Note: Descriptions are shown in the official language in which they were submitted.
MEDICINAI, PRE~R~TION ~O~ ~R~TI~G C~RCINOMAS
~ND PROCESS OF PROD~CIN(, l'~ll. PREPAI~ATIbN
1~3~Zl~
BACKGROUND OF T~-7E INVF.MTI~N
The treatment of carcinomas with cytostatic
agents has been ~,nown for a considerable time. Almost all
cytostatic ac,~ents have the disadvarltage that they are
highly toxic and yenerally speaking also adversely affect
the growth of healthy cells. Further, it has also been
found that the known cytostatic agents are not equally
active on all kinds of carcinomas. Even di~ferent
f~ J
patients having similar carcinomas often exhibit different
responses to one and the sa~e cytostatic agent. In order
to select the optimum cytostatic agent in such cases and
proceed with a selective treatment with the agent thus
determined, Limburg and Krahe have suggested testing tis-
sue cultures taken from the involved carcinoma for sensi-
tivity to various cytostatic agents to establish an onco-
biogram. 5ee Deutsche Medizinsche Wochenschrift, Vol. 89
(1964), page l933.
While the above-mentioned procedure affords the
advantage that the involved organ or or~ans of the patient
are not subjected to the toxic action of cytostatic agent.s
which are not effective on the specific ~ype of tumor, the
patient is still directly affected by a cytostatic agent.
This exposure has proven to be deleterious particularly
when high dosages are administered over a prolonge~1 period
of time.
~13~9
.
It is an object of the present invention to provide a
medicinal preparation which is intended for the treatment of
carcinomas and which is suitable either for use alone, or
jointly with a cytostatic agent, which agent is specifically
effective on the type of tumor involved. The medicinal prepa-
ration should be satisfactorily tolerated by the patient.
According to one aspect of the invention there is provided
a method for isolating, culturing and growing selected carci-
noma cells comprising the steps of: (1) providing a source
of carcinoma cells; (2) culturing and incubating said cells
in a nutrient growth media under cell growth conditions to
produce a cell culture; (3) subjecting a plurality of pre-
determined portions of the cell culture of step (2) to a
plurality of cytostatic agents and observing the visible
morphological damage in each portion; (4) selecting that
cytostatic agent that exhibits visible morphological damage
to said susceptible cells; (5) treating the remaining portion
of the cell culture of step (2) with the cytostatic agent of
step (4) and incubating such that from about 30 to 70~ of
said cells remain intact under microscopic examination and that
said cells are not completely killed but only partially
weakened; (6) washing the weakened cells of step (5) with a
physiologically acceptable solution to remove substantially
completely all of said cytostatic agent from said weakened
cells; and thereafter (7) suspending the cells thus treated in
a physiologically acceptable fluid for a pharmaceutically
1~3~2~
acceptable preparation.
According to another aspect of the invention there is
provided a method for producing a suspension of weakened
selected carcinoma cells that are susceptible to the cyto-
static activity of a selected cytostatic agent comprisingtreating a cell culture of carcinoma cells with a cycostatic
agent to which said cells are sensitive and incubating for
a period of time until microscopic examination indicates that
only about 30 to about 70~ of the thus treated cells are intact,
and thereafter washing the thus weakened cells with a
physiologically acceptable solution to remove said cytostatic
agent.
The objects of the invention are accomplished as will be
apparent from the following description, examples and
appended claims.
DETAILED DESCRIPTION OF THE INVENTION
Carcinoma cells are used as a starting material in the
process according to the present invention and can be re-
covered in a conventional manner. For instance, the cancer
cells can be released by a treatment with trypsin from
surgically explanted tumor tissue; see Limburg et al..,
Deutsche Medizinische Wochenschrift, Vol. 89 (1964), page 1938.
A preferred procedure of recovering carcinoma cells is
explained as follows:
At the beginning of the procedure, any desired malignant
tissue of a tumor is excised under sterile conditions during
- 2a -
~139Z~
a surgical operation. It is then treated with a trypsin
solution and subsequently cultured on mica flakes in ~eighton
tubes. The tissue which has been excised is mechanically
macerated disintegrated under sterile conditions to form a
tissue pulp. Care should be taken to use only firm cancer
tissue which is structurally
- 2b -
~L~ 39Zl~
intact and free from suhstantial admixtures of blood or
dead cells. ~ solution of 0.25~ tryp~in in a phosphate-
buffered physiologica~l salt solution (~BS), which contains
100 units/ml penicillin and 0.1 mg/ml streptomycin, for
sterilization, is added to that pulp, which is subsequent-
ly stirred at 37C. For certain firmer tissues of harder
consistency, the concentration of the trypsin solution ma~
be increased up to 2%. ~fter a~out 20 minutes the re-
leased individual cells, without tissue portions, to the
extent possible, are poured into a second sterile flask.
To interrupt the continued action of trypsin on the cell
suspension which has been poured into the second sterile
flask, the suspension is cooled to ~4C. by means o~ an
ice bath. This sequence of stirring and pouring is re-
peated until there is no more solid tissue in the flaskused for the treatment with trypsin. When the trypsin
treatment has been completed, the cooled cell suspension
is coarsely filtered, preferably through a layer of gauze
that is sterile but has not been treated with any chemi-
cals. The filtrate is centrifuged at about 800 to about1000 revolutions per minute and the separated cell sedi-
ment isolated from the filtrate.
The trypsin treatment may be omitted with carci-
noma cells that are already available in the form of indi-
vidual cells, such as ascites carcinoma cells and leukemiacells.
The cancer cells are next grown to form a cell
culture or cell turf according to known procedures, for
example as in the Limburg et al article in Deutsche r~ed-
izinische Wochensch~ift,mentioned above. The cells arecultured, and the following is an illustration:
-- 3 --
- ~392~
The c~ll s~iment is dilute~ with a liquid nu-
trient to provide a concentration of from 2 to 4 million
viable cells per milliliter and is equally distributed
among about 30 tubes after it has been thoroughly pipet-
ted. One drop is placed on a slide so that the extent ofdilution can be ascertained. The dilution is satisfactory
when about 20 cells can be seen in the field of view under
a magnification power of about 125. For a tube test, 1
milliliter of liquid is filled into each Leightor) tube.
10 Preferably, the liquid nutrient employed has the following
composition:
% By Volume
Tissue culture medium (TCM) according
to Morgan, Morton, Parker (available
commercially from DIFCO) 25.0
Hanks solution (~1% of a phenol red
solution prepared q.s.ing 0.5 g
phenol red up to 20 ml with N/20 NaOH) 55.0
Vitamin MEM solution (10'~) 5-
L-glutamin (10% concentration)5.0
Calf serum 10.0
100 units of penicillin per milliliter and 0.1
mg of streptomycin per milliliter are added to the nu-
'~ trient to ensure its sterility.
The cell suspension is incubated in the incuba~
tor at 37C. The cell metabolism is indicated by the
change of the nutrient color from red to ycllow. 'I'he nu-
trient is continually renewed whenever the metabolism has
been taken place. The growth of the culture is examined
and monitored under a chemist's microscope with reflected
light. Depending on the nature of the starting material,
the cultures have different growth characteristics. For
example, cells from an endometrial or ovarian carcinoma
-~ can grow to form a good cell monolayer within 48 hours Cells
from other malignant growths, such as cells from squamous
cell carcinomas, take up to 120 hours, depending on
the degree to which they have matured.
The test of the cell cultures for sensitivity to
various cytostatic agents is also conducted according to
methods, for instance, as described by Limburg et al.,
Deutsche Medizinische Wochenschrift, mentioned above. An
illustrative procedure is as follows:
Only well grown cell cultures are tested. De-
i.,, ,~
pending on the quantity of the culture material which isavailable, the culture can be tested for sensitivity to
any given cytostatic agent. A single culture can be
tested for sensitivity of from 10 to 20 chemotherapeutic
agents at the same time, which agents are added to respec-
tive tubes in a weight-adequate concentration. Each agent
used for sensitivity testing is used in the same concen-
tration in which it is used in man for continuous or in-
termittent therapy; as a guide see the values stated in
Table 1 that follows. The various cytostatic agents are
prepared in a pharmaceutical form and added to the respec-
tive test tubes. Continuous monitorinq using a chemist' 9
microscope will reveal visable morphological darnage in
most cases after 24 hours and, where antimetabolites are
used, after 48 hours.
In the test in vitro, the following concentra~
tions suitable for therapy are used. These doses are cal-
culated for recomrnended therapy concentrations for humans
-- 5 --
recommended by th~ man~facturers, relate~ to an averagebody wei~ht of 60 kg or to square meters of body surface
and approximately 5000 ml blood v~lu~e, as the case may
be.
TABLE 1
Cytostatic Agent Concentration (Jug/ml)
Cyclophosphamide (Endoxan) 33
Triethylene thiophosphoramide
(Thio-Tepa*) 0.42
Podophyllic acid-2-ethylhydrazide
(Proresid*) 33
VM 26
Vincristin 0.017
Actinomycin D 0.05
Adriblastin
5-Fluoruracil 16.7
Amethopterin (Methotrexate) 2
Using this procedure, the dilution of any other
cytostatic agent can easily be calculated.
IN VITRO SENSITIVITY TESTING
_
The sensitivity or resistance of a culture deri-
ved from cancerous human tissue is determined by a semi-
quantitative method. A culture which is completely resis-
tant will correspond entirely with that in the control
tube. Partial activity is indicated by a partial dissolu-
tiOIl of about 50% of the cells in the field of view. ~igh
sensitivity will be revealed by the fact that the field of
view is free from viable cells and contains only a few
shrunken cells, which have been killed. Only such a re-
sult is regarded as revealing full sensitivity. On anaverage, I have found that at least two cultures treated
* Trade Mark
- 6 -
~ iL39Z~
with different cytostatic atlt?rlts prove to he sensitive.
Only these cultures are to be used for the cell therapy
which will be explained hereinafter. Such tests have been
conducted in more than 3000 individual cases and have
given the unique result that cells from eàch of several
tumors from similar sites and/or of similar histology, but
from different patients, may exhibit greatly different
respollses to the same cytostatic agent, and further that
cells from metastases may differ in response to cells from
the primary tumor.
~he test of carcinoma cell cultures for sensiti-
vity to various cytostatic a~ents may be carried out in
J vivo in the abdomen of rats.
IN VIVO SENSITIVITY TESTING
The cytostatic sensitivity test is carried out
in vivo in diffusion chambers placed into the abdomen of
rats when less than 20 g of tumor material are available.
The cell suspension is prepared in the same manner as in
the in vitro method. The diffusion chamber consists of
two filters, which are placed into two Teflon rings, which
are connected together by screws. The filters act as a
semipermeable membrane. The pore size is selected so that
,J
liquid, electrolyte and proteins can permeate throuqh the
membrane whereas cells such as leucocytes, macropha~Jes and
the like cannot. A typical pore size for the filters is
about 0.25 microns. This chamber is filled with 0.3 ml of
prepared cell suspension.
Imrnediately after their preparation, the cham-
bers are implanted into the peritoneal cavity of inbred
strain BD2 rats. Two chambers are usetl for each animal.
When the chambers have been implanted~ the peritoneum and
the abdominal wall are ~closcc~ ~ ~sing this method, 100~ of
the tumor cells retained on the membrane filters are
grown.
On the fifth day after the imulantation of the
diffusion chambers into the abdomina of the rats, various
cytostatic agents are repeatedly injected into the peri-
oneal cavities of respective rats. Just as in the in
vitro method, the concentration employed is the same as
that used in human therapy. Forty-eight hours after the
last injection, the rats are sacrificed and the diffusion
chambers removed. The mernbrane filters are isolated and
fixed, and the cells are strained accordiny to Papanico-
J laou's method. The cytotoxic activity is determined in
accordance with the above desribed morpholoyic criteria as
in the in vitro test.
CELL WEAKENING TREATMENT
When the cells have been tested for sensitivity
to various cytostatic agents as described above, untreated
carcinoma cells having the same origin as the previously
tested cells are found to have been weakened by the treat-
ment and procedures accordin~ to the present invention.
In order to conduct the process accordiny to my
invention, it is essential that when the optimum cytosta-
tic agent for a given cell culture has been ascertained,
the cells are treated with the selected ayent so that they
are not completely killed but only partly weakened. To
that end, care must be taken that the time of incubation
in the presence of the cytostatic ayent is not too lony.
As would be expected, the time for which a culture is
incubated will vary with different cell cultures and with
the activity of the cytostatic agent. The progressive
weakening can be watche~ un~er a microscope having a mag-
nification of about 125. This can be performed by a skil-
led technician without difficulty. The incubation is pre-
ferably continued until a microscopic examination reveals
that only about 30 to about 70~, and preferably only about
40 to about 60%, of the cells appear to be intact. Incu-
bation times of about 4 to about 15 hours, and particular-
ly of about 6 to about 12 hours, have generally proved ap-
propriate. The incubation i.s preferably effected at a
temperature o~ about 37C.
For the weakening treatment, each
cytostatic agent is used in the same concentration as in
J the above-described test.
The cell weakening treatment is preferably car-
ried out as follows. Cells which have been recovered by
treatment with trypsin, as described above and which have
not been treated with a cytostatic agent, and are suspen-
ded in a nutrient media, placed into Roux bottles and
heated in an incubator to 37C. The particular cytostatic
2~ agent which has given optimum results in the above-men-
tioned test is placed into the bottles in a concentration
;! ~J as indicated in Table 1.
When the cells have been suitahly weak.ened to
the extent desired, the culture is then thoroughly washed
to remove the cytostatic agent. This step serves to at-
tenuate the action of the cytostatic agent on the cells
and mainly to prevent a continucd reaction of the cyto.sta-
tiC agent with the cells or possibly killing the cells. A
phosphate-buffered saline solution is preferred as a wash-
ing fluid although other suitable physiologically compat-
ible solutions may also be used.
ILIh~ c~lls w~ich hav~ ~us ~eerl ~eakerled 'co the ex-
- tent desired, are celltrifuged at about ~OO to about lOO0 r.p.m.
ror 1O minutes and preferably suspended in a physiologic-
ally acceptable fluid, such as physiological salt solution
or calf serum. The concentration of the thus-treated
cells in the resulting pharmaceutical preparations ready `
for administration after dil~tion is preferably about
106 cells per milliliter.
Pharmaceutical pr~ara~ions made according to the
invention are preferably prepared from tunnor cells taken
from the specific patient to be treated. This method will
ensure a particularly high tolerance and optimum activity.
It is also recogni2ed that there may be cases in which it
is not possible to make the preparation from cells taken
from the patient to be treated for various reasons, such
as there is insufficient time available or because persons
skilled in the preparation and testing of cultures grown
from carcinoma cells and equipment required for such work
are not available. In such cases, preparations made from
cells taken from the carcinomas of other patients may be
used. It is, of course, most preferable to treat a
patient with preparations made from cells of carcinomas
which are similar to the carcinoma to be treated. For
instance, patients suffering from an ovarian carcinoma will
be treated with preparations made from cells taken fronn
ovarian carcinomas of other patients.It is also possible
to make mixed preparations, which contain cells from dif-
ferent kinds of carcinomas, such as weaXened cells from
mammary and ovary carcinomas.
The pharmaceucical preparations accordin~ to the
present invention are stable in suspension for about 4
~13~9
- 10 -
~139Z~
wee~s or so at a storage temperature of about -~4C. ~o
increase the shelf life of the preparations, the pharma-
ceutical prep~ations may be quick frozen to temperatures
as low as -80. When deeply frozen the preparations are
stable for about one year. For this purpose, they are
preferably sealed and paekaged together with a slight ex
cess of nutrient solution. The pharrnaceutical prepara-
tions can also be stored for a prolonc3ed time in a lyo-
philized state.
Pharmaeeutical preparations according to the
present invention ean be administered throuc,3h any conve-
'~ J nient and effective route such as by subcutaneous or in-
tramuseular injeetion or by intravenous infusion. Admin-
istration by subcutaneous or intramuscular in~ection is
preferred.
In one preferred treatment method about 1 ml of
a eell suspension aecording to the invention diluted to 10
times its volume and containing about 106 cells are ad-
ministered in each sinr,31e treatment. The treatment is
suitably repeated 5 to 6 tim~s in intervals of about 48
hours. It has been found that treatment with the pharma-
.~
ceutical preparations according to the present invention
will result in a greatly improved tolerance of the patient
to the corresponding cytostatic chernical agents. For thiC;
reason, it is highly desirable to treat a patient with cy-
tostatic agents alone in a conventional manrler according
to known procedures for about 8 days after a sequence c~
treatments with the preparations according to the present
invention. Further details of this aspect will become
apparent from the following clinical stu~ies which serve
to further illustrate and explain the invention.
~ 39Z~
EXAMPLE
The following rea9ents ancl solutions were used:
Washing Solution -- this was prepared by placing
80 ml solution A, 100 ml solution B, 100 ml solution C and
5 ml of an antibiotic solution, into a container and bi-
distilled water was added to make l,000 ml of solution.
Solution A -- was prepared by dissolving the
following in 400 ml bidistilled water:
NaCl 40.0 g
KCl 1.0 g
N ~PO4.2H2O 5~75 9
KH2P04 1. O g
Solution B -- was prepared by dissolving in 500
ml bidistilled water:
CaC12 0.5 9
Solution C -- was prepared by dissolving in 500
ml bidistilled water:
MgC12 . 6H20
Solutions A, B and C were sterilized in an autoclave and
stored at 4C.
Antibiotic Solution
Penicillin (Grunenthal,
Stolberg) 6 mil:Lion I.[J.
Streptomycin (Specia,
Paris) 1 g
were dissolved in 300 ml bidistilled water and stored at
-20C.
- 12 -
Trypsin Solution -- was prepared in a water bath
at 3 7 C . ~139219
Trypsin powder (Serva-
Heidelberg) 25 g
Hanks (BSS) solution
(see below) 475 ml
The solution was filtered under sterile conditions and
then filled in small bottles and stored at -20C. ~or the
trypsin treatment this solution was adjusted to the de-
10 sired concentration with washing solution.
Behring IV Nutrient
Hanks solution (55 ml stock solu-
.~ tion A + 55 ml stock solution B
-to which was added bidistilled
water 1100 ml) 1100 ml
Tissue culture medium solution500 ml
Calf serum 200 ml
Vitamin solution 100 ml
Glutamin solution 100 ml
Antibiotic solution 10 ml
The mixture was filtered under sterile condi-
tions and was then bottled and stored at -20C. Before
t'l :~
use, the nutrient was adjusted to a pH value in the range
of 6.8 to 7.2 with sodium hydrogen carbonate solution (22
g NaHCO3 and 478 ml bidistilled water).
Hanks Stock Solution A -- the following ingre-
dients were dissolved in 250 ml of bidistilled water and
stored at 4C:
- 13 -
NaCl 40.0 g
KCl 2. 0 g ~ g2
MgSO4.7H2O
Mgcl2 6H20
CaC12 0,7 g
Hanks Stock Solution B -- the following mat-
-
erials were dissolved in 240 ml bidistilled water and
stored at 4C:
Na2HP042~120
KH2PO~ 0.3 9
Glucose 5.0 g
Phenol red solution (1%) 10.0 ml
Phenol Red Solution
Phenol red 0.5 9
1/20 N NaOH 15 ml
Phenol red was dissolved in the cold in the sodium hy-
droxide solution within~about 30 minutes and the solution
was then filled up to a volume of 50 ml with bidistilled
water. The solution wa.s stored at 4C.
TCM So_ution
TCM powder (according to
Morgan, Morton and
Parker, Difco Labora-
tories, Detroit, ~.S.) 5.5 g
Hanks Stock Solution A 25 ml
Hanks Stock Solution B 25 ml
Bidistilled water was added to the two stock solutions to
make 500 ml. The powder was dissolved in the resulting
solution.
- 14 -
Glutamin Solution
Glutamin powder (Difco ~139219
Laboratories) 10.0 g
Hanks Stock Solution ~ 6 ml
Hanks Stock Solution B 6 ml
Bidistilled water was added to the stock solutions at a
volume of 100 ml. The glutamin powder was dissolved in
the resulting solution.
Vitamin Solution
Vitamin (MEM 100 x,
Boehringer, Mannheim) 100 ml
Hanks Stock Solution A 50 ml
' j Hanks Stock Solution B 50 ml
Bidistilled water was added to the stock solutions to a
volume of 900 ml and the vitamins were added. The result-
ing solution was then filtered under sterile conditions,
filled into small bottles of 100 ml and stored at -2noc.
Calf Serum
Fetal calf serum (Boehringer, Mannheim) was
stored at -20C. and before use was thawed in a water bath
at 37C~
. ,f
Tissue (50 g) excised from an ovarian carcinoma of
a 46 year old patient in stage T4N5Mlc were manually com-
minuted together with 700 ml of trypsin solution dilutedto a concentration of 0.5% and placed into a lO00 ml
flask. The mixture was agitated for 10 minutes with a
magnetic stirrer. The supernatant liquor was then ~oured
into an ice-cooled flask of the same capacity. The se-
quence of agitating the tissue-trypsin mixture and of
pouring the supernatant liquor was repeated four tim~s
The ~ourth repetition resulted in a clear supernatant
liquor. The combined supernatant liquors and the re~ain-
ing tissue were then filtered through sterile gauze. The
filtrate was centrifuged at 800 to 1000 r.p.m. for 10
minutes. Washing solution (100 ml) was poured onto the
sediment. The resulting mixture was re centrifuged and
spun down.
The resulting sediment was thoroughly pipetted
with 5 ml nutrient and was then filled with nutrient to a
total volume of about 400 ml. After this dilution, the
examination with a Zeiss inverted microscope having a maq-
nification of 125 revealed about 20 to 25 cells in the
field of view. The dilute cell suspension was distributed
among a series of test tubes, 1 ml per tube, and to Roux
bottles, 100 ml per bottle. The growth and metabolism of
the cells were observed and recorded daily for six days.
As soon as the nutrient solution exhibited a color change
from red to yellow, the nutrient solution was renewed.
Good cell growth was observed when the cell suspension had
been incubated at 37C. in an incubator for 6 days.
The cell cultures in the tubes were subjected to
the in vitro test explained above with the cytostatic
agents listed in Table 1 in the concentrations stated
there. After 24 hours the morphological damage was deter-
mined under a microscope. The cell suspension proved sen-
sitive to the following cytostatic a~ents: A~riblastin,
Vincristin and Proresid. All of the remaining listed
cytostatic agents, 6 in this case, were ineffective.
- 16 -
Four Roux bottles which contained the cell sus-
pension obtained by the above method were then prepared.
Adriblastin, Vincristin and Proresid were used to treat
the contents of respective bottles in the concentrations
stated in Table 1. The cell suspension in the remaining
bottle was not treated and served as a contrpl sample.
After the cytostatic agent had been allowed to act on the
cells for 12 hours, the samples were examined under a
microscope and only 60% of the cells were found to be in-
tact. The cells were then separated by centrifugation at800 to 1000 r.p.m. for 10 minutes. 10 ml of fresh nu-
trient were poured onto the sediment. The weakened cells
thus obtained were suspended in the fresh nutrient in a
concentration of 106 cells per ml. The suspension was
stored at 4C.
Test In Vitro
1 ml of an untreated cell suspension, containing
106 cells, was placed into each of a plurality of test
tubes. These cells had been obtained by the above-des-
cribed treatment of the carcinoma tissue with trypsin buthad not been treated with a cytostatic agent. For six
days, 0.3 ml of one of the preparations obtained as des-
cribed above and consisting of cells that had been weak-
ened with Adriblastin, Vincristin and Proresid, respect-
ively, was added daily to each tube. During this test,the tubes were kept in an incubator a~: a temperature of
37C. After 7 to 9 days, about 80 to 90% of the untreated
cells in each tube had been destroyed by tlle preparation
according to the present invention. A.s the cytostatic
agents had been thoroughly washed out of the preparations,
~13~Z~9
it was observed that tl-i.s is the result oE an intercell-
ular reaction.
Clinical Treatment
Based on the results of the above experiments as -*
: i~
well as additional experiments of like character and after ~
,~.
corresponding experiments on strains of cells from animal
tumors, the preparations according to the present inven-
tion were used for clinical therapy. With one exception,
all patients suffered from cancer in terminal stages and
in spite of surgical operations, irradiation treatments
and/or cytostatic treatments were in stages T 4 N3-5 Mlc
in accordance with the classification introduced by the
~nion International contre le Cancer. The average sur-
vival time of such patients is less than 60 days. The
preparations according to the invention were used mainly
for an adjuvant immune therapy in an attempt to strengthen
the immune system as a preliminary to a cytostatic treat-
ment with the corresponding cytostatic agents. In all 22
patients were treated, who had all been fully informed of
their state and the intended course of treatment. Four-
teen of the patients suffered from malignant ovarian tumors
;~J (one of them from mesosigmoid tumor as a recurrent
growth),four ~om mammary carcinomas, one from a pancreas
carcinoma, one from a retothelial sarcoma, one rom a
cecum carcinoma and one from a carcinoma of the corpus
uteri.
Cancerous tissues were excisec] from the patients
by surgical operations and were treated in accordance with
the above-described procedures to formulate the prepara-
tions according to the invention. On alternate days, the
113~Zl~
- lR -
individually prepared cell suspension in a total of ~ to 8
10-ml portions was administered to each patient by intra-
- gluteal injection. Each 10-ml portion contained 106
cells, that is l ml of the preparation in a nutrient solu-
tion and 9 ml physiological salt solution or calf serum. b~'`
In some cases, 1 ml Lidocaine solution (xyloc~ine) in a con-
centration of 0.5% was administered with each 10-ml por-
tion, if desired. In the first sequence, the number of
injections depended on the number of cells which were
available and in most cases this amounted to 5 to 8 injec-
tions in intervals of 43 hours. After a waiting time of 8
to 20 days (with patients in extre~e condition the waiting
J time is at the upper limit), the cytostatic treatment was
carried out in accordance with the individual test result,
in most cases in accordance with the following schedule:
l mg Vincristin by intravenous injection; 6 hours later
Adriblastin by intravenous injection in an amount of 40 mg
per m2 of body surface area; 48 hours later Cyclophos-
phamide or Proresid in an amount of 25 mg per kg body
weight, dissolved in the same concentration in 500 ml of
physiological salt solution. Owing to the toxicity of
J Adriblastin, an interval of 3 weeks was left between two
cytostatic treatment sequences, until a total of 550 mg of
~hat agent had been administered. In case of resistclrlce after test
result, a change was made to the other cytost~tic
agents, such as 5-FU, methotrexate Leukoverin and the
like, in weekly intervals.
This treatment resulted in a subjective and objec-
tive improvement of all patients. Tllere were no loccll or
general secondary effects. Although thc cell suspension
1~L3S'~1~
- 19 -
had to be assumecl to be toxic, its injection did not re-
local or genel~al
sult in/reddening or swelling. A sensation of pain or
similar response to the reaction was not observed. The
improvement wa~ so lasting that in all cases the normally
dangerous cytostatic therapy could be started after an
interval of 20 days, on a maximum.
Four of the twenty-two patients mentioned above
were not given a subsequent cytostatic thcrapy because
they refused it or their tumc~r.s werc apparerltly t.oo advan-
ced. Nevertheless, even these patients had an unusually
long survival time. For instance, a woman of 60 who had a
mammary t~mor larger than a fist and bone metastases and
J also proven brain metastases was subjectetl to a simple
ablatio mammae and given a subseqùent treatment with the
preparation accordiny to the invention. Without additio-
nal therapy she had a survival time of 270 days.
Five patients were also treated with heterolo-
gous carcinoma cell suspensions and showed a very good
tolerance.
The average survival time of all patients is
presently 350 days, which is almost six times the previous
average. Five patients were still alive after 245 to 665
days. Of these, three have had no recurrent yrowtils ant]
two have recurrent growths which are amenable to treat-
ment. All patients treated are in a 9Ood general state.
The result of the treatment with the prepara-
tions according to the invention on two of the twenty-two
patients mentioned above an~ on an atlditional patient will
now be described.
1139;~
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1) A patient of ~6 was admitted to the hospi-
tal when she suffered from mammary ~morson both sides that
had existed for two years, and large abdominal tumors.
The patient was in an extremely poor general state. After
a general pre-treatment, her two breasts, which had been
extensively invaded by cancer, as well as the associated
lymph nodes at the axillae, were removed in two sur~ical
operations in June 1978. Fourteen days later, her uterus
and the two ovar~n ~mors were removed. The remaining can-
cerous tissue in her omentum anc3 on her peritoneum, on
lymph nodes and at other sites could not be surgically rè
moved. The patient was not irradiated but was given 12
J injections with the preparation according to the invention
made fr~m pretreated carcinoma cells from her own body
(prepared in accordance with the above example) and was
subsequently given the combined chemotherapy. I'he patient
recovered completely during the immune therapy, and subse-
quently showed a high tolerance to the cytostatic agents.
After a total of 12 months had passed, the patient reports
that she feels excellent. She is entirely capable of per-
forming her work and has no complaints at all. Her clini-
9 cal examination does not reveal any recurrent growth, nei-
ther abdominally nor in the chest region. At the begin-
niny of the treatment, she was in a tumor stage ~:r4 N5 Mlc.
2) A patient of 32 had a large ~bdominal tumor
when she was admitted to the hospital. Iler uterus ancl the
two adnexes had been removed in another hospita] one year
previous for a treatment of a fairly large ovarian cancer.
She had also received cytostatic treatment for the pre-
vious incident. After relapse, a mesosigma tumor of
1~3g;~1g
- 21 -
almost the size of c ~ ~ was excised, which had
developed as a recurrent growth due to the previous ovarian
carcinoma. It was impossible to excise the mesosigma
tumor entirely in healthy tissue. She had also metastases
at the lymph nodes on the pelvic wall. The patient was
given a post-surgical adjuvant immune therapy consisting
of 12 injections of preparations produced by a treatment
of cells from her own tumors. Because the patient subse-
quently moved away, ~he could not be given a subsequent
cytostatic treatment. The patient returned several times
for subsequent examination. Eleven months from the begin-
ning of the immune therapy she is in an excellent state
without any clinical indication of recurrent growths. The
entire abdomen is free from tumors.
3) A patient of 41, who has not been included
in the above-described group because she is in the Tl
stage, came to the hospital for admittance because she had
a small, possibly cancerous node in the left mammary
gland. The result of mammography, thermography and punc-
ture cytology were positive on the left and doubtful re-
garding another small node on the right tPapanicolaou 3).
0 ; The histology of tumorous tissue excised on the left
breast revealed a scirrhus mammary carcinoma (~fl Nx Mo)
about 2 cm in diameter. This patient's case is reported
separately because she was the only early-stage case. The
patient agreed to a simple mastectomy of the left breast
but refused additional measures particularly on the right
breast. She also refused cytostatic or other subsequent
treatments but agreed to a cell treatment. According to
the present invention, she was given a heterologous cell
~1392~9
treatment with mammary carcinoma cells which had been
obtained from another patient and treated accor ~ g to the
procedures of the present invention. The cell treatment
was performed in accordance with the routine described
above comprising 6 intramuscular injections of 10 ml cell
suspension containing 106 cells each on ~lternate days.
Twelve months after the treatment the patient has no in-
dication of recurrent growth. The suspecte~3 t~mor of the
right breast has completely disappeare~.
It is apparent from the previous investigations
that it was not possible to administer a sufficiently
large dosage in most cases owing to a lack of material.
It would be desirable to give every patient suffering from
a carcinoma, even if it is in an initial stage, a therapy
consisting of a sequence of injections five or six times
every 48 hours within one month, then in each of three
alternate months, then in corresponding months of four
successive half-years and finally in corresponding months
of several successive years. Such treatmellts can be car-
ried out only with heterologous cancer cells but theseshould be taken from similar organs, if possible, in case
no autologous cells are available. ~he heterologous can-
cer cells may be mixed in a ratio of 1:1 with lyophilized
cancer cells, which need not be pretreated.
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1~L3~9
l~ile not wishing to ~e bound by any particular
theory or mode of operation, it appears that the process
of my invention and the products so produced are effective
in the treatment of carcinoma on the following: Based on
S the assumption that malignant tissue growth starts on the
base of an autonomous, progressive and overshooting proli-
feration of body-specific cells, this method of cancer
treatment was developed. The principle of my method
appears, in larye part, to be an amelioration of the immu-
nological starting condition of the patient before the cy-
tostatic therapy itself by repeated injections of ultrafil-
trates of body-specific cytostatically pretreated carcinoma
; cells, as hereinabove described.
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