JP2010184939A - 5'-nucleotidase-resistant derivative of immunostimulating inosine-monophosphoric acid and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inosine-5'-monophosphoric acid derivative which has larger immunostimulating capability, is more stable, and is resistant to 5'-nucleotidase. <P>SOLUTION: A method for producing inosine-5'-monophosphoric acid, resistant to 5'-nucleosidase, and derivatives thereof is provided. By the method, inosine-5'-monophosphoric acid is chemically denatured to be a compound having formula (I) (wherein R is selected from the group consisting of an alkyl group, an alkoxy group, and secondary amino compounds), and the biological activity of inosine-5'-monophosphoric acid is retained in a living body. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates generally to methods of enhancing immune responsiveness by increasing the effectiveness of an immunostimulant, ie, inosine-5'-monophosphate, and the use of the enhancer. More particularly, the present invention relates to the treatment of patients affected by tumors, viruses and intracellular bacterial pathogens and vaccine adjuvants.

(Background of the Invention)
Secondary immunodeficiency is common in cancer, aging, autoimmunity, AIDS, and other viral and bacterial diseases. It has long been thought that the treatment of these secondary immunodeficiencies will lead to an improved prognosis in these diseases. Despite many experimental efforts, only a few levamisole and isoprinosine are only widely approved and clinically used in such treatments. There is a need for more effective drugs of this kind.

  Immune function includes the humoral and cellular parts of the immune system, and these aspects depend on macrophages and granulocytes. These various aspects of immune function can be enhanced or improved by various agents, commonly referred to as immunostimulants. Immunostimulants, including drugs and biological materials, are widely used in the prevention and treatment of human diseases.

  However, recent studies (Sad and Mosmann, 1994; Sieling et al., 1994; Chakkalath and Titus, 1994; Tripp et al., 1994; Bogdan et al., (1991; Fiorentino et al., 1989) suggest that inappropriate stimulation of the immune response may actually facilitate the process of the disease. In both mouse and human models, it has been shown that the cytokine profile of the immune response is modulated, thereby activating one of the T helper (Th) cell subclasses, Th1 or Th2, in response to the pathogen . Th1 cells generally have a pattern of IL-2, IFN-γ and lymphotoxin secretion, while the general secretion pattern of Th2 is IL-4, IL-5, IL-6, IL-9, IL- 10 and IL-13. The Th1 cell cytokine profile is generally associated with resistance to disease, and the Th2 cytokine profile is involved in disease progression. In particular, in connection with intracellular pathogens such as Mycobacterium leprae, Listeria monocytogenes and Leishmania major, the Th1 cell-derived cytokine profile is responsible for the growth of the pathogen. It was shown to be necessary to limit A number of factors determine which subset of T-cells are activated during the immune response and that lead to a Th1 response is that of factors, including activated macrophage-derived IL-12 It is also clear that it is a combination. In these diseases it would be useful to have a means to increase or stimulate this Th1 response. For example, a manic patient does not show this type 1 response, rather the lesion expresses the type 2 cytokine, which is typical of the cell-mediated immunity necessary to resist intracellular pathogens. Humoral response and immunosuppression.

  One group of immunostimulants is derived from purine structures such as inosine or hypoxanthine, such as isoprinosine (molar ratio 1: 3 inosine and p-acetamide-benzoate salt of N, N-dimethylamino-2-propanol). That is, synthetic drugs containing DIP salts).

And NPT 15392 (9-erythro-2-hydroxy-3-nonyl-hypoxanthine):

These compounds are classified as “thymomimetic drugs” (Hadden, 1985) because they stimulate the immune system primarily by acting on thymus-derived (T) lymphocytes. However, they also act on other cells involved in the immune response. Isoprinosine is an example of a medically useful thymic mimetic drug that is approved for human use in many countries around the world. The action of isoprinosine in vitro is similar to that of inosine and is activated by complexation with DIP salts (Hadden, 1978). The action of isoprinosine in vivo is not achieved by inosine or DIP salts alone (Wybran et al., 1982), indicating that complexation and protection of inosine bases are critical for in vivo activity. It suggests.

  The search for more immunologically active molecules of this type for clinical use has accumulated enormous literature.

  U.S. Patent No. 6,099,037 to Godon discloses a complex formed by inosine and an amino-alcohol, which complex has pharmacological activity in prevention against influenza and herpes virus.

  U.S. Patent No. 6,057,031 to Hadden et al. Discloses complexes of purine derivatives (9- (hydroxyalkyl) purines) with amino-alcohol salts of p-acetamidobenzoic acid, which are immune Has regulatory and anti-viral activity.

  U.S. Patent No. 5,057,049 to Hadden et al. Discloses p-acetamide benzoate of 9- (hydroxyalkyl) purines useful as anti-viral factors, immunomodulators and anti-leukemia agents. ing.

U.S. Patent No. 6,057,049 to Giner-Sorolla et al. Discloses esters (purine compounds) having immunomodulatory, anti-viral, anti-tumor and enzyme inhibitor activity.

U.S. Patent No. 6,057,096 to Giner-Sorolla et al. Discloses 9- (hydroxyalkyl) purines useful as immunostimulants, anti-viral factors and anti-leukemia drugs.

U.S. Patent No. 6,057,096 to Giner-Sorolla et al. Discloses purine derivatives having immunomodulatory, anti-viral and anti-tumor activities.

Patent Document 7 and Patent Document 8 granted to Giner-Sorolla et al. Disclose dihydrothiazolopurine derivatives having immunomodulating activity.

  Patent document 9 discloses (heptamine-1-ol) -5'-adenosine monophosphate. Saha et al. (1988) are present in the early stages of splenocyte cultures in stimulating primary humoral immune responses in vitro against T cell-dependent antigens (sheep erythrocytes). The activity is disclosed. Saha et al. (1987) describe the activity of HAA in increasing anti-SRBC PFC activity and antibody titer in ICR male mice. They also disclose the activity of HAA in increasing anti-SRBC PFC activity and antibody titers in rats with primary hypertension.

  Non-Patent Document 1 describes that purines, particularly inosine-containing or inosine-like compounds, experimentally induce precursor T-cell division and mature T-cells, generally in response to infection with intracellular pathogens. In particular, it has been shown to have the ability to mimic the action of thymic hormones, which stimulates the functional response of Th1 cells. An example of these molecules was assumed to contain inosine-5'-monophosphate (IMP) in the most closely examined structure (Non-Patent Document 2).

  It would be beneficial to be able to utilize these immunomodulatory / immunostimulatory compounds for use in the discussion herein below.

  Viral infections and intracellular bacterial pathogens are generally major public health concerns. In both these categories, the bacterial and viral pathogens bypass the host immune system. This is because they are growing in the host cell. The cell-mediated immune (CMI) response by Th1 cells initiated by macrophages is a common mode of host defense or resistance against intracellular pathogens once infection has occurred. An effective antibody response in response to immunization may confer immunity.

  Treatment of bacterial-related diseases with antibiotics is available, but with increasing bacterial drug resistance, it is necessary to consider other methods of treatment. One treatment for infected individuals is to increase the effectiveness of the patient's immune system. In these diseases, sensitized T lymphocytes and activated macrophages are key factors in immunity. Therefore, effective treatments for these diseases should activate macrophages and sensitize appropriate T lymphocytes (Ryan in Stites and Terr, pp. 637- 645 and Mills in Stites and Terr, pp. 646-656).

Intracellular bacterial pathogens include Salmonella, Legionella, Listeria, Mycobacteria and Brucella. Salmonella species are members of the intestinal bacteria and cause the majority of enteric diseases including typhoid fever. S. typhi capsules have a surface capsule antigen and antibodies against this capsule antigen are not protective. In fact, many carriers of typhoid-like diseases have high levels of antibodies against this pathogen.

  Legionella studies indicate that this is an absolute intracellular parasite of macrophages. Listeria is a gram-positive bacterium that causes meningeal infection and sepsis in adults and various infections in newborns. The main role of defense against these pathogens has been shown to be activation of T-lymphocyte-associated macrophages. Research on Brucella-related diseases also revealed that activated macrophages do not protect against antibodies and activated macrophages produced by specifically sensitized T-lymphocytes.

  It would be useful to obtain a drug that is effective against these diseases, activates macrophages and sensitizes appropriate T lymphocytes to the pathogen.

In general, since there are few or no effective anti-viral drugs, protection from viral pathogens remains a major public health objective. Immunization is left as the best source of protection in viral diseases. However, vaccines often do not confer immunity for the following reasons. (1) poor immunogenic viral antigens, (2) less time between immunization and exposure to the virus, or (3) inability of the immunized individual to respond.

  For example, influenza vaccines need to be updated regularly in order for the virus to make changes in antigenicity. The most common vaccines are often not available or are not fully produced before the start of the winter months, which are the peak months of the epidemic. Therefore, those administered with the vaccine may be exposed to the virus before obtaining antibody titers. Furthermore, in patients at higher risk of infection, i.e. older and younger, vaccination compliance rates are low until the epidemic begins. In addition, influenza severely afflicts older and younger people in particular, and also severely afflicts individuals with diseases in the heart-respiratory system. These particular groups often have a low immune response to this vaccine.

  Influenza virus infection suppresses normal lung anti-bacterial protection, and patients with influenza are at a very high risk of developing bacterial pneumonia. It has been found that alveolar macrophages or neutrophils are inhibited during influenza virus infection.

  Thus, means to enhance immune responsiveness to influenza virus infection or resistance to bacterial pneumonia would be useful in treating this disease. One possible way to increase the effectiveness of this treatment is to treat with a substance that has immunostimulatory properties (Hadden et al., 1976; Hadden, 1987).

  Chronic hepatitis B infection is one of the major public health concerns. This hepatitis B virus is responsible for acute and chronic hepatitis and liver cancer. Although this acute disease has a limited progression, the chronic infection persists throughout the host's life. Chronic carriers maintain infectivity throughout their lives. It is estimated that there are over 100 million carriers worldwide.

There is no general highly effective treatment for this disease. Prevention by immunization remains the only solution. Immunization against these high risk factors is decisive (James et al., 1991; Mills, 1991). However, when immunized with the hepatitis B virus surface antigen, almost 2-15% of the vaccinated do not produce antibodies against the hepatitis B virus surface antigens (anti-HBs) and are therefore against this infection. It is known that it will not be protected. In other words, these patients are non-responsive patients (Deinhardt, 1983). To induce strong immunity against hepatitis B,
Various schemes for multiple administration of the antigen formulation have been recommended (Grossman and Cohen, 1991). However, there is still a group of non-responsive patients. Therefore, another means of increasing the immunogenicity of vaccine formulations against hepatitis B is needed to solve this critical public health requirement.

  People with a high risk of infection include patients on continuous hemodialysis treatment (Ferguson, 1990; Walz et al., 1989), HIV-infected and other immunotolerized patients (Hess et al., 1989) most frequently found. People who have come into contact with these groups and those who have never been in contact with the hepatitis B virus have the greatest risk of infection and require immediate and maximum protection from infection. This is particularly critical for health care providers (Grossman and Cohen, 1991). Therefore, patients in these groups who are non-responsive to a commonly available vaccine against hepatitis B are at an even higher risk of infection.

  It would be useful to be able to increase the effectiveness of immunization in this non-responsive patient group. One possible way to increase the effectiveness of prevention is to enhance the immunogenicity of available vaccines by using biologically active substances with adjuvant properties.

It is well known that adjuvants stimulate antibody formation in response to xenoantigens. Adjuvants are defined as compounds that can stimulate an immune response and are therefore defined as a group of immunostimulators (Stites and Terr, 1991). Adjuvants are used to increase immune responsiveness in immunization (Seaman, 1991). For example, in vaccine formulations against hepatitis B, anti-HBs anti-antigenic titer (> 10 IU / l), where HBsAg is generally absorbed onto aluminum hydroxide, thus enhancing the immunogenic effect and preventing infection Has achieved.

However, as mentioned above, there are non-responsive patients who do not respond even to this enhanced vaccine (Celis et al., 1987; Meuer et al., 1989). This lack of protective immunity due to immunization is thought to be due in part to components with immune responsiveness measured at the level of major histocompatibility complex (MHC) (Alper et al., 1989 Walker et al., 1981). “Non-responsive patients” lack the dominant gene of the immune response in the MHC, and as a result, anti-HBs synthesis is at most hardly detectable by commonly applied detection methods. It has been suggested that this occurs at low concentrations (Thomson et al., 1977). Similarly, immune response genes associated with the mouse major histocompatibility complex (H-2) control cellular and humoral responses to determinants on numerous T-cell-dependent antigens, including HBsAg It has been made clear.

The results obtained with the application of the combination of hepatitis B vaccine and various immunomodulators suggest the convenience of this method. This is because treatment with this combination does not respond completely or can reduce the number of people responding with low concentrations of HBs (Celis et al., 1987; Meuer et al., 1989). ). However, there are still people who are encouraged by these data but do not respond to these treatments. For them, it is useful to use an adjuvant-enhanced hepatitis B vaccine, which promotes rapid induction at the maximum concentration of a particular antibody and provides protection in the non-responsive patient population Will enhance the sex.

  Hadden et al. (1983) have studied that purines, particularly inosine-containing or inosine-like compounds, generally induce precursor T-cell division and activate the functional response of mature T-cells, It was revealed that it has the ability to mimic thymic hormone action.

  Isopurinosine has shown some efficacy in the treatment of lethal influenza infection in mice and inhibited lethality in subsequent infection with the virus when a non-infectious dose of virus was administered. Isoprinosine has been approved for use in humans in the treatment of influenza in several countries based on its clinical activity in humans (Glasky, 1985). However, the half-life of isoprinosine in humans is less than 4 hours and is therefore not very effective for in vivo treatment. Isopurinosine is hydrolyzed rapidly resulting in a short half-life in vivo. Inosine in vitro (Hadden, 1978) has properties similar to isoprinosine, but undergoes catabolism rapidly in vivo, and its half-life is further shortened than that of isoprinosine, thus Has no activity (Wybran et al., 1982). It would be useful to obtain a more stable inosine-like compound with greater immunostimulatory capacity.

U.S. Pat.No. 3,728,450 U.S. Pat.No. 4,221,794 U.S. Pat.No. 4,221,909 U.S. Pat.No. 4,340,726 U.S. Pat.No. 4,221,910 U.S. Pat.No. 4,457,919 U.S. Pat.No. 4,510,144 U.S. Pat.No. 4,387,226 JP 58-140100 A

Hadden et al., "Purine Analogs as Immunomodulators", Progress in Immunology IV, (Academic Press, NY), 1983, p. 1393-1408 Willson and Fudenberg, "Controversy About Transfer Factor Therapy Near End?", Immunology Today, 1983, 4: p. 157

(Summary of Invention)
According to the present invention, there is provided a process for the preparation of inosine-5'-phosphate derivatives that are resistant to 5'-nucleotidase, the process comprising the following formula:

And chemically modifying inosine-5′-monophosphate represented by the formula:
The substituent R is selected from the group consisting of alkyl groups, alkoxy groups and groups derived from secondary amino compounds, so that the biological activity of inosine-5′-phosphate is retained in vivo.

The present invention further provides an immunostimulatory composition, the composition being in an amount effective for immunostimulation,
Inosine-5'-monophosphate compound represented by the following formula, resistant to '-nucleotidase and a pharmaceutically acceptable carrier:

Here, R is a moiety that inhibits catabolism of the compound by 5'-nucleotidase, and is selected from the group consisting of alkyl, alkoxy, and secondary amino compounds. In one embodiment, the immunostimulatory effect leads to a T-cell immunostimulatory composition, and in a more specific embodiment leads to T helper cell subset 1 (Th1) cells.

  The present invention also provides an adjuvant for vaccines represented by the following formula:

Here, R is a moiety that inhibits hydrolysis of the compound by 5'-nucleotidase, and is selected from the group consisting of alkyl, alkoxy, and secondary amino compounds.

  In a preferred embodiment, the present invention relates to a vaccine for hepatitis B, which contains a purified viral antigen (preferably recombinant) and an adjuvant for hepatitis B represented by the following formula:

Here, R is a moiety that inhibits hydrolysis of the compound by 5'-nucleotidase, and is selected from the group consisting of alkyl, alkoxy, and secondary amino compounds.

The present invention also provides an immunostimulant 5′-nucleotidase resistant inosine-5′-monophosphate derivative against intracellular bacterial pathogens and viruses, represented by the following formula:

Here, R is an alkyl group having 1 to 6 carbon atoms, or an alkoxy group represented by the formula: —OR ¹ (where R ¹ is an alkyl group having about 1 to 6 carbon atoms). Selected from.

  The present invention further provides a method for treating viruses and intracellular bacterial pathogens in mammals, the method comprising a patient with an infectious disease caused by a pathogen selected from the group consisting of intracellular bacteria and viral pathogens The process of diagnosing is included. Then, according to this method, the identified patient is administered an effective amount of an immunostimulatory 5'-nucleotidase resistant inosine-5'-monophosphate derivative represented by the following formula:

Here, R is an alkyl group having 1 to 6 carbon atoms, or an alkoxy group represented by the formula: —OR ¹ (where R ¹ is an alkyl group having about 1 to 6 carbon atoms). Selected from. In a preferred embodiment, an effective amount of squalane can also be administered.

The present invention further provides the following items:
(Item 1) A method for producing inosine-5'-monophosphate that is resistant to 5'-nucleosidase, wherein inosine-5'-monophosphate is chemically modified to a compound having the following formula: With
A method characterized in that the biological activity of inosine-5′-monophosphate is retained in vivo.

Wherein R is selected from the group consisting of alkyl, alkoxy and secondary amino compounds.
(Item 2) The method according to item 1, wherein the chemical modification step includes condensation with one of an alcohol, an ether and a secondary amino compound.
(Item 3) The method according to Item 1, wherein dicyclohexylcarbodiimide is used as a condensing agent.
(Item 4) The method according to item 2, wherein the alcohol is selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol and heptanol.
(Item 5) The method according to item 2, wherein the ether is selected from the group consisting of methyl ether, ethyl ether, propyl ether, butyl ether, pentyl ether and heptyl ether.
(Item 6) The method according to Item 2, wherein the secondary amino compound is arginine.
(Item 7) The method according to item 2, wherein the secondary amino compound is a peptide linked to a phosphorus atom through its N-terminus.
(Item 8) The method according to item 2, wherein the peptide is selected from the group consisting of ARG-PRO, ARG-PRO-LYS, and ARG-PRO-LYS-THR.
(Item 9) A method for producing inosine-5'-monophosphate resistant to 5'-nucleosidase, comprising the following steps:
(1) a step of protecting nucleotides of 2 ', 3'-isopropylidene inosine;
(2) reacting protected 2 ', 3'-isopropylidene inosine with methylphosphonic acid dichloride;
(3) deprotecting the nucleotide to produce methyl-5′-inosine monophosphate;
A method comprising the steps of:
(Item 10) An immunostimulatory compound represented by the following formula:

Wherein R is a moiety that inhibits hydrolysis of the compound by 5′-nucleosidase;
Selected from the group consisting of alkyl, alkoxy and secondary amino compounds. )
(Item 11) The compound according to Item 10, wherein the alkyl has about 1 to 6 carbon atoms.
(Item 12) The compound according to item 11, wherein the alkyl is methyl.
(Item 13) The compound according to item 10, wherein the alkoxy has the formula: -OR ', and R' is alkyl having about 1 to 6 carbon atoms.
(Item 14) The compound according to item 13, wherein R ′ is methyl.
(Item 15) The compound according to item 13, wherein R ′ is ethyl (item 16), wherein the secondary amino compound is represented by the following formula:

Where R ² is alkyl having a total number of carbons up to about 16
The compound of item 10 shown by.
(Item 17) R ² represents the following formula:

Wherein R ³ is selected from the group consisting of hydrogen, lower C1-C9 alkyl and carboxyl, and R ⁴ is hydroxy, amino, carboxy, secondary alkyl, alkyl-substituted hydroxymethyl, —NHC (NH) NH ₂ , -CONH ₂ ,

N is an integer of 0-4. The compound of item 16 shown by).
(Item 18) The second alkyl group is represented by the formula: —CH (R ⁵ ) R ⁶ , and R ⁵ and R ⁶ may be the same or different, and are independently alkyl having 1 to 3 carbon atoms. 18. The compound according to item 17, selected from
(Item 19) The alkyl-substituted hydroxymethyl is represented by —C (R ⁷ ) (R ⁸ ) OH, and R ⁷ and R ⁸ may be the same or different, and independently represent hydrogen and carbon atoms 1 to 3 18. A compound according to item 17, selected from alkyls, at least one of which is other than hydrogen.
(Item 20) The compound according to item 10, wherein the secondary amino compound is a peptide linked to a phosphorus atom through its N-terminus.
(Item 21) The compound according to item 20, wherein the peptide is selected from the group consisting of ARG-PRO, ARG-PRO-LYS, and ARG-PRO-LYS-THR.
(Item 22) An immunopotentiating composition comprising an immunopotentiating effective amount of a 5'-nucleosidase-resistant inosine-5'-monophosphate compound represented by the following formula and a pharmaceutically acceptable carrier.

Wherein R is a moiety that inhibits hydrolysis of the compound by 5′-nucleosidase;
Selected from the group consisting of alkyl, alkoxy and secondary amino compounds. )
(Item 23) The composition according to item 22, wherein the alkyl has about 1 to 6 carbon atoms.
(Item 24) The composition according to item 23, wherein the alkyl is methyl.
(Item 25) The composition according to item 22, wherein the alkoxy has the formula: -OR ', and R' is alkyl having about 1 to 6 carbon atoms.
(Item 26) The composition according to item 25, wherein R ′ is methyl.
(Item 27) The composition according to item 25, wherein R ′ is ethyl.
(Item 28) The secondary amino compound has the following formula:

Where R ² is alkyl having a total number of carbons up to about 16
Item 23. The composition according to Item 22,
(Item 29) R ² is represented by the following formula:

Wherein R ³ is selected from the group consisting of hydrogen, lower C1-C9 alkyl and carboxyl, and R ⁴ is hydroxy, amino, carboxy, secondary alkyl, alkyl-substituted hydroxymethyl, —NHC (NH) NH ₂ , -CONH ₂ ,

N is an integer of 0-4. 29. The composition according to item 28, wherein
(Item 30) The second alkyl group is represented by the formula: —CH (R ⁵ ) R ⁶ , and R ⁵ and R ⁶ may be the same or different, and are independently alkyl having 1 to 3 carbon atoms. 30. The composition according to item 29 selected from
(Item 31) The alkyl-substituted hydroxymethyl is represented by —C (R ⁷ ) (R ⁸ ) OH, and R ⁷ and R ⁸ may be the same or different and independently represent hydrogen and 1 to 3 carbon atoms. 30. The composition according to item 29, wherein the composition is selected from alkyl and at least one is other than hydrogen.
(Item 32) The composition according to item 22, wherein the secondary amino compound is a peptide linked to a phosphorus atom through its N-terminus.
(Item 33) The composition according to item 32, wherein the peptide is selected from the group consisting of ARG-PRO, ARG-PRO-LYS, and ARG-PRO-LYS-THR.
(Item 34) A T cell immunity enhancing composition comprising an immunopotentiating effective amount of a 5'-nucleosidase-resistant inosine-5'-monophosphate compound represented by the following formula and a pharmaceutically acceptable carrier.

Wherein R is a moiety that inhibits hydrolysis of the compound by 5′-nucleosidase;
Selected from the group consisting of alkyl, alkoxy and secondary amino compounds. )
(Item 35) The composition according to item 34, wherein the T cell is a Th1 cell.
(Item 36) The composition according to item 34, wherein the T cell is derived from an HIV-infected patient.
(Item 37) An adjuvant for a vaccine having the formula:

Wherein R is a moiety that inhibits hydrolysis of the compound by 5′-nucleosidase;
Selected from the group consisting of alkyl, alkoxy and secondary amino compounds. )
38. The adjuvant of claim 37, wherein the alkoxy has the formula: —OR ¹ and R ¹ is alkyl having about 1 to 6 carbon atoms.
(Item 39) The adjuvant according to item 38, wherein R ¹ is selected from the group consisting of methyl and ethyl.
(Item 40) The adjuvant according to item 37, wherein the vaccine is for hepatitis B.
(Item 41) A vaccine against hepatitis B, comprising a purified hepatitis B antigen and an adjuvant represented by the following formula:

Wherein R is a moiety that inhibits hydrolysis of the compound by 5′-nucleosidase;
Selected from the group consisting of alkyl, alkoxy and secondary amino compounds. )
(Item 42) The vaccine according to item 41, wherein the alkoxy has the formula: -OR ¹ and R ¹ is alkyl having about 1 to 6 carbon atoms.
(Item 43) The vaccine according to item 41, wherein R has the formula: -OR ¹ , and R ¹ is selected from the group consisting of methyl and ethyl.
(Item 44) A 5′-nucleosidase-resistant inosine-5′-monophosphate used as an immune system stimulant against intracellular bacterial pathogens and viruses and represented by the following formula.

(Wherein R is
(A) an alkyl group having 1 to 6 carbon atoms, and (b) an alkoxy group having the formula: —OR ¹ , wherein R ¹ is alkyl having about 1 to 6 carbon atoms,
Selected from the group consisting of )
(Item 45) The immune system stimulant according to item 44, wherein R has the group -OR ¹ and R ¹ is methyl.
(Item 46) The immune system stimulant according to item 44, wherein R is methyl.
(Item 47) A method for enhancing the immune response of a mammal in need of immunostimulation treatment, wherein the immunostimulatory effect of 5'-nucleosidase-resistant inosine-5'-monophosphate represented by the following formula: Administering an amount to said mammal.

Wherein R is a moiety that inhibits hydrolysis of the compound by 5′-nucleosidase;
Selected from the group consisting of alkyl, alkoxy and secondary amino compounds. )
48. The method of claim 47, wherein the mammal is infected with HIV.
(Item 49) The method according to Item 47, wherein the mammal has a cancerous tumor.
(Item 50) The method according to item 47, wherein the effective amount of the 5′-nucleosidase-resistant inosine-5′-monophosphate compound is 1 to 50 mg / kg body weight at least daily.
51. A method for treating viral and intracellular bacterial pathogens in a mammal, comprising the following steps:
(1) diagnosing a patient suffering from an infectious disease caused by a pathogen selected from the group consisting of intracellular bacterial and viral pathogens;
(2) a step of administering an effective amount of an immunostimulant 5′-nucleosidase resistant inosine-5′-monophosphate represented by the following formula:
A process characterized by containing.

(Wherein R is
(A) an alkyl group having 1 to 6 carbon atoms, and (b) an alkoxy group having the formula: —OR ¹ , wherein R ¹ is alkyl having about 1 to 6 carbon atoms,
Selected from the group consisting of )
52. The method of claim 51, wherein R has a group —OR ¹ and R ¹ is methyl.
(Item 53) The method according to Item 51, wherein R is methyl.
(Item 54) The method according to item 51, wherein the dose is 1 to 50 mg / kg body weight at least daily. 55. A method for treating viral and intracellular bacterial pathogens in a mammal, comprising the following steps:
(1) diagnosing a patient suffering from an infectious disease caused by a pathogen selected from the group consisting of intracellular bacterial and viral pathogens, and (2) an immunostimulant 5′− represented by the following formula: Administering an effective amount of a nucleosidase-resistant inosine-5′-monophosphate; and

(Wherein R is
(A) an alkyl group having 1 to 6 carbon atoms, and (b) an alkoxy group having the formula: —OR ¹ , wherein R ¹ is alkyl having about 1 to 6 carbon atoms,
Selected from the group consisting of )
(3) administering an effective amount of squalane;
A process characterized by containing.
56. The method of claim 55, wherein R has a group —OR ¹ and R ¹ is methyl.
(Item 57) The method according to item 55, wherein R is methyl.
58. The method of claim 55, wherein the infectious disease being treated is influenza.
59. The 5′-nucleosidase resistant inosine-5′-monophosphate dosage is 1 to 50 mg / kg body weight at least daily and the squalane dosage is at least one day. 56. A method according to item 55, which is 1 to 5 ml.
60. A method for determining patients that would benefit from treatment with 5′-nucleosidase resistant inosine-5′-monophosphate, comprising the following steps:
(1) a step of isolating peripheral blood lymphocytes;
(2) performing an in vitro lymphocyte stimulation assay in the presence of a mitogenic factor and 5′-nucleosidase resistant inosine-5′-monophosphate;
(3) Patients who have a reduced in vitro response to 5′-nucleosidase resistant inosine-5′-monophosphate are not candidates for treatment with 5′-nucleosidase resistant inosine-5′-monophosphate. Identifying the process,
A method characterized by comprising:
(Item 61) The method according to item 60, wherein the mitogenic factor is phytohemagglutinin.
(Item 62) The 5'-nucleosidase-resistant inosine-5'-monophosphate is methyl-5'-inosine monophosphate (methyl-IMP or Me-IMP), methyl-5'-inosine phosphonate (Mp -IMP), ethyl-5'-inosine monophosphate (Ethyl-IMP or E-IMP), arginine-5'-inosine monophosphate (Arginin-IMP or Arg-IMP) and (heptanmin-1-ol)- 61. The method according to item 60, which is selected from the group consisting of 5′-inosine monophosphate (Ha-IMP).
(Item 63) A method for treating a patient having a tumor, comprising the following steps:
(1) a step of administering an effective amount of an immunostimulant 5′-nucleosidase resistant inosine-5′-monophosphate represented by the following formula:

(Wherein R is
From the group consisting of (a) an alkyl group having 1 to 6 carbon atoms, and (b) an alkoxy group having the formula: -OR ¹ and R ¹ is an alkyl having about 1 to 6 carbon atoms. Selected. And (2) administering an effective amount of endotoxin.
(Item 64) The method according to item 63, wherein the endotoxin is selected from the group consisting of a lipopolysaccharide and a Salmonella vaccine.

Other advantages of the present invention will be readily understood and the invention will be more fully understood by reference to the following detailed description, taken in conjunction with the accompanying drawings, in which:
FIG. 1 shows the response of human peripheral blood lymphocytes to the lymphocyte mitogen plant agglutinin as a percentage of the control response (ie proliferation of human peripheral blood lymphocytes) in the culture medium. Existing Mp-IMP (--- ▲ ---), Me-IMP (--- △ ---), IMP (-◇-), E-IMP (-◆-), Arg- It is the graph plotted with respect to the dosage level of IMP (-□-) and Ha-IMP (--- ● ---). FIG. 2 shows normal human lymph against 1-1000 μg / ml MMP (--- ○ ---) and IMP (-● ---) in the presence of 0.5 μg / ml PHA. A graph plotting the dose response curve of the cells, the results being expressed as a ratio to a control consisting of two donors. Fig. 3 shows the dose response curves of control human lymphocytes rich in CD4 ⁺ (--- ○ ---) and CD8 ⁺ (--- ● ---) derived from three donors and the presence of PHA. FIG. 4 is a graph plotted against MIMP (1-200 μg / ml) below. Figure 4 shows 12.5 μM (open bars), 25 μM (shaded bars), 50 μM (black bars) in the presence of various concentrations of MIMP (0.1-100 μg / ml). And a bar graph showing the dose response of control human lymphocytes to peptide gp41 at 100 μM (shortest bar in stippling), control values without peptide are shown as horizontal --lines, and results are tested Representative CPM ± SEM of 5 donors represented. FIG. 5 shows the recombinant interferon alpha in the presence of MMP 1-100 μg / ml, 10 (shaded shaded bars), 100 (shaded bars), and 1000 (solid lines) units / ml. A bar graph of the dose response of control human lymphocytes to inhibitory effects, the control (open bars) is the response to PHA only, and the results are shown in CPM ± SEM. FIG. 6 reverses this action of PGE ₂ (10 ⁻⁵ M), which inhibits the proliferative response of control human lymphocytes (solid bars), and MMP at concentrations of 1, 10 and 100 μg / ml ( (Aya shaded bars) action graph, control (open bars) is PHA response in the absence of MIMP, data is shown in CPM ± SEM. FIG. 7 shows control (c), 15 normal elderly individuals (Ag ⁺ ), 9 elderly individuals with suppressed PHA response (Ag−), 8 ARC patients and 8 AIDS patients. It is a graph which shows the effect | action of the concentration of 100 microgram / ml of MIMP (black bar) on the PHA response (open bar). FIG. 8 shows the response of mouse spleen lymphocytes to the lymphocyte mitogen concanavalin A as a percentage of the control response (ie, proliferation of mouse spleen lymphocytes) versus IMP (- □-), Me-IMP (--- △ ---), E-IMP (-● ---), Arg-IMP (-◆-), Ha-IMP (--- ◇- It is the graph plotted with respect to the dosage level of-) and Mp-IMP (--- ▲ ---). FIG. 9 shows the number of spleen plaque antibody-forming cells (PFC) formed when splenocytes harvested from mice immunized against sheep erythrocytes (SRBC) are stimulated with SRBC (mean ± SEM). The mice were immunized in combination with intraperitoneal administration of Ha-IMP, Arg-IMP and Me-IMP. Figures 10A-C are dose response bar graphs plotting the number of splenic PFCs (mean ± SEM) formed when spleen cells harvested from mice immunized against SRBC were stimulated with SRBC Where the immunization of the mice was performed at various doses of (A) heptamine-1-ol) -5′-inosine monophosphate, (B) arginine-5′-inosine monophosphate and (C This was performed in combination with intraperitoneal administration of methyl-5′-inosine monophosphate. Figures 10A-C are dose response bar graphs plotting the number of splenic PFCs (mean ± SEM) formed when spleen cells harvested from mice immunized against SRBC were stimulated with SRBC Where the immunization of the mice was performed at various doses of (A) heptamine-1-ol) -5′-inosine monophosphate, (B) arginine-5′-inosine monophosphate and (C This was performed in combination with intraperitoneal administration of methyl-5′-inosine monophosphate. FIG. 11 is a dose response bar graph plotting the number of splenic PFCs (mean ± SEM) formed when spleen cells harvested from mice immunized against SRBC are stimulated with SRBC. Here, immunization of the mice was performed in combination with oral administration of various doses of methyl-5′-inosine monophosphate. FIG. 12 shows the response of mouse spleen lymphocytes as a percentage of the control response for the lymphocyte mitogen plant agglutinin (--- ■ ---) and concanavalin A (--- ● ---) (ie (Proliferation of mouse spleen lymphocytes) is plotted against the dose level of methyl-5′-inosine monophosphate. FIG. 13 shows delayed hypersensitivity responses upon stimulation (i.e., increase in mean meat thickness ± SEM) versus dose level of methyl-5′-inosin monophosphate administered at the time of immunization. FIG. 2 is a dose response graph plotted. FIG. FIG. 14 shows the delayed hypersensitivity response (i.e., mean meat) in stimulation with control or administration of methyl-5′-inosine monophosphate upon immunization by administration of control and Me-IMP upon stimulation. It is a bar graph comparing the increase in the thickness (± SEM). FIG. 15 is a line graph comparing the survival when mice infected with Friend leukemia virus (FLV) were treated with control (−) and Me-IMP (−−−). FIG. 16 shows aerosol influenza infection and control (administered PBS 1 day ago,-●-), MMP (administer 100 μg 1 day ago, --Δ--), MMP (100 μg 1 hour ago) ,-○-), MIMP (100 μg administered 1 hour later,-◆-), MIMP (200 μg administered 1 day before,-□-), MMP (200 μg administered 1 hour) ,-◇-), and MMP (200 μg administered for 1 hour,-▽-) is a line graph comparing the survival rate (%) after treatment. FIG. 17 shows aerosol influenza infection and control (administered PBS 1 day before,-●-), control (administer PBS 1 hour later,-○-), MMP (200 μg Administration,-▽-), MIMP (200 μg administered after 1 hour,-△-), MIMP (200 μg + squalane administered 1 day before,-◇-), MMP (200 μg plus squalane after 1 hour) It is a line graph comparing the survival rate (%) after treatment with administration,-◆-).

(Detailed description of preferred embodiments)
It is 5'-nucleotidase resistant (protection-IMP) and is intended to provide a process for the preparation of inosine-5'-monophosphate for use as an immunostimulant. This method chemically modifies inosine-5′-monophosphate or a derivative thereof to be 5′-nucleotidase resistant, but its biological activity in vitro / inosine-5′-monophosphate. Maintains acid profile and extends biological activity for in vivo applications requiring immunostimulation / stimulation. This production method is shown in the following Production Examples 1 to 5. In general, this chemical method involves performing a condensation reaction between inosine-5′-monophosphate or a derivative thereof and an alcohol, ether or secondary amine compound to produce the 5′-nucleotidase active in vivo. Resistant compounds are formed.

The present invention provides a means of stimulating T-cells. In addition, the 5′-nucleotidase resistant inosine-5′-monophosphate derivative functions as an adjuvant within the vaccine to enhance responsiveness to other vaccine components. In a preferred embodiment, the vaccine is a vaccine for hepatitis B.

  In another application of the invention, the protective-IMP can be used to treat tumors, viral infections and intracellular bacterial pathogens. Unexpectedly, a compound has been found in infected individuals that acts to increase immune responsiveness to tumors, pathogen treatment and also functions as an adjuvant in immunization. (Thus, as used herein, the term “treatment” includes treatment and / or prevention).

  By providing a 5'-nucleotidase resistant compound, protection-IMP, the present invention allows the compound to have an in vivo half-life that is effective to enhance the immune response in therapy.

The present invention provides a method for producing inosine-5'-monophosphate that is resistant to 5'-nucleotidase, which comprises inosine-5'-monophosphate represented by the following formula: Provided is a method for producing 5'-nucleotidase resistant inosine-5'-monophosphate by chemical modification as described in:

Here, R is selected from the group consisting of alkyl, alkoxy and secondary amine compounds. The alkyl or alkoxy has 1 to 6 carbon atoms, and in one embodiment is methyl or methyl ester.

The basis of the present invention protects inosine-5′-monophosphate (IMP) from hydrolysis by 5′-nucleotidase, allowing IMP to be used effectively in vivo. In preferred embodiments, the protected IMP is methyl-5'-inosine-monophosphate (methyl-IMP, Me-IMP, MIMP) or methyl-5'-inosine-phosphonate (Mp-IMP). is there. For clarity of discussion, the present invention will be primarily described by one of these two aspects. However, the invention is equally applicable to biologically active IMP protected against any other 5′-nucleotidase.

The alkoxy group in 5′-nucleotidase resistant inosine-5′-monophosphate is of the formula: —OR ^1, where R ¹ is an alkyl group having about 1 to 6 carbon atoms, In embodiments, it is a methyl or ethyl group).

The secondary amine compound in 5'-nucleotidase resistant inosine-5'-monophosphate is represented by the following formula:
-NH-R ²
Here, R ² is a substituted normal alkyl group having about 16 carbon atoms as a whole. This R ² is hydroxyl, amino, carboxy, secondary alkyl, alkyl-substituted hydroxymethyl, —NHC (NH) NH ₂ , —CONH ₂ ,

Selected from the group consisting of Preferably, R ² is represented by the formula: —CH (R ³ ) — (CH ₂ ) _n —R ⁴ where R ³ consists of a hydrogen atom, a lower C ₁ -C ₉ alkyl and a carboxyl group. Selected from the group and in one embodiment R ³ is a methyl group.

R ⁴ is hydroxyl, amino, carboxy, secondary alkyl, alkyl-substituted hydroxymethyl, —NHC (NH) NH ₂ , —CONH ₂ ,

And n represents an integer of 0 to 4, preferably 1 to 3 and most preferably 3.

In embodiments where R ⁴ is a secondary alkyl group, R ⁴ is preferably represented by the formula: —CH (R ⁵ ) R ⁶ , where R ⁵ and R ⁶ are the same or different. Or independently selected from alkyl groups having 1 to 3 carbon atoms. Preferably, the secondary alkyl group has a total of 4 carbon atoms.

In embodiments where R ⁴ is an alkyl-substituted hydroxymethyl group, the group is preferably of the formula: —C (R ⁷ ) (R ⁸ ) OH, where R ⁷ and R ⁸ are the same They may be different and are independently selected from a hydrogen atom and an alkyl group having 1 to 3 carbon atoms, at least one of which is a group other than a hydrogen atom. In one embodiment, both R ⁷ and R ⁸ are methyl groups.

  In the 5'-nucleotidase resistant inosine-5'-monophosphate, the secondary amine compound can be a peptide attached to the phosphorus atom via the N-terminus. In one embodiment, the peptide is selected from the group consisting of ARG-PRO, ARG-PRO-LYS and ARG-PRO-LYS-THR. This formula is one of the preferred embodiments for pharmacologically or immunopharmacologically active peptides in which accidental hydrolysis releases active peptides such as tuftsin (ARG-PRO-LYS-THR). .

Other suitable active peptides are FK565 (heptanoyl-γ-D-glutamyl-L-mesodiaminopineryl-D-alanine), Bestatin ([(2S, 3R) -3-amino-2-hydroxy-4 -Phenylbutyryl] -L-leucine), Imreg (TYR-GLY), Imreg (TYR-GLY-GLY), IL ₁ 163-171 (GLN-GLY-GLU-GLU-SER-ASN-ASP- LYS-ILE), Thymulin (Zn: GLU-ALA-LYS-SER-GLN-GLY-GLY-SER-ASN), Thymopentin (ARG-LYS-ASP-VAL-TYR, ARG-LYS- ASP and ARG-LYS-ASP-VAL), Splenin (ARG-LYS-GLU-VAL-TYR and / or LYS-HIS-GLY).

As described above, the protected derivative of inosine-5′-monophosphate includes a predetermined alcohol or primary amine or peptide and inosine-5′-monophosphate, preferably the presence of a condensing agent such as dicyclohexylcarbodiimide. It can be easily prepared by condensation under. Production Examples 1-5 give examples of such formulations. Suitable alcohols include 1 to 20 carbon atoms such as methyl alcohol, ethyl alcohol, n-propyl alcohol, n-butyl alcohol, n-hexyl alcohol, n-octyl alcohol and n-decyl alcohol.

  Suitable primary amines or peptides are 6-amino-2-methyl-2-heptanol (heptaminol), arginine, aspartic acid, asparagine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, serine, threonine, valine And ARG-PRO, ARG-PRO-LYS and ARG-PRO-LYS-THR and analogs thereof. Suitable methods for producing polyamide-oligonucleotide conjugates are described in the literature of Haralambidis et al. (1990).

The present invention also provides a method of stimulating the immune response of a mammal in need of treatment with an immunogenic stimulant. The method comprises administering to the mammal an immunostimulatory effective amount of a 5′-nucleotidase resistant inosine-5′-monophosphate compound represented by the following formula:

Here, R is a moiety that inhibits hydrolysis of the compound by 5'-nucleotidase, and is selected from the group consisting of alkyl, alkoxy, and secondary amine compounds.

More specifically, the present invention provides a method of stimulating an immune response, wherein the T-cells are 5′-nucleotidase resistant, represented by the following formula in an amount effective for immunostimulation: Stimulated with inosine-5′-monophosphate compound and a pharmaceutically acceptable carrier:

Here, R is a moiety that inhibits hydrolysis of the compound by 5'-nucleotidase, and is selected from the group consisting of alkyl, alkoxy, and secondary amine compounds. In one preferred embodiment, the Th1T-cell subset is preferentially stimulated.

The present invention further provides an immunostimulatory composition, wherein the composition is a 5'-nucleotidase resistant inosine-5'-monophosphate compound represented by the following formula in an amount effective for immunostimulation: And a pharmaceutically acceptable carrier:

Here, R is a moiety that inhibits hydrolysis of the compound by 5'-nucleotidase, and is selected from the group consisting of alkyl, alkoxy, and secondary amine compounds. In one embodiment, this immunostimulation leads to a T-cell immunostimulatory composition, and in a more specific embodiment, leads to T helper cell subset 1 (Th1) cells.

  The present invention also provides an immunostimulatory composition wherein the adjuvant for the vaccine is represented by the following formula:

Here, R is a moiety that inhibits hydrolysis of the compound by 5′-nucleotidase, and is selected from the group consisting of alkyl, alkoxy, and secondary amine compounds. In a preferred embodiment, the alkoxy group is of the formula: —OR ^1, where R ¹ is an alkyl group having about 1 to 6 carbon atoms and more specifically methyl and ethyl groups.

  In one aspect, the invention relates to a vaccine for hepatitis B, which vaccine comprises a purified viral antigen (preferably recombinant) and an adjuvant for hepatitis B represented by the following formula:

Here, R is a moiety that inhibits hydrolysis of the compound by 5′-nucleotidase, and is selected from the group consisting of alkyl, alkoxy, and secondary amine compounds. In a preferred embodiment, the alkoxy group is of the formula: —OR ^1, where R ¹ is an alkyl group having about 1 to 6 carbon atoms and more specifically methyl and ethyl groups.

  Similarly, the present invention relates to inosine-5′-monophosphate, which is resistant to 5′-nucleotidase and functions as an immune system stimulator against intracellular bacterial pathogens and viruses, as shown below: Provides usage of the derivative:

Here, R is an alkyl group having 1 to 6 carbon atoms or an alkoxy group represented by the formula: —OR ¹ (wherein R ¹ is an alkyl group having about 1 to 6 carbon atoms). Selected. In selected embodiments, R and R ¹ are methyl groups.

The present invention further provides a method of treating a viral and intracellular bacterial pathogen in a mammal, the method being affected by an infectious disease caused by a pathogen selected from the group consisting of a virus and an intracellular bacterial pathogen. Diagnosing a patient. In this method, an effective amount of protection-IMP (inosine-5'-monophosphate and its derivatives that are resistant to 5'-nucleotidase) that then functions as an immune system stimulant represented by the following formula: Administering to the identified patient:

Here, R is an alkyl group having 1 to 6 carbon atoms or an alkoxy group represented by the formula: —OR ¹ (wherein R ¹ is an alkyl group having about 1 to 6 carbon atoms). Selected. In selected embodiments, R and R ¹ are methyl groups. In a preferred embodiment, an effective amount of squalane can also be administered, and the amount of squalane to be administered is at least 1-5 ml per day.

  The present invention provides a method for treating a patient suffering from a tumor. The method comprises the steps of administering an effective amount of inosine-5'-monophosphate resistant to 5'-nucleotidase as an immunostimulant as described herein, and an effective amount of endotoxin. For example, comprising administering lipopolysaccharide (LPS), or in a preferred embodiment, administering a Salmonella vaccine administered according to FDA guidelines. In leukemia, an effective amount of inosine-5'-monophosphate resistant to 5'-nucleotidase is used for therapeutic purposes as an immunostimulatory agent, as described herein for FLV leukemia. It should be noted that it can be administered.

  The present invention also provides a method for determining patients who will benefit from treatment with inosine-5'-monophosphate that is resistant to 5'-nucleotidase. This method involves isolating peripheral blood lymphocytes as known in the art and performing an in vitro lymphocyte stimulation assay in the presence of mitogen and protective-IMP. Patients who show a reduced in vitro response to the protective-IMP are not candidates for treatment with this protective-IMP.

  The terms “immunostimulatory agent” and / or “immunostimulatory agent” as used herein refer to an antigen in an individual or test system to which it is administered when administered to an individual or when tested in vitro. And / or a compound that increases the immune response to the compound. Some antigens are weakly immunogenic when administered alone or are harmful to the individual at concentrations that cause an immune response in the individual. The immunostimulatory agent or immunostimulant makes it possible to increase the individual's immune responsiveness to the antigen or compound by making the antigen more immunogenic, or also to enhance the immune system. It is possible to make it more responsive. This immunostimulant / immunostimulatory agent can also affect the immune system such that lower doses of the antigen / compound are required to achieve an immune response in the individual.

Intracellular bacterial pathogens include Salmonella, Legionella, Listeria and Brucella. Salmonella species are members of the intestinal bacteria. Viral pathogens contemplated for treatment in the present invention include but are not limited to influenza, Friend leukemia virus, hepatitis, herpes and HIV.

For the treatment of the diseases discussed above, immunostimulators selected from inosine-5'-monophosphate derivatives that are resistant to 5'-nucleotidase are useful for cancerous tumors, viral infections or intracellular bacterial infections. Will be given after diagnosis of the associated secondary immune deficiency. The immunostimulant will be used in an effective amount and will generally be used at 1-50 mg / kg body weight per day, and in a preferred embodiment 1-10 mg / kg body weight per day. This immunostimulant is administered at the time of the initial diagnosis or determined daily, or according to good medical practice, to the individual patient's clinical condition, site of administration and method, schedule of administration and medical personnel. Will be given in view of other known factors.

  For purposes of the present invention, an “effective amount” is thus determined by considerations known in the field of treatment of secondary immune deficiencies. Here, the treated individual, such as improved survival, faster recovery, amelioration or elimination of symptoms or the development of improvements such as, but not limited to, post-infection complications Suitable antibody titer or high titer for the infectious agent, reduction of tumor mass or suitable for those skilled in the medical arts and well-known in the art. It must be possible to use some other measurement.

In the method of the present invention, the immunostimulatory agent of the present invention, ie a derivative of inosine-5′-monophosphate (
Protection-IMP) can be administered in various ways. The immunostimulant can be administered as the compound itself as a pharmaceutically acceptable salt, or alone or in combination with a pharmaceutically acceptable carrier. These compounds can be administered by the oral, subcutaneous or parenteral routes including intravenous, intraarterial, intramuscular, intraperitoneal, and nasal administration. It is also possible to use grafts of the compound. Patients to be treated are warm-blooded animals and especially mammals including humans.

  It should be noted that human treatment is generally longer than in the case of the mice exemplified here, and the treatment has a length proportional to the length of the disease process and the effectiveness of the drug. . In general, the dose is proportional to body weight, and metabolism and dosage are converted from the animal model illustrated here to human, taking into account such factors as are known in the art.

  The dose may be a single dose or multiple doses over a period of several days in a preferred embodiment.

  When a protected-IMP derivative is administered by the parenteral route, the derivative will generally be formulated into a unit dosage, injectable form (solution, suspension, emulsion). Pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reproduction into sterile injectable solutions or dispersion. The carrier can be a solvent or dispersion medium such as water, ethanol, polyol (eg, glycerol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.

  For example, appropriate fluidity can be maintained by using a coating such as lecithin, by maintaining a predetermined particle size in the case of a dispersion, and by using a surfactant. Non-aqueous vehicles such as squalane, cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, peanut oil, and esters such as isopropyl myristate can also be used as solvent systems for the composition of the compound. . In addition, various additives that enhance the stability, sterility and isotonicity of the composition, such as antibiotic preservatives, antioxidants, chelating agents, and buffers can be added. Inhibition of the action of microorganisms can be ensured by various antibacterial or antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, sodium chloride and the like. Prolonged absorption of injectable dosage forms is made possible through the use of agents that delay absorption, such as aluminum monostearate and gelatin. However, according to the present invention, any vehicle, diluent or additive used must be compatible with the compound.

  A suitable injectable solution can be prepared by formulating the compound used in the practice of the present invention in a predetermined amount of a suitable solvent, if desired, with various other ingredients.

  The protected-IMP derivative pharmacological formulation can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicles, adjuvants, additives and diluents, or in the present invention. The compounds used can be administered parenterally to the patient in the form of sustained release subcutaneous implants or release systems targeted to specific sites, such as polymer matrices, liposomes, and microspheres. Implants suitable for use in the present invention can take the form of pellets that dissolve gradually after implantation, or biocompatible release units well known to those skilled in the art. Such well known dosage forms and units are designed such that the active ingredient is released slowly over a period of days to weeks.

An example of a well-known implant and unit useful in the present invention discloses an implantable microinfusion pump for dispensing drug at a controlled rate, U.S. Pat. U.S. Pat.No. 4,486,194 disclosing a therapeutic device for administration, U.S. Pat.No. 4,447,233 disclosing a drug infusion pump for releasing a drug at a precise infusion rate
U.S. Pat.No. 4,447,224 disclosing a variable flow, implantable infusion device for continuous drug release, U.S. Pat.No. 4,439,196 disclosing an osmotic drug release device having multiple chamber compartments, And U.S. Pat. No. 4,475,196 which discloses an osmotic drug release device. These patents serve as references for the present invention. Many other such implants, release devices, and units are well known to those skilled in the art.

  The protected-IMP derivative pharmacological formulation used in the present invention can be administered orally to the patient. Administration of known methods such as tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like can be used.

  Known techniques that release the protected-IMP and its derivatives orally, intravenously or nasally and maintain biological activity are preferred.

  In one embodiment, the protective-IMP can be first administered intravenously to raise the blood concentration of the protective-IMP to an appropriate level. The level within the patient is then maintained by the oral dosage form. However, other routes of administration including nasal administration are possible depending on the condition of the patient as described above. The amount to be administered of the protection-IMP and its derivatives varies from patient to patient to be treated, ranging from 10 μg / kg body weight per day to 100 mg / kg body weight per day, and preferably 1-10 mg / kg body weight per day. Will vary in range.

Commercially available FDA approved vaccines can be used to prepare the vaccine-adjuvant combinations of the present invention. In addition, the vaccine can be prepared as known per se in the field of vaccine preparation. As an example, a hepatitis vaccine is used. For example, commercially available hepatitis vaccines can be used, or can be prepared using hepatitis surface antigen dosages according to FDA guidelines. The adjuvant is used at a concentration that provides an effective amount, which will generally be in the range of 0.01-100 mg / kg body weight. In a preferred embodiment, the adjuvant will be used at a concentration of 0.01-10 mg / kg body weight (Sosa et al., 1992).

  A vaccine formulation will also be administered and a fixed dose of an inosine-5'-monophosphate derivative, such as Mp-IMP, which is resistant to 5'-nucleotidase will be administered simultaneously.

Furthermore, the adjuvant can be post-administered following the vaccine itself, the simultaneous administration of the vaccine and adjuvant, or the initial administration of the adjuvant-vaccine complex. The adjuvant is used in an effective amount and will generally be at a concentration that gives an amount of 0.01-100 mg / kg body weight. In a preferred embodiment, the adjuvant will be used at a concentration that gives an amount of 0.01-10 mg / kg body weight (Sosa et al., 1992). This adjuvant can be administered within 7 days of the initial administration, daily or as determined in accordance with good medical practice, each patient's clinical status, administration site and method, administration schedule and other known to the medical practitioner. It is administered in consideration of factors.

  For the purposes of the present invention, an “effective amount” is thus determined by consideration as is known in the art of immunization. Here, it is effective in providing measurable anti-viral titers in humans administered the adjuvant and vaccine, and in preferred embodiments in humans that are non-responsive to standard vaccines. There must be.

  The adjuvants and vaccines can be formulated together in unit dose injectable form or alone, as parenteral injection and oral and nasal formulations, including subcutaneous and intramuscular and intraperitoneal administration. Pharmacological formulations suitable for injection include sterile aqueous solutions or dispersions, sterile powders for reproduction into sterile injectable solutions or dispersions. For example, administration of an adjuvant-vaccine combination as a tablet, suspension, solution, emulsion, capsule, powder, syrup or the like can be used. Known techniques that release the formulation of the present invention via oral, nasal or injection while maintaining its biological activity are preferred.

  To determine whether humans that are non-responsive to known vaccines and those immunized according to the present invention are successfully immunized, titers can be determined and viral antigens can be determined. Tests for proliferation in response to can be performed as is well known in the art.

  Isopurinosine has been shown to have some efficacy in lethal influenza-induced treatment of mice, and when administered at a quasi-infectious dose of virus, is lethal during subsequent challenge with the virus. Sex was inhibited (Glasky, 1985). This antifungal activity is probably explained by the adjuvant activity of isoprinosine.

As shown in the examples, the activity of inosine-5'-monophosphate derivatives resistant to 5'-nucleotidase increases survival and the mean survival time in influenza induction is superior to that of isoprinosine. Represents activity. The use of inosine-5'-monophosphate derivative and squalane, which is resistant to 5'-nucleotidase, which provides 100% protection in lethal influenza induction, for any immunostimulant / stimulatory agent, It has not been reported in the prior art. The effect of increasing the survival rate and mean survival time of protected-IMP derivatives after salmonella and influenza challenge (ie, therapeutic efficiency) is inherent in any IMP protected against 5′-nucleotidase Is the action. In these infectious attacks, death occurs suddenly and the mechanism by which the protective-IMP derivatives act is not expected from the overall immunopharmacological profile. The prolongation of inosine-5'-monophosphate derivatives that are resistant to 5'-nucleotidase and the effect of increasing the percentage of components in Listeria have not been reported for any other purine immunomodulators.

Some assumptions can be made regarding the mechanism of the immunostimulatory effect of inosine-5'-monophosphate derivatives that are resistant to 5'-nucleotidase, but the present invention is in no way limited by this mode of action. It should be understood that it is not. Recent insights into the key mechanisms involved in infection-induced survival are included in studies using any intracellular pathogen, such as Toxoplasma, Leishmania, and Listeria. None (Mossman and Coffman, 1989; Scott, 1991; Haak-Frendocho et al., 1992; Trinchieri, 1993; Tripp et al., 1994; Scott ( Scott), 1994; Bogdan et al., 1991; Fiorentino et al., 1989). In this model, Th1 cell responses are promoted by IL-12, mediated by IL-2 and γ-IMP, and blocked by IL-4, and IL-10 protects animals against infections such as Listeria . The Th2 response mediated by IL-4 to IL-10 is associated with lethality due to infections such as Listeria. This model has been shown to be applied to human infection by Mycobacteria leprae (Seiling et al., 1994). Thus, an inosine-5'-monophosphate derivative resistant to 5'-nucleotidase was expected to function by promoting Th1 over the Th2 response. This prediction is further supported by the selective action of inosine-5'-monophosphate derivatives, resistant to 5'-nucleotidase, on delayed hypersensitivity responses, overcoming antibody-mediated responses. (Sosa et al., 1992).

Applicants have shown that in the examples given below, MIMP is active over a wide concentration range and is more sensitive to T cell mitogens such as PHA and to a greater degree of variability in BS such as LPS and pokeweed. FIG. 5 shows stimulating the response of mouse and human lymphocytes to cellular mitogens. The action of MIMP was confirmed for the response of further enriched human CD4 + and CD8 + lymphocytes. Moreover, these results show that the inhibitory action of HIV peptide, IFN alpha, and PGE _{2 on} the PHA response of normal lymphocytes is moderate to moderate, not extreme or toxic The case indicates that it may be reversed. These results also show that the lymphocytes of elderly and HIV-infected individuals can respond to stimulation by the protective-IMP in the presence of PHA if the response is not over-suppressed. The exemplified action of Me-IMP in these studies is considered to be the action of IMP. This is because the actions of Me-IMP and IMP are similar in vitro.

  The selective action of inosine-5'-monophosphate, resistant to 5'-nucleotidase, on T lymphocytes implies a receptor-based interaction. Me-IMP has been shown to induce prothymocyte division markers in addition to the effects described here on mature T lymphocytes (Touraine et al., 1991).

The purine salvage pathway has important implications for T lymphocyte development and function. The deficiency of both adenosine deaminase and nucleoside phosphorylase results in an immunodeficiency syndrome that is deficient in functional T lymphocytes. Several inosine-containing molecules can be suggested to be essential for T lymphocyte development and function. The basis for predicting this exists in the context of transmission factors (Wilson and Fudenberg, 1983). If IMP is part of the transfer factor phenomenon, the protection-IMP
Can mimic non-specific aspects of transfer factor function, possibly via receptors on T lymphocytes. Applicants have confirmed that transfer factor preparation induces a division marker in prothymocytes, ie mimics Me-IMP (Hadden et al., 1986).

  Protective-IMP is regulated with respect to IL-2 action has important implications in the central role played by IL-2 in coordinating cellular immune responses mediated by Th1-type T helper cells Have. In this regard, the main mitogen PHA used in Applicant's studies using protection-IMP has the Th1-pattern of cytokines, namely IL-1, IL-2, γ-IFN and IL-12. Note the selective guidance. Applicants have found that PHA induces IL-10 but not IL-3 or IL-4. Early data show that Me-IMP has only a minor effect on increasing PHA-induced IL-2 or γ-IFN, yet strongly inhibits 1L-10 production. These observations suggest that protective-IMP affects Th1 responses. Th1 response has been shown to be critical in resistance to HIV, cancer, and pathogen infection, ie Toxoplasma, Listeria, and Reishmania (Clerici and Shearer, 1995; Teppler) 1993; Scott and Trinchieri, 1989). Thus, the action of Protect-IMP to make these responses favorable indicates clinical utility in such conditions.

This analysis is supported by in vivo studies with protection-IMP, where DTH is selectively stimulated over PFC responses and AIDS, tumors, and infection induction (Listeria and Salmonella The survival rate in) is increasing. In these studies, Me-IMP was demonstrated to be active by the oral route at doses of 1 mg / kg or less and was non-toxic (oral LD ₅₀ > 5000 mg / kg).

  Thus, clinical application of protective-IMP compounds, such as Me-IMP, is associated with immune recovery in secondary immune deficiency, where T lymphocyte function is compromised and T lymphocytes Numbers are stored reasonably. Such deficiency symptoms have been reported in the relatively early stages of both HIV infection (ARC) and cancer.

In early HIV infection, the T lymphocyte response is thought to be essential in preventing progression to AIDS. The T lymphocyte response is suppressed by products of HIV including gp160, gp41 and TAT (for review see Good et al., 1991). A recent study showed that a retroviral peptide, CKS-17, associated with P-15E, shifted from Th1 to Th2 by inhibiting IL-2 and γ-IFN production and promoting IL-10 production. (Haraguchi et al., 1995). Applicants tested the effect of Me-IMP to reverse the immunosuppressive effect of a 17 amino acid peptide of gp41 that has homology to CKS-17, and if the suppression is mild to moderate, I found out. Me-IMP also increased the PHA response of HIV-infected individuals. These results indicate that Protect-IMP can be used to block the progression of HIV-infected patients to AIDS.

The use of protection-IMP in other viral infections is also disclosed by the present invention. This is because immunosuppression is involved in all types of viral infections studied (Rouse and Horohov, 1986). The immunosuppression that causes high influenza mortality and morbidity in the elderly is due to secondary bacterial infections. Interferon production represents one of the mechanisms by which viruses can suppress immunity. The effect of protection-IMP to reverse the inhibition of IFNα in the PHA response recommends its application in this regard.

Inflammation and physical trauma are known to suppress cellular immune responses, partly mediated by prostaglandins (Davis and Shires, 1986). Of MIMP, the inhibitory effect of PGE ₂ in the PHA response, the ability to reverse the supports its usefulness in these forms of secondary immunodeficiencies.

The above discussion provides a de facto and theoretical basis for the use of inosine-5'-monophosphate derivatives that are resistant to 5'-nucleotidases. The method used with the present invention and its utility can be demonstrated by the following examples.

(General method)
The following materials and procedures were used unless otherwise noted.

(material)
Methyl-5'-inosine-monophosphate (methyl-IMP or Me-IMP), methyl-5'-inosine-phosphate (Mp-IMP), ethyl-5'-inosine-monophosphate (ethyl-IMP or E-IMP), arginine-5'-inosine-monophosphate (arginine-IMP or Arg-IMP) and (heptamine-1-ol) -5'-inosine-monophosphate (Ha-IMP), respectively Prepared according to Examples 1, 2, 3, 4 and 5. The MIMP formulation for use in cell culture was confirmed to be free of endotoxins by the Limulus (Astragalus) lysate assay (Whittaker Bioproducts, Walkersville, MD).

  Inosine-5'-monophosphate (IMP) and adenosine-5'-monophosphate were obtained from Sigma Chemical Co. (St. Louis, MO).

Lymphocyte mitogen plant agglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) were as directed, Burroughs Wellcome (Research Triangle Park, NC), Sigma Chemical Co. Co.) (St. Louis, MO), Gibco (Grand Island, NY) and Murex Diagnostics (Atlanta, GA).

Sheep red blood cells (SRBC) were obtained from Diamedix Corp. (Miami, FL). Hanks balanced salt solution and guinea pig complement were obtained from Gibco. Agarose was obtained from Bacto (Detroit, MI) and DEAE-Dextram was obtained from Sigma Chemical Co. (St. Louis, MO). Cell culture plates (96 and 6 wells) were obtained from Falcon. Ficoll-HYPAQUE was obtained from Pharmacia, Inc. (Piscataway, NJ) and E. coli lipopolysaccharide (LPS) was obtained from Sigma.

  Recombinant IL-2 (rIL-2) was a gift from G. Caspritz from Hoechst Pharmaceuticals (Frankfurt, FRG).

  M. Strand (Jones Hopkins University of Baltimore, MD) synthesized a sequence with 17 amino acids derived from the gp41 part of the gp160 peptide of human immunodeficiency virus (HIV) (Reugg and Strand (Reugg and Strand Strand), 1990), kindly provided us.

Prostaglandin E ₂ is obtained from Sigma Chemical Co. and recombinant interferon α (IFNα, Intron A) is from Schering.
Corp.) (Kenilworth, NJ).

  Human donors were 22 healthy controls (age composition 20-50), 24 elderly (mean age 84 ± 1), 8 HIV-infected pre-AIDS patients (CDC class II-CD4 count 544) and 8 Includes one AIDS patient (mean CD4 count 40).

  Human CD4 + and CD8 + lymphocyte subsets were obtained from normal controls (Applied Immune Sciences, Santa Clara, Calif.) Using industrial panning techniques.

(Cell culture)
All steps related to cell culture were performed under aseptic conditions. General methods of cellular immunology that are not described here are references on cellular immunology techniques such as Michel and Shiigi, Selected Methods in Cellular Immunology. ), WH. Freeman & Co. (NY, 1981) and Stites and Terr, Basic and Clinical Immunology, 7th edition, Appleton & Lange ( Connecticut, Noah Walk, 1991).

(Lymphocyte transformation)
In vitro: Both human peripheral blood lymphocytes (HPBL) and mouse spleen lymphocytes (MSL) were used. Proliferation was assayed by tritiated thymidine incorporation as described by Hadden et al. (1975) and Hadden et al. (1986). For HPBL transformation, PHA and PWM were used at 0.5 μg / ml. For MSL transformation, Con A at 0.5 μg / ml, plant agglutinin (PHA, Murex Diagnostics, Atlanta, GA) at 0.5 μg / ml and lipopolysaccharide endotoxin ( LPS, Sigma, St. Louis, MO) was used at 10 μg / ml. Was added at a concentration of

Mouse splenocytes were obtained from 4-12 month old BALB / c mice by standard procedures (Hadden et al., 1975). These cells are 1.5 per ml of minimal essential medium (Gibco Labs, Grand Island, NY) supplemented with 5% fetal calf serum (FCS, Hyclone Labs, UT, Logan). Cultured in microwell plates at 10 ⁶ cells / ml and in the final pulse, ( ³ H) tritiated thymidine (New England Nuclear, Wilmington, DE, 6.7 Ci / mM; Tested for the proliferative response of the cells to mitogen as measured by uptake of 2.5 Ci / ml), followed by liquid scintillation spectroscopy. Human cells were seeded at 10 ⁴ / ml and cultured for 48 hours using an 18 hour pulse with ³ H-thymidine.

In vivo: Compounds were administered orally by gavage or intraperitoneally. At the end, spleens were obtained as described in Florentin et al. (1982), and cells were obtained with Con A = 0.5 μg / ml, PHA = 0.5 μg / ml or LPS = 0.5 μg / ml. Prepared to examine the proliferative response used.

(Antibody-producing cells)
Direct mouse spleen antibody-producing cells (PFC) were assayed according to the somewhat improved technique of Jerne et al. (1963). Briefly, a group of mice were immunized with SRBC via the intraperitoneal route, and the compounds of interest were administered orally or intraperitoneally as indicated in the results. After 5 days, a spleen cell suspension was prepared and its viability was measured by trypan blue exclusion test. The cells were suspended at 3 × 10 ⁶ / ml. 0.2 ml splenocyte suspension was mixed with 0.8 ml 0.7% agarose and 0.2 ml freshly washed SRBC (6 × 10 ⁵ / ml). This mixture was immediately poured onto the plate and allowed to solidify. After incubation for 60 minutes at 37 ° C. in a humidified air atmosphere containing 5% CO ₂ , the plates were filled with 1 ml of guinea pig complement diluted 1: 5 with Hanks balanced salt water. Hemolytic plaques were counted with an inverted scope at 4x magnification.

(Statistical analysis)
Each experiment was performed 2-10 times as indicated. Four samples from each donor were used at each concentration point. Data are mean ± standard error of the mean (SEM) for each representative experiment
Or for pooled data, expressed as the ratio of control to ± SEM.
Unless otherwise noted, data were analyzed for statistical significance using Student's t-test.

(Immunoassay procedure)
In general, immunoassay EIAs or RIAs were performed using commercially available kits as indicated. EIA can also be developed as known to those skilled in the art. Both polyclonal and monoclonal antibodies can be made and used in the assay. Standard antibody production techniques are well known to those of ordinary skill in the art and generally include Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (Cold Spring). Published by Cold Laboratory), Cold Spring Harbor, NY, 1988. Where appropriate, other immunoassays, such as radioimmunoassay (RIA), may be utilized as is well known to those skilled in the art. Available immunoassays are extensively described in the patent and scientific literature. For example, U.S. Pat.Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533 No. 3,996,345, No. 4,034,074, No. 4,098,876, No. 4,879,219 and No. 5,011,771, and Sambrook et al., Molecular Cloning: A Laboratory Manual See, Cold Spring Harbor, NY, 1988.

(General methods for research using intracellular pathogens)
Depending on the pathogen tested, different strains of mice were used. BALB / c mice weighing 14-16 g were used to test Salmonella and Listeria. For response to influenza virus, the mouse NMRI system was used. These mice were obtained from the Animal Laboratory of the Academy of Medical Science, Russia, and were used in Examples 11-13 described herein.

(General methods for research using hepatitis vaccine: animals)
Various strains of mice, encoded by the Pre-S2 zone S-gene HBV, for immune responses against proteins are arranged in the following haplotype order (Benaceraf and McDevitt, 1972): H -2 ^b > H-2 ^d > H-2 ^s > H-2 ^k > H-2 ^f . DBA / 2 mice do not produce antibodies against HBsAg at high levels when treated with antigen alone (Walker et al., 1981). Accordingly, male mice weighing 16-18 g were obtained from the Animal Laboratory of the Academy of Medical Science, Russia and used in the examples described herein. This mouse line was chosen because they are poor responders to the hepatitis B virus.

(Irradiation protocol)
Experimental mice were irradiated for 4 minutes with an irradiation dose of 25 rad / min (1 rad = 0.01 Gy) using a ⁶⁰ Coγ beam source. Data represent the average of 10 animals in each group.

(Immunoassay procedure)
Serum specimens from immunized mice were tested for the presence of anti-HBs in an enzyme immunoassay (EIA). This test was performed using a diagnostic kit from Roche Diagnostica according to the manufacturer's instructions. The sensitivity of this anti-HBs EIA kit (Roche) is <10 IU / l.

  Antibody concentrations are based on anti-HBs results detected with a standard serum panel (Roche Diagnostics) with the following concentrations: 10 IU / L, 50 IU / L, 100 IU / L and 150 IU / L Measured with the help of a calibrated calibration curve. The results of these two experiments were used for calculating the average value.

(Protocol-Protocol for IMP and HBsAg administration)
HBsAg was administered at a dose of 16 mg / mouse in 0.2 ml PBS (pH 7.4) twice at 2-week intervals between injections.

  Protect-IMP and MIMP were introduced by oral or intraperitoneal route at a dose of 50 mg / kg.

Three schemes were used for MIMP administration. That is,
Scheme # 1: MIMP was administered by oral route by gavage 30 minutes prior to HBsAG introduction.
Scheme # 2: HBsAg and MIMP were administered intraperitoneally.
Scheme # 3: HBsAg and MIMP were administered once intraperitoneally and then MMP was orally administered for 4 days.

  (Production Example 1: Methyl-5'-inosine-monophosphate)

Methyl-5′-inosin-monophosphate was prepared by reacting inosine-5′-monophosphate with methanol using dicyclohexylcarbodiimide as a condensing agent.

To a solution of inosine-5′-monophosphate (2.2 g, 6 mM) in methanol (300 ml) was added tributylamine (1.4 ml, 12 mM) and dicyclohexylcarbodiimide (6.56 g, 30 mM). This solution was maintained at 25 ° C. for 4 days and evaporated to dryness under reduced pressure. 2.3% NaOH solution (20 ml, 11.4 mM) was added to the resulting residue and the resulting suspension was filtered. The resulting precipitate was washed with water (20 ml) and discarded. The filtrate was extracted three times with ether, placed in a column packed with 50 g Amberlite IR / 20 PLUS (NH ₄ ⁺ form) and eluted with water. The UV absorbing fraction was evaporated under reduced pressure and the resulting syrup was dissolved in methanol (20 ml). The solution was poured into acetone (300 ml) and the resulting suspension was washed with acetone and ether and dried over P ₂ O ₅ under vacuum to give a powder product in a yield of 1.4 g (68%). Obtained. The melting point mp of this product was 145 ° C., and UVλmax was 249 nm (pH 5.5, H ₂ O) and 253 nm (pH 10).
Elemental analysis (calculated as C ₁₁ H ₂₀ N ₅ O ₉ P (MW. 397.24))
Calculated value: C, 33.25; H, 5.07; N, 17.63
Found: C, 33.36; H, 5.13; N, 17.34
NMR data DMSO-d ₆ : 3.50 (s, 3H, CH ₃ OP), 3.20-4.70 (m, 7H, ribose), 5.95 (d, 1H, anomeric J-6Hz), 8.12 (s, 1H, C-2 ), 8.40 (s, 1H, C-8), 8.20 (br.s, 5H, NH (CO) and NH ₄ )
(Production Example 2: Methyl-5′-inosine-monophosphate)

  Methyl-5'-inosine-monophosphate was prepared by reacting 2 ', 3'-isopropylideneinosine with methylphosphonic dichloride and then deprotecting the nucleotide.

Methylphosphonic dichloride (0.86 g, 6.49 mM) was added dropwise to a 10 ° C. cold mixture of 2 ′, 3′-isopropylideneinosine (2 g, 6.49 mM) and 50 ml of dry pyridine. After stirring the mixture for 18 hours, ice and water were added to give the protected nucleotide. Hydrolysis of the protected nucleotide was performed using formic acid. HPLC was performed using H ₂ O / methanol. Appropriate fractions were evaporated under reduced pressure to produce methyl-5'-inosine-monophosphate.

  (Production Example 3: Ethyl-5'-inosine-monophosphate)

In the same manner as in Production Example 1, ethyl-5′-inosine-monophosphate was prepared. However, inosine-5
'-Monophosphate was reacted with ethanol using dicyclohexylcarbodiimide as a condensing agent.

  (Production Example 4: Arginine-5'-Inosine-monophosphate)

Arginine-5′-inosin-monophosphate was prepared by reacting inosine-5′-monophosphate with arginine using dicyclohexylcarbodiimide as a condensing agent.

  L-arginine (1.04 g, 6 mM) was added to a solution of inosine-5'-monophosphate (0.55 g, 0.15 mM) in formamide (3 ml). A solution of dicyclohexylcarbodiimide (1.6 g, 7.5 mM) in t-butyl alcohol (10 ml) was added to the mixture and the resulting suspension was heated at 80 ° C. for 8 hours.

The formed precipitate was filtered off, washed with water three times, and the combined filtrate was evaporated to remove t-butyl alcohol. This solution was extracted three times with an equal volume of ether and evaporated under vacuum to give a syrup. Ethanol (30 ml) was added and the resulting precipitate was filtered off to give a very hygroscopic gum. After standing for 10 days, the gum became a solid. mp 130 ° C., UVmax 249 nm (H ₂ O).

  (Production Example 5: (Heptamine-1-ol) -5'-inosine-monophosphate)

  By reacting inosine-5'-monophosphate with 6-amino-2-methyl-2-heptanol using dicyclohexylcarbodiimide as a condensing agent, (heptamine-1-ol) -5'-inosine- Monophosphate was prepared.

To a solution of inosine-5′-monophosphate (1.045 g, 3 mM) in formamide (5 ml) was added 6-amino-2-methyl-2-heptanol hydrochloride. A solution of dicyclohexylcarbodiimide (6.22 g, 30 mM) in butyl alcohol (25 ml) was added to the mixture. The resulting reaction mixture was heated at 80-90 ° C. with stirring for 8 hours. The formed precipitate was filtered off and washed 3 times with 5 ml of water. The precipitate of dicyclohexylurea was discarded and the combined filtrates were evaporated to remove butyl alcohol. This solution was extracted three times with ether and evaporated to dryness under vacuum. The obtained oily residue was suspended in acetone to produce a white hygroscopic substance. This was purified by dissolving in water, treating with activated carbon, and then precipitating in acetone to give a white hygroscopic material. UVmax 249nm (H ₂ O).

Yield of purified material: 0.7 g (53.4%); mp 95 ° C .; empirical formula: C ₁₈ H ₂₈ N ₅ O ₈ P:
% P calculated value 7.03; actual value 5.81
(Production Example 6 Hepatitis B virus surface antigen (HBsAg))
Hepatitis B virus surface antigen (HBsAg; subtypes ad and ay) was purified from antigen carrier plasma according to the following scheme.

(1) First, plasma containing HBsAg diluted with 2 volumes of physiological salt solution at 80 ° C.
Heated in a water bath for 0 minutes. The resulting clot was removed, mechanically disrupted, and denatured protein was removed by centrifugation.

  (2) HBsAg was then precipitated again with polyethylene glycol M 6000 at a final concentration of 15% (wt / vol). The precipitate containing HBsAg was dialyzed against 0.9M NaCl and treated with pepsin (100 mg / ml) and Tween-80 (final concentration 2%).

  (3) The obtained HBsAg solution was ultracentrifuged twice with a linear sucrose gradient. The purified HBsAg sample was dialyzed against 0.9M NaCl, 1 ml was aliquoted and frozen at −60 ° C.

  (4) After being concentrated 50 to 100 times, the obtained sample had the following characteristics.

  The sample consisted only of spherical particles having a diameter of 18 to 25 nm, and neither DANE particles nor HBsAg filaments were present. Negative results were obtained in tests for the presence of HBeAg (EIA kit from Abbott laboratories); DNA-polymerase; HBV-DNA by directed amplification methods; and proteins characterized by normal human plasma.

  The resulting sample was used as the basis for processing an experimental lot of hepatitis B vaccine (Alper et al., 1989).

  Alternatively, hepatitis B vaccine is available from SmithKline Beecham Biologicals and HBsAg is available from Sigma.

Example 1 Test for protection from 5'-nucleotidase activity
Measuring the release of inorganic phosphate according to the method of Ames et al. (1960), the ability to hydrolyze IMP of 5'-nucleotidase (obtained from Crotalus atrox venom, St. Louis, MO, Sigma Chemical Co.) It was investigated by. Nucleotide samples (40-60 nmole) were prepared in a total volume of 100 μl containing 50 mmol HEPES pH 7.3 and 5 mmol MgCl _2.
. Incubate for 10 minutes at 37 ° C. with 02 units of 5′-nucleotidase. The reaction was performed by adding 800 μl of 0.42% 1N H ₂ SO ₄ : 10% ascorbic acid (6: 1, v / v) solution of sodium molybdate, then 0.3 ml of H ₂ O · KH ₂ PO ₄ Stopped. Standards (2-80 nmole) were processed similarly and samples and standards were incubated at 45 ° C. for 20 minutes. Phosphate was determined by absorption at 820 nm. Blanks containing nucleotides but no enzymes were tested in parallel to correct for non-enzymatic hydrolysis. The percentage of hydrolysis was calculated from the exact amount of nucleotide substrate determined by UV absorption using the extinction coefficient e = 12.2 at 249 nm. The results of the four experiments are summarized in Table 1A.

The tested substances were found to be variously resistant to degradation by 5′-nucleotidase. Ha-IMP and Arg-IMP show mild and moderate resistance to hydrolysis, respectively, while both Me-IMP and Mp-IMP are very resistant to hydrolysis as well as E-IMP. was there. None of the 5′-IMP derivatives were found to inhibit IMP hydrolysis, indicating that the compounds are not themselves inhibitors of 5′-nucleotidase.

For IMP, MIMP, and adenosine-5'-monophosphate (AMP), a further combined experiment was designed to determine the sensitivity to degradation by 5'-nucleotidase. The experimental records were as described above except that the first incubation was 20 minutes with 0.1 mM HEPES and 10 mM MgCl ₂ in a total volume of 200 μl. Nucleotide hydrolysis was performed in duplicate for each quantification. The results are shown in Table 1B.

These data indicate that the immunostimulatory effect of the 5′-IMP derivative is an effect of resistance to hydrolysis of the substituted 5′-IMP rather than the specific substituent type.

Example 2 Study Using Human Peripheral Blood Lymphocytes
(Normal HPBL response to stimulation by mitogenic factors)
Normal human peripheral blood lymphocytes (HPBL) were stimulated with the mitogens PHA or PWM. In a series of experiments, Ha-IMP, Arg-IMP, E-IMP, Me-IMP, Mp- for effects that stimulate these responses in the concentration range of 0.1, 1, 10, and 100 μg / ml. IMP and inosine-5′-monophosphate (IMP) were analyzed. Individual blood donors vary in response to these mitogens and compounds, but all five IMP derivatives consistently stimulated the HPBL response to PHA as shown in FIG. IMP itself showed activity comparable to Me-IMP. Arg-IMP, Me-IMP and E-IMP were more active, but Ha-IMP was less active and Mp-IMP was the most active compound. The tested compounds showed little or no effect on the PWM response. None of the compounds stimulated HPBL in the absence of mitogens. Since the PHA response reflects T lymphocyte proliferation and the PWM response reflects B lymphocyte proliferation, the results indicate that 5′-substituted IMP derivatives preferentially enhance T lymphocyte responses. As a representative example, Me-IMP (MIMP) was used in the following experiment.

  FIG. 2 shows the results of the HPBL response to PHA in vitro of two donors in the presence of IMP and MIMP. The response of control lymphocytes to stimulation with PHA for 3 normal donors was not affected by MIMP over a range of concentrations.

  Human peripheral blood lymphocytes average about 80% T lymphocytes and 20% B lymphocytes, 2/3 of T lymphocytes have a CD4 + helper / inducer phenotype, 1 / 3 is the CD8 + suppressor gene / cytotoxic phenotype. Enrichment of CD4 + T cells and CD8 + T cells can be achieved (panning) by removing and reducing other phenotypes (> 98%) using adhesion to flasks coated with monoclonal antibodies. Such enrichment is used with three normal controls (joint use data), and FIG. 3 shows the effect of MIMP on these two cell populations. CD4 + and CD8 + lymphocyte PHA responses are significantly increased by MIMP (p <0.01 for each of the three responses).

(HPBL response to suppression by HIV-derived peptides, interferon α and PGE ₂ )
A synthetic 17 amino acid peptide representing the immunosuppressive site of the intramembrane gp41 portion of human immunodeficiency virus (HIV) was tested on lymphocytes from controls. A representative experiment of the five experiments in which this peptide induces gradual suppression of PHA-induced lymphocyte proliferation at a maximum suppression dose of 100 mM is shown in FIG. The effect of MMP from 0.1 to 100 μg / ml was investigated in combination with inhibition by varying degrees of peptide. When the peptide-induced inhibition was less than 50%, MIMP restored the proliferative response to a near normal range. However, when the suppression was more severe (> 50%), the effect of MIMP was not significant. These data indicate that MIMP can reverse the immunosuppression of HIV-related peptides if the effect is mild to moderate but not severe.

Viral infection is associated with a lymphoproliferative response. FIG. 5 shows the effect of rIFN-α, a virus-inducing mediator that suppresses the in vitro PHA-response of lymphocytes from controls, and when the effect is moderate, ie at IFN-α concentrations of 10 and 100 units / ml. 1,10 to reverse the suppression
The effect of 100 μg / ml MIMP is shown.

Inflammation such as that seen in rheumatoid arteritis is associated with a hypoproliferative response. FIG. 6 shows the effect of PGE ₂ (10 ⁻⁵ M), one of the inflammatory mediators that suppresses the PHA-response of normal lymphocytes, and the effect of MMP (1 to 100 μg / ml) that reverses the suppression. .

(HPBL response in elderly and HIV-infected persons)
FIG. 7 represents the effects of stimulation with 100 μg / ml MIMP compared to the PHA response of elderly and HIV-infected and normal controls (C). Control patient (22) showed normal PHA and MIMP responses. Fifteen elderly patients (Ag +) showed normal PHA response and response to MIME. Nine elderly patients (Ag-) showed a significantly reduced response to PHA and response to MIME. Of nine apparently healthy elderly patients with poor response to PHA, five lymphocyte counts were on average on average.

  Eight HIV-infected persons (ARC) on average showed 50% of the average normal PHA response and responded significantly to MIMP. Eight AIDS patients did not respond. These data suggest that clinical subjects with MIMP treatment should be pre-tested for drug sensitivity in vitro prior to treatment.

  Repeating the above experimental record with 10 and 100 μg / ml Mp-IMP yielded the same results as those obtained for 100 μg / ml MIMP.

Example 3 Study Using Mouse Spleen Lymphocytes
MSL was stimulated with mitogens Con A and LPS. In a series of experiments on the effects of stimulating these responses in the concentration range of 0.1, 1, 10, and 100 μg / ml, Ha-IMP, Arg-IMP, E-IMP, Me-IMP, Mp-IMP and IMP was analyzed. As shown in FIG. 8, Ha-IMP, Arg-IMP, E-IMP, Me-IMP, Mp-IMP and IMP stimulated the proliferative response of MSL to Con A.

  Comparing the data from mouse (FIG. 8) and human lymphocytes (FIG. 1), human lymphocytes are more sensitive to these compounds than mouse lymphocytes.

In further experiments, mouse spleen cells were incubated with PHA or LPS and MIMP (1-100 μg / ml). MIMP did not affect the spleen cell response in the absence of mitogens. In the presence of mitogens, as shown below, MIMP progressively and significantly increased the proliferative response of lymphocytes as measured by the addition of tritium-labeled thymidine. PHA
Since the response of spleen cells to preferentially reflects the T lymphocyte response, the response seen with Con A was confirmed. The LPS response also responds in parallel with the previous experiment.

Data are obtained from 4 different animals and represent 4 fold samples for each concentration. They are expressed as averaged CPM ± SEM, ^* indicates significance of p <0.05, ^** indicates significance of p <0.01.

Example 4 In Vivo Stimulation of PFC Response
(Intraperitoneal (ip) administration)
To determine whether a compound stimulates lymphocytes in vivo, mice are given 1 × 10 ^8.
Immunization is performed by intraperitoneal administration of cell sheep red blood cells (SRBC) and 50 mg / kg body weight of Ha-IMP, Arg-IMP, Me-IMP or IMP, and spleen plaque antibody-forming cells (PFC) are given for 5 days. It was measured later. Ha-IMP, Arg-IMP and Me-IMP significantly stimulated the PFC response (as shown in FIG. 9). There was no significant effect when IMP was compared to controls in 3 experiments at 50 mg / kg body weight (IMP: 12 samples with an average PFC of 197 ± 21; control: 15 samples with an average PFC of 210 ± 17). This indicates that IMP alone has in vivo activity when protected from 5'-nucleotidase activity.

  Ha-IMP, Arg-IMP and Me-IMP dose response data on PFC responses were expressed as shown in FIGS. 10A, 10B and 10C, respectively. All three compounds were optimally stimulated at 50 mg / kg body weight, while Me-IMP was active at low doses.

  In contrast, in vitro data on mouse lymphocyte proliferation, Me-IMP appeared to be the most influential of the compounds tested.

(Oral administration)
To determine if there was activity when administered orally, mice were immunized by intraperitoneal administration of 1 × 10 ⁸ sheep erythrocytes (SRBC) and orally administered Me-IMP. Me-IMP was administered daily when immunized with SRBC antigen and for 5 days thereafter. Splenic plaque antibody-forming cells (PFC) were measured at the end of 5 days. FIG. 11 shows the results for various doses of Me-IMP.

  Me-IMP administered orally with SRBC antigen and daily for 5 days peaked at 50 mg / kg body weight and stimulated the PFC response.

Example 5 In vitro response to mitogens after in vivo administration of Me-IMP
Experiments parallel to those of Example 4 confirm that both PHA and Con A responses of splenic lymphocytes are stimulated by oral administration of Me-IMP.

  Spleen lymphocytes were obtained from mice that were orally administered Me-IMP in the range of 0-100 mg / kg body weight for 5 days and then sacrificed. Spleen lymphocytes were incubated with PHA (0.5 μg / ml) or Con A (0.5 μg / ml) for 48 hours. The culture was pulsed with tritium labeled thymidine for the last 18 hours. FIG. 12 shows the results for various oral doses of Me-IMP.

  These data show that, compared to IMP, the 5'-IMP derivative is a potent adjunct to the T cell-dependent antibody response, and in the case of Me-IMP, it is a potent stimulator of the T lymphocyte proliferation response. Indicates that there is.

  At the doses used, Me-IMP was active both parenterally and orally and was apparently non-toxic. The acute toxicity (LD50) of Me-IMP was 500 mg / kg body weight or more by intraperitoneal administration, and 5000 mg / kg body weight or more by oral administration.

Example 6 Delayed In Vivo Hypersensitivity Stimulation
Mice were immunized by intraperitoneal administration of SRBC at a gradient of 10 ^{6 to} 10 ⁹ cells. Preliminary experiments have shown that the optimal immunization dose of SRBC for delayed-type hypersensitivity is 10 ^{7 to} 10 ⁸ cells. In this study, a dose of 10 ⁷ was selected to immunize the animals.

Four days after immunization, antigen was administered by subcutaneous injection of 10 ⁸ SRBC in 50 μl saline phosphate buffer (PBS) into the left hind paw of each mouse. Vehicle (50 μl) was injected subcutaneously into the right hind paw as a control. The compounds to be tested were injected intraperitoneally either at the time of immunization or at the time of induction. Twenty-four hours after challenge, paw swelling was measured as an increase in foot thickness (left-right) using a technician caliper.

The results are expressed in increments of 0.1 mm as an increase in foot thickness. Characterization of delayed-type hypersensitivity measured in this way has already been described by MacDonald et al. (1979). Figures 13 and 14 show the results of this test. In particular, FIG. 13 shows a dose response plot for Me-IMP when administered at the time of immunization. FIG. 14 shows the response for Me-IMP when administered at the time of immunization (Immunization) and when administered at the time of challenge (Challenge).

  When Me-IMP was administered once at the time of immunization, delayed-type hypersensitivity was significantly stimulated. When Me-IMP was administered once at the time of antigen administration, the effect was not significant. These data indicate that Me-IMP promotes cellular immunity, probably by acting on the afferent limb of the immune response, T helper cells. This is true even at doses that are less than the doses of increasing antibody production, indicating a preferential effect on DTH and Th1 cells that mediate these responses.

Example 7 In vivo treatment with protected -IMP derivatives
(Friend leukemia virus (FLV))
To demonstrate the clinical therapeutic utility of protected -IMP derivatives, BALB / c mice were infected with Friend leukemia virus (FLV). Control mice were treated intraperitoneally with saline solution 3 to 13 days after infection, and mice treated with Me-IMP were treated with 1 mg / kg / day from 3 to 13 days after the day of death. Recorded. FIG. 15 shows that mice treated with Me-IMP have a longer mean survival time (MST). It is satisfactorily significant by the Wilcoxon test (p <0.04)
(Tumor development)
To test the effect of Me-IMP on animals with cancer, a group of Swiss mice (6) was inoculated subcutaneously with a Meth A tumor according to the method of Carswell et al. (1975). After 8 days, when the tumors were about 8 mm in size, the animals were treated intravenously with various immunization doses of various amounts of Me-IMP or an equal amount of saline. After 5 hours, the animals were treated intravenously with 10 μg lipopolysaccharide endotoxin (LPS). Tumor necrosis factor (TNF) levels were analyzed by serum 24 hours after treatment and tumor necrosis (-to +++) was assessed 48 hours later. Complete tumor regression was assessed after 20 days. The results are shown in Table 2.

Table 2 shows that Me-IMP (100) alone or LSP (10) alone is significant for tumor necrosis (all 10 or-), tumor regression (0/12) or survival after 20 days (0/6). There was no effect, but in the case of Me-IMP (100) and LSP (10) significant tumor necrosis (2/3 is ++ or ++), complete tumor regression (2/12) and at 20 days Increased survival (6/6) was induced. Under these conditions, the amount of TNF was higher for Me-IMP treatment and LSP (4 mice) at 24 hours than for saline and LPS (4 mice) controls (4240 vs. <200). . These data are
When used with LSP rather than alone, Me-IMP probably has significant anticancer activity mediated by induction of TNF and related lymphokines.

Example 8 Effect of Protected-IMP on Listeria Infection
(Experimental record)
(infection)
Mouse-adapted bacterial strain L. Monocytes gene EGD (Gamaleya Research Institute Academy Medical Science Russia), mice at a dose of the day of infection (Day 0) to 0.5ml of PBS (pH = 7.4) in 1.7 × 10 ⁴ cells / mouse Were intraperitoneally administered to male BALB / c mice (14 to 16 gm).

(Treatment)
Solutions of various concentrations of MIMP (AGS-36-217) were administered orally or parenterally from 5 days before infection (-5 days) as shown in Table 3.

  (result)

As shown in Table 4, mean survival time (MST) and survivorship of 10 mg / kg body weight by mouth and 1 and 10 mg / kg MIP by intraperitoneal injection 5 days prior to antigen administration The absolute number has increased. MST and survival increased when 0.1-10 mg / kg of MIMP was administered from 5 days prior to challenge, both intraperitoneally and orally.

Example 9 Effect of Protected-IMP on Salmonella Infection
(Experimental record)
(infection)
Mouse-adapted Salmonella typhimurium strain 415 (Gamaleya Research Institute, Russia, Academy
Medical Science) was injected intraperitoneally from mice at a dose of 5 × 10 ⁴ cells in 0.5 ml PBS (pH = 7.4). The dose was determined so that the mean survival time was 2.5 days and the mortality on day 6 was 100%.

(Treatment)
Solutions of MIMP (AGS-36-217) at various concentrations were administered parenterally as shown in Table 5 and the results of treatment are shown in Table 6 below.

As shown in Table 6, all control animals died in 5 days with an average survival time of about 2.5 days. With various treatments of MIMP, MST increased to about 4 days (p <0.01) and long-term survivors also appeared. The case of N3 treatment is most obvious.

(Example 10 Effect of MIMP on influenza virus-induced mortality)
In this model, NMRI mice were challenged with influenza virus by the aerosol method. Influenza virus doses were determined to be 80-100% mortality in control animals with an average survival time of 8-11 days. Control (PBS), MIMP (100
Or treatment with 200 μg / mouse) and squalane (1%) + MIMP (200 μg / mouse) was started 1 day before infection, 1 hour before infection and 1 hour after infection.

  The results are shown in Tables 7 and 8 below. When 200 μg / mouse (5 mg / kg) of MMP was administered intranasally 24 hours (1 day) before antigen administration or around 1 hour, the number of survivors and mean survival time (MST) increased (Table 7). . Survival and MST also increased when MMP was administered only 100 μg / mouse 1 hour before infection (FIG. 16).

  In the second experiment, survival and MST increased when 200 μg / mouse MIMP was administered intranasally with squalane (1%) one day before infection or one hour after infection. As shown in FIG. 17, the mice in these treated groups survived 100%. Squalane alone had no effect.

Example 11 Stimulation of Antibody Response to HBsAg
Using previous studies, DBA / 2 mice were selected as poor responders in antibody production against HBsAg (Walker et al., 1981).

(Normal (control) DBA / 2 mice)
The anti-HBs detection results in the control group treated with HBsAg alone and the group treated with the combination of HBsAg and MIMP as prepared in the examples are shown in Table 9. The following table provides various inoculation plans.
Scheme treatment
1 HBsAg only
2 Oral administration of HBsAg + MIMP (30 minutes before inoculation)
3 HBsAg + MIMP at the same time intraperitoneally, then oral MMP every 4 days
4 HBsAg + MIMP administered intraperitoneally simultaneously
5 Irradiation + HBsAg
6 Irradiation + Scheme N2
7 Irradiation + Scheme N3
8 Control

  The antibody response obtained when HBsAg and MIMP were administered in combination according to various schemes exceeded that of the control group of animals.

(Immuno-protected DBA / 2 mice)
In the second set of experiments, the effect of MIMP on the induction of a specific immune response to HBsAg in immunocompromised DBA / 2 mice exposed to ionizing radiation was identical to that shown for control DBA / 2. Analysis was performed using an inoculation scheme.

  The results of anti-HBs detection in irradiated mice of the control group (administered only with HBsAg) and mice treated with the combination (HBsAg and MIMP using Schemes 2 and 3) are shown in Table 10.

  From the data, it can be seen that in the case of Schemes 2 and 3, anti-HBs significantly increased on day 21, indicating the recovery effect of MIMP on the immune deficient system of these irradiated animals.

The results shown in Example 11 indicate that protected -IMP derivatives can affect the development of a humoral immune response against HBsAg. It was shown that the amount of antibody against HBsAg can be significantly increased when 50 mg / kg MIMP was administered as an intraperitoneal administration alone or as an oral administration alone. After intraperitoneal administration of the antigen, further activity of MIMP did not increase its activity.

Hypotheses regarding the mechanism of adjuvant effect of inosine-5′-monophosphate derivatives resistant to 5′-nucleotidase can be made, but the invention should not be considered limited to this mode of action. An immune response to hepatitis B vaccine was observed in hemodialysis patients and immunocompromised individuals (Walz et al., 1989; Hess et al., 1989, respectively). Seroconversion can be achieved by increasing the vaccine dose or increasing the number of doses (Walker et al., 1981). Soluble interleukin-2-receptor was observed in these patients, and dysfunction of interleukin-2 action due to available IL-2 binding affected the response to hepatitis B vaccine (Walz et al ., 1989; Meuer et al., 1989). Meuer et al. Injected 40 mg of vaccine, followed by 1.2 × 10 ⁵ units of natural interleukin-2 into 10 immune-unresponsive 10 hemodialysis patients. After 4 weeks, 6 out of 10 patients showed seroconversion even though the antibody levels were below normal. MHC-linked immune unresponsiveness to protein or peptide antigens can be overcome by administration of IL-2. The results of these examples suggest that protected -IMP derivatives are realized by T cells as an adjunct and may include interleukin-2.

  Various publications in this application are referenced by citation or number. References not fully cited in this specification are listed below. The complete disclosures of these publications are hereby incorporated by reference into this application to more fully describe the state of the relevant art of the present invention.

  It should be understood that the present invention has been described in an illustrative manner, and that the terminology used is a descriptive term rather than a limiting term.

  Obviously, many modifications and variations of the present invention are possible in light of the above teachings. Accordingly, within the scope of the appended claims, the invention may be practiced other than as described in the specification.

Claims (1)

Invention described in the specification.

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