Inhibition in mice of experimental metastasis of B16 melanoma by the synthetic lipid A-subunit analogue GLA-60.

Antimetastatic effects of the monosaccharic lipid A-subunit analogue GLA-60 against B16-F10 melanomas were investigated. Intravenous injection of 10 micrograms GLA-60 one day or two days before transplantation of 10(5) melanoma cells resulted in a significant decrease in the number of melanoma colonies metastasized into the lung. Intramuscular injection was also effective. Oral administration as well as intraperitoneal and subcutaneous administrations of 100 micrograms GLA-60 were also effective in preventing metastasis of the melanoma. However, these prophylactic effects of GLA-60 on the metastasis were diminished in mice which had been pre-treated with anti-asialo GM1 serum; on the other hand, prophylaxis was not affected in mice pre-treated with anti-Mac 1 antibody. These results suggest that asialo GM1 positive cells, probably natural killer cells, but not macrophages, participate as effector cells in depressing metastasis of the melanoma cells into the lung. In addition, GLA-60 also showed therapeutic potency in depressing metastasis, under a defined condition. The antimetastatic effect of GLA-60 was compared with other immunomodulators.

Non-specific protective activity of synthetic lipid A-subunit analogs against microbial infections is influenced by their 2-N- and 3-O-linked acyl substituents in the D-glucosamine backbone.

Non-specific protective activities against vaccinia virus (VV) and Pseudomonas aeruginosa infections as well as interferon (IFN)-inducing, natural killer (NK) cell and macrophage activation activities of chemically synthesized lipid A-subunit analogs were investigated. The analogs are 4-O-phosphono-D-glucosamine derivatives carrying different 2-N- and 3-O-linked acyl substituents such as (R)-3-tetradecanoyloxytetradecanoyl (C14-O-(C14)), (R)-3-hydroxytetradecanoyl (C14-OH) and tetradecanoyl (C14) groups. Compounds GLA-59 and GLA-60, which possess C14-OH and C14-O-(C14) groups as their acyl substituents, showed stronger IFN-inducing and anti-vaccinia virus activities than GLA-27 and GLA-68, which possess a C14 group instead of the C14-OH group in GLA-59 and GLA-60, although NK cell activation activity was similarly high in all of these compounds. In protective activity against P. aeruginosa infection and macrophage activation activity, GLA-60 and GLA-68, which carry a C14-O-(C14) group at the 3-O-position, expressed higher activities than GLA-27 and GLA-59, which carry the acyloxyacyl group at the 2-N-position. These results indicate that the acyl substituent (whether the counterpart of the C14-O-(C14) group is a C14 or a C14-OH group) and the binding position of the acyloxyacyl group at the 2-N- or the 3-O-position strongly influence the manifestation of antimicrobial and immunomodulating activities in different ways depending on the activity. Among the compounds, GLA-60 satisfied the structure requirements for protection against both VV and P. aeruginosa infections. This compound is a hopeful immunomodulator for prevention against broad microbial infections.

Effect of acyl substituents of synthetic lipid A-subunit analogues on their immunomodulating antiviral activity.

A chemically synthesized lipid A-subunit analogue, GLA-60, 2-deoxy-4-O-phosphono-2-[(3R)-3-hydroxytetradecanamido]-3-O-[(3R)- 3- tetradecanoyloxytetradecanoyl]-D-glucose, has many of the activities of endotoxin but has little toxicity. Then, compounds with various lengths of acyl side chain of the acyloxyacyl group at the 3-O position of GLA-60 were synthesized and evaluated for interferon (IFN)-inducing activity, natural killer (NK) cell activation and antiviral activity. The compounds with acyl side chains between C8 and C15 exhibited significant antiviral activity (inhibition of pox tail lesion formation in vaccinia virus-infected mice), serum IFN-inducing activity and NK cell activation. However, the compound carrying a C2 or a C16 acyl side chain did not exhibit these activities. The compounds with a C13 or C14 acyl side chain showed strong protective against herpes simplex virus type 1 in cyclophosphamide-immunosuppressed mice.

Structure-activity relationship of chemically synthesized nonreducing parts of lipid A analogs.

Some synthetic compounds of the nonreducing part of lipid A were found to preserve significant immunopharmacological activities of the endotoxin, at the same time showing very slight if any endotoxic activity such as pyrogenicity lethality or Shwartzman reactivity. Thus, divers activities of the endotoxin which had earlier been considered intrinsic and inseparable were shown to be separable when certain synthetic analogs of lipid A-subunit are at work. The combination of acyl components as well as phosphorylation and acylation positions in these partial structure analogs of lipid A affect the expression of biological activities of the endotoxin. Moreover, stereospecificities of acyl substituents contribute differently to enhance the various biological activities of the endotoxin. It was remarkable that protective activities of the endotoxin such as enhancing nonspecific resistance against microbial infections and antitumor activity are preserved in lipid A-subunit analogs of small molecular weight of ca. 1,000, which at the same time lose all or most toxic activity such as pyrogenicity, lethality and Shwartzman reactivity. It is hoped that new derivatives of lipid A and/or lipid A-subunit which exert only limited biological activities of endotoxin, whether they be protective or toxic, can be synthesized in order to clarify the structural requirements for expressing a given activity. Such compounds will be useful not only for promoting basic endotoxin research but also for application in clinical medicine. Further detailed experiments on the structure-activity relationships of the newly synthesized 4-O-phosphono-D-glucosamine derivatives binding acyl substituents of varying carbon chain length at the 2-N- and 3-O- positions are in progress.

Enhancement of nonspecific resistance to microbial infections of synthetic lipid A-subunit analogues of GLA-27 modified at the C1 position of the glucosamine backbone.

The C1 position of lipid A-subunit analogue GLA-27, 4-O-phosphono-D-glucosamine carrying N-3-tetradecanoyloxytetradecanoyl(C14-O-(C14)) and 3-O-tetradecanoyl (C14) groups, was S-acetylated, thiolated or phosphorylated. Enhancement of nonspecific resistance to Pseudomonas aeruginosa and vaccinia virus infections of these chemically modified compounds were investigated. Thiolation augmented the nonspecific resistance to P. aeruginosa infection. Protective activity against vaccinia virus infection was reduced by all the chemical modifications. NK cell activity was found not to be effected by S-acetylation, but to be decreased slightly by thiolation or phosphorylation. IFN-inducing activity was reduced remarkably by thiolation or S-acetylation, or completely diminished by phosphorylation, compared with that of GLA-27.

Immunopharmacological activities of 2-keto-3-deoxyoctonic acid-(alpha 2----6)-linked 4-O-phosphono-D-glucosamine derivatives carrying N- and 3-O-acyl substituents.

The immunopharmacological activities of 2-keto-3-deoxyoctonic acid (KDO)-(alpha 2----6)-linked lipid A-subunit analogs, 4-O-phosphono-D-glucosamine derivatives carrying N- and 3-O-acyl substituents, were compared with those of the corresponding analogs without KDO, GLA-27, GLA-47, and GLA-60. Among the analogs tested, GLA-60, a 4-O-phosphono-D-glucosamine carrying N-3-hydroxytetradecanoyl and 3-O-3-tetradecanoyloxytetradecanoyl groups, exhibited the most intensive activities in terms of mitogenicity, adjuvanticity, and mediator (tumor necrosis factor and colony-stimulating factor) induction. Binding (alpha 2----6) of KDO to GLA-60 failed to enhance the activities. Similarly, the activities of GLA-27 and GLA-47 were also decreased by introduction of KDO to the O-6 of the analogs. This indicates that the strengths of the activities of the subunit analogs depend on the kinds of N- and 3-O-linked acyl substituents and not on the presence of the KDO linked to the O-6.

Antiherpes activity of chemically synthesized lipid A-subunit analogue GLA-60 in immunosuppressed mice.

Intraperitoneal administration of 10 micrograms GLA-60, a chemically synthesized lipid A analogue, to mice one day after treatment with 200 mg/kg of cyclophosphamide (CY) significantly increased the number of macrophages, lymphocytes and polymorphonuclear leukocytes (PMNs) in the peritoneal cavity. The intrinsic antiviral activity of macrophages against herpes simplex virus type 1 (HSV-1) as well as natural killer (NK) activity against YAC-1 target cells was stimulated by administration of GLA-60 to CY-immunosuppressed mice. When the mice were administered GLA-60 prior to HSV-1 infection, virus growth was inhibited and the mortality rate of infected mice was reduced. Thus, GLA-60 is a potent immunomodulator achieving its antiviral action through enhancement of nonspecific host defense mechanisms. Combined treatment of GLA-60 with the antiviral agent acyclovir (ACV) resulted in greater protection against HSV-1 in the CY-immunosuppressed mice than did single treatment with either GLA-60 or ACV.

Enhancement of nonspecific resistance to bacterial infections and tumor regressions by treatment with synthetic lipid A-subunit analogs. Critical role of N- and 3-O-linked acyl groups in 4-O-phosphono-D-glucosamine derivatives.

Enhancement of nonspecific resistance against Pseudomonas aeruginosa infection and regression of growth of Meth A fibrosarcoma by chemically synthesized lipid A-subunit analogs, 4-O-phosphono-D-glucosamine derivatives carrying 3-O- and N-linked acyl groups, were investigated. Compounds carrying an (R)-3-hydroxytetradecanoyl (C14-OH) group at the 2-N-position with (R)-3-tetradecanoyloxytetradecanoyl [C14-O-(C14)] or (R)-3-dodecanoyloxytetradecanoyl [C14-O-(C12)] groups at the 3-O-position, termed GLA-60 or GLA-63, respectively, showed strong activity about one-tenth that of natural lipid A. The protective activity of compounds carrying an (R)-3-hexadecanoyloxytetradecanyl group instead of a C14-O-(C14) or C14-O-(C12) group was very weak. GLA-59 carrying the same acyl components as those of GLA-60 but with reversed binding sites showed significant but not so strong protective activity. The activity of compounds possessing a tetradecanoyl group instead of a C14-OH group in GLA-60 or GLA-63 was weaker than that of GLA-60 or GLA-63. Intravenous or intratumoral administration of GLA-59, GLA-60 and GLA-63 induced significant regression of Meth A fibrosarcoma in terms of tumor size, tumor weight and number of cured mice. The activity of GLA-59 was almost equivalent to that of GLA-60. None of the tested compounds exhibited significant pyrogenicity at a dose of 10 micrograms/kg in rabbits.

Enhancement of nonspecific resistance to viral infection by chemically synthesized lipid A-subunit analogs with different backbone structures and acyl groups.

Protection against vaccinia virus infection and induction of interferon (IFN) were investigated in Propionibacterium acnes-primed mice following treatment with chemically synthesized lipid A-subunit derivatives. The antiviral activity was based on the reduction of numbers of tail lesions in mice injected intravenously with the test compounds 1 day before virus infection. GLA-27, a 4-O-phosphono-D-glucosamine carrying 3-O-tetradecanoyl (C14) and N-3-tetradecanoyloxytetradecanoyl [C14-O-(C14)] groups, offered significant antiviral activity. Chemical modifications at the C1 position of GLA-27, e.g. phosphorylation, replacement of OH by an SH, did not cause a significant change in antiviral activity. GLA-57 carrying an N-3-dodecanoyloxytetradecanoyl group showed stronger activity than GLA-27, but GLA-58 carrying an N-3-hexadecanoyloxytetradecanoyl group did not exhibit significant activity. GLA-59 carrying 3-O-3-hydroxytetradecanoyl and N-C14-O-(C14) groups was more active than GLA-27 and GLA-57. GLA-60 possessing the same fatty acid substituents as GLA-59 but in the reversed order was the most active of all compounds tested. This suggests that the nature and position of the acyl substituents are important for achieving the antiviral effects. The (R) isomers of GLA-59 and GLA-60 possessed stronger IFN-inducing activity than the (S) isomers, but no significant difference in antiviral activity was seen between the isomers.

Effect of chemical modification at C1 of the glucosamine backbone of lipid A-subunit analog GLA-27 on manifestation of immunopharmacological activity.

The effect of chemical modification at the C1 position of GLA-27, 4-O-phosphono-D-glucosamine carrying N-3-tetradecanoyloxytetradecanoyl [C14-O-(C14)] and 3-O-tetradecanoyl (C14) groups, was investigated. Replacement by SH or S-acetyl groups of the OH group resulted in the enhancement of mitogenicity but gave rise to a reduction, in macrophage-stimulating ability such as induction of tumor necrosis factor and enhancement of phagocytic and cellular acid phosphatase activities. Bisphosphorylation at C1 and C4 resulted in a slight decrease in mitogenicity or almost complete loss of the macrophage-stimulating ability.

Antitumor and antimicrobial activities of lipid A-subunit analogue GLA-27.

Activities for enhancing nonspecific host resistance against microbial infections and growth inhibition against tumors by a lipid A-subunit analogue GLA-27 were investigated. GLA-27, a 4-O-phosphono-D-glucosamine derivative with 2-N-3-tetradecanoyloxytetradecanoyl and 3-O-tetradecanoyl group, inhibited significantly the growth of solid-type tumors RL male 1 lymphoma and Meth A fibrosarcoma. In the case of RL male 1 lymphoma, 2.5 micrograms of GLA-27 was needed to exhibit the activity, while for Meth A fibrosarcoma, 250 micrograms of GLA-27 was needed. On the other hand, tail lesions induced by intravenous inoculation with vaccinia virus were effectively suppressed in mice injected with 1 microgram of GLA-27 1 day before the viral injection. The antiviral activity of GLA-27 was 100 times higher than that of MDP by ED50. GLA-27 also showed nonspecific protective activity against Pseudomonas aeruginosa infection at a dose of 30 micrograms/mouse, although the activity was far less than that induced by lipid A. GLA-27 did not exhibit toxic activities such as pyrogenicity and Shwartzman reaction.

Antiviral and immunomodulating activities of chemically synthesized lipid A-subunit analogues GLA-27 and GLA-60.

Biological and antiviral activities of chemically synthesized lipid A-subunit analogues, GLA-27 and GLA-60, were investigated with respect to defense mechanisms such as macrophage and natural killer (NK) cell activation and interferon (IFN)-inducing activity. GLA-27, a 4-O-phosphono-D-glucosamine derivative carrying 3-O-tetradecanoyl (C14) and 2-N-3-tetradecanoyloxytetradecanoyl (C14-O-(C14] group, and GLA-60, a similar analogue carrying 3-O-linked C14-O-(C14) and 2-N-linked 3-hydroxytetradecanoyl (C14-OH) groups, strongly inhibited the formation of pox tail lesions and the growth of vaccinia virus at the tail lesion sites in infected mice. The antiviral activity of GLA-60 was about 1000-fold higher than that of muramyldipeptide (MDP), a representative immunomodulator. GLA-27 and GLA-60 had stronger immunomodulating activity than MDP in macrophage activation, NK cell activation and IFN-inducing activity, although it was weaker than natural lipid A. Toxic manifestations such as pyrogenicity, local Schwartzman reaction and lethality were far less pronounced for GLA-27 and GLA-60 than for natural lipid A.

Immunopharmacological activities of chemically synthesized lipid A-subunit analogue GLA-27 combined with muramyl dipeptide via spacers of different carbon chain length.

A lipid A-subunit analogue GLA-27, a 4-O-phosphono-D-glucosamine derivative carrying 3-O-tetradecanoyl and N-3-tetradecanoyloxytetradecanoyl groups, exhibited significant biological activities but no detectable pyrogenicity or local Shwarzman activity. In order to synthesize compounds combining GLA-27 with muramyl dipeptide (MDP), the OH group at the C6 position of GLA-27 was first succinylated. A compound which combined a succinylated GLA-27 (GLA-101) with 1-deoxy N-acetyl-muramyl-L-alanyl-D-isoglutamine methyl ester (1-deoxy MDP) via spacers of different carbon chain lengths of 5, 11 and 15, termed GLA-105, GLA-106 and GLA-107, respectively, and their biological activities were investigated. Intraperitoneal administration of combined preparations with spacers, GLA-105 and GLA-107, induced much higher phagocytic activity in peritoneal macrophages than GLA-27, GLA-101 and 1-deoxy MDP. The activity of GLA-106 did not increase by the combination. In induction of natural killer (NK) activity in peritoneal cells, GLA-105 and GLA-107 were significantly more active than 1-deoxy MDP but only comparable with GLA-27. GLA-101 showed stronger NK activity than GLA-27. The activity of GLA-106 was stronger than 1-deoxy MDP but weaker than GLA-27, GLA-105 and GLA-107. Mitogenic, interferon-inducing and tumor necrosis factor-inducing activities decreased by combining GLA-101 with 1-deoxy MDP. GLA-101, GLA-105 and GLA-107 strongly inhibited the formation of lesions on the tail of mice infected with Vaccinia virus. The activity was almost equivalent to that of GLA-27.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of stereospecificity of chemically synthesized lipid A-subunit analogues GLA-27 and GLA-40 on the expression of immunopharmacological activities.

Tumor necrosis factor (TNF)-inducing, mitogenic, polyclonal B cell activation (PBA), macrophage activation and antiviral activities of chemically synthesized lipid A-subunit analogues, GLA-27 and GLA-40, were investigated. The structure of GLA-27 comprises 4-O-phosphono-D-glucosamine carrying tetradecanoyl and 3-tetradecanoyloxytetradecanoyl [C14-O-(C14)] groups as the 3-O- and 2-N-acyl substituents, respectively. GLA-40 is a 1-deoxy compound of GLA-27. The activities of stereoisomers, (R) and (S) forms at the C3 position of the C14-O-(C14) group, of both compounds were also investigated. TNF-inducing activity of the (S) isomers of GLA-27 and GLA-40 was stronger than that of the (R) isomers while the (R) isomers exhibited stronger mitogenic and PBA activities than the (S) isomers. With respect to macrophage activation such as phagocytosis, acid phosphatase and N-acetyl-beta-D-glucosaminidase activity as cellular lysosomal enzymes and cytostasis, peritoneal macrophages obtained from mice administered i.p. with test samples showed significant activities. Among stereoisomers of GLA-27, the (R) isomer exhibited somewhat stronger phagocytic and lysosomal enzyme activities than those of the (S) isomer while there was no appreciable difference in the activities between the isomers of GLA-40. Significant cytostasis-inducing activity was observed in stereoisomers tested. All of the isomers showed remarkable antiviral activity against vaccinia virus.