CENTER FOR DRUG EVALUATION AND RESEARCH
`
`ABELIQZA l IQN NIJMBER; NBA 20345
`
`L YA
`I AL
`LI
`BIQPHARMACEIITICS REXIEWQ)
`
`
`
`
`
`

`

`.-
`
`NOV l0 l999
`
`CLINICAL PHARMACOLOGY AND BIOPHARMACEUTICS REVIEW
`
`NDA: 20845
`INDJ'
`_
`
`J
`
`DRUG NAME:
`
`Nitric Oxide gas for inhalation
`lNOInaJtTM
`
`SUBMISSION DATES: May 24, 1999
`August 13, 1999
`
`FORMULATION:
`DOSE:
`_
`
`100 a 800 parts per million (ppm) '
`20 ppm up to 14 days
`
`'
`
`INDICATION:
`
`hypoxemic respiratory failure in the term and near-term newborn in
`conjunction with mechanical ventilation
`
`,
`
`SPONSOR:
`nEVIEWER:
`
`=INO Therapeutics, Inc.
`- B. Nhi Nguyen, ammo.
`
`SUBMISSION:
`
`New Drug Application
`
`\ ‘
`
`SUMMARY
`
`The Sponsor is seeking the approval of inhaled nitric oxide (I-NO), 100 and 800 parts per million
`(ppm), for use in hypoxemic respiratory failure in the term and near-term newborn. The sponsor
`submitted a paper NDA that cites, for safety and efficacy, one large randomized, controlled,
`multicenter trial conducted in neonates with hypoxic respiratory failure, one large randomized,
`double-blind, placebo-controlled trial conducted in neonates with primary pulmonary
`hypertension (PPHN), and some other studies and case reports that include a total of 188
`neonates in whom I-NO was used for PPHN. A randomized. double-blind, placebo-controlled
`study examining safety in 155 neonates with PPHN was also cited in the label. Additionally,
`eight studies were cited in the pharmacokinetic section of the label. One of these was the large
`
`Inhaled NO (I-NO), a gaseous blend of nitric oxide (0.8%) and nitrogen (99.2%), is a potent,
`local and selective pulmonary vasodilator. I-NO decreases pulmonary artery pressure and
`increases the partial pressure of arterial oxygen (PaOz) leading to increased blood flow to the
`lungs. I-NO enhances V/Q matching by redistributing pulmonary blood flow away from lung
`regions with low ventilation/perfusion (V/Q) ratios towards regions with normal ratios.
`Between 75-90% of LNG is absOrbed by the alveoli. After inhalation, NO-imrnediately binds to
`hemoglobin. This subsequently produces methemoglobin and nitrate. These are the primary
`‘
`products that enter the systemic circulation. Methernoglobin is primarily metabolized by
`
`

`

`methemoglobin reductase to form hemoglobin and nitrate. Thus, nitrate is the final metabolite
`formed from nitric oxide by all pathways. Nitrates are eliminated principally by the kidney.
`
`RECOWIENDATION
`
`The application does not completely fiilfill the requirement ofthe Office of Clinical Pharmacology
`and Biopharmaceutics since the pharmacokinetic inforr'nation in the target population was not
`submitted. Comments 1-8 and the labeling comment should be forwarded to the sponsor.
`.‘v‘
`
`'
`Pms nus w
`AP 0N oalcmaL
`
`A‘l
`
`
`
`

`

`TABLE OF CONTENTS
`
`Background ................................................................................................. 4
`Summary ofBioavailability I Pharmacoicinetics ..................................................... 4 .
`Comments to the sponsor
`....................... 7
`
`Appendix I (Study Summaries)
`.
`‘
`‘
`Uptake and Distribution
`Borland CDR, Higenbottam TW. A simultaneous single breath measurement of pulmonary
`diffusing capacity with nitric oxide and carbon monoxide. Eur Respir J 1989; 2: 56-63. ......... 9
`..-
`
`Guenard H, Varene N, Vaida P. Determination of lung capillary blood volume and membrane
`diffusing capacity in man by the measurements ofN0 and C0 transfer. Respir Physiol. 1987;
`70:113-120.
`............................'.................................................................... 13
`
`Wennmaim fig Benthiii G, Petersson A-S. Dependence ofthe metabolism ofnitric oxide in
`healthy human whole blood on the oxygenation of its red cell haemoglobin. British Journal of
`Pharmacology. 1992;106:507-503. ...................................................................... 15
`
`Chiodi H, Mohler JG. Effects of exposure of blood hemoglobin to nitric oxide. Environmental
`Research. 1985; 37:355-63. ............................................................................... 17
`
`Metabolism ‘
`Wennmalm A, Benthin G, Edlund A, et al. Metabolism and excretion of nitric oxide in humans:
`an experimental and clinical study. Circulation Research. 1993; 73:1121-1127. .................. 19
`
`.
`INO-Ol and IND-02
`Barefield E et al. A double-blind, randomized, placebo-controlled, dose-response study ofinhaled
`nitric oxide in the treatment of persistent pulmonary hypertension of the newbom ................ 25
`
`Elimination.
`
`Westfelt UN, Benthin G, Lundin S, Stenqvist O, Wennmalm A. Conversion of inhaled nitric
`oxide to nitrate in man. British Journal ofPharmacology. 1995; 114: 1621-1624.
`
`29
`
`Young JD,‘ Sear IW, Valvini EM. Kinetics ofmethemoglobin and serum nitrogen oxide
`production during inhalation of nitric oxide in volunteers. British Journal ofAnaesthesia. 1996; '
`76: 652-56. ...................................................................................................33 -
`
`Appendix 11(other study) ..................................................................... ‘.......... 37
`Appendix III
`"
`,
`Metabolic pathway for inhaled nitric oxide
`Proposed labeling
`
`....................... 40
`................ p........................................ 41
`
`
`
`
`
`

`

`BACKGROUND
`
`The Sponsor is seeking the approval of inhaled nitric oxide (I-NO), 100 and 800 parts per million
`(ppm), for use in hypoxemic respiratory failure in the term and near-term newborn. I-NO is a
`potent, local and selective pulmonary vasodilator that acts from the outer surface of the
`pulmonary vessels. I-NO decreases pulmonary artery pressure and increases the partial pressure
`of arterial oxygen (PaOz) leading to increased blood flow to the lungs. I-NO enhances
`ventilation/perfusion (VIQ) matching, the appropriate contact between alveolar gas and
`pulmonary capillary blood, by redistributing pulmonary blood flow away from lung regions with
`low V/Q ratios towards regions with normal ratios.
`-—.-
`
`Structure
`
`- 13:6 :
`
`nitric oxide
`
`The recommended safe:and effective dose is 20 ppm of INOmax as a constant inhalation for up
`to 14 days until the underlying oxygen desaturation has resolved and the neonate is ready to be
`weaned from lNOmax therapy.
`
`Formulation
`
`I-NO is a gaseous blend of nitric oxide (0.8%) and nitrogen (99.2%)
`
`Deliver System
`The sponsor recommends I-NO be delivered through-an FDA approved NO delivery device, such
`as lNOventTM delivery device. Precise monitoring of inspired NO and N02 should be instituted,
`using a prOperly calibrated analysis device with alarms. This system should be calibrated using a
`precisely defined calibration mixture of NO and N02, such as lNOcalm. Sample gas for analysis
`should be drawn before the Y-piece, proximal to the patient.
`
`SUMNLARY 0F BIOAVAILABILITY I PHARMACOKINETICS
`
`Bioavailability
`Food effects
`It is possible that foods rich in nitrates may interact with I-NO. However, this has not been '
`studied and may not be relevant to the neonatal population. The effect of parenteral nutrition in
`neonates is unknown.
`
`Pharmacokinetics
`Absorption / Uptake ari'd Distribution
`Absorption of I-NO has ranged from 66% to 99% in healthy adult volunteers. The majority of I-
`NO traverses the pulmonary capillary bed where it binds with oxyhemoglobin (60-100% oxygen
`saturated) to form methemoglobin and nitrate (N03). I-NO can also combine with deoxygenated
`hemoglobin to form nitrosylhemoglobin which is rapidly converted into nitrogen oxides and
`methemoglobin upon exposure to oxygen. N0 also undergoes direct conversion to nitrite and
`
`.
`
`
`
`
`

`

`nitrogen dioxide with subsequent oxidation to nitrate and methemoglobin in biological fluids.
`Thus. the end products of LNG that enter the systemic circulation are predominantly
`methemoglobin and nitrate.
`
`.
`Metabolism
`See Appendix III for fun metabolic pathway.
`
`_
`
`The NADH-methemoglobin reductase system accounts for 67-95% of the conversion of
`methemoglobin back to hemoglobin in adults. Neonates have reduced NADH-methemoglobin
`reductase activity compared with adults. ‘Because this is the primary route of metabolism of
`methemoglobin in humans, caution is advised when extrapolating adult data to neonates. Fetal
`hemoglobin may also be more prone to oxidation than adult hemoglobin.
`
`Methemoglobin disposition has been measured in neonates with PPHN during the first 12 hours
`of exposure to 0, 5, 20, and 80 ppm I-NO. All methemoglobin concentrations were significantly
`elevated above placebo, but only the 80 ppm dose group was clinically elevated and associated
`with methemoglobinemia.
`
`Elimination
`In adults,- nitrate is'the predominant NO metabolite excreted in the urine, accounting for >70% of
`the I-NO dose. Nitrates and nitrites are eliminated principally by the kidney at rates approaching
`the glomerular filtration rate. Thus. serum nitrogen oxide (sNOx) concentrations would be
`expected to increase when renal function is impaired; The elimination of nitrates in neonates is
`presently unknown.
`
`Plasma level-dose relationship
`In healthy adult volunteers, there is a dose-dependent increase in plasma nitrate and
`methemoglobin.
`In adults with severe heart failure, there is a dose-related increase in plasma
`nitrate in the systemic and pulmonary arteries. with significantly lower levels in the pulmonary
`arterial plasma compared to the systemic plasma. Twenty minutes after cessation of I-NO, the
`plasma nitrate had dropped in most patients.
`
`.
`
`Nitrate plasma concentrations increase with 24 hours of LNG, while nitrite (N02) Plasma
`concentrations remain unchanged.
`
`
`
`Special populations
`.
`Renal impairment
`No studies have been conducted in renally impaired subjects.
`
`Hepatic impairment
`No studies have been conducted in hepaticaliy impaired subjects.
`
`.
`Drug interactions
`Drug interactions studies have not been conducted in neonates.
`
`
`
`

`

`
`
`Eormulations
`
`The supplies used in the cited studies ranged fi-om 100 to 2000 ppm, and the delivered doses
`ranged fi'om 5 to 512 ppm. None ofthe studies cited used the supply to be marketed to deliver
`the dose to be approved. Even the two studies sponsored by INO Therapeutics, Inc. used the 400
`ppm cylinder to deliver a dose ofLNG 20 ppm. The impact ofthe dlfi‘erent supply concentrations
`used to obtain the delivered dose is unknown.
`
`Delivery system
`Because most ofthe studies submitted fonthis NDA were not sponsored by 1N0 Therapeutics,
`Inc, different delivery systems were used. Additionally, several studies did not provide details of
`their delivery system. Ofthose studies that did provide details, the description ofthe delivery -
`systems ranged from two mask flow regulators, to a tight fitting mask in a non-rebreathing system
`to a leak free face maslg connected to a non~rebreathing valve. It is unknown ifthese difi‘erent
`delivery systems influence the amount of delivered I-NO.
`-
`‘
`
`Assay
`
`
`
`PPEARS Tats-WAY
`A on omanm
`
`
`
`
`

`

`
`
`COMMENTS TO THE SPONSOR
`
`
`
`

`

`
`
`==
`
`1.
`
`‘
`
`LABELING COMIMENT
`
`It should be clearly stated that the uptake, distribution and elimination were determined in
`primarily healthy adults.
`
`Clinical pharmacology briefing held on November 10,1999.
`
`(Fadiran, Hepp, Huang, Marroum, Mehta, Nguyen, Robbie, Selen, Venitzwerelsvescnt)
`”B NhiNguyen, PIT/D
`’ H ”a // ({5ch
`
`RD/RT: Patrick Mari-cum, PhD _.___ /=$/_
`
`CC: NDA 20845, RFD-110, HID-860 (Mehta, Nguyen), CDER document room
`
`
`
`

`

`-_.,
`
`APPENDIX II: CITED STUDIES
`
`A SIMULTANEOUS SINGLE BREATH MEASUREMENT OF PULMONARY
`DIFFUSING CAPACITY WITH NITRIC OXIDE AND CARBON MONOXIDE
`
`INVESTIGATORS: Borland CDR, Higenbottam TW
`STUDY SITE: not specified
`.
`CITATION: Eur Respir J 1989; 2: 56-63.
`-
`STUDY DATES: not specified
`OBJECTIVES: To determine the diffusion and rate of combination of carbon monoxide (CO)
`with hemoglobin and of NC with oxyhernbglobin.
`.
`FORMULATION: The inspired gas mixture containing approximately 40 ppm of NO in %
`helium,
`% CO, % O; in nitrogen was made up in the inspirate bag immediately before each
`test by-adding
`L of 1000 ppm NO in N; to 7L of the standard gas transfer test mixture.
`STUDY DESIGN: open label
`POPULATION: 13 healthy volunteers
`
`PROCEDURE: No more than four single measurements of DL (maximal diffusing capacity)
`were made per person each day. The following were measured: forced expiratory volume in one
`second (FEVI), TLC (whole body plethysmograph), DLCO (pulmonary diffusing capacity
`- measured by carbon monoxide), and DLNO (pulmonary diffusing capacity measured by nitric
`oxide). On each occasion, studies were performed at least two hours after a meal at the same
`time of day.
`r
`
`-:-_.
`
`ASSAYS:
`
`L
`
`j
`
`.1
`
`Diffusing capacity measurement. Standard single breath gas transfer equipment was used to
`sample the inhaled. and exhaled gases for helium (He), CO, oxygen (02) and NO. The equipment
`was calibrated daily. Measurements of DLCO and DLNO were made simultaneously. A single
`breath DI. measurement was performed during inspiration. During expiration, an alveolar
`sample was collected and immediately analyzed. The exact NO concentration at the time of the
`single breath measurement could be calculated knowing the time that had elapsed from filling the .
`inspirate bag (I):
`
`where k is the rate constant for oxidation of NO.-
`
`1/N0,= (IINOO) I (1/2 K(02),)
`
`ANALYSIS: Calculation of DLCO and DLNO. DLCO and DLNO were calculated from
`inspired and alveolar concentrations by the standard method. Within and between day CV was
`
`“-
`
`'
`
`"
`
`
`
`
`
`

`

`
`
`636% for DLCO and 6.4% for DINO. The investigators assumed no alveolar back pressure for
`NO and an exponential rate of decline in alveolar NO in the calculation. These assumptions
`were later justified.
`
`Measurement ofalveolar N0 at varying times of breathhold. Combined DLCO and DLNO b
`measurements were carried out on two subjects but at different breath-hold times ranging from 4—
`10 seconds. The alveolar NO at each time interval was calculated as a fraction of the initial
`concentration.
`
`In eight tobacco smokers and two
`Measurement ofback pressure of N0 and C0 in smokers.
`nonsmokers alveolar concentrations of CO and NO were sampled following a 20 second breath
`hold at TLC. Alveolar concentrations were also measured in one smoker and one nonsmoker
`after rebreathing oxygen for 4 minutes. The smokers' daily tobacco consumption and last time
`smoked were recorded.
`-.
`
`Additional measurements. D]. at varying levels of lung volumes, alveolar oxygen
`concentrations and exercise were measured.
`
`PHARMACOKINETIC RESULTS: The response of the NO analyzer was linear from 1-5
`ppm but for 5-40 ppm a minor departure from linearity was noted, with a slight underestimate of
`NO concentration. Repeated analysis of the same concentration of NO was within 10% at 1 ppm
`and within 1% at 50 ppm. The time to 95% response after a stepwise increase in concentration
`was 24 seconds. No interference was observed with N02 or N20. The figure below shoWs that
`the decline in alveolar NO and CO concentration ov’tér time is logarithmic.
`
`FA‘JFA‘
`
`1 .0D
`
`0.1 D
`
`O
`
`.
`
`5
`
`1D
`_
`Seconds
`
`15
`
`Elv- 1- - Dcdim 'm dual-r NO and CO I'ith varying breath-hold
`time: (one subjeu). Alveolar menu-noon: at end: ti
`(F
`expressed as a fraction at the initial alveolar “Mlmmotl iii)?”
`
`
`
`

`

`
`
`There was no alveolar back pressure for NO detected in either the two nonsmokers or the eight
`smokers after a 20 second breath—hold. This was despite the finding of alveolar CO
`concentrations ranging from 16-34 ppm in the smokers. No alveolar back pressure for NO could -
`be obtainedin the one smoker and one nonsmoker after 4 minutes of rebreathing 02. The
`smoker’s CO back pressure was 70 ppm at an alveolar oxygen concentration of 75%.
`
`DINO exceeded DLCO by a mean ratio of 4.3 i 0.3 (SD).-.Table 1 below shows that the
`presence of CO or NO in the similltaneous single breath test gas mixture did not interfere with
`--.
`the uptake of either CO or NO.
`
`Table 1. - Comparison of Own and Dtco measured together and separately
`——-——-———.—__—.____________________
`Subject
`Dmo
`Duco
`Duo
`Duto
`alone'
`With NO
`done
`with CO
`-———_____.._._______________
`
`1
`2
`3
`4
`5
`
`-_
`
`133 (0.8)
`11.9 (0.3)
`13.7 70.3)
`12.0 (1.1;
`8.9 (0.3)
`
`143 (0.4)
`11.9 (0.3)
`13.5 (03)
`12.0 (1.1)
`8.7 (0.3)
`
`'57.6 (1.0)
`57.9 (5.6)
`53: (5.6)
`49.1 (4.7)
`47.5 (1.6)
`
`59.5 {1.2)
`55.2 (1.6)
`61.0 (0.7)
`50.2 (4.1)
`42.6 (2.8)
`
`'11-: ramming trains are measured With a blurb-hold time
`‘Bxuth-hold time of 10 s.
`of 75 1. Man values (so). three replicate: (mol-min"-ltPa").
`
`The average DLCO was 11.6 :1: 2.4 ml/min/mm Hg and the average DLNO was 49.1 i 10.2
`‘ mllminlmm Hg (table 2).
`
`‘
`
`_
`
`‘.
`
`‘.
`
`Table 2. - Average data {torn which DLCO and 0pm were calculatedWW
`
`3.11:1:de Lime
`0,95
`N0
`cor.
`14:7.
`.
`
`ppm ’WWW
`
`Inhaled
`
`Ezhaled
`
`123(1)
`
`027(0008)
`
`38.90.18)
`
`1170.6)
`
`7310.0
`
`
`
`
`
`0.1 «0.02)10.00) ?_2 (1)WW
`VA
`‘
`Deco
`Duo
`t
`nerd-mh‘hkl’a"
`mirth-Lick"
`
`
`
`
`11.5 {2.4)6.1 (1.5) 49.1 (102)MW
`Mean valuu from 13 mbjects. 3 replicates were taken (:0).
`
`
`
`An increase in alveolar oxygen concentration from an average of 18.6 to 68.5% caused a mean _
`fall in DLCO from 11.3 i 1.2 to 6.5 i 1.2 mmol/min/kPa, but DLNO was unchanged.
`
`The reduction in alveolar volume,.on average from 7 L to 3.9 L produced a significant mean fall
`in DLCO of 8%; 12.9 i_ 1.6 to 11.9 i 1.9 mmollmin/kPa. Carbon monoxide transfer coefficient
`(Koo) rose 48% from 1.9 i 0.2 to 3.1 i 0.2 mmol/min/kPafL. The fall in DLNO was greater;
`34% decline from 54.5 i 7 to 38.6 i 6.6 mmol/min/kPa (p < 0.05). NO transfer coefficient rose
`24% from 7.9 to 10.1 mmoI/minlkPa/L.
`
`ll
`
`
`
`

`

`The increase in DLCO and DLNO is shown in Figure 2. With exercise the DLCO significantly
`increased 45%; 12.3 i 0.7 to 19.5 i 1.7 mmollmin/kPa. DLNO significantly increased 26% with
`exercise; 53.7 i 2.2 to 70 i 9.5 mmol/min/kPa.
`1m
`05-:
`
`’ n:- nun - It» or : cinnamon: an
`(AC! IIIOIVIDIML
`
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`
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`
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`0
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`
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`
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`
`Fig. 2‘. - Increase in Dam and DLCB with nerds: (three mbjeul):
`Rest
`Is
`tamer-steal by the lowest Valuu o( oxygm uptake: um—
`sis: is upru'mled by the maining higher Values.
`-
`
`The rate of decline in concentration of NO in known oxygen concentrations was 7.1 it 10‘lo ppm'
`2rnin'l. This compares favorably to a previously published figure of 7.6 x 10-10 ppm'zmin‘l.
`
`CONCLUSION: These investigators demonstrated that DLNO is four times greater than DLCO
`at rest and at normal 02 levels. Unlike DLCO, DLNO appears to be independent of hyperoxia,
`but DLNO appears to be more dependent on lung volume. DLCO and DLNO increase with
`exercise. Since NO reacts much‘ faster with hemoglobin than CO. DLNO should be influenced
`much less by reacting with hemoglobin. This study supports the sponsor’s claim that a large
`proportion of LNG is absorbed systemically.
`
`
`
`

`

`DETERMINATION OF LUNG CAPILLARY BLOOD VOLUNIE AND NIEMBRANE
`DIFFUSING CAPACITY IN MAN BY THE RIEASUREMENTS OF NO AND CO
`TRANSFER
`
`INVESTIGATORS: Guenard I-I, Varene N, Vaida P
`STUDY SITE: University ofBordeaux, Bordeaux France
`CITATION: Respir Physiol. 1987; 70:113-120.
`.
`.
`.
`,
`STUDY DATES: not stated
`OBJECTIVES: The lung transfer (TL) for a given gas is' related to the membrane difi‘using
`capacity for the gas (Dm) and to the capillary blood volume (Qc). The objective of this study was
`to measure the Dm for N0, and determine Qc.
`_
`.% CO witl' % He;
`FORMULATION: The inspired mixture contained either 8 ppm NO or
`% 02 in N2. To avoid transformation ofNO oxidizing to N02, the mixture was prepared just
`before the experiments.
`STUDY DESIGN: open-label
`POPULATION:
`1.4 healthy subjects (7 men 7 women) that were either non or moderate
`smokers
`.~.
`-
`'
`
`PROCEDURE: NO and CO lung transfer values (TLNO and TLco, respectively) were measured
`using the single breath technique. TLNo measurement. The seated subject breathed through a
`mouth piece in a volumetric body plethysmograph. Inspired and expired gases were separated by
`means of a two-way valve outside the body box. A two-way tap was connected to the inspiratory
`port of the valve. One way was left open to room air, and the other way was connected to a 100
`L rubber balloon containing a mixture with 21% Off-3% He, 8*10".% N0 (Bppm), in N2. The
`expiratory branch of the two-way valve was connected to two electric valves set in parallel. One
`of these was connected to a l L rubber balloon and was open during the time ofthe sample
`collection, while at the same time the other valve was closed.
`
`During the subject’s'full expiration , the two-way tap was tumed to the mixture. The subject
`made a fill] rapid inspired vital capacity and held his/her breath for 3 seconds. Then hefshe made a
`full rapid expiration. The second liter of expirate was collected. The alveolar sample was taken
`, automatically by electrically switching the expiratory valve. TLNO was computed using the
`equation:
`TLNQ = V]! (PB047)t*in FAno(0)/FANO (I), where
`'
`VL lung volume during apnea
`_
`t, the efi'ectivebreath-holding time
`FANO (0), the initial alveolar fi'action
`FANO (t), the alveolar fraction ofN0 at the time of sampling.
`The'effective' breath-holding time Was taken to be the sum of the inspirate time, the real breath-
`holding time and the ex'pirate time to the start of sampling.
`
`TLCO measurements. The inspired mixture contained 21% 02, 2% He, 0.25 % CO in N2. The
`breath holding time was 8 seconds. The sampling and computation of TLco was similar to that
`used for TLNo.
`
`
`
`

`

`SAMPLING: Five successive measurements, at five minute intervals, were made in the seated
`subjects to determine TLNO. Five measurements were made at one day intervals to avoid
`underestimation of mo due to the back pressure of CO.
`
`Assavn’
`
`‘
`
`,
`
`- -
`
`ANALYSIS: NO analysis of the alveolar gas was made immediately after collection from the '
`expiratory valve to avoid any decrease in the NO fraction due to absorption by the balloon rubber
`wall. The fraction of He was then measured.
`
`_l‘
`
`RESULTS: Results are summarized in table 1.
`
`1:11 mini-11.11335! heinmoflhetom 51155:: (SunLlofheasauzm(MLmmmm:mmdmm¢ufigmm NO
`mm(v1.3:VwLTmeamhd-ah“-Tm“mdmmmdum(WW'mmerm-nvmq
`offlefihme mam-m“-Tm-| “WWthim bymaefliduxfl -uk)usu=ed nah-dune L .
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`
`CONCLUSION: The TLNolTlco ratio was ~ 5. This suggests that the specific conductance of
`blood for NO was very high.
`_It may also suggest that NO is not a good indicator for lung transfer
`measurement. The inspired fraction of NO decreased slightly from one experiment to another at
`a rate of 0.5 ppm/h, without apparent NO; formation. This was possibly due to the absorption of
`NO and N02 in the walls of the rubber balloon.
`
`The Qc for men was 78:0 1 13.2 ml. and that for women was 59.5 i 11.6 mL. The capillary
`blood volumes of men and women are significantly different, however after correcting for total
`lung capacity this difference disappears.
`
`This study also supports the sponsor’s claim that a large proportion of LNG is absorbed
`systemically.
`'
`
`
`
`
`14
`
`

`

`DEPENDENCE OF THE METABOLISM OF NITRIC OXIDE IN HEALTHY HUIVIAN
`WHOLE BLOOD ON THE OXYGENATION OF ITS RED CELL HAEMOGLOBIN
`
`' INVESTIGATORS: Wennmalm A, Benthin G, Peterssen A—S.
`STUDY SITE: Gothenburg University, GOthenburg, Sweden
`CITATION: British Journal ofPharmacology. 1992;106:507-508.
`STUDY DATES: not published
`.
`.
`OBJECTIVES: To determine the route by which NO in human blood is converted to nitrate.
`FORMULATION: N0 (AGA Special Gas, final concentration SO-ZOOmM)
`STUDY DESIGN: ex-vivo incubation study
`POPULATION: 20 healthy donors
`1.
`
`PROCEDURE: Portions of venous blood from healthy donors were incubated with NO with or
`without previous oxygenation. O; saturation was estimated. The incubation was interrupted to
`separate the blood into cells and plasma. This was followed by freezing the cell fraction at 70K in
`electron paramagnetic resonance (EPR) tubes. The EPR spectra of the blood cell fraction were
`recorded for methemoglobin and- nitrosylhemoglobin at a microwave frequency of 9.22 GHz and a
`power ofiUmW from about 500 to 3500 gauss with a modulation amplitude of20 gauss. Plasma
`levels of nitrite and nitrate were measured as described below.
`
`. ASSAer'
`
`_
`
`.
`
`7
`
`.1
`_
`'
`1/
`RESULTS: Table l and figure 1 show the basal concentrations of nitrate, nitrite, methemoglobin,
`and nitrosylhemoglobin, as well as these concentrations when incubated with arterialized and ‘
`venous blood mean :l: SE
`
`_——_
`
`_-----
`Arterial ed Arterialized
`Venous
`Venous blood,
`
`
`
`
`
`Plasma nitrite (uM)
`
`Methemoglobin
`
`< l or
`undetectable
`
`undetectable
`
`< l or
`undetectable
`151
`
`< 1 or .
`undetectable
`
`< l or
`.
`undetectable
`260
`
`.
`
`-
`
`92
`
`
`
`'-
`
`
`
`
`
`
`
`
`
`
`
`1.2 :l: 0.3 n 92
`Nitrosylhemoglobin
`units
`
`
`
`;
`
`_
`
`Incubation ofNO with arterialized blood resulted in a dose-dependent increase in plasma nitrate
`and methemoglobin, whereas nitrosylhemoglobin increased minimally. Incubation ofNO with
`venous blood for two minutes caused a parallel increase in plasma nitrate and methemoglobin but
`this increase was ofa smaller magnitude than that seen with arterialized blood. In contrast,
`‘ nitrosylhemoglobin increased markedly. When the incubation was prolonged to 15 minutes,
`concentrations of nitrate and methemoglobin were similar to that obtained with arterialized blood.
`
`,
`
`
`
`IS
`
`

`

`
`
`bIOOd. Nitrosylhemoglobin was unaffected. Incubation of plasma with N0 for 15 minutes
`resulted in semiquantative conversion of nitrite to nitrate in a ratio of 5:1.
`
`Models-dalmatian“
`
`3ng
`Vane-nucleic!”
`
`-.
`
`E
`3"”
`3m
`g
`t
`
`3
`‘5'
`g
`
`to:
`
`c
`
`on man
`.8
`
`_
`
`no new
`I"
`
`Figure 1. Concentrations ofnitrate, methemoglobin, and nitrosylhemoglabin when incubated
`with arterialized and venous blood (mean :L'SE). nitrate (I); methemoglobin (0): nilrosyl
`hemoglobin (A) _
`'-
`-.-_-.
`
`CONCLUSION: Plasma, in comparison to whole blood, was inefficient in converting NO to
`nitrite. This highlights the activity of the blood cell fraction in the inactivation of NO. Since the
`red cells are the most abundant cells in the body where NO can be produced, and the conversion
`of NO to nitrate was found to involve hemoglobin. it appears that the conversion of NO to nitrate
`was more rapid in blood with high compare to low oxygen saturation of hemoglobin. This
`suggests that HbOz acts as an oxygen donor to the NO molecule in it conversion to nitrate.
`
`The present data indicate that NO is readily converted to nitrate and methemoglobin. Nitrate is
`then eliminated by renal excretion, and methemoglobin is convened to hemoglobin by
`endogenous mechanisms. If small amounts of nitrosylhemoglobin are formed, this complex can
`be disintegrated by a high oxygen tension, as in the alveoli capillaries in the lungs.
`
`

`

`
`
`EFFECTS OF EXPOSURE OF BLOOD HEMOGLOBIN TO NITRIC OXIDE
`
`INVESTIGATORS: Chiodi H, Mohler JG
`STUDY SITE: University of Southern California School of Medicine
`CITATION: Environmental Research. 1985; 37:355-63.
`STUDY DATES: not stated
`-
`.
`OBJECTIVES: To describe the reaction between hemoglobin and nitric oxide by an in vitro
`study.
`-
`.
`in vitro study
`STUDY DESIGN:
`POPULATION: humans; no other details provided
`
`less than 48 hours old was used.
`PROCEDURE: Heparinized whole human blood kept at 4°C,
`Blood was exposed to N0 gas according to a flow-tonometry procedure previously described
`(Chiodi et al. In vitro methemoglobin formation in human blood exposed to N02. Environ Res
`1983;30:9-15.). 5.6%‘CO was added to all gas mixtures to keep blood pH within the
`physiological range. A total flow of 800 mIJmin was maintained throughout the experiments.
`Figure 1 shows the amount of methemoglobin, expressed as a percentage of total hemoglobin,
`plotted against the time of nitrosylhemoglobin exposure to a circulating gas mixture (21% 02 and
`5.6% C02 in N2). Nitrosylhemoglobin was obtained by exposing 10 to 20 mL of deoxygenated
`blood to a' continuous flow of 1000 ppm of N0 in N; for 3 hours or to 100% NO for 1 hour. The
`nitrosylhe'moglobin containing blood was then exposed to the O; and C0 gas mixture for 5, 10.
`15, 30; 60, 90, and 120 minutes. Anaerobic blood samples for further analysis were taken at the
`end of each period.
`
`
`
`
`
`'0
`
`/“+.a——+..,/+‘"
`
`Hana an o. + Ilnnn : u
`no.“ In It.
`A
`loan
`(HI Numb-r at Elan-Incin-
`
`"Olin Doeor Il\\ ennui
`
`,.----
`
`...w"
`
`...—- '
`
`
`
`
`
`.Butternut-Mn‘ToutHonulelln
`
`“'
`
`'
`
`
`
`.I
`
`Itulle-IumueIIIOIIOIN‘HII'S
`
`lqutII-ratlon fun. In Inn-II.-
`
`Foo. I. MeIHh formation in blood HUI-lb equilibnled with 21% 0.. 5.6!!- CO, in N}. Previously
`learnenated human bland was enunihr-ted with In continuous flow two enlimln) of Int) ppm NO.
`3.6%- CO, in N, for 1 hr followed by I half-hour [1th with Lfi'h CO, in Na' NOHb—coaulnln; Hand
`In: then equilihrnletl for different lengths or time. as indicated on the Ibeiua. with 2|“ 0, IM 5.6%
`CD, in N, or with IOU:- Co for I hr. Hell-lb was measured with the nnuerobie modification of Evelyn-
`Nutoy method. COHb “I measured In the IL!!! CO-eairneter.
`
`
`
`
`
`U
`
`.0
`
`l7
`
`
`

`

`
`
`APPEARS THIS WAY
`on ORIGINAL
`
`
`
`I8
`
`

`

`
`
`NEETABOLISM AND EXCRETION OF NITRIC OXIDE IN HUNIANS: AN
`EXPERIMENTAL AND CLINICAL STUDY
`-
`
`.
`
`INVESTIGATORS: Wennmalrn A. Benthin o, Edlund A, et a1.
`STUDY SITE: Sahlgrenska Hospital, Gothenburg, Sweden
`CITATION: Circulation Research. 1993; 7321.121-1127.
`‘
`STUDY DATES: Not published
`'
`OBJECTIVES: To determine the metabolism and excretion of NO in humans
`FORMULATION / DOSE:
`Inhalation ofN0 by healthy subjects and in patients with severe heartfailure
`0
`25 ppm in healthy subjects
`_
`'
`.
`o
`20, 40, 80 ppm in severe heart failure patients
`Ex viva studies ofthe degradation of nitrite
`- A final sodium nitrite concentration of 50, 100, and 200 umol/L during incubation
`STUDY DESIGN: Open label study
`'
`POPULATION: Patients participated in only one substudy.
`Inhalation ‘beO by healthy subjects and in patients with severe heartfailure
`0 Eight (four men) healthy nonsmoking hOSpital staff volunteers
`0 Eight (six men) heart transplant candidates
`Ex vivo studies ofthe degradation of nitrite
`- Eight healthy donors
`‘
`Studies on the renal elimination ofN0 metabolite and analysis ofnitrate and nitrite in plasma
`and urine
`_
`.__.__
`0 Eight healthy volunteers (two men) aged 20 to 33 years.
`
`PROCEDURE:
`Inhalation of N0 by healthy subjects and in patients with severe heartfailure. All volunteers '
`were studied in the morning, while seated. After a stabilizationperiod, NO was administered for
`60 minutes in air by mask inhalation. Inhalation depth and frequency were not controlled. Blood
`samples were drawn into heparinized tubes in the basal state and every 10 minutes during‘
`inhalation and were analyzed for nitrate, nitrite, methemoglobin and nitrosylhemoglobin.
`
`-
`
`Heart transplant candidates were studied in the supine position. Nitrodilators were not allowed at
`least 12 hours before NO inhalation. NO was administered in air by mask inhalation in
`subsequent 10 minute periods. Blood samples were drawn similar to that done in volunteers.
`Additional blood samples were collected during the last two minutes of each inhalation period.
`Samples were analyzed for nitrate, nitrite. methemoglobin, and nitrosylhernoglobin.
`
`Ex viva studies ofthe degradation ofnitrite. Venous blood was obtained from eight healthy
`donors and sampled into heparinized tubes. A fraction of the blood was oxygenated prior to
`incubaticn. Eight milliliters of whole blood were incubated at room temperature with sodium
`nitrite for a final concentration of 50, 100, and 200 umol/L. The incubation was interrupted after
`2 and 15 minutes, respectively, to separate the blood into cells and plasma, and to freeze a _
`portion of the cell fraction and plasma fraction.
`
`
`
`1.9
`
`
`
`

`

`Studies on the renal elimination ofN0 metabolite. Volunteers were instructed to avoid
`nonsteroidal anti-inflammatory drugs and excessively salted nutrients for five days prior to the
`study. Voided urine was collected for 24 hours and stored at 4° to 8° C. After the urine
`collection