WO2020264239A1 - Méthodes de diagnostic et de prédiction d'une maladie pulmonaire chronique et d'une maladie intestinale chez des nourrissons prématurés - Google Patents

Méthodes de diagnostic et de prédiction d'une maladie pulmonaire chronique et d'une maladie intestinale chez des nourrissons prématurés Download PDF

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WO2020264239A1
WO2020264239A1 PCT/US2020/039732 US2020039732W WO2020264239A1 WO 2020264239 A1 WO2020264239 A1 WO 2020264239A1 US 2020039732 W US2020039732 W US 2020039732W WO 2020264239 A1 WO2020264239 A1 WO 2020264239A1
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infant
weeks
taken
pma
samples
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Rakesh Patel
Samuel GENTLE
Charitharth LAL
Khandaker AHMED
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Uab Research Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/12—Nitrate to nitrite reducing bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/38—Pediatrics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the invention relates to methods for diagnosing or assessing the risk of developing chronic lung and bowel disease in pre-term infants, as well as guiding the care of pre-term infants, by measuring the nitrate reductase activity in oral microbiome samples from the infant.
  • the present invention overcomes previous shortcomings in the art by providing new tools for diagnosing and predicting risk for developing chronic lung disease and bowel disease in preterm infants.
  • One aspect of the invention provides a method of diagnosing bronchopulmonary dysplasia (BPD) or determining the risk of developing BPD in an infant that is born at a gestational age of about 28 weeks or less (e.g ., a preterm infant or newborn), comprising: measuring nitrate reductase (NR) activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth (e.g., within about one minute to about 96 hours after birth) and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks postmenstrual age (PMA), wherein one of the at least two additional oral microbiome samples is taken at about 29 weeks PMA; and diagnosing the infant as having BPD or determining the infant to be at risk of developing BPD when no spike ( e.g ., no increase) in NR activity is observed in the sample taken from the infant at about 29 weeks PMA relative to NR activity before and/or after 29 weeks
  • NR
  • a second aspect of the invention provides a method of diagnosing pulmonary hypertension (PH) or determining the risk of developing PH in an infant that is a gestational age of about 28 weeks or less, comprising: measuring NR activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; and diagnosing the infant as having PH or determining the infant to be at risk of developing PH when no spike in NR activity is observed in the sample taken from the infant at about 29 weeks PMA relative to NR activity before and/or after 29 weeks PMA.
  • An additional aspect of the invention provides a method of diagnosing necrotizing enterocolitis (NEC) or determining the risk of developing NEC in an infant that is a gestational age of about 28 weeks or less, comprising: measuring NR activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken at birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; and diagnosing the infant as having NEC or determining the infant to be at risk of developing NEC when no spike in NR activity is observed at about 29 weeks PMA relative to NR activity before and/or after 29 weeks PMA.
  • NEC necrotizing enterocolitis
  • a further aspect of the invention provides a method for guiding the care of an infant that is a gestational age of about 28 weeks or less, comprising: measuring NR activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; diagnosing the infant as having BPD or
  • suitable therapeutic and/or prophylactic treatments such as: (a) administering oxygen and/or nitric oxide, (b) administering at least one phosphodiesterase
  • An additional aspect of the invention provides a method for guiding the care of an infant that is a gestational age of about 28 weeks or less, comprising: measuring NR activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; diagnosing the infant as having PH or determining the infant to be at risk of developing PH when no spike in NR activity is observed at about 29 weeks PMA; and guiding the care of the infant that is diagnosed to have PH or to be at risk of developing PH by providing one or more suitable therapeutic and/or prophylactic treatments, such as: (a) administering nitric oxide and/or oxygen, (b) administering a composition comprising at least one phosphodiesterase inhibitor (e.g., sildenafil), (c) administering a composition comprising at least one endo
  • a further aspect of the invention provides a method for guiding the care of an infant that is a gestational age of about 28 weeks or less, comprising: measuring NR activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; diagnosing the infant as having necrotizing enterocolitis (NEC) or determining the infant to be at risk of developing NEC when no spike in NR activity is observed at about 29 weeks PMA; and guiding the care of the infant that is diagnosed to be at risk of developing NEC by providing one or more suitable therapeutic and/or prophylactic treatments, such as: (a) replacing oral feeding with parenteral feeding, (b) removing air and fluid from the stomach and intestine, (c) administering a composition comprising intravenous fluids, d) administering a composition comprising at least one
  • kits comprising one or more reagents for use in assessing the risk of developing BPD, PH, and/or NEC in an infant that is born at a gestational age of about 28 weeks or less and/or for analysing the microbes in an oral microbiome sample taken from an infant that is born at a gestational age of about 28 weeks or less.
  • FIGS. 1A-1C provide representative tracings of nitrate dependent nitrite formation.
  • Fig. 1A nitrite formation from control samples and fresh tongue swabs; (-) indicates isolate to which no nitrate has been added, (+) indicates isolate following nitrate addition.
  • Fig. IB summary of data from all samples.
  • Fig. 1C nitrite formation following 18 hour incubation from tongue swab and air swab (control).
  • Fig. 2A shows oral nitrate reductase activity increases at 29 weeks PMA in preterm infants (p ⁇ O.Olcompared to birth and 27 weeks PMA, and p ⁇ 0.05 relative to 34 weeks PMA. Data analyzed by paired t-test.
  • Fig. 2C shows nitrate reductase activity normalized to CFU.
  • FIG. 3 shows percent relative abundance by microbial genus and species. There were statistically more Rothia and Veillonella at 34 weeks PMA compared to all other time points (p ⁇ 0.03). Analysis performed with 1-way ANOVA with Tukey multiple comparisons test.
  • FIG. 4 provides Spearman correlation metrics demonstrating associations between individual bacteria and NR activity.
  • the microbial abundance of the NR producing bacteria Veillonella and Rothia and the NR-negative bacteria Streptococcus were positively correlated with adjusted NR activity.
  • FIG. 5 shows change in oral nitrate reductase activity as measured between PMA 28 and 29 weeks for pre-term infants that developed BPD with PH, BPD only, or neither after 34 weeks PMA.
  • P-value was determined by t-test between“no BPD nor PH” and“BPD with PH”.
  • N values indicate the number of patients per group.
  • “about X” where X is the measurable value is meant to include X as well as variations of ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of X.
  • a range provided herein for a measureable value may include any other range and/or individual value therein.
  • “about” means ⁇ 1 to 5 days, e.g .,“about 28 weeks” means 28 weeks ⁇ 1-5 days,“about 29 weeks PMA” means 29 weeks PMA ⁇ 1-5 days, “about 30 weeks” means 30 weeks ⁇ 1-5 days, and the like.
  • phrases such as“between X and Y” and“between about X and Y” should be interpreted to include X and Y.
  • phrases such as“between about X and Y” mean“between about X and about Y” and phrases such as“from about X to Y” mean “from about X to about Y.”
  • transitional phrase“consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.
  • the term“consisting essentially of’ when used in a claim of this invention is not intended to be interpreted to be equivalent to“comprising.”
  • the terms “increase,” “increasing,” “increased,” “enhance,” “enhanced,”“enhancing,” and“enhancement” (and grammatical variations thereof) describe an elevation of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more as compared to a control.
  • the term“spike in NR activity” refers to an increase in NR activity at about 29 weeks PMA as compared to a time point prior or after 29 weeks PMA (e.g, at about 28 weeks PMA or earlier than 29 weeks PMA, or about 30 weeks PMA or later than 29 weeks PMA) of at least about 5% (e.g, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 69, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 175, 200
  • the terms“reduce,”“reduced,”“reducing,”“reduction,”“diminish,” and“decrease” describe, for example, a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% as compared to a control.
  • the reduction can result in no or essentially no (i.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.
  • “gestational age” as applied to the age of an infant refers to the time elapsed between the first day of the last normal menstrual period of the infant’s mother and the day of delivery.
  • Chronicological age (or“postnatal” age) is the time elapsed after birth. It is usually described in days, weeks, months, and/or years.
  • postmenstrual age or“PM A” refers to the perinatal period beginning after the day of birth. It is the time el apsed between the first day of the last menstrual period of the mother and birth of the infant (gestational age) plus the time elapsed after birth (chronological age) and is usually provided in number of weeks. As an example, a preterm infant born at a gestational age of 30 weeks who is at a chronological age of 8 weeks would have a PM A of 38 weeks.
  • an infant that is a gestational age of about 28 weeks or less is a preterm infant or preterm newborn having a gestational age of about 21 to about 28 weeks.
  • An infant for which this invention is useful may also be described as having a birth weight of less than about 2500 grams (low birth weight), less than about 1500 grams (very low birth weight), or less than about 1000 grams (extremely low birth weight).
  • oral microbiome refers to the microorganisms that reside in the oral cavity.
  • the oral microbiome refers the microorganisms that reside on the tongue and/or at the posterior dorsum of the tongue.
  • BPD bronchopulmonary dysplasia
  • pulmonary hypertension or“PH” is high blood pressure in the arteries of the lungs (the pulmonary arteries) and affects the right side of the heart.
  • the disease can result in hypoxemia secondary to right-to-left intracardiac shunting of blood.
  • NEC neurotizing enterocolitis
  • GI gastrointestinal
  • ischemic necrosis of the intestinal (large and small) mucosa which is associated with severe inflammation, invasion of enteric gas forming organisms, and dissection of gas into the bowel wall and portal venous system
  • the terms“increased risk” and“decreased risk” as used herein define the level of risk that a subject (e.g ., an infant that is a gestational age of about 28 weeks or less) has of developing BPD, PH and/or NEC, as compared to a control subject (e.g., a subject that exhibits an increase in nitrate reductase activity at about 29 weeks postmenstrual age).
  • the terms“prevent,”“prevents,” or“prevention” and“inhibit,” “inhibits,” or“inhibition” are not meant to imply complete abolition of disease and encompasses any type of prophylactic treatment that reduces the incidence of the condition, delays the onset of the condition, and/or reduces the symptoms associated with the condition after onset.
  • “Diagnosing,”“assessing the risk,”“predicting the risk” or“determining the risk,” as used herein means providing an indication that a subject may be afflicted with or at risk of developing a disease, e.g., a disease such as BDP, PH or NEC, and includes other terms such as screening for a disease, providing a risk assessment for disease, determining
  • responsiveness includes providing an assessment or indication of disease in response to treatment (such as administration of probiotics, antibiotics, oxygen and/or nitric oxide, a phosphodiester inhibitor, a
  • An“effective,”“prophylactically effective,” or“therapeutically effective” amount as used herein is an amount that is sufficient to provide some improvement or benefit to the subject.
  • an“effective,”“prophylactically effective,” or“therapeutically effective” amount is an amount that will provide some delay, alleviation, mitigation, or decrease in at least one clinical symptom in the subject.
  • an“effective,”“prophylactically effective,” or “therapeutically effective” amount can refer to the amount of a composition, compound, or agent that improves a condition in a subject by at least 5%, e.g, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.
  • an“effective,”“prophylactically effective,” or“therapeutically effective” amount in any individual case for any particular agent, compound or composition can be determined by one of skill in the art by reference to the pertinent texts and literature such as Remington, The Science and Practice of Pharmacy (latest edition).
  • microbiota over the tongue dorsum are particularly unique in that several facultative anaerobes residing in the crypts of the tongue dorsum express an enzyme, nitrate reductase (NR) (Duncan et al, Nat. Med. 1(6): 546 (1995)), not found in the human genome.
  • NR nitrate reductase
  • NR containing bacteria catalyze the reduction of nitrate to nitrite, which is a substrate for nitric oxide (NO) via protonation within the acidic gastric environment or further electron reduction through a number of hypoxia sensitive enzymes and proteins in the blood and tissues (Weitzberg et al, Annu. Rev. Nutr. 33 : 129 (2013)). Accumulating evidence suggests that reduction of nitrate to nitrite and NO is an important and parallel pathway, with nitric oxide synthase, to control systemic NO-bioavailability.
  • NO nitric oxide
  • NR significantly impacts systemic, gastrointestinal, and pulmonary vasculature homeostasis.
  • Several studies have demonstrated that nitrate administration significantly lowers systolic and diastolic blood pressures by levels comparable to a single
  • nitrate conversion to nitrite may also serve to limit ischemia-reperfusion injury, endothelial dysfunction, and provide host defense via antimicrobial effects on gut pathogens (Dykhuizen et al, Antimicrob. Agents Chemother. 40(6): 1422 (1996); Xia et al, Chin. Med. J. (Engl.) 119(22): 1904 (2006); Fite et al, Antimicrob. Agents Chemother.
  • mice 48(2):655 (2004); Jadert et al, Redox Biol. 2:73 (2014)).
  • the prevention of right ventricular hypertrophy through nitrate supplementation in mice supports the possible role of enterosalivary nitrate reduction in attenuating pulmonary vascular resistance in mice (Baliga et al, Circulation 125(23):2922 (2012)) [14] with growing evidence to suggest nitrate based therapeutics for modification of pulmonary vascular disease (Koch et al, Free Radic. Biol. Med. 105:48 (2017)).
  • Angiogenesis and vascular resistance play important roles in the development of certain co-morbidities of prematurity in infants.
  • NEC vascular endothelial growth factor
  • a high intestinal vascular resistance pattern in the superior mesenteric artery has been demonstrated in infants that develop NEC (Murdoch et al, Pediatrics 118(5): 1999 (2006)) as well as low splanchnic tissue oxygenation in animal models of NEC (Zamora et al, PLoS One 10(6): e0125437 (2015)).
  • PH a multifactorial disease that develops in around 20% of extremely preterm infants (Bhat et al, Pediatrics 129(3):e682 (2012)) with a mortality risk up to 48% (Khemani et al, Pediatrics 120(6): 1260 (2007)), is influenced by pulmonary vascular growth and function in the developing neonate. A commonality between both NEC and PH is the involvement of small vessels.
  • NR activity in preterm infants and associations with covariates including co morbidities, exposures, and nutrition may be used for diagnosing and predicting the risk of developing BPD, PH and/or NEC in these infants.
  • the present invention provides a method of diagnosing BPD and/or determining the risk of developing BPD in an infant that is bom at a gestational age of about 28 weeks or less, the method comprising: measuring NR activity (e.g ., nM nitrite formed/10 3 CFU/min) in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks (e.g., about 30, 31, 32, 33, 34 weeks) PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; and diagnosing the infant as having BPD and/or determining the infant to be at risk of developing BPD when no spike (e.g, no increase) in NR activity is observed in the sample taken from the infant at about 29 weeks PMA (as compared to the samples taken prior to 29 weeks PMA and after 29 weeks PMA).
  • NR activity e.g ., nM
  • a method of diagnosing PH and/or determining the risk of developing PH in an infant that is a gestational age of about 28 weeks or less comprising: measuring NR activity (nM nitrite formed/10 3 CFU/min) in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks (e.g, about 30, 31, 32, 33, 34 weeks) PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; and diagnosing the infant as having PH and/or determining the infant to be at risk of developing PH when no spike in NR activity is observed in the sample taken from the infant at about 29 weeks PMA (as compared to the samples taken prior to 29 weeks PMA and after 29 weeks PMA).
  • NR activity nM nitrite formed/10 3 CFU/min
  • a method of diagnosing NEC and/or determining the risk of developing NEC in an infant that is a gestational age of about 28 weeks or less comprising: measuring NR activity (nM nitrite formed/10 3 CFU/min) in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken at birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks (e.g, about 30, 31, 32, 33, 34 weeks) PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; and diagnosing the infant as having NEC and/or determining the infant to be at risk of developing NEC when no spike in NR activity is observed at about 29 weeks PMA (as compared to the samples taken prior to 29 weeks PMA and after 29 weeks PMA).
  • NR activity nM nitrite formed/10 3 CFU/min
  • the present invention provides a method of diagnosing BPD, PH, and/or NEC and/or determining the risk of developing BPD, PH or NEC in an infant that is born at a gestational age of about 28 weeks or less, the method comprising: quantifying the bacterial species (e.g ., the number of viable bacterial counts, and/or measuring the DNA associated with the bacteria) in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks (e.g., about 30, 31, 32, 33, 34 weeks) PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; and diagnosing the infant as having BPD, PH and/or NEC and/or
  • quantifying the bacterial species e.g ., the number of viable bacterial counts, and/or measuring the DNA associated with the bacteria
  • Another aspect of the invention provides a method for guiding the care of an infant that is a gestational age of about 28 weeks or less, comprising: measuring NR activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks (e.g, about 30, 31, 32, 33, 34 weeks) PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; diagnosing the infant as having BPD and/or determining the infant to be at risk of developing BPD when no spike (e.g, no increase) in NR activity is observed at about 29 weeks PMA (as compared to the samples taken prior to 29 weeks PMA and after 29 weeks PMA); and guiding the care of the infant that is diagnosed as having BPD and/or to be at risk of developing BPD by providing one or more suitable therapeutic and/or prophylactic treatments, such as: (a) administering a therapeutically effective amount of oxygen and
  • bronchodilator a steroid, and/or a diuretic
  • administering a therapeutically effective amount of a composition comprising at least one probiotic bacterial species/strain, and/or (e) prolongation of pharmacologic exposure to caffeine, thereby guiding the care of the infant.
  • a phosphodiesterase inhibitor that may be administered to the infant diagnosed with or at risk of developing BPD can include, but is not limited to, sildenafil, prostacyclin, iloprost and/or treprostinil.
  • a bronchodilator that may be administered to the infant diagnosed with or at risk of developing BPD can include, but is not limited to, albuterol.
  • a steroid that may be administered to the infant diagnosed with or at risk of developing BPD can include, but is not limited to, hydrocortisone, dexamethasone, and/or prednisone.
  • a diuretic that may be administered to the infant diagnosed with or at risk of developing BPD can include, but is not limited to, furesomide and/or diuril.
  • a probiotic bacterial strain or species that may be
  • administered to the infant diagnosed with or at risk of developing BPD can include, but is not limited to, a bacterial species/strain in the genus Actinomyces, Rothia, Veillonella,
  • the probiotic bacterial strain or species can include, but is not limited to, a bacterial species/strain in the genus Actinomyces, Rothia and/or Veillonella.
  • a therapeutically effective amount of a probiotic bacterium or composition of probiotic bacteria can be for example a daily dose of probiotic bacterium or composition of probiotic bacteria of about 10 4 to about 10 12 CFU, for example 10 5 to 10 10 , or 10 6 to 10 8 , or 10 7 to 10 9 ,or 10 8 to 10 10 total CFUs of bacteria may be used.
  • a daily dose of one or each bacteria may be around 10 8 or 10 9 total CFUs, e.g., 10 7 to 10 10 or 10 8 to 10 10 or 10 8 to 10 9 .
  • Such doses can be in the form of CFU/g or CFU/unit-dosage form.
  • the present invention provides a method for guiding the care of an infant that is a gestational age of about 28 weeks or less, comprising: measuring NR activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks (e.g, about 30, 31, 32, 33, 34 weeks) PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; diagnosing the infant as having PH and/or determining the infant to be at risk of developing PH when no spike in NR activity is observed at about 29 weeks PMA; and guiding the care of the infant that is diagnosed as having PH and/or to be at risk of developing PH by providing one or more suitable therapeutic and/or prophylactic treatments, such as: (a) administering a therapeutically effective amount of nitric oxide and/or oxygen, (b) administering a therapeutically effective amount of a composition comprising at
  • a phosphodiesterase inhibitor that may be administered to the infant diagnosed with or at risk of developing PH includes, but is not limited to, sildenafil.
  • an endothelin receptor antagonist that may be administered to the infant diagnosed with or at risk of developing PH includes, but is not limited to, bosentan.
  • a nitric oxide precursor that may be administered to the infant diagnosed with or at risk of developing PH includes, but is not limited to, L-arginine and/or nitrate.
  • a prostacyclin that may be administered to the infant diagnosed with or at risk of developing PH includes, but is not limited to, epoprostanol, iloprost, and/or treprostinil.
  • a method for guiding the care of an infant comprising: measuring NR activity in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks ( e.g ., about 30, 31, 33, 34 weeks) PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; diagnosing the infant as having NEC and/or determining the infant to be at risk of developing NEC when no spike in NR activity is observed at about 29 weeks PMA; and guiding the care of the infant that is diagnosed as having NEC and/or to be at risk of developing NEC by providing one or more suitable therapeutic and/or prophylactic treatments, such as: (a) replacing oral feeding with parenteral feeding, (b) removing air and fluid from the stomach and intestine, (c)
  • administering a therapeutically effective amount of a composition comprising intravenous fluids comprising intravenous fluids, (d) administering a therapeutically effective amount of a composition comprising at least one antibiotic, (e) administering a therapeutically effective amount of a composition comprising at least one probiotic bacterial species/strain, and/or (f) administering a therapeutically effective amount of a composition comprising L-arginine.
  • an antibiotic that may be administered to the infant diagnosed with or at risk of developing NEC includes, but is not limited to, vancomycin, tobramycin, flagyl, zosyn, and/or fluconazole.
  • a probiotic bacterial strain or species that may be
  • administered to an infant diagnosed with or at risk of developing NEC can include, but is not limited to, a bacterial species/strain in the genus Actinomyces, Rothia, Veillonella,
  • the probiotic bacterial strain or species can include, but is not limited to, a bacterial species/strain in the genus Actinomyces, Rothia and/or Veillonella.
  • a therapeutically effective amount of a probiotic bacterium or composition of probiotic bacteria can be for example a daily dose of probiotic bacterium or composition of probiotic bacteria of about 10 4 to about 10 12 CFU, for example 10 5 to 10 10 , or 10 6 to 10 8 , or 10 7 to 10 9 ,or 10 8 to 10 10 total CFUs of bacteria.
  • a daily dose of one or each bacteria may be around 10 8 or 10 9 total CFUs, e.g., 10 7 to 10 10 or 10 8 to 10 10 or 10 8 to 10 9 .
  • Such doses can be in the form of CFU/g or CFU/unit-dosage form.
  • a method for guiding the care of an infant comprising: quantifying the bacterial species (e.g, the number of viable bacterial counts, and/or measuring the DNA associated with the bacteria in an oral microbiome sample taken from the infant, wherein a first oral microbiome sample is taken after birth and at least two additional oral microbiome samples are taken thereafter until the infant reaches about 30 to 34 weeks (e.g, about 30, 31, 32, 33, 34 weeks) PMA, wherein one of the at least two additional samples is taken at about 29 weeks PMA; diagnosing the infant as having BPD, PH and/or NEC and/or determining the infant to be at risk of developing BPD, PH and/or NEC when no spike (e.g, no increase) in the amount of specific bacterial species is observed in the sample taken from the infant at about 29 weeks PMA (as compared to the samples taken prior to 29 weeks PMA and after 29 weeks PMA); and guiding the care of the bacterial species (e.g, the number of viable bacterial counts, and/or measuring the DNA associated
  • a method for treating an infant that is a gestational age of about 28 weeks or less for BPD, PH and/or NEC, the method comprising diagnosing the infant as having or at increased risk of having BPD, PH and/or NEC by the methods of the invention and treating the infant for BPD, PH and/or NEC.
  • an oral microbiome sample is taken from the mouth of the infant, optionally the tongue of the infant.
  • the oral microbiome sample may be taken from the posterior dorsum of the tongue of the infant.
  • An oral microbiome sample may be obtained from the infant in any acceptable manner.
  • an oral microbiome sample may be obtained by a swab, tongue scraper, suction bulb, and/or cotton pad.
  • a first oral microbiome sample taken after birth may be taken/obtained from the infant within one minute to about 72 hours, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 min, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36, 42, 48, 54,
  • a range of time for which a first oral microbiome sample may be obtained from the infant may be about 5, 10, 15, 30, 45, 60, 90, 120, 180 min to about 10, 15, 24, 36, 48, 60, or 72 hours after birth, and any range or value therein.
  • a first oral microbiome sample may be obtained at about 78, 84, 90, or 96 hours after birth.
  • a first oral microbiome sample may be obtained from the infant from about 24 hours after birth to about 72 hours after birth, optionally at about 45 hours to about 70 hours after birth.
  • the at least two additional oral microbiome samples are two samples, a second sample taken at about 29 weeks PMA and a third sample taken at about 30 weeks PMA, at about 31 weeks PMA, at about 32 weeks PMA, at about 33 weeks PMA, or at about 34 weeks PMA.
  • the gestational age of the infant will determine the timing and number of samples taken from the infant.
  • an infant having a gestational age of about 28 weeks will have a first oral microbiome sample taken at birth (e.g ., about 1 minute to about 96 hrs after birth) and then a second oral microbiome sample taken at about 29 weeks PMA and at least one further oral microbiome sample taken at about 30, 31, 32, 33, and/or 34 weeks PMA.
  • a first oral microbiome sample taken at birth e.g ., about 1 minute to about 96 hrs after birth
  • a second oral microbiome sample taken at about 29 weeks PMA and at least one further oral microbiome sample taken at about 30, 31, 32, 33, and/or 34 weeks PMA will have a first oral microbiome sample taken at birth and then at least a second sample taken at about 29 weeks PMA (and possibly a sample taken at about 26, 27 and/or 28 weeks), and one or more oral microbiome samples taken at about 30, 31, 32, 33, and/or 34 weeks PMA.
  • the at least two additional oral microbiome samples may be obtained at weekly intervals following the first oral microbiome sample obtained after birth until the infant reaches about 30 to 34 weeks PMA.
  • the at least two additional oral microbiome samples may be taken periodically about every 2 days to about every 14 days ( e.g ., about every 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14 days).
  • a sample when taking a sample at about 29 weeks PMA, may be across a series of consecutive days or every other day for ⁇ 1-5 days from the 29 week PMA time point (e.g., samples may be taken every other day or every day for a set series of days that are ⁇ 1-5 days from the 29 week PMA time point, e.g., if October 23 rd is the actual date of the 29 week PMA for a subject, then samples may be taken on October 18 th (-5), 19 th (-4), 20 th (-3), 21 st (-2), 22 nd (-1), 23 rd (0), 24 th ( ⁇ 1), 25 th ( ⁇ 2), 26 th ( ⁇ 3), 27 th ( ⁇ 4), and/or 28 th (+5)) or any subset thereof (e.g., October 18 th (-5), 19 th (-4), 20 th (-3), 21 st (-2), 22 nd (-1), 23 rd (0); 21 st (-2), 22), 22 th
  • the at least two additional oral microbiome samples are taken about 1 min to about 4 hours post-feeding (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • the at least two additional oral microbiome samples are taken about 2 hours post-feeding.
  • the method further comprises culturing the oral microbiome samples for about 5 min to about 36 hours (e.g, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
  • the method may further comprise culturing the oral microbiome samples for about 6 hours to 30 hours prior to determining (measuring) the NR activity. In some embodiments, the method may further comprise culturing the oral microbiome samples for about 12 hours to 24 hours prior to determining (measuring) the NR activity. In some embodiments, the method may further comprise culturing the oral microbiome samples, for about 18 hours prior to determining (measuring) the NR activity.
  • the culturing is under aerobic conditions. In some embodiments, the conditions for culturing include nutrient rich conditions.
  • NR activity is then tested for NR activity. Any method of measuring NR activity may be used. For example, after establishing baseline nitrite levels in the sample (e.g ., cultured sample). The sample (or portion thereof) may be incubated at about 37°C for about 10 min), nitrate may be added to a portion of the sample (e.g., about 10 m ⁇ of 10 mM nitrate to a 90 m ⁇ sample) and the sample mixed (e.g, by vortex). Nitrite formation may be measure using any method known in the art (e.g, triiodide chemiluminescence as previously described (Pelletier et al, Free Radic Biol Med. 41(4):541 (2006)). In some embodiments, nitrite may be measured by Griess reaction by visible spectroscopy or after HPLC purification. In some embodiments, nitrite test strips may be used.
  • nitrite test strips may be used.
  • kits to carry out the methods of this invention.
  • a kit of this invention can comprise reagents, buffers, and apparatus for mixing, measuring, sorting, labeling, etc., as well as instructions and the like as would be appropriate for measuring NR activity and/or for analyzing the microbes present in an oral microbiome sample from an infant.
  • the invention provides a kit for assessing the risk of developing BPD, PH, and/or NEC in an infant, comprising one or more reagents for measuring NR activity in the cultured samples, with optional instructions for the use thereof.
  • the invention provides a kit for microbial analysis of a microbiome sample taken from an infant that is born at a gestational age of about 28 weeks or less, comprising one or more reagents for identifying the microbes present in the oral microbiome sample, with optional instructions for the use thereof.
  • a kit of the invention may comprise three or more
  • the kit of the invention may comprise markers for identifying at least one of a bacterial species/strain in the genus Actinomyces, Rothia, Veillonella, Lactobacillus, Hemophilus, Staphylococcus, Streptoccocus, Enter occocus and/or Prevolella.
  • Samples were taken from the posterior dorsum of each infant’s tongue using sterile wood Fisherbrand cotton-tipped applicators (Cat. No. 23-400-115) by rotating the applicator 360° over the right posterior tongue dorsum. All samples were collected by the same researcher to minimize inter-sampling variability. Samples were then inserted immediately in 1.5 ml of Brain Heart Infusion Broth (Anaerobe Systems, Morgan Hill, CA) and placed on ice. NR activity was measured two times after sample collection: within 2 hours of collection and after 18 hour incubation under aerobic conditions in BHI at 37°C.
  • Standard curves generated on the day of sampling from a nitrite solution of known concentration were then used for final nitrite calculations.
  • One way ANOVA was performed to discern differences in NR activity over time. Mann-Whitney U testing was used to determine the influence of other co-variates with NR activity.
  • CFU colony forming unit
  • NR activity was measured in 190 samples taken from 28 preterm infants between birth and 34 weeks PMA. For samples taken at birth, the mean time of sample collection was 49 ⁇ 18 h, with a median of 48.5 h of life. The average gestational age of the preterm cohort was 25.6 ⁇ 1.6 weeks with an average birth weight of 728 g ⁇ 218 g (other demographic data are displayed in Table 1).
  • FIG. 1A shows representative traces for nitrite formation measured by triiodide mediated reduction to NO
  • FIG. IB summary data from all samples.
  • significant NR activity was observed if tongue swabs were first cultured for 18 hours, and then nitrate-dependent formation of nitrite assessed (FIG. 1A).
  • No NR activity was evident in swabs exposed to air only (FIG. 1C), suggesting that NR expressing bacteria are present on the newborn tongue, but at low levels.
  • Table 1 Demographic Data from Enrolled Preterm Infants (mean ⁇ SD)
  • FIG. 2A plots NR activity normalized to CFU.
  • Preterm infants born via spontaneous vaginal delivery had higher NR activity at birth compared to those born via cesarean section.
  • Samples taken after 48 hour from delivery had higher NR activity compared to samples taken within 48 hours after delivery. Exposure to antenatal antibiotics did not result in statistically different NR activity at birth in preterm infants (p 0.12).
  • FIGS. 2A-2C indicate an increase in NR activity at a PMA of 29 weeks (FIGS. 2A, 2C) that was not associated with altered bacterial count (FIG. 2B), suggesting an increase in specific activity and/or changes in bacterial composition.
  • FIG. 3 shows the relative abundance of bacterial species at each PMA tested.
  • Nitric oxide synthase dependent and independent mechanisms formation of NO in mammals is mediated by nitric oxide synthase dependent and independent mechanisms. The latter involves nitrate-reduction to nitrite by commensal oral nitrate-reducing bacteria.
  • the nitrite formed provides substrate for various NR systems that mediate NO-signaling by hypoxia and pH-dependent mechanisms (Benjamin et al, Nature 368(6471):502 (1994)).
  • hypoxia and pH-dependent mechanisms Benjamin et al, Nature 368(6471):502 (1994)
  • Nitric oxide is a primary vasodilator of the intestinal vasculature (Reber et al, Am. ./.
  • endothelial nitric oxide synthase an enzyme responsible for local NO production within tissue vasculature.
  • eNOS endothelial nitric oxide synthase
  • mice This underscores the low abundance of NR bacteria in the oral cavity from preterm infants and is similar to our prior studies investigating oral NR activity in adult mice (Ahmed et al, Nitric Oxide 66:62 (2017)). We note that in mice, which have similar low NR activities, a nitrate- and oral microbiome dependent signaling has been demonstrated (Ahmed et al, Nitric Oxide 66:62 (2017)).
  • the oral NR activity measured here may not directly relate to any nitrate-dependent activation of NO-signaling cascades, and how much nitrite is needed to elicit NO- dependent effects in the newborn is also unknown.
  • inhaled NO therapy for persistent pulmonary hypertension of the newborn previously thought to have limited effect systemically, doubles serum nitrite levels and increases nitrate levels four fold (Ibrahim et al, J Pediatr. 160(2):245 (2012)). While these levels still remain low, they have been demonstrated to still influence systolic blood pressure in adults (Webb et al, Hypertension 51(3):784 (2008)).
  • NR activity may simply be negligible at birth prior to oral microbiome establishment and serum nitrate and nitrite levels may initially better reflect maternally derived levels.
  • serum nitrate and nitrite levels may initially better reflect maternally derived levels.
  • Streptococcus which was also noted to be associated with NR activity, does not produce NR. However, the relative abundance of Streptococcus has been shown to be as high as 89% in isolates with high NR activity compared to 72% in isolates with low NR activity (Hyde et al, PLoS One 9(3):e88645 (2014)), which may support its role as a commensal organism.
  • microbial contribution of maternal human milk compared to other forms of nutrition such as donor breast milk (which requires pasteurization), formula, and parenteral forms of nutrition may be yet another benefit.
  • These microbiota are of particular importance for NR activity, as it is microbial production of NR allowing bioactivation of nutritional and salivary nitrate.
  • the relative increase in NR activity occurring around 29 weeks PMA may also have physiologic implications for intestinal growth, handling of enteral nutrition, and potentially gastrointestinal pathology.
  • the gastrointestinal tract of the preterm infant has immature motility and an incomplete ability to enzymatically digest and absorb food substrates
  • NO has the largest amount of safety data and is approved for use in term infants with persistent pulmonary hypertension of the newborn.
  • Nitric oxide has been well described as a mediator of pulmonary vascular homeostasis.
  • NO leads to smooth muscle relaxation in pulmonary vasculature via activation of cyclic guanosine monophosphate.
  • pulmonary hypertension there is an incomplete response to signals normally responsible for vasodilatation as well as reduced NO signaling (Tonelli et al, Pulm. Circ. 3(1):20 (2013)).
  • eNOS knockout mice restoration of the gene through an adenoviral vector leads to improved pulmonary arterial pressure (Champion et al, Proc. Natl. Acad. Sci.

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Abstract

L'invention concerne des méthodes pour diagnostiquer ou évaluer le risque de développer une maladie pulmonaire chronique et une maladie intestinale chez des nourrissons prématurés par mesure de l'activité de nitrate réductase dans des échantillons de microbiome oral prélevés chez le nourrisson. L'invention concerne en outre des méthodes pour orienter les soins de nourrissons à long terme sur la base de la mesure de l'activité de nitrate réductase dans des échantillons de microbiome oral prélevés chez le nourrisson.
PCT/US2020/039732 2019-06-27 2020-06-26 Méthodes de diagnostic et de prédiction d'une maladie pulmonaire chronique et d'une maladie intestinale chez des nourrissons prématurés WO2020264239A1 (fr)

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Citations (5)

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US20110144041A1 (en) * 2004-03-12 2011-06-16 Mti Meta Tech Inc. Methods for Treating Inflammatory Bowel Disease
US20140349956A1 (en) * 2012-01-20 2014-11-27 University Of Florida Research Foundation, Incorporated Materials and methods for detecting and/or predicting necrotizing enterocolitis in infants
US20170159108A1 (en) * 2014-05-06 2017-06-08 Is-Diagnostics Ltd. Microbial population analysis
US20170212133A1 (en) * 2014-06-20 2017-07-27 Ino Therapeutics Llc Method of Identifying and Treating Premature Infants at Risk for BPD

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6210918B1 (en) * 1992-10-09 2001-04-03 The Regents Of The University Of California Non-invasive method for detection, diagnosis or prediction of term or pre-term labor
US20110144041A1 (en) * 2004-03-12 2011-06-16 Mti Meta Tech Inc. Methods for Treating Inflammatory Bowel Disease
US20140349956A1 (en) * 2012-01-20 2014-11-27 University Of Florida Research Foundation, Incorporated Materials and methods for detecting and/or predicting necrotizing enterocolitis in infants
US20170159108A1 (en) * 2014-05-06 2017-06-08 Is-Diagnostics Ltd. Microbial population analysis
US20170212133A1 (en) * 2014-06-20 2017-07-27 Ino Therapeutics Llc Method of Identifying and Treating Premature Infants at Risk for BPD

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