Immunologic Research 1998;17/3:345-368

Thymopentin and Splenopentin as Immunomodulators Current Stutus

1Department of Immunology, Sanjay Gandhi Post-Graduate Institute of Medical Sciences, Lucknow, India. 2Division of Biopolymers, Central Drug Research Institute, Lucknow, India. 3Department of Microbiology, RML Awadh University, Faizabad, India.

Abstract Key Words Splenopentin (SP-5, Arg-Lys-Glu-Val-Tyr) and thymopentin (TP-5, Immunomodulation Arg-Lys-Asp-Val-Tyr) are synthetic immunomodulating Splenin corresponding to the region 32-34 of a splenic product called splenin Splenopentin (SP) and the thymic hormone thymopoietin (TP), respectively. TP Thymopoietin was originally isolated as a 5-kDa (49-amino acids) protein from Thymopentin bovine while studying effects of the thymic extracts on neuro- muscular transmission and was subsequently observed to affect differentiation and function. TP I and II are two closely related polypeptides isolated from bovine thymus. A radioimmunoassay for TP revealed a crossreaction with a product found in spleen and lymph node. This product, named splenin, differs from TP only in position 34, aspartic acid for bovine TP and glutamic acid for bovine splenin and it was called TP III as well. Synthetic pentapeptides (TP-5) and (SP-5), reproduce the biological activities of TP and SP, respectively. It is now evident that various forms of TPs were created by proteolytic cleavage of larger proteins during isolation, cDNA clones have been isolated for three alternatively spliced mRNAs that encodes three distinct human T cell TPs. The immunomodulatory properties of TP, SP, TP-5, SP-5 and some of their synthetic analogs reported in the literature have been briefly reviewed.

Dr. V. K. Singh 9 1998 345 Laboratory of Molecular Immunology Humana Press Inc. Department of Immunology 0257-277X/98/ Sanjay Gandhi Post-Graduate Institute of Medical Sciences 17/3:345 368/$14.00 Lucknow - 226 014, India E-mail: vksingh @sgpgi.ren.nic.in Introduction nity and decrease the morbidity of immuno- "hnmunomodulators" are substances that compromised individuals (1-3). Similar have the ability to influence various compo- observations were also made in the case of nents of the immune system. They can be used experimental animals (4,5). One of the thymic therapeutically for correcting pathological hormones called thymopoietin (TP) is a aberrations of the immune response in condi- polypeptide consisting of 49 amino acids (6, 7). tions such as immunodeficiency, chronic TP has pleiotropic biological actions includ- infections, autoimmunity, organ transplant ing effects on neuromuscular transmission and neoplasia, and so on. Such substances are (8,9), T and B cell differentiation (10,11) and often referred to as "Biological Response regulation of the immune response (12-17). Modifiers" as well. The action of immuno- The biological activity to TP is reproduced by modulators may be specific or nonspecific. a synthetic pentapeptide thymopentin (TP-5) The pharmacological modulation of the whose amino acid sequence corresponds to the immune response is essentially dependent region 32-36 of the native hormone (Arg-Lys- upon the dose of the substance applied, the Asp-Val-Tyr). Since the pentapeptide (TP-5) mode of its application, and the prevailing sta- exhibits all the biological activities of TP, the tus of the immune system of the host. former is considered to be the active site of the Endeavors to develop chemically well- native hormone (18). Recently, a closely defined compounds possessing immuno- related pentapeptide called splenopentin modulating properties have resulted in the (SP-5), has been reported to exhibit a particu- identification of several classes of compounds larly interesting biological profile. The amino that can act as or immuno- acid sequence ofSP-5 (Arg-Lys-Glu-Val-Tyr) suppressors. Several peptides and their deriva- corresponds to the segment 32-36 of a splenic tives such as muramyl dipeptide (MDP), hormone called "splenin" (also referred as lauroyl tetrapeptide (LTP), enkephalins and TP-III). SP-5 differs from TP-5 only at posi- thymic hormones, are quite well known for tion 3 due to the presence of Glu residue in their immunostimulating activities. Likewise, place of Asp. SP-5 is known to influence both cyclosporin A, FK-506, and rapamycin are early T and B cell differentiation (19), to well-established immunosuppressive agents. increase the number of antibody-forming cells In addition, many substances such as saponins, in mice after gamma irradiation (20,21). This non-ionic block polymers, monophosphoryl pentapeptide accelerates the repopulation of A and , which stimulate T cell sub- epidermal Langerhans cells at skin sites sets responsible for the manifestation of the depleted with these cells (22,23). Several syn- specific immune responses, are termed thetic analogs of TP-5 and SP-5 have been immunoadjuvants. -1 (IL-1) hap- studied for their immunomodulatory activity pens to be the first that has been used with a view to identify a compound that would as an adjuvant. Administration of -], be more suitable for pharmaceutical applica- (IFN-],) is also known to activate helper T cells tion in terms of stability, biodisposition, and for antibody and delayed-type hypersensi- receptor affinity. Short synthetic tivity responses. sequences of this type may turn out to be ideal In recent years, thymic preparations have candidates as adjuvants for human been widely used for boosting the natural and could also be utilized as immunothera- defense system of the host. Thymic hormones peutic and immunoprophylactic agents in case have been reported to reverse altered immu- of infectious diseases, immunodeficiencies,

346 Singh et al. Table !. Summary of immunomodulatory activities of TP

In vitro studies T cell differentiation Induced prothymocyte to thymocyte differentiation 10 B cell differentiation Inhibited B cell differentiation 11 Lymphocyte proliferation Stronger inducer than synthetic TP-II 48 In vivo studies Neuromuscular transmission Impaired 6 T cells Induced T cell differentiation 50

autoimmune diseases, and cancers. This arti- later found to be wholly crossreactive with cle constitutes an attempt to highlight the TP-I (26). Subsequent studies with synthetic recent developments relating to TP-5 and fragments showed that the pentapeptide SP-5, and to give an idea about their current sequence Arg-Lys-Asp-Val-Tyr, corre- status as immunomodulators. sponding to residues 32-36, was the minimal fragment that reproduced the biologic proper- Thymopoietin ties of TP, both in vitro and in vivo, and this TP is a polypeptide hormone of the thymus synthetic pentapeptide was termed TP-5 (18,19). that induces differentiation of prothymocytes TP is a linear polypeptide hormone secreted to thymocytes and also has secondary by the epithelial cells of the thymus and is effects on neuromuscular transmission (6). Its readily detected in the thymus and absent in immunomodulatoryproperties are summarized control tissues, such as, liver, kidney, thyroid, in Table 1. and muscle (6,27). The isolation of TP-reac- tive material in fetal bovine skin was moni- Isolation and Structure of TP tored by means of a radioimmunoassay. The The similarity of the signs and symptoms of amino acid sequence of this material was myasthenia gravis with those of curare poi- found to be identical with that of TP isolated soning prompted an extensive search for a from the thymus. Experimental evidence sug- curare-like substance in the serum of myas- gests that TP in the circulation is derived from thenic patients or in thymus extracts. How- the thymus and not from the skin, suggesting ever, in the absence of such a substance this that the TP in keratinocytes has a local function, conundrum was solved when the molecules either apocrine or immunoregulatory (28). responsible for the delayed neuromuscular The TP gene is expressed predominantly in impairment in mice were identified following lymphatic tissues. The lymphatic tissue with the injection of thymic extracts or purified the highest levels was observed in thymocytes polypeptides. Two biochemically homoge- and not in thymic stroma. Lower but still sig- neous thymic polypeptides, TP-I and -II were nificant amounts were present in the tonsils, isolated (7). The complete amino acid neck lymph nodes, and small intestines (prob- sequence of TP-II was determined and the bio- ably because of its lymphatic component-- logical activity was shown to reside in seg- Peyer's patches), whereas cultured spleen ment 29-41 by chemical synthesis (24,25). A stromal cells and peripheral blood mono- radioimmunoassay developed for TP-II was nuclear cells displayed a low level of TP

lmmunomodu|ationwith TP-5 and SP-5 347 mRNA. The TP gene expression in all other mRNA expression is demonstrated in lympho- (nonlymphatic) tissues tested was weak, cytes from all the differentiation stages inves- barely detectable, or virtually absent (29). tigated, as well as in the myeloid cell line, Harris et al. (30) reported the isolation of K562. The findings suggest a further expan- cDNA clones for three alternatively spliced sion of the proposed ATP functions (33). mRNAs that encode three distinct human TP. A TP-related peptide gene was identified and Proteins encoded by these mRNAs, named cloned from a human cDNA library (34) and TPc~ (75 kDa), 13 (51 kDa), and y (39 kDa), novel TP responsive proteins 1 and 2, related to contained similar N-terminal regions, includ- signal transduction, were also identified (35). ing sequences nearly identical to that of the TP levels in the serum have been found to originally isolated TP, but had divergent be high in normal humans until the fourth C-terminal regions (31). It is now evident that decade of life, after that the levels decline pro- 49-amino acid TPs were created by proteolytic gressively and are below detection limits by cleavage of a larger protein during isolation 70 yr of age (36). These findings parallel the and represent the N-terminal sequences of known anatomic involution of the thymus and these proteins. The human TP gene maps to emphasize that thymic function does not dis- chromosome band 12q22. The finding of a appear beyond the age of puberty. Limited single thymopoietin gene suggests that vari- studies have shown low TP levels in primary ant forms of TPs purified from thymus in ear- immunodeficiencies and systemic lupus lier publications (7,19) may be related to erythematosus (36-38). posttranslational modifications or amino acid sequencing errors, since the weight of latest TP as a Neuromuscular Regulatory Hormone evidence points strongly to a single TP gene. Attention has been focused on isolation of Other lower molecular weight forms of TPs thymic factors that can influence the activity are detected both intracellularly and extracel- of T lymphocytes. TP was originally isolated lularly in cell culture supernatants and human in relation to myasthenia gravis, since thymi- plasma. These may be derived by proteolysis tis was found to be associated with neuromus- from TPs o~, [~, or y. cular transmission (39). The relationship of Seven mouse TP mRNA transcripts namely experimental thymitis to the neuromuscular a, 13, 13', Y, e, 6, and s, were also isolated. The lesion is determined by thymectomy experi- o~, 13, and Y TP cDNA clones are mouse ments. These determine that adult thymecto- homologs of previously characterized human mized animals are susceptible to other ~, 13, and yTP, whereas TP e, 6, and (y are novel experimental autoimmune diseases and did cDNAs. The mouse TP gene is a single-copy develop an immune response when immu- gene organized in 10 exons spanning 22-kb nized with thymic extracts in complete sequences, located in the central region of Freund's adjuvant. However, thymectomized mouse chromosome 10. The human 13-specific animals immunized with thymic extracts did domain was found to be encoded by 3 exons not develop a neuromuscular lesion, suggest- designated 6a, 6b, and 6c and not by a single ing that the pathogenesis of neuromuscular exon as described previously. These findings impairment in experimental autoimmunethymi- suggest that there may be more human tran- tis involves the release of a substance from the scripts than currently recognized (32). The thymus (40). mRNA expression of this gene in different Using microphysiologic techniques, it was hematopoietic cell lines was studied. TP found that miniature end-plate potential

348 Singh et al. amplitude was increased in thymectomized lular cGMP in a number of human T cell lines animals and decreased in animals bearing mul- that have surface phenotypes corresponding tiple normal thymus grafts, establishing that to mature T cells (46). Furthermore, a radio- the substance in the thymus impairing neuro- receptor assay has been developed that illustrated muscular transmission is a physiologic secre- the interaction of TP with a receptor molecule tion (9). Hence, it was originally termed thymin, isolated from the membrane of a responsive but this name was changed in view of ambigu- human T cell line, CEM. It appears likely that the ity of the term thymin with some other bio- distinct peripheral action of TP on peripheral T logic substances (6,41). cells is mediated by cGMP-mediated actions of TP in inducing T cell differentiation. In Vitro and In Vivo Studies with TP Synthetic bovine TP was found to induce Because the major function of the thymus is expression of Thy 1.2 antigen on T lympho- concerned with immunologicdifferentiation and cytes from athymic mice, which is in agree- regulation, the possibilities remain that TP is an ment with the previous studies on the immunologic hormone with pleiotropic effects. endogenous activity of bovine TP. The bio- These possibilities were resolved by testing logic activity at the skeletal muscle and neu- purified TP or synthetic TP-5 on immune cells. ronal nicotinic acetylcholine receptors could Such studies demonstrated unequivocally that not be confirmed, since it could be related to TP has major immunologic functions. the presence of cobra toxin-like molecule in TP induces the differentiation of early the preparations on natural bovine TP. These T cells (thymocytes) from precursor cells data indicate that synthetic peptides have an (prothymocytes) found in the bone marrow or important role for the evaluation of specificity spleen (10). This action of TP can be readily of the biological activity of the polypeptide. assayed in vitro by the induction of several TP-II, tested for its effect on impaired T lym- thymocyte-specific marker antigens detect- phocyte proliferation by phytohemagglutinin able serologically on the surface of induced (PHA), exhibited a stronger restoring effect than T cells. Selective induction of Tps might have that of a synthetic TP-II (Phe[4F]3-TP) (48). been explained by restriction of TP receptors Purified TP and synthetic TP-5 affect neu- to the prothymocytes. TP inhibited in vitro romuscular regulatory function (19,49). In B cell differentiation (11). TP receptors are vivo studies showed that TP-5 also drive also present on B cells, though the biochemi- T cell differentiation in nude mice that cal circuit inhibits rather than initiates induc- congenitally lack a thymus (50,51). These tion. In contrast, bursopoietin induces induction experiments established a major selective differentiation of B cells but not immunological function for TP, which T cells (42). This suggests a role for TP in the appeared therefore to represent a pleiotropic coordinated interregulation of lymphocyte hormone affecting both immunologic functions subsets, in addition to its better known func- and regulation of neuromuscular transmission. tion as thymic inducer of prothymocytes. Differential regulation of lymphocyte subpop- Thymopentin ulations by TP is brought about by hormone- The pentapeptide Arg-Lys-Asp-Val-Tyr, receptor interactions. TP activates the corresponding to residues 32-36 of TP, was differentiation of T cell precursors (prothy- shown to have the biologic properties of TP mocytes) by a cAMP-dependent mechanism both in vitro and in vivo (18). The retention of (43-.45). TP also induces elevation ofintracel- thymopoietic activity by TP pentapeptide (TP-5)

Immunomodulation with TP-5 and SP-5 349 Table 2. Summary of immunomodulatory activities of TP-5

In vitro studies T cell differentiation Induced prothymocyte to thymocyte differentiation 6 B cell differentiation Inhibited B cell differentiation 11 IgG production Dose-dependent stimulation/inhibition of PWM-induced IgG 57 T cell proliferation Dose-dependent stimulation 59 IL-2 production Enhanced PHA-induced IL-2 synthesis 59 IFN production Stimulated mitogen-induced IFN production 20 NK cell activity Enhanced mouse NK cell activity 60 In vivo studies Neurotransmission Blocked neurotransmission 12 Immunomodulation Restored helper T cell activity 12 Restored tolerance and reduced male skin graft rejection 13,14 Low dose enhanced cytotoxic lymphocyte activity 16 No restoration of immune function by short-term treatment 70 Autoimmunity Switched off autoantibody formation 15 Inhibited antigen-induced arthritis in rats 64 Enhanced OKT4 + cells 65 Immunomodulation in SLE mouse model 66 Increased double-positive and decreased single-positive cells in SLE mouse model 67 Tumors Reduced tumor growth and metastatic spread 69 Tumor prevention and no adverse reaction 68 Delayed onset of tumor-induced immune suppression 17 Leishmaniasis Enhanced Th2 and reduced Thl response 62 Radiation damage Prevented radiation damage and reduced PGE 2 production 71 Thermal burn Improved survival 73 Clinical studies Cancers Breast cancer--reduced immunosuppressive effects 74 Renal cancer--no benefit 75 Head & neck tumour--improved immunologic status 76 --reduced severity 77 Carcinoma--reduced febrile episode 79 Immuuodeficiency Di Georges syndrome--clinical and improvement 80,81 --adherent suppressor cells 82 SCID--increased NK activity 83 Increased T cell proliferation to mitogens, IL-2 production 1 Hypergammaglobulinamia--abrogated suppressor cell activity 86 Atopic dermatitis Normalized T cell number with FcIgG and T8 markers 88 Decreased production of histamine-releasing factor 87 Increased cytotoxic/suppressor cells 89 Safe adjuvant to therapy 90, 92 (continued)

350 Singh et al. Table 2. (continued)

Autoimmunity Rheumatoid arthritis--increased E-rosette cells 93 Immunoregulatory role 97 Increased CD8 § CD11B + T cells 98 CD5 + B cells not affected 99 Scleroderma--decreased CD16 + and CD25 § cells 100 Multiple sclerosis--clinical improvement 101 Infections Hepatitis B--not effective 102 Papilloma, HSV--effective 65 Respiratory infections--reduced incidence 103,104 HIV infection--slowed and arrested disease progression 105 Reduced progression rate in zidovudine-treated patients 106 HIV-associated Candidiasis --all patients benefited 107 Vaginal infection--complete or partial remission 108 Others Radiation--counteracted immunedepression and reduced severity 113 Postsplenectomy infection--prevented infection 110 Cardiac bypass--restored cellular immunity 109 Adjuvant effect--ineffective 117 Myelodysplastic syndrome--good recovery 115 Chronic stress--exerted nonspecific adaptive effects 116 suggests that this pentapeptide may be able to cells is presented as a possible basic mecha- assume a conformation similar to that of the nism of this phenomenon. The biologic active site of native TP (52). Various actions activity of TP is presumably related to the of TP-5 are briefly presented in Table 2. interactions of a specific conformation of resi- dues 32-36 in the intact molecule with the Pharmacokinetics receptor (55). Structure-activity studies on In humans, TP-5 is rapidly cleaved by TP-5 analogs have established a number of plasma proteases (apparent tl/2 = 30 S). In par- criteria essential for biologic activity: ticular, this pentapeptide appears especially 1. A pentapeptide is the smallest fragment of susceptible to enzymes that degrade the pep- TP having full biologic activity of TP on the tide bonds adjacent to Arg and Lys. Cleavage CEM cells; of Val-Tyr bond proceeds more slowly (53). 2. Arginine and aspartic acid are required for It has been reported that intravenously admin- the activity; and istered TP-5 is biologically more effective 3. Lysine, valine, and tyrosine are not essential than the subcutaneous route (54). residues for activity; however, certain substi- tutions are permitted (56). In Vitro Immunomodulation with TP-5 In absence of a primary stimulus, immuno- Immunomodulation is interpreted as a tem- modulation remains physiologically silent, but porary alert in certain parts of the immune results in a modified immune response if cor- system. The activation of immunocompetent responding targets are being stimulated (57).

Immunomodulation with TP-5 and SP-5 351 The activation of the immunocompetent activity of TP-5 may be as a result of preferential cells is one of the first steps in the progression regulation of Thl cells (62). of an immune response (58). Even though activation of such cells could be induced with In Vivo ImmunoregulatoryActions of TP-5 various substances, this does not necessarily Immune imbalance in experimental ani- result in an immune response. As discussed mals could result either from a loss of thymus earlier, TP-5 induces T cell differentiation and function, caused by thymectomy or the invo- inhibits B cell differentiation (6,11). Poke- lution of the thymus that occurs with age or weed mitogen (PWM)-induced IgG produc- from experimental manipulations. In both tion of peripheral blood mononuclear cells instances, the immune imbalance may be (PBMCs) was used as a model for demonstra- either in the direction of hyperresponsiveness tion of a modulatory effect of TP-5 in vitro. or hyporesponsiveness, and TP-5 had been Depending on the concentrations of TP-5 used shown to be immunorestorating in that it could in the cultures, this pentapeptide either stimu- bring the immune system toward normal, lated or inhibited the mitogen-induced IgG whether it is upregulated or downregulated. production. It also influenced prostaglandin Old mice or young thymectomized mice E 2 (PGE2) production and catabolism in cul- have decreased helper T cell activity in that tures stimulated with PWM. Indomethacin they produce low-avidity antibodies to trini- abolished the modulatory effect of TP-5 on trophenol-derivatized antigens (12). This defi- IgG production in this model (57). cit can be restored by injection ofT cells from PBMCs from healthy donors were investi- young animals or alternatively by administra- gated in vitro and observed for the influence of tion of TP-5. Thus, in this model TP-5 cor- different doses of TP-5 on nonstimulated pro- rected the immunodeficiency resulting from liferation, Candida-stimulated proliferation, thymectomy or thymus involution with aging. and PHA-induced interleukin-2 (IL-2) pro- In another model system, young female C3H duction. The study demonstrated the immuno- mice engrafted with male C3H skin grafts modulatory character of TP-5 in two ways: the failed to reject them, although they had the dose-dependency of proliferative responses potential to do so by virtue of the H-Y antigen and the observation that only PHA-induced encoded by the Y chromosome of the male induction of IL-2 could be influenced by mice. This tolerance was based on active TP-5, whereas TP-5 itself will not induce T cell suppressor mechanisms and was lost changes in IL-2 levels (59). with thymectomy or with aging. In this sys- The effect of TP-5 on interferon (IFN)-secre- tem, TP-5 injections restored the state of tol- tion capacity in PBMCs as well as its influence erance and reduced male skin graft rejection on concanavalin A (Con A)-stimulated blasto- (13,14). In experiments designed to enumer- genesis and IFN production was evaluated. ate cytotoxic lymphocyte precursor units after Results demonstrated that TP-5 is capable of immunization, it was found that TP-5 had no inducing IFN production in human PBMC. It effect if the antigen doses used for immuniza- potentiated a mitogen-induced IFN production tion were optimal, but did enhance cytotoxic (20). TP-5 increased natural killer (NK) activity lymphocyte precursor units if they were low of mouse bone marrow and gave indirect support due to sub-optimal antigen doses (16). to the contention that IL-2 is involved in the Mice injected with crossreactive rat eryth- enhancement of NK activity observed after rocytes developed autoantibodies to their own TP-5 treatment (60,61). The immunopotentiating erythrocytes. In this case TP-5-treated mice

352 Singh et al. rapidly switched off autoantibody formation immunopotentiation in the success of com- although titers of anti-rat erythrocyte antibod- bined . The immune func- ies remained unchanged (15). Treatment with tions, such as, mitogen response, cytotoxicity the immunomodulatory thymic preparations to EL-4 lymphoma, and NK activity in Balb/c and T cell-directed immunosuppressive drug, nude mice, could not be restored to full func- cyclosporin A, inhibited the specific immune tional activity by short-term treatment with response against bovine serum albumin and TP-5 (70). the development of autoimmunity against car- Balb/c mice are highly susceptible to Leish- tilage constituents in antigen-induced arthritis mania major infection. TP-5 played an impor- in rats (63,64). Low as well as high concentra- tant role in induction and function of the two tions of the hormone characteristically stimu- subsets of CD4 T cells by producing enhanced lated the OKT4 § after the treatment in levels of IL-2 and IFN-7, but significantly rheumatoid arthritis (65). TP-5 administered reduced IL-4 in mice. Thus, it appeared that subcutaneously to MRL/lpr mice, a well- age-related susceptibility to cutaneous leish- known model of human systemic lupus maniasis was correlated with enhancement of erythematosus (SLE), displayed powerful Th2 and reduction in Thl cell activities (62). immunomodulatory activities (66). The in The enhanced protective effect of IL-lc~ vivo effects of thymic factors (TP, TP-5, when administered along with TP-5 prior to thymolymphotropin, and thymomodulin) on sublethal irradiation in the C57BL/6 mouse the immature lymphocytes in the MRL/lpr was investigated. Results suggested that com- mice induced a significant increase in the bined treatment with TP-5 and IL- 1c~ prevents double-negative T cells, both in the thymus radiation damage in C57BL/6 mice (71). and peripheral lymph nodes, with a concomi- TP-5 reversed the cocaine-induced impair- tant decrease of double-positive T cells in the ment of primary antibody response to sheep thymus and single-positive T cells in the red blood cells (SRBCs), and the effect of the lymph node (67). peptide could be related to the immuno- In a model of immunosuppression induced modulatory activities on T cell functions (72). by transplantable tumor, TP-5 delayed the TP-5 significantly improved survival and onset of immunosuppression induced by the reduced mortality by immunomodulation, tumor (17). There were no adverse reactions particularly in higher doses in mice subjected with TP-5 when the detailed analysis of the to thermal burn or bacterial gavage, or both. immune response was studied during the Pretreatment with TP-5 significantly reduced development and progression of 9,10-dim- PGE 2 concentration after burn and gavage (73). ethyl-l,2-benzanthracene-induced rat mam- mary tumors, and tumor was prevented (68). Clinical Studies with lP-5 Taylor et al. (69) suggested a correlation TP-5 has been tried in various clinical con- between enhanced T-lymphocyte functional ditions. A brief account is given below. parameters and reduced tumor growth and metastatic spread produced by combination Cancer Therapy therapies (TP-5 and 5-fluorouracil) in the There are several reports about the use of spontaneously metastatic murine pancreatic TP-5 in various types of cancer. The adminis- tumor, PAN2. TP-5 has been demonstrated to tration of TP-5 in breast cancer patients led to be a superior immunomodulator, and this reduction of the immunosuppressive side observation supports the postulated role of effects of an adjuvant (74). A

lmmunomodulation with TP-5 and SP-5 353 combination of TP-5 + IFN + IL-2 could not, pressor cells have also been shown in a patient however, be recommended in renal cancer with DiGeorge syndrome (82). Improved NK patients, based on results of 17 patients in cell activity has been shown in patients with whom it was found to be ineffective (75). severe combined immunodeficiency (SCID) Patients with head and neck tumors treated (83). The effect of TP-5 treatment on immune with surgery, chemotherapy, and radio- functions has been investigated in immuno- therapy, who also underwent immunologic compromised aged subjects (84). TP-5 was therapy during 1-yr follow-up, revealed better able to improve the cutaneous delayed hyper- immunologic status at the end of follow-up sensitivity to recall antigens and proliferative when treated with TP-5 compared with other response to PHA, Con A, and PWM, as well as patients (76). A total of 168 patients were stud- PHA-induced IL-2 production. On the other ied to analyze the optimal conditions for the hand, no detectable changes were induced by use of TP-5 in reducing the incidence and TP-5 treatment, either on PWM-induced severity of early and late complications after immunoglobulin synthesis or on lymphocyte radiotherapy of patients with head and neck subsets identified by monoclonal antibodies. cancers. The results demonstrated that TP-5 Enhancement oflL-2 synthesis could be a cru- reduced the incidence of severe reactions in a cial mechanism of immunopharmacologic statistically significant manner in cases of action of TP-5 in aging humans (1). Data indi- irradiation of the hypopharynx and orophar- cate that the in vivo administration of TP-5 to ynx in the oral cavity. Immunomodulatory the immunocompromised aged subjects effects were greater in females than in males enhanced the production of 1L-2 (85). Tp-5 (77). Further, in patients with cancer opera- treatment has been shown to abrogate in vitro tions, the results obtained confirmed the effi- suppressor cell activity in patients with cacy of the proposed immunomodulatory hypergammaglobulinemia (86). treatment: a reduction in the incidence of both Atopic Oermotitis early and late infections, an increase in sur- vival, fall in clinical-oncologic stage to less Results of a clinical trial with 16 children advanced levels, and improvement in perfor- with atopic dermatitis suggest that the clinical mance status (78). In patients with advanced efficacy of short-term TP-5 treatment results carcinoma, TP-5 was associated with reduc- from the decreased production of histamine- tion in febrile episodes as compared with pla- releasing factor and decreased release of cebo, and tolerance to TP-5 was excellent (79). polymorphonuclear leukocyte-derived inflam- matory mediators and may have no relation with Immunodeficiencies antigen IgE immune reaction (87). Patients Several clinical studies evaluated the effec- with atopic dermatitis have deficient relative tiveness of TP-5 in patients with primary and absolute numbers of T cells with FclgG immunodeficiencies. In one study the effect and CD8 markers, and TP-5 treatment is able of TP-5 was studied in 26 patients with pri- to normalize these deficiencies. No significant mary immunodeficiencies. Clinical and effect on IgE serum levels or on spontaneous immunologic improvement was described in in vitro production of IgE-cultured lympho- three patients with DiGeorge syndrome and cytes from the patients was observed (88). In with primary T cell defect. Tolerance was another study, a double-blind clinical trial of described as good, except in patients with 18 patients with atopic dermatitis, it was found hyper-IgE syndrome (80,81). Adherent sup- that younger patients (age <34 yr) responded

354 Singh et al. to TP-5 with a marked improvement in sever- A study was carried out to investigate ity scores than TP-5-treated patients of age whether TP-5 treatment was capable of induc- >34 yr or placebo-treated patients of either age ing changes in the immunologic status of group. TP-5 increased the reduced CD8 § cyto- patients with systemic scleroderma and to toxic/suppressor T cells and prevented the compare any such changes with modifications increase of Ia § cells during pollen season. in clinical conditions. There was a statistically Serum IgE levels were not significantly altered significant decrease of CD16 + and CD25 + in any group (89). Furthermore, it was found lymphocytes compared with pretreatment at that TP-5 could be a safe adjunct to therapy in the end of the first TP-5 treatment cycle (100). patients with severe atopic dermatitis (90-92). A 58-yr-old man with substantially atypical Autoimmune Diseases multiple sclerosis, both in terms of late onset and some characteristics of the course of the In a well-planned study, a total of 15 disease, was treated with TP-5 at a dose of patients with rheumatoid arthritis and 50 mg/d in 3-mo cycles for a total of three E-rosette-forming cells were studied. The cycles. Immunologic tests performed at the increase in E-rosette-forming cells after intra- end of each cycle showed slight but interest- venous treatment with TP-5 was of the same ing modifications in parameters relating to cell degree as the increase obtained after in vitro cycle and HLA antigens, suggesting that the TP-5 incubation of lymphocytes of nontreated patient's immune system was sensitive to patients with rheumatoid arthritis. The in vivo treatment with thymic hormone (lO1). response was also dose-dependent (93). Other clinical studies have confirmed the immuno- Infections regulatory activity and good tolerance of TP-5 TP-5 has been used to control recurrent and suggested that the clinical application of herpes simplex, herpes zoster, and human TP-5 could provide a new approach to the papillomavirus, which were sometimes diffi- treatment of rheumatoid arthritis (94-97). In cult to control by traditional antiviral therapy. another study, treatment with TP-5 was stud- The effect of TP-5 on viral infections appears ied in patients with rheumatoid arthritis and in to be long-lasting even after discontinuation control patients who have different forms of of treatment. The drug was found to be safe chronic synovitis. Treatment was character- and no adverse reactions have been reported ized by an increase of CD8+CD 1 lb + T cells in (65). Strategies for the treatment of chronic the CD8 subset. hepatitis B are currently based on the use of CD5 + B-lymphocytes have an important either antiviral or immunomodulatory agents. role in autoimmune and rheumatic diseases. Results of a randomized trial with 30 patients CD5 cells and other subpopulations of T-lym- indicated that TP-5 treatment was not found to phocytes in peripheral blood before and after be effective in treating chronic hepatitis B (102). therapy with TP-5 were investigated. Lym- TP-5 has been shown to be effective in res- phocyte subpopulations returned to the nor- piratory infections. A total of 80 children, mal range, and the percentage ofCD5 § B cells between the age of 1 and 12 yr, who experi- remained abnormally high (60%), which sug- enced recurrent respiratory infections, were gested that CD5 + B cells can be a useful moni- admitted to a multicenter study and treated with toring index, and confirmed their important TP-5. TP-5 administered subcutaneously for role in the pathogenesis of juvenile rheuma- 5 wk in winter was found to be useful in the toid arthritis (99). treatment of children with recurrent infections

lmmunomodulation with TP-5 and SP-5 355 of the respiratory tract (103). In another study, neously when treated with TP-5 (110). There a survey of 11 clinical cases showed that was no change in perioperative immunity TP-5 reduced the incidence of recurrent respi- related anesthesia and surgical operation when ratory tract infections (104). The clinical effect pretreated with TP-5 (111). The effectiveness appears to be long-lasting as well. of preoperative administration of TP-5 in pre- The efficacy and safety of TP-5 in HIV- venting postoperative infection was evaluated infected patients who had not yet developed in 206 patients with cancer (54 gastric, 152 AIDS was assessed. These patients were colorectal) who underwent an elective major divided into asymptomatic or symptomatic surgery. It was found that administration of groups and were randomized to receive either TP-5 did not significantly reduce the postop- TP-5 or placebo, subcutaneously, in a double- erative infection rate but reduced the severity blind fashion for 24 or 52 wk, three times a of postoperative infection in elderly patients. week. Results of this study indicated that TP-5, TP-5 treatment proved to be a promising by maintaining CD4 + cells, could slow or therapy for the treatment of sarcoidosis of the arrest immune decline and consequent disease skin. Erythema nodosum disappeared in 3 wk; progression at an asymptomatic stage of HIV hilar adenopathy improved or disappeared infection (105). Zidovudine-exposed, pla- more slowly. These results supported the cebo-treated subjects had relatively high pro- effectiveness of TP-5 in sarcoidosis (112). gression rates to AIDS or death, and to It is well known that thymic hormones AIDS-related complex, and these rates were could counteract immunodepression caused reduced by TP-5 treatment. There was no by radiation therapy, preventing and reducing increase in the incidence of adverse reactions the severity and the number of myelotoxic and with TP-5 (106). TP-5 has been tried in 10 HIV- hematologic reactions. This finding was con- positive patients with recurrent or persistent firmed in a clinical study involving 1060 oral candidiasis. All the patients benefited patients who underwent radiation therapy with from topical use of thymopentin, and in all TP-5. TP-5 was found to provide local and cases there was marked increase in salivary general protection against the reactions to secretory IgA, which possibly accounted for irradiation and could be advantageously used the candidiasis improvement (107). for the administration of higher doses of radia- A total of 46 patients affected by human tion therapy (113). papillomavirus plus cervical vaginal infec- Influenza remains a serious cause of illness tions and associated with cervical intra- and death among certain populations. Influenza epithelial neoplasia were treated with TP-5 vaccines in use at present are of limited effec- intramuscularly. Papillomavirus disappeared tiveness because of the high variability of the in all cases. Complete disappearance or only a virus. A study conducted to look for the efficacy partial remission of cervical infection in 82% ofaninfluenza , with TP-5 as an immuno- of cases was observed (108). adjuvant, has emphasized the limited immu- nogenicity of influenza vaccination and the Other Clinical Conditions inefficacy of TP-5 as an immunoadjuvant(114). Combined indomethacin and TP-5 treatment Venditti et al. (115) conducted a study to were found to successfully counteract immune evaluate the efficacy and tolerance of therapy alterations in patients with opportunistic using TP-5, rIFNc~2a, and low-dose arabino- microorganisms (109). After postsplenectomy side in 18 patients with myelodysplastic syn- infections, hyperthermia disappeared sponta- drome. The therapy was well tolerated and

356 Singh et al. Table 3. Summary of immunomodulatory activities of Splenin

In vitro studies T lymphocytes Mitogenic effect 118 In vivo studies Immune response Increased antibody response to erythrocytes 119 Inhibition of IgE production 119 Clinical studies Cancer Rectal cancer--increased immune response 20 Hepatitis Normalized of helper/suppressor ratio 121 Hastened immune homeostasis restitution 122 Angina Immunomodulatory effect 123 Tuberculosis Normalized immune response 124 Ulcerative colitis Improved of regeneration of intestine 125 Hypothyroidism Reduced the decomposition 126

seven patients showed good response to led to these conclusions and that the TP-like treatment. material purified from spleen was TP itself. Chronic stress has been characterized by Important activities of splenin are presented the disintegration of a nonspecific resistance in Table 3. mechanism, which causes the development of immune depression. TP-5 was able to reverse In Vitro and In Vivo Studies with Splenin the effects of chronic stress on the immune A nonprotein factor of the nucleoside origin system (116). was isolated from splenin that had pronounced mitogenic effect on splenic and thymic T-lym- Splenin phocytes. This factor increased the mass of lymphoid organs, promoted restoration of post- As discussed above, radioimmunoassay radiation and steroidogenic lymphopenias (118), detected a TP-like material in bovine spleen and also reproduced the immunomodulatory and lymph node extracts (26) and subse- activity of the native drug for immunoglobulin quently in the skin (117). Because the only synthesis. Premedication with this factor in CBA structural difference between the splenic mice increased primary immune response to product and TP-I and -II, of bovine origin, erythrocytes and suppressed IgE production by was the substitution of Glu for Asp at posi- stimulation of suppressor cells in Balb/c mice tion 34, it was first named TP-III. However, and Wistar rats (119). because of its characteristic properties and tissue of origin it was later called splenin (26). Clinical Studies with Splenin The findings of a single TP gene do not Treatment of patients with cancer of the support earlier suggestions from protein rectum revealed a favorable effect of splenin sequencing that splenin is a distinct molecule on the cellular and humoral arms of the (29-31). Rather it would now seem that errors immune system that was also supported by in interpretation of amino acid sequence data clinical data. This finding made it possible to

Immunomodulation with TP-5 and SP-5 357 recommend splenin as an immunomodulator A comparative analysis of idiopathic in the treatment of patients with cancer of the hypothyroidism outcomes using different rectum (120). therapeutic regimens was studied. The patients Inclusion of tocopherol acetate and splenin were divided into groups that received splenin into treatment of viral hepatitis B ensures a and sodium nucleinate. There were disease marked immunomodulatoryeffect resulting in exacerbations in patients who received no control of T-lymphopenia, normalization of immunoactive drugs. Combined therapy helper-suppressor ratio, reduction of circulat- reduced the number of patients with decom- ing immune complexes, and a tendency position 3.7-fold. Administration of splenin toward restoration of the normal ratio between was also found to bring about a stable idio- separate fractions of immune complexes and pathic hypothyroidism (126). stimulation of phagocytic activity ofmonocytes of peripheral blood. Splenin and tocopherol are Splenopentin therefore recommended in the treatment of As described above, SP-5 is a synthetic hepatitis B (121). Coadministration of splenin pentapeptide sequence (Arg-Lys-Glu-Val- and vilosen was also established to hasten Tyr) derived from the amino acid sequence normalization of clinical indices and immune 32-36 of TP-III (splenin) (7). Its immunomo- homeostasis restitution, thus advocating the dulatory activities are summarized in Table 4. use of these drugs as part of a therapeutic com- plex in the treatment of acute and chronic Pharmacokinetics toxico-allergic hepatitis (122). Usually investigators have used the SP-5 In a recent study, a total of 138 patients with derivative, DAc-SP-5 [(N-acetyl-L-arginyl)- angina and concomittant bronchitis were com- (N-acetyl-L-lysyl)--L-glutamyl-L-valyl-L- pared for immunologic indices. It was found tyrosine]. In human sera, DAc-SP-5 has a half- that a combination of splenin and vilosen life of 40 rain, significantly longer than SP-5. produced a pronounced immunomodulatory Degradation of immunoactive SP-5 and DAc- effect in the primary immunodeficiency states SP-5 in human serum had been investigated caused by recurrences of angina (123). by 1H nuclear magnetic resonance (NMR) Patients with infiltrative pulmonary tuber- spectroscopy. It has been shown that degrada- culosis who developed toxic medicamentous tion of SP-5 occurs as a result of hydrolysis of hepatitis associated with antituberculosis peptide bonds in its N-terminal part, whereas drugs revealed improved clinical parameters in N-acetyl SP-5 those in the C-terminal part and normalized immunologic tests when of the molecule are cleaved (127). treated with combined pharmacotherapy that comprised sodium nucleinate, splenin, and In Vitro Studies with SP-5 quercetin (124). Unlike TP-5, SP-5 does not affect neuro- Enterodes and splenin were used in the muscular transmission. Several immunologic treatment of patients with ulcerative colitis activities such as differentiation of T and (idiopathic proctocolitis) in association with B cell precursors in vitro to mature T cells, routine drug treatment, whereas steroids were both helper and suppressor T cells, were not employed. Results indicated increase of described for SP-5 (14). the therapeutic effect as a result of improve- We undertook the synthesis of various ana- ment of regeneration of the intestine and logs of SP-5 as part of our ongoing program on improvement of the immunity status (125). development of small synthetic peptides as

358 Singh et al. Table 4. Summary of immunomodulatory activities of SP-5

In vitro studies Cell differentiation Induced T- and B cell differentiation 19 NK cell activity SP-5 analogs stimulated NK cytotoxicity 128 HLA gene SP-5 analogs upregulated class l gene 133 In vivo studies Neurotransmission Did not inhibit 19 IFN-7 induction Enhanced IFN-y induction 20 Arthritis Inhibited antigen-induced arthritis 63, 64 Sublethal irradiation Enhanced antibody response 21 Enhanced recovery of leukocyte counts 135 Reconstitution of antibody formation 136,138 Enhanced phagocytic activity 137 Enhanced antibody-forming cells 137 Alcoholic intoxication Normalized antibody and phagocytic activity 139 Foot and mouth disease Protected animals 140 Graft versus host reaction Prevented graft versus host reaction 142 Enhanced engraftment 143 Clinical studies Hypogammaglobulinemia Increased PHA-induced T cell proliferation 144 Immunodeficiency Stimulated lymphocyte proliferation 145 Langerhans cells Accelerated Langerhans cell recruitment 23 Bone marrow toxicity Induced colony formation 146 Psoriatic arthritis Ineffective 147 Learning capacity Significantly increased memory 148 potent and nontoxic immunomodulators and tion 1 in SP-5 by Lys (2) and I>Lys (3) results studied them for their immunomodulary in appearance of significant NK cell activity acivity: Arg-Lys-Glu-Val-Tyr (SP-5), Arg- in vitro. The magnitude of augmentation D-Lys-Glu-Val Tyr (1), Lys-Lys-Glu-Val- by the two analogs of SP-5 was considerably Tyr (2), D-Lys-Lys-Glu-Val-Tyr (3), lower than IFN- 7 but it was quite comparable Arg-Lys-Gly-Val-Tyr (4), Arg-Lys-Gln- to other peptides reported in literature (129- Val-Tyr (5) Arg-Lys-D-Glu-Val-Tyr (6), 132). Similarly, replacement of Val at posi- Arg-Lys-Asn-Val-Tyr (7), Orn-Lys-Glu- tion 4 with Ile (10) results in NK cell activity, Val-Tyr (8), Arg-Orn-Glu-Val-Tyr (9), though not statistically significant. Replace- Arg-Lys-Glu-Ile-Tyr (10), Arg-Lys-Glu- ment of Arg by Orn at position 1 (8) decreased Leu-Tyr (11), D-Lys-Lys-D-Pro-Val-Tyr NK cell activity. Replacement of Lys at posi- (12), and D-Lys-Lys-Pro-Val-Tyr (13). In tion 2 by Orn (9), Glu at position 3 by Gly, Gln, our earlier studies (128), we have reported that D-Glu, Asn, or I>Pro, (4, 5, 6, 7, or 12), Val at SP-5 by itself did not have any effect on NK position 4 with Leu (11) did not lead to analogs cell cytotoxicity. Substitution of Arg at posi- having any NK cell activity (Table 5). Recently,

Immunomodulationwith TP-5 and SP-5 359 Table 5. Effects of SP-5 and its analogs on NK cell cytotoxicity

Arg-Lys-Glu-Val-Tyr (SP-5) 44.7• 48.2• Arg-D-Lys-Glu-Val-Tyr(1) 46.7• 48.8• Lys-Lys-Glu-Val-Tyr (2) 46.5• 60.8• ~ D-Lys-Lys-Glu-Val-Tyr (3) 49.2• 60.7• c Arg-Lys-Gly-Val-Tyr (4) 43.2• 43.8• Arg-Lys-Gln-Val-Tyr (5) 45.8• 43.7• Arg-Lys-D-Glu-Val-Tyr(6) 74.0• 76.0• Arg-Lys-Asn-Val-Tyr (7) 63.0• 62.0• Orn-Lys-Glu-Val-Tyr (8) 59.0• 37.0• Arg-Orn-Glu-Val-Tyr (9) 46.0• 48.0• Arg-Lys-Glu-Ile-Tyr (10) 45.6• 60.3• Arg-Lys-Glu-Leu-Tyr (11) 46.0• 42.0• D-Lys-Lys-D-Pro-Val-Tyr (12) 70.0• 75.0• D-Lys-Lys-Pro-Val-Tyr (13) 35.3• 36.0•

aPeripheral blood mononuclear cells were incubated with 10 4 M concentration of peptides for 18 h for stimulating effectors. Cytotoxicity assay was done in triplicate. Assays with analog (2) and (3) were repeated three times with three different healthy volunteers. bp < 0.01. ~)~ < 0.05.

we have shown that analogs (1), (2), and (3) In Vivo Studies with SP-5 upregulate transcription of the HLA-B7 gene in SP-5 is known to increase mitogen induced the K562 cell line. Mobility shift assays indi- IFN-7 production in vivo (20). Antigen- cate that this transcriptional up regulation of induced arthritis is an experimental model of HLA class I gene may be related to the appear- chronic joint inflammation that shows features ance of novel class I promoter binding factors histopathologically similar to human rheuma- induced in the nuclei of treated cells (133). toid arthritis. Long-term treatment with natu- If these in vitro results are substantiated by ral and synthetic splenic peptide inhibited the in vivo studies, these peptides may provide a development of antigen-induced arthritis in new tool for stimulation of innate host resis- rats. This was demonstrated by a macroscopi- tance, particularly against cancer and viral cally and histologically evaluated decrease in infections. Since IL-2 is known to modulate joint swelling and reduced degree of severity the action of NK cells, the production of IL-2 of synovitis. The drug treatment also by these peptides could well explain the aug- decreased the serum levels of antibodies mentation of NK cells. We have observed that against the specific antigen, methylated analogs (2) and (3) which have NK cell aug- bovine serum albumin, cartilage proteo- mentation potential, also stimulate IL-2 pro- glycans, and collagens type I and Ih The con- duction and CD2R expression (134). clusion drawn from this study was that a

360 Singh et al. long-term treatment with immunomodulatory treatment were established in syngeneic bone thymic and splenic peptides and with T cell- marrow grafted mice after lethal irradiation directed immunosuppressivedrug cyclosporin (136). Sublethally irradiated C57BL/6 Bln A inhibit either the specific cell-mediated and mice treated with SP-5 derivative and humoral immune responses against bovine untreated were compared for their capacity to serum albumin or the development of autoim- produce antibodies against target SRBC. munity against cartilage constituents, or both SP-5-treated mice produced antibodies ear- (64). Treatment with immunomodulatorDAc- lier and in a higher level than the untreated SP-5 reduced the severity of chronic joint animals. Furthermore, SP-5 derivative influ- inflammation and cartilage destruction in rab- enced the phagocytic capability of human bits with antigen-induced arthritis. The level granulocytes in a dose-dependent manner of specific antibodies as well as specific and (137). Sublethally irradiated AB/BIn mice nonspecific cell-mediated immune reactivi- (8 GY), supplemented with syngeneic bone ties, including the proliferative response of marrow cells, were treated with SP-5 deriva- spleen lymphocytes to cartilage proteoglycans tive and compared with untreated for their in treated animals, were lower than in capacity to produce antibodies. In bone untreated arthritic rabbits. Moreover, suppres- marrow cell plus DAc-SP-5-treated animals, sor cell activity, which normally decreased antibody-forming cells were found earlier and during the early phase of inflammation, was in a higher frequency than in mice treated with enhanced and hyperactive helper potential was bone marrow cells only. These findings dem- reduced. These findings suggest that the treat- onstrated that DAc-SP-5 was able to induce ment with DAc-SP-5 normalizes the immune bone marrow cell maturation (138). regulation, which was disturbed in the early SP-5 was administered and the effect phase of inflammation (63). checked in animals with chronic alcoholic In one study, recovery of immunocompe- intoxication. Administration of SP-5 was tence in mice after sublethal irradiation was found to normalize several immunologic pat- shown to be enhanced by DAc-SP-5. The terns: the immune response to the thymus- effects of DAc-SP-5 were verified by splenic dependent antigen, SRBCs, and phagocytic plaque-forming response to a T cell-depen- activity of peritoneal macrophages. Also dent antigen and in the hematopoietic observations of C57BL/6 mice that were char- colony-forming assay (21). The effects were acterized by high level of alcoholic motiva- associated with an accelerated recovery of tion showed that alcohol consumption in mice leukocyte counts in peripheral blood and decreased after administration of SP-5 within spleen without significant changes in the rela- 2 wk (139). tion between leukocyte and lymphocyte sub- The influence of DAc-SP-5 on early protec- populations. Furthermore, in comparison with tion of guinea pigs against foot and mouth dis- control animals, DAc-SP-5-treated mice show ease virus was investigated; it was found that in the first few weeks after exposure, a signifi- 80% protection was achieved if SP-5 could be cantly higher number of bone marrow-derived given before challenge with foot and mouth cells as well as granulocyte-macrophage and disease virus type 01 Lausanne strain (140). macrophage colony-forming cells. Therefore, Immune reconstituting effect in mice with DAc-SP-5 could be a useful substance for immunosuppression was studied after a treating secondary forms of bone marrow continuous treatment with SP-5. A short-term depression (135). Similar effects of DAc-SP-5 therapy induced an increase in antibody forma-

Immunomodulation with TP-5 and SP-5 361 tion that was measurable after 4 wk. The study DAc-SP5. The in vitro effects of SP-5 were showed that only a continuous treatment with studied using the lymphocyte proliferation test SP-5 led to an optimum response, i.e., complete and the detection of immunoglobulin produc- reconstitution of antibody formation (141). tion by lymphocytes. The PHA- and anti-CD3- The influence of SP-5 on the course of graft induced lymphocyteproliferation were inhibited versus host reaction was investigated. It was in young donors and further increased in old found that continuous treatment with SP-5 sig- donors. The biologic activity of human DAc- nificantly prevented symptoms of graft versus SP-5 was shown to be higher in comparison host reaction. The suppression of the antibody with bovine DAc-SP-5 (145). formation was diminished widely, and no loss SP-5 has been shown to accelerate the of spleen weight occurred. Furthermore, dur- repopulation of epidermal Langerhans cells ing the stimulatory phase anti-DNA autoanti- in skin sites deprived of these cells (22). SP-5 bodies were produced in the untreated also accelerated the recruitment of Langer- animals, although the SP-5 therapy prevented hans cells and led to pretreatment levels of this reaction. During the further course of the Langerhans cell density in the skin. These experiment, no increase in autoantibody pro- results indicate that SP-5 could possibly be duction was detected (142). The influence of used to treat disorders (e,g., HIV infection) DAc-SP-5 on engraftment and graft versus in which impaired Langerhans cell density host reaction was studied in different non-H2 and functions could lead to secondary cuta- strain combinations. The engraftment was neous infections (23). more or less enhanced in every case, whereas The influence of SP-5 on bone marrow pro- the situation in the graft versus host reaction genitor cell proliferation has also been exam- was completely different. In one case, SP-5 ined. DAc-SP-5 acted as costimulatory factor did not influence the course of graft versus for recombinant human granulocyte-mac- host reaction much; in the second case the rophage colony-stimulating factor in the symptoms of the graft versus host reaction induction of human bone marrow cell-derived were drastically enhanced leading to high colony formation in vitro. DAc-SP-5 has been mortality. Therefore, it was suggested that hypothesized to support the therapeutic effects before application of DAc-SP-5 to bone mar- of recombinant human granulocyte-macroph- row transplantation in humans to improve the age colony-stimulaing factor (146). engraftment, parameters had to be found that A total of eight patients with confirmed pso- allowed an exact prediction of the influence of riatic arthritis were treated with SP-5 for 12 SP-5 on graft vs host reaction (143). mo. A relief in joint pain could be detected following this treatment both after 6 wk and Clinical Studies with SP-5 after 12 too. In contrast to this, X-ray investi- SP-5 increased the PHA-induced lympho- gations showed a worsening of the disease cyte proliferation in a patient with hypo- after 12 lno. Therefore, SP-5 alone was not gammaglobulinemia. It was demonstrated that considered an efficient drug in patients with the lymphocyte proliferation test could be a psoriatic arthritis (147). suitable method for the estimation of pharma- Martynov et al. (148) investigated seven codynamics of such a peptide after in vivo synthetic peptide fragments of thymic hor- treatment (144). mone using behavioral and electrophysiologi- A study was conducted to determine whether cal methods. SP-5 was shown to increase immunodeficiencies could be influenced by markedly learning capacities and memory.

362 Singh et al. Conclusions and Future Trends . These applications should also be extended to acute infections not In the past 20 yr nonspecific immuno- responding to antibiotic therapies and to modulation has progressed from a "kicking immmunologic dysfunction of aging. the malfunctioning television" approach using It is now amply clear that the immune sys- crude microbial mixtures and extracts to a tem occupies a central position in prevention more soluble strategy with a large collection and control of disease and that immuno- of immunopharmacologically active com- modulators may have a much bigger role in pounds having diverse actions on various therapeutics than merely as adjuvants for vac- components of the immune system alone and cines or as immunosuppressants. There have in conjunction with antigens. Immuno- been significant advances made in the last two modulation seems to have a future in immuno- decades. But the vast potential of immuno- pharmacology, particularly in the context of modulators in disease control, however, has immune restoration in secondary cellular been poorly exploited. Suitable immuno- immunodeficiency and in enhancing the reac- modulators need to be identified that may be tivity of the defense system in chronic infec- useful in control, treatment, and prevention of tions, immunodeficiency, autoimmunity, and different kinds of human diseases. TP-5 and neoplastic diseases. SP-5 have shown promising results and fur- Until recently, the effectiveness ofimmuno- ther investigations are needed to conclude modulatory agents has been generally deter- their beneficial effects. mined in protocols constructed as the conventional protocols that apply to chemo- Acknowledgments therapy, that is, by evaluation of change in survival rate. The development of agents Financial assistance from Council of Sci- modifying immune responses has led to clini- entific and Industrial Research, New Delhi is cal testing in such diverse disorders as chronic gratefully acknowledged. The laboratory and recurrent viral or bacterial infections and infrastructure was provided by JICA grant-in- autoimmune diseases, expanding the realm of aid to the SGPGI projects.

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