CA2031520A1

CA2031520A1 - Cytochalasin compositions and therapeutic methods

Info

Publication number: CA2031520A1
Application number: CA002031520A
Authority: CA
Inventors: Thomas P. Fondy; Dennis Bogyo
Original assignee: Syracuse University
Current assignee: Syracuse University
Priority date: 1989-04-28
Filing date: 1990-04-27
Publication date: 1990-10-29
Also published as: JPH03505744A; EP0429585A4; WO1990013293A1; EP0429585A1

Abstract

ABSTRACT OF THE DISCLOSURE

The present invention is directed to naturally occurring and synthetic cytochalasin compositions and therapeutic treatments utilizing these compositions. More specifically, the present invention relates to certain synthetic analogues of Cytochalasin B (CB) and sustained release formulations containing a cyctochalasin, for example, cytochalasin B and other natural cytochalasins, for example, Cytochalasin D, E, H or J, among others and one or more of its synthetic analogues. This invention also relates to the surprising discovery that the administration of cytochalasins including CB produces transient immunosuppression which is controllable by dose or route of administration and is reversible spontaneously or with the use of IL-2. Thus, a therapeutic regimen of cytochalasins may be used to treat the undesirable hyperimmunity of transplant patients and patients with autoimmune disease. In addition, anti-tumor therapy utilizing CB and other cytoshalasins, and optionally antineoplastic agents other than cytochalasins may be signifantly enhanced by combining the administration of these agents with effective amounts of IL-2 or other lymphokines for reversing the immunosuppression produced during administration of cytochalasins with other antineoplastic agents. This invention also relates to sustained release formulations utilizing liposomes of microcapsules which are effective for delivering high concentrations of cytochalasins and optionally, additional antineoplastic agents to the active site of the tumor without producing undesirable immunosuppression.

Description

W O gO/l3293 2 0 3 1 ~ 2 ~ PCT/US90/02342 CYTOC~ALASIN COMPOSITIONS AND THERAPEUTIC METHODS
s Field of the Invention The present invention i8 directed to naturally occurring and synthetic cytochalasin compositions and therapeutic treatments utilizing these compositions. More 3pecifically, the present invention relates to certain syn~hetic analogues of Cytochalasin B
(CB) and sustained release formulations containing a cyctochalasin, for example, cytochalaRin B and other natural cytochalasins, for example, Cytochalasin D, E, H or J, among others, and one or more of its ~ynthetic analogues. This invention also relates to the surprising discovery that the administration of cytochalasins including CB produces transient i~munosuppresslon which is controllable by dose or roste of administration and iq reversible spontaneously or with the use of IL-2. Thus, a therapeutic regimen of cytochala3ins may be used to treat the undesirable hyperimmunity of transplant patient~ and patients with autoimmune diseaqe.

In addition, anti-tumor therapy utilizing CB and other cytochalasins, ~nd optionally antineopla~tic gents other than cytochalasins may be signlficantly enh~ced by combining the adminlstration of the~e agents with effective amounts of IL-2 or other lympho~ines for rever~ing the im~unosuppression produc~d during administration of cytochalasins or cytochala~ins with other antineoplastic agents.

This invention also relates to sustained release formulations utilizing liposo~es or microcapsules which are effective for delivering high concentrations of cytochalasins and optionally, additional antineoplastic agents to the active site of the tumor without producing undesirable immunosuppression.

W O 90/13293 2 0 3 1 'j 2 ~ PCT/US90/02342 Background of the Inventlon The cytochalasins, membrane- and transport-actlng compounds (also ha~ing cytoskeletal-acting effects), lnclude the congenerlc cytochalasin~ A-M as well as the semi-synthetic derivatives 7,20-di-0-acetyl-cytochalasin B, 7-mono-0-acetyl-cytochalasin B, 21,22-dihydro-cytochalasin B and 21,22-dihydro-cytochalasin A, together with the related chaetoglobosins, constitute a class of more than 24 structurally and functionally related alkaloid metabolites produced by molds (Yahara et al., 1982, J. Cell Biol., 92:69-78, and Rampal et al., 1980, Biochem., 19:679-683). These substances are known to alter a wide variety of cellular functions in many different type3 of normal and neoplastic cell~ and t~ssues in culture (Miranda et al., 1974, J. Cell Biol., 61:481-500) and to exh~bit differential effects in some cases between normal and neoplastic cell types (for references, see Lipski et al., 1987, Anal. Biochum., 161:332-340). The various congeners of these agents alter the biochemistry of fundamental cellular processes controlled by cytoskeletal and plasma membr~le interactions. Some of the cytochalasin~, for example, cytochalasin B (CB) plctured below, the most extensively studied of the congeners, inhibit hexose and amino acid transport in normal and neoplastic cells (Kletzien et al., 1973, J. Biol.
Chem., 248:711-719, and Greene et al., 1976, EYP. Cell. Res., 103:109-117, respectively).

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~CH2 C~
H3C--C8 C~ - R
1 20\
CH2 ~ H
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The cytochalasins are also know~ to alter mlcrofilament morpholo~y (Schllwa, 1982, J. Cell Biol., 92:~9-91), thereby affecting the cellular functions that tepend upon microfilament biochomistr~. Some of the cellular processes sen~itlve to ~he cytochalasins are phagocytosis, pinocyto~ 9, cytokinegi9, secretion, and esocytosis, as well as functlons requlrlng movement and/or intercellular adherence including intracellul~r organelle movement and intercellular transport, cell motillty, transport across tissue barrier~, and a variety of immunological response~.
The cytochalasins have only recently been shown to possess substantial activity against tumor growth and metastasis. Earlier studies on the ln vitro effect~ of the cytochala~ins on transformed (neoplastic) cells showed certain efflcacy. For example, W 0 90/13293 2 ~ 3 1 5 2 0 PCT/US90/0~342 O'Neill (1975, Cancer Res., 35:3111-3115), Medina et al. (1980, Gancer Res., 40:329-333), Relly et al., (1973, Nature New Biol., 242:217-219), and Defendi et al., 1972, Nature New Blol., 242:24-26) showed differential effects between normal and neoplastlc cells, involving nuclear dlvislon and cytokinesls resulting ln multinucleatlon of neoplastic cells. However, the early in vlvo chemotherapeutic studies did not reveal anti-tumor activity against intraperitoneal Ascltes tumors (Katagirl et al., 1971, J.
Antlbiotics, 24:722-723, and Minato et al., 1973, Chem. Pharm.
Bull., 21:2268-2277). In fact, intravenous adminlstratlon of B16F10 cells, treated with CB in vitro showed an increase ln extra-pulmonary metastases (~art et al., 1980, J. Natl. Cancer Inst., 64:891-900).

In spite of the dramatic effects demonstrated by the cytochalasins and related substances on cells and tlssues ln vitro, few studies were conducted with these substances in vivo until only recently because of the lack of availability of sufficient quantlties of these compounds, especially CB. aecent advances in methodologies for producing cytochalasins (see PCT Application US88/02095) have resulted in increased availability of larger quantities of the cytochalasins and in vivo administratlon and blostudies have become more routine.

Certain of these studies have shown that the cytochalaslns, and especially, CB inhlbit the growth and metasta~es of tumors, extend the tumor latency period, and ~xtend host survival ln a murine carcinoma and murine melanoma model system (Bousguet, et al., submitted for publication). These results lead to the proposal that Cytochalasins ~ay function as chemotherapeutlc amplifiers of the activity of known anti-tumor agents by virtue of the effects on cytoskeletal and plasma membrane functlons. In recent studles, cytochalasins have been shown to affect leukocyte-mediated functions in vitro, the differentiation of cytolytic T lymphocytes and also to affect cell motility, cell adherence, phagocytosis and secretion (Wessels, et al. Science, 171, 135, 1971), factors which may affect host immune responses ln vivo. Other studie~ have traced the W O gO/13293 2 0 3 ~ ~ 2 0 PCT/US90/02342 pharmacokinetics of CB in various tlssues, lncludlng the spleen and liver, of mlce ln~ected wlth CB intraperitoneally (Llpskl, et al., 1987, Analytical Blochemlstry 161, 332-340). No study, however, has discussed or detalled the translent lmmunosuppresslve effects of CB
ln ~ivo.

In recent years organ transplants and foreign tissue grafts have become commonly used for treatin8 degenerative dlseases or otherwise saving the lives of patients. However, the introduction of foreign tlssue or a foreign organ ineo a patient is far from a rlsk-free procedure. Often, the tissue or organ is re~ected by the patlent's immune ~ystem, which detects the transplanted organ or grafted tissue as forelgn. Currently-used immuno~uppressive agents, for example, cyclosporin, are effective in reducing the chance of re~ection by the patient, but often at the cost of reducing the patlent'~ overall abllity to ward off infection. In most cases, mortality from re~ection of the transplant is not as high as the mortality rate from infection. The transplant patiene can often die of infectlon usually or otherwise from non-athogenic diseases. In addition, there is often a high lncidence of leukaemia or other malignancy with currently used immunosuppressive agents.

Certain of these currently used agents also find limited use in treating autoimmune diseases. The therapy suffers from the same limitations which occur when immunosuppresslve agents are used to treat transplant or tlssue graft re~ection. The search, therefore, continues for more effective lmmunosuppressivz agents that are effective at controlling a helghtened immune response (hyperim~unity~ ln patient3 with transplanted organs, grafted tissue or an autoimmune disease wlthout affecting the abillty of the patient's sy~tem to fight off infection. The pre~ent lnvention, therfore, al90 relates to the surprl31ng dlscovery that the cytochalasin , especially CB, exhibit significant transient, readily reversible immunosuppression ln vivo.

Cytochalalasins, and especially CB, may become drugs of choice, either alone or ln combination with known antineoplastic agents for W 0 90/13293 2 0 31~ 2 0 PCTIUS90/02342 the treatment of tumors. CB, however, after adminlstration, metabolizes quic~ly to the more highly toxic cytochalas1n A (CA), as well as other metabolltes. Past stud1es have suggostet that CA i9 hlghly reactlve w1th thiol groups, a fact which mlght explaln the high tox~city assoclated w1th 1ts use. It i8 belleved that the metabollc oxldatlon of the 20-hydroxyl group of CB to the 20 keto group ln CA to form the hlghly reactlve enedione system, a chemical system whlch is thought to be highly reactlve with th10ls in enzymes and other proteins, may be primarily respons1ble for the high toxicity associated with the administration of CA. There is therefore a need in the art for an agent or group of agents whlch can take advantage of the pharmacological effects of the cytochalasins to inhibit the growth and metastasis of tumor cells, yet which decrease the general toxicity associated with the metabolism to the enedione system of CA.
The cytochalasins exhibit a number of pharmacological effects, many of whlch may be controlled by the amount and route of administration. These pharmacological effects may also be controlled by formulat10ns utilizing l1posome and microencapsulation techniques, or al~ernatively, by the administration of cytochalasins with, for example, lymphokines.

Liposomes are completely closed lipid bilayer membranes which contain entrapped agueous volume. Liposomes are vesicles which may be unilamellar ~single membrane) or multilammelar (onion-like structures characterized by multiple membrane bilayers, each sepsrated from the next by an aqueous layer). The bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. In the membrane bilayer, the hydrophobic (nonpolar) "tails" of the lipid monolayers orient toward the center of the bilayer, wherea~ the hydrophilic ~polar) "heads" orient toward the aqueous phase. The basic structure of liposomes may be made by a variety of techniques known in the art.

The invention of the present application also relates to the discovery that the use of interleukin-2 (IL-2) in comblnation with the cytochalasins and optionally, other antineoplastic agents, eliminates the immunosuppressive effects of the cytochalaslns.
Interleukin-2, a lymphokine which i9 producet by normal peripheral blood lymphocytes and induces proliferation of antigen or mitogen stimulated T cells after exposure to plant lectins, antigens or other stimuli, was first described by Morgan, D. A., et al.., Science, 193, 1007 (1976). Il-2, in addition to its ability to induce proliferation of stimulated T lymphocytes, also modulates a number of functions of immunocytes in vivo. IL-2 is one of several lymphocyte-produced messenger regulatory molecules which mediate immunocyte interactions and functions.

Ob~ects of the Present Invention It i9 an ob~ect of the present invention to provide a therapeutic method for producing translent immunosuppression beneficial for treating disease states or conditions associated with heightened immune functlon, for eY~rple, autoimmune di~eases or heightened immune response after an organ transplant or tissue graft. It i9 a further ob~ect of the present invention to pro~lde antineoplastic formulations and therapeutic methods which employ cytochalasins in combination with IL-2 and optionally, with additional antineoplastic agents.

It is an additional ob~ect of the present invention to provide formulations ~mploying liposomes or microcapsules and therapeutic methods utilizing these formulatlons for treating neoplasia. It is still a further ob~ect of the present invention to provlde synthetic analogs of CB and therapeutic methods employing these analogs as anti-neoplaQtic agents and immunosuppressive agents. These and othe-objects and advantages of the present invention will become apparent from a review of the detailed description of the present invention.

W 0 90~3293 2 0 ~ 1 ~ 2 ~ PCT/US90/02342 Brief Description of the Flgure~

Flgure 1 shows the maximum tolerated do~age (MTD) for CB
administered to B6D2Fl, CD2Fl or C57Bl/6 via intr~peritoneal (IP), sub-cutaneous (SC) and intravenou~ (IV) routes of admlnlstratlon using different vehicles. This information i8 adapted from Table 2 of U.S. Serial No. 208,199, filed June 17, 1988.

Figure 2 shows the effect of varying doses of CB administered to mice 19 hours before splenectomy on immunosuppression.
Figure 3 shows the effect of varying doses of CB administered to mice 3 hours before ~plenectomy on immunosuppression.

Figure 4 shows the effect of the IP administration of 50 mgJkg on the allogeneic re~ection response in vivo.

Figure 5 shows an absence of suppre 3ion of speciflc cytoto~icity nine days after group~ of mice were treated in vivo with 50 ~$/kg CB at any t~me before or after tumor challenge when compared to vehicle-treated controls.

Figureo 6, 7 and 8 show~ the reversal of immunosuppres~ion produced by addin4 IL-2 after mice were treated with 50 mg/kg of CB
at 19 hours, 3 hours and 72 hours before splenectomy.
Figure 9 compares the cytotoxicity eYhibited by spleen cells which are washed prior to treatment with rIl-2 with the cytotoxiclty exhibited by unwashed spleen cells treated with rIl-2.

Figure 10 compares the immuno~uppression produced by high doses of CB administered in microcapsule~ with the immunosuppression produced by CB at doses varying beeween 2 mg/kg to 50 mg/kg admini~tered IP.

Figure 11 shows the effect of treatment with CB at various times before and after eumOr challenge on tumor-induced splenic enlargemene.

W O 90/13293 2 0 3 1 ~ 2 0 PCT/US90/02342 Figure 12 shows the effect of treatment wlth CB at varlous tlmes before and after tumor challenge on tumor-lnduced splenlc cellularlty.

Brief Descriptlon of the Invention The present 1nventlon relates to pharmaceutical compositlonq and therapeutlc method~ for treatlng mammals, especlally humans, with cytochalasins to reversibly suppress the heightened immune response (hyperimmunity) in autolmmune disease states or followlng organ tranqplants or tlssue grafts. In the lmmunosuppressive method of the preaent lnvention, an immunosuppressive dose comprislng no less than about one flfth the maximum tolerated dosage of a cytochalasin is adminlstered lntravenously, intramuscularly, subcutaneously or orally to a pa~ient sufferlng from hyperlmmunlty.
The present inventlon also relates to intramuscular, intravenous, subcutaneous and oral do~age3 of cytochalasins which may be useful for lnh~biting the gro~th and metastasis of tumors without producing concomltant immunosuppresslve effects. In thls aspect of the present invention, cytochalasins comprising no greater than about one fifth the maximum tolerated do~age for a particular route of adminlstratlon are formulated ~nd adminlstered alone or in combination with other antlneoplastic agents for the inhlbltlon of the growth of tumors.

The presene invention also relaees to the discovery that IL-2 as well as other lymphokine may be used to ellminate the immunosuppressive effects produced by the cytoch~lasins. In this aspect of the present lnvention, pharmaceutical composltlon~ and therapeutlc methods employing cytochalasins and IL-2 ln comblnation are presented. Optionally, an additional antineoplastic agent othe-than a cytochalasin may be included for administration ~ith the cytochala~in and IL-2.

The present lnvention also relate~ to pharmaceutical compos~tlons and therapeutlc methods utlllzing cytochalasins in llposome~ or W 0 90/13293 2 ~ 3 :1 ~ 2 0 PCT/US90/02342 m$croc~psules to provlde sustalned release formulatlons of cytochalasln which deliver large antineoplastic doses of cytochalasin to the site of a tumor without producin6 concomit~nt immunosuppresslve effects. A surprlslng dlscovery of the present lnventlon ls that concentratlon~ of cytochalasln up to about three tlmes the maxlmum therapeutic dosage may be administered in vivo in liposomes or microcapsules without producing the immunosuppresslve effects which exist when cytochalaslns are not administered in liposomes or mlcrocapsules.

The present invention further relates to ~eml-synthetic cytochalasin analogues, pharmaceutlcal compositions containing these analogue~ and therapeutic methods utilizing these analogues which do not readily metabolize to the enedione system of cytachala3in A (CA).

Detailed Description of the Invention The following abbreviatlons and deflnltlons will be employed throughout the speciflcatlon:

BID - administered twice daily CA - cytochalasin A
CB - cytochalasin B
CD - cytochalasin D
CE - cytochalasin E
CMC, CM- cellulose - carboxymethyl cellulose oMC/Tw - carboxymethyl cellulose/Tween Cytochala~ins - synthetic or naturally occurrlng mold-derlved mlcrofllament and membrane-actlng compounds.
DMS0 - dimethyl sulfoxlde E:T ratio - effector ~ell:target cell ratlo FAT - vesiclea made by a freeze and thaw technique GI tract - gastrolntestlnal tract ~PLC - high performance llquid chromatography IC - inhibitory concentrasion; IC50 = 50 percent of cells inhlblted IP - intraperltoneal IV - lntravenous W O 90/13293 2 0 3 1 ~ 2 0 PCT/US90/02342 LD - lethal dose LW - large unilamellar vesicles LUVET - LWs made by the VET technique MLV - multllamellar veslcles MTD - maxlmum tolerated dose ~MR - nuclear magnetic resonance PBS - phosphate buffered saline QID - administered four times daily S~ - subcutaneous SPLV - stable plurilamellar veslcles TAD - tumor appearance day TLG - thin layer chromatography Tw - Tween (polyoxyethylene sorbitan monoalkyl ethers);
surface-acting agents VET - vesicles made by an extrusion technique The present invention relates to a method for producing transient immunosuppression in mammals using cytochalasins. More specifically, it has been dlscovered that the cytochalaslns may be used as transient lmmuno~uppressive agents for treating hyperimmunity a3sociated wlth organ transplants, tissue grafts and autoimmune disease, for example rheumatoid arthritis, lupus, autoimmune diabetes, autoimmune thyroiditi~, autoimmune hepatitis.

In the immunosuppressive method of the present invention, a cytochalasin 18 administered to a mammal in an amount within the ran8e of about one flfth the maximum tolerated dose (MTD) to about the MTD for a particular route of adminlstration, preferably about one half to about the MTD of cytochalasin, and most preferably about the MTD. Any of thc cytochalasins lncluding cytochalasln~ A-M as well as the seml-synthetic derivatlves of cytochalasln, lncludlng 21,22-dihydro-cytochala~ln B, 20-deoxy-cytochalasin B, 20-deoxy-21,22-dihydrocytochalasin B, 20-deoxy-20-fluoro-cyto~halasin B, 20-deoxy-20-fluoro-21, 22-dihydrocytochalasin B and-21,22-dihydro-cytochalasin A, among others, may be used in the immunosuppressive method of the present invention.

W O gO/l3293 PCT/US90/02342 Of COurse~ the numerous cytochalaslns dlffer ln their ability to inhlbit microfilament formation, phagocytosis, cytoXinesi9, secretion and exocytosi~, and conseguently, ln thelr ablllty to produce lmmunosuppresslon. In addltlon, the route of admlnlstration and the pharmacokinetics of the cytochalasln derlvative also play an important role in determining the extent of immunosuppression.
Generally, however, immunosuppressive dosages of natural and semi-synthetic cytochalasins fall within the ran8e of about one fifth to about the MTD for a particular route of administration.

Depending upon the type of cytochalasin used and the route of administration, the daily dosage of cytochalasin effective for producing transient immunosuppression will 8enerallY range from about 0.04 mg/kg to about 150 mg/kg and preferably from about 1.0 mg/kg to about 150 mg/kg. Of course, the concentratlon of cytochalasin used will be varied according to the route of administration and activity of the cytochalasin. By way of example, for administration of a cytochalasin via subcutaneous in~ection, the amount of cytochalasin used generally ranges from about 2 mg/kg to about 150mg/kg and preferably 5 mg/kg to about 150 mg/kg. For administration of the cytochalasin qia intravenous in~ection, the amount of cytochalasin-used will generally r~nge from about 0.1 mg/kg to about 20 mg/kg and preferably 0.25 mg/kg to about 20 mg/kg. For administratlon of a cytochalasin via an intramuscular route, the amount of cytochalasin used will generally ran~e from about 1 m8/k8 to about 50 mg/kg and preferably about 2.5 mg/kg to about 50 mg/kg. Also by way of example, effective concentrations of CE and CD useful in the present invention will generally be much smaller (about one tenth) than the concentration of CB used because of the difference in potency. Most preferably, the cytochalasin~
are administered in an amount equal to about the MTD for a particular route of administration. The table presented in figure 1 presents representative MTD for a number of different CB containing vehicles admi~istered to mice IP, SC or IV.

W O 90/13~93 - 13 - PCT/US90/02342 In formulating the cytochalasins for use as immunosuPpressive agents, typical pharmaceutical formulation solvents and delivery suspensions may be utillzed. The formulat10ns are generally admlnistered in admlxture wlth a pharmaceutlcal carrler selected with regart to the intended route of administration and standard pharmaceutical practice. For parenteral administratlon, the cytochalasin~ may be used ln the form of a sterlle, pyrogen-free aqueous solution which may contain other solutes, for example, salts or sugars such as glucose to maXe the solution isotonic. Any suspension or solvent which is pharmaceutically compatible may be used in the present invention. Of course, the type of suspension or solvent used in the parenteral formulations of the present invention may affect the absorptivity of the cytochalasin. Modifying the formulations to maximize the immunosuppressive effect of the cytochalasins is well within the skill of one of ordinary skill in the formulation arts.

Preferred suspensions for parenteral administration include cytochalasins ln CMC 2%/$ween-20 lX. Preferred solutions include cytochalasins in ethanol/saline (1:2) and DMSO.

The immunosuppressive effect of the cytochalasin used may be affected by the site as well as the route of administration. The transient, reversible immunosuppressive effect of the cytochalasins 18 dependent on timing and the amount of cytochalasin to reach the site of acti~ity. Sites of activity for cytochalasin immunosuppression may include the spleen, bone marrow, lymph nodes, thymus or a transplanted organ or graft site. One of ordinary skill in the art will know to vary the amount, route and site of administration to maximize the concentration of cytochalasin at the slte of immunosuppressive activity. When intravenous administration of cytochalasins is contemplated, care must be taken to choose the site of administration to maximize localization of cytochalasin at the slte of immunosuppres~ion within a short time frame, i.e., about ~S one to twelve hours after-administration, and preferably, one to about three hours after administration.

Immunosuppresslve amounts of cytochalasin may al~o be a~ministered orally in gelatin capsules, powders, syrups, ellxlrs, aqueous solutions, suspensions ~nd the like. Oral dosage forms are preferably administered as immedlate release oral products. An immediate release oral product is one that releases the actlve agent immediately after the product reaches the gut. It iB belleved that orally admlnistered cytochalasins which are formulated as immediate release products may reach certain sites of immunosuppressive activity, for example, the spleen, as readily as the parenterally administered cytochalasins, thus promoting the maximum immunosuppressive effect.

The orally administered products may be administered in combination wlth pharmaceutical carriers and dlluents, for example, lactose, sodium citrate, salts of phosphoric acid, magnesium stearate, starch and talc. A preferred route of oral administration is via immediate release soft gelatin capsules in which the cytochalasin is solubillzed in a lower molecular weight polyethylene glycol, or another solvent, for example, DMSO, or mixtures thereof, 20 80 as to maintain the cytochalasin in a soluble or near-soluble state ln the capsule to maximize dissolution, absorptivity and blood concentration of the cytochalasln after the capsule dissolves ln the 61 tract.

Unlike the immunosuppression produced by traditional immunosupprcs~ive agents or which occur a~ side effects from the administration of most anti-cancer agents, the immNnosuppression produced by cytochalasins is transient and readily reversible. The immunosuppression produced by a bolus dose of cytochalasin generally increases or decreases as a function of the concentration of the cytochalasi~ at the site of immunosuppressive activlty. Therefore, $mmunosuppression produced by cytochalasinY may be carefully controlled with a therapeutic regime~ designed to maxlmize the concentration of cytochalasin at the site of immunosuppression using cytochalasin pharmacokineeic data. Treatment may vary such that an immunosuppressive amount of a cytochalasln may be admlnistered as a bolus dose once or twlce daily via a parenteral route, or orally, W 0 90/13293 2 ~ 3 1 5 2 ~ PCT/US90/02342 once every six to twelve hours (qld or bid). Alternatlvely, the cytochalasins may be adminlstered at the onset of helghtened immunity. Of course, the therapeutic reglmen cho~en wlll vary as a function of the actlvlty, route of adminlstratlon and pharmacokinetics of the cytGchalasin chosen.

The present invention also relates to antineoplastic dosage forms of cytochalasin and methods of treatment which lnhlblt the growth and spread of tumors without producing concomitant immunosuppressive effects. In this aQpect of the present invention, a cytochalasin is admlnistered to a patient in an amount equal to no greater than about one fifth of the MTD, and preferably no greater than about one tenth the MTD. As in the immunosuppressive aspect of the present invention, the type, route of administration and pharmacokinetics of ~he cytochalasin derivative used play an important role in determining the proper dosage. Generally, however, cytochalasin dosages which inhibit the growth and metastasiq of tumors but do not produce concomitant immunosuppression fall within the range of about 0.05 mg/kg to about 30 mg/kg.

In this aspect of the present invention, the cytochalasins may be administered p~rentally or orally in the same m~nner and using the same solvents and other additives that are used in the immunosuppressive aspect of the present in~ention, except that lower dosages are to be used to avoid significant immunosuppression. The cytochalaslns in thi~ aspect of the present invention may be formulated in combination with certain antineoplastic agents other than cytochalasins; however, it is preferred that these agents should not themselves produce signific~nt immunosuppression unless additional therapy for eliminating immuno~uppression i9 also used.

The present invention also relates to the surprising discovery that sustained release formulations comprising cytochalasins and lipo~omes and/or microcapsules in dosages significantly higher than those which produce immunosuppression may be administered to patients without causing appreciable immunosuppression. Dosage forms of cytochalasin comprising at least about the maximum 2 0 31~ 2 0 PC~/US90/02342 tolerated doaa8e~ and preferably up to about three tlmes the maxlmum tolerated doQage for a route of adminlstration formulated ln liposomes or microcapsules are useful for treatlng neoplasla wlthout produclng lmmunosuppression. Although numerous antlneoplastic agents may be formulated slong wlth the cytochalasln in the liposomes or microcapsules, it i9 preferred that only those neoplastic agents which exhibie an absence of immunosuppression should be formulated in combinatlon with cytochalasin unless add~tional therapy for ellminating immunosuppression i9 also used.

Liposomes may be used as the sustalned release dellvery vehicle for the admlnlstratlon of the cytochalasin3 and optionally, an antlneoplastlc agent. Any of the techniques known in the art for makin8 liposomes may be used in thls inYention. For example, the original liposome preparation of Bangham et al. ~J. Mol. Biol., 1965, 12:238-252) which involves suspending phospholipids in an organic solvent, evaporatlng the solvent to dryne~s, addlng an appropriate amount of aqueous phase, allowing the mixture eo "swell"
dispersing the sesulting liposomes wh~ch consist of multilamellar vesicles (MLVs) by mechanical means. Other techniques in~olve the use of small sonicated unilamellar vesicles described by Papahad~opoulos et al. (~iochim. Biophys, Acta., 1968, 135:624-638), and large unilamellar vesicle~.

Unilamellar veslcles may be produced using an extrusion apparatus by a method described in Cullis et al., PCT Publicatlon ~o. WO 87/00238, published January 16, 1986, entitled "Extruslon Technique for Producing Unilamellar Veslcles" incorporated hereln by reference. Vesicles made by thls technigue, called LUVETS, are extruded under pressure through a membrane filter. Vesicles may also be made by an extrusion technique through a 200 nm filter; such vesicles are known as VET2GOs.

Another class of liposomes that may be u~ed are those characterized as having s~bstantlally equal lamellar 901ute dlstrlbution. This cla~ of liposomes is denominated as stable plurilamellar vesicles (SPLV) as deflned in U.S. Patent ~o.

4,522,803 to Lenk, et al., monophaslc vesicle8 as de9cribed ln U.S.
Patent No. 4,558,579 to Fountain, et al. and frozen and thswed multila~ellar vesicles (FATMLV) wherein the vesicles are ex~osed to at least one freeze and thaw cycle; thls procedure 19 descrlbed ln Bally et al., PCT Publlcation No. 87/00043, January 15, 1987, entitled "Multilamellar Liposomes Ha~ing Improved Trapping Efficlencies" whlch are lncorporated hereln by reference.

Any of the methods for maklng llposomes can be used to encapsulate the semi-synthetic or naturally occurrlng cytochalasins. Pharmaceutlcal compositisns of the~e liposomal forms of the cytochalasins can be administered in ~itro or in vivo as de~cribed hereinbelow.

A variety of sterols and their water soluble derivatives have been used to form liposomes; see specifically Janoff et al., U.S.
Patent No. 4,721,612, issued January 26, 1988 entltled "Steroidal Llposomes." Mayhew et al. (PCT Publication ~o. W0 85/00968, published March 14, 1985) described a method for reducing the toYic~ty of drugs by encapsulating them in liposomes comprlslng alpha-tocopherol and certaln derlvatlves thereof. Also, a variety of tocopherols and their water soluble derlvatl~es have been used to form llposomes, see Janoff et al., PCT Publicatlon No. 87~02219, Aprll 23, 1987, entitled "Alpha Tocopherol-Based Vesicles."

Alternatively, when an ionizable antineoplastic agent is e~ployed in combination with cytochalasin, the liposomes can be loaded wlth drug accordlng to the procedures of Bally et al., PCT
Publicatlon No. 86/01102, published February 27, 1986, and incorporated herein by reference. This technigue allows the loading of antlneoplastic agents by creation of a tr~nsmembrane concentration grad~ent across the llposome membranes. This gradient i4 generated by a concentratlon 6ratient for one or more ionic species ~e.g., Na+, Cl-, ~+, Ll+, or H+) across the lipo~ome membranes. Preferably, these lonic gradients are pH (H+) gradients, which dri~e the uptake of the ionizable antineoplastic agent across W 0 90/t3293 2 0 31 r~ PCT/US90/02342 the lipocome membranes. Once the llposomes are loaded wlth the antineoplastlc agent(s) by thls or any other method, pharmaceutlcal formulations can be made whlch can be delivered in vltro or in vlvo as described herelnbelow.

In a liposome-drug delivery system, a bioactive agent such as s drug is entrapped ln or associated ~ith the llposome and then administered to the patient to be treated. For example, see Rahman et al., U.S. Patent No. 3,993,754; Sears, U.S. Patent No. 4,145,410;
Papahad~opoulos et al., U.S. Patent No. 4,235,871; Schneider, U.S.
Patent No. 4,114,179; Lenk et al., U.S. Patent No. 4,522,803; and Fountaln et al., U.S. Patent No. 4,588,578. In the present invention, as mentioned hereinabove, the semi-synthetlc and naturally-occurring mold-derived cytochalasins may be entrapped in or associated with liposomes. Additionally, antineoplastic agent~ or agents which are administered to eliminate the immunosuppressive effects of the cytochalasins or the additional antineoplastic agents A~ described hereinabove can be co-entrapped in liposomes, or entrapped in liposome3 co-administered with those liposomes containing the cytochalasins. Such formulations of liposomes may be administered simultaneou~ly or sequentially. A
preferred method of the preQent invention is to entrap up to three time3 the MTD of cytochala~in alone or in combination with an effective amount of an additional antineoplastic agent. This formulation is then administered to the patient a~ a sustained release form without producing substantial immNnosuppression.

Durin8 preparation of the liposomes, organic solvents may be used to suspend the lipids. Suitable organic solvents for use in the present invention include those with a variety of polarities and dielectric propertie~, which solubillze the lipids, for example, chloroform, methanol, ethanol, dimethylsulfoxide (DMSO), methylene choloride, and solvent mixtures such as benzene:methanol (70:30), among others. As a result, solutions (mixtures ln which the l~pids and other components are uniformly dis~ributed throughout) containing the lipid3 are formed. Solvents are generally chosen on W O 90/13293 2 ~ 3 1 ~ 2 ~ PCT/US90/02342 the baqis of their biocompatabillty, low toYicity, a~d solubilization abilities. Liposomes containlng the pharmaceutlcal formulatlons lncluding cytochalaslns of the present invention may be used therapeutlcally in ma~mals, especlally humans, ln the treatment of neoplasms which require repeated admlnistratlon. The sustained release formulations utllize up to three times the MTD of cytochalasin. Such sustained release composltions are effective against neoplasia without producing substantial concomitant immunosuppression.
~igh dosages of cytochalasins may also be administered as sustained release formulations in microcapsules without producing immunosuppression. A preferred type of microcapsule is that encapsulated in a film-forming polymer made of a mixture of polylactate-glycolate in a weight ratio of about 45:55 to about 55:45, preferably 50:50, and similar materials as taught by U.S.
Patent ~09. 4,675,189, 4,585,482, 4,542,025, 4,530,~40, European Patent Applications 87309286.0 (Publication Number 0,266,119), 87307115.~ (Publication Number 0257915), 81305426.9 (Publication Number 0052510) and 83303605.6 (Publication Number 0129619).
Other formulations well known in the art may al30 be used in formulatin8 sustained release versions of cytochalasin in microcapsules. Still other sustained release formulations readily recognized in the art may be used ln this aspect of the present invention provided that the total amount of cytochalasin included within the formulation does not produce immunosuppression, i.e., is no greater than about three times the MTD of cytochalasin. It is recognized that a large number of sustained release formulations containing cytochalasin may be used provided that the release of cytochalasin is such that otherw~se immunosuppressive leYels of cytochalasin are not reached rapidly at immunosuppressive sites, i.e., within a period of at least about 1 to 12 hours, and preferably within a period of at least about 1 to 24 hours.

Delivery systems other than liposomes or microcapsules may also be used for administering both natural and semi-synthetic cytochalasins as immunosuppressive or antineoplastic agents. Such W 0 gO/13293 2 0 3 1 ~ '~ O PCT/US90/02342 dellvery systems include for example osmotlc pumps, eransdermal patches, infusion pumps, biodegradable polymers, monoclonal antibody-linked systems, suppositorles, rhinile, dragees ~nd troches, amon8 others.

The present lnvention also relates to the use of IL-2 to eliminate the immunosuppression produced by cytochalasins. It has been discovered that the use of IL-2 will reverse the immunosuppressive effects of cytochalasins, providing an effective means of overcoming the immunosuppression that occurs when high doses of cytochalasins alone, or in combinatlon with other antineopla~tic agents, are adminl~tered for the treatment of neoplasia. In this aspect of the present inventlon, the administration of IL-2 to cytochalasin im~unosuppressed lymphocytes will eliminate the i~munosuppression.
Although IL-2 adminlstered any time during immunosuppression will eliminate the i 8 osuppression, to maximize its effect, it is preferred that the IL-2 should be administered in con~unction with cytochalasln or within a short period of time thereafter, for example, within one hour. Although the amount of IL-2 administered will vary depending upon the individual patient, the type of cytochalasin used and the extent of immunosuppression, preferred dosages of IL-2 range from about 5,000 to about lS,000 units per kilogr~m per day. Although IL-2 from any source may be utilized ln this aspect of the present invention, includin8 IL-2 produced by cultivating hum~n perlpheral blood lymphocytes or other IL-2 producing cell lines, the preferred IL-2 is hum~n recombinant IL-2 (rIL-2, available from DuPont Wilmington, Delaware). It is to be recognized that any protein having IL-2 activlty may be used to eliminate the immunosuppressive effects of the cytochalasins and that the term IL-2 lncludes proteins i~ which one or more of the ~mino acids of IL-2 have been changed but in which activity is substantially the same as IL-2. For trea~ment of immunosuppression 3S ln humans, huma~ rIL-2 is preferred.

W 0 90/13293 2 0 3 1 ~ 2 ~ PCT/US90/02342 In cases where the therapeutic method of cytochalasin immunosuppression does not rely purely on the tran~lent reversibility of cytochalasin lmmunosuppres10n for lmmune res~oration, the administration of IL-2 ln appropriate concentrations may be used to restore immunity. Alternatively, the administration of IL-2 may be used to regulate the immunosuppresslve activity of administered cytochalasin. Such administraticn may occur during or after the administration of cytochalasin alone or in comblnation with other antineoplastic agents.

IL-2 may be made by cultivating human peripheral blood lymphocytes (PBL) or other IL-2 producing cell lines. IL-2 may also be made by recombinant DNA technology, which has afforded a means to produce mutein~ and other modified versions of naturally occurring IL-2 which may be used to practice the present invention. A
preferred IL-2, human rIL-2 may be purchased from a number of supplier~ (CetuR Corp., Emeryville, Calif.).

IL-2 may be formulated with cytochalasin using any of the solvents, additives and methods described hereinabove, including liposomes and microcapsules for sustained release formulations, providet that the formulations do not disturb the integrity, stability or activity of the IL-2. A~ explained, IL-2 may be administered in combination with cytochalasin or preferably, shortly thereafter, via parenteral routes of admini3tration, preferably ~ia IV infusion. IL,2 and cytochalasin may also be administered in combination with at least one additional antineoplastic agent. Any number of antineoplastic agents may be used in combination with cytochalssins and IL-2, including dosorublcin, daunorubicin or epirubicin, pyrrolizidine alkaloids, the vinca alkaloids such as vinblastine or vincristine, the purlne or pyrimidine derivatives for esample 5-fluorouracil, among others, the alkylating agent~ such as mitosanthrone, mechlorethamine hydrochloride or cyclophosphamide, platinum compounds such as cis-platinum, folic acid analogs such as methotresate, and the altineoplastic antibiotics such as mitomycin or bleomycin, among others. Any number of the previously described ~olvents, addltives and methods may be used for fonmulating such products.

W O 90~l3293 2 0 3 1 'j 2 0 PCT/US90/02342 The present lnvention also relates to a method snd formulatlons for treatin8 splenome~aly (enlarged splQen) ln human9. The same formulatlons of cytochalasln lncludlng the same amounts of cytochalaoln used to lnduce lmmunosuppresslon are effectlve for treatlng splenomegaly resulting from a hy~erlmmune state. Another aspect of the present lnvention relates to novel seml-synthetic analogues o~ CB whlch are not a9 readlly metabollzed to the enedione system of CA as 19 C8. Synthetlc analogues of the present in~ention have the general structure /c~
~CE~2 C~_ H3C~H ~ca~ R
20\
15 ~ ~
CH
\~ /22 CH ~C=0 \ O~
~ ~
~0 1 CH~ \~

where Bl 18 hyarogen or fluorine, and where the carbon-c~rbon bond alpha, beta to ~1 at C21, C22 ls osldized or reduced. The ~eml-synthetic cytochala~in analogueJ of the present invention are commonly know~ as 20-deoxy-cytoch-la~in, 20-deosy-21,21-dlhydrocytochala~in, 20-deosy-20-fluoro-cytochalasin and 20-deo~y-20-fluoro-21,22-dihydrocytochala~in.

The semi-synthetlc cytochalasins of the present lnvention, unllkc CB, may not be as readily metaboli~ed to the enedione system W O 90/13293 ~O~f520 PCT/US90/02342 of CA, a system which is believed to be responsible for much of the toxicity associated with the administration of CB and CA. The enedione system may irreversibly bind with any number of thiol groups and other nucleophiles in the biological system, thus producing toxic effects. The seml-synthetlc cytochalaslns of the present lnventlon are deslgned to malntaln the actlvlty assoclated with other cytochalasins such as CB, including immunosuppresslon, antineoplaqla and antl-metastasis wlthout readlly metabollzing or convertlng, ln vlvo to the enedlone system of CA.
The semi-~ynthetlc cytochalasins of the present inventlon are advantageously longer-actlng than CB or CA and are designed to reduce the marked deleterious side-effects associated with the administration of CB or CA. The semi-synthetic analogues of the present invention may be made by a number of procedures including total chemlcal synthesis. However, the preferred route of synthesizing these analogues is to chemically modify CA which has been previously isolated from the mold D. dematioidea (A$CC 24346) or which has been prepared by the oxidation of CB to CA using standard prior art methods. Any of the known prior art methods for isolating CA or CB may be used, but lt i9 preferred to utilize a batch ab30rption technigue according to the procedure set forth in PCT Publication No. WO 88/ 10259, Application No. PCT/US88/02095, published December 29, 1988, entitled "Cytochalasin Purification Methods and Compositions", which is incorporated by reference herein. Such a procedure results in sufficient production of CB or CA to provide startin8 material for synthetic modifications to produce compositions of the pre3ent invention. Modification of CA
or CB proceed~ by following synthetic methods readily available ln the art. Ihe followin~ general methods are provided by way of examplo only and it is recognized that the semi-synthetic cytochalasins of the present invention may be produced through a number of synthetic pathways. After isolation from mold or alternatively, after oxidation of CB to CA, to produce 20-deoxycytochala~in, CA is sub~ected to a blocXing procedure ln which the free hydoxyl gr~up is blocked with, for example, a formyl group (85X HCOOH at 60 C for one hour). After blocking the free hydroxyl group of CA, the 20 keto group is sub~ected to a reduction W O 90/13293 2 0 3 1 ~ 2 0 PCT/US90/02342 to produce a hydroxYl at ehe C20 position. The free hydroxyl at C20 is then acti~ated with toluensulfonylchloride to produce the "tosyl"
group at C20. After the "tosyl" group i9 formed, lt 18 tlsplaced with sodium lodide to form the 20-lodo hydroxy blocked cytochalasin.
This analogue is then sub~ected to NaBH4 reduction in solvent and the formyl group 18 cleaved off the hydroxyl group to produce 20 deoxycytochalasin.

To produce 20-deoxy-21,22-dlhydrocytochalasin, the free hydroxyl group of CA is blocked with the formyl group, and the resulting product is sub~ected to a blocking procedure in which the 20-keto group i9 blocked as the ketal with, for example 1,3-dihydroxypropane in acid. The activated double bond at the 21,22 position i9 then reduced in NaBH4 to produce the 21,22 dihydro derivative. After the reduction step, and after removlng the blocking group to form ehe 20 keto group, the 20 keto group may be reduced to form 21,22-dihydrocytochalasin B. Alternatlvely, the free hydroxyl group may be first blocked wlth a ~ormyl group and the 2Q keto group reduced. The resulting free hydroxyl group is tosylated and the tosyl group displaced with lodlne as described hereinabove to produce 20-iodo-21,22-dihydrocytochalasin.
20-iodo-21,22-dihydrocytochalasin may be converted to 20-deosy-20-fluorocytochala~in by simple displacement of the iodo group with fluor~ne (KF or CsF/18-Crown 6 in acetonltrile). The 20-fluoro cytochalasin derivative may be a diastereomeric mixture (raeemic at C20), but the fluoro group ut C20 i8 preferably of the same configurntion as 18 the C20 hydroxyl of CB.

To produce 20-deoxy-20-fluorocytochalasin, the hydroxyl group of CA is blocked with the formyl group, the 20 keto group reduced, tosylated and iodinated as described above. The 20-iodocytochalasin is then converted to 20-deoxy-20-fluorocytochalsin by simple displacement of the iodo group at C20 with fluorine as de~cribed above. The 20-fluoro cytochalasin mlxture may be a dlastereomerlc mixture (the fluorine at G20 is a racemic mixture), but the fluoro group at C20 i~ preferably of the same configuration as is the C20 hydroxyl of CB. As with the other cytochalasins, the semi-synthetic W 0 gO/13293 2 0 3 1 ~ ~ O PCTtUS90/02342 cytochalaslns of the present inventlon may be u~ed as immunosuppressive agents or agents for treating neoplasia with or without IL-2 or additional antineoplastic agents. ~he dosage that i9 to be administered to produce transient, reversible immunosupression i-~ the same as for the other cytochalasins, l.e., at least about one half the MTD for a particular route of administration. Because of their expected longer duration activity, smaller amounts of seml-synthetic cytochalasins than CB are to be used to produced immunosuppression or to treat neoplasia.

The dosage of semi-synthetic cytochalasin for treating neoplasia preferably ranges from about one tenth the MTD to about one flfth the MTD when cytochalasin is to be administered alone or in comblnation with additional antineoplastic agents. To minimize or eliminate immunosuppression produced by cytochalasins adminl~tered at high, immunosuppressive dosage levels, IL-2 may be administered in combination with the semi-synthetic cytochalasins. The semi-synthetic cytochalasins may be administered via intravenous, intramuscular, sub-cutaneous and oral routes, according to the general principles and methods and using the additives and solvents fully described hereinabove. Sustained release formulations comprising very high dosages, e.g., within the range of about the MTD to about three times the MTD of the semi-synthetic cytochalasins of the present invention and liposomes or microcapsules are also contemplated by the present invention. Such dosages in sustained release form provide sufficient sntineoplastic amounts of cytochalasin and produce surprisingly low amounts of immunosuppression.

For purposes of practicing the various aspects of the present invention, it is to be understood that in the treatment of neoplasla or an autoimmune disease state using the pharmaceutical compositlon of the present invention the prescribing physician will ultimately determine the appropriate dosage of the cytochalasin and other agents including IL-2 or-neoplastic agents for ~ given human s~bject and condition, and this can be expected to vary according to the age, weight, and response of the individual as well as the nature .

and severity of the patient's disease or condition. Do8ages would be ultimately determined by the admlnistering phyaiclan accordln8 to the specific cytochalasin used, the circumstances of treatment and the pharmacokinetics of the agent in the patient. The general dosage ranges provided hereln should provide a guidellne to follow in making the final dosage determinations. The dosage of the drug in liposomal or microcapsule form for the treatment of neoplasia without immunosuppression will generally be up to about three times that employed for the free drug. In ~ome cases, however, it may be necessary to administer dosages outside these limits.

The following examples are pro~ided for purposes of illustrating the present invention. These examples sre not to be construed as a limitation on the scope of the invention.

Example 1 Cytochalasin I~murosuppression GENERAL MATERIALS A~D METHODS

CB was eYtracted and purified from cultures of the mold D.
dematioidea (ATCC 24346) utilizing a batch absorption eechnique according to the procedure set forth in PCT Application PCT/US88/02095). Crystalline CB was sub~ected to final purification by preparative ~PLC (75:25 MeOH/H20 on reverse phase C18 DYNAMAXtm silica) followed by recrystallization from CHC13. Purity was shown to be greater than 99% by analytical TLC and HPLC.

DBA/2 and C57BL/6 female mice were purchased from Charles River Laboratories, Wilmington, Massachusetts through the Animal Genetics Branch of the National Cancer Institute and used at 8 to 12 weeks of age.

CB was prepared for injection using emulslfying needles of W O gO/13293 2 0 3 1 ~ 2 0 PCT/US90/02342 decreasing bore diameter from 20 gauge to a final 25 gauge accordlng to the method descrlbed ln Bosquet, et al. submltted for publlcation. Brlefly, suspenslons were achleved by welghlng CB into a syringe, loatlng the vehicle into a aecond 9yrlnge, and ConnectlnB
the syrlnges wlth a double-hubbed emulslfying needle.

The suspenslon was forced back and forth repeatedly through the coupling tube until flow was unlmpeded, then progresslvely the bore diameter was decreased from 21 gauge, to 23 gauge and finally to 25 gauge. Syringes and coupler~ were washed with MeOH and the MeOh wash analyzed on TLC to determine residual CB that was not actually dellvered to the test animals.

The culture media for cell culture was RPMI 1640 medium with HEPES, fetal bovlne serum, sodium pYruvate, gentamycin, penlcillln/streptomycln, MEM non-essential amino aclds and trypan blue, purchased from GIBCO, Grand Island, New York, USA.

RPMI 1640 medium with HEPES was supplemented for the 4-day sensitization cell cultures wi~h 10% fetal bovine serum, 0.1 mg/ml gentamycin, 1% sodium pyruvate, lX non-essential amino acids, lX
glutamine, and 50 uM mercaptoethanol. Medium was immediately sterllized by membrane flltratlon and u~ed immedl~tely or, if not used immediately, was directly sterilized prior to use. RPMI 1640 medium used for the 4 hour Cr cytotoxicity assay was supplemented ~th 5% fetal bovi~e serum and lX peniclllin/streptomycin solution.

P815 mastocy~oma tumor cells were maintained in aqcites form by weekly lntraperitoneal passage into syngeneic DBA/2 mice. Tumor cells used for antigen in 4-day sensitization assays were harvested 72 hours after passage of 5 to 10 x 106 cells. The cell3 were washed, resuQpended in RPMI 1640 medium with HEPES buffer and Y-irradiated on ice in a 35 X 10 mm tissue culture dish at a rate of 200 rad/min and 10 cm target distance for a total of 21 minutes (4200 rad). Cr51 labelled tumor used for targets in a 4 hour cytotoxiciey (LMC) assay were prepared according to the method of Cerottini, et al. (1972 Nature, 237, 272 and 1974 J. Exp. Med., 140 W O 90/13293 2 ~ PCT/US90/02342 703) with the modifications of Orsini, et al. tl977, Cancer Bes., 37, 1719).

For ln vlvo allogenelc challenge, 3 X 107 P815 mastocytoma tumor cells were implanted IP into CS7BL/6 mice previously treated or to be treated with IP CB at concentrations of 50 mg/Xg or with vehicle (CMC/TW- control). The mice were in~ected with this dose at -24, -12. -6, -3, si~ultaneously with, or 24, 48 or 72 hours after implantatior.. 9 days after tumor challenge, splenectomy occurred.
In experiments where effects on resi3tance to allogeneic challenge were to be monitored for an extended period, treated flnimals were maintained for fifty days.

For in vitro analysis of CB effects, spleens were aseptically removed from drug-treated and vehicle-treated (control) mice ae 72 hours! 19 hours or three hours after in~ection with CB. Spleen cells were then forced through coarse (50 mesh) and fine (200 mesh) sterilized stainless steel wire mesh to make a single cell suspension. Cells were washed three times (except where indicated) with BPMI 1640 medium and viability waC determined by trypan blue exclusion.

The 4-day sensitization of splenic lymphocytes in the presence of x-irradiated P815 tumor antigen was carrled out by the method of Cerot~iu~, et al. (1972 Nature, 237, 272 and 1974 J. Exp. Med., 140, 703) with the modlfications of Orsini, et al. (1977, Cancer Res., 37, 1719). Spleen cells were cultured in 17.3 X 35 mm 6- well tissue culture plates (Costar, Cambridge, Ma~s., USA) in a total volume of 2 ml. Each well contained 2 X 107 viable spleen cells and 4 X 105 x-irradiated PB15 cells. At the end of the 4 day culture period effector lymphocytes were recovered from each well by gentle scrapin8 with a rubber policemen, and triplicate cultures were pooled. Cells were washed with RPMI 1640 medium and counted with a hemacytometer or flow cytometer (Coulter Diaenostics, Hialeah, Fla., USA). The cytotoxic activity of sensitized spleen cells recovered from the 4 day cultrures was determined in a st~ndard 4 hour 51Cr release assay as previously described by Bogyo and Mihich, Cancer W O 90~13293 2 0 3 1 ~ 2 0 PCT/US90/02342 Research, 40, 650 (1980). Each cell preparatlon was assayed wlth a mlnimum of four different effector:target cell ratios ran8ing from 100:1 to 6:1. The percentage of 51Cr relea~e was calculated according to the formula:
s 51Cr release = cpm supernatant X 100 ~ 100 cpm supernatant + cpm pellet Later resul~s replaced the formula denominator with maxlmum release of 51Cr from cultures treated with 1% SDS.???????? The percentage of specific 51Cr release represents values obtained with immune effectors minus values obtained wlth control lymphocytes cultured for 4 days in the absence of P815 antigen. Administration - of 50 mg/kg CB IP 19 hours before removal of splenic lymphocytes in 4 experiments consistently impaired the development of cytotoxic T
cells in the allogeneic sensitization assay. The range of inhibition observed waq from 33.2% to 56.5% (E:T ratio = 25/1).

When CB was admlnlstered IP at 19 hours and 3 hours before splenectomy, decreasing, but some inhibition was produced after the admlnistration of 25 mg/kg and 10 mB/kg as shown in Figures 2 (19 hours) and 3 ~3 hours). Two experiments using 5 mg/kg CB failed to show signiflcant immunosuppression. 50 mg/kg CB was administered IP
at 3 hours and 72 hours before splenic lymphocytes were removed for ~ensitization cultures. The ranBe of lnhibition at 3 hours before splenec~omy wa~ 57.0% to ~lmost lOOX as ~hown in Fig. 3. CB
admlnl3tered 72 hours before splenectomy failed to produce consistent im~unosupresslon (data not shown).

Example 2 Responsiveness In Vivo to Allogeneic Tumor Challenge in CB-treated Mice Effect of CB at the maximum tolerated IP bolus dose on allogeneic anti-tumor responsiveness was tested directly ln ~ivo.

wo go/-32g3 2 0 3 1 5 2 0 PCT/US90/02342 C57Bl/6 mice tre-ted wlth CB at 50 mg/kg IP a- a bolus ~uspenslon were challenged wlth 3 X 107 vlable P815 mastocytom- cells IP at varlous times before and after CB treatment. Anlmalg were monltored for thelr abillty to re~ect the allogenelc tumor challenge and for speclflc cytotoxlclty ln spleens taken 9 days after tumor challenge. Figure 4 shows the effect of the IP administration of 50 mg/kg on the allogenelc re~ectlon response. Allogeneic regpOnSlVene99 i9 compromlsed when CB is given 12 or 6 hours prior to tumor challenge to the tent that the allogenelcchallenge is lethal ln some of the animals. Survlvors in these groups appeared to have prolonged allogeneic tumor growth since they showed evidence of abdominal distension and illness up to and beyond day 9 in animals that survived to that point and were maintained for observatlon beyond day 9. Allogeneic tumor growth was also apparent in animals treated with CB 3 hours prior to tumor challenge even though all animals survived the challenge. The time course of this ln vlvo suppression thus showed maximum effect between 3 and 12 hours following CB treatment, and is conslstent with the effects observed in spleens removed from animals 3 hours after CB treatment and tested for allogenelc re~ponsiveness ln vltro.

Spleens ~ensltlzed in vivo by challenge with allogeneic tumor and escl~ed from CB-treated and control groups 9 day~ after tumor challen8e were tested for speclfic allogeneic tumor cell cytoto~iclty in ~ltro. Figure 5 shows no suppresslon of specific cytotoslclty ln any of the groups treated with CB at any time before or after tumor challenge when compared to vehlcle-treated controls.
In the groups treated wlth CB either 6 or 3 hours prior to tumor challenge, groups in which allogeneic tumor growth was continuing at the tlme of spleen excision, specific cytotosicity was increased beyond control levels.

~sample 3 Effects of rIL-2 on Splenic Sensitization Human recombinant interleukin-2 (obtained as a gift from DuPont WO gO/13293 2 0 3 1 ~ 2 0 PCr/US90/02342 Corp, Wilmington, Del., USA) was added eo unwashed cultured splenlc cells from example l at a level of 10 U/ml ln 100 mlcrollters of RPMI 1640 medium at the tlme of culture inltiatlon. Unwashed and washed spleen cells were used in orter to determlne whether restoration of immune-responslveness by rIL-2 could be obtained u~der conditions where any endogenous CB, CB metabolites or suppressive factors remalned present. At the end of the 4 day culture perlod effector lymphocytes were recovered from each well by scraplng. The cells were washed and counted with the hemacytometer and then sub~ected to the 51Cr release assay as described ~n Example l. The addition of 10 U/ml of rIL-2 to the 4 day sensitlzation cultures consistently reversed the immunosuppression produced by prior in vi~o treatment of lymphocytes wlth CB. This was observed for CB administered at 72 hours, 19 hours and 3 hours before splenectomy for all levels of CB administered as shown in flgures 6, 7 and 8. Figure 9 shows that washing the cells before growing the cells in culture results in less immunosuppression when no rIL-2 ~s added to the culture, but that rIL-2 reversed immunosuppression in cells that were not washed before belng grown in culture.

In four experlments in which rIL-2 was added to control lymphocyte cultures without P815 antlgen, cytotoxlc activlty increased between 18.9X and 28.4X (data not shown).

Esample 4 ~ffect of CB on Spleen Weight and Spleen Cellularlty in Unchallenged Spleen Cells To establish the actual responslveness per spleen, the effect of CB at doses of 50, 25, 10, and 5 mg/kg IP on spleen welghts and on spleen cellularity when spleens were har~ested 3 or 19 hours after GB treaement were determlned. There was n~ slgnif~cant effect of CB
treatment on spleen welgh~, with a range from 92 to 110% of the mean spleen weight of matched vehicle-treated controls and no significane dose-respons2 effect as shown in Table 1 below.

WO 90~13~93 2 0 3 1 ~i 2 ~ PCl/US90/02342 Table 1 EFFECT OF CB IP ON SPLEEN WEIGHrS

Time of Treatment (Hours) _3 _3 -19 n-6n=3 n=3 CB IP
(mg/kg) X of Vehicle Controls __ __________________ 98 ~D 101 92 ~D 110 Example S

Sustained Release Formula Effects on Splenlc Sensiti2ation In e~periments where microencapsulated CB (formed from a mixture o~ polylactate-glycolate under contr~ct from Southern ~esearch In~titute- which may al80 be prepared according to the general method taught by U.S. Patent No. 4,389,330) was administered to mice and the animals were reeained for ob~ervation for prolonged periods there was no evidence of apparent toxocity as evidence by the absence of deaths, no obvious signs of illness and the maximum weight loss averaged 3X. Figure 10 ~hows that a dose of CB in microenc~psulated form which is 3-fold above the maximum tolerated dose for IP bolus administration can be ad~inistered to mice without producing significant immuno~uppression either 19 hours or 72 hours following lnjection.

WO 90/13293 2 0 3 1 ~ 2 ~ PCl'/US90/02342 The times u9ed for evaluation of potentlal immunosuppresslon by mlcroencapsulated CB were chosen ba9ed on a determlnatlon of the time course of tissue distribution of the microencapsulated drug. CB
was shown to be released from the mlcrocapsules at a rate sufficlent to produce its maximum concentratlon in the spleen and other organs by 3 days after in~ection (compared to 3 hours by way of 50 mg/kg IP
bolus suspension administration). In spite of the high splenic concentration of CB at 3 days following ln~ection with microencapsulated CB, there is only marginal immunosuppression apparent (Figure 10).
This experiment indicates that sustained release formulations of CB and other cytochalasins may be used as antineoplastic agents at higher concentrations than the MTD without producing substantial immuno~uppressive effects.

Example 6 CB Effects on In Vivo Lymphocyte Sensitizatlon Because CB inhiblts cytotoxic T cell response to tumor sensitiz~tion in vitro, the effects of a single IP administratlon of CB on in vivo response of splenic lymphocytes to prollferating P815 tumors was observed. A single 50 mg/kg IP bolus of CB dld not lmpalr the day 9 or day 10 response of mice in~ected IP with 3 X 107 P815 cells as shown by the results of a 51Cr cytotoxicity assay in Table 1. Thls lack of immunosuppression wa3 consistent regardless of whether CB was admini~tered before or after P815 tumor.

Example 7 CB Effects on Splenomegaly Allogeneic challenge of C57Bl/6 mice with P815 mastocytoma cells produces marked splenomegaly in vehicle-treated controls when spleens are excised and weighed 9 days after allogenelc tumor challenge. The increase in spleen weights is more than 3-fold W O 90/t3293 PCT/US90/02342 2031~'~0 greater than in Animals not challenged with P815 cells. Treatment with CB at various times before and after tumor challenge shows a time-dependent effect on tumor-induced splenic enlargement as shown in Figure 11 and a corresponding effect on splenic cellularlty ~9 shown in Figure 12.

Example 8 Synthesis of 20-deoxy-cytochalasin CA is isolated from the mold D. dematioidea (ATCC 24346) or oxidized from CB isolated from that same mold according to the procedure set forth in PCT Publication No. W0 88/10259. CA is sub~ected to blocking of the hydroxyl group with a formyl group by stirring CA in an excess of 85X HCOOH ae 60 C for one ~our or acetylformate/pyridine at -20 C, isolated by ether extraction followed by evaporation of 301vent. The crude formyl blocked Cytochalasin A i~ then sub~ected to reduction in NaBH4/Dimethylformamide (DMF) or dimethylsulfoxide (DMSO) to produce the formyl blocked derivative having a free hydroxyl group at C20.
The free hydroxyl group at C20 is tosylated (excess toluenesulfonylchloride/pyridine at 37 C overnight) and the tosylated product i3 extracted wlth ether after the excesq pyridine i8 evaporated (several times with ethanol). The tosylated product is then sub~ected to reaction with a 1.5 molar excess of NaI in DMF
or DMSO to displace the tosyl group ~ith an iodo group at C20, which i8 then sub~ected to reductive displacement of the iodo group with ~aBH4 in DMF/DMSO and deblocking of the formyl group under acidic conditions (6~ HCl)) to produce 20-deoxycytochalasin. We note that the 20-iodocytochalasin deri~ative may be used to synthe~ize 20-fluorocytochalasin in Example 9. 20-deoxycytochalasin may be purified from the reaction mixture using preparative HPLC. Ie is sometimes preferable, but more time consuming, to isolate the intermediates of the synthesis using preparative HPLC (75:25 Me0H/H20 on reverse phase-C18 DYNAMAXtm silica gel columns) bef,ore proceeding on to the next synthetic step.

W O 90/13293 2 0 3 1 ~ 2 ~ PCT/US90/02342 Example 9 Synthesis of 20-deoxy-21,22-dlhydrocytochalasin As in Example 8, the free hydroxyl group of CA i8 blocked with the formyl group 85X HCOOH at 60 C or acetylformate/pyridlne at -20 C, and the resulting product is sub~ected to a blocking procedure ln which the 20-keto group is blocked as the Xetal with 1,3-dihydroxypropane or alternaeively with 1,3-dimethoxypropane in toluenesulfonic acid. After extraction in ether~ the ketal-protected derivative is sub~ected to NaBH4 reduction (DMF or DMSO) or Pd/CharcoalJH2 reduction in ethanol to produce the ketal protected 21,22 dihydrocytochalasin. The resulting product is then sub~ected to acid cleavage of the ketal (toluenesulfonic acid in dioxane) followed by blocking of the free hydrosyl group with a formyl group in 85X ~COOH at 60 C for one hour or alternatively, acetylformate/pyridine at -20 C. The 20-keto group is reduced to the hydroxy group, the hydroxy group is tosylated and the tosyl group i8 displaced by iodination (this iodo intermediate may be used to prepare the product of Example 11) reduced according to the method descrlbed in Example 8. The formyl group i8 cleaved in 6N
HCl. The resulting product, 20-deoxy-21,22-dlhydrocytochalasin may be purifled from the reaction mixture using preparative HPLC (as described in esample 8).

2sample 10 Synthesi~ of 20-deoxy-20-fluoro-cytochalasin 20-deoxy-20-fluorocytochalas~n may be produced from 20-iodocytochalasin from example 8 by simple displacement of the iodo group at C20 with RF or CsF/18 Crown 6 (1 Molar equivalent of 18 Crown 6) in acetonitrile followed by removal of the formyl blocking group (6~ ~Cl). Preparative APLC may be performed as described in Example 8.

W O gO/13293 2 0 3 1 ~ 2 0 PCT/US90/02342 Example 11 Synthesis of 20-deoxy-20-fluoro-21,22-dlhydrocytochalasin 20-deoxy-20-fluoro-21,22-dihydrocytochalasin may be produced from 20-iodo-21,22-dihydrocytochalsin from example 9 by slmple displacement of the iodo group at C20 wlth KF or CsF/18 Crown 6 (1 molar equivalent of 18 Crown 6) in acetonitrlle a9 per example 10 followet by removal of the formyl blocking group t6N HCl).

Preparative HPLC may be performed as previously described. It is to be understood that the examples and embodiments described hereinabove are for the purpose~ of providing a description of the present invention by w~y of example and are not to be viewed as limiting the present invention in any way. Various modifications or changes that may be made to that described hereinabove by those of ordinary skill in the art are also contemplated by the present invention and are to be included within the splrit and purview of thi~ application and the following claims.

The invention also contemplates new derivatives of cytochalasins as sho~n at page 22 wherein the formula for the osidized or redu~ed C21, C22 carbon is I

R3 C21r-- B2 wherein R2, R3, R4 and R5 are hydrogen, hydrosy or halogen, wherein R2 and R3 or R4 and R5 taken together are oxygen, or wherein R2 and R4 when taken together are oxygen, and additionally wherein Rl is hydrogen or fluoro.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A composition of the general structure where R1 is hydrogen or fluorine and the carbon-carbon bond alpha, beta to R1 at C21, C22 is oxidized or reduced.

2. A pharmaceutical formulation for treating a patient with neoplasia comprising an antineoplastic disease-treating effective amount of a composition from claim 1 and a pharmaceutical carrier or diluent.

3. The composition according to claim 2 adapted for parenteral administration.

4. The composition according to claim 2 adapted for oral administration.

5. A pharmaceutical formulation for treating a mammal suffering from hyperimmunity comprising a pharmaceutically effective amount of a composition from claim 1 and a carrier or diluent.

6. The formulation according to claim 5 adapted to treat hyperimmunity wherein said composition comprises about one-fifth to about the MTD of said composition for a particular route of administration.

7. The formulation according to claim 5 adapted for parenteral administration.

8. The formulation according to claim 5 adapted for oral administration.

9. A sustained release formulation comprising a composition from claim 1 in an amount ranging from about the MTD to about three times the MTD for a particular route of administration and a sustained release administration vehicle.

10. The sustained release formulation according to claim 9 wherein said vehicle is selected from the group consisting of liposomes and microcapsules.

11. A method of treating a mammal for hyperimmunity in an autoimmune disease state or subsequent to an organ transplant or tissue graft comprising administering an amount of a cytochalasin compound effective for producing immunosuppression.

12. The method according to claim 11 wherein said cytochalasin is administered in an amount equal to about one-fifth the MTD to about the MTD of said cytochalasin for a particular route of administration.

13. The method according to claim 12 wherein said cytochalasin is selected from the group consisting of Cytochalasin A, Cytochalasin B, Cytochalasin D and Cytochalasin E.

14. The method according to claim 13 wherein said cytochalasin is a synthetic cytochalasin selected from the group consisting of the compositions from claim 1.

15. The method according to claim 13 wherein cytochalasin B is administered parenterally in amount ranging from about 10 mg/kg to about 50 mg/kg once or twice daily.

16. The method according to claim 13 wherein said cytochalasin is selected from the group consisting of Cytochalasin D and Cytochalasin E and is administered in an amount ranging from about 1 mg/kg to about 5 mg/kg once or twice daily.

17. A pharmaceutical formulation for treating a patient with neoplasia comprising an antineoplastic disease-treating effective amount of a cytochalasin producing immunosuppression and an amount of interleukin-2 effective to eliminate said immunosuppression.

18. The formulation according to claim 17 wherein said cytochalasin is selected from the group consisting of Cytochalasin A, Cytochalasin B, Cytochalasin D and Cytochalasin E.

19. The formulation according to claim 17 wherein said cytochalasin is selected from at least one of the group consisting of the compositions of claim 1.

20. The formulation according to claim 17 wherein said interleukin-2 comprises about 5,000 units to about 15,000 units per kilogram of said patient.

21. The formulation according to claim 17 further comprising an antineoplastic agent other than a cytochalasin in an amount effective to treat said patient's neoplasia.

22. The formulation according to claim 21 wherein said antineoplastic agent is adriamycin.

23. A method for treating a patient with neoplasia comprising administering the pharmaceutical formulation according to claim 17 to a patient suffering from neoplasia.

24. The method according to claim 23 wherein said cytochalasin is selected from the group consisting of Cytochalasin A, Cytochalasin B, Cytochalasin D and Cytochalasin E.

25. The method according to claim 23 wherein said cytochalasin is selected from at least one of the group consisting of the composition of claim 1.

26. A method of treating neoplasia comprising administering a cytochalasin in an amount effective to treat neoplasia and produce immunosuppresion and an amount of IL-2 effective to eliminate the immunosuppression.

27. A pharmaceutical formulation for treating neoplasia without producing immunosuppression comprising a cytochalasin in an amount equal to at least the maximum therapeutic dosage effective for treating neoplasia and a pharmaceutical delivery vehicle selected from the group consisting of liposomes and microcapsules.

28. The pharmaceutical formulation according to claim 27 wherein said cytochalasin is selected from the group consisting of Cytochalasin A, Cytochalasin B, Cytochalasin D and Cytochalasin E.

29. The pharmaceutical formulation according to claim 27 comprising cytochalasin B and liposomes.

30. The composition according to claim 27 wherein said cytochalasin comprises at least about the MTD and no greater than three times the MTD for a particular route of administration.

31. A method of treating-splenomegaly in a mammal comprising administering an amount of a cytochalasin compound effective to reduce the size and cell number of the spleen.

32. The method according to claim 31 wherein said cytochalasin is administered in an amount ranging from about one half the MTD to about the MTD for for a particular route of administration.

33. The method according to claim 31 wherein said cytochalasin is selected from the group consisting of Cytochalasin A, Cytochalasin B, Cytochalasin D and Cytochalasin E.

34. The method according to claim 31 wherein said cytochalasin is a synthetic cytochalasin selected from the group consisting of the compositions from claim 1.

35. The composition of claim 1 wherein the formula for the oxidized or reduced C21, C22 carbon is wherein R2, R3, R4 and R5 are hydrogen, hydroxy or halogen, wherein R2 and R3 or R4 and R5 taken together are oxygen, or wherein R2 and R4 when taken together are oxygen.

36. The composition of claim 35 wherein R1, R2, R3, R4 and R5 are hydrogen.

37. The composition of claim 36 wherein R1 is fluoro, and wherein R2, R3, R4 and R5 are hydrogen.