Note: Descriptions are shown in the official language in which they were submitted.
~ 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.
~,~7
Bi
~2~ 7
-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.
~2~Z~
--6--
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.
3~Z62~
--7--
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
--8--
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
--1 1--
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.
-12-
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
-13-
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
-15-
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~
-16-
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
-17-
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
-18-
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
-19-
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
-20-
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
-21-
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
-22-
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~
-24-
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
1 5
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~
-25-
.
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
-26-
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
-27-
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
-28-
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
-29-
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~
--30--
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
-31-
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~ ~
-32-
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~
-33-
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.
