Патент USA US3053747код для вставки
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.