United States Patent
`
`[:91
`
`Brusilow et al.
`
`[54] PROCESS FOR WASTE NITROGEN
`REMOVAL
`
`[75}
`
`Inventors: Saul W. Brusilow, Baltimore; Mark
`L. Batshaw, Pikesville, both of Md.;
`Norman 5. Radin, Ann Arbor, Mich.
`
`[73] Assignee: The Johns Hopkins University,
`Baltimore, Md.
`
`[21] App]. No.: 135,635
`
`[22] Filed:
`
`Mar. 31, 1930
`
`Int. C1,! ............................................ ..A61K31/19
`
`[51]
`[521
`[58} Field of Search ....................................... .. 424/317
`
`
`
`[11]
`
`[45]
`
`4,284,647
`
`Aug. 18, 1981
`
`Shih, “The Metabolic Basis of Inherited Diseases", pp.
`362-386, McGraw-Hill, (1978).
`Sherlock, “Diseases of the Liver and Biliary Systems",
`5th ed., pp. 84-106, Blackwell, Oxford, (1975).
`Walser, “The Kidney”, vol. 2, pp. 1613-1642, W. B.
`Saunders, (1976).
`Close, N. Eng. J. Med. 290, pp. 663-667, (1974).
`Lewis, J. Biol. Chem. 18: 225-231, (1914).
`Batshaw et al.. N. Eng. J. Med., 292: 1085-1090 (1975).
`Batshaw et al., Pediat. Res., 12: 22l—224, (1978).
`Ijpma et al., Clin. Chem., 24/3: 489-492, (1978).
`Chaney et aI., Clin. Chem, 8: 130-132, (1962).
`Fleck, et al., Clin. Chimica Acta., 11: 2-12, (1965).
`LaDu et a1., “Fundamentals of Drug Metabolism and
`Drug Disposition", Williams &. Williams, (1971), pp.
`149-186.
`
`Primag; Examiner—Frank Cacciapaglia, Jr.
`Attorney, Agent, or F:'rm—Cushman, Darby & Cushman
`
`[S7]
`
`'
`
`ABSTRACT
`
`A process for controlling waste nitrogen accumulation
`diseases in humans which comprises administering an
`effective amount of at least one compound selected
`from the group consisting of benzoic acid, phenylacetic
`acid and the non-toxic, pharmaceutically-acceptable
`salts of the acids to a human suffering from waste nitro-
`gen accumulation.
`
`9 Claims, 8 Drawing Figures
`
`
`
`[56]
`
`References Cited
`PUBLICATIONS
`
`CA. 3: 33292 (1914).
`CA. 3: 3622 (1914).
`CA. 16: 21655 (1922).
`CA. ".17: 34783” (I912).
`Sherwin et a1.. .1. Biol. Chem, (1919), 40: 259-263.
`James et al., Proc. Royal Soc. London, 13, 132: 25-35,
`(1972).
`McCollum et al., J. Biol. Chem., (1913-14) 16: 295-315;
`321-325.
`Ziter et ai.. Pediat. Res., 2: 250~253, (1968).
`
`1
`
`PAR PHARMACEUTICAL, INC.
`PAR PHARMACEUTICAL, INC.
`EX. 1018
`
`EX. 1018
`
`

`
`U.S. Patent
`
`Aug. 18, 1981
`
`Sheet 1 of6
`
`4,284,647
`
`4
`
`ofiez;
`$1c/72?/17//VF £5:01.79%?/?f£'4-Z-Jr/V//:5
`£94?
`
`(9.5
`
`AP VRPOL/#5 .5&¢A?E(?X>’£/7?”E
`
`.
`
`I
`77¢/EP.-P1562/»{?5.0A35 gP‘PAWr’W/P)’
`5’: (‘£54 «5V/V”’£-5/5-
`
`“"'5§
`07-5
`
`
`
`"<1
`
`6\
`
`id
`
`an;
`7,531 /ACE
`
`4”‘? are
`
`/ 4’-zumMz.?r£5’/J’/E‘Es4’MM: /7190.5?!/4/“E—— --> 0,80214?TE
`‘7f’V/"'7""7N£
`c”r‘€‘/"' “V5
`/W/7196‘//0/Yl7z8/fl/V
`
`0 I
`
`
`
`10/F/V//7‘///V15"
`
`C’/fE’&‘2I A I/VE-
`
`4/ff-47
`
`/75
`
`£’5F'»04E’7"/JFE‘
`
`.424;/NASH
`5,/efi/A,/NE
`
`1.
`
`,4?,«?.5//V/A/05:/cc/More‘
`c’yra5az_
`
`’9
`.«¢‘,x//wficaci-';*£
`
`,z;'c;- - 4:57 Vzéz z/2.74MAPTEKQ dz?-.e'q:22.<a '/=~rae c;a,5_)
`,0; — APEG/N’ /A/0.51/(‘CIA-’r4’5£
`,5; 5 —~ A?,€’6‘-//V/A/0.5 U66 //W C -A36’/17 5)//t//‘A4:-‘r',z.7.-3.-_='
`g p 5 _ J’.-!.?J2.r_‘5’x.7/14 rz -PA/5.5295/«<7.='2'.=' SVA//‘,5/£767.55
`er —-— .51; /1722M’/xv/7.55
`:3 .5 ~—61.£ K//'AM¢'/A/E .S.VN.7’//£7.47-SE
`CC .86? —~,z?z;=7//AH./.1-‘7'042.4 UTE?/2.475
`an 7‘—- c7/Z/W 77-! (‘NE .4‘?/W//V«97'fl?'4‘7/V.‘5/E:!5‘7F?.4?5.¢E'
`0 7c: 4 52 A//' 7/:0/VE 77£’,4?N.s(=.42;a5',0/11')/L .455
`P0 - P501 /NE :3 X/z7»=?5.¢-.:_
`
`2
`
`
`
` ENmyz/,6//Vgzey/I/223555/VP526917(fie:-er/xv,w£
`
`
`
`
`
`U1
`
`4:-
`
`*‘to
`
`

`
`U.S. Patent
`
`Aug. 18, 1981
`
`Sheet 2 of 6
`
`4,284,647
`
`
`
`m\<§.§fi..m%mfimwnx>&§p$<>.m63§$mS
`
`
`
`
`
`6
`5
`4
`//49¢‘/8.5 A?!-’7z:7e _5’£/vzaqrg
`
`3
`
`

`
`U.S. Patent
`
`Aug. 18, 1981
`
`Sheet 3 0f6
`
`4,284,647
`
`E/-7-‘E:/"aFJoJ/I/M15'EA/Zo.4r5
`.z7.a7’M//V/.57-E'A777&A/ av flzflsxwfi
`A7/W/WOA//é//V (E:/£4 //V 7/-/.5‘ (3¢?.5E
`at ..:'2‘fZ?.Z:‘rZ£” J"//£95/V/C’ ,4 /V56Z7/5fc7.5£
`
`/?4?r/5/v7=-A? -
`
`PAr/¢=‘/vr~.B-
`
`asvz?/z/M.E>£/vza/.?7'£
`.5”/>79.
`
`1
`
`£3’
`x
`\5O
`§‘
`§ 40
`:;\
`
`30
`
`20
`
`I 0
`
`O
`
`S §
`
`%§§
`
`Vt
`
`EV
`
`’
`‘S:
`
`o
`
`1
`
`4
`5
`2
`77/WE /5/oo’/E5)
`
`5
`
`6
`
`F/pr
`
`4
`
`

`
`U.S. Patent
`
`Aug. 13, 1931
`
`Sheet 4 of 6
`
`4,284,647
`
`EFFECT OF JOD/-d//Y.-’ PHFN Y1 #657975
`,47.Z?/W//v.5?‘/?é7.77fl/V ./.7/V /-7: #5/WA’ .4/WMWA//Z/A4
`4.»:-'1/.»_.c2 av THE 534.55 0;: /-/EPX?/"'/C” ca/ma?
`
`
`
`
`
`/9445.47%?x=?fl/MON/U/W,:/A»//zzas/zxfz/6
`
`CF00
`
`800
`
`700
`
`600
`
`500
`
`400
`
`300
`
`2 0 0
`
`I00
`
`T
`
`.9” 45.-{’flM'q5 5017/om
`/7;/£Iv >14 A'c’E7'.¢?'E
`
`I
`
`2
`
`5
`
`4
`
`5
`
`6
`
`7”/ME /2742 V5,)
`
`,¢7;;5
`
`5
`
`

`
`U.S. Patent
`
`Aug. 13, 1981
`
`Sheet 5 of6
`
`4,284,647
`
`EFFECT 0/—‘ JQE7/L/M J.‘5’f/VJOAVE
`,4_3',ay//V/_'5;7€,4?f/&’A/ 0/\/'u§‘£‘z.E’L/A7 JZEA’
`/V//'7€&5f'/V //V 7,€54777Ar¢- &’x€£/I4//=7
`
`I40
`
`I20
`
`HO
`
`,2_z.45e.'z89IoHI2
`
`7/Ar/.»<_=(z7»9y.5)
`
`/-79:7
`
`6
`
`

`
`‘US. Patent
`
`Aug. 13, 1981
`
`Sheet 6 of6
`
`4,284,647
`
`/7/E 5-EFL-Tr ac" 50.57/4/M 5'5/vzozzrg
`/917/W/A/.‘577E.47z".-.¢/V :9/V .%0.5M4' .49’/J¢/W49’/v/.*z’/1;’
`,4 5:/5,4 //v 7//.5 ($47.55 JP‘ /EE 3/5:5 .‘53Vrv2:?£’&’/WE
`
`.,5‘¢9.Z7/.//49' .Z'5£"/'lE'0A77‘E'
`
`
`
`/=71/7.;'7‘A7»«?A7M'/3¢6?.A//ww,1!Amas/5//2.=:e
`
`h D 9
`
`300
`
`IQ C)
`
`O
`
`5'
`
`; 0
`
`I 5
`
`2 O
`
`2 5'
`
`7//ms‘ //-/(2‘c'/'85)
`
`/|/d»€A!fll.- RANGE
`
`F/9.6
`
`7
`
`

`
`1
`
`4,2 84, 64?
`
`PROCESS FOR WASTE NITROGEN REMOVAL
`
`The invention described herein was made in the
`course of work under a grant or award from the De-
`partment of Health and Human Services.
`The present invention relates to a process for treating
`humans who suffer from waste nitrogen accumulation
`in the body.
`When the human body is functioning normally, waste
`nitrogen is effectively excreted, primarily in the form of
`urinary urea. However, in certain abnormal situations,
`cg. in the event of kidney or liver failure or inborn
`errors of urea synthesis, waste nitrogen will accumulate
`in the body unless positive steps are taken to avoid this.
`In the case of a urea-cycle enzyme defect. the major
`metabolic abnormality is the inability of the body to
`convert waste nitrogen into urea. As a consequence,
`various nitrogenous metabolites accumulate in the
`body, the most toxic being ammonium although other
`materials such as glut-amine. glutamate and alanine, are
`usually also present.
`Previous therapeutic approaches for treating patients
`with urea-cycle enzymopathies (as well as other nitro-
`gen accumulation diseases) have been designed to re-
`duce the requirements for urea synthesis by quantitative
`and qualitative manipulation of dietary protein, amino
`acids and/or their nitrogen-free analogues. Generally
`speaking, however, the mortality and morbidity of in-
`born errors of the urea-cycle remain high and success
`has been measured more in terms of increased survival
`time than in the elimination of the undesired effects.
`Thus, for example, even with the above cited therapeu-
`tic approaches, it does not appear that children with the
`neonatal form of these diseases who survive past one
`year of age, can normally do so without recurrent epi-
`sodes of hyperamrnonernic coma or mental retardation.
`The present invention proposes to deal with the prob-
`lem of waste nitrogen accumulation caused by urea
`cycle enzyme deficiencies, or other abnormalities in
`body function {e.g. renal or hepatic failure) which cause
`such accumulation. by providing an alternative path-
`way to urea synthesis involving the formation of nitro-
`gen-containing metabolites other than urea which can
`be readily excreted from the body as urinary nitrogen.
`To this end, the invention contemplates converting the
`waste nitrogen into certain amino acid acylation prod-
`ucts for urinary discharge from the body. This is accom-
`plished. according to the invention, by administering an
`effective amount of benzoic acid, phenylacetic acid
`and/or the non-toxic, pharmaceutically-acceptable salts
`of these acids, preferably the sodium salts, to one suffer-
`ing from. or subject to, waste nitrogen accumulation.
`The benzoic acid or salt thereof converts waste nitro-
`gen in the body to hippuric acid. an amino acid acyla-
`tion product, which is readily and effectively excreted
`from the body as urinary nitrogen. Likewise the phenyl-
`acetic acid, or salt
`thereof, causes the formation of
`phenylacetylglutamine, an amino acid acetylation prod-
`uct which is also easily and quickly excreted as urinary
`nitrogen. The invention thus effectively by-passes urea
`synthesis while providing a ready way of excreting
`waste nitrogen from the body.
`Benzoic acid or phenylacetic acid. or salts thereof.
`may be used separately or as mixtures of the acids and-
`/or salts. The amount of acid and/or salt administered
`for present purposes will vary rather widely from case
`to case. Normally. however. the daily dosage of acid
`
`2
`in the range of 100-400
`and/or salt utilized will fall
`mg/ltg body weight for children and from 7.5 to 15
`grams for adults. Generally speaking, the size and fre-
`quency of the dosages given at any time can be varied as
`desired provided the indicated total daily dose is not
`significantly modified. The administration may be car-
`ried out intravenously or orally (e.g.
`in the form of
`sterile injectable solutions, pills. tablets. capsules, solu-
`tions, suspensions or the like).
`It has previously been disclosed that benzoic acid or
`phenylacetic acid as such. or as salts thereof, form hip-
`puric acid and phenylacetylglutamine, which can be
`discharged from the body as urinary nitrogen. Thus, it
`is known from studies by Lewis (J. Biol. Chem.
`I8,
`225-231 (1914)) that benzoic acid or sodium benzoate,
`when orally administered to a healthy man.
`is elimi-
`nated rapidly as urinary hippuric acid. According to
`Lewis, the urinary hippurate nitrogen replaces the uri-
`nary urea nitrogen so that there is little change in total
`urinary nitrogen excretion.
`The Lewis publication, as well as other publications
`on benzoate or phenylacetate metabolism“) are con-
`. cerned with the ability of the body to detoxify benzoie
`acid, phenylacetic acid or their sodium salts and thus
`eliminate beuzoate or phenylacetate from the body.
`There is no disclosure in these prior publications of the
`present concept of using benzoic acid, phenylacetic acid
`or their salts to convert toxic waste nitrogen to urinary
`hippuric acid or phenylacetylglutamine in order to re-
`move waste nitrogen from the body as an alternative, or
`adjunct, to urea synthesis with subsequent urinary ex-
`cretion.
`mI.aDu. B. N.. Mandel. H. Ce. Way. E. L. Fundamentals of Drug
`Metabolism and Drug Disposition. Williams & Wilkins I911.
`It is also noted that Shiple and Sherwin (J. Am.
`Chem. Soc. 44: 618-624, 1922) have disclosed that the
`oral administration of benzoic acid and phenylacetic
`acid results in the formation and excretion of urinary
`hippuric acid and phenylacetylglutarnirle, respectively.
`The hippuric acid or phenylacetylglutamine is formed
`at the expense of urinary urea. The authors indicate that
`following both benzoate and phenylacetic acid adminis-
`tration, the partition of urinary nitrogen was dramati-
`cally altered so that urea nitrogen accounted for as little
`as
`12% of urinary nitrogen and hippurate and
`phenylacetylglutamine nitrogen accounted for as much
`as 60% of urinary nitrogen.
`Shiple and Sherwin, like Lewis, are concerned with
`the detoxification of benzoic acid and phenylacetic acid
`(or salts thereof) when administered to humans rather
`than being concerned with the use of these materials to
`detoxify waste nitrogen accumulating in the body.
`Other publications describing the conversion of
`phenylacetic acid to phenylacetylglutamine when the
`acid is administered to humans include Ambrose et al
`“Further Studies on the Detoxification of Phenylacetic
`Acid". J. Biol. Chem. 1933; 101: 669-675; and James et
`al “The Conjugation of Phenylacetic Acid in Man,
`Sub-Human Primates and Some Non-Primate Species”,
`Proc. R. Soc. Lond. B. I972; I82: 25-35. Of these publi-
`cations, Ambrose et al reported that they found 98% of
`orally administered phenylacetic acid (5-7 g/day for 3
`months in man) was excreted in the urine as phenylace-
`tylglutamine while James et al disclose that they gave
`85 mg/kg of phenylacetic acid to a man and found that
`91% of this dose was excreted in the urine as phenylace-
`tylglutamine. No adverse effects are noted in these
`studies although an earlier paper (Sherwin et al, J. Biol.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`55
`
`60
`
`65
`
`8
`
`

`
`4,284,647
`
`3
`Chem. 1919; 40: 250-263) reported thirst, nausea. and
`dizziness after a single dose of phenylacetic acid.
`The conjugation of benzoic acid with glycine to form
`hippuric acid involves two reactions catalyzed by the
`mitochondrial-matrix enzymes. benzoyl thiokinase and
`a glycine-specific transacylase. Lewis‘s studies showed
`that this pathway can convert 10 g sodium benzoate to
`hippuric acid in less than 24 h in the adult male. Hippu-
`ric acid is well suited for renal excretion because its
`renal clearance is five times the glomerular filtration
`rate.
`
`It will be appreciated from the foregoing that benzoic
`acid and sodium benzoate can be used interchangeably.
`Sodium benzoate,
`like the acid,
`is non—toxic (except
`perhaps in neonatal hyperbilirubinaemia, where in vitro
`experiments suggest that it competes for bilirubin-bind-
`ing sites on albumin) and has been used to treat infants
`with non-ketotic hyperglycinaemia (Ziter et al, Pediat.
`Res. 2: 250-253. i968).
`The synthesis of phenylacetylglutamine using phen-
`ylacetic acid also involves a two-stage reaction which,
`in this case, includes acetylation of glutamine to form
`the desired phenylacetylglutamine, the latter, like hip-
`puric acid, being rapidly excreted by the kidney. Acety-
`lation and excretion of glutamine rather than glycine
`has three advantages: (i) glutamine contains two nitro-
`gen atoms per molecule, (ii) it accumulates in urea-cycle
`enzymopathies. and (iii) it is in equilibrium with gluta-
`mate, the nitrogen donor for urea synthesis.
`The accompanying drawings serve to further illus-
`trate the invention. Of these, FIG. 1 diagrammatically
`shows the pathways of urea synthesis,
`including the
`known points of genetic defects. In all these disorders
`nitrogen accumulates. usually in the form of ammo-
`nium. glutamine, glutamate, and alanine. Furthermore,
`each defect is characterized by the accumulation of the
`specific substrate for the deficient enzyme which may
`be carbamyl phosphate (manifested by accumulation of
`orotic acid), citrulline, argininosuccinic acid. and argi-
`nine. It will be appreciated that the purpose of the in-
`vention is to provide an alternative to the indicated urea
`synthesis for excretion of urinary nitrogen.
`FIGS. 2-8 graphically illustrate various results ob-
`tained by using the invention as discussed in more detail
`hereinafter.
`The invention is also illustrated by the following:
`EXAMPLE I
`
`Patients with urea-cycle enzymopathies were studied
`to determine the effect of sodium benzoate or phenyla-
`cetic acid on urinary nitrogen excretion and to deter-
`mine the effectiveness of sodium benzoate on plasma
`ammonium levels in patients in hyperammonemic coma.
`Urinary nitrogen excretion was studied in a clinically
`stable 17-year old, 40 kgfemale, with carbamyl phos-
`phate synthetase (CPS) deficiency who was maintained
`on a diet containing 2'? g of protein and l800 calories
`while the following protocols were followed. Sodium
`benzoate (6.25 g/d) or phenylacetic acid (6.4 girl) was
`administered orally for 11 and 8 days, respectively.
`Each experimental period was preceded and followed
`by a control period totalling 8-10 days. Daily incom-
`plete twenty—l‘our hour urine collections (the patient
`was episodically incontinent) were made. Urinary urea
`nitrogen, ammonium nitrogen. and creatinine were
`measured by standard techniques. Hippurate and
`phenylacetylglutamine were measured by reverse phase
`liquid chromatography using a Waters C13 column with
`
`4
`a 20% methanol solution of 0.0! M acetate buffer, pH 3,
`as an eluant. Total urinary nitrogen was measured after
`Kjeldahl digestion“) oflll ml urine following which 0.1
`ml aliquots of the digestion mixture (diiuted to 30 ml)
`were analyzed for ammonium by the indophenol reac-
`tionlll. Plasma glycine was measured by automated
`ion-exchange chromatography. Plasma glutamine, glu-
`tamate and alanine were measured by fiuorometric en-
`zymatic techniques”? and plasma ammonium was mea-
`sured either on venous plasma by the Dupont ACAW or
`on capillary plasma by a cation exchange method”).
`“in Fleck and H. N. Munro. Clin. Chim. Acta. I .2 (I965).
`(33.4. L. Chaney and E. P. Marbach. Clin. Chem. 5. I30 (1962).
`'-“M. Batsltaw and s. Brusilow. Pecliat. Res. 12. 221 (1913).
`“is. ‘r. Ijpma. B. G. Blijenberg. B. Leijnse. Clin. Chem. 24.489.
`‘SIM. Batshaw. s. Brusilow, M. Walser. New Engl. J. Med. 292. toss
`(1915).
`FIG. 2 compares the amount and partition of urinary
`nitrogen during the control and sodium benzoate ad-
`ministration periods. The partition of urinary nitrogen
`(mg nitrogen per mg creatinine:SEM) of the patient
`while receiving sodium benzoate is shown in shaded
`bars compared to a control period (open bars). There
`was a 53% increase in total urinary nitrogen excretion
`while the patient was receiving sodium benzoate as
`compared to the control period. Urinary hippurate ni-
`trogen accounted for this increase in urinary nitrogen
`excretion. The plasma concentrations of several urea
`precursors during the control (n=6) and experimental
`periods (n=8) were respectively (,u.M+SEZM): ammo-
`nium, 29.5_-|:l.0 vs 22.9:2.2, p<0.D2; glutamine.
`1675-:49 vs 14221109, alanine, 952il07 vs 90l:9l;
`glutamate, 36: 5.3 vs 2'l'i4.2. The plasma glycine lev-
`els during the control (n=4) and experimental periods
`(n =6) were similar 247:8 vs 294149.
`The effect of phenylacetic acid administration on
`amount and partition of urinary nitrogen is shown in
`FIG. 3. The partition of urinary nitrogen (mg nitrogen
`per mg creatinineiSEM} of the patient while receiving
`phenylacetic acid is shown in shaded bars and com-
`pared with a control period (open bars). There was a
`49% increase in total urinary nitrogen while the patient
`was receiving phenylacetic acid as compared to the
`control period. Urinary phenylacetylglutamine nitro-
`gen accounted for this increase in urinary nitrogen ex-
`cretion. The mean plasma concentrations (pM) or urea
`precursors during the control (n=4) and experimental
`periods (n=3) were respectively: ammonium, 29.3 vs
`17.7; glutamine. 1753 vs 1533; glutamate, 51.5 vs 26.3;
`alanine, 646 vs 670.
`The foregoing results indicate that acetylation of
`glycine by benzoic acid and acetylation of glutamine by
`phenylacetic acid with subsequent renal excretion ofthe
`respective products, hippuric acid and phenylacetyl-
`glutamine, are quantitatively significant alternative
`mechanisms of waste nitrogen disposal in patients with
`inborn defects of urea synthesis.
`Both mechanisms require adequate amounts of the
`natural precursors of the conjugate. i.e. glycine or glu-
`tamine. While the patient received benzoate for 1! days
`there was a significant decrease in the plasma ammo-
`nium level. The plasma glycine level was unchanged
`indicating that de novo glycine synthesis was.
`in this
`case, sufficient for hippurate synthesis. During therepy
`with phenylacetic acid there were apparent decreases in
`the plasma concentrations of ammonium and glutamine
`although the small number of such determinations pre-
`clude statistical evaluation.
`
`ll)
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`65
`
`9
`
`

`
`5
`The foregoing observations indicate that benzoate
`administration can be used in acutely reducing plasma
`ammonium levels during hyperammonemic episodes.
`Accordingly, four patients were given a single dose
`{orally or intravenously) of sodium benzoate (250-350
`mg/kg) during such a hyperammonemic episode. The
`effect of the sodium benzoate on plasma ammonium
`levels in the patients in hyperamtnonernic coma is
`shown in FIG. 4, the patients being: CPS, an 18-year old
`female with partial carbamyl phosphate synthetase defi-
`ciency given 250 mg/kg orally; OTC, a 6-year old fe-
`male with partial ornithine transcarbamylase deficiency
`given 250 mg/kg orally; and OTC, an ll-month old
`male with omithirte transcarbamylase deficiency given
`350 mg/kg intravenously and AS, an 11-month old
`female with argininosuccinic acid syrtthetasc deficiency
`given 300 mg/kg orally. The hatched area in FIG. 4
`denotes normal plasma levels of ammonium (l7~33p.
`M).
`In each case there was a prompt fall in the plasma
`ammonium level and clinical
`improvement following
`administration of sodium bcnzoate. This change is be-
`lieved to be a consequence of the incorporation of am-
`monium or glutamate in the tie novo synthesis of gly-
`cine by one of three pathways; from ammonium via the
`glycine cleavage complex, from glutamate via glyoxy-
`late transarnination or via de novo serine synthesis.
`FIG. 5 shows the course of plasma ammonium level
`in two adults with stable chronic liver disease who were
`each given 5 grams of sodium benzoate. The plasma
`ammonium level
`in umoles per liter is plotted against
`time (hours) after the administration of 5 grams sodium
`benzoate. The results show in one case (patient A) a
`gradual dropping of the plasma ammonium level after
`administration while in the other (patient B), the plasma
`ammonium leveled off after a relatively sharp drop for
`up to three hours after administration. In both cases, the
`results indicated effective control of the plasma ammo-
`nium level using sodium benzoate.
`FIG. 6 graphically shows the course of the plasma
`ammonium level in a 4-year old male child with termi-
`nal hepatic coma. The child was given 5 grams of so-
`dium phenylacetate at about mid-day of day 2. As
`shown, the plasma ammonium level, which was at about
`300p. mols per liter, rose immediately after administra-
`tion of the sodium phenylacetate but
`then dropped
`sharply over the course of the next day to about 50p.
`mols. While the effect of the 5 grams phenylacetate
`administration was not long-lastin g, as evidenced by the
`subsequent increase in the plasma ammonium level over
`the next several days to the point where the patient
`died, the reduction of the plasma ammonium level on
`administration of the sodium phenylacetate is an indica-
`tion that such administration would be effective in the
`treatment of hepatic coma.
`FIG. 7 shows the course of the serum urea nitrogen
`of an adult woman who was given 5 grams of sodium
`benzoate on days 8 and 9 and 10 grams of sodium benze-
`ate on days 10 and 11. As shown, the amount of the
`serum urea nitrogen (measured in milligrams per 100
`milliliters) was substantively reduced on administration
`of the benzoate.
`.
`The data shown graphically in FIGS. 54' indicate
`that sodium benzoate and phenylacetate would be use-
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`65
`
`4,284,647
`
`6
`ful in treating hyperamrnonernia in liver failure and that
`sodium benzoate would be useful in treating uremia. It
`is noted that in the case of uremia the serum urea nitro-
`gen level is the most useful guide as to the state of nitro-
`gen accumulation whereas the ammonium level is the
`most useful indicator in liver disease or urea cycle en-
`zytnopathies.
`The possible application of the present invention in
`the treatment of Reye’s syndrome is shown by FIG. 8.
`This figure illustrates the course of the plasma ammo-
`nium level in a 4-year old girl with Reye’s syndrome
`who was treated with three doses of sodium benzoate
`orally. The doses were 220 mg/kg, 100 mg/kg and 100
`mg/kg at the intervals shown. As indicated, the benze-
`ate markedly reduced the plasma ammonium level in
`less than a day's time from about 550;; mols per liter to
`below 4-Op. mols per liter, i.e. into the normal range.
`It will be appreciated from the foregoing that the
`administration of benzoic acid and/or pltenylacetic
`acid, as such, or in salt form, to form their respective
`amino acid acetylation products (hippuric acid and
`phenylacetylglutamine) for urinary nitrogen discharge,
`according to the invention,
`is of general application
`against diseases and malfunctions involving waste nitro-
`gen accumulation in the body, e.g. urea-cycle en-
`zymopathies, portal-systemic encephalopathy, Reye‘s
`syndrome, and uraemia.
`Having described the invention, what is claimed is:
`1. A process for controlling waste nitrogen accumu-
`lation diseases in humans, caused by an impairment in
`the normal synthesis of urea from ordinary waste nitro-
`gen in the body or in the normal excretion thereof, said
`process comprising administering an effective amount
`of at least one compound selected from the group con-
`sisting of benzoic acid, phenylacetic acid and the non-
`toxic, pharmaceutically-acceptable salts of said acids to
`a human suffering from such waste nitrogen accumula-
`tion disease, the amount of said compound used being
`sufficient to react with the waste nitrogen to form an
`amino acid acylation product for urinary discharge of
`said product.
`2. The process of claim 1 wherein a mixture of hen-
`zoic acid and phenylacetic acid, or salts thereof, is ad-
`ministered.
`3. The process of claim I wherein the administration
`is continued until
`the accumulated waste nitrogen is
`discharged as urinary nitrogen.
`4. The process of claim 1 wherein the human is one
`with a urea-cycle enzymopathy.
`5. The process of claim 1 wherein the human is one
`suffering from uremia.
`6. The process of claim 1 wherein the human is one
`suffering from a hepatic disorder.
`7. The process of claim 1 wherein the human is one
`suffering from Reye's syndrome.
`8. The process of claim 1 wherein the administration
`of the benzoic acid or phenylacetic acid, or salts
`thereof, synthesizes hippuric acid and phenylacetyl-
`glutamine, respectively, and the synthesized product is
`discharged as urinary nitrogen.
`9. The process of claim 1 wherein the salt is sodium
`salt.
`t
`i
`t
`t
`t
`
`10
`
`10