JPH03505744A - Cytochalasin compositions and treatments - 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] Cytochalasin compositions and treatment methods field of invention The present invention provides natural and synthetic cytochalasin compositions and and therapeutic methods utilizing these compositions.

More specifically, the present invention provides certain mature analogues of cytochalasin B (CB). Concerning also cytochalasins, such as cytochalasin B and, inter alia, Other natural cytochalasins such as cytochalasin D, E, H or J and one or more synthetic analogues thereof. be. This invention provides that the administration of cytochalasin containing CB is controlled by the dosage or route of administration. thus creating a transient immunosuppression that can be controlled and reversible either spontaneously or with the use of IL-2. It is also about the amazing discovery of raw zesty shinuru. In this way, the site color Shin no cure! The regimen is recommended for transplant patients and patients with autoimmune diseases. Can be used to treat immunity.

Additionally, CB and other cytochalasins and optionally anti-tumor agents other than cytochalasins. Antitumor treatments that utilize tumor agents include the administration of these substances as cytochalasins or cytochalasins. An amount effective to reverse the immunosuppression that occurs during administration of tocharacin and other antineoplastic agents. significantly enhanced by combining with IL-2 or other lymphokines can be done.

The present invention provides high 28% at the active site of the tumor without causing undesirable immunosuppression. degree of cytochalasin and Fll! Additional resistance! ! An effective link for delivering cancer drugs. It also relates to sustained release formulations that utilize posomes or microcapsules.

Background of the invention Cytochalasia, a compound with membrane and transport effects (also has cytoskeletal effects) Examples include 7,20-di-acetyl-cytochalasi, a semi-synthetic derivative. B, 7-motu〇-acetyllucytochalasin B, 21.22-dihydro - Cytochalasin B and 21.22-dihydro-cytochalasin A, and related In addition to the chaetoglobosin family, the same genus cytocara There are 24 structural and functional compounds produced by molds, including SynA-M. constitute a class of related alkaloid metabolites (Yahara et al., 198 2, J. Ca1l Biol, 92: 69-78 and Rampal et al. , 1980. Biochem, 19:679-683), these The material is suitable for use with many different types of normal and neoplastic cells and tissues in culture. Altering diverse cellular functions (Miranda et al., 1974. J, Ce1l Biol, 61:481-500), and normal and neoplastic cell types. It is possible to show a differential effect between them. (For reference, see Lipski et al., 1987, Anal, Bioc hem, 161: 332-340) are known. of these substances The various homologues are fundamental cells regulated by cytoskeletal and prototypical MIl interactions. Change the biochemistry of the process. Some cytochalasins, e.g. illustrated below Cytochalasin B (CB), which is the most widely studied of its homologues, is and suppresses the transport of hexoses and amino acids in neoplastic cells (respectively, K 15tzien et al., 1973. J, Biol, Che++, 248: 711-719 and Greene et al., 19)6.　Exp, Ca1l　 Res, 103: 109-117).

Cytochalasins are also known to change the morphology of microfibrils (Schli wa, 1982.3. Ce1l Biol, 92+79-91), This affects cellular functions that depend on the biochemistry of the fibrils. Site mustard Some cellular processes that are sensitive to cell-cell organelle movements include cell-to-cell organelle movement and cellular processes. transport, including intervesicular transport, cell motility, transport across tissue barriers, and various immune responses. In addition to functions that require movement and/or cell-cell adhesion, phagocytosis, pinocytosis, cytoplasmic There is division, secretion and exocytosis. The growth of il [cytochalasins] Only recently has it been discovered that it has considerable activity against cancer and metastasis. This is what has happened. Effect of cytochalasins on transformed (new) cells Previous studies of in vitro efficacy have shown some efficacy. For example, O 'Ne1ll (1975, Cancar Res, 35+3111- 3115), Medina et al. (1980, Cancer Res, 40329 -333), Kelly et al. (1973, Nature New Biol,... 242:217-219) and Defendi et al. (1972, Natur@N ew Biol, 242: 24-26> is the nuclear content that becomes multinucleated in neoplastic cells. It showed differentiating effects between normal and neoplastic cells, including clefting and cytokinesis. deer The early research on in-vivo chemotherapeutic methods was carried out by Il! Antitumor activity against intracavitary ascites tumor gender was not disclosed (Katagiri et al., 1971. J, An tibiotics, 24:722-723 and Minato et al., 19 73. Chew, Pharm, Bull, 21: 2268-2277).

In fact, when B16F10 cells treated with CB in vitro are administered intravenously, the lung It was found that extraneous metastasis increased () Tart et al., 1980, J. Nat. 1, Cancer In5t, 64: 891-900).

demonstrated by cytochalasins and related substances to cells and tissues in vitro. Despite their dramatic effects, these compounds, especially CB, are not available in sufficient quantities. Until recently, very little research had been done on these substances in vivo. It wasn't.

Recent advances in methodologies for producing cytochalasins (PCT issue 1Us881) 02095), it has become possible to obtain cytochalasins in larger quantities, and In vivo administration and in vivo studies are becoming more routine.

Some of these studies have shown that cytochalasins, particularly CB, inhibit tumor growth. and suppress metastasis, prolong F1 tumor latency, and mouse cancer and mouse melanoma models. (Bouaquat et al., published by Bouaquat et al. (in progress), these results demonstrate that zuitochalasins have a significant impact on the cytoskeletal and cytoplasmic structures. Known anti-! due to its effect on membrane function! ! Acts as a chemotherapeutic amplifier of tumor drug activity This brings us to the suggestion that it might be possible. Recent research has shown that cytochalasins affect leukocyte-mediated functions in vitro, cytolytic T lymphocyte differentiation, and Cell motility, a factor that may influence host immune responses in vivo, Vessel attachment, phagocytosis and secretion (Vessels et al., 5 cisnce, 171.13 5.1971). In other studies, i.p. Pharmacokinetics of CB in various tissues (including cap 1 and liver) of CB-injected mice research (phar++ acokinetics) (Lipski et al. , 19137. Analytical Biochemistry 161. 332-340) # L/Kashi, the transient immunosuppressive effect of CB in vivo was investigated and has never been studied in detail.

In recent years, H3 treatment for degenerative diseases has been introduced. or otherwise save the patient's life, organ or xeno-tissue transplantation is common. It has come to be used in However, introducing foreign tissue or organs into a patient It is far from a risk-free method to enter any tissue or organ into the brain. It is often rejected by the immune system of the transplanted or transplanted organ. The implanted ill tissue is detected as a foreign object. Currently used immunosuppressants, examples Although cyclosporine is effective in reducing the chance of patient rejection, often at the cost of reducing the patient's overall ability to avoid In most cases, the mortality rate due to transplant rejection is not as high as the mortality rate due to infection. Transplanted ninjas often die, usually from infection, otherwise from non-pathogenic diseases. Furthermore, the morbidity of leukemia or malignancy associated with currently used immunosuppressants is often high.

Some of these currently used agents are used to treat autoimmune diseases. It is also used on a limited basis. This treatment involves administering immunosuppressants to organ or tissue transplants. subject to the same limitations that arise when treating plant rejection. Therefore, infection of the patient's system Ability to repel organ and tissue transplants without having this effect; is effective in regulating hyperimmune responses (hyperimmunity) in patients with autoimmune diseases. Research continues into more effective immunosuppressants. Therefore, the present invention Cytochalasins, especially CB, produce significant, transient, and easily reversible immunosuppression. It's also about a surprising discovery that occurs inside the body.

Cytochalasins, especially CB, can be used alone or in combination with known antitumor agents for tumor treatment. You can choose whether to combine them and make them into medicines. However, CB does not include dosage, etc. Similar to the metabolites of Changes to Past studies have shown that CA is highly reactive with thiol groups and this fact suggests that suggest that this may explain the high toxicity associated with use. . Chemical systems thought to be highly reactive with thiols in enzymes and other proteins is a highly reactive enedione (enedi).

ns) 20-keto group of CB to 20-keto group in CA to form 'ih Metabolic oxidation of the droxyl group is the main cause of the high toxicity associated with administration of CA. It is believed that there may be. Therefore, the pharmacological effects of cytochalasins can be utilized, and the general toxicity associated with the metabolism of CA to enedione systems. An agent or group of agents that reduces tumor cell growth and metastasis is needed. .

Cytochalasins exhibit numerous pharmacological effects, many of which are related to dosage or administration. can be regulated by the pathway. These pharmacological effects are due to liposomes and formulations using microencapsulation technology, or cytochalasins, for example, can be regulated by co-administration with lymphokines.

The riboleme is a completely closed lipid monolayer membrane containing an entrapped aqueous volume It is a kind of thing. Liposomes are unilamellar (single membrane), but also multilamellar (multiple membranous bisections) A ball characterized in that the child membranes are separated from each other by an aqueous layer. The vesicles may be lion-like structures).

Bilayers are two monolayers with a hydrophobic “tail” region and a hydrophilic “head” region. It is configured. In membranous bilayer membranes, the hydrophobic (nonpolar) tails of the lipid monolayer ``heads'' are oriented toward the center of the bilayer, while hydrophilic (polar) heads'' are oriented toward the aqueous phase. It is once oriented. The basic structure of liposomes can be made using various known techniques. Can be done.

The invention of the present application utilizes interleukin-2 (IL-2) as a cytochalasin and its derivatives. The immune system of cytochalasins, especially when used in combination with other antitumor agents, It also concerns the discovery that inhibitory effects are removed. interleukin 2 is produced by normal peripheral blood lymphocytes and is stimulated by plant lectins, antigens or other stimuli. After exposure to the antigen or lymphocyte that induces the proliferation of mitogenic WIT cells, Cain, Morgan, D. A., etc., 5science, 191 ．． 1007 (1976). Proliferation of stimulated T lymphocytes In addition to its ability to induce fII, F-L-2 also induces many functions of immune cells in vivo. B. IL-2 mediates immune cell interactions and functions in some lymphocytes. It is one of the molecules that regulate production and transmission.

Purpose of the invention The purpose of the present invention is to treat patients with high immune function, such as autoimmune diseases or after organ or tissue transplantation. Shinuru therapy that produces transient immunosuppression useful for treating pathologies accompanied by hyperimmune relapse The aim is to provide treatment methods.

Furthermore, it is an object of the present invention to combine cytochalasins with IL-2 and optionally other anti-tumor agents. An object of the present invention is to provide vLr11 tumor preparations and treatment methods for use in combination.

Other objects of the present invention are formulations using liposomes or microcapsules and neoplastic Therapeutic methods utilizing these preparations to treat tumors (naoplasii) It is to provide. Furthermore, the object of the present invention is to provide synthetic analogues of CB as well as these The object of the present invention is to provide therapeutic methods using analogs as antitumor agents and immunosuppressants.

These and other objects and advantages of the invention can be found in the detailed description of the invention. It will become clear.

Brief description of the drawing Figure 1 shows intraperitoneal (IP), subcutaneous (SC) and intravenous ( IV) By route of administration, B6D2F1. administered to CD2F1 or C57B1/6 This finding, indicating the maximum tolerated dose (MTD) for CB, was published in June 1988. This is a revised version of Table 2 of U.S. Application No. 208,199 filed on the 17th. Ru.

Figure 2 shows the administration of CB administered to mice for immunosuppression [1] 19 hours before excision. Show the effect of changing the amount.

Figure 3 shows the effects of changing the dose of CB administered to mice 3 hours before pancreatectomy. It shows the effect on immunosuppression.

Figure 4 shows the effect of intraperitoneal administration of 50 mg/kg on in vivo allogeneic rejection. shows.

FIG. 5 shows groups of mice treated with CB 50 mg/kg at any time before or after tumor administration. Nine days after in vivo treatment, specific cytotoxicity ( 5-specific cytotoxicity).

Figure 6.7.8 shows that 19, 3 and 72 hours before yA evisceration, 50 m g / k, by adding the mouse processed L-2 with the CB of g The resulting reversal of immunosuppression is shown.

FIG. 9 shows i! washed before treatment with rll-2. cells represented by visceral cells Comparison of toxicity with cytotoxicity exhibited by unwashed Ill cells treated with rIl-2. Compare.

Figure 10 shows the immunosuppression caused by high doses of CB administered in microcapsules. by varying the dosage between 2 mg/kg and somg/kg. Compare with the immunosuppression produced by intracavitary CB.

FIG. 11 shows the l! of treatment with CB at various time points before and after tumor administration. Tumor induction Tile mt! The effect on w is shown.

FIG. 12 shows tt tumor induction of treatment with CB at various time points before and after tumor administration. ! ! ! The effect on visceral cellularity is shown.

Detailed description of the invention The present invention relates to hyperimmune therapy in autoimmune disease states or after organ transplantation or tissue transplantation. In order to reversibly suppress the epidemic response (hyperimmunity), cytochalasins are used to treat mammals, It particularly relates to pharmaceutical compositions and therapeutic methods for treating humans. In the immunosuppression method of the present invention In some cases, a dose containing approximately one-fifth of the maximum tolerated dose of cytochalasin is Administer 111 intravenously, intramuscularly, subcutaneously, or orally to patients suffering from the virus.

The present invention prevents tumor growth and metastasis without concomitant immunosuppressive effects. Intramuscular, intravenous, subcutaneous and intravenous administration of cytochalasins can be useful for It also concerns oral dosage.

In this aspect of the invention, about 50% of the maximum tolerated dose for a particular route of administration. Formulated with cytochalasins consisting of less than half of Administer in combination with other antitumor agents.

In order to eliminate the immunosuppressive effects produced by cytochalasins, the present invention It also concerns the discovery that IL-2 can be used in addition to lymphokines. In this aspect of the invention, cytochalasins and ML-2 are used in combination. Pharmaceutical compositions and methods of treatment are provided. Administration of cytochalasin and IL-2 When using M!, other antitumor agents other than cytochalasin should be used. may be included in

The present invention provides significant anti-inflammatory effects on cytochalasins without concomitant immunosuppressive effects. To provide a sustained release formulation of cytochalasin that delivers the tumor dose to the tumor site , pharmaceutical compositions utilizing cytochalasins in liposomes or microcapsules, and The remarkable discovery of the present invention, which also relates to methods of treating cancer, is that cytochalasins The immunosuppressive effect that exists when the drug is not administered in liposomes or microcapsules The cytochalasin concentration can be increased up to approximately three times the maximum therapeutic dose without producing results. It is said that it can be administered into living organisms in posomes or microcapsules. That's true.

Furthermore, the present invention provides semi-synthetic cytochalasin analogs, pharmaceutical compositions containing these analogs. Cytochalasin p, (CA) does not easily change into the endione type due to chemical and metabolic effects. The present invention relates to therapeutic methods utilizing these analogs.

Detailed description of the invention The following abbreviations and definitions are used throughout the specification.

BID - twice daily administration CA-cytochalasin A CB-Cytochalasin B CD-Cytochalasin D CE-Cytochalasin E CMC, CM-cellulose-carboxylmethylcellulose CM C/T w -carboxymethylcellulose n) Cytochalasins - fine am and agricultural compounds derived from synthetic or natural molds DMSO-dimethyl sulfoxide E:T ratio-effect cell:tx cell ratio FAT - Vesicular GI tract produced by freeze-thaw technology - Gastrointestinal tract HPLC-High performance liquid chromatography IC-inhibitory concentration; IC50 = 50% - cent inhibited cells IP - i.p. IV-intravenous LD match death amount LUV-large unilamellar vesicle LUVET-VET technology manufactured by LUVMLV-multilamellar vesicles MTD - maximum tolerated dose NMR - nuclear magnetic resonance PBS - acid buffered saline QID - administered 4 times a day SC-subcutaneous SPLV - stable bilamellar (plurila++ellarl vesicle TAD - tumor Appearance date TLC-thin layer chromatography Tw-Tween (polyoxyethylene sorbitan monoalkyl ether) interface activator Vesicles produced by VET-extrusion technology The present invention uses cytochalasins to The present invention relates to a method for producing transient immunosuppression in mammals. For more details , organ transplants, tissue transplants, and autoimmune diseases such as rheumatoid arthritis, lupus, and autoimmune diseases. Treatment of hyperimmunity associated with autoimmune W disease, autoimmune thyroiditis, autoimmune hepatitis It has been discovered that cytochalasins can be used as temporary immunosuppressants to ing.

In the immunosuppression method of the present invention, the highest tolerated dose ( MTD) to about one-fifth of the MTD, preferably from about one-half to about one-half of the site color. Cytochalasin in an amount up to about the MTD of the cytochalasin, most preferably about the MTD of the cytochalasin. , administered to a mammal. In the immunosuppression method of the present invention, half of the cytochalasin Synthetic derivatives, especially 21,22-dihydro-cytochalasin B, 20-deo xi-cytochalasin B, 20-deoxy-21,2-dihydrocytite Chalasin B, 20-deoxy-20-fluoro-cytochalasin B, 20-deo xy-20-fluoro-21.22-dihydrocytochalasin B and 21.22 -Dihydro-cytochalasin A, any site containing cytochalasin A-M Characins can be used.

Of course, many cytochalasins have a fine fibrous form, which is useful for phagocytosis, cytokinesis, and differentiation. They differ in their ability to interfere with secretion and exocytosis, resulting in immunosuppression. The slimming effect is different. Furthermore, the administration route and pharmacokinetics of cytochalasin derivatives are It also plays an important role in determining the extent of immunosuppression. However, in general, Immunosuppressive doses of natural and semi-synthetic cytochalasins are determined for specific routes of administration. It is in the range of about one-fifth to about λ(TD).

Depending on the type of cytochalasin used and the route of administration, it may produce transient immunosuppression. The daily dose of cytochalasin that is effective for slimming is generally from about 0.04 mg/kg. Approximately 150mg/kg. Preferably from about 1.0 mg/kg to about 150 mg/kg It is within the kg range. Of course, the concentration of cytochalasin used will depend on the cytochalasin. Dosing I! It will vary depending on the route and activity. −For example, cytochalasin When administered by subcutaneous injection, the amount of cytochalasin used is generally about 2 mg/kg to about 150 mg/kg, preferably 5 mg/kg /kg to about 150 mg/kg. cytochalasin intravenously When administered by injection, the amount of cytochalasins used generally ranges from about 0-1 m g/kg to about 2omg/kg, preferably 0.25mg/kg to about 20m When administering cytochalasin by the intramuscular route, it may range up to g/kg. The amount of cytochalasin used generally ranges from about 1 mg/kg to about 50 mg /kg, preferably in the range of about 2.5 mg/kg to about 50 mg/kg. It would be surrounded. Also, - by way of example, effective concentrations of CE and CD useful in the present invention. are much lower than the concentration of CB used (approximately 1/10). It is also preferably abbreviated as MTD for a particular route of administration. Equal doses of cytochalasins are administered. The table shown in Figure 1 shows that the mouse has an IP address, Representative MT for a number of different CBs with excipients administered SC or IV Indicates D.

When formulating cytochalasins for use as immunosuppressants, one generation Typical pharmaceutical formulation solutions and delivery suspensions can be used. The formulation is generally Targeted route of administration and standard! Mixing with the selected pharmaceutical carrier for two-drug practices Administered together.

For parenteral administration, other solutes may be added to make the solution isotonic, such as salts or glucose. in the form of a sterile, pyrogen-free aqueous solution, which may contain sugars such as In the present invention, cytochalasins can be administered using pharmaceutically compatible ! suspensions or solvents can be used. Of course, in the parenteral H formulation of the present invention The type of suspension or solvent used has a dual influence on the absorption of cytochalasin. may maximize the immunosuppressive effects of cytochalasins (to rub the formulation Denaturation can be easily accomplished by a person having ordinary knowledge in the field of pharmaceutical technology. By the way.

Preferred suspensions for parenteral administration include cytochalasins in CMC (2%)/ Cane bean-20 (1%) suspension is mentioned.

Preferred solutions include ethanol/saline (1:2) and cytochalasins. and DMSO solutions.

The immunosuppressive effect of the cytochalasin used depends on the route of administration as well as the site. affected by The transient and reversible immunosuppressive effect of cytochalasins is due to the active site It depends on the timing and amount of cytochalasin reached. cytochalasin immunity Active sites of inhibition include the pancreas, bone marrow, lymph nodes, thymus or transplanted organs or transplants. Examples include explant tissue. Maximizes cytochalasin levels in areas of immunosuppressive activity Those skilled in the art will know to vary the amount, route, and site of administration to achieve the desired results. It's here. If intravenous administration of cytochalasins is intended, immediately from about 1 hour to 12 hours after administration, preferably from 1 hour to about 3 hours after administration. administration to maximize cytochalasin localization at immunosuppressed sites during treatment. Care must be taken to choose the site.

Immunosuppressive doses of cytochalasin are available in gelatin capsules, powders, syrups, and elixirs. Liquid agent, aqueous solution, suspension? I! It can also be administered orally through a moat or the like. The oral dosage form is Immediate release oral product (1 recommendation++adiate r@1sase oral pro Immediate-release oral products, preferably administered as a The active agent is released immediately after formulated as an m-release product; ! Cytochalasins administered IO are similar to cytochalasins administered parenterally. , the immunosuppressive activity easily reaches certain sites, such as the spleen, thus causing excessive immunosuppression. It is believed to promote effectiveness.

Orally administered products may contain pharmaceutical carriers and diluents such as lactose, sodium citrate. Combined with Lithium, Phosphate Salts, Magnesium Stearate, Starch and Talc A preferred route of oral administration is to administer the cytokine in a capsule. Keep the lacine in a soluble or near-soluble state to prevent the capsule from dissolving in the GI tract. Cytochalasin is formulated to maximize the solubility, absorption, and blood circulation of Cytochalasin. low molecular weight polyethylene glycol, or a solvent such as DMSOl or immediate release soft gelatin capsules solubilized in a mixture of

Immunosuppression caused by conventional immune n1M agents or side effects from administration of most anticancer drugs Unlike the immunosuppression that occurs as an effect, the immunosuppression caused by cytochalasins The restriction is temporary and easily reversible. By administering a large pill of cytochalasin, The immunosuppression caused by increase or decrease. Therefore, the immunosuppression caused by cytochalasins is Based on cytochalasin pharmacokinetic data, cytochalasin in immunosuppressed areas Carefully treated with a regimen designed to maximize the degree of I can do it. The immunosuppressive dose of cytochalasin is administered parenterally once a day as a drug. 1 or 2 doses, or 1 sip every 6 to 12 hours (QID or (BID), the treatment may be different, or the cytochalasins may be added to the immune system. It may be administered when the drug becomes high. Of course, 7fjill will be selected! life is chosen It will vary as a function of cytochalasin activity, route of administration, and pharmacokinetics.

The present invention provides an antitumor dosage form of cytochalasin, as well as an associated immunosuppressive effect. Without getting wet! ! It also relates to treatment methods that inhibit the growth and spread of tumors. In this aspect of the invention, the MTD is less than or equal to about one-fifth of the MTD, preferably The present invention involves administering to the patient an amount of cytochalasin equal to about 10 minutes of lpI of D. Type of cytochalasin derivative used, administration, as in immunosuppressive embodiments of Route and pharmacokinetics play important roles in determining the appropriate dosage. but generally inhibit tumor growth and metastasis, but without concomitant immunosuppression. The cytochalasin dosage without is about 0.05 mg/kg to about 30 rng/kg. It is within the range of In this aspect of the invention, significant immunosuppression is avoided. The immunosuppressive mode of the present invention, except that lower doses are used for cytochalasins in the same manner and using the same solvents and other additives as used in can be administered parenterally or orally.

The cytochalasins in this BpJ of the present invention are other than cytochalasins. It may also be formulated in combination with an antitumor agent. However, these agents cause immunosuppression. By itself, it does not produce significant immunosuppression without additional treatment to eliminate it. It is preferable that there is no such thing.

The present invention provides cytochalasins at doses significantly higher than those that produce immunosuppression. Sustained-release formulations containing liposomes and/or microcapsules have shown significant immunosuppression. It is also about the surprising discovery that it can be administered to patients without causing Ru. For administration routes formulated in liposomes or microcapsules, and about the maximum tolerated dose, preferably up to about 3 times the maximum tolerated dose. The dosage form of chalacin treats neonatal tl+iI without producing immunosuppression. It is useful for Numerous anti-tumor agents have been combined with cytochalasins in liposomes or microorganisms. Can be formulated into clocapsules, but additional treatment to remove immunosuppression Cytochalasin is the only antitumor agent that does not show immunosuppression even without the use of cytochalasin. It is preferable to formulate a formulation in combination with

Anti-cytochalasins and discretionary! ! Extended-release delivery for administering anticancer drugs is Therefore, liposomes can be used. Known techniques for making liposomes include: For example, phospholipids suspended in an organic solvent can be used in the present invention. evaporate the solvent to dryness, add an appropriate amount of aqueous phase and allow the mixture to ``swell'', The resulting liposomes, consisting of multilamellar vesicles (MLVs), were prepared by mechanical means. Bangham et al. (J, Mo1. Bial.

, 1965.12: 238-252) is the first method for producing liposomes. So Other technologies include Papahadjopoulos et al. (Biochim, Biophys, ^cta.

, 1968.135, 624-638). Included are those using small lamellar vesicles and large lamellar vesicles.

Unilamellar vesicles were produced using an extrusion device using Cu 11is, etc., PCT publication number WO37100238, published January 16, 1986 , by the method described in the title “Extrusion method for producing unilamellar vesicles”. can be manufactured by Vesicles created using this method are called LUVET. and forced through a membrane filter under pressure. The vesicles were filtered with a 200 nm filter. It can also be made by extrusion through a filter. Such vesicles are VET2 Known as 00.

Lamellar solutes substantially equivalent to other classes of liposomes that can be used Some are characterized by having a distribution. This class of liposomes consists of Li Stable bilaminar as defined in U.S. Patent No. 4,522.803 by nk et al. Fountain et al., U.S. Pat. No. 4,555, also called Fountain et al. 8,579, called monophasic vesicles; Subject to at least one freeze-thaw cycle (this method is cited herein by reference). Ba11Y etc., PCT public number 137/QOO43, January 1, 1987 5th, described in the name “Multilamellar liposomes with improved uptake efficiency” called freeze-thawed multilamellar vesicles (FATMLV).

All methods of making liposomes involve encapsulating semi-synthetic or natural cytochalasins. It can be used to create files. These liposomal forms of cytochalasins The pharmaceutical composition can be administered in vivo or in vitro, as described in .

Various sterols and their water-soluble derivatives are used to form liposomes. It is being Specifically, Janoff et al., U.S. Patent No. 4,721,612 issue, published January 26, 1988, name: “Steroid liposome”, M ayb@w, etc. (PCT publication number WO35100968, published March 14, 1985) is a liposome composed of full 7y-tocopherol and certain derivatives thereof. A method is described for reducing the toxicity of drugs by encapsulating them.

In addition, various tocopherols and their water-soluble derivatives form liposomes. It is used to. Janoff et al., PCT Publication No. 8710221 9. April 23, 1987, name “Alpha-tocopherol-paced vesicles” "reference.

On the other hand, when ionizable antitumor drugs are used in combination with cytochalasins, Baoy et al., PCT Publication No. 861011, which is incorporated herein by reference. 02. Load the drug into liposomes according to the method published on February 27, 1986. be able to. This technology creates a transmembrane gradient across the liposome membrane. By doing so, the antitumor agent is loaded. This gradient is located across the liposome membrane. one or more ionic species (i.e. Na+, Cl-2K+, Li+ or H+) This is caused by the temperature gradient associated with These ion gradients are pH (H+) gradients. Preferably, the incorporation of the ionizable antitumor agent between the liposome membranes to promote By this method or any other method, liposomes can be loaded with antitumor agents. Once loaded, it is possible to create a pharmaceutical formulation that can be delivered in vivo or ex vivo, as discussed later. can be done.

In liposome-drug delivery systems, bioactive agents such as drugs are placed in liposomes. 0 cases of uptake or association with liposomes and then administration to the patient to be treated For example, Rahman et al., U.S. Patent No. 3.993.754; 5ears et al. U.S. Patent No. 4,145,410; Papahadjopoulos et al., U.S. Patent No. 4,235,871; 5cbneider, U.S. Patent No. 4.114, No. 179; Lenk et al., U.S. Pat. No. 4,522°803; and Founta See in et al., US Pat. No. 4,588,578.

In the present invention, as mentioned above, semi-synthetic and natural mold-derived cytochalasins can be incorporated into or associated with liposomes. Furthermore , antineoplastic agents, or cytochalasins administered to eliminate the immunosuppressive effects of or additional anti-tumor agents, such as those mentioned above, can be co-incorporated into the liposomes. These liposomes containing cytochalasin can also be incorporated into liposomes. It can also be administered together with other drugs. Such liposomal formulations can be used simultaneously or over time. A preferred method of the present invention is to administer cytochalasin alone or with By incorporating up to three times the MTD of the potency in combination with additional anti-tumor agents. This formulation can then be used as a sustained release form that does not cause substantial immunosuppression. , administered to the patient.

During the production of liposomes! ! An organic solvent can be used to suspend the jl. Organic solvents suitable for use in the present invention have a variety of polar and dielectric properties. and solubilizes lipids, such as chloroform, methanol, and ethanol. dimethyl sulfoxide (DMSO), methylene chloride and among others , benzene; methanol (70:30). the As a result, the lipid-containing solution (Ilit and other components are uniformly dispersed). A mixture is formed. The solvent is characterized by its biological acceptability, low toxicity and solubilizing ability. Generally selected based on

Liposomes containing the pharmaceutical formulation containing cytochalasin of the present invention can be used in mammals, especially Can be used in humans to treat neoplasms requiring repeated administration. . Sustained release formulations utilize up to three times the MTD of cytochalasin. This kind of Release compositions can be used to combat neoplastic tumors without producing substantial concomitant immunosuppression. It is valid.

As a sustained-release formulation in microcapsules, it can be used in large doses without causing immunosuppression. Tocharacins can also be administered. A preferred type of microcapsule is approximately Polylactate in a weight ratio of 45.55 to about 55.45, preferably 50:50. - film-forming polymers consisting of glycolate mixtures, as well as U.S. Pat. 675.189.4, 585, 482.4, 542, 025.4, 530, 84 0, European Patent Application No. 87309288.0 (Publication number 0,286.11 9), 87307115.3 (Publication number 0257915), 81305426 ．． 9 (Publication number 0052510) and 83303605.6 (Publication number 012 9619). Ru. Other formulations known in the art also provide sustained release of cytochalasin in microencapsulated form. It can be used to formulate products. Cytochalasin contained in the formulation If the total amount of Still other sustained release formulations readily apparent in the art may be included in this aspect of the invention if: can be used.

To prevent immunosuppressive concentrations of cytochalasin from rapidly reaching the immunosuppressive site, i.e. , at least about 1 to 12 hours, preferably at least about 1 to 24 hours. Contains cytochalasin, if cytochalasin is released so as to reach It is recognized that a number of sustained release formulations can be used.

Administration of both natural and semi-synthetic cytochalasins as immunosuppressive or antitumor agents Delivery systems other than liposomes or microcapsules can also be used to . Such delivery systems include, inter alia, osmotic pumps, transfer skin patches, etc. (1Hnsdir alpitch), infusion pump (1nfusion pump) p), biologically degradable polymers, monoclonal antibody binding systems, suppositories, nasal passages Medications (rhinila), dragees and troches are included.

The present invention provides IL-2 therapy to eliminate immunosuppression caused by cytochalasins. It's also about use. The use of IL-2 suppresses the immunosuppression of cytochalasins. The antitumor effect is reversed, and large doses of cytochalasins alone or in combination with other antitumor agents In addition, it is an effective way to eliminate the immunosuppression that occurs when administered for the treatment of new tumors. Provide steps. In this aspect of the invention, IL-2 is used as a cytochalasin immunosuppressant. Administration to lymphotropic cells will eliminate immunosuppression.

During immunosuppression, no matter when IL,2 is administered, the immunosuppression will disappear, but its effectiveness For maximum yield, IL-2 should be administered with cytochalasin or The administered IL- The amount of 2 depends on the individual patient, the type of cytochalasin used and the degree of immunosuppression. Although this may vary, the preferred dosage of L-2 is 1 kg per day. This embodiment of the invention ranges from about 5, OOO to about 15,000 units. , culturing human peripheral blood lymphocytes or other IL-2 producing cell lines. Utilize IL-2 made from any raw material, including IL-2 made by However, preferred IL-2 is human recombinant IL-2 (rIL-2, D available from uPont, Wilmington, and Delaware). Ru. In order to eliminate the immunosuppressive effects of cytochalasins, it is necessary to use Any protein that can be used can be used; the term IL-2 refers to one or more proteins of IL-2. is a protein whose activity is essentially the same as IL-2, although more amino acids have been changed. It should be recognized as including white matter. To treat immunosuppression in humans, Human rIL-2 is preferred.

The treatment method for cytochalasin immunosuppression is to suppress cytochalasin immunity for immune recovery. To restore immunity, if one does not simply rely on the transient reversibility of the IL-2 can be administered at an appropriate temperature. On the other hand, the administered cytoclast rL-2 can be administered to control the immunosuppressive activity of cancer. these during administration of cytochalasin alone or in combination with other antitumor agents; or later. IL-2 is.

For culturing human peripheral blood lymphocytes (PBL) or other IL-2 producing cell lines Therefore, it is made. Engineering L-2 can also be produced by recombinant DNA technology, This provides mutin and natural IL-2 that can be used to practice the present invention. It provides the means to produce modified products. The preferred IL-2 human r IL-2 is available from numerous suppliers (Catus Carp, Emeryville , Calif, ).

Il,,-2 may be gradually reduced if the formulation does not interfere with the integrity, stability, or activity of IL-2. Solvents, excipients and methods as described above, including liposomes and microcapsules for release formulations. Any of the methods can be used to formulate with cytochalasin. explained As described above, L-2 can be used in combination with cytochalasin or subsequently. It can be administered immediately by parenteral route of administration, preferably by intravenous injection.

IL-2 and cytochalasin in combination with at least one additional anti-tumor agent can be administered. Doxorubicin, daunorubicin or evirubicin, bi Lorindine alkaloids, such as vinblastine or vincristine alkaloids, especially purines such as 5-fluorouracil, is a pyrimidine derivative, mitoxantrone, salt Alkylating agents such as acid meclomethamine or cyclophosphamide, cis-platinum platinum compounds such as folic acid analogues such as methotrexate and, among others, any number of anti-inflammatory antibiotics, including anti-inflammatory antibiotics such as tomycin or pleomycin; Oncolytic agents can be used in combination with cytochalasins and lactate L-2. . Any number of the solvents, additives and methods described above may be used in formulating such products. Can be used.

The present invention also relates to methods and formulations for treating macrostasis (Kl! visceral swelling) in humans. There is also. Contains the same amount of cytochalasin used to induce immunosuppression The same cytochalasin preparation that causes the giant 11! How to treat heel syndrome Another aspect of the present invention is that CB is not easily affected by metabolic effects. This paper relates to a new semi-synthetic analogue of CB that does not differ from the enedionic system of CA. The synthetic analogs of the present invention have the following general structure.

Here, R1 is hydrogen or fluorine, and for R1 in C21 and C22, Alpha and beta carbon-carbon bonds are oxidized or reduced.3 Semi-synthesis of the present invention Cytochalasin analogs generally include 20-deoxy-cytochalasin, 2o-deoxycytochalasin, Oxy-21,22-dihydrocytochalasin, 20-deoxy-20-fluoro rho-cytochalasin and 20-deoxy-20-fluoro-21,22-dihyde It is known as rocytochalasin.

The semi-synthetic cytochalasins of the present invention, unlike CB, can be used with the administration of CB and CA. Metabolic effects on the enedione system of CA, a system believed to be responsible for many toxicities. will not change easily.

Endionic systems can contain any number of thiol groups or other nucleophilic groups in biological systems. It irreversibly binds to reagents and produces toxic effects.

The semi-synthetic cytochalasin system of the present invention is metabolized to the enedione system of CA in vivo. Others, such as CB, including immunosuppression, antineoplastic, and anti-metastatic designed to maintain activity in combination with cytochalasins. Book The semi-synthetic cytochalasins of the invention have the advantage of being longer acting than CB or CA. , designed to reduce the extremely harmful side effects associated with administration of CB or CA. 0 Semi-synthetic analogs of the present invention can be made by a number of methods, including complete chemical synthesis. can. However, the preferred route to synthesize these analogs is mold, dem atioidea (ATCC 24346) or standard conventional By chemically modifying CA prepared by oxidizing CB to CA using Is Rukoto. Any known conventional method for isolating CA or CB may be used. PCT publication number WO38/102, which is cited here for reference. 59, Application No. PCT/US88102095. Published December 29, 1988, Batch produced by the method described in the name “Cytochalasin￥R manufacturing method and composition” Preferably, absorption techniques are used. By such a method, sufficient CB or CA will be produced, and raw materials for synthetic modification for producing the composition of the present invention will be provided. Served. Modification of CA or CB can be carried out using the following synthetic method, which is technically easy to use. Proceed according to The general method described below is given by way of example only and does not apply to the present invention. It has been recognized that semi-synthetic cytochalasins can be produced by a number of synthetic routes. You should be aware of this. To produce 20-deoxycytochalasin, from mold After single III or after oxidation of CB to CA, the free hydroxyl group is blotted with, for example, a formyl group. Subject the CA to a block operation (85% HCOOH at 160”C for 1 hour) to . After blocking the free hydroxyl group of CA, the 20 keto groups are reduced to create water at the C20 position. Next, convert the free hydroxyl group at C20 into an acid group using toluenesulfonyl chloride. Activate with C to create a “tosyl” group at C20. After forming “tosyl” group, iodine with sodium to form 2o-iodohydroxy-blocked cytochalasin. do. This analog was then reduced with NaBH4 in a solvent to convert the formyl group into a hydroxyl group. 2O deoxycytochalasin is produced by cleaving it from

In order to produce 20-deoxy-21,22-dihydrocytochalasin, C The free hydroxyl group of A is blocked with a formyl group, and the product is converted into a 20-keto group in acid. For example, blocking operations such as blocking with 1,3-dihydroxypropane as a ketal Next, the activated double bond at position 21.22 was reduced in NaBH4. and prepare the 21.22 dihydro derivative. Removes residues from the reduction process and blocking groups to form the 20-keto group, and then reduce the 2o-keto group to form the 21.22-dihydrosa Itochalasin B is formed. On the other hand, free hydroxyl groups are first blocked with formyl groups. , 20 may reduce the keto group. The resulting free hydroxyl group is tosylated to form tosyl substituting the group with iodine as described above to form 2o-iodo-21,22-dihydrocytyl Manufactures tocharacin. 20-iodo-21,22-dihydrocytochalasin is , simple substitution of iodine group with fluorine (KF or CaF/18-Cr in acetonitrile) own6), it is converted to 20-deoxy-20-fluorocytochalasin. The 20-fluorocytochalasin derivative may be a diastereomeric mixture. (racemic at C20), but the fluoro group at C20 is , preferably has the same steric configuration as the C20 hydroxyl group of CB.

In order to produce 2o-deoxy-20-fluorocytochalasin, as described above, In sea urchin, the hydroxyl group of CA is blocked with formyl group, 20 keto groups are reduced, and tosylation is performed. Then, as described above, the iodo group of C20 is simply iodinated with fluorine. By substituting 20-iodocytochalasin, 20-deoxy-20-phenyl Converts to luorocytochalasin. The 20-fluorocytochalasin mixture is Astereomer mixture m < fluorine at C20 may be a racemic mixture) However, the fluoro group of C20 has the same configuration as the 020 hydroxyl group of CB. and is preferred, fl! As in the case of cytochalasins, the semi-synthetic cytochalasins of the present invention Tocharacins with or without IL-2 or additional antitumor agents , can be used as an immunosuppressant or a therapeutic agent for new tumors. temporary and reversible The dose that should be administered to counteract immunosuppression is similar to that of other cytochalasins. i.e. at least about one-half of the MTD for the particular route of administration. be. Semi-synthetic cytochalasins are expected to have longer-lasting activity; Immunosuppression or new Il! To treat tumors, less than CB is required. A large amount should be used.

When should cytochalasins be administered alone or in combination with additional anti-11% agents? If so, the dose of semi-synthetic cytochalasin to treat neoplastic tumors should be approximately Administered at high immunosuppressive dose concentrations ranging from one-tenth to approximately one-fifth of the MTD In order to minimize or eliminate the immunosuppression caused by cytochalasins For this purpose, IL-2 can be administered in combination with semi-synthetic cytochalasins. Wear. Semi-synthetic cytochalasins can be added according to the -4 principles and methods detailed above. It can be given by intravenous, intramuscular, subcutaneous and oral routes using agents and vehicles. For example, the present invention can be applied to a very low A large amount of the semi-synthetic cytochalasins of the present invention and liposomes or microcapsules are added to Also contemplated are sustained release formulations containing. Administration in such a sustained release form provides sufficient Antitumor doses of cytochalasin are provided, resulting in surprisingly low immunosuppressive doses.

For the purpose of carrying out various aspects of the present invention, the pharmaceutical compositions of the present invention may be used to treat neoplastic or When treating autoimmune conditions, the prescribing physician should determine the appropriate treatment for the given human patient and condition. The final dose of cytochalasin as well as other agents, including IL-2 or anti-tumor agents, This should be understood as determining the nature and severity of the patient's illness or can be expected to vary depending on the condition as well as the age, weight and response of the individual . Dosage depends on the particular cytochalasin used, the treatment setting, and the agent in the patient. The final decision will be made by the administering physician depending on the pharmacokinetics of the drug. here The general dosage range given in It should be given an idle line. for treating neoplastic tumors without immunosuppression. Drug dosages in liposomal or microcapsule form are generally used for free drug. It's probably up to about three times what you can afford.

However, in some cases it may be necessary to administer doses outside these limits. unknown.

The following examples are given for the purpose of illustrating the invention.

These examples should not be construed as limiting the scope of this invention.

Example 1 Cytochalasin immunosuppression General materials and methods PCR by the method described in pcr publication m (PCT/USs s 102095) Using the absorption technique, mold D, dematiooid ea) (ATCC24346) and purified CB. u　 Akira CB hayo (IN HP L C (reverse phase c18 DYNAMAXtm:/ ! subjected to final purification by 75:25 MeOH/H20) on J power and then 9 by analytical TLC and HPLC with recrystallization from CHCl3 at A purity of greater than 9% was observed.

DBA/2 and C57BL/6 female mice were incubated at the National Cancer Institute. Charles/River Laboratory (Will) through the Nimal Genetics Branch Minton, Mass.) and used at 8 to 12 weeks of age.

CB according to the method described in Bosquet et al., submitted for publication. Therefore, we used an emulsifying needle with a reduced hole diameter from 20 gauge to 25 gauge. Prepared for injection. Briefly, the CB is placed in a syringe and the excipient is added to the second syringe. Load the syringes and double-hubbed them. Binding was carried out using an emulsifying needle.

Return this suspension to the original state and repeat the coupling tube until the flow becomes boring. the diameter of this hole from 21 gauge to 23 gauge, and finally to 25 gauge. Reduced to gauge. The syringe and coupler were cleaned with MeOH and the MeO The H washing solution was subjected to TLC to measure residual CB that was not actually transported to the test animals. Ta.

The culture medium for cell culture was HEPES, fetal bovine serum, sodium pyruvate. MEM, gentamicin, penicillin/streptomycin, MEM non-essential amino acids GIBC○, Grand Iceland.

Purchased from New York, USA.

RPMI 1640 medium containing HEPES was mixed with 10% fetal bovine serum. ．． 1mg/ml Gentamicin, 1% Sodium Pyruvate, 1% Non-Essential Amino sensitized for 4 days with acid, 1% glutamine and 50 uM mercaptoethanol. The cells were subjected to sensitization cell culture. Cultivation The soil should be immediately sterilized by membrane filtration and used immediately, or if not used immediately. were sterilized immediately before use. RPMI 1 used for 4-hour Cr cytotoxicity measurement 5% fetal bovine serum and 1% penicillin/streptomycin in 640 medium solution was added.

P815 mastocytoma (+ma) tumor cells were incubated once a week with syngeneic ( syngeneic) D B A / 2 by passing it intraperitoneally into mice. It was held in an ascites shape. 4-day sensitization analysis The tumor cells used as antibodies in Harvested 72 hours after aging. The cells were washed and HEPES buffer was added. Resuspend in RPM11640 medium and incubate for 20 min on ice in a 35 x 10 mm tissue culture dish. Overall 21 minutes (4200r) at a target distance of 1°cm at a rate of 0rad/min ad) X-ray irradiation. Used as target in 4-hour cytotoxicity (LMC) analysis Cerottini et al. (1972 Nature (Nature), 237.272 and 1974 Journal of Expe. Remental Medicine (J, Exp, Med, ), 140.703) The method of 0rsini et al. was improved [(1977, Cancer Research (Can cer　Res,).

37, 1719).

In vivo allogeneic challenge and CB at a concentration of 50 mg/kg or excipients ((:MC/ TW-control) with IC pre-treated or treated with CB intraperitoneally. 57B L/6 mice were intraperitoneally implanted with 3X10'P815 mastocytoma tumor cells. I attached it. 24. 12, 6.3 hours before this drug was inoculated into this mouse. Injections were given at the same time as planting or at 24.48 or 72 hours after planting. Tumor Pancreatic resection was performed 9 days after tumor antigen administration. Tolerance to alloantigen challenge In experiments where the effects of treatment are to be monitored over a long period of time, treated animals It was left as it is for 0 days.

Drug-treated and vehicle-treated (control) mice for in vitro analysis of CB effects. The pancreas was aseptically excised at 72, 19, or 3 hours after CB injection. . The pancreatic cells are then subjected to coarse (50 mesh) and fine (200 mesh) sterilization steps. A single cell suspension was formed by passing through a stainless steel wire mesh. RPMII cells Wash three times with 640 medium (unless otherwise specified) and trypan blue exclusion. Therefore, viability was measured.

Four-day sensitization of pancreatic lymphocytes in the presence of X-irradiated P815 tumor antigen (:6rOttini et al. [1972 Nature, 237 ．． 272 and 1974 Journal of Experimental Medicine (J, Exp, Med, ), 140.703] improvement of the method of 0rsini et al. [(1977, Cancer Res,), 37.1 '719] was added. Pancreatic cells in 17.8x35mm 6-well set 2 ml in a tissue culture dish (Costar, Cambridge, Massachusetts, USA). Cultured in total volume. Each well contains 2x107 viable pancreatic cells and 4x105 of X-ray irradiated P815 cells. At the end of the 4-day incubation period, the go The operating body is removed by gentle scraping by policemen. Lymphocytes were collected from each well and a triple culture was plated. has been passed. Cells were washed with RPMI 1640 medium, and then Coulter Diagnostics, Hialeah, Measured by Fla., USA).

The cytotoxic activity of sensitized pancreatic cells recovered from a 4-day culture was previously determined by Bo gyo and Mihich, Cancer Research (CancerRes, ), 40.650 (1980), the standard 4-hour 51Cr release determined by analysis. Each cell preparation contains at least 4 cells in the range of 100:1 to 6=1. Three different effector:target cell ratios were analyzed. 51Cr The percentage of release is determined by the following formula: % formula % cpm supernatant + cpm pellet calculated according to.

The later results were the maximum release of 51Cr from cultures treated with 1% SDS; The denominator in the above formula has been replaced. ? ? ? ? ? ? ? ? specific 5 The percentage of 1Cr is the control from 4 days of culture in the absence of P815 antigen. Represents the value obtained for lymphocytes minus the value obtained for immunoagonists. . 50 mg/kg CB 19 hours before pancreatic lymphocyte removal in four experiments. Cytotoxic T cell development in allogeneic sensitization assays by intraperitoneal administration of inhibited. The range of this inhibition observed was from 33.2% to 56.6% ( E:T ratio = 25/l).

decreased when CB was administered intraperitoneally 19 hours and 3 hours before tile removal. However, as shown in Figure 2 (19 hours) and Figure 3 (3 hours), 25 m There was some inhibition after administration of g/kg and 10 mg/kg. 5 Two experiments with mg/kH CB failed to show meaningful immunosuppression. I couldn't. 3 h and 1 hr when pancreatic lymphocytes are removed for sensitization culture. Nine hours prior, 50 mg/kH CB was administered intraperitoneally. 3 hours before the tile removal The inhibition ranged from 57.0% to almost 100%, as shown in Figure 3. Ta. Some inhibition occurred after administration of 25 mg/kg and 10 mg/kg . CB administered 72 hours before tile removal produced -penetrating immunosuppression. failed (data not shown).

Example 2 In vivo response to allogeneic tumor challenge in CB-treated mice responsiveness Regarding allogeneic rejection reactivity, the effect of CB at the highest tolerated IP bolus dose was Tested directly within. CB 50 mg/kg is administered intraperitoneally as a bolus suspension. C57B1/6 mice treated with CB by , 3 x 107 viable P815 mastocytoma cells were challenged intraperitoneally at various times.

Ability of animals to reject allogeneic tumor challenge and pancreas 9 days after tumor challenge were monitored for specific cytotoxic effects.

Figure 4 shows the results of intraperitoneal administration of 50 mg/kg regarding allogeneic rejection. Show effectiveness. Alloresponsiveness is a condition in which allogeneic tumor antigen challenge is lethal to those in the animal. CB given 12 or 6 hours before tumor antigen administration to such an extent that Compromises were made when possible. Survived to that point and observed for more than 9 days 9B or more since the surviving animals showed signs of abdominal distension and disease. Survivors in these groups had prolonged allogeneic tumor growth. Conceivable. Allogeneic tumor growth is a condition in which all animals survive challenge. Even cases were also present in animals treated with CB 3 hours before tumor challenge. child This time course in in vivo inhibition is similar to the 3 and 12 hour period leading to CB treatment. showed the greatest effect during and observed in the pancreas removed from animals 3 hours after CB treatment. The results are consistent with those tested for alloresponsiveness in vitro.

The tumor was sensitized in vivo by allogeneic tumor antigen administration, and 9 days after administration of tumor antigen. CB-treated and control group excised pancreata were tested in vitro for specific allogeneic tumors. Tested for cell cytotoxicity.

Figure 5 shows whether before or after tumor antigen administration when compared to vehicle-treated controls. In both groups treated with CB, specific cytotoxic effects were inhibited. In groups treated with CB either 6 or 3 hours before tumor antigen administration, indicating no In this group, allogeneic tumor growth continues to grow until the time of pancreatic resection. However, specific cytotoxic effects were increased above control levels.

Example 3 Effect of rIL-2 on tile sensitization Human recombinant interleukin-2 (Pupont Carp, Wilmingto n, Del, , U, S, A) was used as the initial part of the culture. Sometimes a level of 100/ml in 100 microliters of RP M I 1640 medium was added to the unwashed cultured pancreatic cells of Example 1. Unwashed and washed pancreas, immune response Restoration of sexual activity occurs under conditions where either endogenous CB, CB metabolites or suppressors are present. It was used to measure whether or not it was obtained under the conditions. At the end of the 4-day incubation period, Agonist lymphocytes were found from each well by scraping. wash the cells 51cr release test as described in Example 1. It was attached to. The addition of rIL-210U/m1 to the sensitized culture on the fourth day was CB consistently reversed the immunosuppression that occurred prior to in vivo treatment of lymphocytes. This is the 6th ．． 7 and 8 before tile excision for all levels of CB administered as shown in Figures 7 and 8. observed for CB administered at 2 hours, 19 hours and 3 hours. Figure 9 shows Washing cells in culture before cultivating them introduces γIL-2 into the culture. When not added, the results show less immunosuppression, but γIL-2 was not added during culture. We show that immunosuppression was reversed in cells that were not washed before being grown in cells.

In four experiments, rIL-2 was added to control lymphocyte cultures lacking P815 antigen. The cytotoxic activity increased between 18.9% and 28.4% (data not shown). It has not been).

Example 4 Effect of CB on pancreatic weight and pancreatic cellularity in antigen-naive pancreatic cells To establish the actual responsiveness of the pancreas, the pancreas was treated with CB for 3 or 19 hours. 50.25.10 and 50.25.10 for pancreatic weight and pancreatic cellularity when collected at 　mg　/　k　g　The effect of CB on intraperitoneal drug dose was measured. respond excipients with a range of 92 to 110% of the mean pancreas weight of treated controls; There is no meaningful effect of CB treatment on There was no drug dose-response effect.

Table 1 Effect of intraperitoneal administration of CB on pancreas weight Treatment time (hours) n=6 n=3 n=3 B.I.P. (mg/kg) %Of excipient control 10 98 ND 1015 92 ND 110 Example 5 Effect of sustained release formulation on pancreatic sensitization Microencapsulated CB (as shown in U.S. Pat. No. 4,389,330) Although it can be prepared by a general method, 5other Re5ea polylactate-glycolate under contract with rch In5titiute (formed from a mixture of In the trial, there was no evidence of apparent toxicity as evidence of the absence of mortality and no evidence of disease. There were no signs of deterioration and the maximum weight loss averaged 3%. Figure 10 shows the intraperitoneal cavity. In microencapsulated form, the dose is more than three times the highest tolerated dose for pill administration. CB did not produce significant immunosuppression either 19 or 72 hours following injection. We show that it can be administered to mice without producing any inhibition.

The time used to assess potential immunosuppression by microencapsulated CB is selected based on time course measurements of tissue distribution of microencapsulated drugs. Ta. CB was administered 3 days after injection (50 mg/kg by intraperitoneal suspension administration). fast enough to produce its maximum concentration in the pancreas and other organs (compared to time) It has been shown that it is released from microcapsules at a temperature of 1. Continued microcapsule Despite the high tile concentration of CB 3 days after injection with standardized CB, only Only peripheral immunosuppression appeared (Figure 10).

This experiment demonstrated that sustained-release formulations of CB and other cytochalasins have substantial immunosuppressive effects. It has been shown that it can be used as an anti-neoplastic agent at higher concentrations than the MTD without producing any fruit. It shows.

Example 6 CB effect on lymphocyte sensitization in vivo P81 because CB inhibits cytotoxic T cell responses to tumor sensitization in vitro. 5. Intraperitoneal administration of CB alone on the in-vivo response of pancreatic lymphocytes to tumor growth The effect was observed. Intraperitoneal administration of 50 mg/kg of CB alone is shown in Table 1. As shown in the results of the 51Cr cytotoxicity test, 3X107 P81 5 cells did not impair the 9- or 10-day responses of mice injected intraperitoneally. child The lack of immunosuppression is related to whether CB was administered before or after the P815 tumor. It was consistent.

Example 7 CB effect on macrosomal tumors Allogeneic challenge with P815 mastocytoma cells in C57B1/6 mice When the pancreas was excised and weighed 9 days after allogeneic tumor challenge, vehicle-treated During treatment, a large tumor is produced. This increase in pancreatic weight may cause P815 cells to become antigenic. This is more than three times higher than in non-treated animals. at various times before and after tumor antigen administration. As shown in Figure 11, the treatment with CB caused tumor-introduced pancreas to increase over time. dependent effects and corresponding effects on pancreatic cellularity as shown in Figure 12. show.

Example 8 Synthesis of 20-deoxy-cytochalasin CA was carried out by the fungus Diedematioidea (D , dematioidea) (ATCC 24346) 7')' or by the method described in PCT Application No. W○88710259. It is obtained by oxidizing CB isolated from the same mold. CA for 1 hour at 60°C; in excess 85% HCOOH or in acetyl formate/pyridine at -20°C. By stirring CA, its hydroxyl group is protected with formyl group, and ether extraction is performed. isolation followed by evaporation of the solvent. This crude formyl-protected cytochalasin A Then N a B H,/dimethylformamide (DMF) or dimethyl sulfur Formyl bromine is reduced in phoxide (DMSO) and has a free hydroxyl group at C3. produce a derivative of the molecule. The free hydroxyl group at C1 is tosylated (37 (treated with toluenesulfonyl chloride/pyridine overnight at °C) and The tosylated product was then reacted with a 1.5 molar excess of NaI in DMF or DMSO. and substitute the tosyl group of C1 with an iodo group. Then N in DMF/DMS○ The iodo group was reduced and substituted with aBH4, and the formyl group was removed under acidic conditions (6N MCI). The ru group was deprotected to produce 2o-deoxy-cytochalasin.

The present inventors found that the 2o-iodocytochalasin derivative was 20-fluorinated in Example 9. It is of special note that it can be used to synthesize cytochalasins. . 20-Deoxy-cytochalasin was purified from the reaction mixture using preliminary HPLC. can be manufactured. Before proceeding to the next synthesis step, perform preliminary HPLC (reversed phase-C, Using 75:25MeOH/H, ○) on a DYNAMAXtm silica gel column Although it is sometimes preferable to isolate synthetic intermediates by It is.

Example 9 Synthesis of 20-deoxy-21,22 dihydrocytochalasin As in Example 8. The free hydroxyl groups of CA were removed with 85% HCOOH at 60°C or Blocked with formyl group by acetyl formate/pyridine at °C and obtained The product is subjected to a blocking step. In this step, the 2o-keto group is replaced by toluenesulfonic acid. Among them, 1,3-dihydroxypropane or 1,3-dimethoxypropane is used. blocked as a rule. After extraction with ether, this ketal protected derivative was extracted with Na BH, Pd/charcoal/in reduced (DMF or DMSO) or ethanol H, reduction to produce ketal-protected 21,22 dihydrocytochalasin. do. The resulting product was then subjected to acid cleavage of the ketal (toluene sulfur in dioxane). fonic acid), and the free hydroxyl group was subsequently purified in 85% HCOOH at 60°C for 1 hour. or formyl group block with acetyl formate/pyridine at -20°C. Ru. This 20-keto group is reduced to a hydroxyl group, this hydroxyl group is tosylated, and the tosyl group is iodine. (displace) by hydrogenation (this iodo intermediate is the same as in Example 11) by the method described in Example 8. will be returned. This formyl group is cleaved by 6NHC1. the product obtained, 20-deoxy-21,22-dihydrocytochalasin was analyzed by preliminary HPLC (Example (as described in 8) from this reaction mixture. .

Example 10 Synthesis of 20-deoxy-20-fluoro-cytochalasin 20- from Example 8 From iodocytochalasin, the iodo group at C20 is converted to KF or CsF/1 Simple substitution in acetonitrile with 8 crowns 6 (1 molar equivalent of 18 crowns 6) and subsequent removal of the formyl blocking group (6NHC1) to provide 20 -Deoxy-20-fluoro-cytochalasin can be produced. Reserve H PLC can be performed as described in Example 8.

Example 11 Synthesis of 20-deoxy-20-fluoro-21,22-dihydrocytochalasin From 20-iodo-21,22-dihydrocytochalasin from Example 9, C2 The iodo group in 0 is replaced by KF or CsF/18 crown 6 (18 crown 6 1 molar equivalent) in acetonitrile as in Example 1o. , followed by removal of the formyl blocking group (6N HCI) to Xy-20-fluoro-21,22-cytochalasin can be produced.

Preliminary HPLC can be performed as described above. The fruits described so far The examples and embodiments are intended to provide a description of the invention in illustrative form. However, it is not intended to limit the present invention in any way. As described up to this point by a person skilled in the art, Various modifications or changes that may be made to the above are also contemplated by the present invention. within the spirit and scope of the application and the claims that follow.

This invention also applies to the oxidized or reduced C-carbon shown on page 22. element is expression %formula% (wherein R,, R,, R, and R5 are hydrogen, hydroxy, or halogen, and R 5 and R, or R4 and R1 together are oxygen, or R5 and R4 together is oxygen, and R is hydrogen or fluorine) Also contemplated are novel derivatives of tocharacin.

Figure 1 Eya, agent form, former KG/"IP" CMC/T fart EEN Suspension 50 DMSO solution 　　　　　25 SCC14C/TWEEN Suspension 1 150/ioOD MSO solution 100 IV cMc/Tn[EN suspension 20ErOH/! ! it*　　　　　　　　20/1015*　B6D2F1. (1) 2F 1 or % of vehicle control in C57B1/6 mice % of excipient control Survival% Lu Specified release (%) % of excipient control % of excipient control % of excipient control Specified release (%) % excipient or placebo capsule control Average pancreas weight (mg) 9 days Average lung weight (mg) 9 EI lX10ε [+711 International Survey Report

Claims (1)

[Claims] 1. Composition of the following general formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R1 is hydrogen or fluorine, and for R1 in C21 and C22, rufer, beta carbon-carbon bond is oxidized or reduced). 2. Anti-neoplastic disease-therapeutically effective amount of the composition of claim 1 and a pharmaceutical carrier. or a pharmaceutical preparation containing a diluent for the treatment of patients with neoplastic tumors. 3. The composition according to claim 2, which is adapted for parenteral administration. 4. The composition according to claim 2, which is adapted for oral administration. 5. A composition according to claim 2 containing a pharmaceutically effective amount and a carrier or diluent. A pharmaceutical preparation for treating mammals suffering from hyperimmunity. 6. The composition may contain from about 1/5 to about the MTD for the particular route of administration. 6. The formulation according to claim 5, which is applied to treat hyperimmunity. 7. 6. A formulation according to claim 5 adapted for parenteral administration. 8. 6. A formulation according to claim 5 adapted for oral administration. 9. Claims range from about MTD to about 3 times MTD for particular routes of administration. A sustained release formulation comprising the composition according to scope 5 and a sustained release administration excipient. 10. The excipient is selected from the group consisting of ribosomes and microcapsules. 10. The sustained release preparation according to claim 9. 11. Administering an effective amount of a cytochalasin compound to produce immunosuppression autoimmune disease conditions or severe symptoms following organ or tissue transplantation, including Methods of treating mammals with immunization. 12. Due to the particular route of administration of the cytochalasin, the MT of the cytochalasin The method according to claim 11, wherein the method is administered in an amount equivalent to about 1/5 of D to MTD. . 13. The cytochalasins are cytochalasin A, cytochalasin B, and cytochalasin. The person according to claim 12 selected from the group consisting of Cytochalasin D and Cytochalasin E. Law. 14. The cytochalasin is selected from the group consisting of the compositions of claim 1. 14. The method of claim 13, wherein the method is a synthetic cytochalasin. 15. Cytochalasin B once or twice a day, from about 1 mg/kg to about 50 mg 14. The method of claim 13, wherein the method is administered parenterally in an amount range of /kg. 16. The cytochalasin is a group consisting of cytochalasin D and cytochalasin E. from about 1 mg/kg to about 50 mg/kg once or twice a day. 14. The method of claim 13, wherein the method is administered at ambient temperature. 17. Anti-neoplastic disease of cytochalasin causing immunosuppression-therapeutic effective dose and its immunity and an effective amount of interleukin-2 to remove inhibition. Pharmaceutical preparations for the treatment of patients. 18. The cytochalasins are cytochalasin A, cytochalasin B, and cytochalasin. The product according to claim 17, which is selected from the group consisting of agent. 19. The cytochalasin is at least one member of the group consisting of the compositions according to claim 1. 18. The formulation according to claim 17, selected from: 20. The interleukin-2 ranges from about 5,000 units per kg of the patient to about 5,000 units per kg of the patient. 18. The formulation of claim 17 containing 15,000 units. 21. anti-cytochalasin in an amount effective to treat the patient's neoplastic tumor. 18. The formulation according to claim 17, which contains a live tumor agent. 22. 22. The formulation according to claim 21, wherein the anti-neoplastic agent is adriamycin. 23. Administering the pharmaceutical preparation according to claim 17 to a patient suffering from a neoplastic disease. A method of treating a patient with a neoplastic tumor. 24. The cytochalasins are cytochalasin A, cytochalasin B, and cytochalasin. The person according to claim 23 selected from the group consisting of Cytochalasin D and Cytochalasin E. Law. 25. The cytochalasin is at least one member of the group consisting of the compositions according to claim 1. 24. The method of claim 23, wherein the method is selected from: 26. an effective amount of cytochalasin to treat neoplastic tumors and produce immunosuppression; and administering an effective amount of IL-2 to remove immunosuppression. How to treat tumors. 27. equal to at least the highest therapeutic dosage effective to treat the neoplastic tumor from the group consisting of cytochalasins, and ribosomes and microcapsules in amounts Containing selected pharmaceutical delivery excipients, neoplastic tumors without producing immunosuppression Pharmaceutical preparations for treating ulcers. 28. The cytochalasins are cytochalasin A, cytochalasin B, and cytochalasin. 28. The product of claim 27 selected from the group consisting of Cytochalasin D and Cytochalasin E. Pharmaceutical preparations. 29. The pharmaceutical according to claim 27, which contains cytochalasin B and ribosomes. Medical preparation. 30. The cytochalasin ranges from about 1/2 of the MTD to about MTD for the particular route of administration. 28. The composition of claim 27, administered in an amount range of TD. 31. Cytochalasin compound in an amount effective to reduce spleen size and cell number A method of treating splenomegaly in a mammal comprising administering an agent. 32. The cytochalasin ranges from about 1/2 of the MTD to about MT for the particular route of administration. 32. The method of claim 31, wherein the method is administered in an amount range of D. 33. The cytochalasins are cytochalasin A, cytochalasin B, and cytochalasin. The person according to claim 31 selected from the group consisting of Cytochalasin D and Cytochalasin E. Law. 34. The cytochalasin is selected from the group consisting of the compositions of claim 1. 32. The method of claim 31, wherein the method is a synthetic cytochalasin. 35. The general formula of oxidized or reduced C219, C22, and carbon is the following general formula. A composition according to claim 1. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R2, R3, R4 and R5 are hydrogen, hydroxy or halogen, and R 2 and R3 or R4 and R5 together are oxygen, or R2 and R4 together 36. Claim that R1, R2, R3, R4 and R5 are hydrogen The composition according to range 35. 37. Claim 3 wherein R1 is fluorine and R2, R3, R4 and R5 are hydrogen. 6. The composition according to 6.