**[Review and Evaluation of Pharmacology and Toxicology
Data]{.underline}**

**Division of Medical Imaging and Radiopharmaceutical Drug Products**

**HFD-160**

**Reviewer: Adebayo, A. Laniyonu, Ph.D.**

**[Chemical Name]{.underline}**: N-13 Ammonia

**[CAS Number]{.underline}**: 34819-78-8

**[Structure]{.underline}:**

![](media/image1.png){width="2.4in" height="2.2in"}

**[Drug Class]{.underline}**: Positron Emission Tomography (PET)
Radiopharmaceutical

[**Indication**:]{.underline} Assessment of myocardial blood flow

**[Clinical Formulation (and components)]{.underline}**:

N-13 ammonia will be formulated as a "no carrier added" preparation by
various PET centers, this review therefore is not considering a known
formulation. Reported dose range is 8-25 mCi with specific activity
range of 200-400 mCi per micromole. The worst case scenario would
predict ammonia concentration of 0.125 micromole for the 25 mCi dose.
Different production pathways may impact on the purity and toxicological
profile of N-13 ammonia. Depending on the manufacturing process, likely
impurities and by-products include aluminum or titanium ion. Levels of
less than 1 ppm reported are considered to be of no toxicological
relevance.

**[Route of Administration]{.underline}**: Intravenous

[**Introduction/Drug History**:]{.underline}

There are many published articles concerning the clinical use of N-13
ammonia in conjunction with Positron Emission Tomography (PET) to
measure myocardial blood flow non-invasively \[1,2,3,4\]. PET
radiopharmacueticals are usually manufactured close to the site of use
in hospitals and clinics due to the relatively short half-life of these
products. Different PET centers will therefore be responsible for the
production. Thus unlike a conventional application, this review is not
considering a known formulation. A major purpose of this review is to
review the available scientific literature in an attempt to examine the
scientific basis of use, and to determine whether
Pharmacology/Toxicology studies typically conducted and considered
critical to support the safety of an NDA application have been reported
in the literature. This is not a typical NDA review, because the
experiments upon which the scientific evidence are based were not
conducted with NDA format in mind. Nevertheless, all the articles were
published in peer reviewed journals and the majority of the findings
were reported from multiple laboratories and centers. It is hoped that
the review will (1) identify the concepts that are scientifically valid
and that appear to have general support within the scientific community
and (2) identify the areas where more information is required.

[**Previous clinical experience**:]{.underline} see supervisory medical
officer review.

[**Pharmacology**:]{.underline}

![](media/image2.png){width="4.108333333333333in"
height="3.216666666666667in"}The use of N-13 ammonia in combination with
Positron Emission Tomography for non-invasive measurement of myocardial
blood flow is well documented in both clinical and non-clinical studies
\[5,6,7\]. At a very basic level, N-13 ammonia is functioning as an
extractable diffusion indicator. It diffuses freely across capillary and
cellular membranes and in the myocardium, is metabolically incorporated
into glutamine by the enzyme glutamine synthetase. The N-13 ammonia
essentially becomes trapped as N-13 glutamine in view of the fact that
glutamine is slowly released from the cell. Implicit in this schema are
the assumptions that the level of the enzyme glutamine synthetase is not
the rate limiting step, and that the intracellular and extracellular
N-13 ammonia pools equilibrate rapidly \[8,9\].

Fig 1: Model for the uptake and retention of N-13 ammonia in the
myocardium. Gl-A = glutamic acid; Glu = Glutamine; α-KG = α-
ketoglutarate (From Schelbert et al. 1981, Circulation, 63, 1259-1272).
For ease of mathematical handling and modeling, a three compartmental
model is proposed; Vascular --representing N-13 ammonia in the coronary
capillaries, extravascular- representing N-13 ammonia in the
extravascular compartment , the metabolic comparment represents NM-13
ammonia metabolically trapped as glutamine intracellularly. The glutamic
acid-glutamine is the major trapping mechanism with minor contribution
from the ketoglutarate-glutamic acid reaction.

The uptake of N-13 ammonia is governed by two main factors: the
capillary blood flow and the extraction efficiency, i.e., the avidity
with which the N-13 ammonia is extracted from the blood into the
extravascular space. N-13 ammonia exists in the blood primarily in its
ionic species, the ammonium ion. It is extracted from the blood via
mechanisms including ammonium ion substituting for potassium ion in the
sodium-potassium pump or through passive diffusion across the capillary
and sacrolemmal membranes. The extracted N-13 ammonia is retained by the
myocardium through incorporation into the pool of amino acids. The
trapping by the myocardium results in good contrast myocardial images.
Various studies have demonstrated that at flow rates between 0 to 300
ml/min/100g, there is a linear relationship between N-13 myocardial
tissue concentration and flow. At higher flow rates, a non-linear
relationship ensues, and myocardial blood flow will be underestimated by
the N-13 ammonia method. The underestimation has been attributed to the
fact that under conditions of very high flow, metabolic trapping of N-13
ammonia becomes the rate-limiting step \[5,10\].

![](media/image3.png)

Fig: 2 Relationship between regional myocardial blood flow determined
with the microsphere technique and N-13 ammonia -positron emission
tomography (From Anil Shah et al. JACC 1985, 92-100)

A kinetic approach, separating extraction from retention has been
developed to overcome this limitation \[9,11\]. It was suggested that
N-13 ammonia may not accurately reflect myocardial blood flow during
conditions of low ~p~H, reduced levels of intracellular adenosine
triphosphate, changes in the cardiac transmembrane gradient of ammonia
or inhibition of the activity of glutamate synthetase since N-13
metabolic trapping depends on the activity of the
glutamic-acid-glutamine pathway. Other factors that may impact on the
accuracy of the measurement are the integrity of the myocardial cell
membrane and alteration in cardiac lipid metabolism \[8,12\]. While
these possibilities can not be ignored, Schelbert and colleagues \[8\]
demonstrated that metabolic trapping is relatively constant over a wide
range of hemodynamic and metabolic conditions in dogs.

**[Pharmacokinetics:]{.underline}**

N-13 ammonia rapidly clears from the circulation following intravenous
injection. The results of the study by Lockwood et al. (1979) \[13\],
indicate that ammonia clearance is a first order process at normal, and
moderately elevated ammonia concentrations. N-13 ammonia is the
principal component in the blood during the first two minutes,
decreasing in a species dependent manner to between 25 % (dog) and 50 %
(humans) within five minutes. The elimination half life of N-13 ammonia
in human blood is about 3 minutes while its myocardial half life has
been estimated to be less than 2 minutes. The principal metabolites in
the blood of humans and dogs are N-13 urea and N-13 glutamine,
accounting for over 80% of the metabolites by 10 minutes. N-13 labeled
acidic amino acids such as glutamate or aspartate account for less than
1%. The physical half life of N-13 ammonia is 9.96 minutes.

![](media/image4.png){width="5.997222222222222in"
height="2.879166666666667in"}From Rosenspire et al. (1990) J.Nucl Med
31: 163-167

Quantification of blood flow using N-13 ammonia kinetics requires
accurate determination of the N-13 ammonia activity in the blood. In
view of the fact that these metabolites are rapidly formed, myocardial
blood flow uncorrected for the contribution of these metabolites to the
arterial input N-13 ammonia level would tend to overestimate the
activity that is actually due to N-13 ammonia \[13,14,15\]. In view of
extra cardiac formation of both glutamine and glutamate and the fact
that glutamate is avidly taken up by human myocardium \[16\], the
question arises as to whether the cardiac PET image reflects the sum
total of extracted N-13 ammonia plus N-13 labeled glutamine and
glutamate. However, N-13 glutamate is a very minor metabolite accounting
for less than 1% of the total while Rosenspire et al. (1989) \[14\]
indicated that the uptake of glutamine by the heart is negligible.

N-13 ammonia uptake is mainly by the heart, brain, liver, urinary
bladder, kidneys and skeletal muscle. In both the myocardium and the
brain, N-13 ammonia is removed by single pass extraction becoming
metabolically trapped within the tissues by incorporation into the
cellular pool of amino acids.

The kidney through glomerular filtration excretes urea \[11\].

**[Toxicology:]{.underline}**

No study addressing the toxicology of N-13 ammonia was identified.
However, the pathophysiology of excess ammonia is described in medical
textbooks. Elevation of blood ammonia level secondary to seriously
impaired liver function occur in several clinical situations since the
liver is the major site of ammonia detoxification in the body. The
skeletal muscle becomes an important site for metabolic trapping of
ammonia in cases of advanced liver diseases and portal-systemic shunting
\[13\]. In such cases, experimental data in rats and to some extent in
humans, show increases in the rates of ammonia uptake from the plasma
and of glutamine release by the skeletal muscle in cases of chronic
liver disease and hyperammonemia. \[13\] Moreover, in vitro experiments
using rat skeletal muscle indicate that the concentration of ammonia in
the muscle is rate limiting in muscle glutamine production.

The blood ammonia concentration is less than 35μmol/L. The low level is
important, as ammonia is toxic at high concentration and causes
neurological problems \[17\]. The problem of defining toxic levels is
compounded by technical difficulties in measuring ammonia level. Ammonia
levels more than twice the normal range for a particular laboratory is
considered significant, and a serum level greater than five times normal
is a medical emergency \[18\]. Acute ammonia toxicity in humans rapidly
produces coma, profuse sweating, grunting respiration, vomiting,
diarrhea, and decerebrate rigidity with or without seizure \[18\]. The
LD~50~ of intravenously administered ammonium chloride in rats is
30mg/kg \[19\]. As For the N-13 ammonia-PET procedure, total amount of
ammonia usually administered is less than 0.02μmol/L. This amount of
ammonia will not cause a significant alteration in the circulating level
of ammonia in the body.

**[Carcinogenicity:]{.underline}**

No study addressing the carcinogenicity of N-13 ammonia was identified.

**[Immunotoxicology:]{.underline}**

No study addressing the immunotoxicology of N-13 ammonia was identified.

**[Reproductive Toxicology:]{.underline}**

No study addressing the reproductive toxicology of N-13 ammonia was
identified.

**[Genotoxicity:]{.underline}**

No study addressing the genotoxicity of N-13 ammonia was identified.

**[Overall Summary:]{.underline}**

The purpose of this survey of published articles on N-13 ammonia is two
folds. (1) Examine the scientific basis of its use to measure myocardial
blood flow non-invasively. (2) Determine whether pharmacology/toxicology
studies typically conducted and considered critical to support the
safety of an NDA application have been reported in the literature.

The pharmacological basis of action seems to be well understood. There
is sufficient scientific evidence in the literature to support the use
of N-13 ammonia in conjunction with Positron Emission Tomography (PET)
to measure myocardial blood flow. As stated in the pharmacology section
of this review, N-13 ammonia is functioning as an extractable diffusion
indicator. The mathematical, pharmacokinetics and biochemical principles
involved appear to be rational, and generally accepted within the
scientific community. Impact of confounding factors such as extra
cardiac formation of glutamine and glutamate, extraction efficiency of
N-13 ammonia at high myocardial flow rate and changes in metabolic
conditions on the accuracy of data generated were addressed in the
literature surveyed. This reviewer concurs that there is sufficient
evidence to support the concept that the trapping of N-13 ammonia in the
myocardium as N-13 glutamine is the "pharmacological basis of action" of
N-13 ammonia as used in PET studies to measure myocardial blood flow.

The issue of whether pharmacology/toxicology studies typically conducted
and considered critical to support the safety of an NDA application have
been reported in the literature was examined. No study specifically
addressing the toxicology, carcinogenicity, genotoxicity, reproductive
toxicology or immunotoxicology of N-13 ammonia was identified in the
literature. However, two factors ameliorate the lack of data; Extensive
clinical experience without reports of safety related concerns (see
supervisory medical officer review) and the fact that N-13 ammonia is an
endogenous constituent administered at a very low dose. Relative to
blood concentration, the amount of ammonia contained in PET applications
is small and not expected to pose any problem. N-13 ammonia will be
formulated as a "no carrier added" preparation by various PET centers.
Radiation dose range is 8-25 mCi with specific activity range of 200-400
mCi per micromole. The worst case scenario would predict an ammonia
concentration of 0.125 micromole for the 25 mCi dose. The physical
half-life of N-13 ammonia is 9.96 minutes. Depending on the
manufacturing process, likely impurities and by products include
aluminum or titanium ion. Levels of less than 1 ppm reported are
considered to be of no toxicological relevance.

Overall, N-13 ammonia is used to measure myocardial blood flow in
humans. For such use, only low doses are required which would not be
expected to alter the normal blood concentration of this endogenous
constituent. The pharmacological basis of action is well understood and
clinical evidence suggests that N-13 ammonia as used in the literature
is essentially non-toxic to humans. Although not all data typically
required by pharmacology/toxicology in support of an NDA application can
be identified in the literature survey, it is the considered opinion of
this reviewer that additional non-clinical data are unnecessary at this
time to permit a decision that the drug substance is reasonably safe.

**[Recommendations:]{.underline}**

It is recommended that N-13 ammonia be considered for approval for
measurement of myocardial blood flow

**[Internal Comments:]{.underline}** None

**[Reviewer's Signature:]{.underline}**

Adebayo, A. Laniyonu Ph.D.

­­­­­­­­­­­­­­­­­­­­­­

Date

**[Team Leader Concurrence:]{.underline}**

Nakissa Sadrieh, Ph.D.

­­­­­­­­­­­­­­­­­­­­­­

Date

References:

1\. Gould, L.K., Goldstein, R.A., Mullani, N.A., Kirkeeide, R.L., Wong,
W.H., Tewson, T.J., Berridge, M.S., Bolomey, L.A., Hartz, R.K.,
Smalling, R.W., Fuentes, F. & Nishikawa, A. (1986) Noninvasive
assessment of coronary stenoses by myocardial perfusion imaging during
pharmacologic coronary vasodilation. Clinical feasibility of positron
cardiac imaging without a cyclotron using generator-produced
rubidium-82. J Am Coll Cardiol 7: 775-89

2\. Demer, L.L., Gould, L.K., Goldstein, R.A. Kirkeeide, R.L., Mullani,
N.A., Smalling, R.W., Nishikawa, A.. & Merhige, M.E. (1989) Assessment
of coronary artery disease severity by PET: Comparison with quantitative
arteriography in 193 patients. Circulation, 79: 825-835

3\. Schelbert, H.R., Wisenberg, G., Phelps, M.E., Gould, K.L., Henze,
E., Hoffman, E.J., Gomes, A.. & Kuhl, D.E. (1982) Noninvasive assessment
of coronary stenoses by myocardial imaging during pharmacologic coronary
vasodilation. Am J Cardiol. 49: 1197-1207

4\. Di Carli, M., Sherman, T., Khanna, S., Davidson, M., Rokhsar, S.,
Hawkins, R., Phelps, M., Schelbert, H. & Maddahi, J. (1994) Myocardial
viability in asynergic regions subtended by occluded coronary arteries:
Relation to the status of collateral flow in patients with chronic
coronary artery disease. J Am Coll Cardiol 23: 860-868

5\. Niemeyer, M. G., Kuijper A.. M., Gerhards, L.J., D'Haene E.G. & Van
der Wall E.E. (1993) Nitrogen-13 ammonia perfusion imaging: Relation to
metabolic imaging. Am Heart J 125: 848-854

6\. Storch-Becker, A., Kaiser, K. & Feinendegen L.E (1988) Cardiac
nuclear medicine: Positron emission tomography in clinical medicine. Eur
J Nucl Med 13: 648-652

7\. Meyer G.J., Waters S.L., Cohen H.H., Luxen A., Maziere B. &
Langstrom B.(1995) PET radiopharmaceuticals in Europe: current use and
data relevant for the formulation of summaries of product
characteristics (SPCs). Eur J Nuclear Med 22:1420-1432

8\. Schelbert, H.R., Phelps M.E., Huang, S., MacDonald N.S., Hansen, H.
& Selin, C. (1981) N-13 ammonia as an indicator of myocardial blood
flow. Circulation 63: 1259-1272

9\. Hutchins G.D., Schwaiger M., Rosenspire K.C., Krivokapich J.,
Schelbert, H. & Kuhl, D. (1990) Noninvasive quantification of regional
blood flow in the human heart using N-13 ammonia and dynamic positron
emission tomography imaging. J Am coll Cardiol 5: 1032-1042

10\. Shah, A., .Schelbert, H.R., Schwaiger, M., Henze, E., Hansen, H.,
Selin, C. & Huang SC. (1985) Measurement of regional myocardial blood
flow with N-13 ammonia and positron emission tomography in intact dogs.
J Am Coll Cardiol 5: 92-100

11\. Muzik, O., Beanlands R.S.B., Hutchins, G.D., Mangner, T.J., Nguyen,
N. & Schwaiger, M. (1993) Validation of nitrogen --13-ammonia tracer
kinetic model for quantification of myocardial blood flow using PET. J
Nucl Med 34: 83-91

12\. Rauch, B., Helus, F., Grunze, M., Braunwell, E., Mall, G.,
Hasselbach, W. & Kubler, W. (1985) Kinetics of ^13^N-ammonia uptake in
myocardial single cells indicating potential limitations in its
applicability as a marker of myocardial blood flow. Circulation 71:
387-393

13\. Lockwood, A.H., McDonald, J.M., Reiman, R.E., Gelbard, A.S.,
Laughlin, J.S., Duffy, T.E. & Plum F. (1979) The dynamics effect of
ammonia metabolism in man. J. Clin. Inves. 63: 449-460

14\. Rosenspire K.C., Schwaiger, M., Mangner, T.J., Hutchins, G.D.
Sutorik, A & Kuhl, D.E. (1990) Metabolic fate of N-13 ammonia in human
and canine blood. J Nucl Med 31: 163-167

15\. Krivokapich, J., Barrio, J.R., Phelps, M.E., Watanabe, R., Keen,
R.E., Padgett, H.C. Douglas, A. & Shine, K.I. (1984) Kinetic
characterization of ^13^NH~3~ and ^13^N glutamine metabolism in rabbit
heart. Am. J. Physiol 246: H267-H273

16\. Gelbard, A.S., Benua, R.S., Reiman, R.E., Mcdonald, J.M., Vomero,
J.J. & Laughlin, J.S. (1980) Imaging of the human heart after
administration of L-(N-13)glutamate. J Nucl Med 21:988-991

17\. Oxford textbook of clinical hepatology (1991) Ed: McIntyre N.,
Benhamon, J.P., Bircher, J., Rizzetto, M. & Rhodes, J. Oxford University
Press

18\. Flannery, D.B., Hsia, Y.E. & Wolf, B. (1982) Current status of
hyperammonemic syndrome. Hepatology 2: 495-506

19\. The Merck Index 11^th^ edn (1989), Rahaway NJ: Merck