J. Yuzuru Homma

Biological activities of chemically synthesized analogues of the nonreducing sugar moiety of lipid A

Biological activities of five synthetic lipid A analogues (D‐glucosamine derivatives) were examined to elucidate the structure required for expression of the biological activities of endotoxin. Proclotting enzyme of horseshoe crab activation, interferon‐inducing and tumor necrosis factor‐inducing activities were significantly expressed by an analogue which possesses 4‐O‐phosphoryl, 3‐O‐tetradecanoyl and N‐tetradecanoyloxytetradecanoyl groups. The results obtained with different analogues show that the 4‐O‐phosphoryl and N‐tetradecanoyloxytetradecanoyl groups are important for expression of the above activities. The effect of 6‐O‐acylation in preventing the expression of these biological activities is also suggested. Pyrogenic activity was not detected in any of the compounds tested.

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.

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 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 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.

Differences of chemical structures of Pseudomonas aeruginosa lipopolysaccharide essential for adjuvanticity and antitumor and interferon-inducing activities

Adjuvanticity, antitumor and interferon-inducing activities of a protein rich endotoxin isolated from an autolysate of Pseudomonas aer-uginosa have been demonstrated [l-4]. It has been reported that both antitumor and interferon-inducing activities reside in the lipopolysaccharide portion of the proteinlipopolysaccharide complex [S]. Moreover, chemical structures of the lipopolysaccharide essential for antitumor and interferon-inducing activities were clarified [ 51. Here the relations~p between chemical structures of lipopolysaccharide as well as their derivatives and adjuvanticity were investigated, comparing the results with those of antitumor and interferon-inducing activities previously reported.

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.

Comparison of the effects of a multi-component vaccine and a formalin-killed cell vaccine on protection against enzootic of hemorrhagic pneumonia due to Pseudomonas aeruginosa in mink.

Effectiveness of a multi-component vaccine consisting of the common antigen (OEP) derived from Pseudomonas aeruginosa strain N 10 (serotype E) and toxoids of protease and elastase was compared with that of formalin-killed cells of strain N 10 on protection against enzootic of hemorrhagic pneumonia due to P. aeruginosa in mink. One administration of the multi-component vaccine (100 microgram each of OEP, protease toxoid and elastase toxoid) clearly prevented enzootic of hemorrhagic pneumonia due to P. aeruginosa (serotype G) in mink, while a vaccination of formalin-killed cells was much less effective in preventing an epidemic. The difference in mortality rates between two vaccines was remarkable.

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.