CA1073354A - Glyco/proteinaceous materials derivable from beef liver and other tissues useful for the treatment of toxic and allergic conditions - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Mucoprotides (i.e., complex compounds of protein with carbohydrates and carbohydrate derivatives) derived from animal tissues, for example, mammalian liver by a succession of extractive steps including treatment with acetone to produce three layers or phases of which the upper two are separated and further processed, the bottom layer being discarded. The retained layers are further treated to purify and de-antigenize them. The product is useful for treatment of allergic conditions, as a detoxicant and for treatment of narcotics addiction.

Description

~0~3354 SPECIFIC~TION
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This invention relates to certain aerivatives of 16 animal tissues, nota~ly mammallan ~ r, but also certain other 17 organs, such derivatives having biological activity including 18 e-toxifying effects, utility in ~he treatment of allergic 19 onditions, and the trea~ment of narcotics addiction.
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21 . The invention relates also to similar naturally occur- .
22 ing materials derived fro~ other sources such as yeast. Such 23 materials are identif iable by their infrared transmittance 24 spectrum .
25 .
26 ¦ This invention relates more particularly to proteina-271 ceous derivatives of mammalian livex (an~ o other sources) which

2~¦ has a protide component as the pr~ncipal component but which also 291 has a sigr.ificant carbohydrate compone~t hexeinafter reerred to 30¦ as mucoid, the biological and ~herapeutic properties of these derivatives being dependent on the chemical combi~ations of protide ard ~ucoid.

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1 By the ~erm "protide" is meant not only protein but 2 a~ino acids and oligopolypeptidesO By "mucoid" is meant certain

3 low molecular weight carbohydrates and derivatives such as

4 l¦ hexoses ~for example, glucose, galactose, mannose), sialic acids, hexoseamines such as glucoseamine and galactoseimine, fucose, etc.
6 ;
7 Several groups of wor];ers have, since the 1940's, 8 treated mammalian liver by generally s:imilar procedures starting 9¦ with macerated liver parenchyma - i.e., liver tissue freed o 10¦ fibrous material, fat and blood vessels. In this procedure, by 11 the systematic application of aqueous solutions of inorganic 12 salts as selective precipitants of unwanted (discarded) compo-13 nents, treatment in step-wise manner with acetone and further 14 processing of one or more (but less than all) of the phases resulting from acetone treatment and suitable purification of the 16 selected phase or phases (as, for example, by dlalysis) there 17 have been isolated biologically acti~e components o~ the liver.
18~1 ' 19¦ Far example, Mohamed and Greenberg publishing in 1945 20¦1 in Archives of Biochemistry, vol. 8, p. 349 set forth a proceduxe 21 ¦¦ which is the genesis of all later procedures and which results 22 11 in the isolation of the enz~me arginase.
.23i1 24 Fortini, Blair and Grodsky in U.S. Patent 3,701,768 25 ¦¦ have described a procedure much like that of Mohamed and Greenber~
26l¦ up to a point but with certain differences. For example, the ~711 acetone step is carried out to produce three phases of ~^~hich onlyl -~

28¦¦ the upper layer or phase ls separated and processed a~d the mid-29 I dle and bottom layers or phases are discarded. The separated 30 ¦ upper phase is treated to precipitate a protein component, is ! I
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then subjected to dialysis, then to a heating step to destroy antigens and then to fractionating of the protein component, as by chromatography, to produce a highly purified protein fraction.
Other workers in -this field include Huber (production of Orgotein, see U.S. Patent 3,624,251).
Difficulties encountered in products of such character which have been intended for therapeutic purposes (Fortini et al, and Huber) have included lack of uniformity in that different lots have had greatly different biological activity and further lack of stability.
It is an object of the present invention to provide improved methods of deriving a biologically active, medically useful (for treatment of animals and human beings) material which is more consistent in its significant biological activity and Which is more stable than some, i~ not most or all, of the earlier preparations referred to above.
It is another object of the invention to provide a method of producing biologically active mucoprotide material from mammalian liver and the like which results in higher yields than methods used heretofore as well as minimization of bacterial contamination resulting in pyrogenicity which gives a non-usable clinical product.
Thus, in accordance with the present teachings, a method is provided of producing a biologically active muco-protide product from a natural source such as mammalian liver, mammalian placenta and yeast. The product has anti-allergenic and detoxicant properties. The method comprises providing a dilute aqueous solution of the mucoprotide components of the source material, the solution being a supernate resulting from treatment of the autolysate of the source material to separate proteins which are precipitable by dilute lead ~ -3-~ !

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acetate and dilute ammonium sulfate. The aqueous solution is treated with acetone to produce the formation o three layers. The top and middle layers are separated from the bottom layer and the combined top and middle layers are subjected to heat sufficient to precipitate unstable and antigenic material. The precipitate is separated and discarded.
In accordance with a further embodiment a mucoprotide product is provided which is derivable from liver parenchyma placenta, yeast and other natural sources and consists predominantly of protidic material which consists of protein and protein hydrolysate and contains, based on a total dry weight of mucoprotide products, about 0.1 to 40% of mucoid material. The product is characterized by the ability to prevent and to repair impairment of liver function of rats in accordance with at least one of the Eollowing tests on laboratory rats:
(1) visual examination of liver of sacrificed rats which have been administered allyl alcohol to induce acute intoxication;
(2) BSP clearance by rats which have been administered carbontetrachloride, (3) the product has an infrared spectrum exhibiting a pronounced dip at a wave number between 1000 and 1200.
The a~ove and other objects of the invention will be apparent from the ensuing description and the appended claims.

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,, : ~ '' "' ' ; ' ~ lOq3354 1 We have ~ound that much more consistently active 2 products at higher yields and havi}lg enhanced stability result 3 from a procedure described below.

~ PREPAR~TION -6 A. Starting Materials 7 Mammalian li~er, placenta, spleèn, kidney, pancreas, and pituitary glands are some of the preferred sources of these biologically-acti~e mucoprotides. Liver and placenta are pre-10¦ ferred. However, tests indicate their presence in signlficant 11 amounts in all types of mammalian tissue. The source m~terial 12 should come from healthy animals and should be treated mechani-13 cally to remove extraneous fat, fibrous material, and the li~e, 14 leaving primarily the tissue characteristic o the organ, for :15 example r parenchyma in the case of liver.
:16l ~ ; -I7 ~ I. Autolysis 18 ¦ Defibrinated, decapsulated, defatted young steer 19 liver is obtained from healthy animals under clean conditions -20 I as ~escribed above. The procedure described herein is based 21 upon 3 kilograms of prepared liver (parenchyma). This liver 22 parenchyma is diced and macerated or homogenized in a Waring -23 -blender with 600 ~c. of solution containing 50 grams of sodi~m -24 j~acetate per liter and 15.4 grams of manganous sulphate per liter.
This homogenate is stirred vigorously at room temperature (20 to 26 1 22 C.) for 2~ hours. This homogenate-autolysate is strained - 27 I through one layer of gauge ~80 strainer.
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1 II. ~ulk Removal of Extraneous Protein and 2 Pr~tein Associated Material 31 This strained liquid autolysate is treated with 3000 cc.
4 of 4.575% lead acetate; stirred vigorously for 35 to 40 minutes 51 and let stand for at least l hour at room temperature. The 6 liquid is then centrifuged and the supe:rnatant fluid is adjus,ed to pH 8.3-8.4 with 2.5 liters of 0.2 N NaOH (or whatever amount may be required for this pH adjustment). Let liquid stand for at least 2 hours at room temperature; then clarify liquid by 1~ filtxation and/or centrifugation~ Add to supernatant ~ id one-11 third volume of saturated ammonium sulphate ~780 grams/liter);
12 let stand for 3 to 4 hours at room temperature~ Then ca~efully 13 filter and centrifuge; test for absence of lead. Save clear supernate.

- III. Acetone Treatment and Dialysis of the 16 Selected Phase 1~ Acetone to 35% by volume is added to the lead-free 18 supernate from Step II. The liquid is allowed to stand 4 to 6 19¦¦ hours at room temperature. It is then iltered and centrifuged.
2Q ¦¦ More acetone is then added to the supernate to give 60% acetone 21~ by volume. Let stanA for 6 hours at room temperature. Three ;~
22¦ ~ayers will ~e formed; the bulk of the arginase or arginase-231 associated protein will be in the middle layer. Siphon off the 241 two top layers; discard lowest layer. To the two top layers add 251 a~ equal volume of acetone. A gummy precipitate will form; iet 26¦ stand 4 to 6 hours at room temperature. Isolate the precipitate.
271 The precipitate is dissolved with 0.l M K2HPO4 adjusted to pH 8.5 28!i use 750 to l000 cc. of adjusted buffer for dissolution. Place 29l¦ this solution in dialyzing bags and dialyze against pure wate~
with fre~uent water changes and conLinuous agitation to speed up l l ~ 10~3354 1 dialysis until m2terial in the dialysis bags tests free or 2 minerals (e.g., a conductivity test of not more than l ppm). The 3 time required for dialysis will depend upon the physical param-4 eters of the dialysis system but should be accomplished within

5~ 48 hours to minimize the possibility of significant losses due 61 to a slow disintegration of the product at this stage of isola-7 tion of the product of this invention. Remove solution from the 8 dialysis bags and centrifuge; save the supernate.
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The two top layers separated from the bottom layer 11 before the second acetone addition, lnstead of being~further 12 treated as described above, may be used without further purifi-~3 cation as an edible liver extract for buccal use by lyophilizing -14 them and packaging the resulting dry powder in buccal form as wafers or thin tablets made with an appropriate inert excipient.

~6 The amount of lyophilized product would be determined by 17 bioassay of-the active product ingredient present in these two 18 ! top layers. ~ buccal dosage of ten times the parenteral form 1~ ¦ is presently recommended. Suitable excipients are lactose, 20 I starch and cellulose, and suitable proportions of lyophilized 21 material are 5 to 25% based on total buccal product.

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30!l .
31 1~

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11l IV. De-arti enization and Stabilization 2¦1 Returnins now to the prot~col for the parenteral 3 ¦¦ material, place this supernate in a water bath maintained a~

~1¦ 47~4~ until all extraneous, unstable and antigenic materials - 5 11 are precipitated (large flakes); this is usually accomplished

6~ in 3 to 5 hours. Centrlfuge at high speed until there is , clear supernate. An aliquot of the supernate is removed for determina-8 ¦ tion of concentrat1on of active component and ~or standardi~ation.

91 With some preparations depending on the tissue and species of ~0 ¦ origin, this step of de-antigenization can ~bè elimin~ted; this 1l j is especially true of bovine and human placental preparations. -12 ! As a matter o-f routine practice, however, it is best to gO

13 I through this de-antigen1zation procedure unless a small aliquot 14 ~ of the production batch is first put through this step as a ¦ sa ety test to be certain that no antigenic proteinaceous material ~6 1 i-s pre~ent.
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181i Tne clear supernate resulting from centri-rugation i~
191i sterile-fi~tered in a st~riIizing membrane filtration system 20¦1 and stored in a freezer or lyophilized under sterile conditions.

Zl,¦ ~he yield of concentrate will vary but is usually in the range 221l ~f 2 liters of a 1 to 2% solution, or 20 to 40 grams. A t~pical ~3 li preparation will have a human parenteral dosage range from O.S mg 24l1 to 1.5 mg. per 75 ~g. adult, depending on the biological activity 2$ 11 of a particular batch. The usual parenteral (I.V.) dosage form 26!' is a ~ ccO ampoule. , 27 11, 28. V. Final Purification 29' The final puriflcation of the product is effected by 301 a novel step just prior to ampoulization for clinical usage.

1~ 7 ~33 The concentrate is appropriately diluted to final concentration with pyrogen-free, sterile water and left to stand at room temperature ~65 to 75 F.) for at least 8 days or until no further pinkish precipitate forms. This bulk solution is sterile-filtered to remove the precipitate, reassayed and standardized, and put into final form for clinical usage.

ANALYSIS
Because of the complicated, diverse structural rela-tionships of the group of substances known as mucoprotides, several different approaches had to be used to "tease" these complex molecules apart in order to minimize and/or account for the losses during their hydrolysis or breakdown to their simple compositiona] subunits. The very elaborateness and the necessity for gentle "pretreatment" of the mucoprotides to minimize loss of subunits emphasize the differences of the product of this invention from the simple "pure" protein of the Fortini-Grodsky-Blair patent and the simple metallo-protein of the Huber-Schulte patents. Specifically, a much more gentle and discriminating `;
hydrolytic technique had to be used for the mucoid or carbo-20 hydrate moiety as compared with the conventianal vigorous ' hydrolysis of a pure or simple protein. The general procedure for the qualitative and semi-quantitative pretreatment and analysis of the mucoid or carbohydrate moiety of these muco-protides is similar to the basic procedure described by Richard J. Winzler in the Ciba Foundation symposium "The Chemistry and Biology of Mucopolysaccharides," pp. 258-261, Little Brown & Co., Boston, 1958; for quantitative results, more rigorous analytical methods for the mucoid subunits are used as indicated in the following descriptions.

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The analytical procedure employ0d for the mucoid moiety of the mucoprotides is as follows: A 20 milligrams sample of mucoprotide in its final form (i.e., the active component result-ing from Step V of the preferred preparative protocol) is mixed with 20 milliliters of 1.0 molar hydrochloric acid and the mix-ture heated ~or 15 minutes at 100 degrees Centigrade. This pro-cedure results in the release o~ maximum amounts of sialic acid(s) and fucosé with only about 5~ hydrolysis of the protide moiety. Simultaneously, another 20 milligram sample of muco-protide is treated the same as the first sample but the hydrolysisis continued for 1 hour, thus resulting in the maximum release of hexoses and hexoseamines with only about 15 to 20% hydrolysis of the protide moiety. Aliquots of these treated samples are immediately subjected to dialysis (or ultrafiltration) and the dialysates (or ultrafiltrates) evaporated to dryness, and chroma- ^
tographed, using a descending solvent system of ethyl acetate:
pyridine: water in the ratio of 10:4:3 parts by volume and the resulting chromatogram air-dried and stained with a solution of aniline exalate. The spots are identified by checking positions 2Q with corresponding known standards and quantitated by analysis of the chromatogram by a quantitative analytical comparator and/
or by quantitative ana~lysis o~ the eluate of the individual com-pounds. The specific analytical methods are further detailed in the following references: R.J. Winzler, "Determination o Serum Glycoproteins," Meth_ds of Biochemical Analysis, vol. II, pp. 279-311, Interscience Publishers, N.Y., 1955; Leonard Warren, "The T~iobarbituric Acid Assay of Sialic Acids," Jour al of Biological Chemistry, vol. 23~, pp. 1971-1975 (1959);
C. J. M. Rondle and W. T. J. Morgan, "The Determination of Glucosamine and Galactose-amine," Biochemical Journal, volO 61, _ g _ ~ 7335 pp. 586-589 (1955).

The analytical procedure employed for protide moiety of the mucoprotides is as follows: Because of the presence of carbohydrate or mucoid, excessively harsh or prolonged methods of hydrolysis must be avoided to minimize losses of constitutent amino acids as a result of the so-called "browning reaction"
involving the condensation of released mucoid and protide sub-units to give dark by-products. Mucoprotides of lower carbo-hydrate content (0.1% to 10%) such as those obtained from some mammalian livers can usually be hydrolyzed directly by the pro-cedure described below. Mucoprotides of higher carbohydrate content (10% to 40%) such as those obtained from some mammalian placentas should be subjected to a preliminary mild hydrolysis such as that employed above for the release of carbohydrate sub-units (i.e.~ 1 hour at 100 degrees Centigrade in 1 molar hydro-chloric acid), and then subjected to dialysis or ultrafiltration to remove the bulk of the released carbohydrate or mucoid ` ' materials; such a preliminary hydrolysis step will remove the bulk of the interfering carbohydrate, thus minimizing losses of 2Q amino acids due to reaction with the excessive carbohydrates. ;
The hydrolysis of the protide moiety to the constituent free amino acids is then effected by treating 20 milligrams of protide or protide equivalent (calculated by subtracting the percentage equivalent of carbohydrate if the carbohydrate content is greater than 10% of the intact mucoprotide) with 20 milliliters of triply distilled 6 molar hydrochloric acid in sealed Pyrex glass tubes ~ -at 105 degrees Centigrade for 20 hours. Tryptophan which is destroyed during acid hydrolysis is determined by alkaline hydrolysis using 20 milligrams of protide equivalent in 20 milli-~107335~

liters of 4 normal barium hydroxide at 110 degrees Centigrade for 60 hours. Once hydrolysis is completed, the amino acid mixtures are subjected to conventional ion-exchange analysis (G. R. Tristram .
and R. H. Smith, Advances in Protein Chemist:~~, vol. 18, pp. 227-235, Academic Press, N.Y., 1963).

Typical Analyses are as follows:
Amino Acid Analysis:
Beef Liver Human Placenta Mucoprotide Mucoprotide perce:n:t:;by: ~e:ight percent by weight Alanine 5.3 2.8 Arginine 5.7 7.0 Aspartic acid 7.4 6.0 Cystine 0.6 6.2 Glutamic acid 5.4 7.1 Glycine 6.5 2.4 Histidine 3.2 1.6 Isoleucine 4.0 2.0 Leucine 9.7 5.0 Ly.sine 8.6 3.9 Methionine 1.8 1.6 Phenylalanine 3.4 2.5 Proline 8.8 8.6 `~
Serine 5.5 6.0 Threonine 5.0 5.3 Tryptophan 2.0 0.7 Tyrosine lo 2 2.9 :.
Valine! 7.2 5.5 Amide-NH2 not determined ---NH3 2.9 :

. ' 1~335 Mucoid Analysis:
This is rather variable, both as to total mucoid con-tent and as to the proportions of the various mucoid entities.
The total mucoid content (based on total dry weight of solids) may be as low as 0.1% or as high as 40%. Conditions affecting the total mucoid content are species of animal used as sources of tissue, type of organ or tissue employed, ancl the method of preparation. For example, active mucoprotide preparations ob-tained from mammalian placenta may give a much higher mucoid content than the corresponding mucoprotide obtained from liver or spleen or blood; also the mucoprotides obtained by the improved method of this invention will give mucoprotides with signifi-cantly higher mucoid content than that o~tained by previous ~;
methods. Type of species of animal as well as the health of the animal will limit the fi.nal yields: e.g., human placenta seemsto give consistently higher percentages of mucoid than that obtained from bovine placenta.

Typical mucoid components in typical proportions are as follows:
Sialic Acid(s) 25% to 30%

Hexoseamines (probably acetylated) 25% to 30%

Glucoseamine 15%
Galactoseamine 10%
Hexoses 30% to 25%

Glucose10%
Galactose 12%
Mannose8%
Fucose 1% to 5%

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~ 10~33~4 1 ¦ The material of the present invention is also charac-2 ¦terized by its infrared transmittance spectrum. Figures 1, 2 3 13 and 4 are segments of curves showin~ infrared transmittance 4 ¦of material prepared as described aboveO Ordinates are precentage S Itransmittance and abscissae are in wave numbers. These spectral 6 ¦charts are, respectively, for material prepared for beef liver

7 ¦(Figure 1~, cow placenta (Figure 2), yeast extract ~Figure 3) and

8 ¦commercially available trypsin (Figure 4~ The liver and placen-

9 ¦tal material were prepared as described for beef liver above.

10 ¦TrYPSin was the commercial crystalline material prepared as de-

11 ¦scribed in Hawk's Physiological Chemistry, 14th edition, page 441

12¦ (McGraw Hill publisher). Yeast extract was the ribonucleic acid

13 ¦preparation as described i~ Hawk, op.cit., pages 208 and 209.

14¦ Surprisingly the yeas~ and RNA products contained mucoprotide

15¦ material of the present invention. Th~se products are taken up in

16¦ a solution of the character used for clinical purposes and are

17¦ subjected in solution to de-antigenization and stabilization

18¦ step IV above and preferably also to final purification step ~.
191 . ' .
20¦ As will be seen, each of these materials has a pro-21¦ nounced dip in its infrared spectrum at a frequency close tollOO
22¦ and in all cases between 1000 and 1200. The remainder of the 231 spectrum to the right and to the left of the 1000 - 1200 se~ment, 24I including the spectrum beyond the range shown in the several 251 figures, is typical of extracts prepared by the workers ~Mohamed 26¦ and GreenbPrg; Fortini, Blair and Grodsky and Irons) mentioned 27 ! above. It is the uniaue property of the material of the present 28 invention t~at it has the characteristic transmittance spectrum 29 .

2 ~ 12a.

1~'~3354 1 at 1000 - 1200 as shown in the figures.
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3 The infrared spectrophotometric analyses of the muco-4 protide,preparations were performed on the standard commerci.ally 5 a~ailable Perkin-Elmer 401 recording spectrophotometer ~y com-6 pressing the thoroughly mixed mucoprotide powder and potassium 7 bromide into a standard KBr pellet, the finàl concentration o~
8 mucoprvtide being 1%o The infr~rea spectrophotometer was . I st ~ ~rdize for each c~rve aga rst polysCy ene.

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iO'~33 1 ~50des o~ Ad~inistr~tion and Dosages: !
2 The product of the inven'tion may-be administered by all parenteral routes (intravenous, intramuscular, subcutaneousr 4' intraperitoneal) and by oral administration including buccal. ;, The solution described in Step V may be the form in which it is administered or the solution may be lyophilized and the dry 7 product may be adminlstered in dry form with an excipient such 8 as lactose, starch and cellulose.
'` ` 9 I ` ' ` ' ~ ,' 1~ ¦ ~he consumer product for buccal,use may be in the form of pills, tablets or wafers. An especially advantageous mode 12¦ of a~ninistration is the buccal route wherein a pill, tablet, 131 wafer or aqueous solution aontaining 1~ mg to 15 mg by weight '14 of the active material from Step III is held in khe mouth 15 ¦ between the gums and the cheeks. This route avoids or great]~y 1~ ~ diminishes digestive attack on the active materials; it avoids -17¦ or lessens possible allergic reactions to parenteral administra-18 I tion; it enables the patient to treat himself; and it places 191 these mucoprotides in the category of a nutritional supp?,emeht 2~ (e.g., liver or pancreatic extract) rather than a pure drug or 21 I pharmaceutical. , , 22 I ' ,, ' '23 I Recommended dosages are, as stated above, about 0.5 mg '~4 to 2.0 mg of active material per 50 kilograms of weight. Fre-251~ quency o~ treatment depends upon such factors as type of illness, 261! duration (acute or chronic) of illness, conjunctive therapy,with 27 ¦1 other agents, ag~ and nutritional status o~ patient.

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~ 33 Medical and Veterinar~ Utility:
The material of this invention is useful for treatment and/or prevention of allergic and hypersensitive conditions, of toxic conditions, of untoward side effects of common drugs and for the treatment of drug addiction and alcoholism. The material is especially useful when the condition treated is associated with or results from impairment of liver function as when there has been a deterioration of the liver parenchyma. Examples of conditions that have been or may be successfully treated are effects of addictive drugs such as narcotic analgesics (e.g., heroin and co~aine); alcoholism; and untoward side effects of therapeutic drugs such as penicillin and chloromycetin.

Although various specific posologies have been established for such categories of clinical application, the most favorable prognosis is obtained where the use of mucoprotides is simu~taneously supported by a nu~ritional regimen designed to maximize the rapid restoration of normal liver function by rebuilding the parenchyma of the liver. Accordingly, in more advanced or severely debilitated cases the best clinical responses are obtained by employing an appropriate mucoprotide posology plus a well-balanced diet including intensive supple~
mentation with vitamins, bioflavinoids, and pre-digested protein ti.e., a balanced mixture of free amino acids) to insure rapid repair of damaged liver tissue; such a rapid response and recovery are especially important in alcoholic and narcotic addiction where sudden withdrawal o~ the drug can result in over-whelming patient-management problems and a high rate of recidivism.

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As a specific example, a patient addicted to heroin and who suffered such excruciating withdrawal symptoms that he refused to withdraw from heroin again, was treated by I.V. injec- ;
tion of 1 cc injections twice daily, then at more widely spread intervals, and was put on an adequate diet strongly fortified with vitamins, bioflavinoids and amino acids. Bromosulfothalein was administered at the start of therapy and again after four weeks of therapy. (A normal liver clears bromosulfothalein (BSP) -quickly whereas a damaged liver allows longer retention of BSP.) BSP retention of this patient was 28% at 45 minutes at the com-mencement of treatment and was reduced to 1.9% at 45 minutes~ ;
after four weeks of treatment. Further, after four weeks this patient had no desire for hard drugs such as heroin.

Significance of Mucoid Compone;nt;s;, etc.
We have found that highly purified protein derivativesof mammalian liver, for example, the 97% pure material suggested in Fortini U.S. Patent 3,701,768, is less active and less stable than the mucoprotides of the present invention. Our product is a mixture of mucoprotidic proteinaceous materials and not a single or simple protein such as Orgotein (see, for example, U.S. Patent 3,624,251) or Acutalyn (Sèe Fortini U.S. Patent 3,701,768). E'urther, our product has a significant mucoid com-ponent which is chemically bound in some manner or o-ther to the protidic component. In some manner that is not presently under-stood, this mucoid component is essential for biological and therapeutic efficacy, contributes to stability and results in greater reproducibility of results from one lot to another.

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~Q~33~4 Other Considerations It will be noted that the upper two phases in Step III ~;
are employed in the present invention whereas the middle phase is discarded in accordance with Fortini U.S. Patent 3,701,768.
Further, in prior procedures, for example, that of Fortini, low temperatures and resultant long periods of processing are required, for example, -temperatures of 5 C. and processing steps that require many weeks. In the process of the present invention, room temperatures are employed in most of the steps, the time required for processing is greatly diminished and yields are greater; also, the speed of the improved process is so much greater than the older processes that the chances of bacterial contamination (pyrogenicity) is greatly reduced.

~ cruder form of the product of the present invention is that obtained during Step III and may be used with certain advantages as a buccal product rather than an injectable or parenteral product. The buccal product consists of the top two layers in Step III; this semi-purified material usually contains significant amounts of extraneous proteinaceous contaminants which will usually induce severe anaphylactic responses if used parenterally; hence this crude form is not a practical inject-able product. This crude form of the product may, however, be effective buccally where these anaphylactoid proteinaceous substances (contaminants) will not be absorbed as such by the buccal membranes in the mouth but will eventually be digested by the enzymes of the gastrointestinal tract, whereas therapeuti-cally significant amounts of mucoprotide will be absorbed by the buccal membranes into the body.
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_ 16 -~q~35 BIOAS;SAY

Heretofore persons working the field of derivation of medically useful proteinaceous materials from liver parenchyma and other tissues have striven to achieve a pure protein supposed to be the effective component of cruder products. In accordance ~ith the present invention, a derivative of such tissue is produced (e.g., by a protocol such as described above), which consistently exhibits a significant and measurable ln vivo -~
function, that function being in liver damage due to one or more clearly defined causes and which is susceptible to reliable measurement. The causes are acute allyl alcohol intoxication and carbon tetrachloride poisoning of the liver; the subjects are laboratory rats of an accepted strain for laboratory investi-gation; and the test for effect on liver function is bromosul-fothalein (BSP) clearance or observed liver damage of sacrificeaanimals.

BSP clearance is an accepted criterion of liver func-tion. See, for example, Varley, Practical Clinical Biochemistry, 2~ pp. 293-294, Interscience Publishers, New York, 1958. BSP
administered intravenously is readily detected in the blood stream after administration; it is cleared by the liver; a normal liver will clear the BSP quickly; damaged liver will clear BSP more slowly; and the time required to reduce BSP to a predetermined level is a reasonably quantitative measure of damage to liver function. Examination of the liver of sacri-ficed animals to which the toxic agent has been administered provides a semi-quantitative checkO Details from tests will now be described.

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1 ¦~ (1) Li~~er Damaqe Due to A11v1 A1coho1 Intoxic~tion 2. ~istar male rats, weight range-180 to 200 grams in 3 groups of ~0 are treated with allyl alcohol ad~inistered by 4 mouth in amounts of 0.4 ml of a lQo aqueous solution of allyl alcohol per 100 grams o~ animal weight. A group of animals are 6 treated preventively with a mucoprotide solution of the present 7 invention given subcutaneously at the doses of 1 ml per 100 .
8 grams of animal weight at 48, 24 and 1 hour before admlnistration .
9 of allyl alcohol. Another group are treated c.uratively with the .
same-doses of mucoprotide solution but given I, 6, 24.and 30 11 ho~rs a~ter allyl alcohol administration. All groups are 12 sacrificed 48 hours after allyl alcohol adminlstratlon. A final 13 mucoprotide injection is given 30 hours after intoxication .
14 because liver necrosis becomes clearly~ visible after .36 hours.
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16 ~ ~ ~ The result.s.of three successive illustrative experi-17 ments are summarized in the table below.
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(Acute Alyl Alcohol Intoxicationj, Grams of Damaged Liver Tissue 21 . - . .. . : . : .
22 Experiment No. Control Preventive Curative 23 I ~ - 7.10 0.59 1.30 24 II - 8.00 0.62 1.65 2~ ~ ~ 8.34 0072 . 1.33 26 I . .
.27 I 70-8~ % <20%

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l 18 l ., iO73354 1 : Examination of the post mortem livers clearly revealed 21 extensive perilobular liver necrosis: Mucoprotide treatmen~ !
31 greatly diminished or eliminated the necrosis, whether used 41 prevent^ively or curatively. This hepato protective action of ~ ¦ th~ mucoprotide is clearly shown in the table above.
6 ~ ;
7 ¦ ~2) BSP Clearance after Carbon Tetrachloride Administration I
¦ ~ ~-- -Carbon tetrachloride is administered subcutaneously ¦ twice weekly for three weeks in doses of 0.1 ml per 100 grams 1~¦ of anLmal weight to male Wistar rats of 150 to 200 gram weight -11 ¦ divided into groups of 20 animals. The mucoprotide of-the 12¦ invention is adm.inistered subcutaneously in a dose of 0.5 ml per 13¦ lOG grams of animal weight daily for five days after the end i41 of the carbon tetrachloride administration. Twenty-four hours 1~¦ after the en~ of the mucoprotide treatment BSP is administered 1~¦ to both groups of ànimals tthe treated and ihe untreated or ~71 control groups) intravenously using a solution of 1% BSP at a I I level of Q.5 mg of BSP per 100 grams of animal weight. Blood 19¦ samples are removed after 1 and 10 minutes. The quantity of

20~ B5P--in ~he-serum-is determined by modifying the clinical 211 method describ~d in Varley and the retention values are 22 calculated-with the formula:

24 1! BSP-reten~ion = concentration of BSP at 10 minutes x lG0 25!1 concentration of BSP at 1 minute 26 I The results of three successive typical experiments 27 ¦ are summarized in the following table.

~2~ ~ ,~-.~c ~ 5~ ~ ~ 3 ~ _~

2-9 I .

3~, 1.

33S~

TABLE I I
~SP Retention; (Min~tes) .. . ....................................... .

Experiment No. Control CC14 CC14 & Mucoprotide - . , I 15 - 4 35 - 4 18 + 4 III 17 + 3 28 - 6 17 - 4 100% increase no increase in BSP retention in BSP retention The data in this table indicates that, within experi-mental error, the mucoprotide-treated animals experienced a return to normal liver function as evidenced by the normal BSP
clearance values. Further examination of the rat livers after chronic intoxication with carbon tetrachloride showed a signifi-cant diminution of the central lobular necrosis of hepatocytes in those animals treated with mucoprotide as compared with the untreated controls~ The microscopic evidence is consistent with -the return to normal of the BSP values for overall liver function.

It is to be noted that the first method involves the 20 production and prevention of perilobular liver necrosis whereas ' the second method involves the production and prevention of central lobular liver necrosis. Since liver biopsy has shown that both types of liver damage may be experienced in clinical and veterinary mammalian practice, b~th methods of assay are ~5 employed in the standardization of any specific production batch of mucoprotide.

3~

, 3354 `

¦ Biological activity of the mucoprotide~ prepa ation?-2 ¦ is determined by the minimum amount requlrea to prevent~liver 3 ¦ damage by both o~ the tests described above. Although both the 4l BSP retention test (carbon tetrachloride damage) and the acute 5 alcohol intoxica.ion test are used in the final biological evaluation (bioassay) of a particular production batch of muco-7¦ protide, the BSP retention test is used as the critical test 8 since it is a quantitative and objective test, and is directly 9 related to the corresponding routine clinical laboratory test for evaluating ~iver function. Despite the semi-quantitatlve 11 nature of the alcohol intoxication test, there see~s to--be a -12 good correlation between the two tests.
13 ~
14 The standardization of the cruder buccal form is 15¦ effected by taking out a sma~l aliquot of thLs semi-purified 16l mucoprotide batch and carrying this aliquot through the entire l7i production procedure on a laboratory scale and ~inally assaying 18l the isolated mucoprotides by the two bi~assay methods described lg above. The final dosage of the buccal form is determined by using an aliquot containing ten times the corresponding parenteral

21 I dosage of mucoprotides. The factor of ten times the parenteral 2~ dose was selected by clinical trial and is a reasonable factor in 23 ¦ view of the crudenass of the product and the uncertainties of 24 I absorption by the buccal rou~e of administration as compared to 2~ that of the parenteral route.

Claims (9)

I CLAIM:

1. A method of producing a biologically active mucoprotide product from a natural source such as mammalian liver, mammalian placenta and yeast, such product having anti-allergenic and detoxicant properties, said method comprising the following steps:
(a) providing a dilute aqueous solution of the muco-protide components of the source material, such solution being the supernate resulting from treatment of the autolysate of the source material to separate proteins which are precipitable by dilute lead acetate and dilute ammonium sulfate, (b) treating the said aqueous solution with acetone to cause the formation of three layers, (c) separating the top and middle layers from the bottom layer, (d) subjecting the combined top and middle layers of step (c) to heat sufficient to precipitate unstable and antigenic material and (e) separating and discarding the precipitate resulting from step (d).

2. The method of Claim 1 wherein step (b) is carried out at approximately room temperature.

3. The method of Claim 1 followed by holding the supernate from step (c) at about room temperature for a suffi-cient time for precipitate to form and for precipitation to proceed to completion, separating the precipitate and then packaging the supernate as a final product after further concentration and sterile filtering if needed.

4. A method of producing a biologically active muco-protide product from a natural source such as mammalian liver, mammalian placenta and yeast which comprises providing such source substantially free of tissues such as fat, fibrous material and blood vessels, forming an autolysate of such tissue, subject-ing the autolysate to steps to separate proteins that are precipitable by dilute aqueous lead acetate or by dilute aqueous ammonium sulfate, thereafter treating the aqueous supernatant solution resulting from such separation and freed of such proteins to treatment with acetone to cause the formation of three phases separating the top two phases and converting the top two phases to either a form suitable for buccal use and ingestible by mouth of to a parenteral form suitable for parenteral administration.

5. The method of Claim 4 wherein the separated top layers are subject to lyophilization and are mixed with a suitable buccal excipient.

6. The method of Claim 4 wherein the separated top layers are subject to heat sufficient to precipitate unstable and antigenic material without significantly impairing the activity of the retained, dissolved mucoprotide material to amerliorate liver damage to rats induced by acute allyl alcohol intoxication and to ameliorate the loss of BSP clearance in rates caused by carbontetrachloride injection.

23.

7. A mucoprotide product derivable from liver parenchyma placenta, yeast and other natural sources and consisting predominantely of protidic material consisting of protein and protein hydrolysate and containing, based on total dry weight of mucoprotide product, about 0.1 to 40%
of mucoid material, such product being characterized by the ability to prevent and to repair impairment of liver function of rats in accordance with at least one of the following tests on laboratory rats:
(1) Visual examination of liver of sacrificed rats which have been administered allyl alcohol to induce acute intoxication;
(2) BSP clearance by rats that have been administered carbontetrachloride;
(3) said product having an infrared spectrum exhibiting a pronounced dip at a wave number between about 1000 and 1200, whenever prepared or produced by the process of Claim 1 or by their obvious chemical equivalents.

8. The product of Claim 7 in parenteral form.

9. The product of Claim 7 in buccal form.