* Evaluation of Pulmonary Metastasis:

+++ = more than 10 nodules In the lung.
+ + = more than 5 nodules in the lung
+ = fewer than 5 nodules in the lung.
-- = no metastasis
( ) = date on which new tumor was detected (month/day).

The injections of AMYGDALIN were begun on September 12, 1972, and were completed December 13. 1972, or when the animals died

* Evaluation of Pulmonary Metastasis:

+++ = more than 10 nodules In the lung.
+ + = more than 5 nodules in the lung
+ = fewer than 5 nodules in the lung.
-- = no metastasis
( ) = date on which new tumor was detected (month/day).
° = total number of days that tumor did not grow.

 

THE EFFECT OF AMYGDALIN IN SPONTANEOUS BREAST TUMORS IN MICE CD₈F₁

Translation from the original performed by Br. Kanematsu Sugiura
of the Sloan-Kettering Institute for Cancer Research, New York, USA, 1974

     _{On April 13, 1973, we received 20 female mice CD8F1, with spontaneous breast tumors, from Dr. B.S.Martin of the Catholic Medical Center of Brooklyn and Queens, New York. Fourteen of the 20, or 70%, had two or three spontaneous breast cancers, which indicates that these mice were older than those used in the two previous experiments (September 12, 1972 and February 20, 1973). The primary tumors were also definitely larger than those in the mice of the previous experiments.}

     _{Ten control mice with 19 tumors (between 2.6 x 2.4 cm the largest, and 0.6 x 0.5 cm the smallest) received CMC in daily intraperitoneal injections, and 10 experimental mice with 18 initial tumors (be tween 3.4 x 2.7 cm the largest, and 1.1 x 0.8 cm the smallest) received AMYGDALIN daily by intraperitoneal injection, in dosages of 2,000 mg/kg per day, except Sundays, for four consecutive weeks}

_{Four control and one experimental animals died within seven days after the experiment was initiated and therefore were not included in the results.}

     _{The results obtained are summarized in Tables 1 and 2 (April 19, 1973). It shows that the repeated intraperitoneal injections of AMYGDALIN in dosages of 2,000 mg/kg per day for four weeks, were not able to destroy the spontaneous breast tumors in the mice. All the tumors grew normally in the control mice (see Table 1); nevertheless, there was a strong inhibitory effect in the development of pulmonary met stasis in the mice treated with AMYGDALIN (22%vs. 100%) and the general health and appearance of these animals was much better than that of the control animals.}

_{Breast Tumors CD8F1 (Adenocarcinomas) Control Animal}

SIZE OF TUMOR IN CM	DOSAGE NO.	DURATION OF STUDY	GROWTH OF TUMOR	FINAL SIZE	PULMON METAST*	DIED
1.5 x 1.4 0.9 x 1.3	31	36 days	ALL	2.4 x 2.3 1.8 x 1.9	+	KILLED
2.1 x 1.6 0.8 x 0.8 0.8 x 0.7	16	19 days	ALL	3.0 x 2.4 1.8 x 1.4 1.7 x 1.3	++	KILLED
2.3 x 2.0 2.0 x 1.6	6	7 days			--	YES
2.0 x 1.7	30	35 days	YES	4.1 x 3.3	+	KILLED
2.6 x 2.4 0.6 x 0.5	10	12 days	YES	3.0 x 2.9 0.9 x 0.7	++	YES
1.9 x 1.5	4	5 days	YES		--	YES
1.8 x 2.6 1.2 x 1.2 1.0 x 1.0	13	15 days	ALL	2.1 x 3.1 1.5 x 1.6 1.6 x 1.4	++	YES
1.3 x 1.5 0.9 x 0.8 1.4 x 1.1 (5/10)	30	35 days	ALL	3.1 x 3.5 1.8 x 1.5 1.5 x 1.4	++	YES
1.6 x 1.6	5	6 days			--	YES
2.1 x 2.3 1.9 x 1.7	1	2 days			+	YES

The injections of CMC were begun on April 19,1973 and finalized May 24, 1973, or when the animals died.

Evaluation of Pulmonary Metastasis:

+++ = more than 10 nodules in the lungs ++ = no metatsasis + = fewer than five nodules in the lungs


-- = no mtastasis () = date on which the tumor was detectecd (month/year)

SIZE OF TUMOR IN CM	DOSAGE NO.	DURATION OF STUDY	GROWTH OF TUMOR	FINAL SIZE	PULMON METAST*	DIED
1.4 x 1.3 1.4 x 1.3	12	14 days	ALL	1.4 x 1.7 1.9 x 2.0	--	YES
1.0 x 1.0 1.6 x 1.5	29	35 days	ALL	1.7 x 2.3 2.4 x 3.7	+	KILLED
1.9 x 1.9 1.2 x 1.4 1.7 x 1.1	14	18 days	ALL	2.5 x 2.2 2.0 x 1.5 0.8 x 0.7	--	YES
0.9 x 0.9 0.9 x 1.2 (5/17) 0.8 x 0.6 (5/17)	25	30 days	Stopped 7d°	1.4 x 1.6 1.1 x 1.5 0.8 x 0.7	--	YES
1.6 x 1.4 1.4 x 0.9	17	21 days	ALL	2.0 x 1.4 1.8 x 1.3	--	YES
1.5 x 1.6 1.1 x 1.0	6	6 days	Stopped all	1.4 x 1.6 0.9 x 1.0	--	YES
3.4 x 2.7 1.5 x 1.2	26	30 days	ALL	4.2 x 4.4 2.2 x 1.7	--	YES
1.8 x 1.4	16	19 days	Yes	3.1 x 2.2	+	YES
1.2 x 0.9 1.0 x 1.0	20	25 days	ALL	1.6 x 1.5 2.0 x 1.6	--	YES
1.1 x 0.8 1.1 x 0.7 (5/3)	30	36 days	Stopped 7d° Yes	1.9 x 1.4 1.3 x 1.0	--	SAC.

The injections of CMC were begun on April 19,1973 and finalized May 24, 1973, or when the animals died.

Evaluation of Pulmonary Metastasis:

+++ = more than 10 nodules in the lungs ++ = no metatsasis + = fewer than five nodules in the lungs

-- = no metastasis ( ) = date on which the tumor was detectecd (month/year) Sac. = killed

_{Recently we carried out three separate experiments (Feb. 22, 1974, Mar. 4,1974 and Mar. 11, 1974) concerning the effects of the prolonged treatment with AMYGDALIN of Mexican and German origin (racemic compound), In the growth of spontaneous breast tumors (adenocarcinomas) in female mice CD}8_F1_{. Each group consisted of 10 control animals which received a saline solution of 0.5 cc by intraperitoneal injection, daily except Sundays, and 10 experimental animals which received AMYGDALIN (Mexican or German) in dosages of 2,000 mg/kg per day. The animals were kept on a normal diet (Purina Laboratory Chow) and water.}

_{When the primary tumors became large (generally more than four weeks after the beginning of the experiment and with tumoral dimensions or more than 2.5 cm in diameter), the animals were killed and the negative lungs were given a biological examination to show the presence or absence of metastasis. Nevertheless, if the animals died, the lungs were inspected macroscopically with the aid of a magnifying glass, and histopathologically in order to show metastasis.}

_{The results of the experiment of Feb. 22, 1974 in relation to pulmonary metastasis, are summarized in Table 1, 2, and 3.}

_{The results from the tables show that the intraperitoneal injections of AMYGDALIN in dosages of 2.000 mg/kg per day for four to nine weeks had a strong, inhibitory effect upon the development of pulmonary metastasis.}

_{Control mice: eight positive, two negative or 20% without metastasis. AMYGDALIN (Mexican): three positive, seven negative or 70% without metastasis. AMYGDALIN (German): two positive, eight negative or} 80_{% without metastasis.}

_{This experiment (of Feb. 22, 1974) was repeated (Mar. 4,1974) 'using 30 female mice CD}8_{F1 with spontaneous breast tumors. The control animals received saline solution daily except Sundays and the experimental animals received daily intraperitoneal injections of AMYGDALIN (Mexican or German) in dosages of 2.000 mg/kg per day.}

_{The results obtained in the experiment of March 4,1974, in relation to pulmonary metastasis, is summarized in Tables 4, 5, and 6.}

_{The results from the tables show that the repeated intraperitoneal injections of AMYGDALIN in dosages of 2.000 mg/kg per day for four to nine weeks had a strong, inhibitory effect upon the development of pulmonary metastasis. Control mice: eight positive, one negative or 11% without metastasis. AMYGDALIN (Mexican): two positive, seven negative or 78% without metastasis. AMYGDALIN (German):} three positive, seven negative or 70^% without metastasis.

_{The results from the experiment of March 11, 1974, in relation to pulmonary metastasis are summarized in Tables 7, 8 and 9.}

     _{The results of the tables show that the repeated intraperitoneal injections of AMYGDALIN in dosages of 2.000 mg/kg per day for four to nine weeks had a strong, inhibitory effect on the development of pulmonary metastasis. Control mice: nine positive, one negative or 10% without metastasis AMYGDALIN (Mexican): four positive, five negative or 56% without metastasis. AMYGDALIN (German) three positive, seven negative or 70% without metastasis.}

_{The three present experiments show that the pulmonary anti-metastasis activity of AMYGDALIN of Mexican and German origin seems to be equal - 68^% and 73^% respectively without metastasis, against l4% without metastasis for the control group.}

_{On May 31, 1974, one animal from the control group and two animals treated with Mexican AMYGDALIN, of the original 90 animals, were still alive.}

_{Breast Tumors CD}8_F1 _{(Adenocarcinomas) Control Group}

_{Breast Tumors CD}8_F1 _{(Adenocarcinomas)
Animals treated with Mexican Amigsalin in dosages of 2000 mg/kg/dayControl Group}

_{On May 8,1973, we initiated a new experiment to show the effect of Mexican AMYGDALIN on spontaneous breast cancer and pulmonary metastasis of female mice CD}8_F1_{. At the beginning of the experiment, the animals were approximately five months of age and did not have spontaneous tumors. The animals had at least one pregnancy.}

_{Thirty mice (eight of them pregnant) used as a control group, received daily intraperitoneal injections of 0.5 cc. saline solution (six times per week) for a prolonged period and the other 30 mice (eight of them pregnant) received intraperitoneal AMYGDALIN daily in dosages of 1,000 mg/kg per day for a period equal to that of the control animals. These animals were born in Dec. 1972. When the tumors appeared in the animals, they were allowed to grow to a large size, which took 21 days. The presence of absence of pulmonary metastasis was documented by macroscopic and histological examination. The animals were kept on a regular diet (Purina Laboratory Chow) and water.}

_{When the animals became weakened by the presence of pulmonary metastasis or because of toxemia caused by large tumors (2.0 cm or larger in diameter) they were killed and a macroscopic examination was performed to detect the presence or absence of metastasis.}

_{RESULTS: The daily examination of the animals treated with AMYGDALIN and of the control animals (the last exam was Sept. 30,1974, or 510 days after inItiating the experiment) showed the development of 19 spontaneous breast tumors and two abdominal tumors in the animals of the control group. The first tumor appeared 11-X-73, followed by 8-X11-73, 20-X11-73, etc. (see Table 1). By Sept. 30, 1974, 21 of the control animals had developed tumors, or ⁷O^%. Three of them had a second tumor and of the 18 animals that died or were killed having large tumors, 14 had pulmonary metastasis in various stages, or ^78%. Twelve animals remained alive with or without tumors.}

_{Of the 30 experimental animals, five died from the accidental injection of AMYGDALIN into the intestine shortly after the experiment was started, therefore, these animals were not included in the results.}

_{On Dec. 28, 1973, one of the animals treated with AMYGDALIN developed a spontaneous breast tumor, or rather 79 days after the first tumor was found in the control group, and was followed by 10 more mice with spontaneous, mammary tumors and one abdominal tumor(14-11-74, 20-111-74, 22.111-74, etc.) or that ^48% of the animals showed spontaneous tumors. Twelve animals that died or were sacrificed because of large tumors and great weakness, were given post-mortem examinations and it was found that three animals had pulmonary metastasis, or 25%. Thirteen animals remained alive with or without tumors.}

_{The present study shows that during three quarters of their lives, the prolonged, intraperitoneal injection of AMYGDALIN did not avoid completely the development of spontaneous, mammary tumors in the mice. Nevertheless, there was a definite reduction of breast tumors (70% in thecontrol group against 48% in those treated with AMYGDALIN). Also it was shown that AMYGDALIN has a potent inhibitory ef fect on the development of pulmonary metastasis in mice (75% against 22% in the control animals). The general health and appearance of the animals treated with AMYGDALIN was as good as that of the control animals in spite of the fact that they received injections for 16 months. The mice without tumors treated with AMYGDALIN as well as the control animals without tumors, gained weight. The surviving animals are currently 21 months of age.}

_{KANEMATSU SUGIURA
Sept. 30, 1974}

 

_{- Evaluation of Pulmonary Metastasis:}

_{Table 2.
The Effect of AMYGDALIN on the Development of Breast Tumors (Adenocarcinomas)
and Pulmonary Metastasis in Mice CD}8_F1
_{May 8, 1973 to September 30, 1974}

_{- Evaluation of Pulmonary Metastasis:}

_{EFFECT OF AMYGDALIN ON SPONTANEOUS,
MAMMARY TUMORS IN SWISS ALBINO MICE
Translated from the original performed by Dr. Kanematsu Sugiura
of the Sloan-Kettering Institute for Cancer Research, New York, USA, 1975}

_{This report includes the observations of the effects of the prolonged treatment with AMYGDALIN (Mexican) on the growth of spontaneous breast tumors (adenocarcinomas) in Swiss Webster albino mice (from Taconic Farms, N.Y.). The diagnosis of the tumors was performed by a post-mortem, microscopic examination of the tissues, at the conclusion of the experiment. Occasionally there was complete regres sion of tumors after the injection of AMYGDALIN or saline solution. These undiagnosed tumoral growths were not included in the results. Other spontaneous tumors (not breast) were also not included in the results. The animals were kept on a normal diet (Purina Laboratory Chow) and water. Since we only re ceived from two to five mice with mammary tumors at one time from Taconic Farms, the control and experimental groups were not studied simultaneously. The control animals received 0.5 cc (5) saline solu tion daily. with the exception of mouse 111 which received CMC (carboximetil-cellulose). The experimental animals received peritoneal AMYGDALIN daily except Sundays.}

_{The results obtained in the current study are summarized in Tables 1 and 2. The results of the experiment included in Tables 1 and 2, are in the order in which they occurred. Included were 28 control mice with 35 tumors and two secondary tumors (the largest 2.5 x 2.9 cm and the smallest 0.6 x 0.6 cm), and 35 experimental mice with 37 tumors and five secondary tumors (the largest 2.5 x 1.9cm and the smallest 0.7 x 0.7 cm).}

_{In Table 2 it is shown that the repeated intraperitoneal injections of AMYGDALIN in dosages of 3.000 mg/kg per day for two to eighteen weeks, were unable to destroy the spontaneous, breast cancers in the mice. Nevertheless, they caused the small tumors (of approximately 1.5 cm diameter or less) to stop growing more frequently than what was observed in the control mice (eight of 28 tumors in the control animals stopped growing, or rather, 29% as opposed to 18 out of 35 tumors which ceased growing in the group treated with AMYGDALIN, or 51%).}

_{It also shows that the AMYGDALIN had a potent, inhibitory effect upon the development of pulmonary metastasis in mice: 77% inhibition vs. 7% inhibition in the control animals. Without doubt, the mice with large tumors have pulmonary metastasis and it is possible that these growths were destroyed by the repeated treatment with AMYGDALIN. The general health and aspect of the animals treated with AMYGDALIN was much better than that of the control animals.}

_{The results obtained from the breast tumors of Swiss albino mice are essentially equal to those obtained from the CD}8_F1 _{mice with spontaneous, mammary tumors: the repeated, intraperitoneal injections of AMYGDALIN in dosages of 2,000 mglkg per day, inhibits the growth of small tumors and the development of pulmonary metastasis in mice.}

KANEMATSU SUGIURA

February 10, 1975

_{Table 2.
Breast Tumors (Adenocarcinomas) in Swiss Albino Mice
Animals Treated with AMYGDALIN}