WO2000073257A1

WO2000073257A1 - Derivatives of butyric acid and uses thereof

Info

Publication number: WO2000073257A1
Application number: PCT/US2000/014523
Authority: WO
Inventors: Kathleen Gilbert; E. Kim Fifer
Original assignee: The Board Of Trustees Of The University Of Arkansas
Priority date: 1999-05-28
Filing date: 2000-05-26
Publication date: 2000-12-07
Also published as: AU5165200A

Abstract

The present invention provides a series of compounds having structural formulas (I), (II), (III), (IV) wherein n1 is 1 to 5, n2 is 1 to 4 and m is 1 to 3; X is O or NH; Y is CH2, O, S, NH, NR; R is selected from the group consisting a straight-chain aliphatic group, a branched-chain aliphatic group and an alicyclic group; wherein R' is selected from the group consisting of hydrogen, methyl and ethyl; when Y is O, n1 is not 1; and wherein X and R' are independently optionally substituted at C2, C3 or C4 in compounds of Formula (IV) or a pharmaceutically acceptable salt thereof. Also provided is a method of inactivating antigen-specific T cells in an individual.

Description

DERIVATIVES OF BUTYRIC ACID AND USES THEREOF

BACKGROUND OF THE INVENTION

Cross-R-eference to Related Application

This application claims the benefit of priority of U.S. provisional patent application Serial Number 60/136,579, filed May 28, 1999, now abandoned.

Field of the Invention

The present invention relates generally to the fields of organic chemistry and immunology. More specifically, the present invention relates to derivatives of butyric acid and their use a s inactivators of antigen-specific T cells.

Description of the Related Art

Helper T cells (Th) are regulatory lymphocytes which cooperate with other lymphocytes to expedite an immune response. Generally, helper T cells recognize protein antigens after the antigens have been processed into peptide fragments and have become associated with a class II MHC molecules. Autoimmunity is an immune response directed against self- antigens resulting from the breakdown of the normal mechanisms of self-tolerance that prevent the production of functional self- reactive clones of T cells.

Butyric acid is a naturally occurring four carbon fatty acid found in the gut as a result of fiber fermentation. n - Butyrate is well known as an anti-neoplastic agent and for its ability to induce Gl arrest in virtually all cell types tested and to induce cytodifferentiation of many different transformed cell lines. In murine CD4+ T cells of the T helper type 1 (Thl), anergy is induced when the cells are pretreated with n-butyrate an d antigen thereby becoming unresponsive; i.e., they exhibit a long lasting inability to proliferate or secrete cytokines, to a subsequent stimulation with antigen in secondary cultures (Gilbert & Weigle, J. of Immunology 151(3) 1245- 1254 ( 1993)). I n contrast, Th 1 cells treated with n-butyrate alone, in the absence of antigen stimulation, are totally unaffected by the drug.

Thl cells exposed to n-butyrate in the presence of antigen lose their ability to proliferate to antigen, but retain their ability to proliferate in cultures stimulated with exogenous

Interleukin-2 (IL-2). Therefore, Thl cell unresponsiveness after exposure to n-butyrate is not caused merely by drug toxicity.

Similarly, the inhibitory effects of n-butyrate are not caused by a drug-induced shift in Thl cell proliferation kinetics. The induction of tolerance with n-butyrate is antigen-dependent, and is linked to a decrease in antigen-induced secretion of IL-2. The immunotherapeutic potential for n-buytyrate is limited by its short half-life in vivo (3-6 minutes) (Daniel et ai.,

1989). Even when it is administered by intravenous infusion, n - butyrate is found to be clinically ineffective as an anti-cancer agent (Miller et al, 1987; Novogrodsky et al, 1983) . As n - butyrate contains sodium and as it has a half-life of six minutes, large doses are required resulting in a high dose of sodium administered to a patient. n-Butyrate/tributyrin derivatives with ester and amide functional groups undergo hydrolysis in vivo to release butyrate. This leads to a more sustained release of butyric acid and significantly prolongs the duration of action. These butyrate derivatives also contain an ionizable amino group. This not only allows the compound to be converted to water soluble salts (e.g. hydrochloride), but also avoids the necessity of using the sodium salt of butyric acid which could lead to sodium overload. Therefore, when administered briefly during a n autoimmune response, a member of the bu tyrate/tributyrin family of drugs acts to convert activated self-reactive T cells to a n unresponsive state, but has no effect on the majority of resting T cells/

The prior art is deficient in effective means of using butyrate/tributyrin prodrugs to convert activated self-reactive T- cells during an autoimmune response to an unresponsive s tate without adversely effecting the majority of resting T cells. The present invention fulfills this long-standing need and desire in th e art.

SUMMARY OF THE INVENTION

In one embodiment of the present invention, there are provided a series of compounds having structural formulas

R' O ₃-mN-((CH₂)_n|-CH-X-C-CH₂-CH2-CH3 )n

R'

Y N-(CH₂)_n CH-X -C-CH2-CH2-CH3

I I O , _k R' O

I I I

H₃C-CH₂-CH₂-C- ;sHCH₂)_nrCH-X-C-CH₂-CH₂-CH₃

I l l

I V

wherein nj is 1 to 5, n2 is 1 to 4 and m is 1 to 3; X is O or NH; Y is CH₂, O, S, NH, NR; wherein R is selected from the group consisting of a straight-chain aliphatic group, a branched-chain aliphatic group and an alicyclic group; wherein R' is selected from the group consisting of hydrogen, methyl and ethyl; wherein when Y is O, n j can not be 1 ; and wherein X and R' are independently optionally substituted at C2, C3 or C4 in compounds of Fomula IV or a pharmaceutically acceptable salt thereof.

In another embodiment of the present invention, there is provided a pharmaceutical composition, comprising a compound of the present invention and a pharmaceutically acceptable carrier.

In yet another embodiment of the present invention, there is provided a method of inactivating antigen-specific T cell s in an individual in need of such treatment, comprising the step of administering to said individual an effective amount of compound having the structure

/ \ R I ' M

Y N-(CH₂)_nrCH-X -C-CH₂-CH₂-CH₃

I I

II I

I V

wherein n_j is 1 to 5, n₂ is 1 to 4 and m is 1 to 3; X is O or NH; Y is CH2, O, S, NH, NR; wherein R is selected from the group consisting a straightrchain aliphatic group, a branched-chain aliphatic group and an alicyclic group; wherein R' is selected from the group consisting of hydrogen, methyl and ethyl; and wherein X and R' are independently optionally substituted at C2, C3 or C4 in compounds of Fomula IV or a pharmaceutically acceptable salt thereof.

Other and further aspects, features, benefits, an d advantages of the present invention will be apparent from th e following description of the presently preferred embodiments of the invention given for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention are briefly summarized. above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted; however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope. Figure 1 shows the synthesis of 1 2- (4- morpholinyl)ethyl butyrate (Figure IA), 2-(4-morpholinyl)ethyl butanamide 2 (Figure IB), 2-(4-butanoylpiperazinyl)ethyl butanoate 3 (Figure 1C), 2,2',2"-nitrilotrisethyl trisbutyrate 4 (Figure ID), and l-methyl-4-piperidinyl butanoate 5 (Figure IE). Figure 2 shows that butyrate derivatives inhibit proliferation of Thl cells. KLH-specific Thl cells (clone D3) are stimulated with IL-2 (10% Con A CM) in the presence of different concentrations of n-butyrate or butyrate derivatives. In Figure

2 A Thl cell proliferation is measured after 2 days. I n Figure 2B, the inhibitors are washed out after 24 hours, and th e Thl cells reincubated with fresh IL-2-containing medium. Proliferation of the Thl cells restimulated with IL-2 in th e absence of the inhibitors is measured after an additional 2 days . This experiment was repeated three times, and the proliferation data represents the means of the four experiments.

Figure 3 shows that MEB inhibits primary antigen- specific antibody production in vivo. C57BL/10 mice are injected ip on Day 0 with ovalbumin followed by ip injections of saline, or one of three different butyrate derivatives on Days 1-3. Levels of total anti-ovalbumin antibody in the serum 10 days following antigen administration are measured. *Significantly different from response of mice treated with saline, p < 0.05.

Figure 4 shows that MEB induces antigen-specific T cell inactivation in vivo . C57BL/10 mice are injected ip with ovalbumin/CFA on day 0, followed by a single ip injection of saline or MEB (0.15 mmol) on Days 2 or 3. The mice are reimmunized with ovalbumin sc on Day 10. Isotype-specific anti-ovalbumin antibody generated during the primary immune response (Figure 4A), and the secondary immune response (Figure 4B) are measured in the serum at day 10 and day 16, respectively. (Figure 4C). Lymph node CD4⁺ T cells isolated from the mice 6 days after the second immunization with ovalbumin are stimulated with ovalbumin in vitro, and examined for proliferation. *Significantly different from response CD4⁺ T cells isolated from mice treated with saline, p <0.05. This experiment was repeated, and the proliferation data represents the means of the two experiments.

Figure 5 shows that MEB induces antigen-specific T cell inactivation in vitro. KLH-specific Thl cells (clone D3) are incubated in primary cultures with (Figure 5 A ) MEB (1 mM) and/or IL-2 (10% Con A CM), or (Figure 5B) MEB (ImM) an d/or KLH (50 μg/ml). After 2 days, the Thl cells are isolated from th e primary cultures, rested, and then re-incubated in secondary cultures stimulated with antigen (KLH, 50 μg/ml) or IL-2 (10% Con A CM). Thl cell proliferation in the secondary cultures is measured. * Significantly different from response of Thl cel!s exposed to MEB alone, p< 0.01.

Figure 6 shows MEB-induced T cell unresponsiveness is antigen-specific. (Figure 6 A ) Spleen cells from DBA/2 mice (H-2^d) were stimulated in a primary mixed lymphocyte response (MLR) with irradiated spleen cells or from C57BL/10 mice (H-2^b) in the presence ( O ) or absence (•) of MEB (ImM). As a negative control some wells are stimulated with spleen cells from DBA/2 mice (▼). Proliferation is measured on day 5. (Figure 6B ) After 3 days, some wells in the primary MLR cultures are washed, and rested for a further 2 days. The spleen cells th at were stimulated in the primary MLR with spleen cells from C57BL/10 mice in the presence ( O ) or absence (•) of MEB are then isolated and reincubated with either the original alloantigen (spleen cells from C57BL/10 mice), with a third-party alloantigen [spleen cells from C3H/HeJ mice (H-2^k)], or with IL-2 (Con A CM). Proliferation was measured on day 5 of the secondary MLR. * Significantly different from response of spleen cells not exposed in the primary MLR to MEB, p< 0.01. Figure 7 shows that MEB blocks Thl cell cycle progression in Go/G^ KLH-specific Thl cells (clone C3) are left unstimulated (resting), or are stimulated with IL-2 (10% Con A CM) in the presence or absence of MEB (1 mM). The Th l cells are collected after 4 8 hours, and assayed for DNA content.

Figure 8 shows that MEB sequesters stimulated Th l cells in G₀/G_j. KLH-specific Thl cells (clone C3) were stimulated with IL-2 (10% Con A CM) at the initiation of culture, and then again at 24, 48, and 96 hours (Figure 8A). Some cultures also received a single dose of MEB (1 mM) at times 0, 15, 24 or 3 9 hours after the initiation of culture (Figures 8B, 8C, 8D, and 8E, respectively). The Thl cells were collected at 0, 48, 72, 96 or 1 20 hours after the initiation of culture and assayed for DNA content.

DETAILED DESCRIPTION OF THE INVENTION

The following abbreviations are used herein: KLH: keyhole limpet hemocyanin; PBS : phosphate buffered saline; Con A CM: conditioned medium from rat spleen cells stimulated with Concanavalin A; IL-2: interleukin-2; IL-4: interleukin-4; APC: antigen presenting cells; MEBA: 2-(4-Morpholinyl)ethyl butanamide hydrochloride; BEB: 2-(4-Butanoylpiperazinyl)ethyl butanoate hydrochloride; MEB: 2-(4-Morpholinyl)ethyl butyrate hydrochloride.

As used herein, the term "individual" shall refer to animals and humans.

The present invention provides ester, amide and ester/amide derivatives of n-butyric acid. The general structures of the compounds of the present invention and synthetic schemes of said compounds are shown in Figure 1. These butyrate prodrugs contain an ionizable amino group which, when converted to a water soluble salt, increases the overall aqueous solubility.

The ester and/or amide functional groups undergo hydrolysis to release butyric acid. The optional substitution of th e carbon adjacent to the X group by a methyl or ethyl provides steric hindrance, thereby slowing the in vivo hydrolysis rate and increasing the half-life of the parent compounds. Thus, this sustained release of butyric acid coupled with a longer half-life maintains a butyrate blood level over a longer period of time an d increases the dosing interval.

The butyrate prodrugs and methods of the pre sent invention may be used to inactivate antigen-specific T cells thereby providing immunotherapeutic methods to treat autoimmune diseases, to treat or to prevent other disorders involving an autoimmune component and as anti-cancer reagents . Using butyrate prodrugs is more advantageous than using traditional immunosuppressive drugs. Treatment is short term, rather than long term; the prodrugs of the present invention induce T-cell anergy instead of temporarily suppressing T-cell activity. Additionally, unlike most immunosuppressive drugs, butyrate and its prodrugs are non-toxic.

Autoimmune diseases are characterized by immune cell destruction of self cells, tissues and organs. In systemic autoimmune diseases where lack of information concerning th e self-proteins targeted by the auto-reactive T-cells precludes peptide-based immunotherapies, the time-release characteristics of butyrate prodrugs are beneficial. Representative examples of such autoimmune diseases are rheumatoid arthritis diabetes, multiple sclerosis and systemic lupus erythematosus.

The immunotherapeutic method of the present invention is also useful in terms of allograft and xeno graft transplantation rejection. Concurrent administration of a butyrate prodrug enhances the tolerogenicity of donor cells, an d thereby increases the likelihood of engraftment. The compounds and methods of the present invention may also be used to treat neoplastic diseases. Previously, the u s e of n-butyrate as an anti-cancer reagent was thwarted by its rapid excretion rate. The varied rates of hydrolysis of the butyrate prodrugs of the present invention and their longer half-lives provide a more effective means of treatment. Representative examples of such neoplastic diseases which could be treated using these compounds and methods are renal cancer, ovarian cancer, lung cancer, glioma and leukemia. The methods of the present invention may be used to treat any animal. Most preferably, th e methods of the present invention are useful in humans.

Pharmaceutical compositions are prepared using th e novel prodrugs of the butyrate/tributyrin family of drugs of th e present invention. In such a case, the pharmaceutical or immunogenic composition comprises the novel compounds of th e present invention and a pharmaceutically acceptable carrier. A person having ordinary skill in this art would readily be able to determine, without undue experimentation, the appropriate dosages and routes of administration of the novel compounds of the present invention.

Compounds of the present invention, pharmaceutically acceptable salt thereof and pharmaceutical compositions incorporating such, may be conveniently administered by any of the routes conventionally used for drug administration, e.g., orally, topically, parenterally, or by inhalation. The compounds of th e present invention may be administered in conventional dosage forms prepared by combining the compound with standard pharmaceutical carriers according to conventional procedures.

The compounds of the present invention may also b e administered in conventional dosages in combination with a known, second therapeutically active compound. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable carrier or diluent is dictated b y the amount of active ingredient with which it is to be combined, the route of administration and other well known variable. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The pharmaceutical carrier employed may be, for example, either a solid or a liquid. Representative solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium sterate, stearic acid and the like. Representative liquid carriers include syrup, peanut oil, olive oil, water and th e like. Similarly, the carrier may include time delay material well known in the art such as glyceryl monosterate or glyceryl disterarate alone or with a wax.

A wide variety of pharmaceutical forms can b e employed. Thus, if a solid carrier is used, the preparation can b e tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge. The amount of solid carrier will vary widely but preferably will be from about 25 mg to about 1 gram. When a liquid carrier is used, the preparation will be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable liquid such as an ampule or nonaqueous liquid suspension.

Compounds of the present invention may b e administered topically (non-systemically). This includes the application of a compound externally to the epidermis or th e buccal cavity and the instillation of such a compound into the ear, eye and nose, such that the compound does not significantly enter the bloodstream. Formulation suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of inflammation such as liniments, lotions, creams, ointments, pastes and drops suitable for administration to the ear, eye and nose. The active ingredient may comprise, for topical administration from 0.001 % to 10% w/w , for instance from 1% to 2% by weight of the Formulation. It m a y however, comprise as much as 10% w/w but preferably will comprise less than 5% w/w, more preferably from 0.1 % to l% w / w of the Formulation.

Lotions according to the present invention include those suitable for application to the skin and eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops. Lotions or liniments for application to the skin may include an agent to hasten drying and to cool th e skin, such as an alcohol or acetone, and/or a moisterizer such a s glycerol or an oil such as castor oil or arachis oil.

Creams, ointments or pastes according to the pre sent invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy base. The base may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap, a mucilage, an oil of natural origin such as almond, corn, archis, castor, or olive oil; wool fat or its derivatives or a fatty acid such as steric or oleic acid together with an alcohol such as propylene glycol or a macrogel. The formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as a sorbitan ester or a polyoxyethylene derivative thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin may also be included.

Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions and may b e prepared by dissolving the active ingredient in a suitable aqueou s solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container which is then sealed an d sterilized by autoclaving. Alternatively, the solution may b e sterilized by filtration and transferred to the container by a n aseptic technique. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenymercuric nitrate or acetate (-0.002%), benzalkonium chloride (-0.01%) an d chlorhexidine acetate (-0.01%). Suitable solvents for th e preparation of an oily solution include glycerol, diluted alcohol an d propylene glycol.

Compounds of the present invention may b e administered parenterally, i.e., by intravenous, intramuscular, subcutaneous, intranasal, intrarectal, intravaginal or intraperitoneal administration. The subcutaneous an d intramuscular forms of parenteral administration are generally preferred. Appropriate dosage forms for such administration m ay be prepared b y conventional techniques . Compounds may also be administered by inhalation, e.g., intranasal and oral inhalation administration. Appropriate dosage forms for such administration, such as aerosol formulation or a metered dose inhaler may be prepared by conventional techniques well known to those having ordinary skill in this art. For all methods of use disclosed herein for th e compounds of the present invention, the daily oral dosage regiment will preferably be from about 0.1 to about 100 mg/kg of total body weight. The daily parenteral dosage regiment will preferably be from about 0.1 to about 100 mg/kg of total body weight. The daily topical dosage regimen will preferably be from about 0.01 to about 1 g, administered one to four, preferably two to three times daily. It will also be recognized by one of skill in this art that the optimal quantity and spacing of individual dosages of a compound of the present invention, or a pharmaceutically acceptable salt thereof, will be determined b y the nature and extent of the condition being treated and that such optimums can be determined by conventional techniques.

Suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of inorganic and organic acids, such as hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methane sulphonic acid, ethane sulphonic acid, acetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid and mandelic acid. In addition, pharmaceutically acceptable salts of compounds of the present invention may also be formed with a pharmaceutically acceptable cation, for instance, if a substituent group comprises a carboxy moiety. Suitable pharmaceutically acceptable cations are well known in the art and include alkaline, alkaline earth ammonium and qu aternary ammonium cations.

Thus the present invention is directed toward th e effective use of agents to induce T cell anergy. More specifically, this invention uses ester and/or amide derivatives of butyric acid to inactivate antigen-specific T cells thus providing immunotherapeutic methods of treatment of autoimmune diseases, disorders involving an autoimmune component a n d neoplastic diseases such renal cancer, ovarian cancer, lung cancer, glioma and leukemia, etc. n-Butyrate derivatives designed to possess Gl blocker activity both in vitro and in vivo are synthesized. The ester (MEB) and ester/amide (BEB) derivatives of butyrate are found to suppress IL-2-stimulated proliferation of Th l cells in vitro. Unlike MEB and BEB, the amide analogue of butyrate, MEB A, does not suppress Thl cell proliferation in vitro. The lack of activity of MEBA may be related to the slower metabolic hydrolysis of th e amide bond in MEBA compared to the ester bond in MEB and BEB. When tested in vivo, both MEB and BEB, but not MEBA, are shown to significantly suppress a primary antibody response to a thymus-dependent antigen. Suppression of antibody production reflects inhibition of T cell function and/or B cell function. However, in vivo examination of the effect of MEB on T cell activity revealed that MEB induced antigen-specific unresponsiveness in CD4⁺ T cells. The T cell unresponsiveness induced in mice immunized with ovalbumin and treated with MEB is manifested as an inability of lymph node CD4⁺ T cells to proliferate when stimulated with ovalbumin in vitro. Although this does not negate th e possibility that MEB may also inactivate antigen-activated B cells, it clearly demonstrates that ester analogues of butyrate induce unresponsiveness in antigen- specific CD4⁺ T cells.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.

EXAMPLE 1

Animals

Male C57BL/10, DBA/2 and C3H/HeJ mice at 6 to 8 w k of age were purchased from Harlan Sprague Dawley, Inc (Indianapolis, IN).

EXAMPLE 2 Reagents and antibodies

Inject KLH was purchased from Pierce (Rockford,IL), and n-butyrate was purchased from Sigma (St. Louis, MO).

Butyryl chloride, 4-(2-hydroxyethyl)morpholine, 4 - ( 2- aminoethyl)morpholine, and l -(2-hydroxyethyl)piperazine w ere purchased from Aldrich (Milwaukee, WI).

EXAMPLE 3

Instrumentation

Proton NMR spectra were recorded at 500 MHz on a

Bruker AM500 spectrometer and chemical shifts are reported i n p.p.m. Mass spectra were recorded on a Finnegan TSQ 700 spectrometer (direct exposure probe) at 70 eV electron ionization.

EXAMPLE 4

Synthetic schema of butyrate derivatives l -(4-Morpholinyl)ethyl butyrate fMEB 1

Butyryl chloride (6.07 g, 0.06 mol) was added with stirring to a cooled solution of 5 g (0.04 mol) of 4- ( 2- hydroxy ethyl)morpholine in 20 ml of chloroform over 45 min an d cooling was maintained for 6 h. The mixture was diluted with chloroform (15 ml) and washed three times with 20 ml of 5% sodium carbonate. The aqueous layer was washed with 15 ml of chloroform, and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford 5.37 g (70%) of the ester as an orange liquid. Column chromatography of 500 mg on silica gel 60 (0.063 - 0.200 mm, 1 0 inch, 30 mm inside diameter) was performed using a gradient of ethyl acetate/hexane 1 :3 to ethyl acetate. The recovered sample afforded the following: Η NMR (CDC1₃) _ 0.93 (t, 3H, J = 7.4, CH₃), 1.63 (m, 2H, J = 7.4, CH₃CH₂-), 2.28 (t, 2H, J=7.4, COCH₂-), 2.58 ( ap bs, 4H, ring N-CH₂-), 2.69 (t, 2H, J = 5.7, CO₂CH₂-), 3.74 (t, 4H, J = 4.5, ring -OCH₂-), 4.25 (t, 2H, J = 5.7, NCH₂-); MS m/z 201 (M⁺), 1 30, 113, 100 (Base Peak).

l -(4-MorpholinyPethyl butyrate hydrochloride

A solution of 2.5 g of the crude free base in 60 ml of anhydrous ether was cooled in an ice bath with stirring while 1 4 ml of a cold 1.0 M solution of hydrogen chloride in anhydrou s ether was added dropwise. The resulting white solid was filtered and recrystallized from 60 ml of tetrahydrofuran to yield 1.69 g of white crystals: mp 108.0 -108.3° C.

2-(^'4-Morρholinynethyl butanamide fMEBAI 2

The amide was synthesized in 68% yield in a manner analogous to that described for MEB by treatment of 4- ( 2- aminoethyl)morpholine with butyryl chloride. 'H NMR (CDC1₃) 0.91 (t, 3H, J = 7.4, CH₃), 1.62 (m, 2H, CH₃CH₂-), 2.13 (t, 2H, J = 7.4, COCH₂-), 2.40 - 2.45 (m, 6H, N-CH₂- & ring O-CH₂-), 3.30 - 3.33 (m, 2H, N-CH₂-), 3.66 (t, 4H, ring N-CH₂-), 5.96 (bs, 1H, NH); MS m/z 200 (M⁺), 182, 157, 113, 100 (Base Peak).

2-(4-Morpholinyl)ethyl butanamide hydrochloride

The hydrochloride salt was prepared as described for MEB and recrystallized from tetrahydrofuran to afford hygroscopic crystals: mp 150.6 - 151.1° C.

Synthesis of 2-(4-Butanoylpiperazinyl)ethyl butanoate (BEB) 3

Butyryl chloride (1 1.83 g, 0.1 1 mol) was added dropwise to a cooled solution of 4.84 g (0.04 mol) of l - ( 2- hydroxyethyl)-piperazine in 20 ml of chloroform. Cooling w as maintained for 2 h, and a white precipitate formed. Chloroform (15 ml) was added and the mixture was stirred overnight, w ashed with 210 ml of cold 0.6 N sodium hydroxide solution, then w ashed twice with 50 ml of cold water. The organic fraction was dried over anhydrous sodium sulfate, filtered and, concentrated in vacuo to yield 9.39 g (94%) of a clear yellow liquid. This w as distilled: bp 144-146° C, (0.5 mm Hg) to give 6.08 g (65%) of MEB. Η NMR (CDC1₃) δ 0.897 (t, 3H, J = 7.4, CH₃), 0.901 (t, 3H, J = 7.4, CH₃), 1.59 (m, 2H, CH₃CH₂), 1.61 (m, 2H, CH₃CH₂), 2.24 (t, 2H, J = 7.4, CH₂CO), 2.25 (t, 2H, J = 7.4, CH₂CO), 2.42-2.43 (app t, 2H, J = 5.2, axial CH ester end of ring), 2.43 - 2.44 (app t, 2H, J = 5.2, equatorial CH ester end of ring), 2.59 (t, 2H, J = 5.8, N-CH₂-CH₂-O), 3.41 (app t, 2H, axial CH amide end of ring), 3.57 (app t, 2H, equatorial CH amide end of ring), 4.16 ( t, 2H, N-CH₂-CH₂-O); MS m/z 270 (M⁺), 255, 242, 227, 199, 182, 169 (Base Peak). 2-(4-Butanoylpiperazinyl)ethyl butanoate hydrochloride

The hydrochloride salt was prepared as described for MEB and recrystallized twice from tetrahydrofuran to afford white crystals: mp 130.1 - 130.7° C.

Synthesis of 2.2'.2"-Nitrilotrisethyl trisbutyrate (4)

Butyryl chloride 12.01 g. (0.11 mol) was added dropwise with stirring to a cooled solution of 3 g (0.02 mol of triethanolamine in 20 ml of chloroform. Stirring was continued for 48 h. The reaction mixture was washed three times with 4 0 mL of 5% sodium carbonate. The aqueous layer was washed with 15 ml of chloroform and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to yield 6.77 g (94% of a pale yellow liquid. After conversion to the hydrochloride and back to the free base th e following spectral data were obtained: 'H NMR (CDC1₃) δ 0.90 (t, 9H, J = 7.4, CH₃), 1.60 (m, 6H, J = 7.4, CH₃CH₂CH₂), 2.23 (t, 6H, J = 7.4, CH₂COO), 2.81 (t, 6H, J = 7.4, CH₂N), 4.09 (t, 6H, J = 7.4, OCH,); MS m/z 359 (m⁺), 344, 316, 288, 258, 115 (base).

2.2'.2"-Nitrilotrisethyl trisbutyrate hydrochloride

The hydrochoride salt was prepared as described for MEB, but it was very hygroscopic and readily oiled out. Attempts to recrystallize it were unsuccessful.

l -Methyl-4-piperidinyl butanoate (5

The butanoate ester of 4-hydroxy- l -methylpiperidine w as synthesized by a method similar to that described for MEB b y treatment of 4-hydroxy- l - methylpiperidine with butyryl chloride to afford a yellow oil (97% yield). Distillation (bp 46 - 50°, 0.5 mm Hg) gave a colorless liquid (92%). 'H NMR (CDC1₃) δ 0.92 (t, 3H, CH₂CH₃), 1.58- 1.66 (m, 2H, J = 7.4, CH₂CΗ₃), 1 .66- 1 .7 1 (m, 2H, ring C-3 & C5 ax H), 1.85-1.89 (m, 2H, ring C-3 & C5 eq H), 2.18-2.26 (app t, 2H, ring C2 & C6 ax H), 2.24 (t, 2H, CH₂COO), 2.24 (s, 3H, NH), 2.61 (app br s, 2H ring C2 & C6 eq H), 4.76 (m, 1H, ring C-4 ax H).. MS m/z 185 (m⁺), 114, 98, 97, 96 (base), 82, 70, 57,55.

l -Methyl-4-piperidinyl butanoate hydrochloride The hydrochloride salt was prepared as described for MEB and was recrystallized from tetrahydrofuran to give whi te crystals: m.p. 132.2 - 133.7° C

EXAMPLE 5 Th l cell clones

The KLH-specific were developed in C57BL/10 mice, and characterized as Thl clones based on their ability to secrete IL-2, but not IL-4. The Thl clones were passed every 7-14 day s using KLH, irradiated syngeneic spleen cells as antigen-presenting cells (APC), and IL-2-containing Con A CM using a previously described protocol (Gilbert et al, 1990).

EXAMPLE 6 Suppressive Effects of Butyrate Prodrugs in vitro and in vivo

Prior to testing the suppressive effects of butyrate prodrugs in vivo, the ability of n-butyrate to inhibit a primary antibody response in mice was examined. As shown in Table 1 below, n-butyrate itself did not suppress a primary antibody response by more than 40-50%.

TABLE 1

Anti-HGG antibody in serum of individual mice Experiment 1 Experiment 2 n-B utyrate n-B uty rate

Control tre ated Control treated

56 29 15 4

270 83 6 9

1 1 8 24 13 31

1 26 202 40 12

1 8 3 91 39 6 x= 150±80 x=86±72 x=23±1 6 x= 12±l l

Aggregated human gammaglobulin (HGG) was injected (100 μg) into mice ip on day 0, followed by ip injections of 5.5 mg n - butyrate on days 2-5 (Experiment 1) or 2-6 (Experiment 2) . Levels of anti-HGG antibody were measured by ELISA on day 9.

EXAMPLE 7 Butyrate derivatives reversibly inhibited IL-2-induced proliferation of Thl cells

To test the ability of the butyrate derivatives to

4 suppress T cell activity in vivo, Thl cells [(5 x 10 cells/well in 9 6 well plates (Costar)] are stimulated with 10% IL-2-containing Con A CM in the presence of various concentrations of different butyrate derivatives. In some cultures, Thl cell proliferation is measured after two days. In other cultures, the Thl cells are washed after 24 hours, and fresh IL-2-containing medium is added. Proliferation is measured in the washed, IL- 2- restimulated cultures after an additional 2 days.

As shown in Figure 2, the ester and es ter/amide derivatives of n-butyrate, MEB and BEB respectively, are comparable to n-butyrate in their ability suppress IL-2-induced proliferation of Thl cells. In addition, similar to n-butyrate- treated T cells, T cells treated with butyrate derivatives regained their ability to proliferate to IL-2 once the compounds are w ashed out of the cultures. This later observation means that the cell cycle blocking effects of the MEB and BEB are not due to dru g - induced toxicity.

EXAMPLE 8

Butyrate derivatives inhibited antibody production to thymu s - dependent antigen in vivo The butyrate derivatives are tested for their ability to suppress lymphocyte activity in vivo. C57BL/10 mice ( 5 mice/group) are injected ip with 100 μg of ovalbumin i n conjunction with complete Freund' s adjuvant on Day 0. In one experiment, the mice also receive one ip injection per day of th e butyrate derivatives (0.091 mmol) on Days 1-3. Serum samples are obtained 10 days after the initial injection with ovalbumin, and tested for the presence of anti-ovalbumin antibodies using a n ELISA. To perform the ELISA 96 well plates (Costar 3595) are first incubated with ovalbumin (100 μl/well of 100 μg/ml in PBS) overnight at 4°C. The plates are then washed 4 times with PBS and 0.5% Tween 20, blocked with 1% fetal calf serum for 3 0 minutes at 37°C, and washed again. Individual serum samples are added (diluted 1/100 or 1/1000 in PBS), and the plates are incubated for 2 hours a t 20° C. The plates were next washed 7 times with PBS/Tween, and alkaline phosphate (AP)- labeled goat anti-mouse IgG, IgA, IgM (H + L) (Zymed) is added (1/1000) for 1 hour at 20° C. The plates are again washed 7 times with PBS/Tween, alkaline phosphate substrate (1 mg/ml) is added. After 10 minutes, Ig levels are quantified by an ELISA reader (absorbance 405 nm). The concentration of anti-ovalbumin is determined by comparison with a standard curve obtained using mouse anti-ovalbumin antibody (Sigma; St. Louis, MO). To measure isotype-specific anti-ovalbumin antibodies, s erum samples (diluted at 1/300 or 1/1000) are incubated on th e ovalbumin-coated plates as described above. After washing, biotinylated detecting antibodies directed against mouse IgG_2a (rat IgG, , clone R19-15), IgG_2b (rat IgG_2a , clone R12-3) IgG, (rat IgG, , clone A85-1), or IgM (rat IgG_2a , clone R6-60.2) (all purchased from PharMingen, La Jolla, CA) areadded at 2.5 μg/ml for one hour at 20°C followed by AP-labeled ExtrAvidin (Sigma) for one hour a t 20°C and AP substrate. Ig levels are presented as CD measurements .

When anti-ovalbumin levels in the serum of the mice are tested 10 days following administration of the antigen, MEB is shown to significantly decrease by 65% the ability of the mice to generate a primary antibody response to a thymus-dependent antigen as compared to control mice treated with saline (Figure 3). Mice treated with a second butyrate derivative, BEB, also produced significantly less antigen- specific antibody than control mice. In contrast, the third butyrate derivative, MEBA, is unable to suppress antigen-specific antibody production in vivo. These results show that the ester and the ester/amide derivatives of butyrate suppressed lymphocyte function both in vitro and in vivo, while the amide analogue of butyrate was ineffective both in vitro and in vivo.

EXAMPLE 9

Butyrate derivatives inhibited antigen-specific T cell responses i n v ivo

If the butyrate derivatives suppressed antibody production to a thymus-dependent antigen by inactivating th e antigen-specific CD4⁺ T cells required for B cell help, then th e butyrate derivatives need only be present during an early stage during which the CD4⁺ T cells would otherwise be activated b y antigen. To demonstrate that short term exposure to butyrate derivatives alters the T cell response to antigen in vivo, male C57BL/10 mice (5 per group) are injected ip with 100 μg of ovalbumin in conjunction with complete Freund' s adjuvant on Day 0, followed by a single ip injection of saline or MEB (0.15 mmol) o n Day 2 or 3. On day 10 the mice receive 100 μg ovalbumin in conjunction with incomplete Freund' s adjuvant sc at the base of the tail. After an additional 7 days, cells from the periaortic an d mesenteric lymph nodes were enriched for CD4⁺ T cells b y negative selection (Griffin et al, 1998). The T cells are then incubated at 1 x 10⁵ in half-area (100 μl /well) 96 well plates (Costar 3696) along with 2 x 10⁵ irradiated (2000R) spleen cells from untreated C57BL/10 mice as APC, and various concentrations of ovalbumin. Proliferation is measured on day 4 by assessing

₃ incorporation of [ H]-TdR after a 12 hour pulse.

Isotype-specific anti-ovalbumin antibody is measured to more precisely delineate the effect of MEB on antigen-specific antibody production. In addition, to look more directly on the effect of MEB on CD4⁺ T cells, lymph node CD4⁺ T cells isolated from mice 6 days after the second immunization with ovalbumin are examined for their ability to proliferate to ovalbumin in vitro. Treatment with a single dose of MEB significantly decreased the production of IgG_2a and IgG_2b anti- ovalbumin antibody during the primary antibody response (Figure 4A). IgG, and IgM anti-ovalbumin antibody production is also decreased, albeit not dramatically, if MEB is administered on d a y 2, but not on day 3, following immunization.

An evaluation of the antibody response generated by a second exposure to ovalbumin reveales that IgG_2a and IgG_2b anti- ovalbumin antibody remain dramatically low (decreased by a t 80% compared to controls) in mice treated with MEB on either d ay 2 or day 3 following their initial immunization with ovalbumin (Figure 4B). IgGl anti-ovalbumin production during th e secondary antibody response is also significantly decreased, while the IgM anti-ovalbumin antibody production following reimmunization with ovalbumin is unaffected by the initial treatment with MEB. The MEB-induced decrease in IgG antigen- specific antibody production correlates with a significant loss of antigen-specific proliferation observed in the CD4⁺ T cells isolated from antigen-primed mice treated with MEB on day 2 or day 3 following immunization (Figure 4C). Taken together even a brief exposure to the butyrate derivative MEB in vivo can induce antigen-specific unresponsiveness in CD4⁺ T cells. EXAMPLE 10

Butyrate derivative induced antigen-specific inactivation in CD4⁺ T cells in vitro

To demonstrate the ability of MEB to induce antigen- specific unresponsiveness in CD4⁺ T cells in vitro, it is necessary to determine whether antigen is required for MEB-induced T cell anergy. Thl cells are treated with MEB in the presence or absence of antigen or exogenous IL-2. The Thl cells are then removed from the primary cultures, washed free of MEB, and re- stimulated with antigen or IL-2 in secondary cultures; tolerized Thl cells are characterized by the fact that although they lose their ability to proliferate when restimulated with antigen, their continued expression of IL-2 receptors enables them to proliferate when stimulated with exogenous IL-2. Figure 2 shows that Thl cells treated with IL-2 an d

MEB, although blocked in primary cultures, retain their ability to proliferate in response to IL-2 once the MEB has been w ashed from the cultures. Thl cells pretreated with IL-2 and MEB also retain their ability to proliferate to antigen once MEB has been washed from the cultures (Figure 5A). In contrast to Thl cells pretreated with IL-2 and MEB, Thl cells pretreated with antigen and MEB lose their ability to proliferate in antigen-stimulated secondary cultures (Figure 5B). The fact that the Thl cells pretreated with antigen and MEB, although unable to respond to antigen, can still proliferate in secondary cultures stimulated with exogenous IL-2, suggests that the lack of antigen responsiveness in these Thl cells is not due to a loss of viability. Thl cells incubated in primary cultures with MEB alone, or in medium alone retain their ability to proliferate in response to antigen stimulation in secondary cultures. This result shows that antigen- activated, but not IL-2-activated, Thl cells become unresponsive to a subsequent stimulation with antigen following exposure to MEB.

EXAMPLE 11

MEB induction of alloantigen-specific T cell unresponsiveness

The ability of MEB to induce alloantigen-specific T cell unresponsiveness is shown by incubating spleen cells from DBA/2 mice (H-2^d) (2.5 x 10⁵ in 200 μl /wells) with stimulator cells [2.5 x 1 0⁵ irradiated (2000R) spleen cells from C57BL/10 mice (H-2^b)] . MEB (ImM) is added to some wells of the mixed lymphocyte reaction (MLR) 24 hours after the initiation of culture. After a n additional 3 days, the MLR cultures are washed, and rested for a further 2 days. The T cells from the MLR are then isolated an d reincubated at 2.5 x 10⁵ /well with either the initial alloantigen (spleen cells from C57BL/10 mice), or with a third-party alloantigen [spleen cells from C3H/HeJ mice (H-2^k)]. Proliferation in both the primary and secondary MLR is measured on day 5 b y

₃ assessing incorporation of [ H]-TdR after a 12 hour pulse. MEB addition blocked spleen cell proliferation in a primary one-way MLR (Figure 6A). More interestingly, splenic T cells incubated with MEB during the primary MLR lose their ability to proliferate when restimulated with the initial alloantigen in a secondary MLR that does not contain MEB. However, the T cells treated with MEB in the presence of th e initial alloantigen are able to proliferate when restimulated with a third-party alloantigen or exogenous IL-2. In contrast to the T cells isolated from the MEB- treated MLR, T cells isolated from a primary MLR that does not contain MEB, are able to proliferate when restimulated with the initial alloantigen or with a third-party alloantigen. Thus, MEB-induced unresponsiveness is not generalized, but occurs only in those T cells which are simultaneously stimulated with antigen.

EXAMPLE 12 Butyrate derivative blocked activated Thl cells in G,

The ability of the butyrate derivative to induce Th l cell anergy is examined using a protocol previously developed for n-butyrate-induced T cell tolerance (Gilbert and Weigle, 1993 ) .

5

Briefly, Thl cells are incubated in primary cultures at 2.5 x 1 0 cells/ml, along with MEB (ImM), KLH (50 μg/ml), and 5 x 1 0⁶/ m l irradiated syngeneic spleen cells as APC. Alternatively, the Th l cells are incubated in primary cultures containing MEB and IL- 2 (10% Con A CM). Control primary cultures receive MEB and APC, but no antigen or IL-2. After incubation for 24 h at 37 C, the cells in the primary cultures are harvested, washed free of MEB, and

5 reincubated at 2.5 x 10 ml in secondary cultures without MEB. The Thl cells in the secondary cultures are stimulated with 10%

6 IL-2-containing Con A CM, or with 5 x 10 /ml irradiated syngeneic spleen cells as APC, and KLH. After 2 days in the secondary cultures, the Thl cells are assessed for proliferation (pulsed with

[³H]-TdR for 12 h).

Since MEB appeared to suppress antigen-specific T cell responses both in vivo and in vitro, further characterization of its mechanism of action was conducted. n-Butyrate-induced T cell tolerance has been linked to the ability of the compound to block cell cycle progression of T cells in G, (Gilbert and Weigle, 1993). Although it was shown that MEB, as well as other butyrate derivatives, inhibited the proliferation of activated Thl cells, i t was not known where in the cell cycle this inhibition occurred. A n analysis of DNA content showed that similar to n-butyrate, essentially all the Thl cells stimulated with IL-2 in the presence of MEB remained in G₀/G, (Figure 7).

EXAMPLE 13

Butyrate derivative sequestered activated Thl cells in G^ G_j^ If the ability of butyrate and its derivatives to induce

Thl cell tolerance is linked to their ability to block antigen- activated Thl cells in G, , their therapeutic importance is enhanced if MEB induces G, cell cycle blockade regardless of when in the cell cycle the compound is added. MEB is added to cultures of IL- 2- stimulated Thl at various time points. In the absence of MEB, it is shown that approximately 50% of IL-2-activated Thl cells h av e exited G, by Day 2 (Figure 8). In contrast, if MEB is added at th e initiation of culture, over 97% of the IL-2-stimulated Thl cells remain in G₀/G , for the duration of the experiment (120 hours). I f MEB is not added until 15 hours after the Thl cells are stimulated with IL-2, the initial cell cycle progression is similar to that seen in the absence of inhibitor, but by 96 hours, 79.3% of the IL- 2- stimulated Thl cells are blocked in G₀/G,, compared to only 56.9% in control cultures. If MEB is added 24 hours instead of 15 hours after IL-2 stimulation, 89% of the Thl cells are blocked in G, phase by 48h, and the cell cycle profile looks very similar to that obtained if MEB is added at the initiation of culture. Finally, even if MEB is added as late as 39 hours after IL-2 stimulation, 87% of the Thl cells (as compared to 49.9% of controls) are sequestered in G₀/G, when DNA content is measured at 72 hours. Very similar cell cycle kinetics are obtained when n-butyrate instead of MEB is added to cultures of IL-2-stimulated Thl cells (data not shown).

Taken together, this data suggests that if activated Th l cells are exposed to MEB when the Thl cells are still in G₀/G,, MEB-induced cell cycle blockade is immediate and dramatic. Similarly, if MEB is added at 24 hours, the time at which th e majority of Thl cells have apparently completed one cell cycle an d are back in G,, an immediate and effective cell cycle blockade is again observed. If MEB is added at a time (e.g. 15 or 39 hours after stimulation) when at least some of the activated Thl h av e already exited G,, the Thl cells have to cycle back to G, in order to become susceptible to MEB-induced cell cycle blockade, b u t eventually essentially all the activated Thl cells are blocked in G, by MEB.

Therefore, MEB induces eventual G, sequestration of activated Thl cells no matter when it is added during the cell cycle. This suggests that MEB will be effective in treating a n ongoing T cell response, a valuable characteristic of a n immunotherapeutic agent. Many methods of inducing antigen- specific T cell unresponsiveness for the treatment of autoimmunity are very useful in preventing the initiation of th e disease process, but are much less effective in treating an already established autoimmune response (Bai et al, 1998; Meyer et al, 1996; Gaupp et al, 1997). In addition, use of butyrate derivatives to treat autoimmune disease does not require identification of th e specific autoantigens targeted by the self-reactive lymphocytes . Theoretically, the butyrate derivatives would inactivate any CD4⁺ T cell that w as simultaneously being stimulated with antigen, thus encompassing all autoreactive CD4⁺ T cells activated in response to any self-antigens.

Thus, the short-term use of butyrate derivatives can be used in vivo to induce antigen-specific inactivation of at least the Thl cell-like subset of CD4⁺ T cells, thereby providing th e basis for a novel method of immune intervention with potential for the treatment of autoimmune disease. Such a treatment regimen has definite advantages over most existing immunotherapies which consist of long-term use of drugs th at induce generalized immune suppression and may produce significant clinical side effects.

EXAMPLE 14

DNA analysis To examine DNA content, Thl cells were fixed i n prechilled 70% ethanol overnight at 4 C. The fixed Thl cells w ere next washed in PBS, resuspended in 1 ml of staining buffer containing RNase (lmg/ml; Sigma) and propidium iodide (50 μg/ml; Sigma), incubated for 20 minutes in the dark at 20°C, an d analyzed by flow cytometry using a FACSCalibur (Becton Dickinson, Moutain View, CA). The data were analyzed using th e ModFit DNA analysis program (Verity Software House).

EXAMPLE 15 In vivo administration of butyrate prodrugs

Effective suppression in vivo of CD4⁺ T cells and th e corresponding T cell-induced antibody response is dependent o n when the prodrugs are administered. Butyrate has a half-life of six minutes; the butyrate prodrugs of the present invention possess a half-life in the range of several hours. No toxicity is observed in the present study when mice were treated with MEB at a dose which approximated 0.7 g/kg/day. Even if administered at high doses for extended periods of time it seems unlikely that MEB would be toxic. Concerns about possible sodium overload and lack of efficacy have precluded studies examining the potential toxicity of high doses of n-butyrate. However, th e arginine salt of butyrate was shown to be nontoxic in human s even when perfused at doses as high as 2 g/kg/day (Perrine et al, 1994). The efficacy of a single dose of MEB suggests that shori- term use of the compound would be effective, thus eliminating any possible toxicity associated with long term use.

MEB is able to inactivate CD4⁺ T cells in vivo even if administered in a single dose. If MEB works in vivo as it does in vitro, i.e. by inducing anergy in antigen-stimulated T cells, i t would be necessary for MEB to be present only during the narro w window of time when T cell stimulation by antigen occurs in vivo. Since T cell activation in draining lymph nodes has been shown to occur 2-3 days following immunization of naive mice (Garside e t al, 1998; MacLennan et al, 1997), MEB was administered in a single dose on either day 2 or day 3 following administration of the antigen, in this case ovalbumin. Both primary and secondary anti-ovalbumin antibody production is inhibited in the MEB- treated mice. However, not all the isotypes of anti-ovalbumin antibody are suppressed equally. IgM anti-ovalbumin is slightly inhibited during the primary antibody response, and is totally unaffected during the secondary antibody response. Since, th e requirement for antigen-specific T cell help during the production of IgM is less stringent than that needed for the production of IgG subclasses of Ig (Steele et al, 1996), this finding would suggest that MEB is better at suppressing specific T cell responses than it is at inhibiting non-specific T cell help or B cell activity.

EXAMPLE 16 Effect of MEB treatment on antigen-specific antibody production

Although a single dose of MEB has little effect o n antigen-specific IgM production, this treatment regimen suppresses primary IgG_2a and IgG_2b anti-ovalbumin antibody production, and blocks the generation of the memory T cells required for a secondary IgG_2a or IgG_2b anti-ovalbumin antibody response. MEB also decreases the generation of memory T cells required for a secondary IgG, antibody response. However, th e effect of MEB treatment on IgG, antibody production is less profound than the effect of MEB on IgG_2a or IgG_2b. IgG, production is dependent on IL-4, and thus largely driven by Th2 cells, while IgG_2a production is enhanced by IFN-γ, and thus driven by Thl cells (Stevens et al, 1988). The relationship between IgG_2b and a particular CD4⁺ T cell subset is less well-defined, but since IL-4 suppresses IgG_2b (Kuhn et al, 1991 ), it is not unlikely that Thl cells rather than Th2 cells promote IgG_2b production in vivo. Consequently, it is possible to interpret the differential effect of MEB on isotype-specific antibody production in vivo b y postulating that Thl cells are more susceptible than Th2 cells to MEB-induced unresponsiveness. The results showing that MEB induced antigen-specific unresponsiveness in Thl cells in vitro underscore the likelihood that Thl cells, both in vitro and in vivo, are susceptible to MEB-induced tolerance. The suggestion that Th2 cells are less susceptible than Thl cells to MEB-induced unresponsiveness is in accordance with other methods of inducing T cell tolerance which have similarly documented th e relative resistance of Th2 cells to tolerance induction (Gilbert e t al, 1990; Williams et al, 1990). The fact that the memory IgG, antibody response is suppressed to some degree in mice treated with MEB suggests that although Th2 cells may be somewhat resistant to MEB-induced unresponsiveness, somewhat longer exposure to MEB, or perhaps higher doses of MEB may be expected to more completely suppress Th2-mediated IgG, production.

MEB-induced T cell unresponsiveness was not generalized, but was reserved for T cells that w ere simultaneously stimulated with antigen. Unlike Thl cells exposed to both antigen and MEB in vitro, Thl cells exposed to MEB alone, or to MEB and IL-2, did not lose their ability to respond to a subsequent antigen challenge. Along these same lines, splenic T cells stimulated in vitro with an alloantigen in the presence of MEB lost their ability to proliferate in response to a subsequent challenge with the initial alloantigen, but retained their ability to proliferate when stimulated with a third-party alloantigen. Taken together, these results underscore the antigen specificity of MEB- induced T cell unresponsiveness.

The following references are cited herein: Bai et al., ( 1998) Complexities of applying nasal tolerance induction as a therapy for ongoing relapsing experimental autoimmune encephalomyelitis (EAE) in DA rats . Clin Exp Immunol 111 : 205-210.

Daniel et al., (1989) Pharmacokinetic study of butyric acid administered in vivo as sodium and arginine butyrate salts. Clin. Chim.Acta 181 : 255 - 264.

Garside et al., (1998) Visualization of specific B and T lymphocyte interactions in the lymph node. Science 281 : 96-99.

Gaupp et al., ( 1997) Modulation of experimental autoimmune neuritis in Lewis rats by oral application of myelin antigens. J Neuroimmunol 79: 129-137.

Gilbert et al., (1990) Thl and Th2 clones differ in their response to a tolerogenic signal. J. Immunol. 144: 2063-2071.

Gilbert et al., (1993) Thl cell anergy and blockade i n Gla phase of the cell cycle. J. Immunol. 151 : 1245- 1254.

Kruh J, Defer N and Tichonicky L (1992) Molecular an d cellular action of butyrate. CR. Seances Soc.Biol.Fil. 186: 12-25.

Kuhn R, Rajewsky K and Muller W ( 1991 ) Generation and analysis of Interleukin-4-deficient mice. Science 254: 707.

MacLennan et al., (1997) The changing preference of T and B cells for partners as T-dependent antibody responses develop. Immunol. Rev. 156: 54-66.

Meyer et al., (1996) Suppression of murine chronic relapsing experimental autoimmune encephalomyelitis by the oral administration of myelin basic protein. J Immunol 157: 4230- 4238.

Miller et al., (1987) Clinical pharmacology os sodium butyrate in patients with acute leukemia. Eur.J. Cancer Clin.Oncol. 23 : 1283-1287.

Novogrodsky e t al., (1983) Effect of polar organic compounds on leukemic cells. Butyrate-induced partial remission of acute myelogenous leukemia in a child. Cancer 51 : 9 - 14.

Perrine et al., (1994) Butyrate derivatives. New agents for stimulating fetal globin production in the β-globin disorders . Am. J. Pediatr. Hematol. Oncol. 16: 67-71.

Steele et al., (1996) Two levels of help for B cell alloantibody production. J Exp Med 183: 699-703.

Stevens et al., (1988) Regulation of antibody isotype secretion by subsets of antigen-specific helper T cells. Nature 334 : 255 -258.

Williams M E, Lichtman A H and Abbas A K ( 1990) Anti-CD3 antibody induces unresponsiveness to IL-2 in Thl clones but not in Th2 clones. J. Immunol. 144: 1208- 1214.

One skilled in the art will readily appreciate that th e present invention is well adapted to carry out the objects an d obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within th e spirit of the invention as defined by the scope of the claims.

Claims

WHAT IS CLAIMED IS:

1. A compound of the structure

R₃-mN- (CH₂)_n|-C^'H-X_'C ϊ?-CH₂-CH₂-CH₃ „

I wherein nj is 1 to 5; m is 1 to 3; X is O or NH;

R is selected from the group consisting of hydrogen, a straight-chain aliphatic group, a branched-chain aliphatic group and an alicyclic group; and

R' is selected from the group consisting of hydrogen, methyl and ethyl; or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, which is 2, 2', 2"- nitrilotrisethyl trisbutyrate or a pharmaceutically acceptable salt thereof.

3. A pharmaceutical composition, comprising a compound of claim 1 and a pharmaceutically acceptable carrier or diluent.

4. The pharmaceutical composition of claim 3 , wherein said compound is is 2,2',2"-nitrilotrisethyl trisbutyrate or a pharmaceutically acceptable salt thereof.

5. The compound of the structure

-CH₂-CH₂-CH₃

II wherein n, is 1-5; X is O or NH; Y is CH₂, O, S, or NR; wherein R is selected from the group consisting of hydrogen, a straight-chain aliphatic group, a branched-chain aliphatic group and an alicyclic group; and

R' is selected from the group consisting of hydrogen, methyl and ethyl; wherein when X is O and Y is O, τi_\ is not 1 ; or a pharmaceutically acceptable salt thereof.

6. The compound of claim 5, which is 2- (4- morpholinyl)ethyl butanamide.

7. A pharmaceutical composition, comprising a compound of claim 5 and a pharmaceutically acceptable carrier or diluent.

8. The pharmaceutical composition of claim 7 , wherein said compound is 2-(4-morpholinyl)ethyl butanamide or a pharmaceutically acceptable salt thereof.

9. The compound of the structure

O R' O

H₃C-CH₂-CH₂-C-N N-(CH₂)ni-CH-X-C-CH₂-CH₂-CH₃

III wherein nj is 1 to 5; n2 is 1 to 4;

R' is selected from the group consisting of hydrogen, methyl and ethyl; and X is O or NH; or a pharmaceutically acceptable salt thereof.

10. The compound of claim 9, which is 2 - (4- butanoylpiperazinyl)ethyl butanoate.

1 1 . A pharmaceutical composition, comprising a compound of claim 9 and a pharmaceutically acceptable carrier or diluent.

12. The pharmaceutical composition of claim 1 1 , wherein said compound is 2-(4-butanoylpiperazinyl)ethyl butanoate or a pharmaceutically acceptable salt thereof.

13. The compound of structure

IV wherein n2 is 1 to 4; X is O or NH;

R is selected from the group consisting of hydrogen, a straight-chain aliphatic group, a branched-chain aliphatic group and an alicyclic group; and R' is selected from the group consisting of hydrogen, methyl and ethyl; wherein X and R' are independently optionally substituted a t C2, C3 or C4; or a pharmaceutically acceptable salt thereof.

14. The compound of claim 13, which is l -methyl-4- piperidinyl butanoate.

15. A pharmaceutical composition, comprising a compound of claim 13 and a pharmaceutically acceptable carrier or diluent.

16. The pharmaceutical composition of claim 15 , wherein said compound is l -methyl-4-piperidinyl butanoate or a pharmaceutically acceptable salt thereof.

17. A method of inactivating antigen-specific T cells in an individual in need of such treatment, comprising the step of administering to said individual an effective amount of a compound of structure

R' O

R3-mN- (CH₂)_n|-CH-X^C-CH₂-CH₂-CH₃ ^ wherein nj is 1 to 5; m is 1 to 3;

X is O or NH;

1 8. The method of claim 17, wherein said compound is 2,2',2"-nitrilotrisethyl trisbutyrate or a pharmaceutically acceptable e salt thereof.

19. The method of claim 17, wherein inactivation of antigen-specific T cells is useful in the prophylaxis or therapeutic treatment of autoimmune diseases, disorders involving a n autoimmune component or neoplastic diseases.

20. The method of claim 19, wherein said autoimmune diseases are selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, diabetes, an d multiple sclerosis.

21 . The method of claim 19, wherein said disorders involving an autoimmune component are selected from the group consisting of allograft transplantation rejection and xenograft transplantion rejection.

22. The method of claim 19, wherein said neoplastic disease is selected from the group consisting of renal cancer, ovarian cancer, lung cancer, glioma and leukemia.

23. A method of inactivating antigen-specific T cells in an individual in need of such treatment, comprising the step of administering to said individual an effective amount of a compound of structure

R' O

I I

Y N-(CH₂)_nrCH-X -C-CH₂-CH₂-CH₃

II wherein _\ is 1-5; X is O or NH;

Y is CH2, O, S, or NR; wherein R is selected from the group consisting of hydrogen, a straight-chain aliphatic group, a branched-chain aliphatic group and an alicyclic group; and R' is selected from the group consisting of hydrogen, methyl and ethyl; or a pharmaceutically acceptable salt thereof.

24. The method of claim 23, wherein said compound is 2-(4-morpholinyl)ethyl butanamide or a pharmaceutically acceptable salt thereof.

25. The method of claim 23, wherein said compound is 2-(4-morpholinyl)ethyl butanoate or a pharmaceutically acceptable salt thereof.

26. The method of claim 23, wherein inactivation of antigen-specific T cells is useful in the prophylaxis or therapeutic treatment of autoimmune diseases, disorders involving a n autoimmune component or neoplastic diseases.

27. The method of claim 26, wherein said autoimmune diseases are selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, diabetes, an d multiple sclerosis.

28. The method of claim 26, wherein said disorders involving an autoimmune component are selected from the group consisting of allograft transplantation rejection and xenograft transplantion rejection.

29. The method of claim 26, wherein said neoplastic disease is selected from the group consisting of renal cancer, ovarian cancer, lung cancer, glioma and leukemia.

30. A method of inactivating antigen-specific T cells in an individual in need of such treatment, comprising the step of administering to said individual an effective amount of a compound of structure

I I I wherein nj is 1 to 5; n2 is 1 to 4; X is O or NH; and

3 1 . The method of claim 30, wherein said compound is 2-(4-butanoylpiperazinyl)ethyl butanoate or a pharmaceutically acceptable salt thereof.

32. The method of claim 30, wherein inactivation of antigen-specific T cells is useful in the prophylaxis or therapeutic treatment of autoimmune diseases, disorders involving a n autoimmune component or neoplastic diseases.

33. The method of claim 30, wherein said autoimmune diseases are selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, diabetes, an d multiple sclerosis.

34. The method of claim 30, wherein said disorders involving an autoimmune component are selected from the group consisting of allograft transplantation rejection and xenograft transplantion rejection.

35. The method of claim 30, wherein said neoplastic disease is selected from the group consisting of renal cancer, ovarian cancer, lung cancer, glioma and leukemia.

36. A method of inactivating antigen-specific T cells in an individual in need of such treatment, comprising th e step of administering to said individual an effective amount of a compound of structure

IV wherein n2 is 1 to 4; 0 ^' X is O or NH;

R is selected from the group consisting of hydrogen, a straight-chain aliphatic group, a branched-chain aliphatic group and an alicyclic group;

R' is selected from the group consisting of hydrogen, methyl 5 and ethyl; wherein X and R' are independently optionally substituted at positions 2, 3, or 4 of the ring structure; or a pharmaceutically acceptable salt thereof.

37. The method of claim 36, wherein said compound 0 is l -methyl-4-piperidinyl butanoate or a pharmaceutically acceptable salt thereof.

38. The method of claim 36, wherein inactivation of antigen-specific T cells is useful in the prophylaxis or therapeutic 5 treatment of autoimmune diseases, disorders involving a n autoimmune component or neoplastic diseases.

39. The method of claim 36, wherein said autoimmune diseases are selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, diabetes, and multiple sclerosis.

40. The method of claim 36, wherein said disorders involving an autoimmune component are selected from the group consisting of allograft transplantation rejection and xenograft transplantion rejection.

41 . The method of claim 36, wherein said neoplastic disease is selected from the group consisting of of renal cancer, ovarian cancer, lung cancer, glioma and leukemia.