JP2591725B2 - Immunomodulators containing 8-substituted guanine derivatives - Google Patents

Description

Detailed Description of the Invention Related Application This application is a continuation-in-part of Serial No. 439,846, a pending application filed November 9, 1982.

TECHNICAL FIELD The present invention relates to a modulator of an animal cell response, and more particularly to an immunomodulator including a low molecular weight derivative of guanine.

BACKGROUND ART An animal's immune system is composed of a number of components, each of which is independent and / or cooperating with an animal subject (host).
Act to counteract, eliminate or neutralize substances recognized by the immune system as foreign substances. Generally, but not necessarily, substances that are recognized as foreign by the immune system are foreign to the host.

Examples of such foreign substances are infectious bacteria, by-products of their cellular activity, virus particles and their proteins, proteins injected by insect needles, and the like. In autoimmune diseases such as rheumatoid arthralgia, the host's immune system recognizes proteins produced by the host, ie, autologous proteins, as foreign substances.

The main agent of the immune system is leukocytes, which
Thymus-derived lymphocytes (T cells), lymphocytes produced by bone marrow (B cells), those that produce enzymes that produce oxidizing agents such as hydrogen peroxide, which have cytotoxic effects on neutrophils, especially bacteria. And macrophages that pass foreign substances or antigens to T cells and also produce a protein called interleukin-1 that helps convert T cells to T helper cells.

Complement, a complex mixture of proteins that acts on foreign substances in an ordered and step-by-step manner
nt) also play a major role in the immune response.

B cells can be distinguished from T cells by, inter alia, the presence of immunoglobulins on their membrane surface. Immunoglobulins function as antibodies.

There are five known classes of immunoglobulins, distinguished as IgA, IgD, IgE, IgG and IgM based on the five antigenically distinct heavy protein chains that form part of the immunoglobulin molecule. B cells also carry non-immunoglobulin cell markers, including complement receptor (C
R), the receptor for the Fc portion of immunoglobulin (FCR),
Included are antigens associated with the I region (Ia) and a group of differentiation antigens (Lyb1-7) that are distinguished by all antisera and are associated with various aspects of B cell maturation and activation. These markers help distinguish B cells by phenotype.

B-cell immunoglobulins act on foreign substances or antigens, whereas T cells and especially helper T cells are necessary to stimulate B cells to divide and differentiate into antibody-secreting cells for humoral immunity. It is believed that. Suppressor T cells contribute to the regulation of humoral immunity, while cytotoxic T cells and delayed-type hypersensitive T cell mediators are the major agents of cell-mediated immunity.

T cells are Lyt1,2 and 3 associated with T cell function.
And antigens called. Lyc helper T cell precursor
t1 ^+, 2 ^-, 3 ^- a phenotype. It is these cells that normally participate in the activation and regulation of B cells.

Helper T cells are known to assist in the activation and differentiation of immunoglobulin-secreting B cells after the initial message has been received from an antigenic agent activated by the B cells. However, the manner in which T cells provide B cells with a second message of activation and differentiation is controversial.

Guanosine-3,5'-cyclic monophosphate (cGMP)
Has been inferred to be a naturally occurring agent that provides the necessary second message. 8-bromoguanosine-
3 ', 5'-Cyclic monophosphate (8-BrcGMP) has been found to be a weakly synthesized intracellular lymphocyte mitogen.

The immune response can be modulated by artificial suppression (immunosuppression) or by subaru (immunosubaru). Immunosuppression or artificially induced reduction of immunoreactivity can be achieved in six general ways: (1) administration of antigens, (2) administration of specific antisera or antibodies, (3) other biological Use of agents such as anti-lymphocyte antiserum, (4) use of drugs or hormones,
(5) irradiation, and (6) surgical removal of lymphoid tissue. Immunization involves the administration of agents that increase the rate at which the immune response proceeds, increase the intensity or extent of the response, sustain the response, or evolve the response to another non-immunogenic substance.

Agents known to promote the immune response are commonly referred to as adjuvants and can be divided into two general categories: (1) those that provide general subjugation, ie, cells and cells against various antigens. A substance that enhances both the humoral immune response and (2) a substance that gives specific substantivity, that is, a substance that enhances a specific response only to a certain antigen.

Substances that can act as adjuvants can be divided into the following categories: (1) Water and oil emulsions, such as Freu
nd supplements, (2) synthetic polynucleotides, (3) hormones, drugs and cyclic nucleotides, (4) endotoxins, (5) lymphokines and monokines such as interleukins.

Substances that can specifically promote the immune response are transfer factors obtained from human peripheral leukocytes, and dialyzable leukocyte extract (DLE). Transfer factors have been shown to have some effect in patients with immune deficiencies and have potential effects in cancer patients and patients with limited immune deficiency. However, much work remains to be done on this particular substance.

In certain diseases and physiological conditions, such as X-related agammaglobulinemia, senile and drug-induced immunosuppression, B cell activation and differentiation is absent and / or present only at low levels, and Decreases immune response. These diseases and conditions represent an immunosuppressive state. Now, the activation and differentiation of the sub-population, if it can occur, tends to beneficially reduce symptoms and / or improve the condition of the patient.

The state of immunity can be exemplified by the state of the body after the vaccine injection. There, the immune response is already enhanced by the antigenic reaction, but can be even more advantageously increased to improve the degree and / or duration of the immunization.

Neoproliferative cell proliferation, including primary tumors and metastatic disease, causes cells to replicate without normal regulated constraints and / or at a much higher rate than is normal for unaffected cells It can be described as an expression of cell message. One Common Chemotherapy for Neoproliferative Diseases, General Cytotoxicity Affects Rapidly Replicating Neoproliferative Cells Greater Than Normal Cells, thereby Selectively Killing Rapidly Replicating Neoproliferative Cells Hoping that
Contacting a substance that is generally cytotoxic with low levels of neoplastic cells. Disadvantages of such treatments include the general loss of many bodily functions such as weight and hair loss, stomach and bowel modulation.

Interferon is a type of soluble small protein that inhibits virus growth. These proteins can be found in almost any animal virus grain or other viral agent, and
For example, produced by cells infected by bacterial products as well as agents such as monoclonal antibodies. Interferons are cell-specific, not virus-specific. The ability to increase the amount of interferon present in animals in the absence of a naturally occurring (pathogenic) microbial, interferon-inducing agent would be beneficial for promoting the animal's general resistance to virus infection .

In an autoimmune disease, a host recognizes its own protein as an antigen and, in essence, produces antibodies against its own tissue to attack itself. Autoimmune diseases are the result of excessively active production of anti-autoantibodies, since animals normally make some antibodies to themselves (anti-autoantibodies) and their effects are suppressed or made resistant. It is believed that the body mechanisms that provide normal suppression and / or tolerance to it do not respond well. Excessive production of such anti-autoantibodies can be considered analogous in antibody level to excessive cell proliferation exhibited by neoplastic cells.

Salicylates such as acetylsalicylic acid (aspirin) are the main chemotherapeutic agents for treating autoimmune diseases such as rheumatoid arthralgia. Salicylate is thought to act to reduce the symptoms of rheumatoid arthralgia by inhibiting prostaglandin production and thus its effect on affected tissues. Chemotherapeutic agents such as gold, mercaptopurine and D-penicillamine have been used to treat rheumatoid arthralgia, however, some such agents have toxic or teratogenic side effects and Use has been linked to lymphoma and possible infection. Accordingly, it would be advantageous to provide a chemotherapeutic agent that can be used to successfully treat an autoimmune disease without the side effects described above.

Lymphkins and monokins are immunogenic proteins made by lymphocytes and cells of the monocyte macrophage lineage, respectively. One monokin, interleukin-1, is made by macrophages when they are stimulated with mitogens or antigens. Interleukin-1 is usually required to make a primary antigen response.

Interleukin-1 aids in the production of interleukin-2 in T cells. Interleukin-2 is a growth factor for T cells and helps convert to T helper cells. That is, induction of interleukin-1 production,
Or similar to that made by interleukin-1,
Inducing a protein responsive activity on T cells is beneficial for enhancing the immune response, especially in the absence of macrophages or defective interleukin-1 production.

BRIEF SUMMARY OF THE INVENTION Animal cells are administered by administering an immunomodulator comprising as an active ingredient an effective amount of an 8-substituted guanine derivative 9-1 'linked to an aldose having 5 to 6 carbon atoms in the aldose chain. It has been found that the response can be adjusted. A specific example of an animal cell response in which antigen-specific humoral immune responses leading to strong complementarity, T cell turnover factor-like (TRF-like) activities and immune reconstitution activities can be modulated according to the present invention. is there. The guanine derivatives include 8-oxoguanosine, 7-methyl-8-oxoguanosine, 8-methoxyguanosine, 8-mercaptoguanosine (8-MGuo),
Selected from the group consisting of -mercapto-7-methylguanosine and 8-bromoguanosine (8-BrGuo). As used herein, the term "modulation" refers to the subversion and suppression of animal cell responses in vitro and / or in vivo.

The cell response modulating composition of the present invention, ie, the immunomodulator, also contains a diluting amount of a physiologically acceptable carrier. The immunomodulators of the present invention can be used to elicit various, but related, results depending, inter alia, on the mode of administration, the dosage and the number of cells of the subject to be administered. The active ingredient can be present in a carrier, for example, as a suspension of a solid 8-substituted guanine derivative in a solid or liquid carrier or as a solute in a carrier.

That is, by contacting leukocytes such as T lymphocytes, macrophages, neutrophils or B lymphocytes with the immunomodulator of the present invention, the immune response of these leukocytes is regulated. Modulation of a B lymphocyte (B cell) response can be performed by providing the B cells with an antigen before or simultaneously with contacting the B cells with an immunomodulator.

A B cell immune response can also be modulated by providing the B cells with an antigen and then contacting the cells so treated with an immunomodulator with an additional amount of the antigen. These steps can be performed in an environment in the substantial absence of T lymphocyte helper activity. Immune response modulation can also act on senescent B lymphocytes that show a reduced immune response before being contacted with the immunomodulator of the present invention.

The growth of neoplastic cells can be inhibited by contacting such cells with an immunomodulator of the invention. Contacting an animal cell with an immunomodulator of the invention induces the production of interferon even in the absence of a naturally occurring microbial interferon-inducing agent. Similar treatment of macrophages with the immunomodulators of the present invention in the absence of T cell activity gives T cells an activity similar to interleukin-1 in the absence of antigen, whereas in the presence of IL-2 Contacting a T cell with an immunomodulator of the invention confers a synergistically enhanced proliferative activity on the T cell. Contacting B lymphocytes of a host with an autoimmune disease with an immunomodulator of the invention suppresses the autoimmune disease.

The immunomodulators of the present invention are also useful for subcutaneous production of antibodies, eg, monoclonal antibodies from B cells. Here, for example, the immunomodulator of the invention is contacted with B cells in the immunized animal to obtain an increase in antigen-specific B cells used for cell fusion to form hybridomas. Monoclonal antibody-producing hybridoma cells can also be contacted in vivo or in vitro with an immunomodulator of the invention to enhance the production of monoclonal antibodies from these cells.

The immunomodulator of the present invention may be used on cells in vivo as well as in vitro.

The immunomodulator of the present invention may be administered subcutaneously, in the form of tablets or capsules or in the form of a liquid such as a slurry, suspension or solution.
It can be administered intraperitoneally or orally.

The present invention has several benefits and advantages.

One of the significant benefits of the present invention is that its use provides a second message required for B lymphocyte activation and differentiation in response to the first (antigenic) message.

Another benefit of the present invention is that this activation and differentiation triggers protein production as in the case of immunoglobulin secretion from B cells.

Yet another benefit of the present invention is that immunosuppressive or immunodeficient conditions and symptoms can be ameliorated and / or alleviated by use of the immunomodulatory agents of the present invention, respectively.

Another benefit of the present invention is that its use can confer improved immunity to a host with the activity of otherwise normally functioning B cells.

A particular advantage of the present invention is that a subcutaneous immune response can be triggered in the absence of T helper cell activity. That is, a sublimated immune response is described in a T-cell independent system.

Another advantage of the present invention is that it inhibits neoproliferative cell growth and stimulates interferon production in the presence or absence of naturally occurring microbiological interferon inducers,
And to prohibit autoimmunity.

Still other benefits and advantages of the present invention will be apparent to one skilled in the art from the following description, examples, accompanying drawings and claims.

Personalized expressions, such as sending and receiving messages by and to chemicals and cells, are used for descriptive purposes to help understand the observed phenomenon.

BRIEF DESCRIPTION OF THE DRAWINGS The drawings forming part of the present disclosure will be described.

FIG. 1 illustrates the sublimation of the primary antibody reaction by 8-MGuo. 10 ⁷ Viable CBA / CaJ spleen cells from SRBC
Was cultured in the presence or absence of Escherichia coli while changing the increase rate of 8-MGuO stepwise. After 4 days of culture, PFC was directly measured on SRBC. Results are arithmetic mean of triplicate cultures ± standard error (S.
E.).

FIG. 2 illustrates the sublimation of the primary antibody reaction by 8-BrGuo and 8-BrcGMP. 10 ⁷ Viable CBA / CaJ spleen cells from SRBC
Were cultured with and without increasing 8-BrGuo or 8-BrcGMP. Direct PFC for SRBC after 4 days of culture
Was measured. The results are represented as in FIG.

FIG. 3 illustrates the sublimation of the secondary IgM antibody reaction by 8-MGuo. 10 ⁷ Viable SRBC-added CBA / CaJ spleen cells at 0.3 mM
Was cultured with or without the presence of 8-MGuo at different SRBC concentrations. The results are shown as in FIG.

FIG. 4 illustrates the sub-progression of a secondary IgG antibody reaction by 8-MGuo. 10 ⁷ Viable SRBC-added CBA / CaJ spleen cells at 0.3 mM
Was cultured in the presence or absence of 8-MGuo at different SRBC concentrations. After 4 or 5 days of culture, the indirect PFC for SRBC was measured. The results are shown as in FIG.

FIG. 5 illustrates the subversion of the antibody response to the TI-1 antigen by 8-MGuo. 10 ⁷ Viable CBA / CaJ spleen cells at 0.3 mM
Was cultured with or without TMG-LPS concentration in the presence or absence of 8-MGuo. After 3 days of culture, PFC was directly measured for TNP. The results are shown as in FIG.

FIG. 6 illustrates the sublimation of the antibody response to the TI-2 antigen by 8-MGuo. 10 ⁷ Viable CBA / CaJ spleen cells at 0.3 mM
In the presence or absence of 8-MGuo, the concentration of TNP-AECM-ficoll was changed while culturing. After 3 days of culture, PFC against TNP was measured. The results are shown in FIG.

FIG. 7 illustrates the subversion of the antibody response in the absence of intact T cells. 5 × 10 ⁶ Viable CBA / CaJ B cells were cultured using SRBC in the presence or absence of 5% MLC supernatant and / or 0.3 mM 8-MGuo. After 4 days of culture, PFC was directly measured on SRBC. The results are shown in FIG.

FIG. 8 illustrates the dynamic change due to the addition of 8-MGuo.
10 ⁷ Viable CBA / CaJ spleen cells were cultured in the presence or absence of SRBC on successive culture days with the addition of 0.1 mM or 0.3 mM 8-MGuo. After 4 days of culture, PFC was directly measured on SRBC. The results are shown in FIG.

FIG. 9 illustrates the increase in antibody response of immunodeficient mice by 8-BrGuo. 10 ⁷ Vi from male or female NCF ₁ mice
able spleen cells were cultured with 2 × 10 ⁵ or 2 × 10 ⁶ SRBC in the presence or absence of 0.3 mM 8-BrGuo. After 4 days of culture, PFC was directly measured on SRBC. The results are shown in FIG.

FIG. 10 illustrates the ability of 8-MGuo to induce blast transformation.

A. ⁴ × 10 ⁵ Viable CBA / CaJ spleen cells were treated with various 8-MGu
Cultures were performed at o concentration and evaluated for [ ³ H] uptake or embryo development as described in the “Materials and Methods” section below.

Results are the mean ± SE of 5 (TdR ingestion) or 4 (embryonic development) cultures.

B. Micrograph of unstimulated cells.

C. Photomicrograph of cells cultured in 1 mM 8-MGuo for 2 days.

FIG. 11 illustrates the ability of 8-MGuo to promote DNA synthesis in cultures from innate athymic mice. 4 × 10 ⁵
Viable C57BL / 6J nu / nu (−−−−) or nu / + (−−
---) Spleen cells were treated with various concentrations of 8-MGuo for 2 days or 1 day.
The cells were cultured using μg / ml ConA. The results are shown as in FIG. 10A.

FIG. 12 illustrates the ability of 8-MGuo to activate B- and T-rich lymphocyte populations. 4 × 10 ⁵ Viable C
BA / CaJ thymocytes (----), spleen B cells (----), spleen T cells (...), or spleen B
And a 1: 1 mixture of T cells (-.-) were cultured at various concentrations of 8-MGuo for 2 days. The results are shown in FIG. B
Cells were only 4% of the ConA response of unseparated cells, and T cells were only 5.9% of the LPS response of unseparated cells.

FIG. 13 illustrates the ontogeny of the mitogenic response of 8-MGuo. 4 days after birth (----) or 56 for adults
4 × 10 ⁵ Viable CBA / CaJ from mice on day (−−−−)
Splenocytes were cultured at various concentrations of 8-MGuo for 2 days. Results are expressed as the ratio of cpm in 8-MGuo containing cultures to control cultures.

FIG. 14 illustrates the ability of 8-MGuo to activate effluent and residual cells from a Sephadex G-10 column.

A. ⁴ × 10 ⁵ Viable CBA / CaJ spleen cells were cultured before (−) or after (...) passing through a G-10 column. The third group is 5%
G-10 efflux cells supplemented with A. spleen adherent cells (----). These three groups of cells were cultured at various concentrations of 8-MGuo for 2 days. The results are shown in FIG.

B.4 × 10 ⁵ Viable CBA / CaJ non-separated (−), G-10 outflow (・
・ ・) Or G-10 attached (− ・ −) cells are 8-MGuo for 2 days
At various concentrations. The results are shown in FIG.

FIG. 15 shows that 2 × 10 ⁷ SRBC were added to a group of 4 CBA / CaJ mice.
Figure 3 shows the results when different concentrations of ip and insoluble 8-BrGuo were injected. After 7 days, PFC was measured directly on SRBC.

FIG. 16 shows that 6 × 10 ⁶ SRBCs were added to the group of 4 CBA / CaJ mice.
ip and 3 days later various amounts of insoluble 8-MGuo
Shows the results of the injection. Direct PFC against SRBC was measured after 5 days.

FIG. 17 shows the results of a group of four CBA / CaJ mice injected with SRBC ip followed by saline (NS), 8-BrGuo, or Guo. Direct PFC against SRBC was measured after 7 days.

FIG. 18 shows that groups of four CBA / CaJ mice
The results when C ip and saline or 25 mg 8-MGuo ip were injected are shown. Direct PFC against SRBC was measured after 5 days.

Figure 19 is a four animals of NCF ₁ male or 6 × Groups of female mice 10 ⁶
Shown are the results of injection of one SRBC ip followed by saline or 25 mg of insoluble 8-BrGuo. Direct PFC against SRBC was measured after 7 days.

FIG. 20 shows the ability of 8-MGuo to replace T cells in primary antibody reactions in vitro. 4 × 10 ⁶ Viable C
BA / CaJ splenic B cells were cultured with and without SRBC at various concentrations of 8-MGuo. 4 days later direct to SRBC
PFC was measured. Results are as arithmetic mean of triplicate cultures ± standard error (circled) or as control (ie without 8-MGuo)
It is expressed as the ratio of experiment to culture (marked with ●).

FIG. 21 shows the ability of 8-MGuo to alter T cells in the primary antibody response of innate athymic mice. 10 ⁷ Viab
le C57BL / 6J nu / nu or nu / + spleen cells were cultured with or without SRBC in the presence or absence of 8-MGuo. Four days later PFC was measured directly against SRBC. The results are shown as the arithmetic mean of three cultures ± standard error.

FIG. 22 shows that IL-independence of 8-MGuo T cell turnover ability. 10 ⁷ Viable CBA / CaJ spleen cells were cultured with or without SRBC in the presence or absence of 3 × 10 ⁻⁴ M 8-MGuo. Various concentrations of cyclosporin A were added at the start of the culture. Four days later PFC was measured directly against SRBC. The results are shown as the arithmetic mean ± standard error of the difference between the three experiments and the control (control = 220 ± 59 PFC) (left panel). The right figure shows each reaction in the absence of cyclosporin A.
The response to BC is shown as normalized to 100%.

FIG. 23 shows the synergy between MLC supernatant and 8-MGuo. 5 × 10 ⁶ Viable CBA / CaJ spleen B cells were cultured in the presence of various amounts of MLC supernatant with or without SRBC. One set of cultures (-) used 3-MGuo 3 × 10 ^-4 M and the other (-)
−−−) was performed without using. Four days later PFC was measured directly against SRBC. Results are shown as the arithmetic mean ± standard error of the difference between the three experiments and the control (control = 88 ± 3 PFC).

FIG. 24 shows the induction of immunoglobulin secretion in cells from young and old mice. 5 × 10 ⁶ Viable C57
B1 / 6J spleen cells in 1 ml volume, medium only, 0.1 mg / ml LP
The cells were cultured for 3 days in the presence of S or 10 ⁻³ M 8-MGuo. Results are shown as the arithmetic mean of the differences between the three experiments and the control (control = 62 PFC / culture (young cells), 3PFC / culture (old culture)).

FIG. 25 shows the supplemental action of 8-MGuo and LPS in aged mice. 10 ⁷ Viable spleen cells from aged C3H / HeN mice were injected with 10 ^-3 M 8-Guo or 0.1 mg / ml LPS in a volume of 1 ml
For 4 days with or without 2 × 10 ⁶ SRBCs. Results are shown as the calculated mean of three cultures ± standard error.

FIG. 26 shows the relative TRF-like activity of 8-MGuo and LPS in aged mice. 4 from old C57BL / 6J mice
× 10 ⁶ Viable spleen B cells in 1 ml volume of 2 × 10 ⁶ SRBCs
In the presence or absence of ^10-3 M 8-MGuo, 0.2 mg / ml LPS
Alternatively, the cells were cultured for 4 days with supplementing only the culture medium. The results are shown as the arithmetic mean of three cultures ± standard error.

In FIG. 27, 4 × 10 ⁴ Viable tumor cells or 5 × 10 ⁵ normal spleen cells were cultured for 2 days in the presence of various concentrations of 8-MGuo. Cultures should be 1 micron Ci for the last 24 hours of culture.
Labeled with [ ³ H] TdR. The results are shown in FIG.

Figure 28: 10 ⁶ Viable CBA / CaJ spleen cells were transformed with [ ¹⁴ C] GMP
Various concentrations of 8-B in serum-free medium with 0.1 micron Ci
Cultured for 3 hours in the presence of rGuo. At the end of the culture period, cells were centrifuged, the supernatant was collected, supplemented with 1 mM unlabeled Guo or GMP, and analyzed by TLC on PEI cellulose plates. Spots corresponding to GuO or GMP were scraped and beta emission counted.

FIG. 29 shows the in vivo response to human gamma globulin (HGG) in A / J mice injected with 400 micrograms of heat-aggregated HGG and various amounts of 8-BrGuo. The number of indirect (IgG) anti-human gamma globulin plaque forming cells per spleen cell was determined 6 days after immunization with a minimum of 11 mice per data point. Results are shown as arithmetic mean ± standard error.

FIG. 30 shows the ability of guanine derivatives to promote mitogenesis. 4 × 10 ⁵ Viable CBA / CaJ spleen cells were cultured with various concentrations of guanine derivatives for 2 days. The results are shown as in FIG. 10A.

FIG. 31 shows the T cell turnover factor for murine B cells (TR
F) shows a difference in B cell fission and polyclonal B cell activation compared to activity. SJL or CBA / CaJ B cells were cultured for 2 days (left figure), 3 days (middle figure) or 4 days (right figure)
The culture was continued for a while. After 24 hours of culture, fission cultures are labeled with [ ³ H] TdR for an additional 24 hours and harvested using the assay described above for FIG. In the middle figure, the direct
Measure PFC, while direct PFC to SRBC on the right
It was measured as in the figure.

FIG. 32 shows 8-MGuo-induced subcutaneous progression of the primary human immune response to SRBC as an antigen.

Wysocki and Sato, Proc. Natl. Acad. Sci. USA 75: 2844
(1978), using the method of Seragen, Inc. of Boston, MA
"Panning" on a plate commercially available from
2 lost cells with of H ₂ histamine receptors by
× 10 ⁶ Viable human peripheral blood lymphocytes in a volume of 1.0 ml, 1
The cells were cultured for 6 days in a medium containing 0% heat-inactivated fresh autologous plasma. 5 × 10 ⁶ SRBC and / or
Or there is a final concentration of 1 × 10 ⁻³ molar 8-MGuo (+
) Or removed (-). PFC was evaluated by the speckle test described above. Results are reported as the arithmetic mean of three cultures ± standard error.

DETAILED DESCRIPTION OF THE INVENTION Table I provides a list of abbreviations used herein.

Table I shown Term Meaning AECM N-(2-aminoethyl) carbamylmethyl CAMP adenosine 3 ', 5'-cyclic monophosphate benzoate ATS rabbit anti-mouse thymocyte serum B Cells Chizui derived lymphocytes 8-BrcGMP 8- Bromoguanosine 3 ', 5'-cyclic monophosphate 8-BrGuo 8-Bromoguanosine Con A Concanavalin A CR complement receptor cGMP Guanosine 3', 5'-cyclic monophosphate EA Erythrocyte antibody complex EAC Erythrocyte antibody complement complex FcR Fc receptor FCS fetal calf serum Guo guanosine 8-HGuo 8-haloguanosine [ ³ H] TdR Deoxyribosyl thymidine labeled with tritium Ia antigen Antigens controlled by immunoreactive genes Immunoglobulins A, D, E, G and M IL Interleukin IL-1 Interleukin-1 IL-2 Interleukin-2 L PS Bacterial lipopolysaccharide Lyb 1-7 Lymphocyte antigen on murine B cells Lymphocyte antigen on Lyt murine T cells 8-MGuo 8-mercapto guanosine MHC Main histocompatibility complex MLC Mixed leukocyte culture 5'NT 5 ' -Nucleotidase PFC spot-forming cells PHA phytohemagglutinin RBC red blood cells RIA radioimmunoassay SRBC sheep red cells SE standard error T cells thymus-derived lymphocytes TCA trichloroacetic acid TI-1,2 Reaction TNP 2,4,, 6-trinitrophenyl TRF T cell replacement factor Reported mitogenic (mitogenic) agents cGMP and
During the study of the effects of 8-BrcGMP, the present inventor has determined that when a low molecular weight guanine derivative is present in an effective amount as an active ingredient in an immunomodulator, including a diluent amount of a physiologically acceptable carrier, the animal cell Have been found to have a significant effect in regulating the response of Antigen-specific humoral immune responses resulting in potent adjuvant action, TRF-like activity and sub-activity of immune reconstitution activities are particular examples of animal cell responses that can be modulated according to the present invention.

This new class of immunomodulators includes 8-oxoguanosine, 7-methyl-8-oxoguanosine, 8-methoxyguanosine, 8-mercaptoguanosine (8-MGuo),
Selected from the group consisting of -mercapto-7-methylguanosine and 8-bromoguanosine (8-BrGuo).

Physiologically acceptable carriers, dosages, specific compositions, routes of administration, and the like for useful immunomodulators of the invention are described below.

Lymphocyte Activation The present inventors have unexpectedly found that per molecule ingested,
8-BrGuo is about 1.5 times more active than 8-BrcGMP as a B cell activator.
It has been found that it is more effective on the order of about 2 (digits) and that exogenous cGMP itself has poor mitogenic capacity. Goodman and Weigle, Proc. Natl. Ac
ad.Sci.USA, 78: 7604 (1981). Included here by reference.

Induction of immunoglobulin secretion 8-BrcGMP has little effect on inducing precursor B cell maturation to produce polyclonal immunoglobulin secretion. However, per molecule absorbed by cells, 8-BrGuo
Is about 2 to about 2 in both serum-free medium and medium containing 5% FCS.
Biologically more powerful on the order of 2.5. The enhanced polyclonal immunoglobulin secretion induced is also dose-dependent in the presence and absence of serum. Guo and cGM
P suppresses the polyclonal response of B cells to 8-BrGuo. Goodman and Weigle, J. Immunol., 128: 2399 (198
Please refer to 2). This is incorporated herein by reference.

Other guanine derivatives such as 8-oxoguanosine,
8-Methoxyguanosine, 7-methyl-8-oxoguanosine, 8-MGuo and 8-mercaptomethylguanosine also, like 8-BrGuo, induce immunoglobulin secretion in spleen cells as shown in Table II.

Resistance to T cell metabolism 8-BrGuo is not metabolized in B cells and does not interfere with the enzymatic treatment of Guo. These findings indicate that 8-substituted guanine derivatives are used by cells like their unsubstituted homologs, or they cannot be considered to be metabolized by cells as well.

Goodman and Weigle, J. Immunol, 129: 2715 (1982) are hereby incorporated by reference.

The single cell suspension of CBA / CaJ spleen cells used in this study was prepared as described in Goodman et al., J. Immunol., 121: 1905 (1978). Red blood cells are lysed with 0.83% ammonium chloride and T lymphocytes are Goodman
And Weigle, J. Exp. Med., 145: 473 (1977), using a monoclonal anti-thy-1.2 antibody and complement to remove. The resulting B cell population was purified under serum-free conditions by RPM as described in Goodman et al., Supra.
Cultured in medium based on 1640.

Make a transmissive labeled compound, Goodman and Wigle, P
Acad. Sci. USA, supra, roc. Natl. Acad. Sci. USA, thin film chromatography of lymphocyte extracts was performed.

FIG. 1 shows the effect of 8-MGuo as a supplement in the primary antibody response to SRBC evaluated in vitro. At optimal concentrations, immunomodulators containing this nucleotide derivative enhanced the response to SRBC by an order of magnitude or more. This effect is dose dependent and has the greatest effect in the range of 0.3 mM 8-BrGuo for the studied culture. The antibody reaction (2725PFC) has a subunit of the specific reaction to SRBC (253PF
C) and polyclonal reaction to 8-MGuo (540PFC)
Cannot be explained by the additional effects of

By comparison, even at concentrations as high as 3 mM, 8-B
rcGMP did not increase the primary response to SRBC (second
Figure). In contrast, 8-BrGuo significantly enhances the antibody response to this antigen. Data for the polyclonal response (ie, no antigen) on day 4 are shown for comparison, again indicating that the observed adjuvant effect was not due to polyclonal activation of B cells.

The adjuvant effect of 8-MGuo-containing immunomodulators that act on cells with and without antigen experience is also of interest.

The effects of 8-MGuo-containing immunomodulators on secondary IgM and IgG responses to SRBC are shown in FIGS.

Both reactions were significantly enhanced by contacting the cells with an immunomodulator containing this nucleotide derivative. Ig
The M (direct) response is 12.2 times higher and the TgG (indirect) response is
7.2 times higher. This adjuvant effect was dependent on the concentration of antigen added to the culture. The nucleotide concentration that elicited the secondary reaction with the highest peak observed was about the same as the optimal dose (0.3 mM) for the primary IgM reaction for the tested culture.

The efficacy of the immunomodulator of the present invention on cells from humans is demonstrated by the complementarity of 8-MGuo on human peripheral blood lymphocytes in the presence of a particular antigen, SRBC. These results are shown in FIG. As can be seen from FIG. 32, human peripheral blood lymphocytes were treated with 8-MG in the presence of a specific antigen.
When brought into contact with the immunomodulator of the present invention containing uo, direct anti-
The primary response to the antigen is approximately 6 times higher as measured by SRBC PFC.

The ability to enhance antibody responses to T-independent antigens also
It was tested to see if the supplemental effect of 8-MGuo works mainly on T helper cells or on B cells making antibodies. At antigen doses below that induce polyclonal activation, the response to TNP-LPS (an antigen of the TI-1 class) was increased about 3-fold, as shown in FIG. In contrast, TNP-AECM-f
The response to icoll (antigens of the TI-2 class) was increased 8.9-fold as shown in FIG.

A study in which intact T cells were replaced by supernatants from mixed lymphocyte cultures (MLCs) containing soluble T helper activity showed that the target of 8-MGuo subcellular effect was antibody-producing cells or T cells I knew Britton, Scand.
J. Immunol., 1:89 (1972). FIG. 7 shows antigens and MLC
The antibody response generated by the B cells in the presence of the supernatant
9 shows that when 8-MGuo-containing immunomodulators are added and brought into contact with cells, they increase by more than one order of magnitude. The data in FIG. 7 shows that this nucleotide is
4 shows that T helper cells can be surrogated in the absence of MLC supernatant.

The nature of the signal provided by the supernatant factor and the nucleotide-containing immunomodulator seems likely to be distinct when both were added simultaneously to the culture, as significant synergy was observed. That is, the adjuvant effect of 8-MGuo is not mediated by intact T cells, but can act directly on sensitive B cells or on antigen presenting cells.

Certain supplements, such as LPS, lose their ability to sublime when added to cultures more than one day after the antigen. See Ortiz-Ortiz and Jaroslow, Immunology, 19:38 (1970). But 8
The sensitivity of -MGuo to the adjuvant effect was not limited to the beginning of the culture period, as can be seen from FIG. Significant sublimation of the basic antibody response occurred even when the culture was supplemented with nucleotide derivatives on the third day of the four day culture. Thus, the adjuvant effect appears to act on events that occur relatively late in the reaction.

One of the important possible uses of adjuvants is in the amelioration of immunodeficiency conditions. To see if 8-BrGuo open to this point is valuable, to evaluate their effect on the antibody response of immunodeficient (CBA / N × CBA / CaJ ) F 1 mice.

The basal antibody response to thymus-derived antigens was observed to be very low in defective (male) animals as compared to control (female) animals, as seen in FIG. 9 and reported by others. Was. For example, Janeway and Barthold, J. Im
munol., 115: 898 (1975). However, supplementing the culture with an immunomodulator containing an 8-substituted guanine derivative, 8-BrGuo, as an active ingredient, resulted in a significant subcutaneous (approximately 20-fold) response produced by cultures containing immunodeficient cells. This sublimation raises the response above that achieved with normal cells alone, and is in the range of enhanced responses produced by normal cells in the presence of 8-BrGuo and small doses of antigen.

The above results provide a distinct secondary message when the immunomodulator of the invention is contacted with the cells, which is
Indicates that it does not depend on the presence of cell signals. The above results also indicate that the concentration that induces maximal fission and polyclonal immunoglobulin secretion is on the order of about 0.5 (half an order of magnitude).
It shows a particular, dose-dependent increase in antibody response at low concentrations. (Goodman and Wwigle, Proc.Natl.Acad.Sc
iUSA and J. Immunol., 128: 2399, supra).

While the immunomodulators of the present invention are useful for enhancing fission, polyclonal response and co-potentiation as described above, it is believed that these three properties are obtained from at least two different pathways, It has to be mentioned that fission and polyclonal reactions are often the result of simultaneous reactions, whereas adjuvant effects are often different. For example, Goodman et al . , J. Exp. Med., 147: 800 (1978);
McIntire et al. , J. Immunol., 117: 674 (1976); and Hoffma.
n et al . , J. Exp. Med., 146 (1977).

The separation of the above three properties can be further illustrated by looking at FIG. There, normal (CBA / Ca
J) It is seen that mice respond to the pleiotropic effects of 8-MGuo in assays for three properties. However, abnormal mice (SJL) are proliferated or polyclonal (non-specific)
It cannot react with 8-MGuo for an antibody reaction, but does react in the presence of a particular antigen (SRBC).

SRBC as an antigen to give a T-like signal
In separate studies with SJL B cells in the presence and absence of 8-MGuo, fission and TRF-like activity were both 8
-It was found to show dose dependency on MGuo concentration. However, fission was increased only about 4- to 5-fold, while TRF-like activity was increased by about 140-fold, followed by increasing the SRBC concentration by 0.01 in the same concentration range of 8-MGuo ( ^10-5 to ^10-3 mol). The percentage drops to about a 40-fold increase when kept constant.

Also, the observed dose-dependent nuclear fission is only seen in the presence of certain antigens.

Further, it must be noted that fission continued to increase over the dose range of 8-MGuo studied, but TRF-like activity peaked within that concentration range. The results further indicate that fissionogenic activity does not account for the observed TRF-like activity.

Although the immune response was observed to be subcutaneous at all doses of antigen, the degree of subcutaneous is usually maximal at optimal or near optimal antigen concentrations. Our results indicated that the supplemental action of 8-MGuo or 8-BrGuo was synergistic with the antigen and not due to the sum of the antigen-specific and polyclonal (non-specific) responses.

Sublimation of antibody production by 8-MGuo involves not only naive, antigen-inexperienced B cells, but also antigen-experienced, or memory, B cells. That is, the primary IgM
And secondary IgM and IgG responses were enhanced by contacting the culture with an immunomodulator containing an effective amount of 8-MGuo as an active ingredient.

8-MGuo is thought to sub-prime the primary humoral immune response by acting directly on B cells and / or antigen-providing cells. That is, the use of this nucleotide derivative
The antibody response to the T-independent antigen, ie, the response involving B cells and antigen presenting cells, was enhanced. Also, immunomodulators, including 8-MGuo, show their adjuvant effects in cultures initiated in the absence of complete functional T cells. MCL for T cells
When replaced by the T cell helper activity contained in the supernatant,
The ability of 8-MGuo to increase the antibody response was not reduced.
Furthermore, the synergy observed between soluble T cell signals and immunomodulators containing guanine derivatives indicates that the signals provided by each are qualitatively different. This synergy was observed over a range of supernatant concentrations, indicating that guanosine derivatives are not simply providing more of the MLC-like signal. A similar degree of synergism has been shown to allow such B cell cultures to undergo ML in the presence of antigen.
C was observed when complementing the T cells but not the supernatant (which is actually derived from the T cells) and contacting with a guanine derivative-containing immunomodulator of the invention.

The T cell-mediated effect of 8-MGuo's adjuvant effect is not dependent on the T-independent observation of its adjuvant effect. That is, the existence of the T cell-independent phase is not related to the existence of the T cell-dependent phase. That is, a more essential subversion by guanosine derivative-containing immunomodulators resulted in lower doses of T- and T-independent type 2 antigens than T-independent type 1 antigens (more completely T-cell independent) ( (T cell dependent state). This indicates the presence of a T cell dependent component.

The test of adjuvant titer is its ability to restore the immune response of an immunodeficient body. That is, CBA / N mice were used as a murine model of X chromosome-related (X-related) primary B cell immunodeficiency. This pedigree is Lyb3 / 5/7
It is considered that the functional activity of the subpopulation of mature B lymphocyte T cells having the antigen is defective. Huber et al., J.Ex
p.Med., 145: 1 (1977); Ahmed et al ., J. Exp.Med., 145: 101.
(1977); and Subbarao, J. Immunol., 122: 2279 (197
See 9). Of NCF ₁ male mice with this defect, inability to react normally to T-dependent antigen (Janeway and Barth
old, supra) is improved to levels above control animals by supplementation of the culture with 8-BrGuo.

Details of the above results are described in "Materials and Methods, Part A". (Goodman and Weigle, J. Immunol. 130: 2580 (198
3)).

The results described above in this section show that the T cell-like signal
Being sent to the B cell by contacting the cell with an immunomodulator of the invention in the presence of an antigen, and wherein this signal is synergistic with certain other signals derived from the T cell or its product. Is shown.

The ability of β-interferon and 8-MGuo to produce an enhanced T cell mimic signal in B cells In the course of studying the effect of C8 derivatized nucleotides on B lymphocyte function, B cells in the presence of antigen A relationship was observed regarding the ability of nucleotides to send T cell-like signals. Subsequently, T cells-and macrosurge-
The T cell-like eliciting effect of 8-MGuo on B cells and antigens was studied in the presence of supernatant containing various cytokins from different sources.

Cytokine was obtained as a supernatant free of cells from CBA / CaJ and (C57B1 / 6J × DBA / 2 ) mixed lymphocyte cultures between F ₁ mice. Such supernatants are, inter alia, interleukin-1, interleukin-2, B
Includes cell growth factors, T cell replacement factors, and interferons. Co-culture of B cells in the presence of antigen and these supernatants with 8-MGuo, as seen in FIG.
Or results in a synergistic response (antigen-specific antibody-secreting cells) compared to that seen with B cells and antigen in the presence of supernatant alone.

Work is ongoing to identify cytokins involved in synergistically enhancing the antigen-specific response of purified B cells in the presence of 8-MGuo. Interferons in particular are promising in this regard, and initial results using β-interferon are shown in Table III below.

As shown in Table III, murine beta interferon was 8
-Can provide T cell-like triggering signals for B cells and antigens as well as -MGuo. However, in the presence of both 8-MGuo and β-interferon, a clear synergistic effect of the evoked response is observed. About 1 × 10 ^-5 to about 1 × 10
^The subcutaneous immune response observed in vitro by contacting an immunomodulator of the invention containing a ^-3 molar concentration of an 8-substituted guanosine derivative with a cell, such as a B cell, in the presence of the antigen, is indicative of the immune response A synergistic improvement can be achieved if the modulator also comprises about 1 × 10 ^{3 to} 5 × 10 ⁹ units / l of interferon, such as β-interferon. More preferably, the concentration of interferon in vitro is about 1 × 10 ⁴
約 5 × 10 ⁶ units / l.

For in vivo administration to humans, the dosage of interferon may be from about 1 × 10 ³ to about 5 × 10 ⁹ units per day, more preferably from about 1 × 10 ⁴ to about 5 × 10 units per day, in single or repeated administration.
× 10 ⁶ units must be used. For administration to animals, the dosage will be proportional to that of humans based on the weight of the animal. Effective dosages of the 8-substituted guanine derivatives of the invention, such as 8-MGuo, used in combination with an interferon, such as β-interferon, are described below. These dosages are typically based on the weight of the human or animal being treated.
It is in the range of about 1 to about 1000 mg / kg. Studies with α and β interferons and other cytokins are ongoing and are likely to show similar synergies.

Characterization of Lymphocyte Subpopulations Activated with 8-Sulfidoguanine Derivatives FIG. 10 shows CBA / CBA cultured under serum-free conditions with immunomodulators containing various concentrations of 8-MGuo. Fig. 3 shows the results of culturing spleen cells from CaJ mice. The graph shows [ ³ H] TdR uptake and germinal vesicle conversion measured on day 2 of culture, where peak values are normalized. Representative areas of stimulated and unstimulated cells can be seen in FIGS. 10B and C. The results, shown in FIGS. 10A and C, show that immunomodulators, including 8-sulfide substituted guanine derivatives, induced lymphocyte activation (as an indicator of proliferation) in a dose-dependent manner.

Spleen cells from innate athymic (nu / nu) C57BL / 6J mice and their heterozygous (nu / nu)
+) Splenocytes from littermates were cultured in serum-free medium in the presence of immunomodulators containing various concentrations of 8-MGuo. A response to a known T lymphocyte cytogen, ConA, was elicited against a control of T cell contamination. As seen in Figure 11, 8-MGuo is a heterozygous littermates of C57BL / 6J nude mice and their heterozygotes.
In both cases, dose-response graphs of [ ³ H] TdR ingestion are given in parallel to those seen in CBA / CaJ mouse cultures. The absence of functional T cells from contaminating these populations is indicated by the lack of stimulation induced by ConA in nu / nu cultures. This is shown at the right end of FIG.

Rich in B cells cultured under serum-containing conditions. Also T
First cell-rich CBA / CaJ thymocyte and spleen cell culture effects
See Figure 2. The plot in FIG. 12 shows that B lymphocytes are 8-M
It shows a strong proliferative response to Guo, while neither thymocytes nor spleen T cells showed a significant response. However, both thymocytes and spleen T cells
A strong response to the cell mitogen ConA was observed.

The cell surface phenotype of 8-MGuo-reactive cells was determined using spleen lymphocytes depleted with cells with various surface markers, as described in more detail in Materials and Method B. Was. Remove cells with surface IgM heavy chains,
When the same cells were then re-depleted, the resulting depleted population did not respond to 8-MGuo or LPS, but responded well to T-cell mitogen, ConA (Table I).
V, A). Applying the same approach to cells with surface IgD heavy chains using the monoclonal product of the 10-4.22 cell line, the population with cells with surface IgD heavy chains removed is smaller than 8-MGuo However, it was found that a significant response could be obtained (Table IV, B). Non-adhered populations were observed to be moderately responsive to LPS, but sufficiently reactive to ConA.

Splenocytes divided into FcR ⁺ and FcR ^- populations by the EA-rosetting method show that FcR ^- cells are almost unresponsive to 8-MGuo, whereas FcR ⁺ cells respond well. (Table IV,
C). Splitting spleen cells into a population that has or lacks a receptor for the complement (ie, C3) can be performed by EAC-
Achieved by rosetting. CR ⁺ cells were removed (de
The pleted) population showed a small but significant response to 8-MGuo. The CR ⁺ subset was observed to be highly responsive to immunomodulators containing this nucleotide derivative (Tables IV, D).

Reactivity to 8-MGuo was evaluated in a CBA / N immunodeficiency model using the mice described above for this purpose. Here, the culture of the immunomodulator containing 8-MGuo of using cells of NCF ₁ female mice resulted in strong response. For culture in cells from NCF ₁ male mice, only moderate reaction was observed.

The ^{CBA / CaJ (H-2 k} ) and ^{BALB / c (H-2 d} ) human organ cells, using various dilutions A.TH anti A.TL antiserum (anti-Ia ^k) 8-M
Cultures were performed in the presence or absence of immunomodulators containing optimal concentrations of Guo to determine whether the response to 8-M Guo was sensitive to inhibition by anti-Ia antibodies. As can be seen from Table V below, high concentrations of the antiserum exhibited non-specific inhibitory effects, while specific inhibition was observed at lower dilutions. The incomplete suppression observed indicates that a significant number of 8-M Guo-reactive cells lack surface Ia or tolerate such low concentrations that cell activity is not suppressed by antibody binding.

IgD ^-, Ia ^-, CR ^- or Lyb 3/5/7 ^- for low-level reactions caused to prove that no attributed to positive few contaminating cells surface markers described above, the nuclear fission Hara The ontogeny of the response (mltogenic response) was directly evaluated using CBA / CaJ mice of various ages. The data in FIG. 13 shows that prior to the expression of these markers on the cell surface, at 4 days of age the stimulation index was approximately 20-25% of that generated in culture of 8 weeks old mice.

To demonstrate the role that the response to 8-M Guo plays in response to immunomodulators containing this guanine derivative as an active ingredient,
Evaluated before and after passing through Sephadex G-10 resin. Cells effluent from this column cannot react to 8-M Guo, and adding non-adherent cells to irradiated spleen adherent cells will re-react their response to 8-M Guo. Did not build. This is shown in FIG. 14A. 14th
Panel B shows that the cell population adherent to Sephadex G-10 is 8-M Gu
o indicates that it is rich in reactive cells.

8-M Guo stimulates normal spleen B cells as well as spleen cells from innate athymic (nu / nu) mice lacking functional T cells. The presence of T cells in the B cell culture was 8-
He did not subsidize the fission reaction to M Guo. The effect of 8-M Guo appears to be a non-specific antigen in which a large proportion of spleen B cells responded to it by germinal transformation. Also, the presence of a specific antigen is not required.

Lymphocytes that respond mitogenically to 8-M Guo are exclusively B cells and appear to have a predominantly mature surface phenotype. Almost all reactive cells have a surface phenotype of IgM ⁺ Fc
R ⁺ Thy1.2 ^- characterized by. Many of the cells also
Surface Ia antigen, IgD heavy chain, C3 receptor, and
It appears to have the Lyb 3/5/7 antigen. Another group of Bs lacking all of the above markers or having them at very low density
Cells can also be entrained in the reaction.

Details of the above results can be found in Materials and Method B below. (Goodman and Weigle, J. Immunol, 130: 551 (1983
reference).

In Vivo Modulation of Immune Response FIG. 15 shows that immunomodulators containing 8-M Guo suspension
Figure 5 shows a marked immunopotentiating effect on the primary antibody response to SRBC at in vivo observed when injected into CBA / CaJ mice 30 minutes after injection of the antigen. Relatively high doses, about 25 mg per animal (about 1 g / kg), were well tolerated by animals. FIG. 16 shows that when the administration of 8-M Guo was delayed until about 3 or 4 days after the administration of the antigen, the concentration that elicited the greatest adjuvant effect was about 300 μg (0.01 g / kg) per mouse.
To about 2 orders (two digits).

FIG. 17 shows that the substitution at position 8 of the guanine derivative is important for the induction of mltogenicity and polyclonal immunoglobulin secretion as described above. In other words, CBA / CaJ mice still have S
RBC is injected intraperitoneally (intraperltoneally: lp)
Then injected with normal saline (NS), either 8-Br Guo or Guo. As can be seen from FIG. 17, NS and Guo produced the same low primary antibody response. On the other hand, 8-Br Guo produced a significant antibody response.

FIG. 18 shows the antigen dose dependence of the mice described above on a constant level of the co-effect of 8-M Guo (25 mg / animal) injected ip, together with the case of ip injection of NS as a control. This figure shows that at all levels of antigen injection, there was a subversion of the immune response, but the subaru increased with increasing degree of basal response. That is, 8-
M Guo increases the response about 2- to 3-fold at low antigen doses, but increases response by about an order of magnitude at optimal doses.

FIG. 19 shows the efficacy of the immunomodulator of the present invention comprising 8-Br Guo as an adjunct in ameliorating in vivo immune deficiency conditions. This in vivo illustration corresponds to the in vitro improvement described above in FIG.

Of immunodeficient (CBA / N × CBA / CaJ ) F 1 mice to the antigen of SR
Inject BC ip then saline or 25 mg / animal 8-Br Guo
Were injected ip. FIG.
The basal antibody response to thymus-dependent antigens is almost zero in defective (male) animals compared to control (female) animals. This is the case with Janeway and Barthold,
Supra, and by Schner et al., J. Immunol, 123: 477 (1979). This figure shows that the injection of 8-M Guo shows a marked sublimation (> 20-fold) for this response. This regulatory subaugmentation causes higher levels of response in defective male mice than those achieved by normal female mice with antigen alone,
Equivalent to the enhanced response produced in normal mice with Br Guo.

The above results indicate that the optimal effect is about 25 mg / mg when injected shortly after the antigen, i.e., within about 24 hours of the antigen injection.
At the dosage level of the animal (about 1 g / kg) and the administration is performed at least about 3-4 hours after the animal cells have been given the antigen in vivo.
About 300 μg / animal (about 0.01 g / k
g weight).

The remarkable adjuvant effect of the immunomodulators of the present invention, which occurs late in the course of the ongoing reaction, suggests that this immunomodulatory effect is not attributable to the clonal expansion of cells that respond to the antigen. Rather, it is believed that the immunomodulator of the present invention provides a final trigger signal to a population of cells whose activity would otherwise not be able to progress beyond intermediate stages and would eventually be discarded.

This result further indicates that the immunomodulator of the present invention is more effective when cells are contacted at a time after antigen challenge, consistent with the in vitro studies described above. There, these immunomodulators brought about equal or better results when contacted with the cell culture approximately three days after the antigen than when initially added to the culture.

The immunomodulator of the present invention enhances the cellular response at all doses of the antigen, but the degree of Subaru progression is usually maximal at optimal or near optimal concentrations. It was shown that the adjuvant effect of the immunomodulator of the present invention is not the sum of the antigen-specific reaction and the non-specific (clonal) reaction.

The in vivo adjuvant effect of the immunomodulator of the present invention further includes:
Exemplified by the ability to elicit an antibody response in mice with X-related primary B cell immunodeficiency. The above results show that mice with this genetic defect respond normally to T-dependent antigens when their cells are contacted by a complementary injection of an immunomodulator of the invention.

That is, the immunomodulator of the present invention exerts a strong adjuvant action when administered in vivo. In addition, its use
They are beneficial in that they are non-microbial in nature, do not require injection of necrotic oil emulsions, and offer new possibilities for enhancing ongoing immune responses.

Details of the above results are given in Materials and Method Part C. (Goodman and Weigle, Proc. Natl. Acad. Scl. US
A., 80: 3452 (1983)).

A secondary in vivo mouse response to human gamma globulin (HGG) as an antigen was also studied. A total of 11 A / J mice, 5 control mice and 6 treated mice, were subjected to DEAE Sephadex (Pharmacia Fine Chemical Chemical) according to the procedure of Chiller and Weigle, J. Immunol. 110: 1051 (1973).
s, Plscataway, NJ), heat-aggregated HGG40
0 μg was injected intravenously in vivo. Control mice were then injected intraperitoneally with a 10 mg / ml solution of carboxymethylcellulose in saline (0.15 molar NaCl). Treated mice were 25 m insoluble suspension of 8-Br Guo in the same CMC solution.
g was injected intraperitoneally. HGG heat-aggregated in mice on day 10
Was administered intraperitoneally. On day 14, mice were evaluated for specific antibody-secreting cells against HGG according to the procedure of Chiller and Welgle, J. Immunol., Supra. The results obtained were 2064 in the control group (HGG + CMC).
There were 8947 PFC / hilt in the group contacted with PFC / hilt and HGG and 8-Br Guo in CMC. These two numbers are the arithmetic mean of each group.

The primary response to HGG with repeated administration of nucleotides was also examined in the above manner. HGG4 aggregated by heating
00 μg was administered intravenously in vivo to 19 mice on day 0. On days 0, 2, 4, 6, and 8, 4 mice were injected intraperitoneally with carboxymethylcellulose, 5 mice received a dose of 30 μg of 8-Br Guo in CMC, and 5 mice received 8 μg of CMC.
-Br Guo 300 μg, 5 animals were injected intraperitoneally with 3000 μg 8-Br Guo in CMC. On day 10, the mice were sacrificed, the number of PFC / spleen was determined, and the data is shown in Table VI below.

As shown in FIG. 29, primary P of mice against HGG
The effect of a single dose of 8-Br Guo on the FC response was also studied. A / S mice were injected intravenously on day 0 with 400 μg of heat-aggregated HGG intravenously. After 3 hours, 8-Br suspended in carboxymethylcellulose solution or CMC solution
Guo was injected. On day 6, spleens are removed and PF against HGG
Examine C. The bars in FIG. 29 are the standard errors, and the data points are from left to right on 12, 11, 12, 12, 12 and 13 test mice. FIG. 29 shows that the mouse in vivo PFC response to human gamma globulin protein increases steadily with increasing 8-Br Guo dose.

T Cell Surrogate Activity The ability of the immunomodulator of the present invention to surrogate T cells in an antibody response to a T-dependent antigen is shown in FIG. Here, B cells generated in vitro by treatment with the monoclonal anti-thy1.2 + complement were cultured with or without SRBC in the presence of immunomodulators containing various concentrations of 8-M Guo. The data in FIG. 20 shows that under these conditions
This shows that the isolated B cell culture cannot react against the antigen unless complemented with 8-M Guo. 8-
The response modulated by M Guo is dose-dependent as well as antigen-dependent. Also, this response cannot be attributed to non-specific polyclonal activation of B cells (lower curve). SRBC
Of normal spleen cells to about 600 to about 100 per culture
0PFC.

The ability of the immunomodulator of the present invention to provide an alternative source of T cell-like signals is shown in Figure 2.1, where the present invention reconstitutes the primary antibody response of cells from innate athymic (nu / nu) mice. The ability of immunomodulators is seen. Here, heterozygous (nu / +) littermates are controls.

Supplementation of these cultures with concentrations as low as 0.1 mM of 8-M Guo not only reconstitutes the primary antibody response of cells from nu / nu animals, but also elicits additional adjuvant effects. The resulting ultimate response is nu / + against antigen alone
It is significantly larger than the response of the culture and may be comparable to the extent of the response generated by nu / + cultures supplemented with both antigen and the immunomodulator of the invention. These results indicate that mature T
The presence of cells has been shown to be unnecessary for the activity of the immunomodulator of the invention.

The cultured cell population above appears to have maintained precursor T helper cells. In order to suppress the IL-2 dependent growth of such precursors as well as the development of IL-2 in the context of a primary immune response, whole spleen cell populations were optimized for 8-M Guo.
The antigen was cultured in the presence or absence of an immunomodulator containing Various concentrations of cyclosporin A were added at the indicated dosage ranges to suppress IL-2 production. Bunjes
Eur.J.Immnal., 11: 657 (1981).

The data on the left of Fig. 22 shows that cyclosporin A is SR
It shows that the spleen response to BC progressively interferes until it falls below background levels. Cultures supplemented with 8-M Guo resist this suppressive effect of cyclosporin A, thereby indicating that this effect is IL-2 independent.

In the right panel of FIG. 22, the spleen cell response was normalized with the response to SRBC in the absence of cyclosporin A as 100%.

Of a B cell population containing antigens for various amounts of MLC supernatants (which may include IL-2 or other lymphokins),
The reaction in the presence or absence of -M Guo is shown in FIG. The data in FIG. 23 shows that the MLC supernatant could support a significant response to the antigen in B cell culture, but the extent of this response was increased several-fold in the presence of 8-M Guo. That is, administration of the immunomodulator of the present invention to cells induced specific antibody formation by a mechanism different from that of MLC lymphokin.

The immunomodulator of the present invention and a lymphocyte derived from a T cell,
B cell populations generated in vivo and then generated by in vitro treatment at the beginning or 72 hours after culture were added. The results of this measurement indicate that these cultures do not lose their ability to respond to IL-2-related lymphokins when lymphokin addition is delayed, but correspond to 8-Br Guo added during culture. Is shown. The results are shown in Table VII below. Also, the addition of such an IL-2 preparation at the start of the culture and the addition of 8-Br Guo 72 hours after the start of the culture produces additional effects,
Furthermore, it has been demonstrated that these two classes of adjuvants work by different mechanisms.

The above data indicate that the immunomodulators of the present invention provide T cell-like signals to B cells that are primed, otherwise completely replacing T cell requirements under conditions of T-dependent responses. Is shown. That is, recruitment of 8-M Guo-containing immunomodulators to T cell cultures depleted of T cells bearing thy1.2 replaces the need for T helper cells in generating a primary antibody response to SRBC. . This is what Harwell et al., JE
xp. Med., 152: 893 (1980), spleen cells were obtained by in vitro treatment with monoclonal, anti-thy1.2 and complement, or by in vivo injection of ATS followed by ATS, anti-thyroid. Correct irrespective of whether T cells were removed by in vitro treatment with thy1.2, anti-Lyt 1 and anti-Lyt 2 and the complement. The TRF-like activity of 8-M Guo-containing immunomodulators is clearly dose-dependent,
It cannot be explained by non-specific polyclonal activation in B cells.

The possibility that the immunomodulators of the present invention induce T helper precursors to mature and thereby contribute to the observed response hinders IL-2 production that could contribute to the clonal expansion of such cells. The use of cyclosporin A in the amounts indicated in the light for the above was ruled out by the aforementioned measurements. (See Bunjes et al., Supra). Cyclosporin A was able to completely abolish the response of spleen cells to SRBC in the absence of 8-M Guo, whereas the response to exposing these cells to antigen in the presence of 8-M Guo was A
It was resistant.

That is, the TRF-like effect of 8-M Guo is completely IL-2 independent. Also, the supplemental expansion induced by contacting the cells with an 8-M Guo-containing immunomodulator with a culture medium containing T cells and antigen but without 8-M Guo is also T cell-independent and IL-dependent. -2 independent. Thus, the immunomodulator of the present invention served as a proxy for complete T cells or soluble T cell-derived molecules in the primary humoral response to the antigen.

The above results also indicate that the mechanism of action of the immunomodulator of the present invention was distinct from T cell-derived lymphokins and the T cell surrogate (or B cell stimulatory) activities involved therein. This was demonstrated by the synergistic effect of T-helper factor generated in 8-M Guo and MLC supernatant. There, the anti-SRBC PFC reaction supported by the supernatant was 8-M
It was increased about 2 to about 3 times by the addition of the Guo-containing immunomodulator.

8-Br Guo immunomodulators also work with dynamic properties that are quite different from the dynamic properties of IL-2-related lymphokins, where IL-2 was co-chromatographed on a DEAE-Sephadex column. The preparation containing lymphokin was completely ineffective when the addition to the culture was delayed, while the cells were treated with 8-Br 72 hours after the start of culture.
Contact with immunomodulators, including Guo, enabled the generation of specific responses to antigens.

Also, add the above-described lymphokin preparation first, then
When 8-Br Guo was added late, the reaction obtained was the sum of the individual reactions supported by each agent. This indicates a lack of synergy with IL-2-related lymphokines, indicating that MLC
It is believed that the effects of other lymphoids in the supernatant, such as interferon, can be attributed (see Table III above). Therefore, the immunomodulator of the present invention is T cell independent and IL
Cultivate -2 independent antigen-specific responses, but their mechanism of action must be essentially different from that of TRF activity of IL-2-related lymphokins, and their transient characteristics are Sc
more closely resembles a T cell surrogate that acts with a delay as described by himpl et al., Nature (New Biol.), 237: 15 (1972).

Experimental details of the above work are described in Materials and Methods, Part D (Goodman and Weigle, J. Immunol., 130: 2042 (1
983)).

Modulation of humoral immune response in immunodeficient aged mice. The relative ability of 8-M Guo and LPS to activate murine B lymphocytes to immunoglobulin secretion is young and old.
It was studied in the culture of spleen cells from C57BL / 6J mice. The results of these studies, shown in FIG. 24, indicate that both 8-M Guo and LPS effectively induced polyclonal immunoglobulin secretion in spleen cells taken from young adult mice (discontinuous bars). Cells from aged mice reacted strongly when contacted with immunomodulators, including 8-M Guo, but did not respond to LPS (hatched line). The response of cells from old animals to 8-M Guo is less than that of young animals, while the response of old cells to 8-M Guo is greater than the response of young cells to LPS.

LPS has previously been shown to have a strong adjuvant effect on primary antibody responses in vitro. Chiller et al., Proc. Natl. A
cad. Scl. USA, 70: 2129 (1973); Skidmore et al., J. Immun.
ol, 114: 770 (1975). The adjuvant effect of the immunomodulator of the present invention has been shown above. The relative ability of LPS and the immunomodulator of the present invention to act as an adjunct for the primary antibody response to SRBC, as shown in Figure 25, indicates that the statistically meaningless response to SRBC in the absence of the adjunct Only possible was evaluated in cultures of spleen cells from aged C3H / HeN mice.

As seen in FIG. 25, neither 8-M Guo nor LPS shows a significant polyclonal effect under these conditions. The lack of polyclonal effects of the 8-M Guo-containing immunomodulators is believed to be due to differences in dosage and kinetic requirements for optimal polyclonal and antigen-specific responses. However, 8-M Guo
Addition of the antigen to the culture in the presence of E. coli allows the development of a pronounced response to the antibody, to a degree from young adult mice to the antigen in the absence (but not the presence) of 8-M Guo. Cell response. Addition of LPS to cell cultures stimulated with antigen from aged mice failed to consistently restore their response to antibodies.

TRF-like activities for 8-M Guo (described above) and LPS
In vitro (Sjoberg et al., Eur. J. Immunol., 2:
326 (1972)), whereby normal spleen B cells or n
Splenocytes from u / nu mice were able to generate an antibody response to T cell antigen in the presence of any of these activators. The relative TRF-like activity of these two agents was evaluated in cultures of B cells from aged C57BL / 6J mice.

FIG. 26 shows that B cells alone cannot respond to each of the antigens or LPS, and that these agents do not elicit a greater response when present together in culture. However, supplementing B cells with antigen and 8-M Guo elicits the development of a significant PFC response to the antibody, which may be comparable to that produced by antigen alone from cells of young adult mice. Under these conditions, a moderate polyclonal response to the immunomodulator containing the 8-substituted guanosine derivative in the presence of the antigen is evident, probably due to a 3-fold higher concentration of 8-M Guo used in these experiments. These data clearly show that the elimination of the need for normal T cells for response to SRBC is not simply a consequence of the synergistic effect between polyclonal response and background.

The data above show that administration of the immunomodulators of the invention to animal cells demonstrates antigen-specific as well as non-specific humoral immunization in cell cultures from aged mice whose immune response has diminished prior to administration. It clearly shows that sex can be regenerated. Polyclonal immunoglobulin secretion
It is very effectively induced by the immunomodulators of the invention, but poorly, if any, by LPS. This difference is believed to be due to the rapid uptake of the 8-substituted guanosine derivative into cells and the independence of its effect on membrane function.
That is, transduction of signals across the membrane appears to limit the reactivity of cells from aged rats.

This membrane defect, however, appears to distinguish between antigen-specific and non-antigen-specific signals. This is indicated by data from studies of adjuvant action and T cell surrogateness. In these cases, the antigen alone caused no or little antibody response. LPS, with or without antigen, is also
moto et al., failed to elicit an antibody response as previously reported in J. Immunol., 166: 294 (1976). 8-M Guo alone caused a low level of polyclonal reaction after 4 days in culture. However, when the 8-substituted guanosine derivative was combined with the antigen, a significant response to the antigen occurred.

The degree of response to the administration of the immunomodulator of the invention together with the antibody eliminates common explanations such as the background and the effect of a non-specific response. These results indicate that the antigen does not signal B cells from aged mice.
This initial signal is required for all antigen-specific reactions, but not enough for T-dependent antigens. The data above shows that aged T cells, like LPS, give ineffective second signals. It is believed that the effectiveness of the immunomodulator of the present invention, including the 8-substituted guanosine derivative, to send a second signal can be attributed to its ability to bypass membrane-dependent events.

T cells cannot be seen to play an important role in the immune reconstitution effects of the immunomodulators of the invention in aged mice. The B cell population and macrophages are
As long as the immunomodulator of the present invention provides a second signal similar to a T cell, it reacts with an antigen in the absence of a functional T cell,
The extent of the reaction in the presence or absence of cells is generally comparable. T cells from aged mice function poorly in humoral responses (Morgan et al., Cel. Immunol., 63:16 (19
81): Krogsrud et al., J. Immunol., 118: 1607 (1977)) and
Hardly produce IL-2 (Miller et al., Eur. J. Immunol., 1
1: 751 (1981)] provides a reasonable basis for understanding the above observations.

The assessment of survival values against hosts, which modulate an inadequate immune response, has advanced to intensive studies on increasing systemic immunity. The importance of this approach is most apparent because of the immune deficiencies often associated with the aging process. Already many people have a wide range of immune-related diseases. The above results in aged mice indicate that one link in the event chain leading to the induction of B lymphocyte function (ie, membrane function), one link of the present invention that shorts one link known to be defective in the aged animal. Demonstrate the ability of immunomodulators.

The experimental conditions used to obtain the above results are described in Materials and Method E below.

Adjuvant effect by oral administration The adjuvant effect of the immunomodulator of the present invention administered by in vivo ip injection is described above. The results in this section relate to the adjuvant effect of the immunomodulator of the present invention administered orally through a tube extended into the stomach of the animal. These results were obtained using the materials and conditions described in Part C of the Method.

SRBC was injected ip and PFC measurements were taken 7 days after the first ip injection of SRBC. The immunomodulator of the invention, including 8-M Guo, was administered periodically within the same 24-hour period as the SRBC antigenic dose, or within the next 72 hours. The data from these measurements is shown in Table VIII below.

As can be seen from the above data, administration of the immunomodulator of the present invention can be achieved by contacting the cells within the same 24 hour period that the cells were exposed to the antigen, or within 72 hours thereafter. , Giving an enhanced primary response to the antigen. As mentioned above, adjuvant action is improved when there is a delay of at least three days between exposing the cell to the antigen and contacting it with the immunomodulator of the present invention.

Activation and differentiation by 8-bromoguanosine derivatives Studies on cell activation and differentiation (mitogenesis) are described in Goodman and W.
USA, Proc. Natl. Acad. Sol. USA, supra, using a procedure similar to that described in 8-bromo-2'-O-.
Methylguanosine, 8-bromo-2 ', 3', 5'-tri-
Performed with O-acetylguanosine and 8-bromo-2'-deoxy-guanosine. Non-brominated guanosine derivatives were obtained from Sigma Chemical Company, St. Louis, Missouri. Bromination was performed as described in the cited reference and gave a single spot on a polyethylene imine cellulose plate as described in the cited reference. Cell culture is performed in the presence of the 8-substituted guanine derivative for up to 24 hours, then [
^[3 H] TdR was administered, cultured for another 24 hours, and collected. [ ³
[H] TdR measurements were performed as described in the references cited above. The data for the 8-Br Guo derivative described above is shown in Table IX below.

The data above strongly indicates that derivatives on the aldose moiety of 8-substituted guanine derivatives confer cell activation and differentiation similar to 8-Br Guo.

Further comparisons were made as described in Table X and FIG. 30 using the procedure described above for mitogenesis. To get the data here, Goodmon and Weigle,
The assay procedure of J. Immunol, 130: 551 (1983) was used. This is quoted and included here. Table X shows that various oxo and mercaptoguanine derivatives promote mitogenesis. FIG. 30 further shows that 8-M promotes mitogenesis.
9 shows the performance of Guo and 8-thio- (β-hydroxyethyl) Guo.

The mitogenic effect on immunoglobulin secretion and polyclonal activation is a co-additive effect for antigen-specific responses and
Although mentioned that there is no need for concordance compared to TRF-like activity, 8-substituted guanine derivatives are useful because they are mitogenic as exemplified in Table X, and as shown in Table II Provides polyclonal activation and, like 8-M Guo and 8-Br Guo, also act as a cofactor for antigen-specific reactions and TRF-like activities.

Suppression of Growth of Neoproliferative Cells The previous studies focused mainly on cell growth stimulation and adjuvant effects obtained by administering the immunomodulator of the present invention to various cells, especially leukocytes. The results relate to the inhibition of neoproliferative cell proliferation obtained by contacting the neoplastic cells with the immunomodulator of the present invention. More particularly, cultured lymphoid B cells, neoplastic macrophages and hybridoma cell cultures were cultured in the presence of various amounts of 8-MGuo, using the same cell culture without 8-MGuo as a control . Non-neoplastic spleen cells are also 8-MGuo
Cultivated in the presence and absence of E. coli and used as a comparison to neoplastic cell cultures.

The results of these culture studies are shown in FIG. Where,
[ ³ H] TdR uptake in the presence of 8-MGuo separated by the same uptake and control cultures is plotted against 8-MGuo concentration for each of the 4 cultured cell types. 27th
The figure shows that spleen cells are stimulated to proliferate in the presence of 8-MGuo, while growth of each of the three types of neoplastic cells is approximately
It is shown that the concentration is greatly suppressed by 8-MGuo concentration exceeding 0.1 mM. Examination of the kinetics of ingestion shows that administration of the immunomodulator of the invention to neoplastic cells causes cytotoxicity in one step of the metabolism of the cells, rather than lysis.

The mechanism of suppressing the growth of newly grown cells by contacting the cells with the immunomodulator of the present invention is unknown. However, since neoplastic growth can be described as the expression of a cellular message that causes cells to replicate much faster than normal, the immunomodulators of the present invention replace these rapidly replicating cells with cells that replicate too fast. It may even spur them so fast that they die.

Induction of Interleukin-1 Similar Activity IL-1 produced by macrophages assists in the production of IL-2 in T cells. IL-2 is a growth factor for T cells and aids in the conversion of T helper cells.

Supernatants from irradiated spleen cell cultures (here only macrophages survive the radiation process [Gorczynski,
J. Exp. Med., 134: 1201 (1971)] and is supplemented with immunomodulators of the invention containing various concentrations of 8-MGuo. )
It has been found that thymocytes can be made to proliferate in the same manner that thymocytes proliferate in the presence of IL-1. Ultimately, the immunomodulators of the present invention are believed to stimulate what can be termed IL-1 mimic activity, where macrophage supernatants exhibit a thymocyte response similar to that exhibited by thymocytes in the presence of IL-1 Trigger. Table XI below
Data show the effect obtained by contacting thymocytes with IL-1 free supernatant, ie, exhibit IL-1 similar activity.

The data above shows radioactive thymidine
Shows that it was incorporated into thymocytes in the presence of a spleen cell supernatant containing the immunomodulator of the present invention.

Induction of endogenous interferon production. Spleen cells were isolated from Goodman and Weigle, J. Immunol., Supra.
The cells were cultured for 24 and 48 hours as described in the literature cited above, after which cells were harvested and supernatants from the harvested cell cultures were examined for interferon production. This test revealed that relatively high levels of interferon were produced by assays with suppression of virus spot formation [Armstrong, Appl. Miorokio., 21: 723 (1
972)].

Mitogenic agents are known to stimulate interferon production in the absence of a viral interferon inducer. However, such mitogenic interferon inducers are unacceptable in animals and therefore cannot be used. On the other hand, the immunomodulator of the present invention is well tolerated,
Therefore, it is effective for inducing endogenous interferon production in the absence of a naturally occurring microbiological interferon production inducer and for providing a supplementary effect for interferon production in the presence of an interferon production inducer. . Also, based on the ability of the immunomodulator of the present invention to enhance the cellular response to the inducing substance (adjuvant action), such immunomodulators may likewise be expressed in the presence or absence of interferon inducers. It is thought to increase production.

Enhanced 5'-nucleotidase activity in immunodeficiency conditions As mentioned above, an important use of adjuvants is in amelioration of immunodeficiency conditions. The hypogammaglobulin mia associated with X (mias) exhibits such an immunodeficient condition, wherein the males exhibit reduced lymphocyte surface ect-5'-nucleotidase (5'NT) activity.

The data in FIG. 28 shows that lymphocytes of CBA / CaJ mice were 8-M
5 shows the percentage of 5'NT activity when contacted with an immunomodulator of the invention, including Guo, on a percentage basis. Similar results were obtained using human lymphocytes prepared by one of the present invention. These data further demonstrate the ability of the immunomodulators of the present invention to reconstitute immune when used for contact with immunocompetent cells.

One of the most active agents to induce the secretion of neutrophilic enzymes, the release of lysosomal enzymes from neutrophils, is the complement component C5a. Chenoweth
And Hugli, Proc. Natl. Acad. Sci., USA, 75: 3943 (197
First settle with dextran according to the method of 3) and f
Discharge through the icoll gradient, then Han at 4 × 10 ⁶ cells / ml
Human neutrophils (neutrophils) resuspended in k's balanced salt solution and then contacted with the immunomodulator of the present invention containing 8-MGuo
^- Phils) has release an amount of Risosomaru enzyme elevated.

The observed enhancement of lysosomal enzyme secretion was achieved by using the optimal concentration of complement component C5a when the concentration of 8-MGuo used in the immunomodulator of the present invention was about 1 to about 10 mM. Almost equal. Enzyme secretion is Chenowet
h and Hugill, supra.

The active ingredients of the present invention can be administered orally or in conventional dosage unit compositions, since the immunomodulator in unit dosage form consists of a physiologically acceptable carrier and an effective dosage unit of the active ingredient. It is administered parenterally.

The term unit dosage form herein refers to physically discrete units suitable as unit dosages for humans or other warm-blooded animals, each unit being a required physiologically acceptable carrier, such as a diluent. Or the required amount of the active ingredient calculated to produce the desired therapeutic effect when combined with the vehicle. Details of the novel unit dosage forms of the present invention include (a) the unique properties of the active ingredients and the particular therapeutic effect to be achieved, and (b) such activities for therapeutic use in humans and animals. It is dictated by and directly dependent on the limitations inherent in the art of blending the components. Examples of suitable unit dosage forms according to the present invention are tablets, capsules, pills, powder packets, granules, composites of any of these, such as enveloped paper, and solutions and suspensions.

The amount of active ingredient to be administered depends on the age, weight,
It depends on the particular condition to be treated, the frequency of administration and the route of administration. The dosage range is about 1 to about 1000 mg per kg of body weight,
More preferably from about 5 to about 250 mg, most preferably from about 10 to
It can be about 100 mg. The dose for adult humans ranges from about 50 to about 50,000 mg per day, with a single dose or 3 or 4 doses.
It is given as a divided dose. Dosage for livestock
The dose will be proportional to the weight of the animal as compared to a human adult.

Example 1: Tablet Tablets are mixed formulated from the following ingredients: Ingredients: parts by weight 8-BrGuo 5.0 Lactose, powdered 35.4 Corn starch, dry 33.0 miniaturized SiO ₂ 5.6 Polyvinylpyrrolidone 0.6 Magnesium stearate 0.4 80.0 8-BrGuo Is lactose, corn starch 25.0 parts by weight, S
Completely mixed with 4.0 parts of iO ₂ . The resulting mixture is then homogenized with a 5% solution of polyvinylpyrrolidone in ethanol. The wet material is then passed through a 1 mm mesh screen to make granules. The obtained granules are dried in a drying cabinet at 60 ° C.
Let dry for 24 hours. Pass the dried granules again through a 1 mm mesh screen. 70 parts of the granules obtained are mixed in a suitable mixer with a mixture (previously passed through a 1 mm mesh screen) consisting of the balance of SiO ₂ , balance of corn starch and magnesium stearate. The mixture thus obtained is then treated with 50 mg of 8-BrG, each weighing 800 mg.
Press into tablets containing uo.

Example 2: Starch capsules Capsule contents are prepared from the following ingredients: parts by weight: 8-MGuo 50.0 lactose 450.0 corn starch 500.0 1000.0 Lactose is gradually added to 8-MGuo and mixed. When all the lactose has been added, the resulting mixture is combined with corn starch. The mixture obtained is then filled into capsules having 1.0 g of the formulation. Each capsule contains 50 mg of 8-MGuo.

Example 3: Tablets As many as 10,000 tablets, each containing 50 mg of 8-BrGuo, are made from the following amounts of the following types of ingredients: 8-BrGuo 500 g dicalcium phosphate 1000 g methylcellulose, USP (15 cps) 60 g talc 150 g corn starch 200g Magnesium stearate 10g Mix 8-BrGuo and dicalcium phosphate well and use No.8 screen (U.
Granulate through S.Std.Sieve Series) and dry carefully. Dry the granules using a No. 12 screen (USStd. Sieve S
eries), mix well with talc, starch and magnesium stearate and press into tablets.

These tablets are useful for oral administration at a dosage of 1 to 3 tablets every 8 hours to enhance antibody production.

Example 4: Injectable preparation A sterile preparation, suitable for intracavitary injection and containing 75 mg of 8-MGuo per ml is made from the following component types and amounts:

8-MGuo 7 g Benzyl bezoate 200 ml Methyl parapen 1.5 g Propyl paraben 0.5 g Cottonseed oil, qs to 1000 ml Total amount of this sterile preparation 1-3 ml is injected into the body cavity once a day to enhance humoral immunity.

Example 5: Aqueous preparation for oral use An aqueous preparation for oral use containing 50 mg of 8-BrGuo in each 5 ml (1 teaspoon) is made from the following ingredients: 8-BrGuo 55 g methylparaben, USP 0.75 g Propyl paraben, USP 0.25 g Saccharin sodium 1.25 g Cyclamate sodium 0.25 g Glycerin 300 ml Tragacanth powder 1.0 gg Orange oil flavor 1.0 g FD.and C.orange dye 0.75 g Deionized water, appropriate amount To a total amount of 1000 ml per day Two to four teaspoon doses are useful for subcutaneous humoral immunity.

Materials and Methods Part A Mice Male CBA / CaJ mice, 8-16 weeks old, were purchased from Jackson Lab
Oratory, purchased from Bar Harbor, ME. The breeding nucleus of CBA / N mice is the Animal Production Section, National I
Supplied by nstitutes of Health, Bethesda, MD. All mice were treated with the wayne Lab Biox F6 pellet (Allied Mi
lls, Inc., Chicago, IL) and chlorinated water (acidified to pH 3.0 with HCl).

Application for secondary in vitro antibody reaction Mice were sheep erythrocytes
0.1 ml of a 100% suspension of (SRBC) was injected intraperitoneally. Six to eight weeks after application (Priming), mice were treated with SRBC
The same dose was administered intraperitoneally (ip) and was used 7 days later.

Antigen pooled SRBC is available from The Colorado Serum Co., Den
ver, Co. TNP-LSP was obtained from Dr. John M. Fidler. TNP-AECM-ficoll is from Biosearch, Inc., San Rafael, C
Purchased from A.

Lymphocyte culture The serum-containing culture medium used in these experiments was made as follows: 90.9 ml of RPMI 1646 (Flow Labor
atories Inc., Rockville, MD), 0.1 ml of 100x glutamine, 1.0 ml of 100x sodium pyruvate, 1.0 ml of 50x.
Non-essential amino acids, 1.0 ml of 1.0 M HEPES ⁴ buffer (Microbio
logical Associates, Bethesda, MP), 1.0m of water containing 10 ⁴ units of penicillin G and 10 ⁴ [mu] g streptomycin
l, including 5.0ml of Supportive lot of fetal calf serum (FCS) 1
00ml. Populations enriched in spleen cell suspensions and spleen B cells,
Made as described by Goodman et al., J. Immunol., 121: 1905 (1978).

For assessment of primary humoral immune response to SRBC, 10 ⁷
Murine spleen cells were cultured in 1.0 ml of a medium containing 5% FCS for 4 days in the presence of various concentrations of the indicated antigen. For the assessment of secondary humoral immune response to SRBC, 10 ⁷ spleen cells from primed mice were treated with various concentrations of SRBC at 5% F
The cells were cultured in 1.0 ml of CS-containing medium for 4 or 5 days. The cells are
37 ° C in a culture dish (3008, Falcon Plastics, Oxnard, CA)
10% CO _{2 in} air using a tissue culture box (CBS Scientific, Del Mar, CA) locked at a frequency of 7 cycles / min.
Cultured in a humidified atmosphere.

Mixed lymphocyte culture supernatant 1.5 × 10 ⁷ CBA / CaJ spleen cells were cultured with 1.5 × 10 ⁷ BDF ₁ spleen cells at 37 ° C. in a humidified atmosphere of 10% CO ₂ in air at 5.0 ml volume for 4 days in a volume of 5.0 ml. . Cells were pelleted by centrifugation and the supernatant medium was subjected to 0.22μ filtration before use.

Assay for spot-forming cells (PFC) PFC-secreting antibodies to SRBC were determined by Jorne and Nordin, Scien.
ce, 140: 405 (1963), using a variant of the haemolytic speck assay, evaluated 4 days after culture.

Part B Mice 8-12 week old CBA / CaJ mice are available from Jackson Labor
Bought from atory, Bar Harbor, ME. 8-12 weeks old NC
F ₁ , C57BL / 6J nu / nu and nu / + male mice were Scripps Cli
Obtained from a mouse breeding facility of the nic and Research Foundation, La Jolla, CA. All mice are Wayne Lab Blox
F6 pellets (Allied Mills, Inc., Chicago, IL) and 3.0
Housed in chlorinated water acidified with HCl to pH.

Culture agents The components of the serum-free culture medium used in this study are described in Goodman et al., J. Immunol., 121: 1905 (1978). For serum-containing media, SupportiveFCS
Replaced with 5% of the volume of PMI1640 and the HEPES buffer was omitted. E. coli 055: B5LPS is from Difco Laboratories, Detr
Bought from oit, MI. Concanavalin A is Miles-Yeda
Ltd., Rehovot, Isreal. 8-MGuo, Vega Bioche
micals, Tucson, AZ and Sigma Chemical Co., St. Louis
s, bought from MO. A..TH anti-A.TL antiserum was Dr.JGRa
y, JR., Immunology, Allergic and Immunologic Diseases
Dr. DCShrefflor through Program, NIAID, Bethesda MD
Obtained from.

Cell preparation Splenic and thymocyte suspensions were made as described by Goodman et al., Supra. T lymphocyte-enriched spleen cells are described by Julius et al., Eur. J. Immunol, 3: 645 (1973).
It was made by passing through a nylon wool (NW) column according to the instructions. A population enriched in B cells was 10 ⁸ spleen cells from monoclonal anti-Thy1.2 (New England Nuclear, Bosto
n, MA) at 30 ° C. for 30 minutes. Treated cells were centrifuged at 280 G for 10 minutes to remove antibody, and cells were resuspended in a 1: 6 dilution of CBA RBC-absorbed guinea pig complement at 37 ° C. for 45 minutes. The cells were then washed and cultured as described above. Adherent cells are
Ly and Mishell were removed through a column of Sephadex G-10 as described in J. Immunol. Methods, 5: 239 (1974). S
For ephadexG-10 adherent cells, open the column in a Petri dish,
Collected by vigorous pipetting. After disposing of the beads, the suspended cells were washed three times and used for culture.

Separation of CR and FcR lymphocytesCells that have receptors for complement
And Hayward, Proc. R. Soc. Lond. B. 187: 65 (1974), Paper
sI and II, separated by rosetting procedure. Briefly, 1 ml of RPMI with 10% FCS was added to NH ₄ Cl treated CBA
/ CaJ spleen cells were combined with 2 ml of 25 × 10 ⁶ / ml and 1 ml of a 5% suspension of rabbit anti-SRBC IgM and SRBC previously sensitized with mouse complement at optimal titers. The mixture was gently agitated at 20 ° C for 30 minutes, pelleted by centrifugation, and incubated at 4 ° C for 10 minutes. The pellet was then slowly resuspended, allowed to come to room temperature, and layered gently on a isopaque / ficoll gradient in a silicon tube. The gradient was centrifuged at 1200 G for 30 minutes, pelleted, and the interplanar cells collected separately. Rosettes to 0.
Lysed with 83% NH ₄ Cl, cells were counted, washed and used in culture. Fc receptor-bearing cells are EA rosetting
(Parish and Hayward, supra) and then proceed as described above,
Rabbit anti-SRBC except fresh mouse supplement
Isolation was achieved by replacing IgM with rabbit anti-SRBC IgG. Enrichment was verified by microscopic examination of the separated fractions. EAG- and EA-non-rosetting fractions were each 1% rese.
Contains ttes. Cells that bound four or more red blood cells were determined to be positive for the particular marker.

Removal of IgM heavy chain and IgD heavy chain-bearing cells In order to deplete lymph cells carrying IgM heavy chains (haevy chains) or IgD heavy chains from spleen cells, Wysocki and Sato, Proc. Natl. Acad. Sci. , USA, 75: 2844 (1978)
Panning procedure was used. Briefly, 10 ml of rabbit anti-mouse IgM heavy chain (50 μg / ml, Bionetics, Kensington, MD) or 10-4.22 supernatant (50 μg / ml) is added to 15
Disaggregation was performed by ultracentrifugation at 0,000 G for 60 minutes, and the supernatant was slowly transferred to a 100 × 15 mm polystyrene microbiological Petri dish. After 1 hour at 20 ° C., the supernatant was decanted, the plates were gently rinsed 4 times with PBS, and 3 ml of NH ₄ Cl treated spleen cells (10 ⁷ / ml) were added to the Petri dishes at 4 ° C. After 40 minutes, the plate was rotated slowly and incubated for a further 30 minutes. Non-adherent cells were pooled from those obtained from two additional spin rinses in PBS. Adherent cells were removed with rubber policeman when used. Non-adherent cells are unresponsive (anti-IgM) or slightly responsive (anti-IgD) to LPS, but respond well to ConA.

Lymphocyte culture Murine spleen cells were cultured in microculture plates (no. 3546, Costar, Cambridge, Mass.) At a cell density of 4 × 10 ⁶ viable cells / ml in a volume of 0.1 ml.
Microcultures were cultured at 37 ° C. in a humidified atmosphere of 10% CO ₂ in air. Cultures were grown in 8 μl nutrient solution (Mishell and Du
tton, J. Exp. Med. 126: 423 (1967)) daily.

Measurement of DNA synthesis During the last 24 hours of culture, cells were treated with 1.0 μCi of [3H] T
dR / culture (5 Ci / mM, Amersham Radio Chemicals, Amersh
am, England). Small culture
Bramdel cell harvester, Model M 24V (Biological Res
earch and Development Laboratories, Rockville, MD)
And collected on glass fiber filter strips.
Transfer the filter disc to a plastic scintillation vial, cover with liquid scintillation cocktail,
Counting was performed on a ckman LS-230 liquid scintillation counter.

Germ Cell Counting Histological preparations were generated on microscope slides from individual lymph cultures with the aid of cytocentrifugation. In order to simplify the calculation of neopyroninous germ cells,
Mc Manus and Mowry, Staining Methods, Harper and Row,
Slides were smeared using the methyl green pyronin Y technique of New York (1960), pp. 76-78. The result is 4
It is expressed as the arithmetic mean of standard cultures ± standard error.

Part C mice 8-16 week old CBA / CaJ male mice are available from Jackson Lab
Oratory, bought from Bar Harbor, ME. The breeding nucleus of CBA / N mice is the Animal Production Section, National Institut
Supplied from tes of Health, Bethesda, MD. All mice were treated with Wayne Lab Blox F6 pellets (Allied Mills, In
c., Chicago, IL) and chlorinated water acidified to pH 3.0 with HCl.

Injected mice were treated with various concentrations of washed SRBC in saline.
The suspension was injected ip. At different times thereafter, different amounts of 8-MGuo or 8-BrGuo were injected ip. These nucleoside derivatives were insoluble in normal saline and were particularly effective when used as suspensions. For oral feeding studies, mice were inserted a poly (propylene) catheter from mouth to stomach and a measured amount of the adjunct composition was introduced therethrough.

Assay for spot forming cells (PFC) The number of PFCs that secreted antibodies to SRBC was determined by Jerne and No.
Evaluated 5-7 days after antigen injection using a modified haemolysis test of rdin, supra.

D part mouse 8-16 weeks old C57BL / 6J nu / nu and nu / +, CBA / Ca
J and BDF ₁ male mice, Scripps Clinic and Research F
Sounding, La Jolla, California. All mice were injected with Wayne Lab BloxF6 pellets (Allied Mills, Inc., Chicago, Illinois) and HCl.
Grew up in chlorinated water acidified to H3.0.

Tissue culture agent Pooled SRBC is available from Colorado Serum Compa
Bought from ny, Denver, Colorado. 8-MGuo and 8-BrGuo
Bought from Sigma Chemical Company, St. Louis., Missouri. Mier and Gallo, Proc. Natl. Acad. Sci., USA, 77:
IL created by DEAE fractionation as described in 6134 (1980)
-2 preparation is from Callaborative Research., Ion., Waltham, M.
A, obtained from

Lymphocyte Culture The culture medium containing serum and the preparation of a population enriched in spleen suspension and spleen B cells used in these studies are described in Goodman and Weigle, J. Exp. Med., 145: 473 (197
7). In one experiment, B cells were obtained from ATS (lot 15038, Microbiological A
60 μl of ssociates) are injected in vivo and then in vitro ATS, monoclonal anti-thy 1.2, monoclonal anti-Lyt 1.1 and monoclonal anti-Lyt 2.1 (New England)
Nuclear) and a mixture of rabbit and guinea pig complement, Harwell et al., J. Exp. Med., 152: 893 (198
Made by 0). For evaluation of the primary humoral immune response to SRBC, 10 ⁷ Nezumihi visceral cells or 4-5 × 10 ⁶ Nezumihi organ cells, in 1.0ml of 5% FCS-containing medium for 4 days 24
Dented plastic culture tray (No.3524, Coster, Cambri)
Dye, MA) in the presence or absence of the indicated antigen. Cells were cultured at 37 ° C. in a humidified atmosphere of 10% CO ₂ in air using a tissue culture box locked at a frequency of 7 cycles / min.

Measurement of Cell Antibody Productivity The direct spot forming cell (PFC) response to SRBC was assessed 4 days after culture using a modified hemolytic spot test of Jerne and Nordin, supra.

Mixed lymphocyte culture (MLC), each 2.5ml of manufacturing CBA / CaJ and BDF ₁ human organ cells supernatants were incubated with ⁶ × 10 6 / ml in 60mm petri dishes. Four days after the start of culture, the culture was transferred to a tube, centrifuged, and the supernatant medium was collected.

Part E Mice Male 8 / 16-week-old or 2-year-old C57BL / 6J and C3H / HeN male mice were purchased from Scripps Clinic and Research Foundation, L.
a Bought from a mouse rearing facility in Jolla, California. All mice use Wayne Lab Blox F6 pellets (Allied
(Mills, Inc., Chicago, Illinois) and water acidified to pH 3.0 with HCl.

Antigens and activators Pooled SRBCs are available from Colorado Ser
um Company, Denver, Colorado. Bacterial lipopolysaccharide extracted by Boivin technique (LP
S) 055: B5 is Difuco Laboratories, Detroit, Michigan
Bought from 8-MGuo is available from Sigma Chemical Company, St.
Bought from Louis, Missouri.

Lymphocyte culture The culture medium containing serum and the method for preparing spleen cell suspensions and individuals enriched in spleen B cells used in this experiment are described in Goodman and Weigle, J. Exp. Med., 145: 473 (1977).
It is described in. For induction of polyclonal immunoglobulin secretion, spleen cells were plated on a 24-well plastic plate (No. 3524, Coster, Cambridge, Mass.) At 5 × 10 ⁶ vi.
The cells were cultured in a 1.0 ml volume at a cell density of able cells / ml. Culture plates were cultured at 37 ° C. in a humidified atmosphere of 5% CO ₂ in air. For evaluation of primary humoral immune response to SRBC, 10 ⁷ murine spleen cells or 4-5 × 10 ⁶ murine spleen B
Cells were cultured in 1.0 ml of medium containing 5% FCS for 4 days in the presence or absence of the indicated antigen. Cells were cultured in a culture tray as described above at 37 ° C. in a humidified atmosphere of 10% CO ₂ in air using a tissue culture locked at a frequency of 7 cycles / min.

Measurement of Cellular Antibody Production The direct plaque forming cell (PFC) response to SRBC was assessed after 4 days of culture using a modified hemolytic plaque assay of Jerne and Nordin, supra.

The above is for describing the present invention and is not intended to limit the present invention. Numerous changes and modifications can be made without departing from the true spirit and novel concept of the invention.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Weigle William O. 92014 Delhi, California, USA 9750 Del Mar Lutete La Parc 13750 Apartment Sea (56) Reference Proc. Natl. Acad. Sc i. U. S. A. Vol. 78, No. 12, p. 7604-08 (1981) Journal of Immunology, Vol. 128, No. 6, p. 2399-2404 (1982)

Claims (2)

(57) [Claims] (1) 8-oxoguanosine, 7-methyl-8-
Oxoguanosine, 8-methoxyguanosine, 8-mercaptoguanosine, 8-mercapto-7-methylguanosine and 8-bromoguanosine, an effective amount of an 8-substituted guanine derivative selected from the group consisting of a physiologically acceptable carrier substance. An immunomodulator suitable for the subcellular promotion of an antigen-specific humoral immune response of T cell-independent B lymphocytes, together with a dilution of 2. (a) 8-oxoguanosine, 7-methyl-8-oxoguanosine, 8-methoxyguanosine, 8
An effective amount of an 8-substituted guanine derivative selected from the group consisting of mercaptoguanosine, 8-mercapto-7-methylguanosine and 8-bromoguanosine; and (b) an effective amount of interferon, a physiologically acceptable carrier substance. An immunomodulator suitable for subcutaneous promotion of antigen-specific humoral immune response of T cell-independent B lymphocytes, together with a dilution.