throbber

`
`ENE
`
`DR
`
`EAL AT
`
`AD
`
`ARH
`
`APPLICATI! IN N! l M BER: NDA 20845
`
`PHARMA
`
`L
`
`Y
`
`IE
`
`
`
`

`

`2' MCDONALD
`
`DCTIBIQQT
`
`1
`
`REVIEW AND EVALUATION OF PRECLINICAL PHARMACOLOGY
`AND TOXICQFOGY:_‘-.DATA
`
`NITRIC OXIDE
`0h_meda Pharmaceutical Products Division Inc
`110 Alle'ri" Road
`Liberty Corner, NJ 07938-0804
`
`Narendra B. Oza, PH. D.
`October 9, 1997
`
`
`
`5.
`
`
`
`

`

`TABLE or CONTENTS
`
`Safety Pharmacology:
`
`7
`
`'
`
`'
`
`1.
`
`‘ 2.
`
`Acute cardiovascular evaluation of NO in the dog. Has'sler CR et al.
`1994. (Report RDR-0064-DS)
`*
`-
`'
`
`Eiectrocardiographic evaluation of N0 in the dog. Hassler OR at al.
`1994. (Report RDR-0087-DS)=
`_
`._.-.-
`
`Pharmacokinetics:
`
`.
`
`_
`
`7
`
`3.
`
`Phannacokinetic modeling of methemoglobin concentration--time
`data in normal dogs inhaling 80, 160, 320 or 640 ppm nitric oxide.
`Wilhelm JA, 1996.
`(Report RDR-0075-DD)
`
`'
`
`Toxicology:
`
`'
`
`‘5;
`
`
`
`-
`
`‘
`‘
`
`:—
`
`4.
`
`5.
`
`6.
`
`7.
`
`8.
`
`9.
`
`Page
`
`5 ,
`
`7
`
`8
`
`10
`
`13
`
`13 .
`
`14
`
`18
`
`7-day range-finding study of nitric oxide in the rat via inhalation,
`Hassler CR et al. 1994 (Report RDR-0062-DS)
`
`Pathology: 7-day range-finding study orfvznitric oxide (NO) in the rat
`via inhalation, Tofl JT and Singer AW, Dec. 1996 (Supp.#SCQ40063)
`EM study: 7-day range-finding study of nitric oxide in the rat by
`inhalation. Mann P. et al. August 1996 (RDR-0149-DS)
`
`-
`
`28-day exposure with recovery of nitric oxide in the rat by inhalation,
`Hassler OR et ai, 1994, (RDR-OOBS-DS)
`
`Pathology: 28-day exposure with recovery of nitric oxide in the rat via
`inhalation, Singer AW., December 1996. (RDR-0152-DS)
`
`EM study: 28-day exposure with recovery of nitric oxide in the rat by
`inhalation. Mann P.. et al. August 1996 (RDR-0150-DS)
`
`18 7
`
`4
`
`Review-of Pertinent Literature
`
`Summary and Recommendations
`
`-
`
`20
`
`27
`
`
`
`

`

`
`
`1NDA #2o,a45
`
`OCT I 0 I997
`
`REVIEW AND EVALUATION OF Preclinical PHARMACOLOGY
`‘
`-
`AND TOXICOLOGY DATA
`
`.'- Na'rendra B. Oza, PH. D.
`' October 9. 1997
`
`‘-
`
`--
`
`ORIGINAL NDA:
`
`20,845
`
`w-
`
`CENTER RECEIPT DATE:
`REVIEWER RECEIPT DATE. ---
`
`1‘
`
`.Ignéftjljtéé? ‘
`-'=~lf:.}~..uulyi 1, 1997
`
`SPONSOR:
`
`I
`
`‘-
`
`OhmedaInc.,Pharmaceutical Products Div.
`110 Allen Road, PO Box 804, Liberty Corner,
`NJ 07938 (908) 604 7722
`
`DRUG PROPRIETARY NAME:
`
`l-NOTM (Nitric Oxide)
`
`‘* __
`
`GENERIC NAME/CODE NAME:
`
`N IA
`
`STRUCTURE I NATURE:
`MOLECULAR WEIGHT:
`FORMULATION:
`
`._
`"
`
`I
`
`N=Qj Evanescent gas
`30
`Formulated in 100, 400 and 1600 ppm nitric
`
`-
`
`oxide with nitrogen (N2) as the balance
`
`RELEVANT IND'S:
`
`.
`
`F
`
`_
`
`L’
`
`'
`
`'-
`
`'
`
`I J
`
`_JN|H IND for reSpIratory hypoxia
`
`. PHARMACOLOGICAL CLASS:
`
`Selective Pulmonary Vasodilator
`
`INDICATION:
`DOSAGE:
`
`'
`
`"
`
`Reapiratory Hypoxia of the newborn
`5-30 ppm for up to 14 days
`
`MODE OF ADMINISTRATION:
`
`Inhalation -
`
`
`
`

`

`
`
`:Pharmacodynamlcs:
`
`Amman;
`
`in
`NO is an endogenous. potent vasodilator substance. After its production , e.g.,
`vascular endothelial cells. the NO diffuses intosmooth muscle cells. binds with the
`heme moiety of soluble guanylate cyclase and thus activates the enzyme. This
`enzyme increases intracellular cyctic guanosine 3',5'-monophosphate (cGMP) from its
`precursor, guanosine triphosphate (cGTP). The resulting increase in intracellular
`cGMP concentration leads to vasodilation.
`
`,wr'
`
`B.m ‘
`
`‘1‘
`
`NO. synonymous with EDRF (Endoth‘elium-Derived Relaxing Factor). is biosynthesized
`from L-Arginine by NO-Synthase primarily in endothelial cells.
`It
`is now becoming
`apparent that macrophages. neurons and many other cell types may also have a
`capability for local production of NO.
`
`NO is an evanescent compound with half life of 41 seconds. NO is known not to be
`transported in the vascular bed and thus actions and interactions of NO are short term
`and local; NO has great affinity for hemoglobin. Following a very rapid binding with
`NO, haemoglobin is converted into methemoglobin (MHB) and the NO is inactivated.
`MHB is an abnormal, nonfunctioning ferric hemoglobin that is incapable of binding
`oxygen or carbon dioxide. Since MHB is unable to bind or release oxygen. any
`increase in its concentration can lower oxygen saturation in the blood and thus can
`become fatal. MHB level of 70% is usually fatal although a survival has been reported
`even after 81% level of MHB. The major enzyme responsible for the reduction of MHB
`is the NADPH dependent MHB reductase which is present in the erythrocytes.
`Spontaneous reduction of MHB is generally slow and methylene blue can serve as a
`cofactor by donating an electron and thereby increasing the amount of available
`NADPl—l.
`In vivo increase of NADPH can greatly accelerate the reduction of MHB.
`
`The sponsors have proposed to monitor MHB levels and to terminate the inhalation of
`NO if the MHB level should exceed 5%.
`
`.
`
`
`
`

`

`G. Animal §tudies on Inhaled N9;
`
`assler R et al
`'nhalation
`t e do vi
`0 NO i
`1. Acu e cardio ascular evaluati
`d
`e 0 #SC 4 0
`4-D 'Ba
`eLabs
`19 4 Ohmeda
`o
`DFi-
`Location of Data:
`Vol. 2.8, p. 634 (final report) and Vol. 2.5. p. 42 (summary)
`
`The purpose of this study was to evaluate in anesthetized laboratory beagles the
`physiological. pharmacological and toxicological effects resulting from acute
`inhalation of nitric oxide (INO). Methods:
`In companSOn with control dogs exposed to
`21% 02 in N2, the effects of 6 hr inhalation of 80 - 640 ppm NO were evaluated in
`groups of anesthetized. instrumented (inclusivelofecardiac catheterization) dogs. In
`addition, animals were studied at 80 pprrtzusi'ngythe same exposure device intended
`for clinical use. Continuous data were collected during exposures for systemic arterial
`and left ventricular pressures-minutetyolume, tidal volume, respiratory rate, pulmonary
`resistance. pulmonary compliance .and pulmonary arterial pressure. Hourly
`determinations wer‘e made for cardiac output, ECG, MHB concentration and arterial
`blood gases. The concentration of NO and N02 were measured at the animal and the
`levels were as shown in the following Table 1.
`
`Table 1: Protocol design of the Test Groups
`
`
`
`Results:- The first physiologic response to NO exposure was dose-dependent, hourly
`increase in MHB concentration as shown in Table 2. This increase was concentration
`and duration related and statistically significant. p < 0.05, at 320 and 640 ppm.
`
`
`
`
`
`

`

`
`
`Table 2
`.
`Group average percent Methemoglobin
`
`
`
`-----m
`-—---m -
`
`
`
`One of the three dogs treated with 640 ppm NO died with electrocardiographic
`changes. The ECG changes, found in all the NO-treated animals, consisted of Left
`ventricular depolarization (2 dogs 80 ppm and 1 at 160 ppm), Sinus tachycardia (3
`animals), Junctional rhythm (2 animals at 80 and 160 ppm) and R on T phenomena (1
`animal) at 640 ppm. The data suggested ventricular irritability and sinus tachycardia in
`dogs exposed to NO.
`
`Arterial oxygen tensions (P302) declined throughout the exposure period and were
`significantly different from controls after 2 hrs exposure in the 320 and 640 ppm
`groups. Minute volume increased as a function of increased respiratory rate in the 320
`& 640 ppm groups in an apparent response to the decreased oxygen tension.
`Systemic pressures decreased late in. the experiment in the 320 & 640 ppm groups. A
`trend towards increasing heart rates was observed in all the dose groups, including 80
`& 160 ppm.
`,
`
`
`lead to
`Conclusions Occurrence of premature ventricular depolarization (PVD's)
`further investigate the potential of inhaled nitric oxide to cause the PVDs.'The earliest
`response amongst many physiological changes was in the test-drug-dependant
`production of MHB; a concern which is prominent throughout this submission.
`
`
`
`

`

`
`
`:g. Electrocardiographic evaluation of HQ in the dog yia inhalation. Hassler OR at a[,
`da
`e
`-
`-
`e abs
`tu
`eo #N 1
`1A
`Location of data: Volume 2.9, p. 804 (report) and Volume 2.5, p. 49 (summary)
`
`This was a follow-up study to determine it IMO produces abnormalities of cardiac
`conduction in normal unanesthetized dogs and to re-determine if the previously
`observed PVDs were incidental to anesthesia and cardiac catheterization.
`
`Methods
`Six beagles, alsex, were surgically implanted with ECG radiotelemetry
`transmitters and tracheal fistulas such that -the. conscious animals coutd be
`administered air or test drug during Spontaneous breathing while sling restrained
`them for up to five hours. ECG data were continuously collected during the treatment
`which lasted five hours . Following a baseline obtained with ambient air. the animals
`were treated for the first hour with‘1a-cohtrc5lle'd'air mixture of 21% 02 and 79 % N2.
`Blood hemoglobin and 'MHB"were=Tneasured hourly. Clinical observations and body
`weights were recorded regularly. All' animals received nine treatments. 4 hrs each,
`which included: ambient air baseline. air control, and treatments with lNO at 40-.320
`ppm.
`In one of the treatments (No. 2) a malfunction was found in the NO delivery
`system éwhich resulted in a higher delivery of NO to test animals. Estimates of WC
`delivery were. therefore. calculated for treatment no. 2. The last 320 ppm treatment
`was preceded and followed by controiled air treatment. Two animals were treated per
`day with at ieast one week between treatments.
`
`
`Fiesults With one exception (this animal had a prior abnormality. of Wolf-Parkinson-
`White syndrome). INO up to 320 ppm did not affect cardiac conduction, rate or rhythm
`in normai. conscious. spontaneously breathing dogs including the treatment group
`no.2 in which instrument malfunction was noticed. The PVDs observed in the previous
`study were not foUnd under these experimental conditions. The sponsors conciuded
`that INO was unlikely to cause cardiac abnormalities.
`-
`
`the sponsor 'noted that: “the blood MHB
`Regarding methemoglobinemia,
`concentrations increased with increasing INC-exposures above 80 ppm with
`magnitude of the increases similar to those seen in the previous study”. In fact, further
`calculation of the data reveal that animals exposed to 40 ppm NO also appear to
`increase MHB concentration. Although the increase in MHB caused by 40 and higher
`ppm of NO does not appear to cause apparent lung damage (see study no. 7), there
`are proponents that any level of MHB may be undesirable. The sponsor has defined
`severe methemoglobinemia at 740% MHB.
`In neonates 5% MHB was considered
`clinically significant. ltappears that there may be a fair degree of tolerance of MHB
`due to improvements in perfusion and recovery of collapsed lung tissue. Furthermore,
`methemoglobinemia can be reversed by termination of INO and/or by iv infusion of
`vitamin C or methylene blue. Despite these favorable options. it appears prudent to
`abide by the recommendation of the sponsor to monitor MHB constantly and terminate
`lNO when MHB level reaches 5%, particularly in view of the fact that the treatment is
`intended for PPHN.
`
`
`
`

`

`bommem; Excepting the facts that the 320 ppm dose was cushioned with pre and
`post inhalation of air and that only two animals were used per dose. the data is
`agreeable with a notion that the physiological abnormalities observed in the previous
`experiment were related to anesthesia and catheterization. Blood MHB concentration
`of dogs treated with 40 ppm lNO was not different from the controls suggesting a
`reasonablesafety of low dosages.
`'
`.
`-
`
`APPEARS THIS WAY
`0N ORIGINAL-'4'
`
`“mun-wwu-w-u»
`
`m"itr'srx'w’W-t'w‘a'm‘-'M"''-‘~"‘1
`
`
`*"tp-rtrihuHEN”#5154.."
`
`I{v-t,3"i‘plfi1"_v',.'-'.‘
`
`

`

`fharmacokinetics:
`
`ai
`o
`cen raion--irne data in
`c
`lobi
`e e
`od l'
`aco i etic-
`3 '
`dogs inhaling so. 150, 320 or me ppm nitric oxide. Wilhelm 43, 1995'.
`(Ohmeda PPD
`meat-amp)
`
`Location of Data: Vol. 2.8, p. 756- (report) and Vol. 2.5, p.264 ( summary)
`The purpose of this follow-up (see cardiovascular evaluation in # 1) study was to
`describe phannacokinetics of inhaled nitric oxide in dogs. The animals of this study
`are exactly the same as those repoged in review #1:?
`
`Methods: The data listed in table 2 “(of review titf'Were utilized to derive a model :
`1
`,.
`.
`I“. I...
`. .~‘-'.
`
`Vm CNO
`1
`
`,
`
`-
`-—-------'-' 0N0 (1' e'ke'l) + Chase
`
`C =
`
`‘1
`
`"
`
`where C = percent MHB. 0N0: NO exposflre concentration, K.3 = MHB elimination rate
`constant, Vm & Km = rate of MHB formation and Chase = baseline MHB level.
`
`flesults: Using this model and the kinetic data of table 2, the following parameters
`were estimated:
`
`0.0038
`
`Half-life (min)
`
`'
`
`Model-predicted, steady state percentage of MHB (Css) and MHB formation rates
`were:
`.
`
`Ppm NO exposure
`
`Predicted CSS (%MHB)
`
`
`
`
`_-_-m
`_-_m
`
`Rate of MHB increase
`_
`(%/min)
`
`
`
`
`
`

`

`Based upon the estimated elimination half—life of approx. 3 hrs, the time to reach
`steady state %MHB was approximated at 12 - 15 hrs. In a similar study performed in
`human volunteers, elimination half-life was approximated at 1 hr and the time to reach
`steady state was approximated at 4-5'hrs.
`-
`'
`
`Conclusion; These data are obtained from the plasma samples of study #1 which
`was “discarded”'because of artetact arising from anesthesia and oatheterization. Thus
`the kinetic data. as well, can not be conveying any-thing meaningful.
`
`w
`
`-
`
`.
`
`:.-..‘
`
`APPEARS tHIsiwAv
`on ORlGINAL
`
`10
`
`
`
`

`

`:Toxioology:
`
`4
`
`-da
`
`ran e-fi di
`edP
`
`ia'n laio
`9
`“Mo oxide'
`3 d of
`o D-
`-'ae absu
`
`assle C etal
`eort#S 40 3.
`
`Location of Data:
`
`.
`
`Study No./ Date:
`GLP Compliance:
`Animals:
`
`Volume 2.6, p. 283 (repc'lrt); Volume 2.5, p. 139
`(summary)
`_
`-
`Report No. RDR-IbOBD7-Dé‘; November. 1994
`Yes
`_-__-_’=-_"-::-"'
`L:-
`-._ M'IFZSprague-Dayvmy Rats/SD:CDBFi
`
`“:3; -‘
`
`Administration:
`
`=
`
`inhalation
`
`bosageé
`
`Groups:
`
`'
`
`'
`
`o (20% 02 + 80%‘N2 ), so. 200, 300. 400 or 500 ppm
`
`NO + 21% 02 + Balance of N2
`
`Rats were exposed for 6 hrs/day at approximately the
`same time each day for periods of 1. 3, or 7
`consecutive days at 5 concentrations of NO and air
`control ( Stime points, 2 sexes, 5 rats/sex/time point
`and 6 treatments) as follows:
`
`. _
`
`
`
`
`Dose
`
`Air control
`GL1
`
`80 ppm
`Gl‘.2
`
`200 ppm
`Gr.3
`
`' 300 ppm
`GL4
`
`11
`
`Animals
`M
`E
`5
`5
`5
`5
`5
`5
`
`5
`5
`5
`
`5
`5
`5
`
`5
`5
`5
`
`'5
`5
`5
`
`5
`5
`5
`
`5 _
`5
`5
`
`Treatment
`Date
`1
`3
`7
`
`1
`3
`7
`
`1
`3
`‘ 7
`
`1
`3
`7
`
`
`
`

`

`Observations:
`
`‘ Results:
`
`-
`
`"its ,
`
`g.
`
`400 ppm
`Gr.5
`
`500 ppm '
`Gr. 6
`'
`
`-
`
`5
`5
`5 _
`
`5
`5
`5
`
`5
`5
`5
`
`5
`5
`~ 5
`
`1
`3
`7
`
`1
`3
`7
`
`Rats were observed for morbidity and mortality twice
`daily throughoUt the 7 days. Body weights were
`recorded once pregstudy and on days 1, 3 and 7 of
`the’ exposures-periods. A gross necropsy was
`performed on all surviving animals on days 2. 4 and
`{Bia‘n‘dfififief the last exposure of each dosing phase.
`A complete necropsy was performed on animals
`found dead or moribund. Histopathology was
`performed on tissues of the respiratory tract.
`
`Moflaiim There were no modalities in air control, 80
`ppm and 200 ppm groups. All the animals died on
`Day 1
`in the groups treated with 400 and 500 ppm
`NO. Twenty six animals of the 300 ppm group died
`during days 1 & 2 and the four surviving were
`sacrificed on termination date. The survivors had
`cyanotic or bi’uish ears. nose and feet.
`
`Clinical observations: Exposure-related discoloration
`(bluish tint) of the skin in the 300 ppm _gr and red
`nasal discharge were noted in all the drug-treated
`animals.
`
`of
`ercentage
`Meihemogiobinemia; P
`MHB concentration of all the groups are shown in
`the following table no. 3.
`Table 3
`
`Percent Methemoglobin:
`
`
`
`

`

`has:
`
`M
`
`From this data the sponsors concluded that “80 ppm
`animals were not distinguishable from the control".
`Although it appears that way, one can not ignore the
`high-reading of 3.7 % MHB obtained with 80 ppm NO
`on day 7 (see table 3). Thus it seems that 80 ppm is
`alright for 3 days but not;fo‘r.7 days. The NO-induced
`methemoglobinemia was associated with increases
`in blood urea nitrogen...potassium and aspartate
`amino transferase, particularly in animals treated at
`ppm of 300‘&_ higher.
`There weré‘lsporadic changes (decrease in total
`protein,‘ increase in blood glucose,
`increase in
`tore'altininef-etc) in some parameters which did'not
`establish a trend and could not be related to
`treatment.
`_
`-
`Light Microscopy: Several rats had a focal-multifocal,
`minimal inflammatory foci andlor microgranulomas in
`the lung ‘parenchyma. Since these changes‘were
`found also in the controls, the sponsors concluded
`that these changes were random. spontaneous and
`unrelated to agent exposure.
`9mg; The sponsor attributes most of the
`toxicity of N9 to the formation of MHB. There are no
`fewer than a dozen different excitatory agents
`(histamine, prostaglandins, bradykinin, SRS etc)
`thought to act as humeral mediators for the onset or
`reversal of pulmonary inflammation. None of them
`are either measured or taken into account. Although
`the sponsor suggests that “dosages higher then 80
`ppm promote methemoglobinemia in SD rats”, the
`
`
`
`
`
`
`
`
`
` 5. 7-da ' an e-findin 5 ud' ' ' ' ' " ' '
`Singer AW, December 199;: Einal Begort [Ohmeda EPD Beport RDB-let-DS;
`Supplemental Repofi to Bagelle study #SQ24OOQS),
`'
`Location of data: Volume 2.9, p. 936 & Volume 2.5, p.149.
`
`This is an Anatomic Pathology part of the toxicology study outlined in the preceding.
`
`Extemal carcass and internal organs were examined with a particular attention to the
`respiratory tract. The lungs were examined for. petechiae. macroscopic foci of
`inflammation, edema and possible discoloration due to methemoglobinemia.
`
`13
`
`

`

`Abnormal morphology, oral and nasal passages and evidence of exudate, foam or
`edema were also recorded. Begum; No insult was found in the epithelium of the
`upper respiratory tract which is more vulnerable to inhaled toxicants. Light microscopy
`did not reveal loss or damage to cilia and ciliated cells. No evidence of toxicity or
`alteration was found in the mast cells. There was also no evidence of toxicity in
`terminal bronchioles. Qommegr;
`It is somewhat surprising that the respiratory tract was
`unaffected despite a 100% mortality and a high degree of methemoglobinemia found
`at higher dosages.
`Is it possible that light microscopy-is unable to detect these
`changes ? Fortunately an answer was found in the EM data! which follows.
`
`6. Electron. microscopy study [epoch 7-
`’ a b i hala io
`a
`e a
`
`Id: 4‘
`Location of Data: Volume—2.9, p‘. 8775(report) & Volume 2.5, p. 152 (summary .
`
`This study examines the ultrastructural effects on rat bronchiolar and respiratory tissue
`samples obtained from thetoxicology study (#80940063) described above.
`
`F’
`1'-
`
`_
`'
`
`2
`
`Lung specimens of the male rats (5 / group), shown in the following table 4, were
`examined by electron microscopy. In choosing these samples one wonders why the
`low dose, 80 ppm, and the fatal dosages were excluded. The selection of mid-dose,
`200 ppm, is difficult to rationalize because the actual clinical use may be well under 80
`-160 ppm.
`
`Table 4
`
`
`
`
`The lung 'sections were examined at the Laboratory for advanced electron and light
`optical methods. College of Veterinary medicine, N. Carolina state university, Raleigh.
`A low power and a high power photomicrographs were taken on coded specimens
`and were decoded atter the evaluation by a pathologist. Results;
`lntersticial edema
`has been reported as an early change produced by exposure to relatively low
`concentration of N02, In this study. the incidence ofedema was greater relative to
`control but similar for both 1 and 7-day-animals. The severity of edema was graded .
`
`'
`
`14
`
`

`

`“moderate“ in animals sacrificed after 1 day and “slight/mild” in animals sacrificed after
`7"days. This observation indicated signs of transient oxidative injury to the respiratory
`epithelia at 200 ppm NO and 2.2 ppm N02 in rats. Qqaclusioa; Exposure of rats to
`200 ppm NO and 2.2 ppm N02 promotes oxidative insult to respiratory epithelia. The
`sponsor graded it to “lowest-observable-effect level”. Whether such an insult was
`apparent also in the low dose groups remains unknown.
`-
`
`(report) &Volume 2.5, p. 166
`
`irico ide in the atb inhalation. HasslerC e
`0
`ec vs
`7. 28-da e osure w'
`al, 1994 Final Begon. (thedg EPQ Regen RQR-QQQS-DS; Battelle §1udy Began
`#SCQ4DQ§fll
`.
`Location of Data:
`Vol’Eirneflzfi'f-pgilgt
`‘iflflfrfl
`'
`1‘lqepert. No. HDR-OOBS-DS; November. 1994
`
`Study No./ Date:
`
`_
`
`GLP Compliance:
`Animals: I
`
`'
`
`.
`Yes
`M/F Sprag'ue-Dawley Rats/SD:CDBR
`
`I
`
`.
`
`Administration:
`
`Inhalation
`
`Dosage:
`
`.
`
`._
`_
`
`'
`
`.
`-
`
`0 (air), 40, 80, 160, 200 and 250 ppm NO in 21% 02
`administeredfi hrs/day,7 days/week through nose
`only delivery system of
`inhalation. Nose only
`inhalation system allowed the administration of high
`concentration of NO without excessive oxidation to
`other oxides,
`in particular N02. ‘This' was
`accomplished by diluting the NO into a nitrogen
`atmosphere and adding oxygen immediately prior to
`administration. The test article concentrations were
`measured using chemiluminescent monitor which
`determines NOI NOx (NOx =tota| oxides of nitrogen).
`A solenoid valve was used to direct the sample flow
`through the catalytic convertor to measure NOx or
`bypass the converter to measure NO. The oxygen
`level was monitored using Airway Gas Monitor. Both
`instruments were calibrated before and during the
`experiment. Each level of the exposure group was
`monitored for NO. NOx and 02 in a continuously
`rotating sequence from the lowest to the highest
`dose groups. Each exposure level was monitored at
`least five times during the daily 6 hr exposures. The
`
`15
`
`

`

`effective concentration of inhalants was as described
`in table No. 5.
`
`Table no. 5
`
`
`
`N02
`02
`Nox
`NO
`MiSD MiSD M180 M250
`ppm
`ppm "
`%
`ppm
`NA
`NA
`21.
`NA
`-_
`310.9
`
`.
`
`.
`
`1. ‘Air
`Cont.
`
`'
`
`
`
`.
`
`.
`
`.
`
`
`2. 40
`"ppm
`
`
`'79.
`'
`..-.-.- 923.9
`4.160
`159.5 :1:
`ppm
`10
`
`40.
`5:1.4
`80.
`413.9
`161.1 i
`10.1
`
`
`
`
`
`
`
`
`
`21.
`510.8
`
`21.
`3i0.7
`
`21.2 a:
`1.1
`
`21.4 a:
`0.9
`
`0210.4
`
`O.6:t0.4
`
`1.6 i 0.6
`
`2.6 i 0.9
`
`2.3 i 3.4
`
`
`
`5. 200
`ppm
`
`5. (a)
`200
`ppm
`
`196 i
`6.4
`
`200.6 i
`6.6
`
`198.6 1:
`2.2
`
`200.9 i
`4.0
`
`6. 250 247.0:
`ppm
`" 20.9
`
`250.5 :1:
`21.2
`
`21.
`6i0.7
`
`3.5 :l: 0.9
`
`Observations:
`
`Results:
`
`1O [sex/gr allocated for terminal sacrifice on Day 29
`5 [sex gr allocated to 28 day recovery period.
`Additional 8 M 81 5 F were added to 200 ppm group
`because of instrument malfunction that occurred. .
`
`Rats were exposed for 6 hrs/day, 7 days / week. The
`concentration of NO, NOx and 02 were measured as
`described above. Body wts.. food consumption.
`clinical observation and clinical pathology'
`determinations were made periodically. Gross
`necropsy was performed on
`all
`animals.
`Histopathology was performed on all animals of the
`control and 200 ppm groups.
`
`Mortality: There were no deaths in 40. 80 & 160 ppm
`groups. One F in 200 ppm (gr. 5a), and 19 of the 30
`in 250 ppm group died within the first two days of
`
`16
`
`

`

`“is;
`
`exposure. An instrument malfUnction (although this
`malfunction is stated here very casually. it reaffirms
`my doubts that a similar malfunction may be
`impossible to avoid) resulted in overdosing by 32%
`which. apparently, killed 6 animals in the 250 ppm
`and 10 in the 200 ppm groups. Another point of
`disturbance was in the 'fact
`that,
`if
`it was an
`instrument malfunction .to' overdose by 32%, I would
`have expected more deaths in the 250 ppm group
`rather than the 200 ppm group unless one interprets
`that NO doses __o_i 200 ppm and higher can be
`randomlyjata'lo, A'group of 13 animals was added as
`a replacement "for 200 ppm but not for the 250 ppm
`1‘ under~.an.~argument that mortalities were high at 250
`ppm. In fact mortalities were higher in the 200 ppm
`group. Of the replacement, 1 F died on day 1 and 1
`M died on day 15th of the exposures.
`'
`Rapid respiration. ataxia, lethargy and blue & pale
`skin texture were noteworthy clinical observations
`which, according to sponsor, were primarily limited to
`the 200 & 250 ppm exposure groups. Body weights
`and food consumption were unremarkable. Clinical
`pathology revealed dose-related elevations
`in
`methemoglobin; “the only toxicologically significant
`finding” asper the sponsors.
`It is noted that MHB
`levels were higher only when blood samples were
`taken immediately after the NO exposure; As
`expected, theselevels had returned to normal 24 hrs
`after or after 28 days of continuous treatment.
`Gross necropsy revealed a pale ten to brown
`coloration in the lungs of the animals treated with
`200 & 250 ppm NO. Organ weights were
`unremarkable. Light microscopic evaluations did not
`reveal NO-related damage.
`.
`Methemoglobinemia: The most significant reason of
`death was methemoglobinemia (as shown in Table
`6) resulting in histotoxic anoxia.
`
`17
`
`
`
`

`

`Table 5: NO induced Methemoglobinemia
`
`
`
`If one disregards the time factor (days 7-56) of
`determination. normalized average % MHB
`appears to increase with NO as calculated below :
`-
`
`Table 6a: NormalizatiOn of Methemoglobinemia
`
`
`
`
`
`The sponsors state that “the effects were not readily
`discernible at exposures of 80 ppm and lower (see
`Table
`6) dosage”.
`'I
`think
`the
`trend of
`methemoglobinemia is apparent even at the smallest
`dose as shown in Table 6a. In comparison with air
`control, the average MHB appears 118% and 213%
`' at 40 & 80 ppm respectively.
`In view of the high
`affinity of NO for Hb,
`i will be surprised If MHB was
`-not formed. in the absence of fatality, at the best,
`this
`data
`indicates
`that
`rat
`is
`tolerant
`of
`methemoglobinemia invoked by 40 - 80 ppm NO.
`
`18
`
`

`

`rl
`
`Methemoglobinemia appears to
`anglugiog;
`occur even at the lowest tested dose. Although there
`were no fatalities amongst the low dose groups,
`concerns on MHB continue to persist.
`
`8. 28-day exposure with recovery of nitric oxide in the [at via inhalation. Singer AW“
`December 1996, Final Bepofi (theda PPD Report E.DBng-QS)
`Location of Data: Volume 2.10. p. 1004 (report) and Volume 2.5, p. 171 (summary).
`This is an Anatomic Pathology part 5i thetoXicolzogyfstudy outlined in the preceding.
`
`External carcass and internalorgansywere-examined with a particular attention to the
`respiratory tract. The lungs were examinedior petechiae, macroscopic foci of
`inflammation. edema and possible discoloration due to methemoglobinemia.
`Abnormal morphology. oral and nasal passages-and evidence of exudate, foam' or
`edema were also recorded. flesults; No insult was found in the epithelium of the
`upper respiratory tract which is more vulnerable to inhaled toxicants. Light microscopy
`did not reveal ioss or damage to cilia and ciliated cells. No evidence 'of toxicity or
`alteration was found in the mast cells. There was also no evidence of toxicity in
`terminal bronchioles. Qommegt:
`It is comforting to note the absence of abnormal
`morphology in this study. However a possibility can not be excluded that
`light
`microscopy was unable to detect finer changes as in the previous study.
`
`o nitric oxide int a re
`osure with ecove
`stud '2 -da 6
`9_ Electron microsco
`.by inhalation; Mann P. et al. August 1996 Final Began #541-902 (Ohmeda PPD
`Report FiDR-OiSO-DS: )
`_
`.
`Location of data: Volume 2.9, p.909 (report) &_ Volume 2.5. p.174 (summary)
`
`Ultrastructural effects of NO exposure were investigated in bronchiolar, transitional
`and respiratory tissue samples of the animals of the foregoing toxicology study (no.
`
`RDR-0063-DS). Methods Lung specimens from 5 M rats were processed from groups
`1 & 5 (see table 6) exposed-at 0 and 200 ppm NO respectively. It is unclear why the
`groups with low dosages, 40 & 80 ppm, were not examined. Tissue sections were
`prepared _using standard processing techniques for electron microscopy. Four grids
`were examined from each lung lobe. Terminal bronchioles and adjacent alveoii were
`examined and photographed when both areas were present. All
`the
`photomicrographs of the control animals were examined first to establish background
`.changes and then randomized with the treated animals, coded. and then given to a
`pathologist for evaluation. flesults; The ultrastructural changes included spreading of.
`type II cells, decreased number / size of cilia in ciliated respiratoryrepithelial cells,
`electron lucent areas in clara cells and altered shapes or decreased height of clara
`
`‘19
`
`
`
`

`

`cells. Since these changes were found in treated animals as well as in controls, the
`sponsor does not relate them with the drug treatment. quclusjoa; Since only the low
`doses of INO may have a chance for therapeutic use, I would have liked to see the EM
`data of low dose groups just to reassure that the findings are similar.
`
`3;.
`
`ir-W‘E‘”?-,"7
`
`APPEARS THIS WAY
`0N ORIGINAL
`
`
`
`20
`
`
`
`

`

`:liteview of pertinent literature:
`
`The sponsors have cited a number of references from which a selected few.
`addressing the issues of efficacy and safety are reviewed. Since the division has
`asked the Pharmacology Reviewer to comment on Tolerance and Withdrawal,
`this
`topic is also included in the following review of literature:
`_
`
`Efficacy;
`
` ertension in
`
`
`
`The aim of this study watts-investigate- the feasibility of administering NO to 1-2 day
`old piglet (neonatal model), to evaluate the dose response characteristics and to
`determine the'time‘course for effect of NO on hypoxic pulmonary vasoconstriction. -
`Methods; Newborn piglets were instrumented to allow measurements of cardiac
`index, pulmonary arterial pressure and systemic arterial pressure. Pulmonary
`hypertension was induced by reducing the fraction of inspired oxygen to 0.12 40.14.
`The animals were then given 5-80 ppm NO. Besglts; All concentrations of NO were
`associated with a rapid decrease in pulmonary arterial pressure and pulmonary
`vascular resistance. Cardiac index increased and systemic vascular resistance
`decreased. once again at all dosages. Very important finding was in the fact that there
`was no significant difference noted betweenthe various doses of nitric oxide. Stated
`in other words, the pulmonary vasodilator response of 5 ppm was the same as that of
`. 80 ppm NO. The investigators further stated, and l quote, that “ although there appears
`to be a consistent maximal effect with 80 ppm in the newborn piglets, we found no
`statistical advanta e in
`oin be ond 5
`m in terms of the absolute decrease 'in PAP
`and reduction of PVB". Thus, assuming that human newborns may be approximately
`twice the weight of piglets (approx. 1.8 kgm), there may not be a need to exceed 10
`ppm NO; a maximum approvable dose. Conclusion; The main concern of NO-therapy
`is toxicity due to methemoglobinemia and pulmonary injury due to N02_ Both of these
`issues are likely to be diluted out by the use of very low levels, eg 5 ppm, of NO.
`Furthermore, as stated in_the following publication by Romand et al, all of the
`pulmonary vasoconstriction is not reversible by [NO anyway, then why risk the safety
`by administering higher dosages of NO ?
`'
`Although there are other studies (eg. Rich et al. J. Appl. Physiol.. 75: 1278-1284, 1993
`which used isolated rat lung model) suggesting dose-dependant vasodilatory effects
`up to 1000 ppm NO. those studies have employed either isolated organs or have
`employed adult animals. The data generated in newborns animals are somewhat
`direct and provide a better guideline despite the absence of symptoms of PPHN. The
`preclinical animal studies. thus, indicate that whatever beneficial effect is likely to be
`derived from the NO therapy.
`is indeed offered by low doses INO. Until additional
`evidence is available on safety, it may be prudent to use the very low doses of INC.
`
`21
`
`
`
`

`

`
`
`
`
`In this paper Jacob et al confirm above reviewed observations of Etches et al. The
`data demonstrated pulmonary vasodilatory properties of N0'_ in piglets with hypoxia
`induced by hypoxic ventilation. The antihypertensive response of 5 ppm NO did not
`improve further with a higher dose of" 40 ppm. Thus. the findings in piglets appear in
`contrast to other observations made in sheep and rat which suggest dose-dependant
`improvement of vasodilation. Despite this divergence of data, and unless proven
`otherwise (by animal models whigh truly replicaterhu'rnan infants).
`I would tend to
`hypothesize that the [N0 therapy in human" infants may show all the total benefits at
`low dosages without further improvements at higher dosages (similar to that found in
`the present and the previous study-efv-Etches et al) and recommend the use of mo
`therapy at / around 10 ppm. Of course, the review of the Medical Officer can
`supersede this recommendation if he/she has a more compelling, direct evidence.
`
`everses H oxic Pul ona
`(iii) Inhaled Nitric oxide a ial
`dog: Remand et al, J. AQQL Physio!“ 75(3). 1359-1355. 1gg4,
`
`vasoconstriction in the
`
`The purpose of this study was to characterize interaction between NO and hypoxic
`pulmonary vasoconstriction (HPV) in a canine model. The authors hypothesized that
`NO inhalation would completely reverse the increase in pulmonary vasomotor tone
`during hypoxia and that the time course of this response may follow zero-order
`kinetics. Methods: Arterial and venous systemic and pulmonary pressures, flow-probe
`derived cardiac output, nitrosyl-haemoglobin (NO-Hb) and Methemoglobin (MHB)
`were determined in hypoxic pulmonary vasoc

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