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Патент USA US3053747

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United States Patent 0
Patented Sept. 11, 1962
2
1
amples are adrenal, gonadal and thyroid hormones, vari
3,053,737
ous phenols and alcohols bearing a free hydroxyl group,
various amines such as aniline, p-phenetidine, and p-tol
uidine. The administration of these aglycones to certain
species of animals and to- humans leads to the excretion of
the corresponding glucuronide in the urine.
(2) Glucuronic acid.—Glucuronide formation takes
N-ACETYL-p-AMINOPHENOL ANTIINFLAM
MATORY STEROID COMPOSITIONS
Willard J. Johnson, % Frank W. Homer Ltd.,
Box 959, Montreal, Quebec, Canada
No Drawing. Filed Nov. 9, 1959, Ser. No. 851,513
Claims priority, application Canada Sept. 4, 1957
7 Claims. (Cl. 167-77)
place in the liver. Uridinediphosphate glucuronic acid
(UDPGA), the “active” form of glucuronic acid has been
This invention relates to a pharmaceutical preparation 10 shown to participate in glucuronide synthesis. The reac
tion involves the transfer of glucuronic acid from UDPGA
to the aglycone, and is catalyzed by an enzyme, glucuronyl
transferase. When the aglycone is an alcohol or phenol
More particularly the present invention is directed to the
the reaction can be schematized as follows:
inhibition of the glucuronylation of steroids so as to effect
a decreased rate of inactivation of administered steroid 15
glucuronyl
‘for inhibiting the glucuronylation of therapeutically active
compounds which become glucuronylated in the body.
and consequently, increased biological efficacy.
R-OH + UDP-glucuronic acid -—->
transferase
Steroids are administered for various purposes including
R O——glucuronlc acid + UD P
the treatment of rheumatoid arthritis, gouty arthritis, os
The same enzyme catalyzes the transfer of the glu
teoarthritis, allergic arthritis, bursitis, myositis, ?brositis,
bronchial asthma, rheumatic fever, allergic diseases and 20 curonic acid moiety of UDPGA to a wide range of agly
cones to form the corresponding glucuronides.
in?ammatory ocular diseases. The effectiveness of this
The concentration of free hydrocortisone in the blood
treatment is however hampered by rapid inactivation and
plasma of normal human subjects ranges from 6 to 25
excretion of the aministered steroid. Thus, Migeon et al.
micrograms per 100 ml., with a mean of 15 14.5 (4). The
J. Clin. Endoc. and Metab. 16, 1137, 1956 found that
hydrocortisone, administered intravenously to man disap 25 amount of this hormone present in the plasma represents
an equilibrium between formation of hydrocortisone by
peared rapidly from the blood. Fifteen to twenty min
the adrenals and inactivation by conjugation with glucu
utes after the injection only 12 to 15% of the dose was
ronic acid in the liver. Inhibition of the latter process
present as free steroid in the estimated total plasma vol
tends to increase the level of circulating hydrocortisone.
ume.
The inactivation and excretion of adrenal and gonadal 30 Similarly, inhibition of glucuronide formation tends to
maintain the plasma level of administered hydrocortisone.
steroid hormones is controlled mainly by the liver, inas
E. M. Glenn in Endocrinology, 64, 373, 1959, states “A
much as the liver converts these steroids to compounds
slower rate of inactivation or excretion of a compound
'more readily excreted by the kidneys than is the parent
from the organism necessarily implies an enhanced bio
steroid. The bulk of urinary steroids are present as
water soluble conjugates of glucuronic acid and sulfuric 35 logical e?icacy, since more steroid becomes available to
responsive tissues for a longer period.”
acid. Thus, as reported by Peterson, R. E., and Wyn
By introducing, along with the steroid, a competitive
gaarden, J. B., Am. N.Y. Acad. Sci. 61, 297, 1955, it has
glucuronylation inhibitor the glucuronylation of the ste
been found that 94 percent of administered hydrocorti
sone can be accounted for through urinary excretion, and 40 roids or its derivative can be inhibited in vivo thus delay
ing inactivation of the steroid and making it effective in
2 percent through the feces. Of the urinary excretion,
smaller doses. The extent of inhibition will depend, theo
practically the entire quantity appears as a water solu
retically, on the ratio between inhibitor and substrate
ble conjugate, mostly as the glucuronide which possesses
steroid.
no physiological activity. A recent study reported by
Hartiala Acta Physiologica Scandinavia, Vol. 42, Suppl. 45 The steroids with which this invention is concerned in
clude those steroids, both natural and synthetic, which are
145 IX Scand. Physiol. Congress in Stockholm 1957 has
subject to glucuronylation, for example, cortisone, hydro
shown that the intestinal mucosa also contributes to the
cortisone, prednisolone, prednisone, 9a-?uorohydrocorti
sone, triamcinolone, dexamethasone, 6a-?uorohydrocorti
sone, 6a-?uoroprednisolone, 604-9a-di?uoroprednisolone,
glucuronide conjugation of steroids.
iFurther experiments are reported by Hechter, 0.,
Caspi, F. E., and Frank, H., Endocrinology 60, 705, 1957,
6u-methylprednis0lone, 9a-fluoro-16a-methylprednisolone,
who infused cortisone at a constant rate into the portal
vein of a dog. It was found that only about 4% of the
9oz - ?uoro - 16a - methylprednisone, and 9zx-?L101‘O-16a
methylhydrocortisone.
infused cortisone emerged from the liver ‘as unaltered
cortisone or hydrocortisone. The corticoid disappearance
The competitive glucuronylation inhibitors which can
as a result of circulation through the liver was not due to 55 be used for the purposes of this invention are selected from
the group consisting of soluble non-toxic compounds hav
accumulation or retention of the administered corticoid
ing the formula R—A where R is the active group and
in either liver or bile. Further studies indicated that the
is at ‘least one member selected from the group consist
major portion of the administered corticoid was rendered
ing of hydroxyl, and amino groups and A is selected from
These, and other studies, indicate that as the rate of 60 the group consisting of benzene, pyridine-carboxylic acid,
acetanilide, N-substituted acetanilide, propionanilide, N
transformation of steroids to their respective glucuronides
substituted propionanilide and benzylacetamide.
is extremely rapid the reactions in which the hormone
Examples of suitable glucuronylation inhibitors include:
participates in order to exert its unique physiological ac
highly water soluble, “probably by conjugation.”
tivity must occur within a short time following either
the secretion or introduction of the steroid into the body. 65
This invention is based on the idea of increasing the
physiological eifect of endogenous or exogenous steroids
which are inactivated in the body by glucuronylation by
All glucuronides are composed of two moieties:
(1) The aglycone 0r glucuronic acid accept0r.-—Ex
HILQOH
(N-acetyl-para-amlnophenol [acetaminophen] )
HN~C O-OH,
retarding their glucuronylation through the use of a com
petitive glucuronylation inhibitor.
OOOHa
70
l
our-@011
(para-hydroxybenzylacetamide)
3,053,737
3
kg. of body weight. N-acetyl-p-aminophenol was given
CO--CH:
@cm?iiawatgm.
CH3
by stomach tube at a single dosage level of 250 rng./kg.,
irrespective of the hydrocortisone dosage. An interval of
at least three days was allowed between successive injec
tions of hydrocortisone to obviate the possibility of carry
over of plasma hydrocortisone from one experiment to
the next. A given dose of hydrocortisone was injected
into a rabbit and the plasma hydrocortisone was deter
Ha
(N- [ 2- ( methylphenethylamino) ~propy1 ] -N-acetyl-para
amiuophenol)
mined after one or two hours. Three days, and in some
10 experiments 5 days later, N»acetyl-p-aminophenol was ad
CH3
ministered to the same rabbit, followed by the same dose
(N—[2- (methylphenethylarnino ) -pro)pyl] -N-prop1ony1-para
of hydrocortisone previously used, and the plasma hydro
amlnophenol
cortisone level again determined. Thus, the plasma levels
C O-CH;
(“MEPCMLGOH
_/
of free hydrocortisone in individual rabbits were deter
15 mined in the presence and absence of N—acetyl-p-amino
phenol, in which case the rabbits were serving as their
|
CH;
own controls.
(N- [1-methyl-2~piperidlnoethyl] -N-ucetyl-para-aminophenol)
The results are shown in Table I. It may be seen that
when N-acetyl-p-aminophenol is administered concur
C 0- CH1 CH3
rently with hydrocortisone, the plasma concentration of
free hydrocortisone is more than 200 percent of that which
.
obtains when hydrocortisone alone is given.
Ha
(N~[1-methyLZ-piperidinoethyl] -N-propionyl-para-amino
phenol)
Table I
OF‘ N-ACETYLp-AMINOPHENOL ON PLASMA
25 EFFECT
LEVELS OF ADMINISTERED HYDROCORTISONE AT
VARIOUS DOSAGES AND TIME INTERVALS
Hydrocortisone ! Plasma
(salicylhydroxamie acid)
1
Rabbit number
30
Br
Hydrocor-
Time
tisone dose
after
mgJkg.
dosage,
hrs.
C O-NHOH
OH
(?-bromosalicylhydroxamlc acid) ; O-aminophenol; 5-bromo
salicylamide; pyrogallol; pyrocatechol; and
HaN-QC O-NHOH
OH
(para-aminosalicylhydroxamic acid)
This invention in its broadest aspect is of general ap
plication and is not limited to particular inhibitors.
N-acetyl-p-aminophenol is particularly suitable as a
rig/ml.
acetyl-p-aminophenol is recovered in the urine in the con
jugated form, mainly as glucuronide but partly as ethereal
sulfate; only 4 percent appears unchanged.
Experiments were carried out with the rabbit as a test
animal to establish the effect of N-acetyl-p-aminophenol
on the plasma levels of injected hydrocortisone.
No detectable amount of hydrocortisone is present in
rabbit blood plasma due to the fact that the rabbit adrenal
secretes corticosterone rather than hydrocortisone as the
With NAPA
(250 mgJkg.)
2. 44
9. 0
4. 54
7.67
4. 57
6. 31
10. 4
8. 59
7. 60
9. 41
25.00
13. 5
7. 09
315. 07
5. 25
2. 20
3. 90
6. 40
4. 40
2. 30
9.30
4. 20
10. 20
10. 10
9. 4O
9. 70
3. 95
fact that it gives rise to a high rate of glucuronide forma
tion. Eigthy-?ve to ninety-six percent of ingested N
sone and cannot be construed to arise, for instance, from a
Without
INAPAZ
3. 20
competitive glucuronylation inhibitor by virtue of the
major hormone. The hydrocortisone plasma levels found
after injection consist solely of administered hydrocorti
levels
,
7. 10
‘8. 56
4. 97
14. 72
5. 81
4. 68
5. 44
6. 94
5. 42
11. 15
23. 06
13.31
17. 62
13.87
12. 23
13.89
6. 85
515. 02
2. 80
1. 50
4. 15
2. 65
2. 15
a3. 40
1 The ‘Silber-Porter procedure as modi?ed by Peterson et ul
(Analytical Chem. 29, 144, 1957] was used for the determi
nation of hydrocortisone. This procedure measures free, but
not conjugated, hydrocortisone.
‘stimulatory effect of N-acetyl-p-aminophenol on the adren 60
\als. This has been veri?ed by preliminary experiments
in which it was found that three days after a single intra
peritoneal injection of a 100 mg./kg. dose of hydrocorti
2 NAPA'N-acetyl-para-aminophenol.
3 225.24% of control.
4 216% of control.
5 220% _of control.
“ 158% of control.
‘sone into rabbits there was no detectable hydrocortisone
It is known (Cantarow and Trumper, Clinical Bio
in the blood plasma. It was also found that the admini 65 chemistry, Saunders, Philadelphia, 1955, page 423) that
three structural features are essential for biological ac
stration of a single 250 mg./kg. dose of N-acetyl-p-amino~
.tivity of adrenocortical steroids (corticoids):
(1) A 17,2l-dihydroxy-ZO-ketone side chain at C17
(2) A A4-3-keto grouping at Ring A
its metabolites do not interfere with the determination of 70 (3) A hydroxyl (OH) or ketonic (=0) group at Cu
phenol alone, does not give rise to detectable hydrocorti
sone in the plasma. The latter also furnishes additional
evidence that administered N-acetyl-p-aminophenol and
plasma hydrocortisone.
EXAMPLE 1
Hydrocortisone was administered by intraperitoneal in
jection at dosages of respectively 100, 50, 25 and 10 mg./
These three structural features are retained in all active
synthetic derivatives of cortical hormones, whatever other
changes may be made in the molecule to alter the degree
of activity. The Porter-Silber reaction is speci?c for the
17,2l-dihydroxy-ZO-ketone side chain and the isoniazid
‘3,053,737
6
5
(INH) reaction of Umberger (Analyt. Chem. 27, 768,
1955), which involves the formation of isonicotinylhy
All pretreatment blood samples were drawn about the
same time (9:30 am). Normal 17-OHCS values,
drazones of A4-3-ketocorticosteroids, is speci?c for the
6-25 mg./ 100 cc.
'
The results of those studies are summarized below in
A4-3~keto group in Ring A. The amounts of ‘free Porter
Silber
aliquotsand
of INH-reactive
plasma from rabbits
materialstreated
were with
determined
hydrocorin 5 Table
1150,“ alone ‘and wlth hydrocortlsone Plus N'acetylfP‘
Table IV
PLASMA 17-HYDROXYCORTIGOSTEROID (17-011os) LEV
aminophenol. In each case the values for INH-reactive
gps ililsiyréglgéigigTslggkgwilgrckgiéé‘p IADR1L141¥§Isg1g€I1Io§
materials were essentially the same as those obtained by
the Porter-S?ber method. These results, shown in Table 10
II below indicate that the increased plasma levels of 17OHCS obtained after concurrent administration of hydro-
(NAPA), AND A COMBINATION OF BOTH DRUGS
I
‘
'
.
_
.
'
-.
J
-p-
P
0
“OHCS plasma
levels
cortisone and N-acetyl-p-aminophenol are comprised
mainly of. biologically
active steroids since they retain
.
the essential groupings.
Patient
Drug dose
Pretreiat6151s.
after
men
osage
15
‘lg/100
‘lg/10o
ml.
ml.‘ ,
Table II
COMPARISON
BETWEEN
PORTER-SILBER
AND
INH-
Male 38..... NAPA 1,0o0'mg. (a) _______________ __
12
15
Hydrocortisonc 20 mg. (b); ________ __
s
16
REACTIVE 17-01103 IN PLASMA FOLLOWING ADMINIS-
' TRATION OF 25 MGJKG. HYDROOORTISONE INTRAPERITONEALLY
.,
Hydrocortisone 20 mg. plus NAPA
20
1,000 mg. (a+b) ................. __
.
Male ‘24
11
a
16
i
Porter-Silber
INH
_moms), ‘ta/m1.
(ALB-K05), ugJml.
Without
NAPA
With
Without
NAPA,
NAPA
25° mg-lkg-
96
Male
,
With
3. s4
4. 50
1. 53
1. s9
2. 91
5. 34
2.81
5. 31
2.81
8.62
1. 59
8.06
5. 575:1.2
13
10
1,1;
Female 25..
a. 59
4. 05
1. 93:3
30
25 Female 38"
NAPA,
25° mg-lkg-
1. 54
1.87
1 2. 31¢. 32
l?
I
17 >
22
19
15
10
24
15
38
a
30
5.25=l=.1
_
'
_
~
_
It is seen from the above table that the administration
of N-acetyl-p-aminophenol conjointly with h-ydrocortisone
1Mean=|= S.E.
to 5 human subjects gave rise to plasma hydrocoi'tisone
EXAMPLE 11
concentrations which were twice those attained when
By means of the procedure described in Example I, the
effect of N-acetyl-p-aminophenol on the plasma concentrations of various steroids was ascertained. It was found
that the plasma concentrations of prednisolone, 6-methylprednisolone, triamcinolone (9a-?uoro-16a-hydroxypred-
35 hydrocortisone alone was ‘administered. l-lydrocortisone
was employed in these experiments as ‘an example of a
typical adrenocortical steroid, and similar results would
be expected if hydrocortisone were replaced by predniso
lone, triamcinolone, or any other similar steroid currently
40 employed as a therapeutic agent,
nisolone) and dexamethasone (9u-?uoro-l6ot-methylpred
nisolone) increased in a manner similar to that previously
EXAMPLE IV
found in the case of hydrocortisone, when the above
EFFECT OF N ACETYL
AMINOPHENOL
(\IAPA
ON
steroids ‘are individually administered to rabbits concur-
PLASMA LEVELS OF AD?iNISTERED TRlAMlclNoioNE
rently with N-acetyl-p-aminophenol. The results of these 45
studies are summarized in the :following Table III.
(QwFLUORO-l?a-HYDROXYPREDNISOLONE) IN RABBITS
Triamcinolone plasma
Table III
Drug dosage,
Rabbit No.
EFFECT OF N-ACETYL-p-AMINOPHENOL (NAPA)
triamcino-
ON
lone, mgJkg. mgJkg.
PLASMA LEVELS OF EXOGENOUS 17-HYDROXYOORTIOOSTEROIDS (17-0OHs) IN RABBITS
50
body wt.
17-01105 plasma levels 1
Steroid
Dosage
10
0. 74
10
1.35
10
1.07 __________ __
(mg.[kg.) dosage, Without
With
hours
NAPA I
NAPA
10
10
10
2
4
5
2. 56
1.98
o. 50
5. 31
3.52
0. 95
Do___1___-ai1_.___-
25
15
2
2
5.42
4_ 01
11.25
7,29
__
Do ____________ __
15
Percent of
control
after
Prednisolone ______ .Do.-D0_
Dexamethasone.
,.g./m1.
Time
(pg/ml.) (pg/ml.)
Triamcinolonen
evcls
NAPA,
Percent
10
184
.......... -.
1.78
174
of
10
1. 35 __________ __
control
10
1.91
10
1.40
10
1.86
140
__________ __
133
236
183
189
157. 7
177
186 60
1o
2
2
4.05
8.30
12. 50
7.02
175
i5
2
10.51
17.97
171
155
1 Determined by the Silber-Porter procedure as modi?ed by Peterson
The following are examples of compositions according
'
'
'
.
to thls mvenuon'
_ _
Composition 1
.
Milligrams
'
_
et al. (Analyt. Chem. 29, 144, 1957). The steroids shown in the table
Predmsolone .
have in common an a-ketol side chain and are known co1lectively_as
N-acetyl-p-aminophenol ____________________ __ 500.0
17-hydroxycorticosteroids. Each value shown is the average ot4rabbits.
Wheat Starch
B N-acetyl-p-aminophenol, 250 mgJkg. body wt. administered orally
30 minutes before the steroid.
charge, otherwise normal in all respects.
45 0
_
Magnesium stearate _______________________ ..
EIQKMPLE III
70
The eifect of the conjoint administration of N-acetyl-p_
aminophenol with hydrocortisone on plasma hydrocorPmdnlsolone _
tisone concentrations was determined on human subjects.
All ?ve patients were post-fracture cases awaiting dis-
1, 0
'
5.5
Composition 2
2-5
N'acetY1'P'amln0Phen01 -------------------- -- 500-0
Wheat starch
‘
v
45.0
75 Magnesium stearate ___________________ __.____
5.5
3,053,737
7
8
Composition 3
ingredients cortisone and N-acetyl-p-aminophenol in a
6 -imethylpr'ediiisolone ______________________ _ _
ratio of between 1:10 and 1:1000.
3. A therapeutic composition comprising as its active
1 .0
N-acetyl-p-aminophenol ___________________ _‘__ 500.0
Wheat starch _____________________________ ___
Magnesium stearate _____________________ __‘__
ingredients prednisolone ‘and N-acetyl-p-aminophenol in
45 .0
5.5
a ratio of between 1:10 and 1:1000.
4. A therapeutic composition comprising as its active
ingredients prednisone and N-acetyI-p-arninophenol in
Composition 4
a ratio of between 1:10 and 1:1000.
Triamcinolone ____________________________ __
5. A therapeutic composition comprising N-acety1-p~
‘1.0
N-acetyl-p-aminophenol ____________________ __ 500.0
Wheat starch _____________________________ __
45.0
Magnesium stear-ate _______________________ __
5.5
10
aminophenol and 6a~methylprednisolone
6. A therapeutic composition comprising N-acetyl-p
aminophenol and dexamethasone.
7. A therapeutic composition comprising N-acetyl-p
Composition 5
aminophenol and triamcinolone.
Dexamethasone ___________________________ _-
0.5
References Cited in the ?le of this patent
N-acetyl-p-arninophenol ____________________ ___ 500.0
Wheat starch
___
45.0
Magnesium stearate _______________________ __
5.5
Corte et a1. (58): P.S.E.B.M., 97:4, pp‘. 751-755,
April 1958.
Corte et a1. (57): Canadian Pharm. 1., vol. 90, pp.
Although the ratio of steroid to inhibitor is given by 20 436-438, July 1957.
way of example from 1:200 to 1:1000 the ratio can be
Denko et a1.: P.S.E.B.M., v01. 95, pp. 483-484, July
varied from 1:10 to 1210,000.
1957.
This application is a continuation-in-part of my co
pending application Serial No. 757,465 '?led August 27,
1958, now abandoned.
I claim:
1. A therapeutic composition ‘comprising as its active
ingredients hydrocortisone and N-acetyl-p-aminophenoi
in a ratio of between 1:10 and 1:1000.
Roskam: J.A.M.A., 161:7, p. 661, June 16, 1956.
C& E News, 34:51, p. 6186, 'Dec. 17, 1956.
Batterman et 211.: Federation Proceedings, vol. 14, pp.
316-317, March 1955.
Wallenstein et al.: Federation Proceedings, vol. 13, p.
414, March 1954.
Cornely et al.: J.A.M.A., 160:14, pp. 1219-1221, Apr.
2. A therapeutic composition comprising as its active 30 7, 1956.
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