CA1262907A - 3-¬n-phenylacetylaminopiperidine|-2,6 dion and process of synthesizing same - Google Patents
3-¬n-phenylacetylaminopiperidine|-2,6 dion and process of synthesizing sameInfo
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- CA1262907A CA1262907A CA000475098A CA475098A CA1262907A CA 1262907 A CA1262907 A CA 1262907A CA 000475098 A CA000475098 A CA 000475098A CA 475098 A CA475098 A CA 475098A CA 1262907 A CA1262907 A CA 1262907A
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- antineoplaston
- fraction
- dion
- phenylacetylaminopiperidine
- reaction mixture
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Abstract
3-[N-PHENYLACETYLAMINOPIPERIDINE]-2,6 DION AND PROCESS
OF SYNTHESIZING SAME
Abstract Disclosed is a compound 3-[N-phenyl-acetylamino-piperidine]-2, 6-dion and the pharmaceutically acceptable salts thereof, and the process for synthesizing same, which process comprises the steps of providing a quantity of L-glutamine, providing a quantity of phenylacetyl halide, mixing together the L-glutamine and phenyl acetyl halide in a weakly alkaline aqueous solution to provide an aqueous reaction mixture, reacting the reaction mixture for a period of time to form the reaction product 3-[N-phenylacetylamino-piperidine]-2, 6-dion, and recovering from the reaction mixture the product 3-[N-phenylacetylaminopiperidine]-2, 6-dion, and when desired preparing the pharmaceutically acceptable salts thereof.
OF SYNTHESIZING SAME
Abstract Disclosed is a compound 3-[N-phenyl-acetylamino-piperidine]-2, 6-dion and the pharmaceutically acceptable salts thereof, and the process for synthesizing same, which process comprises the steps of providing a quantity of L-glutamine, providing a quantity of phenylacetyl halide, mixing together the L-glutamine and phenyl acetyl halide in a weakly alkaline aqueous solution to provide an aqueous reaction mixture, reacting the reaction mixture for a period of time to form the reaction product 3-[N-phenylacetylamino-piperidine]-2, 6-dion, and recovering from the reaction mixture the product 3-[N-phenylacetylaminopiperidine]-2, 6-dion, and when desired preparing the pharmaceutically acceptable salts thereof.
Description
~ Z BURG:003 3-[N-PHENYLACETYL~INOPrPERIDINE]-2,6 DION AND PROCESS
~ OF SYNTHESIZING SAME
-This applic~ on is a division or Canadian Serial No.
403,789, filed May 26, 1982.
The presen~ invention relates generally to medicinal compositions and the use thereof; and more par~icularly, it relates to biologically active peptide compositions useful in the treat~ent of human neoplastic dlsease.
Investigations into the presence of physiologically or pathologically active peptides in urine have been on going for the past 80 yearsO Biologically active poly-peptides have been isolated from urine which have demon-strated hormone like activity or regulation of biological function. Examples of biologically active polypeptide compositions isolated from urine include growth factors, pituitary hormones, and kinins.
The practically infinite variety of peptides that can be formed~by the combination of the twenty common amino acids has prompted many investigators to suggest that peptides may constitute a system carrying informa-tion from cell to cell and organ to organ. Following this view on the regulatory significance of peptides, researchers have isolated urinary peptides which exert an influence o~ blood pressure, behavior modification, cardiovascular regulation, and smooth muscle activity.
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~ OF SYNTHESIZING SAME
-This applic~ on is a division or Canadian Serial No.
403,789, filed May 26, 1982.
The presen~ invention relates generally to medicinal compositions and the use thereof; and more par~icularly, it relates to biologically active peptide compositions useful in the treat~ent of human neoplastic dlsease.
Investigations into the presence of physiologically or pathologically active peptides in urine have been on going for the past 80 yearsO Biologically active poly-peptides have been isolated from urine which have demon-strated hormone like activity or regulation of biological function. Examples of biologically active polypeptide compositions isolated from urine include growth factors, pituitary hormones, and kinins.
The practically infinite variety of peptides that can be formed~by the combination of the twenty common amino acids has prompted many investigators to suggest that peptides may constitute a system carrying informa-tion from cell to cell and organ to organ. Following this view on the regulatory significance of peptides, researchers have isolated urinary peptides which exert an influence o~ blood pressure, behavior modification, cardiovascular regulation, and smooth muscle activity.
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-2--Accordingly~ it has been considered by a number of researchers that neoplastic growth may be zontrolled by naturally occurring biochemical defense mechanisms. The immunological process has most often been attributed with antineoplastic ac~ivity (see for example, Aoki et al, Prog. Exp Tumor Res., 19:23~ 1974). There are, however, other possible mechanisms.
It has been suggested that neoplasia is a disease of cell differentiation. Given the large number of differen-tiating cells and assuming the possibility of error in the program for differentiation, groups of abnormally growing cells ca~ often arise under the influence of carcinogenic factors. Without a reliable mechanism for "normalizing~
such erroneously developed cells, the organisms would not live very long. Such a mechanism should be able to correct the growth of newly developed neoplastic cells and direct them into normal differentiation pathways. It is Appli-cantls belief that peptides are ideal compounds ~o func-tion as information-carrying molecules regula~ing cell differentiation.
In recent years, Applicant has described a number of medium-sized peptides derived from human urine, which demonstrate inhibition of DNA synthesis and mitosis in cultures of various neoplastic cells without significant inhibition of normal cell replication [see Burzynski, Physiol. Che~. Phys., 5:437 (1973); Burzynski et al, Fed.
Proc~, 32:766 (1973); Burzynski et al, Physiol. Chem.
Phys., 8:13 (1976); Burzynski et al, Fed. Proc., 35:623 ~1976); Gross et al, Physiol. Chem. Phys., 8:275 (1976);
and Burzynski et al, Physiol. Chem. Ph~s., 9:485 (1977)1.
The active compounds, but heretofore unidentified discrete compounds, from these fraction~ have been given the working name ~antineoplastons". Applican~ has defined ,~
~2~;ZY~1~)'7 antineoplastons as substances produced by a living organism that protect it against development of neoplastic growth by a non-immunological process which does not significantly inhibit the growth of normal tissues.
Although some polypeptides have been synthesized which demonstrate antineoplastic properties (see de Barbieri et al, Boll. Chim. Farm., 111:216, 1972), applicant is not aware of prior art describing small sized (less than 10 amino acids), low-molecular weight polypeptides which have been isolated and identified from tissues or body fluids that exhibit antineoplastic activity significantly higher than inhibition of normal cell growth. Nor is applicant aware of prior art describing the peptide, 3-[N-phenylacetylaminopiperidine]-2, 6-dion, or its use as an antineoplastic agent.
The invention in this divisional application pertains to the composition 3-[N-phenylacetylaminopiperidine]-2, 6-dion, and the pharmaceutically acceptable salts thereof, and the process for synthesizing same, which process comprises the steps of providing a quantity of L-glutamine, providing a ~uantity of phenylacetyl halide, mixing together the L-glutamine and phenyl acetyl halide in a weakly alkaline aqueous solution for a period of time to provide an a~ueous reaction mixture, including the reaction product 3-[N-phenylacetylaminopiperidine]-2, 6-dion, and isolating the composition from the reaction mixture. When desired pharmaceutically acceptable salts of the composition may be prepared. The composition inhibits the growth of neoplastic cells in a host having neoplastic disease.
The invention also disclosed herein and claimed in the parent application (Canadian Serial No. 403,789) pertains to low molecular weight substances (MW less than 2-5000) useful in the treatment of human neoplastic disease are isolated and concentrated from human urine. The isolation procedures involve initial ultrafiltration operations separating lower molecular weight compounds (less than) ~ ~6Z5~
2000-5000 M~) from higher molecular weight compounds and proteins. Following the fil-tration and ultrafiltration operations, the resulting urine ultrafiltrate containing the lower molecular weight compounds is then subjected to diverse sequential separational procedures yielding in particular an antineoplaston frac-tion comprising small sized peptide compounds ~less than lO amino acids).
According to one sequential separational process, the urine ultrafiltrate is acidi~ied, filteeed again, 1~ and subjected to high performance liquid chromatography employing a silica gel C-18 column. The fraction detected and collected as refractive index peak after elution with 450 ml water is referred to as antineoplaston fraction Al.
According to a second sequential separation process, the urine ultrafiltrate is acidified, filtered again, and passed through a polymeric resin adsorbent column. Eluates are collected from the polymeric resin column corresponding to three sequential washes: water, water and methanol, and a final water wash. The combined eluates from these washes are acidified and then further purified by C-18 bonded phase silica gel chromatography. The colored fractions developed by a methanol wash are collected and combined to constitute antineoplaston fraction A2.
According to a third sequential separation process, the urine ultrafiltrate is acidified, filtered again, adsorbed onto a polymeric resin adsorbent, and subsequently eluted from the resin with an alkaline solutionO The alkaline eluate is acidified to pH 2.5 and the oxidized.
The oxidized fraction is further puriied to antineoplaston fraction A3 by adsorption chromoatography on silica gel C-18.
According to a fourth sequential separation process, urine is first acidified and then oxidized. The oxidized solution is filtered and separated into antineoplaston ~raction A4 by passage through silica gel C-18 chromato-graphy phase~ The colored fraction eluted with methanol is collected and termed antineoplaston raction A4.
According to a fifth sequential separation process, the urine ultrailtrate is acidified and then eluted feom silica gel C-18 by a methanol wash. The c~lored portion of the methanolic eluate is collected and termed antineo plaston fraction A5.
Further in accordance with the present invention, the common component of each of the antineoplaston fractions was isolated to homogeneity using high performance liquid chromatography and thin layer chromatography. A common component of each antineoplaston fraction A1 to A5 was identified as 3-[N-phenylacetylaminopiperidine]-2, 6-dion.
Further in accordance with the present invention, a method for synthesizing the major active component, 3-[N-phenylacetylaminopiperidine]-2, 6-dion is provided, which comprises the steps of reacting L-glutamine and phenyl-acetyl chloride together followed by several extraction operations to isolate the produce, 3-[N-phenylacetylamino-piperidine]-2, 6-dion, from by-products.
Upon hydrolysis of 3-[N-phenylacetylaminopiperidinel-2, 6-dion there are yielded the degradation products~ phenyl-acetyl glutamine and phenylacetic acid.
The antineoplaston fractions, 3-[N-phenylacetylamino-piperidine]-2, 6 dion and degradation products are useful in the treatment of human neoplastic disease.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 represents a chromatograph of antineoplaston fraction A1.
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Figure 2 represents a chromatograph of antineoplaston fraction A2.
Figure 3 represents a chromatograph of antineoplaston fraction A3.
Figure 4 represents a chromatograph of antineoplaston fraction A4.
t0 Figure 5 represents a chromatograph of antineoplaston fraction A5.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
.. . . . . .. ~
The invention will be described in terms of preferred embodiments known to the Applicant at the time of this application which represent the best mode corresponding to the isolation, purification and implementation of urine antineoplaston fractlons and synthetic antineoplastons exhibiting antineoplastic activity.
In accordance with such preferred embodiments, start-ing material for the preparation of each antineoplaston fraction is urine pooled from healthy subjects. Typically, ~5 the amount of urine required to elaborate useful ~ields of a desired antineoplaston fraction A1-A5 range from about 2000-3000 liters. Usable yields extracted from 2000-3000 liters of urine are on he order of 100-800 grams of dry matter for each of the respective antineoplaston fractions.
Pooled urine specimens may be lyophilized to a dry powdered form if ~he extraction, isolation and purification pro-cesses are not to be accomplished immediately. Typically, however, the isolation and purification of the respective antineoplaston fractions are performed immediate]y utiliz-ing the freshly pooled urine.
It has been suggested that neoplasia is a disease of cell differentiation. Given the large number of differen-tiating cells and assuming the possibility of error in the program for differentiation, groups of abnormally growing cells ca~ often arise under the influence of carcinogenic factors. Without a reliable mechanism for "normalizing~
such erroneously developed cells, the organisms would not live very long. Such a mechanism should be able to correct the growth of newly developed neoplastic cells and direct them into normal differentiation pathways. It is Appli-cantls belief that peptides are ideal compounds ~o func-tion as information-carrying molecules regula~ing cell differentiation.
In recent years, Applicant has described a number of medium-sized peptides derived from human urine, which demonstrate inhibition of DNA synthesis and mitosis in cultures of various neoplastic cells without significant inhibition of normal cell replication [see Burzynski, Physiol. Che~. Phys., 5:437 (1973); Burzynski et al, Fed.
Proc~, 32:766 (1973); Burzynski et al, Physiol. Chem.
Phys., 8:13 (1976); Burzynski et al, Fed. Proc., 35:623 ~1976); Gross et al, Physiol. Chem. Phys., 8:275 (1976);
and Burzynski et al, Physiol. Chem. Ph~s., 9:485 (1977)1.
The active compounds, but heretofore unidentified discrete compounds, from these fraction~ have been given the working name ~antineoplastons". Applican~ has defined ,~
~2~;ZY~1~)'7 antineoplastons as substances produced by a living organism that protect it against development of neoplastic growth by a non-immunological process which does not significantly inhibit the growth of normal tissues.
Although some polypeptides have been synthesized which demonstrate antineoplastic properties (see de Barbieri et al, Boll. Chim. Farm., 111:216, 1972), applicant is not aware of prior art describing small sized (less than 10 amino acids), low-molecular weight polypeptides which have been isolated and identified from tissues or body fluids that exhibit antineoplastic activity significantly higher than inhibition of normal cell growth. Nor is applicant aware of prior art describing the peptide, 3-[N-phenylacetylaminopiperidine]-2, 6-dion, or its use as an antineoplastic agent.
The invention in this divisional application pertains to the composition 3-[N-phenylacetylaminopiperidine]-2, 6-dion, and the pharmaceutically acceptable salts thereof, and the process for synthesizing same, which process comprises the steps of providing a quantity of L-glutamine, providing a ~uantity of phenylacetyl halide, mixing together the L-glutamine and phenyl acetyl halide in a weakly alkaline aqueous solution for a period of time to provide an a~ueous reaction mixture, including the reaction product 3-[N-phenylacetylaminopiperidine]-2, 6-dion, and isolating the composition from the reaction mixture. When desired pharmaceutically acceptable salts of the composition may be prepared. The composition inhibits the growth of neoplastic cells in a host having neoplastic disease.
The invention also disclosed herein and claimed in the parent application (Canadian Serial No. 403,789) pertains to low molecular weight substances (MW less than 2-5000) useful in the treatment of human neoplastic disease are isolated and concentrated from human urine. The isolation procedures involve initial ultrafiltration operations separating lower molecular weight compounds (less than) ~ ~6Z5~
2000-5000 M~) from higher molecular weight compounds and proteins. Following the fil-tration and ultrafiltration operations, the resulting urine ultrafiltrate containing the lower molecular weight compounds is then subjected to diverse sequential separational procedures yielding in particular an antineoplaston frac-tion comprising small sized peptide compounds ~less than lO amino acids).
According to one sequential separational process, the urine ultrafiltrate is acidi~ied, filteeed again, 1~ and subjected to high performance liquid chromatography employing a silica gel C-18 column. The fraction detected and collected as refractive index peak after elution with 450 ml water is referred to as antineoplaston fraction Al.
According to a second sequential separation process, the urine ultrafiltrate is acidified, filtered again, and passed through a polymeric resin adsorbent column. Eluates are collected from the polymeric resin column corresponding to three sequential washes: water, water and methanol, and a final water wash. The combined eluates from these washes are acidified and then further purified by C-18 bonded phase silica gel chromatography. The colored fractions developed by a methanol wash are collected and combined to constitute antineoplaston fraction A2.
According to a third sequential separation process, the urine ultrafiltrate is acidified, filtered again, adsorbed onto a polymeric resin adsorbent, and subsequently eluted from the resin with an alkaline solutionO The alkaline eluate is acidified to pH 2.5 and the oxidized.
The oxidized fraction is further puriied to antineoplaston fraction A3 by adsorption chromoatography on silica gel C-18.
According to a fourth sequential separation process, urine is first acidified and then oxidized. The oxidized solution is filtered and separated into antineoplaston ~raction A4 by passage through silica gel C-18 chromato-graphy phase~ The colored fraction eluted with methanol is collected and termed antineoplaston raction A4.
According to a fifth sequential separation process, the urine ultrailtrate is acidified and then eluted feom silica gel C-18 by a methanol wash. The c~lored portion of the methanolic eluate is collected and termed antineo plaston fraction A5.
Further in accordance with the present invention, the common component of each of the antineoplaston fractions was isolated to homogeneity using high performance liquid chromatography and thin layer chromatography. A common component of each antineoplaston fraction A1 to A5 was identified as 3-[N-phenylacetylaminopiperidine]-2, 6-dion.
Further in accordance with the present invention, a method for synthesizing the major active component, 3-[N-phenylacetylaminopiperidine]-2, 6-dion is provided, which comprises the steps of reacting L-glutamine and phenyl-acetyl chloride together followed by several extraction operations to isolate the produce, 3-[N-phenylacetylamino-piperidine]-2, 6-dion, from by-products.
Upon hydrolysis of 3-[N-phenylacetylaminopiperidinel-2, 6-dion there are yielded the degradation products~ phenyl-acetyl glutamine and phenylacetic acid.
The antineoplaston fractions, 3-[N-phenylacetylamino-piperidine]-2, 6 dion and degradation products are useful in the treatment of human neoplastic disease.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 represents a chromatograph of antineoplaston fraction A1.
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Figure 2 represents a chromatograph of antineoplaston fraction A2.
Figure 3 represents a chromatograph of antineoplaston fraction A3.
Figure 4 represents a chromatograph of antineoplaston fraction A4.
t0 Figure 5 represents a chromatograph of antineoplaston fraction A5.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
.. . . . . .. ~
The invention will be described in terms of preferred embodiments known to the Applicant at the time of this application which represent the best mode corresponding to the isolation, purification and implementation of urine antineoplaston fractlons and synthetic antineoplastons exhibiting antineoplastic activity.
In accordance with such preferred embodiments, start-ing material for the preparation of each antineoplaston fraction is urine pooled from healthy subjects. Typically, ~5 the amount of urine required to elaborate useful ~ields of a desired antineoplaston fraction A1-A5 range from about 2000-3000 liters. Usable yields extracted from 2000-3000 liters of urine are on he order of 100-800 grams of dry matter for each of the respective antineoplaston fractions.
Pooled urine specimens may be lyophilized to a dry powdered form if ~he extraction, isolation and purification pro-cesses are not to be accomplished immediately. Typically, however, the isolation and purification of the respective antineoplaston fractions are performed immediate]y utiliz-ing the freshly pooled urine.
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It is to be noted that standard precautlons against bacterial contamination are taken throughout and the preparations are routinely checked for pyrogenicity, toxicity and sterility assay according to standard tech-niques~ Pyrogen free sterile water is employed through-out the final steps and all procedures are performed at -~ ambient room temperature unless stated otherwise.
. Isolation and Purification of Anti-neoplaston Fraction Al from Human Urine Reconstituted lyophilized urine (redissolved in deionized or distilled water) or freshly pooled urine is first physically filtered through paper, membrane, or cartridge filter having an average pore size of 3~ . The first filtrate is then filtered through a second filter having an average pore size of O.~p . These filtering steps are performed to separate suspended particulate or sedimented ~atter from the urine fluid.
Next, the prefiltered urine is submitted to ultrafil-tration. Desirably, ultrafiltration is accomplished through a hollow ~iber system, preferably an Amico~ ~r Romico~Msystem filter having a molecular weight cut off of about 5000 daltons. For the purpose of ultrafiltration, any other ultrafiltrati~n membrane or hollow fiber ultra-filter may be employed having a molecular weight cut off suitably in the range of 5000 to 2000. Such ultrafiltra-tion serves to remove materials having molecular weights greater than 2000 to 5000 depending on the selected filter employed.
The ultrafiltrate is acidified with concentrated acid, suitably hydrochloric or sulfuric acid, added slowly while vigorously Rtirring until ~he solution reaches a pH
ranging from ~ to 3, preferably pH 2.5. The acidified ultrafiltrate is then filtered through a 0.2~ filter to remove any precipitated particulate matter~
High performance liquid chromotography techniques are used to further purîfy and concentrate the designated anti-neoplaston fraction A1. A sample, suitably 250 ml (the amount, of course, depends upon the capacity of the sys-tem), of the acidified ultrafiltrate is introduced into a high performance liquid chromatography column, desirably a Waters Pre~M500 HPLC system utilizing a PrepTM500 C-18 silica gel cartridge column (bonded phase type silica).
The system is also equipped with a refractive index detec-tor. However, any suitable means of detection such as UV
photometry, ion detector, etc. is suitable. Antineoplas-ton fraction ~2 is eluted with deionized or distilled water and is characterized as the component peptide fràc-tion having a refractive index peak occurring after pas-sage of approximately 450 ml of water through the column.
The resulting antineoplaston fraction A1 is collected and concentrated by rotary evaporation under reduced pressure and then further freeze dried by lyophilization.
Antineoplaston fraction A1 can be used in a wide variety of pharmaceutical forms including, but not limited to, intravenous, intramuscular, subcutaneous, intracavital and intratumor injections, capsules and tablets for oral administration, rectal suppositories and solutions and sprays for topical use.
B. Isolation and Purification of Anti-neoplaston Fraction A2 from Human Urine Urine which has been prefiltered and ultrafiltered according to the description directed to the preparation ~L2~29~7 g of antineoplaston fraction A1 is acidified with concen-trated acid, typically hydrochloric acid. The acid is added slowly to the ultrafiltrate solution while vigor-ously stirring until the solution reaches a pH ranging from about 1 to about 2, preferably pH 1.5.
The ultrafiltrate from the above step is introduced onto a chromatography column containing a polymeric resin adsorbent; preferably Amberlite XA~ polymeric resin adsorbent, a product of Rohm & Haas Co., Philadelphia, Pennsylvania. Any other material similar in chemical structure or physicochemical properties may be substituted for the Amberlite adsorbent. The active antineoplastic materials are eluted from the column by employing sequen-tial washings comprising a first wash of deionized or reverse osmosis water (W1); a second wash of a mixture of water and methanol (for example, 8% volume/volume) (M);
a third wash of deionized or reverse osmosis water (W2);
a fourth wash of 4% sodium hydroxide aqueous solution (N); and a fifth wash of deionized or reverse osmosis water until the pH of the eluate is in the neutral range.
Eluates W1, W2 and M are collected. The pH of the solu-tions of Wl, W2 and M is adjusted to about 2.5 with dropwise addition of acid, for example, sul~uric acid.
The eluates W1, W2 and M from the previous step are passed through a chromatographic column packed with silica gel Prep C-18 (bonded phase type silica gel) available from Waters Associates, Whatman or other companies. The column is initially washed with deionized or distilled water and then eluted with methanol. Three brownish-yellow colored fractions appear as bands, designated MWl, MW2 and MM. Colored fractions MWl, MW2 and MM are col-lected independently and each fraction is concentrated on a circulatory evaporator to a 1 liter volume. Each i2~7 --1 o--fraction is further evaporated to dryness either by rotary evaporation or by freeze drying. Dry fractions MW1, ~W2 and MM are suitable for pharmaceutical application sepa-rately or mixed together. The mixture of them is termed antineoplaston fraction A2, and the mixture is suitable for pharmaceutical administration in the same variety of formulations and routes of administration described for antineoplaston fraction A1.
C. Isolation and Purification of Anti-neoplaston Fraction A3 from Human Urine Antineoplaston fraction A3 is isolated from urine during the same operation as the isolation and purifica-tion of antineoplaston fraction A2. P~refiltration, ultrafiltration, acidification, XAD-~ adsorption and elution is identical to that practiced for the isolation of antineoplaston fraction A2.
Fraction N eluted from the XAD-8 column with 4~
sodium hydroxide is collected and the p~ is adjusted to 2.5 with acid, desirably sulfuric acid. Fraction N is then subjected to oxidation operations. The oxidation of fraction N is preferably accomplished by dropwise addition of a saturated aqueous solution of potassium permanganate until the violet color of potassium permanganate disappears.
After the oxidation operation, fraction N is filtered sequentially through a ~ and 0. ~ filter and the clear filtrate is further separated by C-18 chromatography.
This chromatographic step is repeated in the same manner as described for the isolation of antineoplaston fraction A2. The colored band visible on the column designated MN
is eluted with methanol. The colored band MN is collected and evaporated to dryness of freeze dried. This fraction ~LZ~9~7 is called antineoplaston fraction A3 and is suitable for direct pharmaceutical application. Antineoplaston ~rac-tion A3 can be used in the same variety of pharmaceutical formulations and routes of administration as listed for antineoplaston fraction A1.
D. Isolation and Purification of Anti-n oplaston Fraction A4 from Human Urine The pH of reconstituted or fresh urine is adjusted to 2.5 with acid, suitably sulfuric acid. Contents of the urine are then oxidized by mixing the urine with a satu-rated solution of potassium permanganate in water until the violet color of potassium permanganate disappears.
After oxidation, the treated urine is filtered and the clear filtrate is separated by C-18 chromatography per-formed in the same manner as described for the isolation of antineoplaston fraction A2.
The colored band visible on the column is designated fraction U and is eluted with methanol and evaporated to dryness or freeze dried. This fraction called antineo-plaston fraction A4 is suitable for pharmaceutical appli-cation in the same variety of pharmaceutical formulations and routes of administration as described for antineo~
plaston fraction A1.
E. Isolation and Purification of Anti-neoplaston Fraction A5 from Human Urine The prefiltration, ultrafiltration, and acidi~ication of reconstituted or fresh urine is repeated as described above for the preparation of antineoplaston fraction A1.
The acidified material is filtered again through a 0.2 filter to remove any precipitated or sedimented residue.
This filtrate is then introduced to a chromatographic column filled with C-18 bonded phase type silica gel, ~available for example from Waters Associates or Whatman~.
Other silica gel, packings having the same physicochemical properties may be substituted for the C-18 column packing.
The column is initially washed with deionized, dis-tilled water and then eluted with methanol. A colored methanolic fraction is collected, which is evaporated to dryness or freeze dried The dry raction is labelled antineoplaston fraction A5. Antineoplaston A5 is suita-ble for pharmac~utical application in the same variety of formulations and routes of administration as described for antineoplaston fraction A1.
F. Chromatographic Characterization of Antineoplaston_ ractions Al-A5 For purposes of identifying each of the derived anti-neoplaston fraction, a chromatographic fingerprint wasdeveloped. Each of the derived antineoplaston fractions from the respective above-described processes was subjected to high performance liquid chromatography, a Waters Prep 500 HPLC system equipped with a Prep 500 C-18 bonded phase 2~ type silica gel column and refractive index detector.
Each antineoplaston fraction was developed according to the same sequential wash routine~ First, a predetermined amount of reconstituted antineoplaston fraction was introduced to the columnO Typically the antineoplaston fraction in powdered form was reconstituted with distilled water.
Following introduction of the antineoplaston fraction sample, a first wash with 1000 ml of water was passed through the column. This water wash was followed with 1000 ml of an acetic acid solution, pH 2.5, wash. ~inally, 1000 ml of water was passed through the column and 600 ml o~ methanol. As the eluates exited the column, the detector measured and recorded the apparent refractive index of the components eluted within an exiting solvent.
With reference to the figures, a characteristic chromatograph for each of the antineoplaston fractions is illustrated. The figures illustrate the relative refrac-tive index corresponding to components present in the eluates exiting the column at the relative elution volumes, corresponding to passage of each of the solvent washes.
It will be appreciated by those familiar with chromato-graphic development, that each chromatograph represents a resolution of peak distribution characteris-tic for a particular mixture. A chromatograph serves as a finger-print analysis of the mixture or in thisinstance a finger-print for the antineoplaston frac'cions. It is therefore apparent, that the respective product antineoplaston fractions purified according to the methods of the inven-tion, will exhibit the characteristic chromatographcorresponding to the figures illustrated herein when developed according to theconditions described above. It will also be appreciated by those skilled in the art of chromatography, that the relative heiyht of the peaks will vary with the concentration of the component elements present in each fraction, however, the distribution of the peaks will not vary substantially from batch to batch of each fraction.
Referring now to Figure 1, a chromatograph is depicted wherein antineoplaston fraction Al exhibits a discrete sharp peak in the region of first water wash. Further, Z9~7 antineoplaston fracti~on A1 exhibits a broad peak distribu-tion comprising a series of moderately defined peaks con-centrated in the region of the end of acetic acid wash and of the second water wash. In addition there are sharply defined peaks occurring after 450 ml of methanol wash.
Figure 2 depicts the chromatograph of antineoplaston fraction A2, wherein a series of sharply defined peaks are apparent in the region of the end of acetic acid wash and extend into the region of the second water wash.
Further, there are sharply defined peaks in the methanol wash.
Figure 3 depicts the chromatograph of antineoplaston fraction A3, which exhibits a small peak in the initial water wash and a concentrated band of peaks in the region of the end of acetic wash and extending into the second water wash. In addition, there are well deined peaks in the methanol wash.
Referrin~ now to Figure 4, there is depicted a chromatograph of antineoplaston fraction A4 which exhibits a small pea~ in the initial water wash and a broad peak resolved in the acetic acid wash and second water wash.
Further, there are sharp peaks in the methanol wash.
Next, turning to Figure 5, there is a chromatograph of antineoplaston fraction ~5O The chromatograph depicts a small peak in the initial water wash and a broad peak comprising a series of moderately defined peaks in the end ~f acetic acid wash and extending into the second water wash. There are also well defined peaks in methanol wash.
* * *
b;2~7 Further attempts were made to isolate and identify tl1e component elements of each antineoplaston fraction.
The component elements of antineoplaston fractions A1-A5 were separated by high performance liquid chromatography on a column packed with sulfonated polystyrene. A system developed by Glenco Scientific Inc. for amino acid analy-sis was used. The elution was effected by 0.2M citrate bufers at three different values of pH, namely 3.25, 3~80 and 4.10 and at temperatures form 50C to 70C. The ~o changes of buffers and temperature were selectively con-trolled according to Glenco Scientific, Inc. pro~ram for amino acid analysis.
The eluates were reacted as they came off the column with ninhydrin at 110C to yield absorption peaks simul-taneously measured and recorded at 470m~ and 440m~ . In order to standardize the procedure, a mixture of 18 amino acids was separated establishing the retention times pre-sented in Table 1 op~
TABLE 1. Standard Retention Times of Amino Acids Amino Acids Retention Times (Minutes) Aspartic Acid 18.0 Threonine 21.5 Serine 22.7 Glutamic Acid 26.0 Proline 30.0 Glycine ~ 38.4 Alanine 38.9 Cysteine 42.0 Valine 47.0 Methionine 49.0 Isoleucine 52.2 Leucine 53.8 Tyrosine S9.2 Phenylalanine 64.0 Lysine 72.0 Ammonia 78.0 Histidine 80.2 Arginine 93~5 Each of the antineoplaston fractions A1-A5 were separated into discrete homogeneous components under the same conditions as the standardized amino acid mixture.
Table 2 summarizes the retention times of the discrete components making up each of the heterogeneous antineo~
plaston fractions.
iZ9~;)7 TABLE 2. Retention Time of Alpha-Amino Components of Antineoplaston Fractions A1-A5 -Antineoplaston Fraction Retention __ (mM~L of alpha-amino_nitrogen~ _ Time (Min.) A1 A2 A3 A4 AS
0.12 1.57 0.24 1.97 0.20 0 0.78 0.05 0.82 0 26 0 0.22 0 0.75 0.13 1~37 2.44 0.25 0 0.59 0008 48 0.12 0.47 0.30 0.77 0.20 53 0 0.12 0.04 0.43 0 63 0 0.23 0 0.63 0.10 71 0 0.17 0.07 0.30 1576 0.13 0.14 0.45 0.87 0.13 79 3g.50 9.39 4.44 16.35 14.88 82 0.22 1.01 0.92 1.09 0.21 0.07 0.62 0.65 0.45 0.14 101 0 0.01 0 0.10 0 ?0 -The preparations of antineoplaston fractions A1, A2, A3, A4 and A5 do not contain either amino acids or proteins.
The peaks recorded during analysis of the antineoplaston fractions correspond to the different compounds reacting with ninhydrin, namely amino acid derivatives and peptides.
As denoted in Table 2, the relative concentration of each component compound within the particular antineoplaston fraction is measured relative to the reactivity between ninhydrin and the alpha-amino nitrogen of the component compound. In accordance with the chromatographic analysis described in this section, each antineoplaston fraction exhibits a significantly prominent peak at a retention time corresponding to 79 minutes. Further, in accordance ~62~1J'7 with the methods of preparing each anitneoplaston fraction embodied by the present invention, the relative concentra-tion of the component corresponding to a 79 minute reten-tion time is at least two times the relative concentration of any other residual component compound. It is to be appreciated, however, that the relative concentration of each component compound constituting an antineoplaston fraction will vary with the source of urine. Indeed, depending on the source of urine several of the component compounds within an antineoplaston fraction might not be evident. Applicant makes no representation that each component compound within an antineoplasto~n fraction possesses antineoplastic activity; rather, antineoplastic activity has been demonstrated for each antiplaston frac-tion A1-A5 and for the component compound characterized by a 79 minute retention time obtained by the above-described separation technique.
The major common active component of antineoplaston ~0 fraction A1, A2, A3, A4 and A5 was finally purified by high performance liquid chromatography and thin layer chromatography. Applicant has termed this compound anti-neoplaston A10. Its chemical structure was determined by mass spectrometry, 13C~NMR) spectroscopy, and infrared spectrometry. The structure is depicted below and termed 3-[N-phenylacetyl~aminopiperidinel-2, 6-dion.
~,o I ~
o ~I~NH
~_/ H
~LZ~Z9~37 G. Synthesis of Antineoplaston A10, 3-lN-phenylacetylaminopiperidine3-2, 6-dion Sodium bicarbonate (4.7 mole) and L-glutamine (2.3 mole) were dissolved in water ~13.5 liters). Phenylacetyl chloride (3.0 mole) was gradually added to the reaction mixture and vigorously stirred for 90 minutes. After completed reaction, the pH of the solution was adjusted to 2.5 with acid and the solution filtered. The filtrate was extracted twice with dichloromethane and the lower organic layers were discarded. The upper aqueous layer had the pH
adjusted to 7O0 with base, typically lN NaOH. The upper layer was then further purified by mixing with Norit ~M(209) (available from American Norit Co., Jacksonville, Florida).
The mixture was filtered after 30 minutes contact with Norit A. The resultant filtrate was evaporated and the residue redissolved in methanol. The recovered methanolic solution was filtered and freeze dried or evaporated. rrhe dried residue was redissolved in water and the pH adjusted to 2.5 suitably with ~C1. Two layers were formed after standing at room temperature. The lower layer was heated until it turned dark brown. This viscous brown layer was redissolved in methanol wherein crude antineoplaston A10 precipitated upon cooling. The crude antineoplaston A10 was redissolved in hot methanol and Norit A was added to remove any color. The solution was filtered while hot.
On cooling, white crystals of antineoplaston A10 formed.
The structure of the synthetic material was elucidated by mass spectrometry, 13C(NMR) spectroscopy and infrared spectrometry was found to be identical to the maior common active component of antineoplaston fractions A1, A2, A3, A4 and A5 having a 79 minute retention time.
~L2~;~907 Antineoplas~on A10 is most suitable ~or phaEmaceuti-cal applications in the form of salts, sodium salt being : preferred. ~n order to prepare sodium salt, antineoplas-ton A10 is suspended in ethanol and heated with a solution : 5 of sodiu~ hydroxide water until all material is dissolved.
The reaction mixture is then freeze dried. The solid resi-. due is kept at room temperature until the salt cyrstallizes out. Ethanol is added and stirred. Filtration yields a white crystalline solid corresponding to the sodium salt of antineoplaston A10. Suitable solutions for parenteral administration are prepared by dissolving sodium salt of antineoplaston A10 in pyrogen free water up to a concentra-tion of lOOmg/ml and adjusting the pH to 7Ø
~. Degradation Products of Antineoplaston A10 The initial hydrolysis of antineoplaston AlO yields phenylacetyl glutamine, and when carried further produces phenylacetic acid:
f~Z ~11 H 1 N /~ /~ O H
3-[N phenylacetylamino- N-phenyl acetyl piperidine]2, 6-dion glutamine 3~ ~0.
O /~1 H 2 (~ .~
Phenylacetic acid Glutamine ~L~62907 Phenylacetyl glutamine was first described by Thier-felder and Sherwin [see J. Physiol. Chem. 94:1 (1915)~ as a constituent of normal human urine. In later investiga-tions of the compound phenylacetyl glutamine was shown to have a slight effec~ on the growth of murine tumors [see Lichtenstein et al, Israel J. Med. Sci., 13:316 ~1977)3 but there was no indication that the compound was useful in the treatment of human cancer. The sodium salt of phenylacetic acid was used by Neish in the treatment of Rd/3 sarcoma in rats but failed to inhibit tumor growth.
Indeed, the results suggested that the treatment with phenylacetic acid caused some enhancement of tumor growth ~see Neish, Experientia, 27:860 ~1971)]. Applicant has demonstrated in his clinical studies that phenylacetyl glutamine alone and a mixture of phenylacetyl glutamine and phenyl acetic acid are each useful in the treatment of human cancers.
A mixture of the sodium salts of phenylacetyl gluta-mine and phenyl acetic acid in the ratio 1 to 4 is thepreferred formulation for use in the treatment of human cancer.
Solutions for parenteral administration are prepared by reconstituting the respective chemicals in form of sodium salts in pyrogen free water to the desired total concentration, for example 100 mg/ml. The p~ of the solution is adjusted to 7.0 with 1N NaOH or lN HCl.
Sterilization of the reconstituted solution is done by 33 filtration according to the guidelines of the U.S. Phar-macopeia. The sterility of the material is tested as required by the rules and regulations of the Food and Drug Administration, Section 610.12. The resulting sterile formulations are suitable for parenteral injections.
~ZS29q~7 I. Pharmaceu~ical Applications for Anti-neoplaston Fractions A1-A5, Antineo-~laston A10, and Degradation Products Solutions for parenteral administration are prepared by reconstituting each respeCtiYe antineoplaston fractions, antineoplaston A10 and degradation products in pyrogen free water to a desired convenient concentration, for example, 100 mg/ml. The pH of the solution is adjusted to 7.0 with lN HCl or lN NaOH.
Sterilization of the reconstituted solution is done by filtration according to the guidelines of the U.S.
Pharmacopeia. Alternatively, the lyophili~ed powders of the respective antineoplaston fractions can first be gas sterilized, for example with ethylene oxide, and the ; powders subsequently incorporated into the pyrogen free water which, of course, itself is sterile. The sterility of the material is tested as required by the Rules and Regulations of the Food and Drug Administration, Section 610~12. The resulting sterile formulations are suitable for intravenous, intramuscular, subcutaneous, intracavital and intratumor injection.
If the reconstituted lyophilized antineoplaston frac-tion is not to be used immediately, the prevention of microbial proliferation can be attained by the addition of various antibacterial and antifungal agents to the antineoplaston solutions, ~or example, parabens, chloro-butanol, benzyl alcohol, phenol, sorbic acid, thimerosal, and the like. In many instances it will be desirable to include isotonic agents to the injectable solutions, for example~ sugars or sodium chloride.
~L2~
The antineoplastic activity of the antineoplaston fractions A1-A5, antineoplaston A10 and degration products was first evaluated experimentally by observing the cytostatic effects the preparations would have on a tumor line as compared to the overall toxicity the preparation would have on experimental animals. Accordingly, the preparation having the greatest cytostatic activity and the lowest animal toxicity are said to have the better antineoplastic activity, or therapeutic effectiveness.
The cytostatic activity of antineoplaston fraction A1 was tested in a culture of human carcinoma of the breast line MDA-MB-231 obtained from M.D. Anderson Cancer Institute, Houston, Texas. MDA-MB-231 is a fast growing line of human breast cancer established by Cailleau et al, J. Natl. Cancer Inst., 53:661 (1974). The estimated .
doubling time of these cells is 18 hours when grown in the original medium described by Beall et al, Physiol. Chem.
Physics, 8:281 (1976). ~rieflyr to summarize the preferred medium and method, the cells are grown in monolayers at 37C in Leibovitz L-15 medium supplemented with 20% fetal bovine serum, 1.6~ g/ml glutathione, 0.25 U/ml insulin, 100u g/ml disodium carbenicillin and 10~ g/ml gentamycin.
For the bioassay, antineoplaston fraction A1 was dis-solved in the above-described medium at four different concentrations selected arbitrarily with the range of 0.5 to 50 mg/ml. Monolayer cultures were incubated with the antineoplaston fraction A1-containing medium for 96 hours.
The cells were counted by visual method at 24 hour inter-vals. Control cultures were grown in the standard medium without added antineoplaston fraction A1.
~Z6~
Antineoplaston fraction Al in concentrations of 5 mgfml produces a cytostatic effect in such h~lman breast carcinoma cultures. The cytostatic effect is determined as a stable number of cells (within the limits from 80% to 120~) counted after 24 hours from incubation and persist-~
ing for at least an additional 48 hours.
The cytostatic concentration of the antineoplaston fraction A2-A5 and antineoplaston A10 and degradation products were determined in the same manner described above. The cytostatic concentration for each antineo-plaston fraction is as follows:
AntineoplastonCytostatic Concentration Fraction Al 5 mg/ml Fraction A2 5 mg/ml Fraction A3 5 mg/ml Fraction A4 2 mg/ml Fraction A5 2 mg/ml A10 2 mg/ml Phenyl acetyl glutamine10 mg/ml Phenyl acetyl glutamine and Phenyl acetic acid (1:4 mixture) 3 mg/ml ~
Above these concentrations all antineoplaston fractions produce cytotoxic effect in human breast carcinoma cul-tures.
Acute toxicity studies on experimental animals reveal that antineoplaston fraction A1 has very low toxicity.
For example, experiments involving ~wenty-five HA/ICR
swiss mice injected intraperitoneally with antineoplaston fraction Al resulted in a LD50 if 1.35 g/kg. The autopsy ~LZ6X~
.
and microscopic studies of the tissues of the animals which died during the experiment revealed congestion of the liver and marked pulmonary edema. The animals which survived were kept for one week under close observation and were noted to carry on normal activity. After a week~, a select number of the mice were sacrificed. The autopsy and microscopic examination of the tissues of these animals were identical to those of control, uninjected subjects.
Acute toxicity studies involving antineoplaston fractions A2-A5, antineoplaston A10 and degradation products were carried out in the same manner described above. The respective LD5Q for mice for each fraction is as follows:
Antineoplaston LD50 Fraction Al 1.35 g/kg ~0 Fraction A2 3.55 g/kg Fraction A3 3.55 g/kg Fraction A4 5.33 g/kg Fraction A5 5.11 g/kg A10 10.33 g/kg Phenyl acetyl glutamine2O90 g/kg Phenyl acetyl glutamine and Phenyl acetic acid (1~4 mixture) 2.83 g/kg J. Clinical ~valuation of Antineoplaston The definitions of remission associated with neoplas-~ic disease are as follows: complete remission is the disappearance of all clinical evidence of disease and partial remission is reduction by at least 50~ in the sum ~;~6~ 7 of the products of two perpendicular diameters of all measurable disease lasting at least four weeks. Patients are considered stabilized if measurable tumor regression occurs but does not meet the criteria for partial remission.
In accordance with the methods in the present inven-tion, human neoplastic disease was treated with the various antineoplaston fractions. For each neoplastic disease studied, each tested antineoplaston fraction, antineoplaston A10 and degradation product, phenylacetyl glutamine, and a combination of phenylacetyl glutamine and phenylacetic acetic was efective to some extent in aiding ~ the regression of tumors. As would be expected, some fractions or compositions exhibited more effectiveness for some forms of neoplasia than other fractions.
.
The dosage of the selected antineoplaston fraction for the treatment of the indicated neoplastic condition depends on the age, weight and condition of the patient;
the particular neoplastic disease and its severity; and the route of administration. A dose of from about 0.5 to about 1~ g/m2/24 hr. or a total dose of from about 0.9 to about 20 g given in divided doses of up to 6 times a day embraces the effective range for ~he treatment of most neoplastic conditions for any one of antineoplaston fractions A1-A5, antineoplaston A10, and degradation products.
EXAMPLE I
To date, fourteen patients with advanced cancer have been treated with antineoplaston fraction A2 and followed for up to one year. The preferable route for the adminis-tration of the preparation is intraven~us injection given 6Z~3~7 every 12 hours through a catheter inserter into the sub-clavian vein. Direct intrapleural or intraperitioneal injections can also be given. The average dose given was 0.85 g~m2 and the maximum was 2.2 g/m2 intravenously every 12 hours. Full dose intravenous treatment with antineoplaston fraction A2 was usually given until the complete remission was obtained and then continued for at least 6 weeks to eradicate any remaining microscopic disease. Afterwards, IV injections were discontinued and the maintenance treatment was started. Maintenance treatment consisted of IM injections of 2 to 3 ml of 50 mg/ml antineoplaston fraction A2 initially given every other day. If no sign of cancer recurred, the frequency of IM injections was reduced every 6-8 weeks, tapering from one injection every third day to one injection once a week.
In the group of 14 patients treated with antineoplas-ton fraction A2 four obtained complete remission. These four cases included undifferentiated large cell carcinorna of the lung, stage III (T3NOMO~; poorly differentiated metastatic carcinoma of the liver with unknown primary;
and two cases of recurrent transitional cell carcinoma of the bladder, grade II. In an additional case involving adenocarcinoma of the breast with multiple bone and liver metastases, stage IV ~TONOM10SS, ~EP) a complete remission of large liver metastases and stabilization of bone metastases was obtained. There was also one additional case of transitional cell carcinoma of the bladder, grade II in which complete remission was obtained with help of antineoplaston fraction A2. In this case antineoplaston fraction A2 was given as a maintenance treatment after the complete remission was obtained with antineoplaston A.
~ILZ~9~7 Partial remission was obtained in three cases: peri-toneal mesotheliomal; carcinoma of the breast with multiple bone metastases, stage IV, TONOM10SS; and squamous cell carcinoma of the esophagus with multiple lung metastases, stage III, T3NOM1PUL.
Stabilization of the disease was obtained in four cases which included glioma, stage IV; adenocarcinoma of the kidney with multiple lung metastases, carcinoma of the breast with multiple bone metastasest stage IV, TONOMlOSS:
and carcinoma of the breast with lymph node involvement, stage IV, TON3MO.
Overall response rate to the treatement with antineo-plaston fraction A2 is 93% with only one patient (7%) showing continued progressive disease. The treatment is very well tolerated. Few side effects were evident includ-ing stimulation of the growth of epidermis, stimulation of bone marrow and very infrequent fever. Stimulation of the growth of epidermis was evident after 3 weeks of treatment ; as more rapid growth of nails and a thicker skin on the palms. Stimulation of the bone marrow was shown as ele-vated white blood and platelet count. These side effects are beneficial in most cases because of poor healing of the skin and myelosuppression present in large number of cancer patients.
EXAMPLE II
The following case history illustrates a successful method of treatment employing antineoplaston fraction A3 for the treatment of adenocarcinoma of the prostate with bone metastases, stage IV (RONOMlOSS, G3).
;Z~Q7 Treatment of a 72 year old white male was initiated with antineoplaston A (see Burzynski et al, PhYsiol._Chem Phys. 9:485, 1977) for three months. The initial treatment with antineoplaston A resulted in the stabilization of the disease and some questionable decrease o the size of the~
metastases.
After three months antineoplaston A was discontinued and the patient ~as started on antineoplaston fraction A3.
He received a first injection of 1 ml of antineoplaston fraction A3, 100 mg/ml given through a subclavian catheter followed with 3 ml of normal saline and 250 units of heparin. The dose was further increased up to 5 ml of the same preparation given intravenously every 12 hours. Such a treatment regimen corresponds to the dosage 0.47 g/m~/24 hour. After approximately 7 months the frequency of the injections was decreased to 2 ml of antineoplaston fraction A3, 100 mg/ml, given intramuscularly every third day, and finally after 4 months the dosing regimen was further decreased to 2 ml of the preparation administered intra-muscularly once a week.
The treatment with antineoplaston fraction A3 resulted in the complete remission of the bone metastastes as judged by bone scans. The treatment was very well tolerated with-out any apparent side effects.
Patient is continuing a maintenance treatment with antineoplaston fraction A3, and has not shown any recur-rence of his cancer at the present time.
~Lz6zg~
EXAMPLE I I I
Phenly acetyl glutamine in the form of its sodium saltwas given in two routes of administration - intravenous and oral. The observed effective dosage range was between 1.1 g/m2/24 hr. to 3.0 g/m2/24 hr. IV and 0.5 g/m2/24 hr. to 2.87 g/m2/24 hr. po. The intravenous injections were usually given in divided doses preferably every 6 hours.
Oral preparations were given in the form of 500 mg capsules ~very 12 or every 6 hours. Six patients were treated with the sodium phenyl acetyl glutamine. Complete remission was obtained in two cases which included squamous cell car-cinoma of`the larynx, ~tage II and large cell undifferen-tiated carcinoma of the lung with lymph node and liver metastases, stage III. Stabiliæation of the disease was observed in one case of adenocarcinoma of the lung, stage III. Progression of the disease was noticed in three patients who were suffering on adenocarcinoma of the sig-moid colon with multiple liver metastases, stage IV and adenocarcinoma of the colon with multiple metastases, stage IV and carcinoma of the breast with multiple lung and bone metastases, stage IV. The treatment was usually started with intra~enous injections and continued until complete remission was obtained. Afterwards, the maintenance treat-ment was implemented by using the oral preparation. Oraladministration of phenyl acetyl glutamine sodium salt often produced mild irritation of the stomach, which was relieved by the concomitant administration of antacids preparations.
EXAMPLE IV
In clinical studies involving the therapeutic assess-ment of phenyl acetyl glutamine and phenyl acetic acid a 2~3~)7 1:4 mixture of sodium salts was selected as the base for-mulation. This mixture reconstituted in sterile, buffered water was primarily administered through the intravenous route in the dose range from 0.24 g/m2/24 hr. to 5.3 g/m2/24 hr. The daily amount was usually given in divided doses preferably every 5 hours. Ten patients with various advanced neoplastic conditions were evaluated.
There was only one case in which complete remission was obtained but the phenyl acetyl glutamine and phenyl acetic acid mixture was used after patient received radia-tion. Therefore the beneficial effect of the treatment could be due to the combination effect from radiation and chemotherapy. This patient was suffering on carcinoma of the uterine cervix, stage lA. There were four cases of partial remission obtained during the treatment with the mixture. In three of these cases there was no other con-ventional treatment given in addition to the mixture.
These cases included: carcinoma of the breast with mul-tiple bone metastases, stage IV, lymphocytic lymphoma,stage IVr and chronic myelocytic leukemia. In an addi-tional case of adenocarcinoma of the lung with multiple brain metastases, stage III the treatment with phenyl acetyl glutamine and phenyl acetic acid mixture was given after radiation therapy. Stabilization of the disease was seen in three cases wh~ch included carcinoma of the sigmoid colon with multiple liver metastases, stage IV, glioma (primary malignant brain tumor) and carcinoma of the larynx with multiple lung metastases, stage IV.
* * *
Implementation of the disclosed antineoplaston frac-tions A1-h5, antineoplaston A10, phenylacetyl glutamine ~2~Z9~ ~
and a combination of phenylacetyl glutamine and phenyl-acetic acid has been directed with success in the regres~
sion of tumors associated with human cancer of esophagus, breast cancer, bladder cancer, colon cancer, large cell undifferentiated carcinoma of the lung, mesothelioma, adenocarcinoma and squamous cell carcinoma of the lung, oat cell carcinoma, brain metastases, bone metastases, lung metastases, prostate cancer, pancreas cancer, lym-phatic lymphoma, uterine cervix cancer, primary malignant brain tumor.
Further, each of the antineoplaston fractions A1-A5, antineoplaston A10, phenylacetyl glutamine, and combina-tions o phenylacetyl glutami.ne and phenylacetic acid are useful in the treatment of other forms of neoplastic disease including myelocytic leukemia, cancer of the larynx, cancer of the uterus, lymphoma, cancers of the colon and sigmoid.
* *
The foregoing description of the invention has been directed to particular examples of the extraction of antineoplastic peptide fractions from human urine for purposes of explanation and illustration. It is to be understood however, that many modifications and changes in the product compositions, the processes for extracting the antineoplaston fractions, the synthesis of antineo-plaston A10, and methods of using the same can be made in the implementation and utilization of the present inven-tion without departing from the scope of invention defined in the claims. For example, it is contemplated that pep-tides having antineoplastic activity can be extracted from sources other than urine, such as, blood, saliva, organ or tissue samples. It is to be understood that Applicant has ~2~
directed his fractionation processes to urine based on the economic feasibility of obtaining a large volume of the fluid which is necessary to derive usable amounts oE the antineoplaston fraction normally present in such fluid at very low concentration. However, it will be appreciated -by those skilled in the art after considering this specif-ication that other tissue fluids or tissue samples also contain minute amounts of peptides exhibiting antineoplas-tic activity, and that such peptides can be extracted by modification of the processes described herein.
It is to be noted that standard precautlons against bacterial contamination are taken throughout and the preparations are routinely checked for pyrogenicity, toxicity and sterility assay according to standard tech-niques~ Pyrogen free sterile water is employed through-out the final steps and all procedures are performed at -~ ambient room temperature unless stated otherwise.
. Isolation and Purification of Anti-neoplaston Fraction Al from Human Urine Reconstituted lyophilized urine (redissolved in deionized or distilled water) or freshly pooled urine is first physically filtered through paper, membrane, or cartridge filter having an average pore size of 3~ . The first filtrate is then filtered through a second filter having an average pore size of O.~p . These filtering steps are performed to separate suspended particulate or sedimented ~atter from the urine fluid.
Next, the prefiltered urine is submitted to ultrafil-tration. Desirably, ultrafiltration is accomplished through a hollow ~iber system, preferably an Amico~ ~r Romico~Msystem filter having a molecular weight cut off of about 5000 daltons. For the purpose of ultrafiltration, any other ultrafiltrati~n membrane or hollow fiber ultra-filter may be employed having a molecular weight cut off suitably in the range of 5000 to 2000. Such ultrafiltra-tion serves to remove materials having molecular weights greater than 2000 to 5000 depending on the selected filter employed.
The ultrafiltrate is acidified with concentrated acid, suitably hydrochloric or sulfuric acid, added slowly while vigorously Rtirring until ~he solution reaches a pH
ranging from ~ to 3, preferably pH 2.5. The acidified ultrafiltrate is then filtered through a 0.2~ filter to remove any precipitated particulate matter~
High performance liquid chromotography techniques are used to further purîfy and concentrate the designated anti-neoplaston fraction A1. A sample, suitably 250 ml (the amount, of course, depends upon the capacity of the sys-tem), of the acidified ultrafiltrate is introduced into a high performance liquid chromatography column, desirably a Waters Pre~M500 HPLC system utilizing a PrepTM500 C-18 silica gel cartridge column (bonded phase type silica).
The system is also equipped with a refractive index detec-tor. However, any suitable means of detection such as UV
photometry, ion detector, etc. is suitable. Antineoplas-ton fraction ~2 is eluted with deionized or distilled water and is characterized as the component peptide fràc-tion having a refractive index peak occurring after pas-sage of approximately 450 ml of water through the column.
The resulting antineoplaston fraction A1 is collected and concentrated by rotary evaporation under reduced pressure and then further freeze dried by lyophilization.
Antineoplaston fraction A1 can be used in a wide variety of pharmaceutical forms including, but not limited to, intravenous, intramuscular, subcutaneous, intracavital and intratumor injections, capsules and tablets for oral administration, rectal suppositories and solutions and sprays for topical use.
B. Isolation and Purification of Anti-neoplaston Fraction A2 from Human Urine Urine which has been prefiltered and ultrafiltered according to the description directed to the preparation ~L2~29~7 g of antineoplaston fraction A1 is acidified with concen-trated acid, typically hydrochloric acid. The acid is added slowly to the ultrafiltrate solution while vigor-ously stirring until the solution reaches a pH ranging from about 1 to about 2, preferably pH 1.5.
The ultrafiltrate from the above step is introduced onto a chromatography column containing a polymeric resin adsorbent; preferably Amberlite XA~ polymeric resin adsorbent, a product of Rohm & Haas Co., Philadelphia, Pennsylvania. Any other material similar in chemical structure or physicochemical properties may be substituted for the Amberlite adsorbent. The active antineoplastic materials are eluted from the column by employing sequen-tial washings comprising a first wash of deionized or reverse osmosis water (W1); a second wash of a mixture of water and methanol (for example, 8% volume/volume) (M);
a third wash of deionized or reverse osmosis water (W2);
a fourth wash of 4% sodium hydroxide aqueous solution (N); and a fifth wash of deionized or reverse osmosis water until the pH of the eluate is in the neutral range.
Eluates W1, W2 and M are collected. The pH of the solu-tions of Wl, W2 and M is adjusted to about 2.5 with dropwise addition of acid, for example, sul~uric acid.
The eluates W1, W2 and M from the previous step are passed through a chromatographic column packed with silica gel Prep C-18 (bonded phase type silica gel) available from Waters Associates, Whatman or other companies. The column is initially washed with deionized or distilled water and then eluted with methanol. Three brownish-yellow colored fractions appear as bands, designated MWl, MW2 and MM. Colored fractions MWl, MW2 and MM are col-lected independently and each fraction is concentrated on a circulatory evaporator to a 1 liter volume. Each i2~7 --1 o--fraction is further evaporated to dryness either by rotary evaporation or by freeze drying. Dry fractions MW1, ~W2 and MM are suitable for pharmaceutical application sepa-rately or mixed together. The mixture of them is termed antineoplaston fraction A2, and the mixture is suitable for pharmaceutical administration in the same variety of formulations and routes of administration described for antineoplaston fraction A1.
C. Isolation and Purification of Anti-neoplaston Fraction A3 from Human Urine Antineoplaston fraction A3 is isolated from urine during the same operation as the isolation and purifica-tion of antineoplaston fraction A2. P~refiltration, ultrafiltration, acidification, XAD-~ adsorption and elution is identical to that practiced for the isolation of antineoplaston fraction A2.
Fraction N eluted from the XAD-8 column with 4~
sodium hydroxide is collected and the p~ is adjusted to 2.5 with acid, desirably sulfuric acid. Fraction N is then subjected to oxidation operations. The oxidation of fraction N is preferably accomplished by dropwise addition of a saturated aqueous solution of potassium permanganate until the violet color of potassium permanganate disappears.
After the oxidation operation, fraction N is filtered sequentially through a ~ and 0. ~ filter and the clear filtrate is further separated by C-18 chromatography.
This chromatographic step is repeated in the same manner as described for the isolation of antineoplaston fraction A2. The colored band visible on the column designated MN
is eluted with methanol. The colored band MN is collected and evaporated to dryness of freeze dried. This fraction ~LZ~9~7 is called antineoplaston fraction A3 and is suitable for direct pharmaceutical application. Antineoplaston ~rac-tion A3 can be used in the same variety of pharmaceutical formulations and routes of administration as listed for antineoplaston fraction A1.
D. Isolation and Purification of Anti-n oplaston Fraction A4 from Human Urine The pH of reconstituted or fresh urine is adjusted to 2.5 with acid, suitably sulfuric acid. Contents of the urine are then oxidized by mixing the urine with a satu-rated solution of potassium permanganate in water until the violet color of potassium permanganate disappears.
After oxidation, the treated urine is filtered and the clear filtrate is separated by C-18 chromatography per-formed in the same manner as described for the isolation of antineoplaston fraction A2.
The colored band visible on the column is designated fraction U and is eluted with methanol and evaporated to dryness or freeze dried. This fraction called antineo-plaston fraction A4 is suitable for pharmaceutical appli-cation in the same variety of pharmaceutical formulations and routes of administration as described for antineo~
plaston fraction A1.
E. Isolation and Purification of Anti-neoplaston Fraction A5 from Human Urine The prefiltration, ultrafiltration, and acidi~ication of reconstituted or fresh urine is repeated as described above for the preparation of antineoplaston fraction A1.
The acidified material is filtered again through a 0.2 filter to remove any precipitated or sedimented residue.
This filtrate is then introduced to a chromatographic column filled with C-18 bonded phase type silica gel, ~available for example from Waters Associates or Whatman~.
Other silica gel, packings having the same physicochemical properties may be substituted for the C-18 column packing.
The column is initially washed with deionized, dis-tilled water and then eluted with methanol. A colored methanolic fraction is collected, which is evaporated to dryness or freeze dried The dry raction is labelled antineoplaston fraction A5. Antineoplaston A5 is suita-ble for pharmac~utical application in the same variety of formulations and routes of administration as described for antineoplaston fraction A1.
F. Chromatographic Characterization of Antineoplaston_ ractions Al-A5 For purposes of identifying each of the derived anti-neoplaston fraction, a chromatographic fingerprint wasdeveloped. Each of the derived antineoplaston fractions from the respective above-described processes was subjected to high performance liquid chromatography, a Waters Prep 500 HPLC system equipped with a Prep 500 C-18 bonded phase 2~ type silica gel column and refractive index detector.
Each antineoplaston fraction was developed according to the same sequential wash routine~ First, a predetermined amount of reconstituted antineoplaston fraction was introduced to the columnO Typically the antineoplaston fraction in powdered form was reconstituted with distilled water.
Following introduction of the antineoplaston fraction sample, a first wash with 1000 ml of water was passed through the column. This water wash was followed with 1000 ml of an acetic acid solution, pH 2.5, wash. ~inally, 1000 ml of water was passed through the column and 600 ml o~ methanol. As the eluates exited the column, the detector measured and recorded the apparent refractive index of the components eluted within an exiting solvent.
With reference to the figures, a characteristic chromatograph for each of the antineoplaston fractions is illustrated. The figures illustrate the relative refrac-tive index corresponding to components present in the eluates exiting the column at the relative elution volumes, corresponding to passage of each of the solvent washes.
It will be appreciated by those familiar with chromato-graphic development, that each chromatograph represents a resolution of peak distribution characteris-tic for a particular mixture. A chromatograph serves as a finger-print analysis of the mixture or in thisinstance a finger-print for the antineoplaston frac'cions. It is therefore apparent, that the respective product antineoplaston fractions purified according to the methods of the inven-tion, will exhibit the characteristic chromatographcorresponding to the figures illustrated herein when developed according to theconditions described above. It will also be appreciated by those skilled in the art of chromatography, that the relative heiyht of the peaks will vary with the concentration of the component elements present in each fraction, however, the distribution of the peaks will not vary substantially from batch to batch of each fraction.
Referring now to Figure 1, a chromatograph is depicted wherein antineoplaston fraction Al exhibits a discrete sharp peak in the region of first water wash. Further, Z9~7 antineoplaston fracti~on A1 exhibits a broad peak distribu-tion comprising a series of moderately defined peaks con-centrated in the region of the end of acetic acid wash and of the second water wash. In addition there are sharply defined peaks occurring after 450 ml of methanol wash.
Figure 2 depicts the chromatograph of antineoplaston fraction A2, wherein a series of sharply defined peaks are apparent in the region of the end of acetic acid wash and extend into the region of the second water wash.
Further, there are sharply defined peaks in the methanol wash.
Figure 3 depicts the chromatograph of antineoplaston fraction A3, which exhibits a small peak in the initial water wash and a concentrated band of peaks in the region of the end of acetic wash and extending into the second water wash. In addition, there are well deined peaks in the methanol wash.
Referrin~ now to Figure 4, there is depicted a chromatograph of antineoplaston fraction A4 which exhibits a small pea~ in the initial water wash and a broad peak resolved in the acetic acid wash and second water wash.
Further, there are sharp peaks in the methanol wash.
Next, turning to Figure 5, there is a chromatograph of antineoplaston fraction ~5O The chromatograph depicts a small peak in the initial water wash and a broad peak comprising a series of moderately defined peaks in the end ~f acetic acid wash and extending into the second water wash. There are also well defined peaks in methanol wash.
* * *
b;2~7 Further attempts were made to isolate and identify tl1e component elements of each antineoplaston fraction.
The component elements of antineoplaston fractions A1-A5 were separated by high performance liquid chromatography on a column packed with sulfonated polystyrene. A system developed by Glenco Scientific Inc. for amino acid analy-sis was used. The elution was effected by 0.2M citrate bufers at three different values of pH, namely 3.25, 3~80 and 4.10 and at temperatures form 50C to 70C. The ~o changes of buffers and temperature were selectively con-trolled according to Glenco Scientific, Inc. pro~ram for amino acid analysis.
The eluates were reacted as they came off the column with ninhydrin at 110C to yield absorption peaks simul-taneously measured and recorded at 470m~ and 440m~ . In order to standardize the procedure, a mixture of 18 amino acids was separated establishing the retention times pre-sented in Table 1 op~
TABLE 1. Standard Retention Times of Amino Acids Amino Acids Retention Times (Minutes) Aspartic Acid 18.0 Threonine 21.5 Serine 22.7 Glutamic Acid 26.0 Proline 30.0 Glycine ~ 38.4 Alanine 38.9 Cysteine 42.0 Valine 47.0 Methionine 49.0 Isoleucine 52.2 Leucine 53.8 Tyrosine S9.2 Phenylalanine 64.0 Lysine 72.0 Ammonia 78.0 Histidine 80.2 Arginine 93~5 Each of the antineoplaston fractions A1-A5 were separated into discrete homogeneous components under the same conditions as the standardized amino acid mixture.
Table 2 summarizes the retention times of the discrete components making up each of the heterogeneous antineo~
plaston fractions.
iZ9~;)7 TABLE 2. Retention Time of Alpha-Amino Components of Antineoplaston Fractions A1-A5 -Antineoplaston Fraction Retention __ (mM~L of alpha-amino_nitrogen~ _ Time (Min.) A1 A2 A3 A4 AS
0.12 1.57 0.24 1.97 0.20 0 0.78 0.05 0.82 0 26 0 0.22 0 0.75 0.13 1~37 2.44 0.25 0 0.59 0008 48 0.12 0.47 0.30 0.77 0.20 53 0 0.12 0.04 0.43 0 63 0 0.23 0 0.63 0.10 71 0 0.17 0.07 0.30 1576 0.13 0.14 0.45 0.87 0.13 79 3g.50 9.39 4.44 16.35 14.88 82 0.22 1.01 0.92 1.09 0.21 0.07 0.62 0.65 0.45 0.14 101 0 0.01 0 0.10 0 ?0 -The preparations of antineoplaston fractions A1, A2, A3, A4 and A5 do not contain either amino acids or proteins.
The peaks recorded during analysis of the antineoplaston fractions correspond to the different compounds reacting with ninhydrin, namely amino acid derivatives and peptides.
As denoted in Table 2, the relative concentration of each component compound within the particular antineoplaston fraction is measured relative to the reactivity between ninhydrin and the alpha-amino nitrogen of the component compound. In accordance with the chromatographic analysis described in this section, each antineoplaston fraction exhibits a significantly prominent peak at a retention time corresponding to 79 minutes. Further, in accordance ~62~1J'7 with the methods of preparing each anitneoplaston fraction embodied by the present invention, the relative concentra-tion of the component corresponding to a 79 minute reten-tion time is at least two times the relative concentration of any other residual component compound. It is to be appreciated, however, that the relative concentration of each component compound constituting an antineoplaston fraction will vary with the source of urine. Indeed, depending on the source of urine several of the component compounds within an antineoplaston fraction might not be evident. Applicant makes no representation that each component compound within an antineoplasto~n fraction possesses antineoplastic activity; rather, antineoplastic activity has been demonstrated for each antiplaston frac-tion A1-A5 and for the component compound characterized by a 79 minute retention time obtained by the above-described separation technique.
The major common active component of antineoplaston ~0 fraction A1, A2, A3, A4 and A5 was finally purified by high performance liquid chromatography and thin layer chromatography. Applicant has termed this compound anti-neoplaston A10. Its chemical structure was determined by mass spectrometry, 13C~NMR) spectroscopy, and infrared spectrometry. The structure is depicted below and termed 3-[N-phenylacetyl~aminopiperidinel-2, 6-dion.
~,o I ~
o ~I~NH
~_/ H
~LZ~Z9~37 G. Synthesis of Antineoplaston A10, 3-lN-phenylacetylaminopiperidine3-2, 6-dion Sodium bicarbonate (4.7 mole) and L-glutamine (2.3 mole) were dissolved in water ~13.5 liters). Phenylacetyl chloride (3.0 mole) was gradually added to the reaction mixture and vigorously stirred for 90 minutes. After completed reaction, the pH of the solution was adjusted to 2.5 with acid and the solution filtered. The filtrate was extracted twice with dichloromethane and the lower organic layers were discarded. The upper aqueous layer had the pH
adjusted to 7O0 with base, typically lN NaOH. The upper layer was then further purified by mixing with Norit ~M(209) (available from American Norit Co., Jacksonville, Florida).
The mixture was filtered after 30 minutes contact with Norit A. The resultant filtrate was evaporated and the residue redissolved in methanol. The recovered methanolic solution was filtered and freeze dried or evaporated. rrhe dried residue was redissolved in water and the pH adjusted to 2.5 suitably with ~C1. Two layers were formed after standing at room temperature. The lower layer was heated until it turned dark brown. This viscous brown layer was redissolved in methanol wherein crude antineoplaston A10 precipitated upon cooling. The crude antineoplaston A10 was redissolved in hot methanol and Norit A was added to remove any color. The solution was filtered while hot.
On cooling, white crystals of antineoplaston A10 formed.
The structure of the synthetic material was elucidated by mass spectrometry, 13C(NMR) spectroscopy and infrared spectrometry was found to be identical to the maior common active component of antineoplaston fractions A1, A2, A3, A4 and A5 having a 79 minute retention time.
~L2~;~907 Antineoplas~on A10 is most suitable ~or phaEmaceuti-cal applications in the form of salts, sodium salt being : preferred. ~n order to prepare sodium salt, antineoplas-ton A10 is suspended in ethanol and heated with a solution : 5 of sodiu~ hydroxide water until all material is dissolved.
The reaction mixture is then freeze dried. The solid resi-. due is kept at room temperature until the salt cyrstallizes out. Ethanol is added and stirred. Filtration yields a white crystalline solid corresponding to the sodium salt of antineoplaston A10. Suitable solutions for parenteral administration are prepared by dissolving sodium salt of antineoplaston A10 in pyrogen free water up to a concentra-tion of lOOmg/ml and adjusting the pH to 7Ø
~. Degradation Products of Antineoplaston A10 The initial hydrolysis of antineoplaston AlO yields phenylacetyl glutamine, and when carried further produces phenylacetic acid:
f~Z ~11 H 1 N /~ /~ O H
3-[N phenylacetylamino- N-phenyl acetyl piperidine]2, 6-dion glutamine 3~ ~0.
O /~1 H 2 (~ .~
Phenylacetic acid Glutamine ~L~62907 Phenylacetyl glutamine was first described by Thier-felder and Sherwin [see J. Physiol. Chem. 94:1 (1915)~ as a constituent of normal human urine. In later investiga-tions of the compound phenylacetyl glutamine was shown to have a slight effec~ on the growth of murine tumors [see Lichtenstein et al, Israel J. Med. Sci., 13:316 ~1977)3 but there was no indication that the compound was useful in the treatment of human cancer. The sodium salt of phenylacetic acid was used by Neish in the treatment of Rd/3 sarcoma in rats but failed to inhibit tumor growth.
Indeed, the results suggested that the treatment with phenylacetic acid caused some enhancement of tumor growth ~see Neish, Experientia, 27:860 ~1971)]. Applicant has demonstrated in his clinical studies that phenylacetyl glutamine alone and a mixture of phenylacetyl glutamine and phenyl acetic acid are each useful in the treatment of human cancers.
A mixture of the sodium salts of phenylacetyl gluta-mine and phenyl acetic acid in the ratio 1 to 4 is thepreferred formulation for use in the treatment of human cancer.
Solutions for parenteral administration are prepared by reconstituting the respective chemicals in form of sodium salts in pyrogen free water to the desired total concentration, for example 100 mg/ml. The p~ of the solution is adjusted to 7.0 with 1N NaOH or lN HCl.
Sterilization of the reconstituted solution is done by 33 filtration according to the guidelines of the U.S. Phar-macopeia. The sterility of the material is tested as required by the rules and regulations of the Food and Drug Administration, Section 610.12. The resulting sterile formulations are suitable for parenteral injections.
~ZS29q~7 I. Pharmaceu~ical Applications for Anti-neoplaston Fractions A1-A5, Antineo-~laston A10, and Degradation Products Solutions for parenteral administration are prepared by reconstituting each respeCtiYe antineoplaston fractions, antineoplaston A10 and degradation products in pyrogen free water to a desired convenient concentration, for example, 100 mg/ml. The pH of the solution is adjusted to 7.0 with lN HCl or lN NaOH.
Sterilization of the reconstituted solution is done by filtration according to the guidelines of the U.S.
Pharmacopeia. Alternatively, the lyophili~ed powders of the respective antineoplaston fractions can first be gas sterilized, for example with ethylene oxide, and the ; powders subsequently incorporated into the pyrogen free water which, of course, itself is sterile. The sterility of the material is tested as required by the Rules and Regulations of the Food and Drug Administration, Section 610~12. The resulting sterile formulations are suitable for intravenous, intramuscular, subcutaneous, intracavital and intratumor injection.
If the reconstituted lyophilized antineoplaston frac-tion is not to be used immediately, the prevention of microbial proliferation can be attained by the addition of various antibacterial and antifungal agents to the antineoplaston solutions, ~or example, parabens, chloro-butanol, benzyl alcohol, phenol, sorbic acid, thimerosal, and the like. In many instances it will be desirable to include isotonic agents to the injectable solutions, for example~ sugars or sodium chloride.
~L2~
The antineoplastic activity of the antineoplaston fractions A1-A5, antineoplaston A10 and degration products was first evaluated experimentally by observing the cytostatic effects the preparations would have on a tumor line as compared to the overall toxicity the preparation would have on experimental animals. Accordingly, the preparation having the greatest cytostatic activity and the lowest animal toxicity are said to have the better antineoplastic activity, or therapeutic effectiveness.
The cytostatic activity of antineoplaston fraction A1 was tested in a culture of human carcinoma of the breast line MDA-MB-231 obtained from M.D. Anderson Cancer Institute, Houston, Texas. MDA-MB-231 is a fast growing line of human breast cancer established by Cailleau et al, J. Natl. Cancer Inst., 53:661 (1974). The estimated .
doubling time of these cells is 18 hours when grown in the original medium described by Beall et al, Physiol. Chem.
Physics, 8:281 (1976). ~rieflyr to summarize the preferred medium and method, the cells are grown in monolayers at 37C in Leibovitz L-15 medium supplemented with 20% fetal bovine serum, 1.6~ g/ml glutathione, 0.25 U/ml insulin, 100u g/ml disodium carbenicillin and 10~ g/ml gentamycin.
For the bioassay, antineoplaston fraction A1 was dis-solved in the above-described medium at four different concentrations selected arbitrarily with the range of 0.5 to 50 mg/ml. Monolayer cultures were incubated with the antineoplaston fraction A1-containing medium for 96 hours.
The cells were counted by visual method at 24 hour inter-vals. Control cultures were grown in the standard medium without added antineoplaston fraction A1.
~Z6~
Antineoplaston fraction Al in concentrations of 5 mgfml produces a cytostatic effect in such h~lman breast carcinoma cultures. The cytostatic effect is determined as a stable number of cells (within the limits from 80% to 120~) counted after 24 hours from incubation and persist-~
ing for at least an additional 48 hours.
The cytostatic concentration of the antineoplaston fraction A2-A5 and antineoplaston A10 and degradation products were determined in the same manner described above. The cytostatic concentration for each antineo-plaston fraction is as follows:
AntineoplastonCytostatic Concentration Fraction Al 5 mg/ml Fraction A2 5 mg/ml Fraction A3 5 mg/ml Fraction A4 2 mg/ml Fraction A5 2 mg/ml A10 2 mg/ml Phenyl acetyl glutamine10 mg/ml Phenyl acetyl glutamine and Phenyl acetic acid (1:4 mixture) 3 mg/ml ~
Above these concentrations all antineoplaston fractions produce cytotoxic effect in human breast carcinoma cul-tures.
Acute toxicity studies on experimental animals reveal that antineoplaston fraction A1 has very low toxicity.
For example, experiments involving ~wenty-five HA/ICR
swiss mice injected intraperitoneally with antineoplaston fraction Al resulted in a LD50 if 1.35 g/kg. The autopsy ~LZ6X~
.
and microscopic studies of the tissues of the animals which died during the experiment revealed congestion of the liver and marked pulmonary edema. The animals which survived were kept for one week under close observation and were noted to carry on normal activity. After a week~, a select number of the mice were sacrificed. The autopsy and microscopic examination of the tissues of these animals were identical to those of control, uninjected subjects.
Acute toxicity studies involving antineoplaston fractions A2-A5, antineoplaston A10 and degradation products were carried out in the same manner described above. The respective LD5Q for mice for each fraction is as follows:
Antineoplaston LD50 Fraction Al 1.35 g/kg ~0 Fraction A2 3.55 g/kg Fraction A3 3.55 g/kg Fraction A4 5.33 g/kg Fraction A5 5.11 g/kg A10 10.33 g/kg Phenyl acetyl glutamine2O90 g/kg Phenyl acetyl glutamine and Phenyl acetic acid (1~4 mixture) 2.83 g/kg J. Clinical ~valuation of Antineoplaston The definitions of remission associated with neoplas-~ic disease are as follows: complete remission is the disappearance of all clinical evidence of disease and partial remission is reduction by at least 50~ in the sum ~;~6~ 7 of the products of two perpendicular diameters of all measurable disease lasting at least four weeks. Patients are considered stabilized if measurable tumor regression occurs but does not meet the criteria for partial remission.
In accordance with the methods in the present inven-tion, human neoplastic disease was treated with the various antineoplaston fractions. For each neoplastic disease studied, each tested antineoplaston fraction, antineoplaston A10 and degradation product, phenylacetyl glutamine, and a combination of phenylacetyl glutamine and phenylacetic acetic was efective to some extent in aiding ~ the regression of tumors. As would be expected, some fractions or compositions exhibited more effectiveness for some forms of neoplasia than other fractions.
.
The dosage of the selected antineoplaston fraction for the treatment of the indicated neoplastic condition depends on the age, weight and condition of the patient;
the particular neoplastic disease and its severity; and the route of administration. A dose of from about 0.5 to about 1~ g/m2/24 hr. or a total dose of from about 0.9 to about 20 g given in divided doses of up to 6 times a day embraces the effective range for ~he treatment of most neoplastic conditions for any one of antineoplaston fractions A1-A5, antineoplaston A10, and degradation products.
EXAMPLE I
To date, fourteen patients with advanced cancer have been treated with antineoplaston fraction A2 and followed for up to one year. The preferable route for the adminis-tration of the preparation is intraven~us injection given 6Z~3~7 every 12 hours through a catheter inserter into the sub-clavian vein. Direct intrapleural or intraperitioneal injections can also be given. The average dose given was 0.85 g~m2 and the maximum was 2.2 g/m2 intravenously every 12 hours. Full dose intravenous treatment with antineoplaston fraction A2 was usually given until the complete remission was obtained and then continued for at least 6 weeks to eradicate any remaining microscopic disease. Afterwards, IV injections were discontinued and the maintenance treatment was started. Maintenance treatment consisted of IM injections of 2 to 3 ml of 50 mg/ml antineoplaston fraction A2 initially given every other day. If no sign of cancer recurred, the frequency of IM injections was reduced every 6-8 weeks, tapering from one injection every third day to one injection once a week.
In the group of 14 patients treated with antineoplas-ton fraction A2 four obtained complete remission. These four cases included undifferentiated large cell carcinorna of the lung, stage III (T3NOMO~; poorly differentiated metastatic carcinoma of the liver with unknown primary;
and two cases of recurrent transitional cell carcinoma of the bladder, grade II. In an additional case involving adenocarcinoma of the breast with multiple bone and liver metastases, stage IV ~TONOM10SS, ~EP) a complete remission of large liver metastases and stabilization of bone metastases was obtained. There was also one additional case of transitional cell carcinoma of the bladder, grade II in which complete remission was obtained with help of antineoplaston fraction A2. In this case antineoplaston fraction A2 was given as a maintenance treatment after the complete remission was obtained with antineoplaston A.
~ILZ~9~7 Partial remission was obtained in three cases: peri-toneal mesotheliomal; carcinoma of the breast with multiple bone metastases, stage IV, TONOM10SS; and squamous cell carcinoma of the esophagus with multiple lung metastases, stage III, T3NOM1PUL.
Stabilization of the disease was obtained in four cases which included glioma, stage IV; adenocarcinoma of the kidney with multiple lung metastases, carcinoma of the breast with multiple bone metastasest stage IV, TONOMlOSS:
and carcinoma of the breast with lymph node involvement, stage IV, TON3MO.
Overall response rate to the treatement with antineo-plaston fraction A2 is 93% with only one patient (7%) showing continued progressive disease. The treatment is very well tolerated. Few side effects were evident includ-ing stimulation of the growth of epidermis, stimulation of bone marrow and very infrequent fever. Stimulation of the growth of epidermis was evident after 3 weeks of treatment ; as more rapid growth of nails and a thicker skin on the palms. Stimulation of the bone marrow was shown as ele-vated white blood and platelet count. These side effects are beneficial in most cases because of poor healing of the skin and myelosuppression present in large number of cancer patients.
EXAMPLE II
The following case history illustrates a successful method of treatment employing antineoplaston fraction A3 for the treatment of adenocarcinoma of the prostate with bone metastases, stage IV (RONOMlOSS, G3).
;Z~Q7 Treatment of a 72 year old white male was initiated with antineoplaston A (see Burzynski et al, PhYsiol._Chem Phys. 9:485, 1977) for three months. The initial treatment with antineoplaston A resulted in the stabilization of the disease and some questionable decrease o the size of the~
metastases.
After three months antineoplaston A was discontinued and the patient ~as started on antineoplaston fraction A3.
He received a first injection of 1 ml of antineoplaston fraction A3, 100 mg/ml given through a subclavian catheter followed with 3 ml of normal saline and 250 units of heparin. The dose was further increased up to 5 ml of the same preparation given intravenously every 12 hours. Such a treatment regimen corresponds to the dosage 0.47 g/m~/24 hour. After approximately 7 months the frequency of the injections was decreased to 2 ml of antineoplaston fraction A3, 100 mg/ml, given intramuscularly every third day, and finally after 4 months the dosing regimen was further decreased to 2 ml of the preparation administered intra-muscularly once a week.
The treatment with antineoplaston fraction A3 resulted in the complete remission of the bone metastastes as judged by bone scans. The treatment was very well tolerated with-out any apparent side effects.
Patient is continuing a maintenance treatment with antineoplaston fraction A3, and has not shown any recur-rence of his cancer at the present time.
~Lz6zg~
EXAMPLE I I I
Phenly acetyl glutamine in the form of its sodium saltwas given in two routes of administration - intravenous and oral. The observed effective dosage range was between 1.1 g/m2/24 hr. to 3.0 g/m2/24 hr. IV and 0.5 g/m2/24 hr. to 2.87 g/m2/24 hr. po. The intravenous injections were usually given in divided doses preferably every 6 hours.
Oral preparations were given in the form of 500 mg capsules ~very 12 or every 6 hours. Six patients were treated with the sodium phenyl acetyl glutamine. Complete remission was obtained in two cases which included squamous cell car-cinoma of`the larynx, ~tage II and large cell undifferen-tiated carcinoma of the lung with lymph node and liver metastases, stage III. Stabiliæation of the disease was observed in one case of adenocarcinoma of the lung, stage III. Progression of the disease was noticed in three patients who were suffering on adenocarcinoma of the sig-moid colon with multiple liver metastases, stage IV and adenocarcinoma of the colon with multiple metastases, stage IV and carcinoma of the breast with multiple lung and bone metastases, stage IV. The treatment was usually started with intra~enous injections and continued until complete remission was obtained. Afterwards, the maintenance treat-ment was implemented by using the oral preparation. Oraladministration of phenyl acetyl glutamine sodium salt often produced mild irritation of the stomach, which was relieved by the concomitant administration of antacids preparations.
EXAMPLE IV
In clinical studies involving the therapeutic assess-ment of phenyl acetyl glutamine and phenyl acetic acid a 2~3~)7 1:4 mixture of sodium salts was selected as the base for-mulation. This mixture reconstituted in sterile, buffered water was primarily administered through the intravenous route in the dose range from 0.24 g/m2/24 hr. to 5.3 g/m2/24 hr. The daily amount was usually given in divided doses preferably every 5 hours. Ten patients with various advanced neoplastic conditions were evaluated.
There was only one case in which complete remission was obtained but the phenyl acetyl glutamine and phenyl acetic acid mixture was used after patient received radia-tion. Therefore the beneficial effect of the treatment could be due to the combination effect from radiation and chemotherapy. This patient was suffering on carcinoma of the uterine cervix, stage lA. There were four cases of partial remission obtained during the treatment with the mixture. In three of these cases there was no other con-ventional treatment given in addition to the mixture.
These cases included: carcinoma of the breast with mul-tiple bone metastases, stage IV, lymphocytic lymphoma,stage IVr and chronic myelocytic leukemia. In an addi-tional case of adenocarcinoma of the lung with multiple brain metastases, stage III the treatment with phenyl acetyl glutamine and phenyl acetic acid mixture was given after radiation therapy. Stabilization of the disease was seen in three cases wh~ch included carcinoma of the sigmoid colon with multiple liver metastases, stage IV, glioma (primary malignant brain tumor) and carcinoma of the larynx with multiple lung metastases, stage IV.
* * *
Implementation of the disclosed antineoplaston frac-tions A1-h5, antineoplaston A10, phenylacetyl glutamine ~2~Z9~ ~
and a combination of phenylacetyl glutamine and phenyl-acetic acid has been directed with success in the regres~
sion of tumors associated with human cancer of esophagus, breast cancer, bladder cancer, colon cancer, large cell undifferentiated carcinoma of the lung, mesothelioma, adenocarcinoma and squamous cell carcinoma of the lung, oat cell carcinoma, brain metastases, bone metastases, lung metastases, prostate cancer, pancreas cancer, lym-phatic lymphoma, uterine cervix cancer, primary malignant brain tumor.
Further, each of the antineoplaston fractions A1-A5, antineoplaston A10, phenylacetyl glutamine, and combina-tions o phenylacetyl glutami.ne and phenylacetic acid are useful in the treatment of other forms of neoplastic disease including myelocytic leukemia, cancer of the larynx, cancer of the uterus, lymphoma, cancers of the colon and sigmoid.
* *
The foregoing description of the invention has been directed to particular examples of the extraction of antineoplastic peptide fractions from human urine for purposes of explanation and illustration. It is to be understood however, that many modifications and changes in the product compositions, the processes for extracting the antineoplaston fractions, the synthesis of antineo-plaston A10, and methods of using the same can be made in the implementation and utilization of the present inven-tion without departing from the scope of invention defined in the claims. For example, it is contemplated that pep-tides having antineoplastic activity can be extracted from sources other than urine, such as, blood, saliva, organ or tissue samples. It is to be understood that Applicant has ~2~
directed his fractionation processes to urine based on the economic feasibility of obtaining a large volume of the fluid which is necessary to derive usable amounts oE the antineoplaston fraction normally present in such fluid at very low concentration. However, it will be appreciated -by those skilled in the art after considering this specif-ication that other tissue fluids or tissue samples also contain minute amounts of peptides exhibiting antineoplas-tic activity, and that such peptides can be extracted by modification of the processes described herein.
Claims (6)
1. A process for synthesizing the compound 3-[N-phenylacetylaminopiperidine]-2, 6-dion, and the pharmaceutically acceptable salts thereof comprising the steps of:
providing a quantity of L-glutamine;
providing a quantity of phenylacetyl halide;
mixing together the L-glutamine and phenyl acetyl halide in a weakly alkaline aqueous solution for a period of time to provide an aqueous reaction mixture including the reaction product 3-[N-phenylacetylaminopiperidine]-2, 6-dion; and isolating 3-[N-phenylacetylaminopiperidine]-2, 6-dion from the reaction mixture and when desired preparing the pharmaceutically acceptable salts thereof.
providing a quantity of L-glutamine;
providing a quantity of phenylacetyl halide;
mixing together the L-glutamine and phenyl acetyl halide in a weakly alkaline aqueous solution for a period of time to provide an aqueous reaction mixture including the reaction product 3-[N-phenylacetylaminopiperidine]-2, 6-dion; and isolating 3-[N-phenylacetylaminopiperidine]-2, 6-dion from the reaction mixture and when desired preparing the pharmaceutically acceptable salts thereof.
2. The process according to claim 1 wherein the reaction mixture contains sodium bicarbonate.
3. The process according to claim 1 wherein the phenyl acetyl halide is phenyl acetyl chloride.
4. The process according to claim 1 wherein the step of recovering 3-[N-phenylacetylaminopiperidine]-2, 6-dion comprises:
acidifying the reaction mixture;
providing an immiscible organic solvent in contact with the aqueous reaction mixture:
extracting into the organic solvent the excess reagents and by-products; and recovering the compound 3-[N-phenylacetylamino-piperidine]-2, 6-dion from the aqueous reaction mixture.
acidifying the reaction mixture;
providing an immiscible organic solvent in contact with the aqueous reaction mixture:
extracting into the organic solvent the excess reagents and by-products; and recovering the compound 3-[N-phenylacetylamino-piperidine]-2, 6-dion from the aqueous reaction mixture.
5. A pharmaceutical composition for inhibiting growth of neoplastic cells in a host having neoplastic disease comprising a carcinostatically effective amount of 3-[N-phenylaminopiperidine]-2, 6-dion, or pharmaceutcially acceptable addition salts thereof.
6. A composition of matter consisting essentially of 3-[N-phenylacetylaminopiperidine]-2, 6-dion, or pharmaceutically acceptable addition salts thereof.
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| US279,728 | 1981-07-02 | ||
| US06/330,383 US4470970A (en) | 1981-07-02 | 1981-12-15 | Purified antineoplaston fractions and methods of treating neoplastic disease |
| CA000403789A CA1188218A (en) | 1981-07-02 | 1982-05-26 | Purified antineoplaston fractions and methods of treating neoplastic disease |
| CA000475098A CA1262907A (en) | 1981-07-02 | 1985-02-25 | 3-¬n-phenylacetylaminopiperidine|-2,6 dion and process of synthesizing same |
| US330,383 | 1989-03-29 |
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