TiPS - September 1990 [Vol. 11] 11 Pace-Asciak, C. R. (1984) Biochim. Biophys. Acta 793, 485--488 12 Ojeda, S. R., Urbanski, H. F., Junier, M-P. and Capdevila, j. (1989) Amt. NY Acad. Sci. 559, 192-207 13 Fitzpatrick, F. A. and Murphy, R. C. (1989) Pharmacol. Rev. 40, 229-241 14 Weiss, R. H., Arnold, J- L. and Estabrook, R. W. (1987) Arch. Biochem. Biophys. 252, 334-338 15 Belardetti, F., Campbell, W. B., Falck, l- R., Demontis, G. and Rosolowski, M. (1989) Neutral 3, 497-505 16 Samuelsson, B. and Funk, C. D. (1989) I. Biol. Chem. 264, 19469-19472 17 Lindgren, J-A., H6kfelt, T., Dahl6n, S-E., Patrono, C. and Samuelsson, B. (1984) Proc. Natl Acad. Sci. USA 81, 6212-6216 18 Shimizu, T., Takusagawa, Y., Izumi, T., Ohishi, N. and Seyama, Y. (1987) I. Neurochem. 48, 1541-1546 19 Shimizu, T. and Wolfe, L. S. (1990) ]. Neurochem. 55, 1-15 20 Dumuis, A., Sebben, M., Haynes, L., Pin, I-P. and Bockaert, J. (1988) Nature 336, 68--70 21 Wolfe, L. S., Pellerin, L., Drapeau, C.

373

22 23 24 25 26 27 28 29 30 31 32

and Rostworoski, K. (1990) /. Neural Transm. 29, 29-37 Lazarewicz, 1. W., Wroblewski, J. T., Palmer, M. E. and Costa, E. (1988) Neuropharmacology 27, 765-769 Felder, C. C., Kanterman, R. Y., Ma, A. L. and Axelrod, J. (1990) Proc. Natl Acad. Sci. USA 87, 2187-2191 Piomelli, D., Shapiro, E., Feinmark, S. Iand Schwartz, i. H. (1987) I. Neurosci. 7, 3675-3686 Piomelli, D. et al. (1987) Nature 328, 38-43 Shapir,-,, E. ct al. (1988) Cold Spri~g Harbour Syrup. Quant. Biol. 53, 425--433 Dennis, E. A. (1987) Drug Dev. Res. 10, 205-220 Carlen, P. L. et al. (1989) Brain Res. 497, 171-176 Buttner, N., Siegelbaum, S. A. and Volterra, A. (1989) Nature 342, 553-555 Moore, S. D., Madamba, S. G., loels, M. and Siggins, G. R. (1988) Science 239, 278-280 Schweitzer, P., Madamba, S. and Siggins, G. R. (1990) Nature 346, 464--467 Keyser, D. O. and Alger, B. E. (1989) Soc. Neurosci. Abstr. 15, 178

Immunomodulatory activity of small peptides Vassil St Georgiev The activity of the immune system can be modulated by a wide variety of natural and synthetic peptides. Here, Vassil St Georgiev summarizes the actions of some of the immunostimulatory and immunosuppressant small pelJtides that have shown most promise as therapeutic agents. Some are already in use as vaccine adjuvants or to prevent graft rejection. There are now indications that these peptides may also be of benefit in conditions in which the immune system is compromised, in autoimmune disease and in cancer. A large number of natural and synthetic peptides have been evaluated for their immunomodulatory effects. These derive from (or are modifications of) sources as diverse as bacterial cell wall peptidoglycans, microbial and fungal metabolites, peptide hormones, fragments of immunoglobulins and other plasma proteins and colostrum and milk proteins. Although in many cases little is known of the mechanism of action, the clinical potential of many of these peptides has clearly been demonstrated. Some of the less well-characterized peptides may also prove to be useful; their properties are summarized in Table I. V. St Georgiev is Vice-President of Chenlical Sciences, Division of Life Sciences, Orion Research and Technologies Corporation, PO Box 463, Tampa, FL 33601-0463, USA.

lmmunostimulatory peptides Many peptides have been isolated that have nonspecific stimulatory effects on the immune system. Work is under way to develop these peptides as adjuvants in vaccines and as therapeutic agents for conditions including immune deficiencies and cancer. M u r a m y l peptides Freund's complete adjuvant killed mycobacterial cells emulsified in mineral oil - has long been k n o w n to be a powerful adjuvant of bumoral and cellmediated i m m u n e reactions. The smallest biologically active unit of bacterial cell wall peptidoglycan capable of replacing whole Mycobacteria in Freund's complete adjuvant is muramyl dipeptide (MDP) t. In addition to its immuno•

33 Bug, W., Role, L., Siegelbaum, S. A. and Simmons, L. (1989) Soc. Neurosci. Abstr. 15, 177 34 Bley, K. R. and Tsien, R. W. (1990) Neuron 2, 379-391 35 De Camilli, P., Benfenati, F., Valtorta, F. and Greengard, P. Annu. Rev. Cell Biol. (in press) 36 Man-Son-Hing, H., Zoran, M. l., Lukowiak, K. and Haydon, P. G. (1989) Nature 341,237-239 37 Piomelli, D. et al. (1989) Proc. Natl Acad. Sci. USA 86, 8550-8554

FMRFamide: Phe-Met-Arg-Phe-NH2 hepoxilin A~: 8-hydroxy-11,12-epoxyeicosatrienoic acid hepoxilin B3: 10-hydroxy-ll,12-epoxyeicosatrienoic acid 12-HETE: (12s)-hydroxyeicosatetraenoic acid 12-HPETE: (12s)-hydroperoxyeicosatetraenoic acid 12-KETE: 12-ketoeicosatetraenoic acid MK886:3-[1-(p-chlorophenyl)-5-isopropyl3-tert-butylthio-lH-indol-2-yl]-2,2dimethylpropanoic acid

adjuvant activity, manifested by a potent stimulation of the production of antibodies against antigen administered simultaneously2, MDP stimulates nonspecific resistance against bacterial, viral and parasitic infections, and against tumors by stimulating cytotoxic macrophages and natural killer cells 2, and increases the duration of slow-wave sleep 2. However, it also has other, largely undesirable biological effects such as pyrogenicity, transient leukopenia, sensitization to endotoxin and induction of arthritis, granuloma a n d u v e ~ t i s 2.

muramylmoiety 4

HN'

',

',,

6 C H2OH

O

R1

R2

R4 O

L-alanine moiety

CR3

0II

~-isoglutamine moiety

MDP In order to determine which structural features of the molecule of MDP are important for biological activity, congeners with changes in the muramyi, c-alanyl and o-isoglutamyl moieties have been tested in vitro and in vivo i (Table IIL

19qo,ElsevierSciencePublishersLtd.(UK) I)~65- 6147/90/$02.00

TiPS - September 1990 [Vol. 11]

374 TABLE I. Immunomodulatingactivities of small peptides

Structure of peptide

Origin

Activity

• Peptide fragments of immunoglobulins Thr-Lys-Pro-Arg (tuftsin) related to the Fc region of leukokinin Thr-Lys-Pro tuftsin congener Asp-Ser-Asp-Pro-Arg

related to the Fc region of human IgE

e Plasma protein fragments and analogues Gly-Pro-Thr-Gly-Thr-Gly-Ser-Lys- degradation of prealbumin with Cys (PAP) 6 Mguanidine-HCI PAP-Pro PAP-Pro-Leu His-Pro-Ala-Glu-Asn-Gly-Lys Fibrinogen-derived peptide

synthetic PAP congener synthetic PAP congener identical with the 13-19 segment of ~2-microglobulin derived from the 42 kDa C terminus of the Ac~chain of fibrinogen

e Colostrum- and milk-derived peptides VaI-Glu-Ser-Tyr-VaI-Pro-Leuderived from PRP (proline-rich Phe-Pro polypeptide); related ~oovine colostrai immunoglobulins VaI-Glu-Pro-Ile-Pro-Tyr identical with the C terminus of human l~-casomorphin-9, an opioid peptide found in human casein Gly-Leu-Phe derived from human and bovine caseins Leu-Leu-Tyr derived from human and bovine caseins • Enzyme fragments Peptide P-1 • Myelopeptides - 2 kDa myelopeptides

Ref.

enhances the phagocytic properties of monocytes and macrophages reduces IL-1 production by rat macrophages, and chemotaxis and IgE-specific expression in human alveolar macrophages undergoing clinical trials for topical use in the treatment of ocular and nasal ~llergies

a

restores the sensitivity of splenic E-rosette-forming cells of adult-thymectomized mice to azathioprine-induced immunosuppression twice as active as PAP twice as active as PAP suppresses in vitro the sheep erythrocyte-induced E-rosette formation by human blood lymphocytes inhibits the proliferation of human and murine lymphocytes induced by phytohemagglutinin, pokeweed mitogen and allogeneic cells

d

b c

d d e f

modulates the antibody responses of mice to sheep erythrocytes and affects the maturation of T-ceU precursors in vitro immunostimulating activity on murine macrophages and human monocytes stimulates phagocytosis in vitro stimulates phagocytosis in vitro

pepsin-derived fragment of honey bee venom phospholipase A~

downregulates the immune responses of patients with anaphylactic sensitivity to honey bee venom

from supematant of bone marrow cell cultures

enhance antibody production at the peak of the immune response by two- to threefold

"Najjar, V. and Fridkin, M. (1983) Ann. NY Acad. Sci. 419, 1-273; b Auriault, C. et al. (1985) Int. J. Immunopharmacol. 7, 73-79; ©Hahn, G. S. (1986) Nature 324, 283-284; "Burton, P. M. et aL (1987) Int. J. Immunopharmacol. 9, 297-305; =Abiko, T. et al. (1979) Biochem. Biophys. Res. Commun. 86, 945-952; f Plow, E. F. and Edgington, T. S. (1986) J. Immunol. 137, 1910-1915 gWieczorek Z. etal. (1979) Immunology36, 875--881; "Parker, F. etal. (1984) Eur. J. Biochem. 145, 677-682; =Berthou, J. etaL (1987) FEBSLett. 218, 55-58; i Litwin, A. etal. (1988) Int. Arch. Allergy AppL Immuno/. 87, 361-366; k Petrov, R. V. et at. (1986) PatoL Fiziol. Eksp. Ter. 7-12

Modification of the D-lactyl residue at C-3 of the muramyl moiety produces two compounds, n o r - M D P and 3 ' - n - p r o p y l - M D P , which have reduced toxicity. NorMDP has been used in 'adjuvant 65' with a malaria antigen, and as an emulsion in squalene-Arlacel in an antifertility vaccine 3. The cyclic osidic structure is essential for activity; only the D-giuco-, Dmanno- and D-galacto-MDP congeners are active 1. Changing the configuration of L-alanine usually abolishes activity. When alanine is replaced with 2-aminobutyric acid, the resulting compound, [0~-Abul]MDP, has antibacterial properties and activates alveolar macrophages. The 6-O-stearoyl derivative of [0~-

Abul]MDP when encapsulated in liposomes arrests the development of pulmonary metastasis 4 and acts synergistically with antimonate meglumine (glucantime) against visceral leishmaniasis and with amphotericin B against murine candidiasis 5. [Thrl]MDP, in which alanine is replaced by threonine substantially increases humoral and cellular responses when applied in a pluronic polyol formulation, and may prove useful as an adjuvant for vaccines 6. The presence of three structural features in the D-isoglutamyl moiety is important for activity: (1) the D-configuration of :-he amino acid residue; (2) the presence of two methylene groups separating the two carboxyls; and

(3) the two carboxyl functions (free or substituted). These are all essential for stimulating nonspecific resistanceL Two congeners - m u r a b u t i d e and m u r a m e t h i d e - have shown therapeutic promise. A third derivative, M T P PE, is currently being studied in the clinic. When administered in liposomes, MTP-PE eradicates pulmonary metastasesT; it also induces tumoricidal properties in h u m a n monocytes s, and when applied prophylactically it protects against viral infections even after intranasal application 9. During the last several years, a n u m b e r of muramyl peptides, especially MDP, have been found to act synergistically with other immunomodulating agents. For

TiPS

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September 1990 [Vol. 11]

example, MDP has synergistic effects with trehalose dimycolate in an in vitro guinea-pig hepatocarcinoma model 2 and against influenza and tuberculosis infections in mice 2. A combination of mycobacterial cell walls and trehalose dimycolate is being used in the USA for treatment of ocular carcinoma in veterinary medicine 2. The possibility of using synergistic combinations of liposome-encapsulated MDP and macrophage-activating factor for in situ activation of murine alveolar macrophages 10, and MDP and recombinant mouse or human interferon-~/for activation of ar, titumor properties in human blood monocytes and mouse macrophages 11, have also been investigated. The tumor-necrotizing activity of toxic and detoxified endotoxins towards subcutaneous Meth A fibrosarcoma transplants in syngeneic BALB/c mice was markedly enhanced by simultaneous i.v. injection of MDP (Ref. 2). The mechanism of action is still largely unknown, but it is clear that muramyl peptides act on macrophages and B lymphocytes 12. However, there is no evidence for the presence of MDP receptors at the surface of these cells. When radiolabeled MDP was injected i.v. in mice, 95% of it was recovered unchanged within 2 h in the urine lz. Clinical evaluation of several muramyl peptides is currently under way. Murabutide has already completed phases I and II trials as an immunoadjuvant for a tetanus vaccine2. Nor-MDP is being used as an adjuvant in trials of a WHO-sponsored antifertility vaccine consisting of an oligopeptide corresponding to the amino acid sequence 109-145 of the C-terminal region of the f~subunit of human chorionic gonadotropin conjugated to a protein carrier diphtheria toxoid. Active

[c.,(~.j ,J?.co,~.,

375 TABLE II. Structures of MDP and congeners Compound

MDP Nor-MDP 3'-n-Pr-MDP FFhrl]MDP [o~-Abul]MDP Murabutide Muramethide

R1

R2

R3

R4

CH3 H n-C4H9 CH3 CH3 CH3 CH3

CH3 CH3 OH 3 CH(CHa)OH C2Hs CH3 OH3

NH2 NH2 NH2 NH2 NH2 OC4Hg-n OCH 3

OH OH OH OH OH NH2 NH 2

immunization of baboons inhibited fertilization 3. An anti-influenza vaccine comprising synthetic hemagglutinin and B-30-MDP in m~celle~ ha~ b~en reported to be effective in mice 2. A vaccine against feline leukemia virus containing [Thrl]MDP as an adjuvant has been marketed in the USA for veterinary use 2. • Lipopeptides Lipopeptide molecules containing a covalently bound fatty acid (e.g. pimelautide and RP56142) have considerable immunomodulating activity in vitro and in vivo 12. The immunomodulating properties are retained only in isomers containing 2,6-diaminopimelamic acid in the L,L-configuration. Compound FK156, together with its more potent heptanoyl-oalanine derivative FK565 exerted broad immunostimulating active.ties 12. A synthetic tripalmitoyl pentapeptide compound representing the N-terminal portion of a lipopeptide isolated from the outer membrane of E. coil elicited a mitogenic activity in mouse B lymphocytes and polycionai activation of immunoglobulin production]3,]t. • Bestatin and derivatives A compound isolated from Streptomyces olivoreticuli ~5- bestatin increases interleukin 1 (IL-1) production by macrophages and IL-2 production by mitogen-stimu-

R)NH2s CIH2CH(CH3.)2 CH2--CH-CHCONHCHCOOH OH

\,

uo'4~

bestatin

/ NHCOCH~

OH I

CH~CHC- L- Ala - D - Glu - NH: O

B-30-MDP

lated lymphocytes by suppressing the activity of membrane-associated aminopeptidase B and leucine aminopeptidase ~". Its

immunomodulating properties are manifest by stimulation of cellmediated immunity and enhanced antibody production. At doses of 0.1-100 pg per mouse, given orally or i.p. at the time of immunization, bestatin elevates the delayed-type hypersensitivity response to sheep erythrocytes in mice. The sconfiguration of the (x-carbon of the 3-amino-2-hydroxy-4-phenylbutanoyl moiety proved to be absolutely necessary for the inhibition of aminopeptidase activity and enhancement of the delayedtype hypersensitivity response. Bestatin also has antitumor activity, and in cancer patients (at OH B st CH3CHCH2CH :CHCO-VaI-Val- Asp f I CH3 NH2

amastatin HO

NH z

forphenicine 30 mg per day, orally) increases the usually reduced levels of T-cell population and natural killer cell activity, in what appeared to be an imm,morestorative mechanism of action. p-Hydroxybestatin (a 4-hydroxyphenyl metabolite of bestatin) is five- to ten-fold more potent in inhibiting the activity of aminopeptidase B (Ref. 15). It also augments the delayed-type hypersensitivity reaction to sheep erythrocytes or oxazolone in mice, and increases the number of antibodyforming cells in the spleen I-~. Two related microbial products - amastatin and the previously unknown amino acid forphenicine - both increase the popu-

TiPS- September 1990 [Vol. 11]

376 lation of cells producing antibodies against sheep erythrocytes =7,1s. in addition, forphenicine markedly enhances the delayed-type hypersensitivity response to sheep erythrocytes and arrests the development of Gardner iymphosarcoma and basosquamous carcinoma in mice.

• Thymic peptides and synthetic analogues Peptide hormones isolated from thymic extracts or from serum are known to induce specific T-ceU markers on precursor immature lymphocytes and to stimulat o Tcell functions. The amino acid sequences of three thymic hormones, thymopoietin (49 amino acid residues), thymosin ~1 (28 amino acid residues) and thyinulin (nine amino acid residues) have been fully characterized. There is no apparent homology between them; thymulin also binds readily to Zn 2+ (Ref. 19). A synthetic pentapeptide, thymopentine C/P-5), which corresponds to the biologically active amino acid sequence 32-36 (ArgLys-Asp-Val-Tyr) of thymopoietin, mimics the activity of thymopoietin. Two other synthetic analogues TP-3 and TP-4 (amino acids 32-34 and 32-35, respectively), possess similar activity20. 21, Splenin is a 49-amino-acid spleen polypeptide that differs from thymopoietin only by substitution of Glu for Asp at position 34; and possesses similar immunoregulatory activity. When given to uremic patients, splenin restores to normal the levels of B cells ~. The synthetic peptide splenopentin (SP-5), representing the biologically active site of splenin, differs from TP-5 only in substitution of Glu for Asp. Both compounds enhance the H-Y rejection response of young thymus-intact female C3H/HeJ mice to syngeneic male skin, but neither affects the response of splenectomized female mice 23. Furthermore, while TP-5 lowers the raised H-Y rejection response of thymectomized female mice, SP-5 does not. Thus, although SP-5 and TP-5 are structurally similar, their immunoregulatory effects in vivo may differ, and depend on the immune status of the recipient 23. Recently, splenin has been used to correct immune deficiencies in patients with tuberculosis and syphilis24.25.

Bursin (Lys-His-Gly-NH2), a Bcell differentiating hormone isolated from the bursa of Fabricius of chickens, potentiates the phenotypic differentiation of both avian and mammalian pre-B cells 26. Thymic peptides are currently being used to treat a variety of pathologies, including primary immune deficiencies (severe combined immune deficiency and DiGeorge's syndrome), immune deficiencies in elderly patients, AIDS and AIDS-related complex, recurrent herpes simplex, chronic active viral hepatitis B, lepromatous leprosy, active rheumatoid arthritis and malignancies (lung and colorectal cancers and hypernephroma) 19. • Transfer factor Dialysable material that is extracted from sensitized humans and other vertebrates is termed transfer factor. When transfer factor from sensitized donors is administered to unsensitized recipients, the recipients acquire the ability to express the cell-mediated immune responses of the donors 27. Transfer factor is a polypeptide with molecular weight of 4-6kDa, which is sensitive to most proteases but resistant to trypsin, chymotrypsin and leucine aminopeptidase and to nucleases. Kirkpatrick 27 proposed that it consists of polypeptide chains linked by disulfide bridges; however, the presence of nucleic acids, ribose and phosphodiester groups has not been ruled out. The key function of transfer factor is its ability to transfer delayed cutaneous hypersensitivity to previously unsensitized recipients. Lymphocytes from recipients of transfer factor respond to antigens in vitro by producing lymphokines (with lFN-y-like activity), proliferating, and expressing cytotoxic activity. It appears that all the immunological properties of transfer factor are antigen specific and concordant with the immunological responses of the transfer factor donor 27. Transfer factor has been used in treatment of patients with chronic mucocutaneous candidiasis (in conjunction with antifungal drugs), and herpes simplex infections, and children with leukemia apd no immunity to varicella-zoster (with transfer factor obtained from immune donor). Only one of the 16 transfer-factor-treated

children exposed to developed chickenpox.

infection

• Leukocyte dialysates Leukocyte dialysates contain a variety of components, distinct from transfer factor, that modulate the expression of delayed-type hypersensitivity reaction when injected intradermally with an antigen to which the recipient is sensitive 2s. IMREG-1 is one such peptide, which in contrast to transfer factor does not transfer delayed-type hypersensitivity response from antigen-sensitive donors to nonsensitive recipients, but rather amplifies the responses of recipients who were already sensitive to particular antigens 2s. In vitro, in the presence of antigen or mitogen, IMREG-1 enhances the production of various biological response modifiers and lymphokines (migration-inhibition factor, leukocyte-inhibition factor), and the antigen-induced expression of receptors for IL-2 on CD4 + cells 28. In an open phase II trial with patients with AIDS or AIDSrelated complex IMREG-1 was reported to restore delayed-type hypersensitivity reactions to recall antigen in patients who had been anergic prior to treatment, to decrease the rate of destruction of CD4 + cells and to clear candidal infections 28.

lmmunosuppressivepeptides Immunosuppressive peptides and their analogues are now being used to prevent graft rejection; they may also have a role in treating autoimmune disease.

• Ciclosporin and congeners The best-studied immunosuppressive peptide, ciclosporin (cyclosporine) is a fungal metabolite isolated from Tolypocladium inflatum Gams. It is a neutral, hydrophobic, cyclic peptide composed of 11 amino acid residues, which 10

11

1

2

3

M e L e u - - M e V a l -r- M e B m t ~ A b . -r- S a r 9 M , eLeu

I

i

I D-Ala -- Ala --'- MeLeu

8

7

6

; V a l ---' M e L e u

.5

4

ciclosporin with the exception of D-alanine (at position 8) and the non-chiral sarcosine (at position 3), all possess

TiPS - September 1990 [Vol. 11]

377

Glossary Adjuvant 65. An adjuvant consisting of an emulsion of peanut oil with mannide monooleate, stabilized with aluminium monostearate. S-antigen. 'Soluble' antigens; an incomplete noninfectious internal component of virus appearing early in a viral infection. EAC rosette-forming cell. A lymphoid cell that forms rosettes with sheep erythocytes in the presence of antisheep-erythrocyte antibody and complement, by binding of the erythrocytes to C3b receptors. EAC rosette formation is used as a marker for B cells, although phagocytes possess C3b receptors and can, therefore, also form EAC rosettes. E-rosette-forming cell. A lymphoid cell that can form spontaneous rosettes with sheep erythrocytes. E-rosette formation is accepted as a T cell marker in humans and mammals.

the L-configuration. One of the amino acids, the (4R)-4-I(E)-2butenyl]-4,N-dimethyl-L-threonine (MeBmt) was previously unknown. Ciciosporin has rigid conformation in which the amino acid sequence 1-6 adopts an antiparallel B-pleated sheet conformation containing three transannular hydrogen bonds, and is significantly twisted to form a type II' B-turn between sarcosine at position 3 and N-methylleucine at position 4. The remaining hydrogen b o n d is of y-type and holds the backbone of the molecule in a folded 'L' shape. The sequence MeVa111-MeBmt1-Abu2Sar3 is essential for i m m u n o suppressive activity. Ciclosporin is a potent and selective immunosuppressive agent affecting both humoral and cellular responses 29. Ciclosporin binds to the same cell-surface b i n d i n g protein as prolactin; this protein is not identical with the antigen recognition site or the T-cell receptor. It has also been suggested that ciclosporin intercalates into the lipid matrix of the plasma membrane. After internalization, ciclosporin binds to the protein cyclophilin in the cytoplasm 29. Following penetration into the nucleus, ciclosporin is thought to inhibit transcription of lymphokine genes without affecting the bulk of other genes. GranelliPiperno et al. 3° proposed that it acts on a transcriptional control site shared by IL-2, IFN-y and cmyc genes, thus suppressing the mRNA synthesis for those proteins. As a result, ciclosporin in-

Leukocyte-migration inhibition factor. A lymphokine with protease activity that can suppress migration of neutrophils. Migration-inhibition factor. A lymphokine that inhibits the migration of macrophages. There are at least two forms: one of 65 kDa with pI 3-4; and the other of ~25 kDa with pl ~5. Phytohemagglutinins. Lectins isolated from Phaseolus vulga~a or P. communis that bind N-acetyi-[$-ogalactosamine residues and thus cause agglutination of certain erythrocytes. In soluble form, they are powerful mitogens for T ceils. When pres,mted on insoluble matrices, they may act as a mitogetn ~'o.rB cells. Pokeweed mitogen (PWM). A group of ~i'.'e mitogens known as Pa 1-5, and obtained from Phytolacca americana. While Pal activates both B and T cells, Pa2, Pa3 and Pa4 only activate T cells.

fluences the early cellular events that take place in an immunocompetent T cell in response to antigenic stimulus. It inhibits the antigen signal transduction by suppressing the reception of the message triggered by antigen recognition, thus halting the cell cycle in the Go or early G1 phase 29. Furthermore, ciclosporin inhibits the enhanced incorporation of unsaturated fatty acids that occurs in the vicinity of the signal-generating plasma m e m b r a n e domains of activated T and B cells, thereby preventing synthesis of the second messengers that stimulate m R N A synthesis al. There is evidence that the immunosuppressire effects of ciclosporin are also mediated by the displacement of prolactin by ciclosporin from its binding sites on the lymphocytes 32. Since its initial use in kidney and bone m a r r o w transplantations, ciclosporin has been used widely in clinical medicine to prevent graft rejection following organ and bone-marrow tramsplants. One major advantage of ciclosporin theray)y is that it does not require pretransplant tra:lsfusions, splenectomy and maintenance dialysis therapy; trr:atment with ciclosporin is superior to previous therapies in prolonging the survival of patients while reducing complications sucil as myelosuppression and s~eroid morbidity. In recent years, ciclosporin has also found application in the treatment of a u t o i m m u n e diseases 29. The drug seems to be highly effective in preventing the develop-

merit of a number of autoimmune conditions in animal models, especially when administered during the induction phase of the disease. In the clinic, ciclosporin has been successfully used" in the treatment of patients with posterior uveitis whose lymphocytes responded to S-antigens in vitro. Treatment of autoimmune conditions related to type I diabetes mellitus is another area for which the usefulness of ciclosporin is being explored 33. Changes in the essential sequence MeVal11-MeBmtl-Abu2Sar3 disturb the integrity of the type II' [~-turn and often significantly weaken immunosuppressive activity 34. Structural modifications of MeBmt have usually resulted in inactive compounds. Modifications of the 2-aminobutyric acid residue do not always inactivate the drug, but any configurational and/or structural changes to methylvaline are detrimental. The synthetic congener [norVal2Jciclosporin has similar immunosuppressive properties but diminished nephrotoxicity. Another synthetic derivative, [Val2]dihydrociclosporin seemed to have a different spectrum of immunosuppressive activity; it did inhibit some types of cellmediated immune responses, but the humoral immunity and allograft rejection remained largely intact. Such activity would make this compound useful in autoimmune diseases where T cells are involved 34. The analogue 8-5-49 enhanced resistance towards Leishmania maior when admin-

378

TiPS - September

istered p r o p h y l a c t i c a l l y to BALB/c mice.

c.

MeLeu-- MeVal-;-N- C~ C -;- Abu~- Sar

r e c e i v i n g FK506 (Ref. 38). T h e widespread vasculitis may be s p e c i e s specific for d o g s 38. It is thought that the N-ketomalonyipipecolate moiety of FK506, having three sequential keto g r o u p s m a y a c c o u n t for its i m m u n o s u p p r e s s i v e effect. References

9

MeLeu

I

D- A l a - - A l a -..-..~MeLeu, ' Val'~-MeLeu 8

"~

6

$

4

B-5-49

A e b i et al. 35 h a v e a c c o m p l i s h e d t h e s y n t h e s i s of several conform a t i o n a l l y r e s t r i c t e d iactam a n a l o g u e s of ciclosporin a n d e x a m i n e d t h e role of t h e t y p e II' ~ - t u r n o n biological activity. In b o t h in v i t r o a n d in v i v o experiments FKS06, a novel non-

HO~.

.H

C i % O ~ "~ ~

CH3 ..~

A

.0 C~

'"lc"f Y. Y T ii

%..%...0 .o,s....... 0%,00 FK506

oca,

r"'