigators at each center if such reactions developed. All adverse reactions were reported to the principal investigator, the independent monitoring committee (see below), the study center's institutional review board, and the Food and Drug Administration. Significant adverse events included the following: death, sepsis, drug-related adverse events, prolongation of hospitalization, overdose, and disabling injury. If a significant adverse event was considered possibly associated with the study drug, then this was grounds for discontinuation of the study. One case of drug overdose occurred in the study; this did not result in any other adverse events. In that case, a 10-fold higher concentration of the study drug (CCK-OP) was given, which was recognized by the investigational drug service after the drug had been administered. Despite this larger amount of drug, no hemodynamic changes or feeding intolerance was noted. In addition, the pain score (see below) did not change, compared with predosing scores. The randomization code was broken for this patient; however, the patient remained in the study because of an intent to treat all recruited patients. A full report of this adverse event was made to all institutional review boards and the Food and Drug Administration.
Grading of pain among premature neonates used the Premature Infant Pain Profile system.21 Feeding intolerance had to be associated consistently (2 times) with the administration of CCK-OP, within a 1-hour period, to be considered related to administration of the study drug. To assess alterations in blood pressure, standard blood pressure curves were used and a 20% decrease from a previously normal mean, within 10 minutes after CCK-OP administration, was defined as CCK-OP-induced hypotension.
Independent Monitoring Committee
An independent monitoring committee was established to monitor the study for safety and to determine whether a statistical difference had been achieved between the 2 groups for the primary outcome measure. This committee examined data for adverse events after the first and second thirds of recruited patients had completed the study. The committee was charged with determining whether administration of study drug or placebo resulted in an increase in the number of adverse events. The study was not terminated early.
Statistical Methods
Data were summarized with descriptive measures, including means and SDs. Logarithmic transformations were also used for data that had skewed distributions with a long upper tail. Baseline values were compared between groups with a 2-sample t test for continuous measures and a 2 test for dichotomous measures. A comparison between study centers was also performed, to ensure that similar groups of patients were studied. A standardized scoring system, the Score for Neonatal Acute Physiology, was computed for each neonate.22,23 The scores were found to be similar among study sites.
The primary analysis used the intent-to-treat approach. The primary outcome measures were compared between treatment groups with a 2-sample t test for continuous measures and a 2 test for dichotomous measures.
To assess whether CCK-OP was effective over time, a repeated-measures analysis of variance was fitted to the postrandomization CB levels. A dummy variable for treatment group and a term for interaction between the treatment group and the week of treatment were included in the models. Also, multivariate regression analysis was performed for comparisons of the postrandomization CB levels between treatment groups, controlling for potential confounding variables that might influence the development of either PNAC or elevated CB levels. These latter variables included inclusion criteria at enrollment (prematurity, NEC, or surgical group), gestational age, and use of taurine. A total of 216 cases with complete sets of covariates were included in these models. Because several of the covariates were highly correlated, backward elimination was used to identify a minimal subset of covariates.
RESULTS
Baseline Characteristics
Primary Outcome Measure
CB results are shown in Table 2 and Figure 2. No significant difference was noted in maximal levels of CB between the placebo and CCK-OP groups. Because of the skew of the distribution in each group, logarithmic transformation of the data was performed. This analysis on the logarithmic scale also failed to demonstrate any significant differences between the 2 groups (Table 2). Levels were then plotted over weeks of the study (Fig 2). Although CB levels among infants in the CCK-OP group were consistently lower than those in the placebo group, these differences were not significant. Of the 3 cohorts of patients (premature, NEC, and surgical), patients who were stratified to the surgical group (operative NEC, gastroschisis, or atresia) were at significantly (P < .017) greater risk of developing PNAC (52% had CB levels of 5.0 mg/dL), compared with those stratified to the premature group (28%). To address stratification as a potential confounding factor, a general linear model that included as covariates the infants' diagnosis and degree of prematurity and whether they received a taurine-conjugated PN solution was fit to the CB data. This analysis also showed no significant impact on the use of CCK-OP to lower CB levels (P = .68). Because of the large number of preterm infants, compared with the other 2 cohorts (169 preterm infants, compared with 72 other patients), treatment effects were estimated separately for the premature group and the surgical and NEC groups. The estimates of the treatment effects trended toward CB reduction, and were –0.13 ± 0.18 for the premature group and –0.43 ± 0.98 for the NEC group. Both of these represented small effect sizes and were not significant. In addition, it would be highly unlikely that much larger numbers of patients would influence this result.
Because total PN (TPN) duration might have an influence on the efficacy of CCK-OP, reanalysis was performed for infants who received TPN for >14 days and >25 days. At 14 days, 126 patients remained on <50% enteral nutrition; 118 of these had sufficient data for complete analysis. The estimate of the treatment effect for infants who received TPN for >14 days was 0.1423 (95% confidence interval: –0.3223 to 0.6069), with a P value of .5450. At study day 25, 72 patients remained in the study, and 70 patients had sufficient data for complete analysis. For patients who received TPN for >25 days, the treatment effect for CCK-OP was 0.0813 (95% confidence interval: –0.9666 to 1.1293), with a P value of .8772.
Secondary Outcome Measures
The overall incidence of adverse events was quite high in our study. This did not appear to be attributable to the effect of the drug, however. In fact, we intentionally recruited high-risk patients to yield a greater number of patients who would develop PNAC. Sepsis occurred for 51% of all enrolled patients. The incidence of sepsis did not differ statistically between the study groups (51% in the CCK-OP group and 49% in the placebo group). Mortality rates were also high in this series but that was not surprising, because of the selected population of neonates who required long-term PN; again, no differences were noted between the CCK-OP and placebo groups (15% in each group).
Administration of CCK-OP did not show any immediate adverse side effects. Specifically, there was no change in the incidence of pain, intolerance of oral feeding, or hypotension that could be linked clearly with the period of time around drug administration. Lengths of ICU and hospital stays did not differ between the study groups (Table 3). In addition, the times to 50% enteral feeding and 100% enteral feeding did not differ between the groups (Table 3).
Ultrasonography was performed for a subgroup of patients, to assess the presence of sludge in the gallbladder. Evaluations were performed at baseline and at 2 weeks (Table 4) if the patient remained on either study drug or placebo. The presence of sludge in the gallbladder at 2 weeks showed no correlation for patients receiving study drug versus those receiving placebo. Biliary sludge was noted for 58% of patients in the CCK-OP group and 42% of those in the placebo group (P = .33). Interestingly, with stratification of patients between the premature group and other cohorts, a significantly (P < .0001) greater percentage of infants with NEC and surgical problems developed gallbladder sludge (64.7%), compared with premature infants (20.5%).
DISCUSSION
A number of studies have linked the development of PNAC to several risk factors. Perhaps the first association was the finding of an inverse relationship between the incidence of PNAC and both gestational age and birth weight.2,24–27 In a series reported by Beale et al,28 50% of infants who were <1000 g at birth developed PNAC, whereas the incidence decreased to <10% among infants weighing >1500 g. The longer PN is used for neonates, the greater risk they have of developing PNAC; virtually 100% of neonates developed PNAC if they received PN for >8 weeks.24 Another potential risk factor that may worsen the degree of PNAC is the occurrence of sepsis.29 The precise pathogenesis of PNAC is unknown, although a number of causes have been proposed.27,30,31 The process may well be multifactorial. Among these causes is a potential lack of essential factors in TPN, including choline.32 In addition, the administration of PN leads to mucosal atrophy, with a loss of epithelial barrier function.33 This can lead subsequently to endotoxins and proinflammatory cytokines entering the liver, with resultant hepatic injury.34 This may explain why the addition of enteral antibiotics has shown efficacy in the treatment of PNAC.35 In addition, a number of bile canalicular transport proteins have been shown to be disturbed with PN, and these may contribute significantly to the development of PNAC.36 Finally, a loss of enteral nutrition leads to a decline in the expression of a number of gastrointestinal hormones, including CCK, which may also account for the observed decline in bile flow and biliary sludge with TPN administration.16 In addition to this lack of understanding regarding the pathogenesis of PNAC, currently there are no known clearly effective pharmacologic modalities to treat or to prevent this condition.5
The clinical implications of PNAC include increased rates of sepsis, cirrhosis, and death. In one series, the incidence of sepsis was 56% among infants with PNAC, compared with 13% among neonates receiving PN without PNAC.3 Liver failure occurs with end-stage PNAC. In one review, infants with PNAC had a 31% mortality rate, compared with a 3% mortality rate for those receiving PN without cholestasis.3 Among a series of patients with short-bowel syndrome receiving long-term PN, 16 of 23 children (70%) had direct bilirubin levels of >2.5 mg/dL).37 If infants maintained a CB of 3.0 for >3 months, there was an associated 78% risk of mortality.
Current modalities used to treat cholestatic jaundice involve primarily early initiation of enteral feeding. Unfortunately, for many infants the intestinal tract either is too immature or, because of a variety of pathologic disorders, is without sufficient functional integrity to tolerate feedings. Other important care aspects include prevention of sepsis, confirmation that the patient is not being overfed (prevention of hepatic steatosis), and early cycling of PN. Medical options that have been suggested or used include the removal of certain trace elements, such as copper and manganese, from the PN.7 Both of these elements are excreted primarily in bile. Depositions of copper have been observed in cholestatic livers, and manganese deposits have been found in the brains of individuals receiving PN.1
Ursodeoxycholic acid has demonstrated usefulness in some cases of intrahepatic cholestasis, including primary biliary sclerosis, and for patients with cystic fibrosis.38 Its mechanism of action includes the exchange of hydrophobic bile acids for hydrophilic ones, leading to an improvement in bile flow. Although some studies suggest that it may be potentially effective in the care of neonates with PNAC,39 a prospective, randomized, controlled trial with tauroursodeoxycholic acid failed to demonstrate any benefit in preventing or reducing the severity of PNAC.5 Other modalities to treat PNAC include the use of enteral antibiotics. These antibiotics may cause a reduction in intraluminal bacteria, which may lead to the development of bacterial translocation and subsequent sepsis. Furthermore, bacteria produce endotoxins, which are associated with proinflammatory cytokines. Cytokines such as tumor necrosis factor- and interleukin-1 are associated with chronic liver injury. Clinical use of oral antibiotic therapy has shown some promise,35 but its use may lead to the development of resistant organisms.
CCK has the ability to induce gallbladder contraction and to increase intrahepatic bile flow.40,41 Because of these actions, administration of CCK has been thought to be efficacious in the prevention or treatment of PNAC. Studies with rodents receiving PN demonstrated that CCK was beneficial in reducing the degree of cholestasis.42 In another study, the use of CCK-OP was investigated with prepubescent rabbits maintained on PN. That study demonstrated that there was a reduction in periportal inflammation and fibrosis among CCK-OP-treated rabbits.43 In addition, significant increases in basal bile flow rates and clearance of sulfobromophthalein were noted in the CCK-OP-treated group.
Patients with short-bowel syndrome may also have low CCK-OP levels, compared with control patients, which suggests that a deficit in CCK-OP may be common among patients receiving long-term PN.44 This suggests that loss of intestinal length or function may affect CCK levels and may be one reason why patients in the NEC and surgical cohorts of the current study had significantly more biliary sludge. CCK-OP has been used to prevent the formation of biliary sludge among adult patients receiving long-term (>21 days) PN.12 In addition, 2 clinical reports showed that CCK-OP might be beneficial in reducing direct bilirubin levels among infants who have already developed PNAC, potentially contributing to resolution of the process.13,15 In those 2 reports, reductions of direct bilirubin levels were noted for several infants. However, the extent of the decreases was quite variable, and reductions did not occur among infants with evidence of cirrhosis.
The current study was prompted by the findings for a prospectively studied group of 21 neonates treated prophylactically with CCK-OP, compared with 21 historically treated neonates matched with respect to gestational age, diagnosis, and duration of PN.16 Two severities of PNAC were examined, low (direct bilirubin levels of >2.0 mg/dL but <5.0 mg/dL) and high (>5.0 mg/dL). Although no difference in the incidence of PNAC was seen for infants with low-severity PNAC, a significant reduction in the number of neonates with high-severity PNAC was observed in the CCK-OP-treated group, compared with the untreated group (9% and 38%, respectively). The study was preliminary but provided sufficient data to predict the needed size of the current prospective randomized trial.
In the current study, we attempted to use CCK-OP to prevent the development of PNAC. Despite a lack of statistical difference in the incidences of PNAC between the 2 groups, it was interesting to note that there was a persistent trend of lower levels of CB in the CCK-OP group. However, because of the large SDs in CB levels, no statistical difference could be noted between the study groups. It is conceivable that we did not achieve a statistical difference because of a type II error (insufficient number of patients). However, on the basis of our power analysis, we do not think this is the case. We certainly do not think that another study with larger numbers of patients would be warranted. We also considered the fact that an insufficient dose of CCK-OP might have been used in this study. The dose selected was 2 times higher than the standard dosage based on weight. In a previous study by our group, dosing in this range was effective in decreasing bilirubin levels among patients who had already developed PNAC.13 This dose was as efficacious as higher doses of CCK-OP and was associated with very few or no drug-related adverse events. Although CCK-OP might have had some effect in increasing bile flow (ie, the numerically lower bilirubin levels), other factors that likely contribute to PNAC (see above) may be more than CCK-OP treatment can overcome. The fact that biliary sludge did not decrease with CCK-OP treatment might be attributable to the fact that infants respond to CCK-OP differently than do adults. It might also suggest that other factors, aside from a lack of CCK, contribute to sludge. In fact, studies in mouse models suggested that PN might alter bile composition by downregulating bile canalicular transport proteins.45 Therefore, PNAC may continue to progress, despite the actions of CCK-OP. Clearly, this would be consistent with the general concept that PNAC is a multifactorial process.
CONCLUSIONS
CCK-OP was not effective in preventing the development of or reducing the severity of PNAC. Clearly, greater understanding of the pathogenesis and treatment of this devastating disease process is needed. It is hoped that additional studies can identify therapeutic strategies to treat or to prevent this process.
ACKNOWLEDGMENTS
This work was supported by the Food and Drug Administration (grant FD-R-001449-01).
We thank the following participants: independent study monitors: Carol Braunschweig, PhD, and Mary Fran Sowers, PhD; statistical design: Anthony Shork, PhD; study monitoring: Janet Lord, Jennifer Thibault, RN, Jill Knox, Stephanie Adams, RN, Virginia McCann, RN, Sarah Borror, and Jason Rytelewski.
We thank the following individuals for assistance in the performance of this study: Robert Schumacher, MD, James Padbury, MD, Arlet G. Kurkchubasche, MD, Lisa Judge, MD, and Judith Ivacko, MD. We thank the following individuals for investigational drug services: Roberta Tankanow, RPh, Helen Tamer, RPh, Chuck Bork, RPh, Bill Johnston, RPh, and Kathleen Truelove, RPh.
Additional support in Toledo, Ohio, was from the Frederick M. Douglas Foundation.
FOOTNOTES
Accepted Sep 28, 2004.
No conflict of interest declared.
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