Objective. To assess the efficacy of Sn-mesoporphyrin (SnMP), a potent inhibitor of bilirubin production, in: a) moderating the need for phototherapy (PT) in full-term breastfed infants with plasma bilirubin concentrations (PBC) of ≥256.5 μmol/L and ≤307.8 μmol/L (≥15 mg/dL and ≤18 mg/dL, respectively) that were reached between ≥48 and ≤96 hours of age; b) diminishing the time required for the PBC to decline to ≤222.3 μmol/L (≤13 mg/dL) (closure of the case); c) decreasing the number of bilirubin determinations required for monitoring hyperbilirubinemia.
Study Participants. Healthy full-term breastfed infants with a PBC between ≥256.5 μmol/L and ≤307.8 μmol/L (≥15 mg/dL and ≤18 mg/dL, respectively) reached between 48 to 96 hours of age.
Design/Methods. After obtaining informed consent from the parents, infants were randomized to either the SnMP (6.0 μmol/kg birth weight, single dose, intramuscular) group or the control group. The infants' PBCs were followed by daily measurements either in the hospital or at discharge as outpatients until the hyperbilirubinemia had subsided (PBC ≤222.3 μmol/L [13mg/dL]). The total number of newborns enrolled in the study was 84; the SnMP group comprised 40 infants; the control group comprised 44 infants. The groups were similar in sex ratio, birth weight, gestational age, PBC, and age at enrollment. All infants were breastfed. Phototherapy was initiated at a PBC of 333.5 μmol/dL (19.5 mg/dL).
Results. SnMP entirely eliminated the need for supplemental PT to control hyperbilirubinemia; in contrast, of the 44 control infants, 12 required treatment with PT (27%) when their PBC reached or exceeded the level (333.5 μmol/dL; 19.5 mg/dL) at which time the use of PT was dictated by hospital guidelines. None of the 40 SnMP-treated infants reached a PBC of 19.5 mg/dL. SnMP also markedly diminished the median hours to case closure (SnMP: median, 86.5 hours; minimum/maximum, 24/216 hours; controls: median, 120 hours; minimum/maximum, 72/336 hours); and significantly reduced the number of bilirubin determinations required for clinical monitoring of the infants (SnMP: median, 3; minimum/maximum, 1/9; controls: median, 5; minimum/maximum, 3/11). No adverse effects of SnMP use were observed.
Conclusion. A single dose of SnMP proved effective in controlling severe hyperbilirubinemia in full-term breastfed newborns with high bilirubin levels between 48 and 96 hours. In addition, SnMP eliminated the need for PT and reduced the use of medical resources in the clinical treatment of this problem as well as the related, important and painful, emotional costs for both mothers and infants.
- AAP =
- American Academy of Pediatrics •
- HO =
- heme oxygenase •
- SnMP =
- Sn-mesoporphyrin •
- PT =
- phototherapy •
- PBC =
- plasma bilirubin concentration •
- BW =
- birth weight •
- GA =
- gestational age
Hyperbilirubinemia remains the most frequent clinical problem pediatricians must deal with during the newborn period, and under certain circumstances may cause severe brain damage even in healthy term newborns.1 The American Academy of Pediatrics2 (AAP) has provided recommendations to aid in the evaluation and treatment of these infants; nevertheless, the guidelines remain somewhat ambiguous as indicated by the disclaimer “the AAP has attempted to describe a range of acceptable practices, recognizing that adequate data are not available from the scientific literature to provide precise recommendations.” Early discharge has increased the confusion associated with the problem of hyperbilirubinemia. As a result, concerns for newborns have arisen because of the difficulty in accurately predicting which infants may be at risk of bilirubin-induced brain damage; in addition, intensive follow-up clinic programs for monitoring plasma bilirubin levels in this neonatal population are not universally available.3
The demonstration that a synthetic heme analog could, by acting as a competitive inhibitor, strongly inhibit the activity of heme oxygenase (HO), the rate-limiting enzyme in heme catabolism,4 and concurrently suppress naturally occurring or experimentally-induced hyperbilirubinemia in animals and in humans5–20 has opened the way to a new therapeutic approach to the treatment of newborn jaundice. Sn-mesoporphyrin (SnMP) is the most potent and innocuous HO inhibitor developed to date17 and has undergone extensive clinical study. The only side effect observed has been a transient, nondose-dependent erythema that disappeared without sequelae in infants who received phototherapy (PT) after SnMP administration.18 The use of HO inhibitors such as SnMP to suppress the production of bilirubin in the immediate postnatal period seems inherently more logical than attempting to control elevated plasma bilirubin concentrations (PBC) in newborns by methods (ie, PT and exchange transfusion) that attempt to dispose of excessive bilirubin only after the bile pigment has already been formed and entered the circulation.
We have in a previous report shown that in our Argentine study population, 7.4% of healthy term infants reached a serum bilirubin concentration of ≥291 μmol/L (≥17 mg/dL) a level, at which according to current AAP guidelines, PT should be considered.2 Even in our hospital when newborns are allowed to reach a PBC of 342 μmol/L (20 mg/dL) before starting intensive PT, this approach neither eliminated the use of PT nor reduced the number of clinical and laboratory tests required to monitor the course of hyperbilirubinemia.21 In both cases the economic and emotional costs are high.22 The uncertainty of correctly identifying those infants who will require PT and those who will have to return to the hospital for treatment of hyperbilirubinemia because of early discharge23 supports the use of an inhibitor of bilirubin production, such as SnMP, as a pharmacologic means for preempting the development of severe hyperbilirubinemia in this large population of newborns.
The study was approved by the Institutional Review Boards of the collaborating institutions: Rockefeller University and the Hospital Materno Infantil Ramon Sarda. In addition, the use of SnMP as an investigational new drug in newborns is approved by the United States Food and Drug Administration (IND 29 462), and the The Argentina Food, Drug, and Technology Administration (ANMAT [Disposicion No. 3166]).
Study Population and Design
The enrolled neonates were delivered at the Hospital Materno Infantil Ramon Sarda between November 2, 1996 and November 26, 1997. To be included in the study, a) infants had to be healthy and delivered between ≥38 and ≤41 weeks gestational age (GA) after an uncomplicated pregnancy with a PBC between ≥256.5 μmol/L (15 mg/dL) and ≤307.8 μmol/L (18 mg/dL) 48 to 96 hours after birth; and b) informed consent had been obtained from the parents. We excluded infants with: a) any congenital anomalies (heart, central nervous system, gastrointestinal, renal, chromosomal); b) evidence, or strong suspicion, of congenital infection (cytomegalovirus, rubella, herpes, toxoplasmosis, syphilis, hepatitis, human immunodeficiency virus infection, and Chagas) (Infection was confirmed by chemical and radiologic testing and clinical examination. Screening tests during pregnancy were used to detect intrauterine infection.); c) neonatal complications (asphyxia, respiratory distress syndrome, metabolic and/or thermoregulation or hemodynamic alterations at birth); d) birth weights that were less than the 10th or more than the 90th percentile for their GA; e) maternal use of phenobarbital during the last month of pregnancy; f) venous hematocrit ≥65%; g) significant bruising or large cephalhematomas; or h) hemolytic disease. We considered hemolytic disease to include Rh or ABO incompatibility and a positive Coombs' test or a serum bilirubin level increasing by more than 8.5 μmol/L (0.5 mg/dL) per hour. ABO incompatible infants who were Coombs-negative and had no rapid bilirubin rise were included in both the control (n = 4) and SnMP-treated (n = 6) groups.
After parental permission was obtained, infants were randomly allocated to the treatment (SnMP) group or to the control group by a simple randomization method using a table of random numbers. Infants were excluded after randomization if they met the exclusion criteria. The randomization number of the excluded infant was assigned to the next eligible infant. All healthy term infants were fully breastfed and did not receive supplements. On discharge, mothers were reminded not to expose their infants to sunlight. It should be noted that in Argentina infants are not customarily exposed to sunlight after birth and return home; thus our instructions were reinforcing normal practice.
Routine screening of cord blood for blood group, Rh, and Coombs is conducted at the Hospital Materno Infantil Ramon Sarda for hemolytic disease exclusion. Two experienced investigators evaluated each infant daily for the presence of jaundice. Based on their clinical judgment, a PBC test was obtained daily. If the PBC reached between ≥256.5 μmol/L (15 mg/dL) and ≤307.8 μmol/L (18 mg/dL) 48 to 96 hours after birth, consent was sought from the parents to enroll their infants in the study.
We determined the bilirubin level in all the patients included in the trial at predetermined intervals based on previous bilirubin measurements. The measurements were made every 24 hours if the PBC was between 222.3 μmol/L and 290.7 μmol/L (13 and 17 mg/dL) and every 12 hours if the PBC was >290.7 μmol/L (>17 mg/dL). All newborns were followed, either during hospitalization or at discharge as outpatients, until a PBC of ≤222.3 μmol/L (≤13 mg/dL) was reached. At the Sarda Hospital the average length of stay for an uncomplicated newborn is 2 days and for infants delivered by cesarean section it is 5 days. The study was completed for each enrolled newborn with a direct bilirubin reading and a complete hemogram that included white blood cell count, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin count, red cell distribution width, platelet, and mean platelet volume determinations.
After permission was obtained, infants were assigned at random to either the SnMP-treated or the control group. Infants in the treated group received a single intramuscular dose of SnMP (6 μmol/kg birth weight [BW]) 48 to 96 hours after birth. Single-dose vials of 24 μmol/mL were prepared at Rockefeller University Hospital and stored at 4°C in the dark as previously described.18 Liver function tests (serum glutamic pyruvic transaminase and glutamic–oxaloacetic transaminase) were performed at day 12 and at 3 months of age. Placebo drug was not administered to control infants.
Total bilirubin in serum was determined using a Bilitron Elvi 444 bilirubinometer (Elvi Electronica, Milan, Italy) with a 20-microliter sample obtained by a heel stick. The determination consists of readings at two wavelengths, 461 nm, which corresponds to the maximum absorption of bilirubin and 551 nm, which is the absorption peak of hemoglobin. The concentration of bilirubin (mg/dL) in the sample that the meter automatically measures is obtained by the difference of the two optical densities. A bilirubin standard of known bilirubin concentration was used to calibrate the instrument. This standard met the following criteria: a) the bilirubin concentration is 323 μmol/L (18.9 mg/dL) in a solution containing 6% human serum albumin, and b) the mM extinction coefficient of the standard is in the range of 59.1 to 62.3. The coefficient of variation is 5%. The determination of direct bilirubin was made by the colorimetric method of Lathe and Ruthven.24 The coefficient of variation is 5%.
If the PBC of a newborn reached 331.5 μmol/L (19.5 mg/dL), PT was administered by exposing the infants to Special Blue lamps (Philips F20T12/BB, Philips, Fairmont, WV). Each PT unit contained six 20 watt lamps. The infants were naked, with their eyes covered, and their positions supine or prone and were turned every 4 hours during PT. PT was administered for at least 24 hours and was discontinued when the PBC reached ≤13.5 mg/dL. Exchange transfusion was indicated when the PBC reached 425 μmol/L (25 mg/dL); this is the usual PBC for the use of this procedure at the Hospital Materno Infantil Ramon Sarda.
The following variables were measured in both the SnMP-treated and controls groups: a) the number of infants whose PBC reached 331.5 μmol/L (19.5 mg/dL) the level at which PT was routinely administered; b) the maximum PBC attained; c) the number of PBC measurements between enrollment and closure (when PBC reached a level ≤222.3 μmol/L [13 mg/dL]); d) the time required for the PBC to decrease to a level of 13 mg/dL; e) the hours of PT; f) the number of infants requiring ET; and g) clinical evaluation of the infants. After administration of SnMP the infants were observed for any systemic effects of the compound. In addition all infants were observed for the development of erythema.
All infants enrolled in the study were scheduled for two formal follow-up examinations. The first examination at 3 months includes medical and nutritional history, a general pediatric examination, a hematologic examination (identical with hemogram completed at case closure), and if required, biochemical tests. The second examination at 18 months will include a detailed medical, neurologic, and neurodevelopmental assessment using the Bailey Scale of Infants Development.
The following variables were examined to determine if the SnMP-treated and the control groups were comparable: BW (g), GA (weeks), sex (number, %) history of jaundice in siblings (number, %), PBC at enrollment (mg/dL), age at enrollment (hours), and abnormal weight loss, >9% between 48 to 72 hours and >12% between 73 to 96 hours (number, %).
Sample Size and Outcome Variables
We used the requirement for PT as the major outcome variable. PT was initiated if the serum bilirubin level reached 331.5 μmol/L (19.5 mg/dL). It was estimated, based on our experience with this population,25 that this would occur in 20% of infants in the control group and 2% in the SnMP-treated group. Based on those assumptions, we required a minimum of 42 patients in each group to have a 90% chance of showing a significant difference between groups (P < .05; one-tailed). The sample size calculations were based on the formula of Lwanga and Lemeshow26 that is used to compare small differences in independent samples (Fisher's exact test).
Data were analyzed using Statistix for Windows (96 Analytical Software). Whenever possible, numerical variables were compared between the control and SnMP treatment groups using the Student'st test for independent samples. Normal error distribution was evaluated using both graphical methods (normal probability plots and box-plots) and the Shapiro Wilk's test. Homogeneity of variance was evaluated through the F-test when the assumption of normal distribution was not rejected. In those numerical variables in which the hypothesis of normal distribution was rejected at the 0.05 level, the Mann-Whitney test based on ranks was used. Thus, BW (g), GA (weeks), age at enrollment (hours), were compared with the ttest, and PBC at enrollment (mg/dL), maximum PBC (mg/dL), number of PBC measurements, time (hours) until closure (PBC ≤13 mg/dL), were compared with the Mann-Whitney test. The χ2 test for independence was used to compare categorical data that included sex, history of sibling with jaundice, and requirement for PT. Abnormal weight loss was compared between the two groups through the use of Fisher's exact test.
During the period from November 2, 1996 until November 26, 1997, 103 infants were considered to be candidates for this study according to the criteria for enrollment described in the “Methods” section. Nine parents refused to allow their infants to participate in the clinical trial. Ten newborns were excluded after randomization for the following reasons: 1 infant because of hemolytic disease, 1 infant because of infection, and 8 infants in whom the bilirubin level measurements were not made according to the protocol design. Thus, a total of 84 infants were enrolled, 40 of whom were randomly assigned to the SnMP group and 44 to the control group. There were no significant differences between the groups in sex ratio, BW, GA, hematocrit, PBC at enrollment and age at enrollment, weight loss, and history of jaundice in siblings (Table 1).
We found significant statistical differences between the study groups in the outcome variables (Table 2). The administration of a single dose of SnMP entirely eliminated the need for PT; none of the 40 infants in the SnMP-treated group required supplemental PT, whereas 12 of 44 (27%) infants in the control group received PT. In addition, SnMP significantly reduced the maximum serum bilirubin level reached when compared with the control group (Table 1). We explored the question of whether SnMP affected the distribution of maximum PBCs. It is clear (Fig 1) that the distribution of maximum PBC was shifted to the left by SnMP treatment despite the fact that 12 of the 44 control infants required PT (P = .0043, Mann-Whitney test). The median time required between enrollment and closure was significantly reduced by SnMP administration from 120 hours in the control group to 86.5 hours in the SnMP-treated group (P < .0001) (Table 2). In addition the number of bilirubin measurements between enrollment and closure was reduced from (median) 5 to 3 by SnMP treatment (P < .0001).
There were no systemic or local reactions to the intramuscular injections of SnMP. A particular scrutiny was made for the appearance of erythema; none, however, was observed in any of the SnMP-treated (none of them of course required PT) or the control group of newborns. There were no differences in liver function tests conducted at 12 days and at 3 months of age between the control and SnMP-treated infants; in both groups all tests were normal (results not shown).
The present study clearly demonstrates that a single dose of SnMP, administered to healthy full-term breastfed newborns at a time when hyperbilirubinemia is becoming severe (15 to 18 mg/dL), eliminates the need for PT and reduces both the number of bilirubin measurements and the length of time the infants are under clinical care for the problem of hyperbilirubinemia. Administration of SnMP was well-tolerated and no side effects were observed. These findings are consistent with the results of our previous studies18 ,19 that have demonstrated the efficacy of SnMP in controlling neonatal hyperbilirubinemia before plasma bilirubins reached levels potentially toxic to the central nervous system; and they extend the effectiveness of SnMP in controlling hyperbilirubinemia to a new and large population of newborns.
We selected newborns with a PBC ≥15 mg/dL for this study, based on the observation that 15 mg/dL represents the 95th percentile for the breastfed population.27 In addition, infants were enrolled at 48 to 96 hours of age to provide a more homogeneous sample in which to examine the effect of SnMP and to reduce those variables (eg, age-related maturity) that could interact with and confound the course of hyperbilirubinemia. In a previous controlled clinical trial to compare the effect of four different interventions on hyperbilirubinemia in healthy breastfed term newborns, a total of 125 of 1685 (7.4%) newborns reached a serum bilirubin concentration of ≥291 μmol/L (≥17 mg/dL), 3.5 days (range, 2 to 8 days) after birth.21 When the newborns were allowed to reach plasma bilirubin levels approaching 342 μmol (20 mg/dL) before starting intensive PT, 24% reached or exceeded this level and required hospitalization. This approach did not eliminate the need for PT or the number of clinical laboratory tests required until the plasma bilirubin levels decreased; ie, the problem was reduced but not solved. By contrast a follow-up study of jaundice during hospitalization and in the outpatient clinic in all healthy breastfed newborn infants born in the Ramon Sarda hospital during the month of January 1995 showed that 4.9% of infants who clinically had no evident jaundice at discharge (average hours of hospital stay, 55 ± 14.2 hours) reached a PBC of 290.7 μmol/L (17 mg/dL) during follow-up.25 The problem, therefore, has been shifted from the neonatal units to the outpatient setting, and the necessity for adequate and close follow-up of all newborns is clearly essential.
The uncertainties faced by clinicians regarding: a) what is considered to be a threatening level of bilirubin in the circulation, b) the inability to identify those infants who will reach a PBC at which PT is required, and c) the problem of infants lost from follow-up programs, leads us to propose the more general use of the therapeutic approach taken in this study that is designed to inhibit bilirubin production and thus preempt the development of severe hyperbilirubinemia. Thus, by interdicting the production of bilirubin for a short period immediately after birth it would be possible to eliminate many of the uncertainties regarding bilirubin toxicity and treatment thresholds that complicate the treatment of neonatal hyperbilirubinemia. This approach offers important advantages to both the newborns and to the clinicians involved in their care.
A related concern, difficult to quantitate but clearly understandable, is the emotional impact on the mother of having a sick infant. This entails frequent traveling to and from the hospital, additional blood sampling for bilirubin determinations, and separation from the infant if rehospitalization is required. This situation contrasts with the current practice of encouraging mother-infant interaction and bonding. The long-lasting effects of these changes in clinical practice are not known, but as Kemper et al22 reported, despite education from pediatricians and nursing staff, many mothers of healthy full-term infants consider jaundice a serious illness. This is expressed by their concern and behavior and this population of jaundiced infants is considered to be vulnerable by their mothers. Indeed the appearance of behavioral, functional, and developmental disabilities with age may partly reflect the impact of the currently used clinical interventions on the mother-infant relationship. Based on the results described above, they could be avoided by suppressing bilirubin production with SnMP, or a related HO inhibitor, thus eliminating the need for PT to treat progressive hyperbilirubinemia.
This research was supported by NICHHD contract NO1-HD-5-3234 and gifts from the Ablon Foundation and the Renfield Foundation.
We thank the medical and nursing staff of the Hospital Materno Infantil Ramon Sarda for their support; Drs Maria del Carmen Perego (Chief Laboratory Unit) and Alba Smirnoff for help with biochemical measurements; and Mrs Liliana Orellana, biostatistician, for her assistance. We thank Lisa Meyer for her assistance in the preparation of this manuscript.
We are deeply grateful to all the mothers and infants who made this trial possible.
- Received June 25, 1998.
- Accepted September 18, 1998.
Reprint requests to (A.K.) Rockefeller University, 1230 York Ave, New York, NY 10021.
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- Copyright © 1999 American Academy of Pediatrics