JPS61502123A - Pharmaceutical products with antitumor activity; use of pharmaceutical products or pharmaceutical compositions for antitumor therapy - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Pharmaceutical products with antitumor activity; pharmaceutical products or drugs for antitumor therapy Use of chemical compositions The present invention provides pharmaceutical products with anti-tumor activity as well as pharmaceutical products for anti-tumor therapy. and uses of the compositions and pharmaceutical compositions.

Endotoxins - essential components of the cell walls of Gram-negative bacteria - are composed primarily of lipids and polysaccharides. It is sometimes called lipopolysaccharide (LPS) or glycolipid because it is made of It has many kinds of antitumor activities, including induction of hemorrhagic necrosis and tumor regression in Ru.

However, these are unsuitable for clinical use due to their high toxicity.

On the one hand, detoxified endotoxins such as non-toxic lipids xA undergo acid hydrolysis with hydrochloric acid. Thus (Wes tpha) and 1.;Angew by deritz.

Chen+. Vol. 66, p. 407 (1954)], or 1.50 kGy"Co By irradiation with r-rays (Fj: st, etc., Infect, Imn+un+, Vol. 16, 8 (pp. 26-31, 1977), and its toxicity is considerably low, making it an excellent candidate for endotoxins. Preferably comparable synthetic non-toxic analogues (Kotant et al., Tnfect, Immun, Vol. 41, pp. 758-773, 1983; ISO et al. + Agri e, Biol, CheIll, vol. 45, pp. 1523-1.525, 1981 Year; Behling et al. IJ, Immunology Vol. 17, p. 847, 1976i Ponpipom et al., J. Med. Chem. Volume 23, 1184 ~1188 pages, 1980) were also obtained. However, unfortunately, this low toxicity The lower one is also accompanied by a considerable decrease in antitumor activity.

Rtbi et al. (Cancer Immunol, Immunother, 7 Volume, pages 43-58, 1979; Cancer Re5earch Volume 41, pp. 2654-2657, 1981; Cancer Immunol. Imm another, vol. 12, p. 91-96, 1982), the detoxified endotoxin composition When a substance is administered intralesionally (i.e., intratumorally), the detoxified endotoxin composition Trehalose simicolic acid, muramyl dipeptide or bacterial peptide fraction If you add salt and oil, you can eliminate 1ine-10 hepatocellular carcinoma induced by carcinogens. It was discovered that strong antitumor activity was observed in guinea pigs implanted subcutaneously. . Additionally, the addition of muramyl dipeptide clearly increased the mortality rate due to endotoxin. However, the combination of detoxified endotoxin and muramyl dipeptide causes death and early sleep in test animals. It was also discovered that it does not cause any harmful phenomena such as.

However, due to the need for simultaneous and intralesional administration of oil, Rib It is impossible to directly transfer the research results obtained in guinea pigs to drugs. Ru. Muramyl dipeptide (MDP, chemical name: N-acetylum) used by Ribi et al. ramyl-L-alanyl-D-isoglutamine) is specific for soluble particulate antigens. is a well-known immunoadjuvant for the formation of targeted antibodies (Chedjd et al., Pr. ogr, AllergL, Vol. 25, pp. 63-105, 1978).

Addition of mineral oil, for example incomplete Freund's adjuvant, It is known to be a good adjuvant for cell-mediated immune responses (Chedid Etc+J, Reticuloendothel, Soc+ vol. 26, 631-64 0, 1979), MDP itself is not immunogenic. In other words, MDP is a disease various non-specific mechanisms of the immune system, including resistance to drug substances and interleukin production. Can stimulate effector mechanisms (Parant, Springer Sem1n . Immunopathol.

2, pp. 101-11.8, 1979; Wood et al., Ce11. ■Il1m unol.

70@, p. 407, 1982). However, MDP alone has significant antitumor activity. does not have

In summary, the following properties of MDP are known; A, ability to form specific antibodies; Stimulation (humoral adjuvant) B, stimulation of specific cellular immunity (cell adjuvant) C 8 Stimulation of non-specific resistance interferon-induced (lymphocyte and/or single-cell macrophage (tar) E, interleukin induction (leukocyte factor) F, macrophage activation G1 fever (heat-induced), induction of temporary leukopenia (lack of circulating white blood cells) ), induction of joint inflammation (arthritis) and increased susceptibility to endotoxin toxicity. Some of these properties are related to the use of oil.

Various non-pyrogenic congeners and precursor forms of MDP have been described (Kiso et al., C. Arbohydrate Re5earch volume 90, C8-C11.

1981; Lederer+ Prog, Immunol, Volume IV, 1 pp. 194-1121, 1980; Krueger et al., J. Exp, Me. d, vol. 159, pp. 68-76, 1984; Durette et al., Carboh ydrate Re5earch Volume 108, Pages 139-147, 19.82 ;　Matsumoto etc. +　Infectionand　lmff1unit y39, pp. 1029-1040, 1983; Ogawa et al., Infecti on and Immunity, Vol. 35, pp. 612-619.

1982; 5aiki et al., Infection and Immunity Volume 39, pages 137-141, 1983; Chedid et al., Proc, N atl, Acad, Sci.

USA Vol. 73, pp. 2472-2475; Lederer, J., Med. Chew, vol. 23, p. 819, 1980; MatsuIlloto et al. Infection and Im+1unity32@, pp. 748-758, 1 981; Tsuj ia+ot.

etc.+ Microbiol, Io+ll1uno1. Volume 23, 933-93 6 pages, 1979; Durette et al., J. Med, Chew, 25 Vol., pp. 1028-1033.

1982; Lefrancier et al., J. Med, Chell, 25 Vol. 87-90, 1982; Masihi et al., Infection an d Immunity Volume 43.

pp. 233-237, 1984; Kusumoto et al., Bull, Chew , Soc.

Japan Vol. 52, pp. 1665-1671, 1979; Arnon et al., P roc, Natl, Acad, Set, USA Vol. 77, 6769-67 72 pages.

(1980). The properties of all these known congeners and precursor forms vary considerably. It shows gender. Thus, compared to MDP itself, the congener 6-0-(2- Tetradecyl-hexadecanoyl)-N-acetylmuramyl-L-alanyl-D - Isoglutamine is a strong stimulator of cellular immunity and a weak stimulator of nonspecific resistance. homologue 6-0-acetyl-N-acetylmuramyl-L-alanyl-D- Isoglutamine is active as well as a stimulator of nonspecific resistance and can be used as an adjuvant. is comparable to MDP; the congener Na-(N-acetylmuramyl-L-a Ranyl-D-isoglutamine)-N'-stearoyl lysine is more effective than MDP. It is a stimulus with strong non-specific resistance.

detoxified endotoxin (or its synthetic non-toxic or acceptably toxic congener) body) and muramyl dipeptide (or its non-pyrogenic or acceptable pyrogenic combinations of congeners or precursor drug forms) with the necessary oil and trehalogen according to Ribi et al. - without using trehalose dimycolate administration modes other than intralesional administration, in particular intravenous, intramuscular and/or subcutaneous administration forms. When used in formulas, a synergistic effect regarding antitumor activity occurs, with a substantial increase in toxicity. There's nothing wrong with that.

Accordingly, the present invention includes one or more pharmacologically acceptable vehicles, solvents, diluents and and/or an adjuvant; and (a) at least one detoxified endotoxin or or synthetically non-toxic or acceptably toxic analogues thereof; and (b) ramyl dipeptide (MDP) or its non-pyrogenic or acceptable pyrogenic form. Congeners or precursor drug forms that are toxicologically acceptable on the one hand and effective antitumor on the other hand. Concerning a pharmaceutical product having anti-tumor activity, contained in an active amount .

In one aspect of the invention, the pharmaceutical product is suitable for intravenous, intramuscular or subcutaneous administration. It has a single solution form. However, in the preferred G-like of the present invention, pharmaceutical The product is at least one detoxified endotoxin or a non-toxic synthetic product. or solutions suitable for intravenous, intramuscular or subcutaneous administration of congeners of acceptable toxicity; and MDP or its non-pyrogenic or acceptable pyrogenic congeners or precursors. having a form consisting of another solution suitable for intravenous, intramuscular or subcutaneous administration in a propellant form; . 1 by administering separately, i.e. by administering in different locations , an excess of antibodies against component (a) resulting from the immunoadjuvant activity of (b). formation as well as neutralization of the effect upon subsequent injection or possible anaphylaxis to (b) Anaphylactic reaction.

can be prevented.

The invention further provides that the pharmaceutical product according to the invention has anti-II! Use in 1TIk therapy Regarding.

More particularly, the present invention provides MDP or its non-pyrogenic or acceptable exothermic in combination with the use of pharmaceutical products consisting of sex congeners or precursor drug forms; Both are non-toxic or acceptable by detoxifying endotoxin or its synthesis. Regarding the use of pharmaceutical compositions consisting of toxic congeners in antitumor therapy, such Antitumor therapy uses both types of acids, on the one hand toxicologically acceptable and on the other hand effective. Administer drug-active amounts to different sites simultaneously or nearly simultaneously.

Both of the above-mentioned acids are suitable for intravenous, intramuscular and/or subcutaneous administration in solutions, especially aqueous solutions. Preferably, it is a liquid. However, other routes of administration are also possible, e.g. intraperitoneal injection. .

Next, the present invention will be explained based on examples.

On castration In this example, individual BAL B/c mice with the same genetic composition were tested. Endotoxin composition detoxified against subcutaneous (sc) MethA fibrosarcoma grafts The effect of intravenous (iv) infusion of an aqueous MDP solution in combination with was studied. ratio For comparison, MDP alone, detoxified endotoxin alone, and MDP The effects of liquid and saline solutions were also studied.

For the experiment, 11-week-old female BAL B/c mice were used. MethA sarcoma of LB/c origin is reported by the Clinical Research Center (Igiko ns), Risu, Mido. Obtained from Les Sexes, Harrow Ward) and maintained in ascitic form by continuous intraperitoneal subculturing. did.

MDP is the Pasteur Production Institute (Institut Pa5teurProdu). ction) (Marnes-Souler Conquette, France). colon Surface 0111: B4 endotoxin (LPSw) (Difco Laboratory es, Detroit, Michigan, USA) is a phenol/water extraction method (Westpha 1st Prize+Angew, Chew, Volume 66, Pages 407-417, 1954 and Z., Naturforsch, Vol. 76, pp. 148-155, 1952). Manufactured by

Heptoseless (Re) mutant Salmonella t yphimurium) (Ribi IIIlmuno Chew, Inc. Purified toxic or detoxified endotoxins from Rib i et al. Cancer Immunother, Volume 12, pp. 91-96, 19 Manufactured and refined as described in 1982. In short, phenol/chloroform Extract endotoxic glycolipids from cell walls with petroleum/petroleum ether and prepare finely divided gel. fractionation by chromatography, and further fractionation by Sephadex LH-20. and purified. In this way, it is free of phospholipids and nucleic acids and contains less than 0.3% protein. A content of non-aggregated endotoxic glycolipids was obtained. Detoxification of purified endotoxin is 0. I It was carried out by hydrolysis in N HCl at 100°C. The resulting composition is It is 1/1000 times less toxic as measured by the mortality rate of chicken embryos, and in fact K Purified endotoxin phosphate without DO (keto-3-deoxyoctonate) It had a phosphate content of about 1/2 of the content.

MDP was dissolved in pyrogen-free saline. Endotoxin is 0.5% triethyla Endotoxin 2×10.4n+J), diluted with saline *0-5ml The entire amount was injected.

3 x 10' viable MethA cells were subcutaneously injected into the abdomen of mice, and 9 days later, Tumors were treated when they had a diameter of approximately 7.5 mm.

On day 11, necrosis was measured and expressed as 100 times the ratio of the necrotic area to the average diameter of the tumor. did. The incidence of necrosis stained brightly and darkly is shown separately. Tumor size within 2 days after injection If the size did not increase, growth inhibition was recorded. Tumor within 19 days of treatment Complete healing was recorded when the tumor disappeared completely. Data are mean above SEM (= standard error of the mean).

The results are summarized in Tables A and B below.

Table-A *Flat turtle direct±SEM Table - Kazushi *10 mice cured of toxic and detoxified endotoxin of Salmonella Typhimurium R,e Five of them died within 24 hours.

In addition, intravenous toxic and detoxified Salmonella Typhimurium Re endotoxin alone or in species. Toxicity was measured when mixed with various amounts of MDP. The results are shown in the attached figure. 5 Mau Several groups of students were given laboratory food and water ad libitum at a constant temperature (18°C) and They were reared in a humid atmosphere. 24 hours (no spot) and 48 hours (black spot) after injection Changes in measured body weight were measured immediately before injection and expressed as mean ± SEM (vertical line ) expressed in proportion to individual body weight. Numbers in parentheses indicate death after 24 and 48 hours. Shows the total number of mice killed. Diarrhea and nausea were assessed 5 hours later. These are the ratings The number of mice with symptoms is shown. The expression nd means not measured.

In the experiment whose results are shown in Table A, BAL B with MethA tumors for 9 days /c Injection of 25 μg of E. coli endotoxin in 100% of cases induced obvious tumor necrosis, but inhibited growth of most tumors and inhibited tumor growth in 30% of mice. It was found that complete healing occurred. The lower the dose (the lower the dose, the better in all aspects) Activity decreased. Although MDP alone did not induce Ill tumor necrosis at various doses, This resulted in some regression. Add 3 to 100 μg of MDP to 10 μg of E. coli endotoxin This results in a significant enhancement of all measured parameters of endotoxin antitumor activity. Became. No clear relationship between dose and effect was observed, with the exception of the degree of necrosis. There wasn't. MD, Even if the P dose is kept constant at 30 μg and the endotoxin dose is reduced. , no obvious decrease in antitumor activity occurred.

In the experiments whose results are shown in Table B and Figures, detoxified Salmonella typhimurium Re at various doses was tested. Endotoxin causes minimal necrosis but has little effect on tumor growth. found.

Although the toxic endotoxin showed considerable activity even at doses as low as 10 μg, MD Addition of P was often lethal.

However, the combination of detoxified endotoxin composition and MDP has very strong antitumor activity. None of the combinations studied produced death or diarrhea and increased MDP doses. Approximate clothing was observed only when Weight loss was reduced by increasing the dose of both components. However, the loss was less than 10%. Body after 48 hours in all cases The weight was higher than after 24 hours (which seems to indicate recovery. As shown in the figure , any dose of toxic endotoxin, with or without MDP. However, MDP surprisingly prevented diarrhea to a certain extent. There was found. Weight loss is substantial, sometimes greater than 15% (after 48 hours) The weight loss was greater after 24 hours.

Increasing the MDP dose reduces weight loss, indicating that tissue loss as a result of shock This is thought to suggest that fluid accumulation occurs within the pores. Death was observed at high doses . LD of toxic endotoxin alone.

is higher than 160 μg per mouse, but MDP 30 pg and 24.6. co g (by the Spearman-Karber method modified by Nowotny) Calculated, Ba5ic Exercises in Immunochemis try pages 303-304, 1979, Berlin, Slinger Verlag) .

Example■ In this experiment, the time and route of administration for endotoxin and MDP were studied in an animal experiment. The same types of mice and tumors as in Example I were used in the experiment.

In addition to E. coli 0111:B4, E. coli 089 endotoxin (LPSw) and the same endotoxin A radiation-detoxified preparation (150 kcy, &OCO γ-ray irradiation) was used. Both of these rFr6deric Joliot-Curie J National Institute of Radiobiology and Radiological Science Obtained from Dr. Bertok, professor at the Biomedical Research Institute (Budapest, Hungary) This is what I did. This detoxification method uses Fus, Infect, Ia+m, etc. Un, vol. 16, p. 26-31, 1977; detoxified preparations It is commercially available under the trade name "Tolerin".Female CF Y-random LD of both these formulations in the kit. The values are 2■/- and 21■/kg, respectively. Unless otherwise indicated, these preparations are prepared in pyrogen-free saline solution. 0.5 mj! Injected intravenously as

On day 0 of the experiment, 3 x 10' viable MethA cells were injected subcutaneously into the abdomen of the mouse. Ta. Unless otherwise indicated, treatment begins on day 9 (when the tumor has a diameter of approximately 7.5 when) started. The degree of necrosis was measured by light and dark staining on the 111th day, and the necrosis area and Expressed as 100 times the average tumor diameter ratio. The diameter is measured with a caliper. Ta. Growth inhibition was recorded if the tumor did not grow within 2 days of treatment.

Complete cure was recorded if the tumor completely disappeared within 19 days of treatment. De Data are expressed as mean above SEM. Analysis of significance was Student's t test. It was carried out by P values greater than or equal to 0.05 were considered not significant.

The results are summarized in Tables C-F below.

Table--q a, Endotoxin B. coli 011 in BALB/c mice bearing MethA grafts for 9 days. 1:LPSw from B4)3 u gb8 Light and dark necrosis was measured on day 111. necrosis Degrees are expressed as Nao Hirahori±SEM.

c, p<0.05. compared to all other treatments in experiment l.

d, p<0.05. compared to treatment with endotoxin alone.

The tumor size did not increase within 2 days after e3 injection.

f. Tumor completely disappeared within 19 days of treatment -1 Antitumor activity of MDP and endotoxin B. coli 0111:B4) co-injected via various routes. Ten BALB/C tumors with cutaneous ``FMethA'' tumors in their abdomens were treated for 1.9 days. Groups of mice were given 3 μg of endotoxin and 30 μg of MDP subcutaneously on the soles of their feet. and intravenously or by other routes.

2. Necrosis was measured on the 111th day. The degree of necrosis is expressed as mean ± SEM.

5, p<0.05 compared to corresponding treatment but without MDP.

Once in From 5 BAL B/c mice with MethA grafts for 1.9 days 3 μg of endotoxin B. coli 0111:B4 (LPSW) and MD 30 μg of P or saline was injected subcutaneously into the left and right soles, respectively.

2. Measured on day 111, necrosis degree is expressed as mean value ± SEM.

3. Tumor size did not increase within 2 days after injection.

4. The tumor completely disappeared within 19 days of treatment.

Table F Radiation detoxified endotoxin (Tolerin) and E. coli 0 normal endotoxin 1 ．． BALB/c mice bearing MethA grafts were treated with endotoxin formulation alone for 9 days. Or, it was mixed with 30 μg of MDP and injected intravenously as a 0.51 saline solution.

2. Recorded on day 11, necrosis is expressed as mean above SEM.

3. Tumor size did not increase within 2 days after tL administration.

4.1 Tumor completely disappeared within 19 days of Tsukuda.

5, p<0.05. compared to treatment without MDp.

The experiment involved suboptimal E. coli 0111: 843 μg for MethA-containing mice. Simple intravenous injection of an appropriate amount clearly causes a high incidence of light-colored necrosis! wake up, then press 6 on the mouse resulting in tumor growth inhibition in 0% and complete tumor disappearance in 20% of mice. and showed.

MDP alone caused only incidental necrosis and regression.

Administering a mixture of the two produces a significantly wider range of dark colors than treatment with either component alone. It resulted in a 100% incidence of necrosis and a high incidence of complete regression (60%). Endotoxicity Administration of MDP 1 day or 2 days before or 1 day after plain administration increases the antitumor activity of endotoxin. was not strengthened. However, administration of MDP and endotoxin within 4 hours of each other administration It produced the same effect as simultaneous administration.

Simultaneous administration of endotoxin and MDP by intravenous and subcutaneous routes, respectively, may result in combined intravenous administration. It produced an antitumor activity comparable to that obtained by administration. By subcutaneous route The endotoxin injected as a whole showed antitumor activity when administered intravenously at the same time as MDP. I didn't. However, when combined treatment is performed using the opposite route, similar severe necrosis can occur. and tumor growth inhibition, but associated regression was infrequent. left Simultaneous injection of endotoxin and MDP via the subcutaneous route into each of the right and left soles of the foot can cause necrosis and high rates of necrosis. endotoxin alone was not effective in this case either. It was.

Table F shows that a 25 μg dose of E. coli 089 endotoxin induced significant necrosis and 20% regression. It shows what you did. Tolerin" had lower activity at the same dose. Although the quality has low activity at low doses (10 μg), MD The addition of P resulted in a higher The antitumor activity was enhanced to a low value.

Example■ This example shows differences in pyrogenicity, adjuvant action and non-specific resistance stimulation ability. We further investigated the potentiating effect of MDP using several MDP congeners known to to be analyzed. Two other synthetic formulations with adjuvant action in aqueous vehicles (Snippe et al., Int, Arch, Allergy Appl, Im n+unol+volume 65, page 390.

(1981), i.e., it is a good adjuvant for inducing antibody production. , Pluronic Polyol L121 and an adjuvant that promotes the induction of cell-mediated immunity. dimethyldioctadecylammonium bromide (DDA), which is a Their activity was compared with MDP and MDP congeners.

As in Example 1, female BAL B/c mice approximately 11 weeks old were used. The immunogenic MethA fibrosarcoma, which has the same genetic composition, was developed at the Clinical Research Center. (Harrow, Middlesex, UK) and by serial IP subculture. The ascites was maintained.

Endotoxin (LPSW from E. coli 0111:B4) was purchased from Dirco Laboratories (Mission, USA). dimethyldioctadecyl ammonium Romid (DDA) is from Eastw+anKodak (USA, New York State) , Rochester); Pluo27li/Polyol L121 is BAS F Wyandotte Co.

(Wyandoft, Michigan, USA), MDP is Pa5teur. Is it BioM Research Institute (France, Marnes-La-Coquesote; name: MDP(F))? N-acetylmuramyl-L-alanyl-isoglutamine (MDP (LL)) is from Calbiochem-Behring (California, USA). Purchased from U.Jola, Runia. MDP called MDP (J) and , N"-(N-acetylmuramyl-alanyl-D-isoglutaminyl)u6- Styaroyl-L-lysine (MDP-Lys (L18)) is Yokoy Kindly provided by Dr. Ama and Dr. A. Inoue (Daiichi Pharmaceutical Co., Ltd. 2, Tokyo, Japan). 6-0-(2-tetradecylhexadecanoyl-N-acetyl muramyl-shi-alanyl-D-physoglutamine (B 30-MD P> and 6- 〇-acetyl-N-acetylmuramyl-shi-alanyl-D-isoglutamine (L 2-MDP) is Professor S. Kotani and Professor T. 5hiba (Faculty of Dentistry, Osaka University). MDP-Lys (LlB ) and L30-MDP, all drugs were dissolved in pyrogen-free saline. Ta. MDP-Lys (LlB) and 83'O-MDP are usually used for clinical nonionic activity. 5% (W/V) N1kkolHCO-60 (Nikoh Che micals, Tokyo, Japan) and 0.03M phosphorus containing 4% (w/v) glucose. Usually dissolved in acid salt buffer. If necessary, add N1kkol solution or saline. I diluted it further. In one experiment, Mullard 5onifier 76 After sonication for 5 seconds at maximum power by Type 85/2, MDP-Ly s(L18) was administered as a saline solution. Do the same for DDA and L121 stock solution of endotoxin and MDP in saline (Stock 5olut). ion) was stored in small aliquots at -20°C. Do not give instructions to others Finally, a solution of MDP congeners DDA and L121 was prepared immediately before use.

3 x 10' viable MethA cells were subcutaneously injected into the abdomen of mice, and 9 days later ( Tumor diameter ± 7.5 fl), one or more formulations in vehicle/saline solution 0.5 ca Mice were treated by intravenous injection as l.

The degree of necrosis stained with IJ sun and moon light and dark was measured with a caliper, and the necrosis area and Expressed as 100 times the ratio of the mean tumor diameter. A temporary regression period may occur depending on the size of the tumor. Depending on the number of times the tumor size is smaller than or the same as the tumor size in the treatment molar. It was expressed as follows.

Complete regression was recorded when the tumor completely disappeared within 19 days of treatment.

Data are expressed as mean ± SEM, and significance was analyzed using Student's t test. It was carried out by P values greater than or equal to 0.05 were considered not significant.

The results are summarized in Table G-K below.

Table G a, BALB/c mice bearing MethA sarcoma grafts were injected with 3 μg of endotoxin for 9 days. Germany or MDP homolog 60nIIIol (MDP60nmol#30μg) Combined with this, the total amount is 0.5mj! It was administered intravenously as a saline solution. However, MDP - Lys (L 18) and B2O-MDP are 3% N1kkol solution in saline solution 0.5 Injected as of.

b, Necrosis stained brightly and darkly was measured every 11 days. Necrosis degree is the mean value ± SEM Describe it.

C, p<0.05 compared to treatment with endotoxin alone.

d. Complete regression was recorded if the tumor disappeared within 19 days of treatment. After treatment tumor size is smaller than or equal to the tumor size on the day of treatment for more than 1 day 8. If the condition was not met, it was considered to be a temporary regression. Duration is expressed as mean days±SEM.

-1 Enhancement of endotoxin-induced tumor necrosis and regression by various doses of MDP and various congeners. a, 3 μg of endotoxin was administered to BALB/c mice bearing MethA sarcoma grafts for 9 days. 0.5-total saline alone or in combination with graded doses of MDP congeners as a solution or as a 0.1χN1kkol solution in saline (M D P -L ys (Li8) and B2O-MDP) were administered intravenously. Solutions of all MDP congeners are It was prepared the day before use and stored at -20°C.

b, c, d, see footnotes to Table G.

Table ■ B2O-MDP and Mop- as N1kkol or saline solutions at various concentrations tysa, endotoxic to BALB/c mice bearing Meth A sarcoma grafts for 9 days 830-MDP or MDP-Lys (Ll In combination with B), the total amount is 0.5 + n j! Finally 3 or 0.1X (7) It was administered intravenously as a saline solution containing Nikkol. MDP-Lys (LlB) is It was also administered as a saline solution after brief sonication.

b, c, d1100 See footnotes.

table view Effect of MDP (LL) on endotoxin-induced tumor necrosis induction and regression a, 9 days BALB/c mice bearing MetbA sarcoma grafts received endotoxin 3 μg alone or M Mixed with DP congener, total amount 0.5n+j! It was administered intravenously as a saline solution.

b, c, d, see footnotes to Table G.

Table K Effects of DDA and L121 on endotoxin-induced tumor necrosis induction and regression a. 9 days F 3 μg endotoxin alone in BALB/c mice bearing IethA sarcoma grafts; ! L121. Mix with DDA or MDP as a 0.511z saline solution I gave it an intravenous injection.

b, c, d, see footnotes to Table G.

result MDP and congeners result in endotoxicity in tumor-bearing mice against endotoxin-induced necrosis and regression Administration of 3 μg intravenously resulted in moderate necrosis and 60% and 2% of the tumor, respectively. 0% temporary and complete regression occurred (Table G).

All MDP congeners induced only occasional necrosis and regression. endotoxin Addition of either MDP congener to the Necrosis occurred. Their efficacy in enhancing the incidence of subsequent regression varied considerably. Inside 830-MDP in 3% N1kkol when compared to treatment with toxin alone Particularly potent, MDP and L2-MDP are mildly active, 3% N1kk MDP-Lys (L18) in OL showed no effect at all. between congeners Different amounts of the formulation were compared with respect to their enhancing activity in order to be able to differentiate between Table H). For all formulations, decreasing the dose decreased the enhancement of tumor damage. 0. MDP, L2-MDP and MDP-Lys (LlB) in 1% N1kkol are equal However, L30- frozen in 0.1% N1kkol MDP had only minimal activity. MDP in experiments 1 and 2 The difference in activity between Lys(L18) and L30-MDP is due to Nlkkolti degree and/or or due to changes in storage conditions, N1kkol solutions with various concentrations The effects of these freshly prepared congeners were studied (Table 1). 3%N1kk The effect of 60nmoAB-30MDP in OL was similar to that observed in experiment 1. (Table G), which was observed in Experiment 2 when the N1kkol concentration was reduced to 0.1%. (Table H). 3%N1kkol or 0.1%N 'B30-MDP 6 nmoj as any solution of 1-kkol! for volume moderately enhanced necrosis but maximally enhanced regression. 0.1%N1kkol The new formulation of MDP-Lys (L18) as a solution or saline solution is highly had similar activity (Table 1).

The effect of MDP (LL) on endotoxin-induced tumor damage is shown in Table J. MDP (L-L) has an enhancing effect on necrosis and counteracts the induction of degeneration by endotoxin. It even showed a tendency to

Effects of DDA and L121 on endotoxin-induced necrosis and regression In other experiments, endotoxin-induced necrosis and regression MD of synthetic adjuvants DDA and L121 in enhancing toxin-induced tumor damage We investigated whether it could be a substitute for P (Table K). Both adjuvants are It caused little tumor damage and little enhancement of endotoxin-induced tumor necrosis. but , endotoxin-induced tumor regression was enhanced by addition of L121. L121 and M Combined administration of DP resulted in moderate necrosis and occasional regression. L12] and MDP The combination of endotoxins resulted in extensive necrosis and complete death in all treated mice. Complete healing occurred. However, this treatment method is difficult to determine based on the animal's deep presbyopia and hypothermia. It was extremely harmful.

Consideration MDP homologs L2-MDP and MDP-Lys (L1B) are MDP It appeared to be a good enhancer of endotoxin-induced tumor necrosis as well.

On a molar basis, 830-MDP as a 3% N1kkol solution is low in this respect. showed activity (Table G-1). On the other hand, L30-MDP has a suitable concentration of N1kk MDP and L2-MDP have roughly equal activity if applied as an ol solution. MDP-Lys (L18) is a better regression enhancer than It had low efficacy.

MDP (LL) had no potentiating activity (Table J). These observations are Somewhat distinct properties of MDP have been shown to be associated with endotoxin-induced necrosis and regression. , consistent with our findings that induction of tumor necrosis and regression are separate events.

Regarding whether each phenomenon of tumor damage is immune independent or immune dependent, The nonspecific nature of MDP and its congeners is associated with enhanced necrosis and the loss of specific immunostimulatory properties. I would like to suggest that this is related to line reinforcement. Necrosis appears very quickly Therefore, there is no possibility that a specific immune stimulation mechanism is involved in the enhancement of necrosis. child This means that non-MDP adjuvants DDA and L12] have almost no enhancing activity. This is supported by the observation that there is no Stimulation of nonspecific bacterial resistance leads to tumor necrosis This appears to be a general property of the most effective enhancers. However, B30-MDP Although it cannot fully stimulate nonspecific resistance, it is thought that it can still enhance tumor necrosis. Things are 1. This suggests that these parameters are not clearly correlated.

The macrophage activation properties of B30-MDP and DDA are unequal in their ability to enhance necrosis. 1. There is no relationship between the ability to activate macrophages and the ability to enhance tumor necrosis. It seems so.

Regarding endotoxin-induced tumor regression, the best enhancer, B30-MDP, is present in aqueous solution. All tested MDP congeners stimulate the induction of both antibody formation and delayed hypersensitization. appears to be the most potent immune adjuvant. specific cell-mediated immunity Whether the stimulating ability is related to the ability to enhance endotoxin-induced tumor regression is questionable for the following reasons. I doubt it. : fl) D D A did not stimulate regression (Table K), but DD The pattern of immunoadjuvant action of A is similar to that of B30-MDP. The incidence and extent of necrosis induced by DDA were 6 nmol B30-MD. (Tables 1 and K); (2) various MDP and various congeners as a solution do not stimulate a delay in tumor regression, but (3) The induction of delayed exaggeration by MDP in oil is usually caused by antigens. It is questionable whether 30-MDP stimulates cell-mediated immunity as it requires mixing with It is.

On the other hand, the adjuvant nature of antibody formation is mechanistically linked to enhanced tumor regression. You can't ignore that. MDP and congeners as aqueous solutions are separate from antigens. When administered by this route, specific antibody formation can also be stimulated. moreover , another unrelated pure humoral adjuvant, L121, when administered in combination with endotoxin. induced a fairly long-lasting tumor growth inhibition in the (Table K); some of this latter may be due to L121 enhancing necrosis. Although this is not possible, this is thought to be due to the fact that it can promote the next regression. However, endotoxic It is not clear that enhanced antibody formation is involved in the enhanced tumor regression induced by the tumor. It seems like that. The reason for this is that the T-cell immune system is completely degenerated and the T-cells alone or Existence of concomitant immunity thought to be mediated by clophage-associated T cells This is because it turned out that this is necessary. However, specific anti-MethA antibodies The role cannot be ignored considering the data below.

Histologically confirmed delayed skin reactivity to MethA is due to viable Met This indicates that the antibody is transmitted by the serum of mice immunized with hAill cells. It has been suggested that this is mediated by Furthermore, anti-Meth antibodies In vitro and in vivo, Me It was found that the growth of thA was inhibited. For these reasons, MethA-containing materials Does administering MDP together with endotoxin to mice increase the amount of specific antibodies? , and if so, does this play a role in increasing the incidence of regression? It seemed useful to test whether

B30-MDP and MDP-Lys (L18 ) seems to depend on the addition of the optimal amount of the nonionic surfactant N1kkol. It seemed to me. This is believed to have implications for the biological efficacy of such agents. It will be done. In this regard, the effectiveness of conjugating lipophilic MDP congeners to liposomes The research on the results is noteworthy. Fiddler et al. (Proc, Natl, Acad, S ci, US 78, p. 1680, 1981), these agents are C57BL. Be effective in non-specific eradication of B16 melanoma spontaneous metastasis in /6 mice It shows.

In conclusion, MDP and its various congeners increase endotoxin-induced tumor necrosis and regression. and that these effects are in addition to other known agents of such agents. It has been discovered that biological effects cannot yet be attributed.

Giving m Older MethA tumors are generally insensitive to induction of tumor regression by high doses of endotoxin (Berendt et al., J. Exp. Med, vol. 148, 1550 and 1 560 pages, 1978).

This is due to the induction of T suppressor lymphocytes by large immune tumors (N orth, Adv, 1m+auno1. Volume 35, pages 89-155.

(1984) showed that administration of 25 μg of the toxic endotoxin resulted in complete tumor regression for 15 days. It has been confirmed that rows are not induced (B1oks+++a, etc.).

Eurj, Cancer C11n, Onco1.20, pages 397-403, 1984.

Table L), but necrosis is significantly induced. The data from the table shows that low doses of toxic Not only did endotoxin and MDP induce significant necrosis, but they also inhibited MethA tumors for 15 days. It has also been shown to induce complete regression of tumors. Tumor-containing animals for 13 days Pretreatment with high dose of cyclophosphamide (3■/mouse = 150■/ksr) and then treated with a combination of toxic or detoxified endotoxin and MDP for 15 days. A high incidence of complete regression occurred.・ Cyclophosphamide itself caused only growth retardation. low dose cyclophospha Mido (0.9■/mouse) had no effect on tumor growth and was associated with toxic endotoxin and MDP. did not enhance the antitumor effect of the drug. Highly selective elimination of T suppressor lymphocytes Low doses of cyclophosphamide (0.3 μ/mouse) induce toxic endotoxin and MDP It also had no enhancing effect on the activity of (data not shown).

person - once 15 days of treatment with toxic or detoxified endotoxin for Meth A III ulcers. Effect of cyclophosphamide pretreatment on the antitumor effect of combination with MDP1 in the abdomen Cyclophoresis was performed on BAL, B/c mice bearing Meth A tumors for 3 days. Sfamide (C) or saline was administered intravenously. Day 155: Escherichia coli 0111:B4 The toxic endotoxin from Salmonella Typhimurium Re (Tax) or the detoxified endotoxin from Salmonella Typhimurium Re (Tax). Detox) was combined with 30 μg of MDP and administered intravenously as a saline solution.

a. (ratio of necrotic area to tumor area) x 100, (SEM above mean value) b, complete cure No increase in tumor size after treatment in mice showing no healing (SEM on average number of days) C1 Growth rate of these tumors was significantly delayed compared to saline-treated controls.

Last 111! The data from the following tables M and N indicate that the combination of MDP and endotoxin is present in two lymphoid tumors. showed that endotoxin alone was more effective than endotoxin alone.

In '7 mice cured from MOPC315 tumor, lymph glands and axillary lymph glands were mainly affected. Metastases were found in the lymph glands after a short time. At this time, it is still completely localized within the skin. Surgical removal of MOPC315 tumors for 13 days resulted in metastases at least as frequently These metastases were probably not induced by treatment. Seem.

Table M 8 days after subcutaneous implantation of SL2 tumors into DBA/2 mice with the same genetic composition. Effect of toxin/MDP SL2 cells (T cell lymphosarcoma) IXIO that can grow in the abdomen of DBA/2 mice ' was injected subcutaneously. On day 8 (tumor diameter ±7-8.5 fl), E. coli 0111: Endotoxin (3 μg) and/or MDP (30 μg) from B4 in saline solution. I gave it an intravenous injection.

a, (ratio of necrosis surface to tumor area) x lOO5 (mean ± SEM) b, complete cure No increase in tumor size after treatment in non-healing mice (mean days ± SEM) The intensity of C1 necrosis is weak.

Suichi N 12-day MOPC315 plasma in genetically identical BALB/C mice Effect of combination on cell tumor B A L B / c Can grow in the abdomen of mice MOPC315 cells 1×1 OS were injected subcutaneously. Day 122 Tumor diameter ±12. 5m) with toxic endotoxin (Tox) from E. coli 0111:B4 or murine typhoid. Detoxified endotoxin (Detox) from the fungus Re is mixed with MDP3 (1 μg) and It was administered intravenously as a saline solution.

a. These treatments are lethal to all mice.

(Average number of days ± SEM) d, These mice showed distant metastases after primary tumor healing.

C3 One of these mice showed distant metastases after primary tumor healing.

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Claims (6)

[Claims] 1. One or more pharmacologically acceptable vehicles, solvents, diluents and/or agents a juvant and (a) at least one detoxified endotoxin or synthesis thereof; and (b) muramyl dipeptide (M DP), or a non-pyrogenic or acceptable pyrogenic homolog or precursor thereof. form, on the one hand, to be toxicologically acceptable and, on the other hand, to have effective antitumor activity. 1. A pharmaceutical product having antitumor activity containing in an amount of 2. Claim 1 as a single solution for intravenous, intramuscular or subcutaneous administration pharmaceutical products. 3. at least one detoxified endotoxin or its synthetic non-toxic or acceptable solutions for intravenous, intramuscular or subcutaneous administration of toxic congeners, and MDP or The non-pyrogenic version of this drug is an acceptable pyrogenic congener or precursor form intravenously, intramuscularly. A pharmaceutical preparation according to claim 1, comprising a separate solution for intra- or subcutaneous administration. product. 4. Antitumor therapy with a pharmaceutical product according to any of claims 1 to 3. Use for. 5. MDP or a non-pyrogenic or acceptable pyrogenic congener or precursor thereof at least one detoxifying compound in combination with the use of a pharmaceutical composition consisting of a drug form. Drugs consisting of endotoxins or synthetic non-toxic or acceptably toxic congeners thereof In the use of chemical compositions for anti-tumor therapy, both compositions are considered toxicologically acceptable. can be applied simultaneously to different sites in such amounts as to have effective anti-tumor activity on the other hand. or actual simultaneous administration. 6. The two compositions are solutions for intravenous, intramuscular and/or subcutaneous administration. Uses as described in Scope No. 5.