IE49181B1 - Treatment of malignancies by administering sarcosine,its derivatives and mixtures - Google Patents

Description

There is a great need for improving the chemotherapy of malignant tumors. Heretofore tumors were treated substantially with anti-neoplastic chemotherapeutics, among them, particularly antimetabolites, alkylating and intercalating substances and plant ingredients. Since these preparations influence not only the tumor cells, but also other rapidly proliferating systems of the organism, e.g. blood forming centres, skin and appendage formations, mucous membranes and the germina epithelium, the therapeutic effect is limited by undesired side effects.

The increase of the chemotherapeutic effect without increasing the toxic effect is therefore a desirable goal.

The present invention provides N-lower alkyl glycines with 1 to 4 carbon atoms in the N-alkyl part in the form of acid amides as well as sarcosine anhydride as sole active substance, in mixtures with each other or in combination with known tumor inhibiting compounds for use in the treatment of tumors.

The invention also relates to a process for the preparation of agents for use in the treatment of tumours wherein (A) at least one member of the group N-lower alkyl glycine having 1 to 4 carbon atoms in the N-alkyl part in the form of acid amide, sarcosine and sarcosine anhydride is combined with (B) a known antitumor agent, by bringing an effective amount of each of (A) and (B) into intimate contact with each other and isolating the product obtained thereby. Such antitumor agents are for instance listed in Ullmann's Enzyklopaedie der Tech, Chemie, 4th Edition, Vol. 9, Pages 705-714 under the heading Cystostatica.

Of special interest in this connection are anti-neoplastic alkylating agents, intercalating agents and antimetabolites.

The process consists in combining (A) and (B) into intimate contact, preferably in solution, generally in amounts of 2:1 to 50:1 parts by weight, especially 2:1 to 10:1 parts by weight of (A) and (B). The resulting combination which could be termed an addition product or double salt, is isolated in a manner known per se, for instance by vacuum drying, lyophilization or any convenient method and formulated into units of an effective amount in a usual manner, for example, encapsulated or granulated, if desired together with usual fillers, excipients and/or other convenient auxiliary agents.

Due to the high toxicity of most of the cystostatica it is often more convenient to formulate the preparations according to their toxicity LD/5O by combining (A) and (B) in such a manner that generally 1/10 to 1/?Q Of the LD./50 of (B)is combined with 1/20 to 1/100 of the LD/50 of (A) (LD/60 in mg/kg) to make unit dosages. The preferred units have a great margin of safety and thus contain apout 1/10 to 1/20, especially about 1/15 of th® ^/50 of (B) combined with 1/20 to 1/40 especially ]/§5 to 1/30 of the LD/50 of (A). fr It may sometimes be preferred to encapsulate (A) and (B) in such a manner that one of the components (A) or (B) is partly encapsulated separately in a long time capsula which in its turn is encased in a capsula common to the full preparation so that one part of the combination is given off to the body later than the other part. Methods for this type of encapsulation are well known in the art. In proceeding in this manner only part of the reactants (A) and (B) are reacted before encapsulation while part of (A) or (B) is encapsulated separately. 48181 Thus, for example, a more than double increase of the therapeutic tumor inhibiting action of Adriamycin or cyclophosphamide is achieved in transplantable tumor without an increase of the toxic side effect by injecting sarcosine at selected time intervals after administering Adriamycin or cyclophosphamide Since sarcosine as an amino acid has an extremely low toxicity, it has no toxic ef.fect in doses considered to be practical.

The use of sarcosine and/or the above-mentioned sarcosine derivatives in combination with the above mentioned antineoplastic chemotherapeutics actually increases the therapeutic effect synergistically without increasing the toxic side effects. It is preferably administered sequentially, giving the anti15 neoplastic compound first, and the sarcosine or the sarcosine derivative at a later time. However, with the administration of intercalating substances, the simultaneous administration of both substances is advisable.

It is of interest that tumors implanted intra-cerebrally 2u (into the brain), which are not influenced by cyclophosphamide, can be cured in over 50% of the test animals by a combination of cyclophosphamide and sarcosine.

This gives rise to the possibility for a considerable unexpected improvement in this and other therapeutic combinations for the treatment of tumors.

This property of sarcosine and certain sarcosine derivatives seems to be rather specific, since sarcosine, sarcosine anhydride (sarcosine AH), N-methylglycinamide §pd N-propyl glycinamide show a tumor-inhibiting effect in various transplanted 10 tumors, while creatine an isourea-derivative of sarcosine, is interestingly enough ineffective, although creatinine, the anhydride of creatine has a tumor-inhibiting effect.

The following examples will illustrate the invention.

The animal tests were carried out on mice and rats.

Example 1.

Effect of sarcosine and Ν-alkyl' glycinamides' alone and in combination with Endoxan, another fumor-’inhlbitihg compound (results in table 1 to 3) >3 A use of sarcosine and various sarcosine derivatives in the treatment of Leukemia P.388 showed that the curative · effects achieved were similar to those obtained with Endoxan (cyclophosphamide) used as a positive control substance (as seen in table 1, N-propanol glycinamide (series 2 of table 1) which were included in tahle 1 as a reference substance like Endoxan in series 10 and 11), proved to be of little effect regarding the healing rate (5 out of 10 animals cured) and the ILS (life extension of the treated series, compared to the control series in %), though it is closely related to N-propyl glycinamide.

But sarcosine in a single dose of 270 mg/kg proved to be comparable to Encoxan used as a positive control both regarding the curative effect (11 out of 15 animals cured) and the survival rate (see series 10 and 11). In a repetition of the sarcosine dose of 270 mg/kg after 48 hours (series 4), there was a slight increase of the curative effect and survival rate. Sarcosine, anhydride, (in the table designated as sarcosine AH, series 5,6), used in equimolar dosage as sarcosine, had a comparatively lower chemotherapeutic effect as a single dose (series 20 5). Only when two doses of sarcosine anhydride were given within hours, (series 6) did the curative effect and the survival rate increase. But N-methylglycinamide yielded optimum effect with a single dose, (series 7), which effect could not be increased by administration of two equal doses (series 8). N-propyl gly7 cinamide (series 9), like N-methyl glycinamide, had a clear chemotherapeutic effect with a single dose.

The dosage for the individual preparations can be varied. Extensive tests with both sarcosine and sarosine anhydride showed 5 in the meantime that the optimum dosage for sarcosine was in a range of 150 to 270 mg/kg, while for sarcosine a'tihytiride the best chemotherapeutic effect is with a dose.of 2O0<-32Q mg/kg.Increases beyond the indicated range do not seem fo lead to an increase in chemotherapeutic effect.

The use of an equitoxic dose of sarcosine and its derivatives must also be taken into consideration, as the following statements will show. In the tests described here, Endoxan (cyclophosphamide) was used as a reference substance, and it therefore seems to be of interest to make a comparison between the chemotherapeutically used Endoxan dose of 30 and 60 mg/kg respectively and the lethal dose of 50 (LD/50) which is for Endoxan 450 mg/kg in subcutaneous application.- Based on the lowest therapeutically used dose of Endoxan, this would yield a factor of 15, that is, the 15 fold dose of 30 mg/kg Endoxan would correspond to the Liyso of Endoxan. For sarcosine the LD/50 is 6500 mg/kg.

The difference between the smallest dose of sarcosine S 18 1 in this test (270 mg/kg) and the LD-/50 of sarcosine would yield a factor of 23, that is, a 23-fold dose of the therapeutically used dose of sarcosine would correspond to the Λ^/δΟ of sarcosine. For sarcosine anhydride, the LD/50 would he 8300 mg/kg. In the test system used (Leukemia P 388), sarcosine and its derivatives offer substantial advantages over Endoxan, because of their lower toxicity. In a comparison test of sarcosine, . sarcosine anhydride and creatinine in Leukemia L 1210, sarcosine anydride and creatinine showed an even slightly better tumor-inhibiting effect than sarcosine.

As mentioned above, there is a very Interesting and therefore preferred field of application for sarcosine and the above-mnetioned derivatives in the combination with other tumor inhibiting compounds, particularly with alkylating and inter15 calating'substances. Among the alkylating substances we mention particularly cyclophosphamide (Endoxan) and 1,3-bis-(2-chloroethyl) 1-nitro-isourea as well as l-(2-chloroethyl)-3-cyclophexyl)-lnitroisourea and other chloroethyl-l-nitroisourea derivatives, and among the intercalating substances particularly Adriamycin. 2o Dactinomycin, Cohlorambucil and Melphalan. For the combination with metabolites Methotrexate can he employed.

Sarcosine and sarcosine derivatives with a free carboxyl group or basic nitrogen, which still is available for the formation of a salt, can be used as a common salt, e.g., alkali or ammonium salt, which usually improves the solubility.

As a further example, the comination of sarcosine with Endoxan may be used in the treatment of Lewis Lung'carcinoma in. mice. The Lewis Lung carcinoma is a bronchial carcinoma in mice, which leads after implantation in the musculature of the hind leg in a relatively short time to extensive metastatic formation in the lung. This results in a tumor model which no longer 0 corresponds to the biological behavior of tumors in man as far as metastatic formation is concerned.

Table 2 shows that Endoxan was given in a single increasing dose (80, 120, 160 mg/kg) as well as in three doses of' mg/kg per week and 120 mg/kg per week. (Series 2-6). With a single dose of 80 mg/kg Endoxan (Series 2) the healing rate was of 2 out of 15 animals, the survival rate 110%.

In a combination of 80. mg/kg Endoxan and 270 mg/kg . sarcosine, given sequentially in an interval of 6 hours (Series 7), 10 out of 15 animals were cured, the survival rate in the 20 Endoxan-sarcosine combination (Series 7) xvith the chemotherapeutic effect of double the Endoxan dose (160 mg/kg, shows that the curative effect (Series 5) (4 out of 15 animals) is clearly lower. A calculation of the difference of the cured animals, which received 80 mg/kg Endoxan (Series 2), compared to the Endoxan-sareosine combination (Series 7) in the four-panel test with given 2a/v> 0.5 show a statistically significant difference. The significance of the time interval between the administration of Endoxan and sarcosine can be seen from series 8 and 9. An extension of the time interval to.8 hours leads to a slight increase of the curative effect and of the survival rate (Series 8), while a reduction of the time interval to 3 hours (Series 9) leads to a clear decrease of the curative effect and survival rate.

While there are considerable differences between the Lewis Lung carinoma and the Ehrlich carcinoma or sarcoma 180 regarding the doubling time of the tumor and the generation time of the tumor cells, the time interval between Endoxan or other alkylating substances of 6 hours proved to be optimal, the general range being between 4 and 18 hours.

Table 3 shows the results of the combinations with Endoxan (cyclophosphamide) and sarcosine. The treatment started after an average weight of the tumor of 3,5 g had been obtained 20 in order to provide more diffcult test conditions. As a tumor model the Ehrlich carinoma in mice was used an a treatment period of 4 weeks was selected. Endoxan’ has no curative effect with a dose of 120 mg/kg (Series 2); but there is clear tumor-inhibiting effect. When the Endoxan dose was doubled to 240 mg/kg (Series 3) 5 out of 15 animals were cured. Sarcosine in monotherapy (Series 4) he? pO tumorinhibiting effect. However, the combination of ssreosine-Endoxan (Series 5) leads to a significant synergistic curative effect (10 out of 15 animals). Since Endoxan was used in ell cqmbina10 tions in a dose of 120 mg/kg, it can be seen that the sarcosineEndoxan combination clearly exceeds the curative effect.of the double Endoxan-dose in Series 3. The simultaneous administration of sarcosine and Endoxan Series 6 .shows no significant difference compared to the Endoxan monotherapy (Series 2), But the Endoxan-sarcosine combination with a time interval of 3 hourse Series 7 shows a slight increase of the curative effect. A significant difference compared to series 3 (Endoxan monotherapy) is found in the Endoxan-sarcosine combination, where the combination partners were used in a time interval of 6 hours Series 8 (12 out of 15 animals cured), When a longer time interval (18 hours) is used (Series 9) between the administration of Endoxan and sarcosine the curative effect diminishes again. Conspicuous differences are also found in a comparison of the body weight differences between the start and the end of the test within the series treated with Endoxan (see last column). We can see clearly that the loss of body weight in the Endoxansarcosine combination is less than in the Endoxan series.

As it can be seen from table 3, the optimum time interval between known neoplastic agents and sarcosine derivative is 3 to 6 hours, since the weight loss of the test animals are here at a minimum.

Example 2.

Effect of Adriamycin as an additional compound, alone 10 and in combination with sarcosine (results in tahle 4' and 5), z Table 4 shows that Adriamycin given intravenously in a* dose of 2.5 mg/kg has no curative effect in sarcoma 180 and causes no marked inhibition of the average tumor weight; the average tumor weight was inhibited by less than 50% compared to the control (Series 2). The simultaneous Adriamycin-sarcosine combination (Series 3) had the effect that 6 out of 10 animals were cured and there was a significant inhibition of the tumor weight in those not cured.

In Table 5 Adriamycin was also used but in a higher dose (8 mg/kg iv.) in sarcoma 180 (Series 2). With this dose there is a significant inhibition of the tumor growth, compared to the control series; the curative effect in this series, however, (1 out of 15 animals cured)· was accompanied by toxic side effects (1 out of 15 animals died). But the sareogine5 Adriamycin combination with a time interval leads po a significant increase of the curative effect (9 out of 16 animals cured), without any death rate by toxic effects. A simil'ay trend is found in a comparison of the body weight differences pf the treated series. It is conspicuous that the body weight difference between the start and the end of the test is pel&tfvely high in all control series. This can be accounted for by the pachexia caused by the progressive and inhibited growth of the tumor.

Additional tests show that combination with other i tumor-inhibiting (anti-neoplastic) chemopherapeutics are also possible. The advantage of these combinations is that a synergistic unexpected anti-neoplastic effect is achieved without an increase in toxicity since sarcosine and its derivitives have a low toxicity. This offers the possibility of combining preparations for the simultaneous administration of sarcosine and/or sarcosine derivatives, particularly with intercalating substances, primarily the combination sarcosine-Adriamycin.

The tests described in Example 1 are compiled in tables 1, 2, 3, and the results of the tests described in Example 2 are compiled in Tables 4 and 5.

TABLE 1 Series Ko. Preparation Single Dose mg/kg Total Dose mg/kg Cured Died ILS % Average Survival Rate 1 Control - - - 15/15 - 7-10,73-14 2 N-Propanol- glycinamide sc. 450 450 5/15 10/15 16,03 10-12,7-16 3 Sarcosine sc, 270 270 11/15 4/15 135,47 10-12,5-13 4 after 48h Sarcosine sc. Sarcosine sc. 270 270 270 270 13/15 2/15 157,22 10-12-14 5 Sarcosine AH sc. 260 260 6/15 9/15 85,15 13-13,1-14 6 after 48h Sarcosine AH sq, Sarcosine AH sc. 260 260 260 260 8/15 7/15 106,89 13-13,28-15 7 N-Methyl- glycinamide sc. 318 318 9/15 6/15 112,48 10-12-13 8 N-Methylglycinamide sc. 318 318 after 48h N-Methylgly- . cinamide sc. 318 318 8/15 7/15 100,02 10-11,71-13 9181 TABLE 1 CONT 9 N-Propylglycinamide sc. 380 380 9/15 · 6/15 113,73 10-12,3-15 10 Endoxan sc. 30 30 ' 9/15 6/15 126,15 14-15,66-17 11 Endoxan sc. 60 60 10/15 5/15 107,51 14-18,4-22 Strain: ®2®6^1 Tumor: P 388 Transplant- mode: ip Transplant-date: Start of Therapy: Treatment days : Duration of Treatment End'o'f Test: Number of Animals : in series 16. 5. 1978 17. 5. 1978 1 x/Week Week 9. 6. 1978 48181 TABLE 2 Series No. Preparation Single Dose mg/kg Total Dose mg/kg Cured Died ILS % Average Survival Rate 1 Control - - - 15/15 - 26-27-29 2 Endoxan sc. 1 x Treatment 80 80 2/15 13/15 110,9 37-53,0-68 3 Endoxan sc. 1 x Treatment 120 120 1/15 14/15 60,5 36-40,7-55 4 Endoxan sc. 120 360 3/15 12/15 139.3 47-59.6-61 5 Endoxan sc. 1 x Treatment 160 160 4/15 11/15 135.9 47-55,6-70 6 Endoxan sc. 80 240 15/15 92,5 43-56,8-60 7 Endoxan sc. 80 240 10/15 5/15 187,4 44-56,8-60 after 6 h Sarcosine sc. 270 810 8 after 8 h Endoxan sc. Sarcosine sc. 80 270 240 810 9/15 6/15 169,7 36-50,1-60 9 after 3h Endoxan sc. Inosine sc. 80 200 240 600 5/15 10/15 147,1 47-56,8-63 TABLE 2 CONT, Strain: Tumor: Transplant- mode: Weight of tumor at Start of Treatment: Transplant date: Start of Therapy: Treatment days : Duration of therapy End of Test No. of Animals in series: Lewis-Lung-Carcinoma i.m. 1,2 g - Start of Therapy after 5 days 1. 3. 1978 6. 3. 1978 1 x Weekly 1 every 3 weeks 30. 4. 1978 TABLE 3 Series No. Preparation Single Dose mg/kg Total Dose mg/kg Cured Died 0 Tumor Weight 0 Body Weight Difference 1 Control - - - - 16,9 -21,3 7. 2 Endoxan 120 480 - - 3,3 -15,3 7, 3 Endoxan 240 960 5/15 - 2,4 -19,3 7. 4 Sarcosine 280 1120 - - 14,7 -21,1 % 5 Sarcosine sc. 280 1120 after 4 h Endoxan sc. 120 480 10/15 1,4 - 7,7 7. 6 Endoxan + Sarcosine 120 280 480 1120 2/15 - 3,2 - 7,1 7. 7 after 3 h Endoxan sc. Sarcosine 120 280 480 1120 4/15 - 2,4 - 4,5 7. 8 after 6 h Endoxan sc. Sarcosine 120 280 480 1120 12/15 - 2,5 - 2,8 % 9 after 18 h Endoxan sc. Sarcosine sc. 120 280 480 1120 6/15 - 3,1 - 7,3 7. 9 18 1 TABLE 3 CONT.

Strain: Swiss Tumor : Ehrlich-dpi. Transplant - mode: i.m. Weight of Tumor at Start of Treatment: 3,8 0 g Transplant date: 3. 3. 1977 Start of Therapy: 8. 3. 1977 Treatment days : 1 x Weekly Duration of therapy 4 Weeks End of Test 4. 4. 1977 No. of Animals in series : 15 Mice 48181 TABLE 4 Series No. Preparation Single Dose mg/kg Total Dose mg/kg Cured Died $ Tumor Weight 0 Body Weight Difference 1 Control - - - 14,7 -26,3 7. 2 Adriamycin i.v. 2,5 10 - 8,9 -17.0 7, 3 Adriamycin + Sarcosine i.v; 2,5 200 10 800 6/15 2,9 -10,1 7, Strain: Swiss Tumor: Sarcoma'180 Transplant - Mode i.m. Weight of Tumor at Start pf Treatment: 1,2 g Transplant date: 21. 1. 1977 Start of Therapy: 24. 1. 1977 Treatment days: 1 x Weekly Duration of Therapy: 4 Weeks End of Test 26.2. 1977 No. of Animals in series: 15 Mice TABLE 5 Series No. Preparation Single Dose mg/kg Total Dose mg/kg Cured Died P Tumor Weight P Body Weight Difference 1 Control - - - - 15,1 -14,3 % 2 Adriamycin i.v. 8 24 1/15 1/15 4,4 - 9,9 % 3 after 2 h Sarcosine i.v. Adriamycin i.v. 180 8 540 24 9/15 - 5,9 - 3,5 % Strain: Tumor: Transplant - Mode Weight of Tumor at Start of Treatment: Transplant Date: Start of Therapy: Treatment days : Duration of Treatment End of Test: No.of Animals in series: Swiss Sarcoma 180 i.m. o,7 φ 29. 11. 1976 1.12. 1976 1 x Weekly 3 Weeks 28. 12. 1976 Mice Example 3.

Comparison of the chemotherapeutic effect of sarcosine with sarcosine anhydride (sarcosine AH) as well as Endoxan in intramuscularly implanted DS-carcinosarcoma. (Table 6).

Therapy was started with a far advanced tumor growth (weight of 4.5 - 5.0 g). The resistance of the tumor to Endoxan (cyclophosphamide) can be seen from series 2. The difference between the tumor weight of the control series (Series 1) and the Endoxan-treated series (Series 2) i.e. 28.6 g/20.1 g is very small.

But sarcosine in a single subcutaneous administration of 180 mg/kg per week had a curative effect in 8 out of 10 rats (Series 3). Interestingly enough as seen in Series 4 it is not possible to improve the curative or tumor-inhibition effect by administering 180 mg/kg sarcosine three times a week subcutaneously. Sarcosine AH (sarcosine anhydride) has a curative effect in out of 10 rats with a single subcutaneous administration of 290 mg/kg. (Series 5). The same result was obtained with 290 mg/kg sarcosine AH administered subcutaneously three 20 times a week (Series 6), It seems that sarcosine and sarcosine anhydride intervene in preliminary stages of the purine synthesis, and differences in the extent of the effect of sarcosine and sarcosine anhydride appear, depending on the type of tumor, if both preparations are used in equimolar amounts. The great sensitivity of the DS-carcinosarcoma to sarcosine and sarcosine anhydride opens up the possibility of clarifying the action pf . the biochemical mechanism and to bring the question of the lack of toxicity even with high doses compared to normal, proliferating systems (e.g. blood forming centers in the b0i}8 marrow.) closer to a solution.

The results are compiled in table 6.

Example 4.

Comparison of sarcosine anhydride with Endoxan in the DScarinosarcoma of the rat. (Table 7) It is known from numerous data in the literature (e.g.

Brock, N.: Experimental Basis of Cancer Chemotherapy., Chemotherapy 7_, 19-50 (1976), that Endoxan has little effect on the DS-carcincsarcoma. In the present test, Endoxan and sarcosine anhydride were tested for comparison. It should be pointed out that Endoxin is barely tolerated with a daily subcutaneous injection of 2 x 2000 mg/kg. It was found that Endoxin in a dose of 60 2° mg/kg has a curative effect in 2 out of 10 animals, while· the other animals showed practically no inhibition of the tumor growth, as it can be seen from a comparison of the average weights of the control - and Endoxan series (Series 1,2). Sarcosine anhydride in a dose of 290 mg/kg sc. (3 times a week) effect a complete remission of the tumor in 8 out of 10 animals.

The inhibition of the tumor in the two uncured animals 5 seems insignificant.

The results are compiled in Table 7.

Example 5.

Comparison of the chemotherapeutic effect of various doses of VM 26, and sarcosine anhydride, as well as their combination in the ckiploid, intracerebrally implanted Ehrlich as cited tumor, (Table 8) VM 26, an., epipodophylltoxin derivative, which is a 4dimefchyleplpodophyllotoxin-thenylidene-glucoside, is used in acute myeloid leukemia, in neurogenous tumors and malignant 5 lymphomas; particularly in neurogenous tumors the results obtained seem to be of interest.

The tests showed the following; VM 26, whose clinical field of application are brain tumors and Hodgkins disease, has in the intracerebrally implanted Ehrlich ascites tumor, a, chemotherapeutic effect, depending on the dose (Series 2-4) which leads to a pronounced cure (10 out of 15 mice) with an intravenous injection of 12 mg/kg per week. The combination of VM 26 (6 mg/kg) and sarcosine anhydride (290 mg/kg) (Series 5) surpasses With the same dose the curative effect of the three-times higher WOhOtherapy with VM 26. All animals were cured. But if th© time interval between the combination partners is between 3 and 6 houps (Series 6,7) the curative effect of the VM 26-BarcOSine anhydride combination diminishes. Sarcosine anhydride Ih a si-Ugle dose, as it is used in the combination with VM 26, has no effect on the death rate (Series 8). A dose of sarcosine anhydride applied on five successive days (Series 9) leads to a cure in 13 out of 15 mice, while a single dose of 1150 mg/kg sarcosine anhydride 15 (Series 10) leads to a cure in only 8 out 15 mice. Sarcosine anhydride is tolerated without any loss of weight with a daily subcutaneous injection of 2 x 2500 mg/kg over 14 days, while VM 26 injected intraveneously only once a week in a dose of 18 mg/kg can be given without severe toxic damages. In addition to the 20 combination effect of sarcosine anhydride and VM 26, it seems to be of interest that, when sarcosine anydride is given alone in a higher dose or daily, a clear curative effect is achieved.

TABLE 6 + Series No. Preparation Single Dose mg/kg Total Dose mg/kg Cured Died 0 Tumor Weight 1 Control - - - - 28,6 + 1.7 2 Endoxan i.v. 60 120 - - 20,1 + 1,0 3 Sarcosine sc. 180 360 8/10 - 19,6 + 1,5 5 Sarcosine sc. 3 x a week Mon.Wed.Fri. 180 720 8/10 - 20,7 + 1,3 5 Sarcosine AH sc.290 580 10/10 - - 6 Sarcosine AH 3 x weekly sc. 290 1160 10/10 Mon. Wed. Fri.

Strain: SD Rats Tumor: DS Carcinoma Transplant-Mode intramuscular Weight of Tumor at Start of Treatment: 4,5 - 5,0 grai Transplant Date : 12.3.79 Start of Therapy: 21.3.79 Duration of treatment: 3 weeks End of Test: 2.4.79 No. of Animals in series: 10 Rats Z7 TABLE 7 Series No. Preparation Single Dose mg/kg Total Dose mg/kg Cured Died ρ ί Tumor Weight 1 Control - - - 16,2 + 1,48 2 Endoxan sc 1 x a week ' 60 180 2/10 15,3 + 2,1 3 Sarcosine AH sc 3 x a°week 290 2320 8/10 12,4 + 1,2 . . . J«7 Strain: Sprague-Dawley Tumor: DS-Carcinosarcoma Transplant- Mode: im. Weight of Tumor at Start of Treatment: 0 1,5 Grams Transplant Date: 15.2.79 Start of Therapy: 21.2.79 Treatment Days :-' Mon. Wed. Fri. Duration of Treatment: 3 Weeks End of Test: 9.3.79 No. of Animals in series: 10 4918 TABLE 8 Series No. Preparation Single Dose mg/kg Total Dose mg/kg Cured Died Average Survival Rate 1 Control - - 0/15 15/15 6- 6.5-8 2 VM 26 iv. 6 12 3/15 12/15 6- 9,3-13 3 VM 26 iv. 12 24 4/15 11/15 7-9,7-13 4 VM 26 iv. 18 36 10/15 5/15 7-11,8-13 5 VM 26 iv. 6 12 + 15/15 0/15 Sarcosine AH sc. 290 580 6 VM 26 iv. 6 12 3h later 13/15 2/15 6- 7,5- 9 Sarcosine AH sc. 290 580 7 VM 26 iv 6 12 6“ later 9/15 6/15 6-10,1-13 Sarcosine AH sc, 290 580 8 Sarcosine AH sc. 290 580 0/15 15/15 6- 7,6- 9 9 Sarcosine AH sc. 5 x 290 2900 13/15 2/15 10-11,5-13 10 Sarcosine AH sc. 1150 2300 8/15 7/15 6-11,7-14 TABLE 8 CONT. Strain: Swiss Mouse Tumor: Ehrlich-Carcinoma Diploid Transplant - Mode: intracerebral Transplant - date: 6.3.79 Start of Therapy: 7.3.79 Treatment days : 1 x a Week Duration of Treatment: 2 Weeks End of Test: 13.3.79 No. of Animals in series: 15 Mice Example 6.

Comparison of the combination between VM 26 with sarcosine or sarcosine anhydrid (Sarcosine AH) in the intramuscularly implanted diploid Ehrlich ascited tumor. (Table 9).

' $ In this example in which sarcosine and sarcosine anhydride were used in equimolar doses it was found that, in contrast to the intracerebrally implanted Ehrlich ascites tumor, the optimum effect appears with a time interval of the combination partners (VM 26 with sarcosine or sarcosine anhydride) of 6 hours.

Sarcosine and sarcosine anhydride with a single weekly administration in a dose as in the combination, effects no significant tumor-inhibition. VM 26 in monotherapy causes in .the dose range used increasing tumor inhibition with increasing dosage (Series 2-4). The combination of VM 26 and sarcosine is clearly superior to the VM 26 monotherapy, both with regard to tumor inhibition and to the curative effect (Series 5-7), an Increase of the curative effect can also he seen in the combination with increasing VM 26 dose. Similar results can be achieved with the combination VM 26-sarcosine anhydride, where the curative effect increases more 2θ in the lower and medium dosage range than with the highest VM 26 dose (Series 8-10). It is possible, also, from the foregoing- Ί that sarcosine anhydride has a curative effect in intramuscularly Implanted Ehrlich ascites tumors, with a higher dose of daily administration.

The results are set forth in Table 9.

Example 7.

Comparison of the effect of c'isplatinum with sarcosine alone and in combination. (Table 10).

Cisplatinum (cis-dichlordiamine platinum, CPDD for short), is of particular importance because of the long-term remissions achieved with it (e.g. up to five years) after copipletion of the therapy in testicle tumors, osteoid sarcomas and prostatic carcinoma.

In the diploid Ehrlich carcinoma, which was implanted intramuscularly in female Swiss mice, the treatment with cisplatinum in various doses as well as with cisplatium-sarcosine combinations was started 4 days after the tumor transplant with an average tumor weight of 2.5 g. As a postive control, 120 mg/.kg Endoxin were injected subcutaneously, which was given in intervals ]5 of 14 days like the various intravenously injected cisplatinumdoses and their combinations with sarcosine. The results in the table show that Endoxin (Series 2) has a clear tumor-inhibiting effect. Cisplatinum (Series 3-5) in a dose of 8 mg/kg achieves significant tumor-inhibition and complete tumor remission. The simultaneous combination of cisplatinum with sarcosine (Series 6-8) proved superior to the monotherapy with cisplatinum. Depending on the dose of cisplatinum used, there is a clear increase of tumor inhibition and of the curative effect. 48181 Even with a dose of 4 mg/kg cisplatinum, inhibition of tumor growth of more than 50% is achieved in a combination with sarcosine (Series 6) to lead finally to a complete cure in 10 out of 15 animals in a combination of 8 mg/kg cisplatinum and sarcosine (Series 8) and to more intensive tumor inhibition in those not cured than with cisplatinum in monotherapy in the same dose (Series 5). Sarcosine given alone in intervals of 14 days has no chemotherapeutic effect (Series 9). This would require much higher doses in shorter intervals. Apart from the improvement of the chemotherapeutic effect of cisplatinum by Sarcosine, other effects (reduction of the deposit of platinum in the tubules of the kidneys, one of the main problems in the application of cisplatinum) may be possible.

It will be seen from the foregoing that sarcosine, and its derivitives including creatinine are effective compounds.in the primary as well as auxiliary treatment of various forms of malignancies. In particular, it is observable from the foregoing that the use of sarcosine in an auxiliary manner with other known tumor inhibitors provides a synergistic, multiple effect not obvious and not obvious and not expected from the individual use of either the known inhibitor or the sarcosine alone. It will be apparent to those skilled in the art, that the tests set forth in this discription and the examples of the use and application pf the present invention can readily be applied to known malignancies since those malignancies and therapies set forth io the examples are known for an equivalent or similar behavior in humans. . 49181 Example 8 Preparation 1 To a solution of 12 g Endoxane in 500 ml ethanol a saturated aqueous solution of sarcosine, containing 28 g sarcosine, is added under vigorous stirring at room temperature. The reaction mixture is further vigorously stirred and then after 10 to 15 minutes lyophi1i zed.

The residue is then in a manner known per se pressed to tablets, made into a pill, encapsulated or formulated into an injection preparation. The unit dose contains an endoxane amount of 50 mg, which is usual for this compound, combined with 117 mg of sarcosine.

Example 9 Preparation 2 In the same manner as shown in Example 8, 18 g sarcosine, in the form of a concentrated aqueous solution, are reacted with 8 g adriamycine dissolved in 500 ml ethanol By lyophilization the addition product is isolated. A 48181 unit dose contains the adriamycine/sarcosine complex in a ratio of 50 mg adriamycine to 1125 mg sarcosine.

Example 10 Preparation 3 g sarcosine anhydride in 250 ml water are added to a 2 percent ethanolic solution of 6 g VM 26. After a few minutes of vigorous stirring and then standing the resulting solution is lyophilized and in a manner known per se, formulated into an injection preparation, containing per unit dose 30 mg of VM combined with 1450 mg of sarcosine anhydride.

Example 11 Preparation 4 g cisplatine in the form of a 1.5 percent aqueous solution, which at the same time contains 0.9 percent of NaCl, is added under vigorous stirring into an aqueous solution of 178g sarcosine in 500 ml water. After 5 to 10 minutes of further vigorous stirring the reaction product is lyophilized and in a manner known per se, formulated into an injection preparation, containing 80 mg, CPDD, combined with 1780 mg sarcosine per unit.

All percentages given are weight percent.

TABLE 9 + Series No. Preparation Single Dose mg/kg Total Dose mg/kg Cured Died 0 Tumor Weight 0 Body Weight Difference 1 Control - - - - 13,9 + 3,2 - 12,4 2 VM 26 iv. 5 15 0/15 0/15 5,8 ± 1,9 3 VM 26 iv. 10 30 0/15 0/15 3.1 i 1,6 4 VM 26 iv. 15 45 1/15 0/15 2.6 ± 1,5 - - 5 VM 26 . after 6hrs. iv. 5 ' 15 0/15 0/15 1.8 Sarcosine sc. 178 534 + 0,9 6 VM 26 after 6hrs. iv. 10 30 5/15 0/15 0.9 Sarcosine sc. 178 534 + 0,5 7 VM 26 after 6hrs. iv. 15 45 13/15 0/15 0,3 Sarcosine sc. 178 534 ' ± θ-3 8 VM 26 after -6hrs. iv. 5 15 3/15 0/15 1.0 Sarcosine AH sc. 284 852 + 0,4 9 VM 26 after 6hrs. iv. 10 30 8/15 0/15 0,5 Sarcosine AH sc. 284 852. ' ± 0,3 10 VM 26 after 6hrs. iv. 15 45 10/15 0/15 0,6 Sarcosin AH sc. 284 852 + 0,3 491&1 TABLE 9 CONT. Strain: Swiss Mouse Tumor: Ehrlich-Carcinoma Diploid Transplant - Mode: intramus cular Weight of Tumor at start of Therapy: 0 1,5 Grams Transplant - Date: 9.3.79 Start of Therapy: 12.3.79 Treatment days: 1 x a Week Duration of Treatment: 3 Weeks No. of Animals in series: 15 Mice • TABLE 10 Series No. Preparation Single Dose mg/kg Total Dose mg/kg Cured Died + 0 ~ Tumor Weight Grams 0 Body Weight Difference 1 Control - - - - 14,3 '+ 3,6 - 23.2 τ 2 Endoxan sc. 120 360 - - 4.8 + 2,3 - 4.4 % 3 CPDD iv. 4 12 - - 11,1 + 4.1 — 11,6 % 4 CPDD iv. 6 18 7,8 ± 3,4 - 9,1% 5 CPDD iv. 8 24 1/15 - 4,3 + 3-,5 - 8,8 % 6 CPDD + 4 iv. 12 6,8 + 4,5 - 3,8 7=. Sarcosine 178 534 7 CPDD + 6 iv. 18 2/15 4,5 + 3,2 - 3,2 7= Sarcosine 178 534 8 CPDD 8 24 2,4 iv. 10/15 - ± 2,1 - 4,2 7» Sarcosine 178 534 9 Sarcosine 178 534 - - 11,2 + 3,3 - 12,1 7= 48181 TABLE 10 ’ · CONT.

Strain : Swiss Tumor: Ehrlich - Carcinoma Transplant - Mode: im. Weight of Tumor at Start of Therapy·. 2,5 g Transplant - Date: 12.1.79 Start of Therapy: 16.1.79 Treatment days: 1., 3., 5. Weeks Duration of Treatment 6 Weeks End of Test 19.2.,79 No. of Animals in series: 15 Mice

Claims (13)

1. N-lower alkyl glycines with 1 to 4 carbon atoms in the N-alkyl part in the form of acid amides as well as sarcosine anhydride as sole active substance, in mixtures with each other or 1n combination 5 with known tumor inhibiting compounds for use in the treatment of tumors.

2. Sarcosine anhydride, N-methylglycineamide and N-n-propylglycine amide for use in the treatment of tumors in accordance wi$h claim 1.

3. Sarcosine in combination with known tumor inhibiting compounds for use in the treatment of tumors. 10

4. Antineoplastic alkylantiene, intercalating substances and/or antimetabolites as known tumor inhibiting compounds in combinations used in the treatment of tumors in accordance with claims 1 and 3.

5. Agents with tumor inhibiting activity and/or which synergistically support tumor inhibiting compounds for use according to claims 1 to 4, 15 comprising a content of N-lower alkyl glycines with 1 to 4 carbon atoms in the N-alkyl part in the form of acid amide as well as sarcosine anhydride alone, as mixtures or together with known tumor inhibiting compounds as active components with usual carrier- and auxiliary-substances if desired. 20

6. Agents with tumor inhibiting activity and/or which synergistically support tumor inhibiting compounds for combined use according to claims 1 to 4, comprising a content of sarcosine mixed with known tumor inhibiting compounds as active components with usual carrier- and auxiliarysubstances if desired.

7. Process for the preparation of agents according to claims 5 and 6 for use in the treatment of tumors, wherein a) at least one member of the group N-lower alkyl glycine having 1 to 4 carbon atoms in the N-alkyl part in the form of 5 acid amide, sarcosine and sarcosine anhydride is combined with b) a known anti-tumor agent, by bringing an effective amount of each of a) and b) into intimate contact with each other and isolating the product obtained thereby.

8. Process according to claim 7, wherein a) and b) are 1° combined by 2:1 to 50:1, preferably 2:1 to 10:1 parts by weight.

9. Process according to claim 7 or 8, wherein 1/10 to 1/20 of the LDg 0 of b) are combined with 1/20 to 1/100 of the LD^q of a) in a standard dose.

10. Process according to claims 7 to 9, wherein the product is 15 encapsulated or granulated, if desired with further addition of active substance a) or b) in a form with delayed release in the body.

11. Process for the preparation of agents according to claim 5 or 6, substantially as described herein with reference to the Examples.

12. Agents with tumour inhibiting activity and/or which 20 synergestically support tumor inhibiting compounds whenever prepared by a process as claimed in any of claims 7 to 11.

13. Compositions with tumor inhibiting activity and/or which synergestically support tumor inhibiting compounds substantially as described herein with reference to the Examples.