METHOD OF IDENTIFYING AND TREATING PREMATURE INFANTS AT RISK FOR BPD

Abstract

Methods for identifying premature infants at risk for developing bronchopulmonary dysplasia and/or most likely to benefit from administration of inhaled nitric oxide for prevention of bronchopulmonary dysplasia (BPD). Methods for treating premature infants identified as at risk and/or likely to benefit are provided. also provided are methods for identifying premature infants that are not at risk for developing bronchopulmonary dysplasia and/or unlikely to benefit from administration of inhaled nitric oxide for prevention of bronchopulmonary dysplasia, and methods for avoiding risks of toxicity and undesirable side effects associated with inhaled nitric oxide therapy comprising administering only non-iNO treatment modalities to these infants.

Claims

1. A method for identifying a premature infant that is likely to have a therapeutic response to inhaled nitric oxide comprising: a) obtaining a blood or tissue sample from the infant prior to initiation of inhaled nitric oxide therapy; b) analyzing the blood or tissue sample for presence of a genetic mutation in a gene encoding a nitric oxide synthase that results in decreased endogenous synthesis of nitric oxide; c) determining whether the genetic mutation is present or absent, wherein presence of the genetic mutation indicates that the infant is likely to have a therapeutic response to inhaled nitric oxide.

2. The method of claim 1, wherein the genetic mutation is T-786C or G894T in the eNOS gene.

3. The method of claim 1, wherein the blood or tissue sample is analyzed using single nucleotide polymorphism analysis or restriction fragment length polymorphism analysis.

4. The method of claim 1, further comprising treating the infant with inhaled nitric oxide to prevent development of bronchopulmonary dysplasia when the genetic mutation is present.

5. The method of claim 1, wherein the premature infant is less than 30 weeks gestational age.

6. A method of avoiding risks associated with administration of inhaled nitric oxide to premature infants at risk for developing bronchopulmonary dysplasia comprising: a) testing the infant for one or more of (i) a plasma total concentration of nitrate and nitrite; (ii) a concentration of nitric oxide in exhaled ambient or nitric oxide-free air; and (iii) presence of a genetic mutation in a gene encoding nitric oxide synthase that results in decrease endogenous synthesis of nitric oxide; b) determining whether the total concentration of nitrate and nitrite in the plasma is at least about 50 μM, whether the concentration of nitric oxide in the exhaled air is about 2.5 ppb or higher and/or whether no genetic mutation in a gene encoding nitric oxide synthase that reduces endogenous synthesis of nitric oxide is present; and c) treating the infant only with non-inhaled nitric oxide modalities to prevent development of bronchopulmonary dysplasia if the total concentration of nitrate and nitrite in the plasma is at least about 50 μM, the concentration of nitric oxide in the exhaled air is about 2.5 ppb or higher, and/or the genetic mutation is not present.

7. The method of claim 6, wherein the premature infant is less than 30 weeks gestational age.

8. The method of claim 6, wherein the genetic mutation is T-786C or G894T in the eNOS gene.

9. The method of claim 6, wherein the blood or tissue sample is analyzed using single nucleotide polymorphism analysis or restriction fragment length polymorphism analysis.

10. The method of claim 6, further comprising treating the infant with inhaled nitric oxide to prevent development of bronchopulmonary dysplasia when the total concentration of nitrate and nitrite in the plasma is at least about 50 μM, when the measured concentration of nitric oxide in the exhaled air is less than about 1.5 ppb, and/or when the genetic mutation is present.

Description

DETAILED DESCRIPTION

[0022] Before describing several exemplary embodiments of the invention, it is to be understood that the invention is not limited to the details of construction or process steps set forth in the following description. The invention is capable of other embodiments and of being practiced or being carried out in various ways.

[0023] It has been discovered that certain biomarkers present in premature infants are predictive of the risk of developing BPD and/or the likelihood of a therapeutic response to iNO to prevent BPD. As used herein, the term “therapeutic response to iNO” means that iNO administration increases the rate of survival without bronchopulmonary dysplasia (BPD) at 36 weeks of post menstrual age compared to the rate of survival with development of BPD at 36 weeks of post menstrual age in an untreated premature infant population. As used herein, the term “premature infant”, “preterm infant” and the like means infants born at less than 30 weeks gestational age. The biomarkers identified are of three types: a) the concentration of metabolites of NO, such as nitrite and nitrate, in blood or plasma, b) the concentration of NO in exhaled air, and c) the presence or absence of a mutation in the eNOS gene that decreases endogenous NO production, or any combination thereof.

[0024] A first method for identifying premature infants that are at risk for developing BPD and/or are likely to have a therapeutic response to treatment with iNO comprises measuring the concentration of one or more nitric oxide metabolites in the plasma of the infant prior to iNO therapy, wherein a concentration of the metabolite that is lower than a normal concentration of the metabolite prior to treatment with iNO indicates that the infant is at risk and/or is likely to have a therapeutic response to treatment with iNO. A modification of the first method may be used for identifying premature infants that are not at risk for developing BPD and/or are unlikely to have a therapeutic response to treatment with iNO, so that non-iNO treatment modalities may be administered and the risks of toxicity and undesirable side effects of iNO therapy can be reduced. In this modification, if a concentration of the metabolite is normal or higher than a normal concentration of the metabolite prior to treatment with iNO, iNO is not administered. Typically, in such cases only non-iNO treatment modalities will be administered for prevention of BPD. By way of example, the metabolite that is measured is nitrite and/or nitrate.

[0025] Several suitable tests are available for quantitating nitrite and/or nitrate in plasma. For example, the Nitrate/Nitrite Colorimetric Assay Kit (Cayman Chemical Company, Ann Arbor, Mich.), the Nitrate/Nitrite Assay (Kamiya Biomedical Company, Seattle, Wash.), and the Total Nitric Oxide and Nitrate/Nitrite Parameter Assay Kit (R&D Systems) are all suitable for use in the invention as instructed by the manufacturer. It has been found that a total concentration of nitrite and nitrate in plasma of about 50 ?AM or more correlates with reduced risk of developing BPD and with non-responsiveness to iNO therapy. A total concentration of nitrite and nitrate of less than about 50 !AM, including concentrations that are below the limit of detection in the assay used (effectively 0), indicates that the premature infant is at risk for developing BPD and/or is likely to have a therapeutic response to treatment with iNO. In specific examples, the total concentration of nitrate and nitrite from about 0-45 [tM, about 5-40 [tM, about 10-40 [tM, about 10-30 1 AM, about 20 [tM, or below the detection limit of the assay indicates that the infant is at risk for developing BPD and/or is likely to have a therapeutic response to treatment with iNO. In specific examples, a total concentration of nitrate and nitrite at least about 50 RM, greater than about 60 [tM, or about 60-185 indicates that the infant is not at risk for developing BPD and/or is unlikely to have a therapeutic response to treatment with iNO.

[0026] A second method for identifying premature infants that are likely to have a therapeutic response to iNO therapy comprises measuring the concentration of NO in exhaled air while the infant breathes ambient air or air having all naturally-occurring NO removed. The measurement of NO concentration in exhaled air is taken prior to treatment with iNO. If the concentration of NO in air exhaled by the infant is lower than a normal concentration of exhaled NO, the infant is at risk for developing BPD and/or is likely to have a therapeutic response to iNO treatment for prevention of BPD. Typically, healthy pre-term infants exhale about 2.5 ppb of NO when breathing ambient air. However, concentrations of NO in exhaled air from about 0.5-1.5 ppb indicate that the infant is at risk for developing BPD and/or is likely to have a therapeutic response to iNO administration. In a particular embodiment a concentration of exhaled NO that is about 1.5 ppb or less, about 1 ppb or less, for example 0.5-0.9 ppb, indicates that the infant is at risk and/or is likely to have a therapeutic response to iNO administration. A modification of the second method may be used for identifying premature infants that are not at risk for developing BPD and/or are unlikely to have a therapeutic response to treatment with iNO, so that non-iNO treatment modalities may be administered and the risks of toxicity and undesirable side effects of iNO therapy can be reduced. In this modification, if a concentration of NO in exhaled air is normal or higher than a normal concentration of NO prior to treatment with iNO (about 2.5 ppm or above), iNO is not administered. Typically, in such cases only non-iNO treatment modalities for prevention of BPD will be administered.

[0027] Several suitable instruments and devices are available for measuring the concentration of NO in exhaled air, and can be adapted for use with preterm infants. The analytical instrument should have a sensitivity for gas samples of at least 0.5 ppb and a range of at least 0.5-5 ppb. For example, exhaled breath samples may be analyzed using the Sievers Nitric Oxide Analyzer (NOA 280i) (GE Analytical Instruments, Boulder, Colo.).

[0028] A third method for identifying premature infants that are at risk for developing BPD and/or are likely to have a therapeutic response to iNO administration comprises, prior to initiation of iNO therapy, analyzing a blood, plasma, serum, orally component of blood, plasma or serum, tissue, or other sample, tracheal aspirates or the like, for the presence of a mutation in a gene encoding a nitric oxide synthase (e.g., eNOS, nNOS or iNOS), wherein the mutation results in decreased endogenous synthesis of nitric oxide. Any known mutation or genetic anomaly in a nitric oxide synthase gene can be analyzed, provided it results in decreased endogenous synthesis of nitric oxide. Examples of mutations suitable for analysis include the T-786C mutation (a T-*C mutation at position −786 in the 5′ flanking region of the eNOS gene) and G894T (a G-*T mutation at position 894 of the eNOS coding sequence that results in a Glu.sup.298-*Asp polymorphism in the enzyme). Testing for the T-786C mutation is commercially available through diagnostic laboratories such as Molecular Diagnostics Laboratories (MDL, Covington, Ky.). In addition, any of the known laboratory methods for analysis of single nucleotide polymorphisms (SNPs), insertions, deletions and genotyping is suitable for use in the invention, e.g., melting curve analysis of polymerase chain reaction products or polymerase chain reaction-restriction fragment length polymorphism analysis. A modification of the third method may be used for identifying premature infants that are not at risk for developing BPD and/or are unlikely to have a therapeutic response to treatment with iNO, so that non-iNO treatment modalities may be administered and the risks of toxicity and undesirable side effects of iNO therapy can be reduced. In this modification, if no mutation that reduced endogenous synthesis of nitric oxide is detected in a gene encoding a nitric oxide synthase, endogenous synthesis of nitric oxide will be normal and iNO is not administered. Typically, in such cases only non-iNO treatment modalities for prevention of BPD will be administered.

[0029] The absence of a mutation in a gene encoding a nitric oxide synthase that results in reduced endogenous synthesis of nitric oxide (indicating normal NOS activity) correlates with reduced risk of developing BPD and/or with a failure of therapeutic response to treatment with iNO for prevention of BPD. The presence of a mutation that reduces endogenous synthesis of nitric oxide due to impairment of NOS activity indicates that the infant is at risk for developing BPD and/or is likely to have a therapeutic response to iNO treatment.

[0030] Any of the foregoing methods for identifying premature infants that are at risk for developing BPD and/or are likely to have a therapeutic response to iNO therapy for prevention of BPD can be used in connection with methods for preventing BPD in premature infants. In these methods, prior to initiation of iNO therapy, premature infants requiring mechanical ventilation are evaluated as discussed above for concentrations of nitric oxide metabolites in their plasma, concentrations of exhaled nitric oxide while breathing ambient air or air from which substantially all naturally-occurring nitric oxide has been removed, and/or a mutation in a gene encoding NOS that results in decreased endogenous synthesis of nitric oxide. If the result of one or more of the tests indicates that the infant is at risk for developing BPD and is likely to have a therapeutic response to iNO administration, iNO therapy is initiated. Typically, if one or more of the tests indicates that the infant is not at risk and/or is unlikely to have a therapeutic response to iNO administration, administration of iNO will be avoided and the infant may be treated only with one or more non-iNO modalities for prevention of BPD. These identification tests provide improved information for weighing the risk vs. benefit of treating the infant with iNO, thereby reducing the risk of toxicity and undesirable side effects of the treatment. In addition, the identification tests substantially increase the likelihood of efficacy of iNO treatment for the individual premature infant.

[0031] Any treatment protocol known in the art for administering iNO to premature infants to prevent BPD is suitable for use in the present invention, after determining that the infant is at risk for developing BPD and/or is likely to have a therapeutic response to iNO administration. Generally, one or more of the tests to identify the premature infant that is at risk and/or is likely to have a therapeutic response to administration of iNO are performed from birth to about 14 days after birth. In certain embodiments, the one or more tests are performed between birth and about 5 days after birth. In specific embodiments, the one or more tests are performed immediately after birth, on the day of birth, before the third day after birth, or before the fifth day after birth.

[0032] It has been reported that on the third day after birth there is no difference in exhaled NO in premature infants that subsequently develop BPD and those that do not. Accordingly, particularly for evaluation of exhaled NO, the evaluation is generally performed at a time between birth and the third day after birth to obtain the most accurate results.

[0033] In general, administration of iNO to premature infants that have been identified as at risk for developing BPD and/or as likely to have a therapeutic response begins at least five days after birth. Treatment of these infants may begin 5 to 14 days after birth, 7 to 21 days after birth, or 5 to 7 days after birth. Administration of iNO generally continues for 20-30 days after it is initiated. In specific treatment protocols, administration of iNO may be continued for at least 24 days.

[0034] For example, the protocol disclosed by R. A. Ballard, et al. (2006. N. Eng. J. Med. 355:343-353) is a particularly useful treatment method. In this study, premature infants between 7 and 21 days of age requiring ventilator support and at risk for developing BPD received decreasing concentrations of nitric oxide, beginning at 20 ppm, for a minimum of 24 days. The initial dose of nitric oxide was administered for 48-96 hours, and subsequent doses of 10, 5, and 2 ppm were administered at weekly intervals thereafter. The authors report that administration of iNO according to this protocol improves the pulmonary outcome for premature infants who are at risk for BPD with no apparent short term adverse effects.

[0035] A further example of a suitable administration protocol is that of M. D. Schreiber, et al. (2003. N. Engl. J. Med. 349:2099-2107), in which premature infants undergoing mechanical ventilation for respiratory distress syndrome were treated with iNO at 10 ppm on day 1, followed by 5 ppm for six days.

Examples

[0036] A multi-center, double blind, placebo-controlled randomized clinical trial was conducted to examine the efficacy of iNO in preterm infants less than 30 weeks gestational age and less than 1250 grams who required mechanical ventilation or positive pressure support on days 5 to 14 after birth. A secondary objective of the trial was to examine the pharmacokinetics (PK) of nitric oxide in preterm infants using plasma nitrite and nitrate (NOx) as surrogate biomarkers. Continuous iNO administration was begun at 20 ppm by administration into the inspiratory limb of the ventilator circuit in the mechanically ventilated subject using an INOvent® delivery device. Therapy was continued for 24 days, following a dose reduction schedule (10 ppm after 72 hours of treatment, 5 ppm on day 10). If an infant was extubated before 24 days, therapy was continued via nasal continuous positive airway pressure or nasal cannula to complete the protocol.

[0037] Because of the challenge to measure free NO in systemic circulation, the PK assessment was done via plasma concentration data of NOx (nitrite and nitrate). In addition, methemoglobin (another metabolite of NO) was used as a secondary variable for PK evaluation. This evaluation was done to understand the relationship between pharmacokinetic behavior of iNO and clinical outcomes. The sampling protocol included pre-treatment time points (less than 2 hours before initiation of NO dosing), as well as a variety of time points during iNO therapy and after discontinuation of iNO therapy.

[0038] A liquid chromatographic/mass spectrophotometric (LC/MS) assay method was developed and qualified for measurement NOx in human plasma. The quantitation of NOx was performed using the .sup.15N-stable isotope labeled nitrate as an internal standard. Nitrate reductase and NADH was added to the samples for enzymatic reduction of nitrate to nitrite in plasma. Upon completion of the reduction reaction, acetone was added to precipitate plasma proteins. The alkylating reagent, pentafluorobenzyl bromide (PFBBr), was subsequently added to the samples to convert nitrite to a chemically stable pentafluorobenzyl derivative. After completion of derivatization, plasma supernatant was injected into the LC/MS for analysis. Plasma concentrations of total nitrite and nitrate were measured and reported as NOx using this assay method. The instrumental analysis of the nitrite and NOx samples was performed using reversed-phase high performance liquid chromatography (HPLC), coupled with mass spectrometric (MS) detection. A triple-quadrupole mass spectrometer was used to acquire single-ion recording (SIR) data, and was operated in the negative electrospray ionization mode for the detection of the nitrite derivative (PFB-NO.sub.2) and the .sup.15N-labeled internal standard derivative.

[0039] The NOx results for subjects that had a therapeutic response to iNO treatment are shown in the following Table:

TABLE-US-00001 Subjects who had a response with Nitric Oxide Treatment Subject Race Treatment Primary Lab Test Lab Test  425301 WHITE NITRIC Y <LLOQ uM  427801 WHITE NITRIC Y <LLOQ uM 1821202 BLACK NITRIC Y 77.5 uM 3412801 WHITE NITRIC Y <LLOQ uM Mean NOx level 19.4 uM Values <LLOQ (below the limit of quantitation of the assay) were assumed to be zero (0)

[0040] The NOx results for subjects that did not have a therapeutic response to iNO treatment are shown in the following Table:

TABLE-US-00002 Subjects who DID NOT have a response with Nitric Oxide Subject Race Treatment Primary Lab Test Lab Test  203801 HISPANI NITRIC N 62.3  uM  414501 WHITE NITRIC N <LLOQ uM 1110501 BLACK NITRIC N <LLOQ uM 1623801 BLACK NITRIC N 184    uM 3631502 WHITE NITRIC N <LLOQ uM 3632801 HISPANI NITRIC N <LLOQ uM 3635401 WHITE NITRIC N 76.8  uM 3917501 WHITE NITRIC N 62.7  uM Mean NOx Level 49.8  uM Values <LLOQ (below the limit of quantitation of the assay) were assumed to be zero (0)

[0041] As can be seen from the results above, treatment with iNO was not effective in subjects with NOx levels from 62.3 uM to 184 uM. The calculated mean NOx concentration for the non-responding population was 49.8 uM. It can be concluded that a NOx concentration in plasma of about 50 1 AM or above indicates that the subject is less likely to have a therapeutic response to iNO administration.

[0042] Conversely, the results show that treatment was therapeutically effective in subjects having an average NOx concentration of about 20 [NI or less. It can be concluded that a NOx concentration in plasma of less than 50 μM, e.g., about 10-30 μM, indicates that the subject is more likely to have a therapeutic response to iNO administration.

[0043] Reference throughout this specification to “one embodiment,” “certain embodiments,” “one or more embodiments” or “an embodiment” means that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrases such as “in one or more embodiments,” “in certain embodiments,” “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily referring to the same embodiment of the invention. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments.

[0044] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It will be apparent to those skilled in the art that various modifications and variations can be made to the method and apparatus of the present invention without departing from the spirit and scope of the invention. Thus, it is intended that the present invention include modifications and variations that are within the scope of the appended claims and their equivalents.