Nitric Oxide in Bronchiolitis

אשר טל' פרופ

Nitric oxide (NO)

Nitric Oxide (NO)

A small lipophillic free radical gas molecule

Endogenous produced by various cell types

Has a short half-life in the circulation

NO and its related reactive intermediates:

Anti-microbial abilities – acting in multiple

mechanisms

An immune regulator – affecting innate and

adaptive immunity

Nitric Oxide Synthesis

NO is catalyzed by nitric oxide synthetases (NOS) which

produce NO during the oxidation of L-arginine into citrulline.

eNOS – endothelial NOS

nNOS – neuronal NOS

iNOS – inducible NOS

iNOS

expressed mainly in immune cell

Up-regulated during inflammation (involving IL-1ß, TNFà,

IFNg, or LPS or lipoteichoic acid

The role of NO in immuno-regulation

At low concentration NO

Regulate vascular tone and blood pressure

Anti-oxidative

Anti- coagulative

Anti-apoptotic

NO exerts multiple regulatory actions on immune cells

Inhibit mast cell activation

Regulate natural killer cells function

Enhance or inhibit neutrophil activation

At high concentration, NO poses anti-microbial, anti-viral, and anti-fungal activity in multiple mechanisms:

NO lipophillicity

NO instability in an oxygen environment promotes the formation of toxic intermediates

NO is able to bind or alter proteins and enzymes

NO acts at multiple compartments: intracellular and extracellular

BacteriaGramstaining

Latent Period*

(hrs)-2.5 Log10

(hrs)LD100(hrs)

S. aureus ( ATCC) Positive 3 3.3 4

P. aeruginosa (ATCC) Negative 1 2.1 3

MRSA Positive 3 4.2 5

Serracia sp. Negative 4 4.9 6

S. aureus (Clinical) Positive 3 3.7 4

Klebsiella sp. #1 Negative 3 3.5 6

Klebsiella sp.#2 Negative 2 4.1 5

Klebsiella sp. #3 Negative 3 5.1 6

S. maltophilia Negative 2 2.8 4

Enterobacter sp. Negative 4 5.3 6

Acinetobacter sp. Negative 4 5 6

E. coli Negative 3 4.2 5

Group B Streptococci Positive 1 1.5 2

Average N/A 2.77 3.82 4.77

SD N/A 1.01 1.17 1.30

Mycobacterium smegmatis Positive 7 9.2 10

In vitro Antimicrobial Activity

• Nitric oxide-producing salts inhibit replication cycle of the SARS virus (Akerstrom, et al. J. Virol. 2005, Chen, et al. 2004. Clin Infect Dis.) andInfluenza A (Rimmelzwaan J Virol 1999.).

• Gaseus Nitric oxide-reduces Influenza infectivity. (Regev-shoshani,

Nitric Oxide 2013.).

Gaseous nitric oxide reduces influenza infectivity in vitroRegev-Shoshani et al. / Nitric Oxide 31 (2013) 48–53

RSV

NITRIC OXIDE

Agriculture and Agriculture etAgri-Food Canada Agroalimentaire Canada

Paired t-test p < 0.01

IRT

Tem

pera

ture (

0F

)

101

101.5

102

102.5

103

103.5

104

104.5

1 3 4 5 6 9 10

Day

NO Started NO Started

Fig. 6: Effect of NO given Prophylactically or

at the time of IRT Early Detection on

IRT Temperatures

Paired t-test p < 0.01

0

1

2

3

4

5

6

7

1 3 4 5 6 9 10

NO StartedNO Started

Day

Fig. 5: Effect of NO given Prophylactically or at the

time of IRT Early Detection on clinical

Infection score (industry standard signs)

Cli

nic

al

Sco

re

Phase I – Human subjects

Safety and Tolerability of Nitric Oxide Given intermittently via inhalation to infants with acute bronchiolitis – A

prospective, double-blind, randomized, controlled trial (Phase IIa)

Objectives

To assess the safety and tolerability of nitric oxide inhalation treatment

To assess the efficacy of nitric oxide inhalation treatment compared to supportive treatment (O2)

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Bronchiolitis– Study Design

Inclusion criteria

Age 2 to 12 months

Clinical Score >6 to 10

Parents signed an informed consent

Exclusion criteria

Post prematurity (<36w)

Known CLD

Genetic disease, CHD

Bronchiolitis– Study Design

Nitric oxide formulation delivered via inhalation

5x/day for 30 minutes

Two treatment groups (1:1)

Group 1 –Treatment group - receives intermittent (5 x 30 minutes, a day) inhalation of 160 ppm NO in addition to supportive treatment (O2 ), for up to 5 days

Group 2 – Control group -receives ongoing inhalation of supportive treatment (O2 )

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Stage 2: Study (NO/control) treatment

Stage 1: Screening +

Randomization

Prospective, Double Blind, Randomized Single-Center,

Evaluation of the Safety and Tolerability of Nitric Oxide Given

Intermittently via Inhalation to infants with Bronchiolitis

1st day 1st day-up to 5th day 14th day 21st day

Visit 1

Stage 3: Follow-up

• Clinical score assessment

• NP and OP Swabs for

bacterial carriage • NP Wash for RSV

• Physical exam

• Vital signs

• NO 160ppm /control x5/day

• Clinical score assessment

• Vital signs

• Physical exam

• NP Wash for RSV

• Safety data

• Clinical evaluation

• Questionnaire

30th day

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Primary End points- Safety

To determine the % MetHb associated with inhaled nitric oxide

To determine adverse events associated with inhaled nitric oxide

Secondary End points- Efficacy:

To compare the Length of hospital stay (LOS) in days

To compare the Rate of clinical score improvement

To compare the Length of oxygen treatment in hours

End points

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Clinical Score (Modified from Tal 1983)

Score: mild, <5; moderate, 6-10; severe, 11-12

ScoreRespiratory Rate

WheezingSaO2

(room air)Accessory

muscle use< 6 mths ≥ 6 mths

0 40 30 None ≥95% None

141-55 31-45

End expirationWith Stethoscope

92-94% +

256-70 46-60

Insp. & ExpirationWith stethoscope

90-92% ++

3>70 >60

Audible without Stethoscope

≤89% +++

Supportive O2 Tx

(n = 22)

NO + Supportive Tx

(n = 21) Demographics

14/813/8Male / Female

5.5

2.0 – 11.9

4.1

2.0 – 8.7

Age (months)

Median

Range

6.5 (0.7)5.35 (3.3)Weight (Kg), Mean

(SD)

39.3 (1.1)38.9 (1.6)Gestational Age,

Mean (SD)

NO in Bronchiolitis - Results

Supportive Tx

(n = 22)

NO+supoprtive Tx

(n = 21)

Viral PCR

Nasal wash

1617RSV

22RSV + corona

22hMPV

1AdenoV +Influenza A

2None

Results - Nasal wash

Supportive Tx

(n=22)

NO+Supportive Tx

(n=21)

Severe Adverse

Events

10Respiratory Failure

11Pneumonia

20Acute Otitis Media

02Diarrhea

01Prolonged fever

Results - Safety

NO in Bronchiolitis- Safety

20

NO in Bronchiolitis - Safety

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Mean Methemoglobin (%) levels before and after treatment

Results- Tolerability

A total of 156 NO inhalations were given (Mean 7.4 , Range 2–16)

4 patients prematurely discontinued treatment

• 2 non cooperative parents (1 in each group),

• 1 was transferred to PICU (respiratory failure) - Control group

• 1 with 2 episodes of Met-Hb >5% - NO group

NO in Bronchiolitis- EfficacyPreliminary Results

24

All data presented in this presentation is addressedto modified intention to treat (mITT*) population –

40 patients that completed the study.

Results - Length of hospital stay (LOS)

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Kaplan-Meier analysis: Length of hospital stay (LOS in hours)

Results - Length of hospital stay (LOS)

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Kaplan-Meier analysis: Length of hospital stay (LOS in hours)

Length of hospital stay (LOS)

27

LOS (in hours) was calculated from O2 start inhalation to removal from study/ discharge from hospital

LOS

(hours)

NO and

supportive

treatment (O2)

Supportive

treatment

(O2)

P-value

N 19 21

0.1153

Mean 42.36 55.73SD 17.48 42.78

Min 14.50 16.00Median 45.08 42.45

Max 74.25 169.78

Change in clinical score

Mean Clinical Score Changes by Assessment and Treatment

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Change in clinical score

Kaplan-Meier analysis: Change in clinical score (by assessment #)

Change was defined as the decrease of at least 2 points from baseline in clinical score

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Time to achieve 92% saturation

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Time (days) to achieve 92% saturation

NO and supportive

treatment (O2)Supportive treatment (O2) P-value

N 21 22

0.1426

Mean 3.12 4.27SD 1.89 3.01

Min 1.20 1.10Median 2.20 2.30

Max 8.00 10.00StdErr 0.41 0.64

Time to achieve 92% saturation*

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Kaplan-Meier analysis: Time to achieve saturation >92%

*Equal to O2 treatment

Time to achieve 92% saturation*

32

Kaplan-Meier analysis: Time to achieve saturation >92%

*Equal to O2 treatment

Mean change in body temperature by clinical assessment showing the decrease in body

temperature for subjects in both groups.

Change in body temperature (0C)

Conclusions

Intermittent high-dose NO inhalations is safe and

tolerable in infants with acute bronchiolitis

Efficacy – preliminary data on small number of

patients show promising signals of efficacy…

To be continued…

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