because of its anticoagulant activity, poor absorbance, and instability (Abrams et al., 1989; Lorentsen et al.,. 1989; Flexner et el., 1991). Lentinan (branched (1~3)-~.
Antiviral Chemistry & Chemotherapy (1995) 6(4), 271-280
Sulfated alkyl oligosaccharides inhibit human immunodeficiency virus in vitro and provide sustained drug levels in mammals H. Nakashima,":" K.lnazawa,1 K.lchiyama,1 M.lto,1 N. lkushlma," T. Shoji,2 K. Katsuraya," T. Uryu,3 N. Yamamoto," A. S. Juodawlkls" and R. F. Schinazi5 ,* 1Department of Microbiology, Yamanashi Medical University, 1110 Shimokato Tamaho-cho, Nakakomagun, Yamanashi, Japan. "cemre; Research Laboratories, Dainippon Ink and Chemicals, Inc., Chiba, Japan. 31nstitute of Industrial Science, University of Tokyo, Tokyo, Japan. "Depertmem of Microbiology, Tokyo Medical and Dental University, Tokyo, Japan. "veterene Affairs Medical Center and Department of Pediatrics, Laboratory of Biochemical Pharmacology, Emory University School of Medicine, 1670 Clairmont Road, Decatur, Georgia 30033, USA.
Summary This study provides an estimate of the relative antihuman immunodeficiency virus (HIV) activities of synthetic sulfated alkyl oligosaccharides in vitro and of their mechanism of action, and an assessment of the levels of alkyl oligosaccharides in small mammals. The antiviral activities of the compounds against several human immunodeficiency virus type1 and type·2 strains were determined in human CD4+ cells, including primary lymphocytes and macrophages. laser flow cytometry and a cell-based syncytium assay were used to elucidate the antibinding/fusion properties of the oligosaccharides. The sulfated alkyl laminarioligosaccharide Dl-110 was shown to be the most potent and selective antiHIV agent in culture with a median inhibitory concentration of 0.2 JlM in primary human lymphocytes. This compound did not markedly interact with the CD4+ receptor on lymphocytes at 50 JlM, but demonstrated potent anti-syncytium properties in vitro at submicromolar concentrations. Dl-110 had no anti-coagulation activity at 38 JlM. Mice, rabbits and beagle dogs were given an intravenous injection of test comReceived 21 December, 1994; revised 3 March, 1995; accepted 6 March, 1995. *For correspondence. Tel. 404·728·7711; Fax 404-728-7726.
© 1995 Blackwell Science Ltd
pounds and the drug levels in serum were quantified. When 32 mg kg-1 of Dl-110 was administered to mice, significant antiviral concentrations in serum were achieved even 12 h after treatment. Similarly, prolonged antiviral effects were noted in rabbits and dogs 24 h after injection of Dl·11 O. The half·life of Dl110 in mice, rabbits and dogs was estimated to be 5 h. Dl·110 and some of its derivatives are promising candidates for further evaluation of the prophylaxis and therapy of HIV infections. Key-words: AIDS; antiviral agents; CD4; HIV; oligosaccharides. Introduction Sulfated polysaccharides, such as dextran sulfate, heparin and pentosan polysulfate, are potent inhibitors of the replication of human immunodeficiency virus (HIV) in vitro through interference with viral binding to CD4 receptors (Nakashima et el., 1987a,b; Ito et sl., 1987; Saba et ei., 1988a,b). Of these, dextran sulfate has not been clinically utilized as a therapeutic agent against HIV infection because of its anticoagulant activity, poor absorbance, and instability (Abrams et al., 1989; Lorentsen et al., 1989; Flexner et el., 1991). Lentinan (branched (1~3)-~ glucan) sulfate (Yoshida et al., 1988) and curdlan (linear (1 ~3)-~-glucan) sulfate (Kaneko et si., 1990) showed high anti-HIV potencies and relatively low anticoagulant activities. The biological activities of these compounds were closely related to the molecular weight and the degree of sulfation (Nakashima et al., 1989). We recently reported the synthesis of sulfated alkyl oligosaccharides with potent anti-HIV activities in vitro (Uryu et al., 1992, 1993). These compounds were composed of hydrophilic sulfated oligosaccharide portions and hydrophobic long alkyl portions, with structures and properties characteristic of surface-active agents. The anti-HIV activities of compounds with molecular weights of 1500-3000 were almost equivalent to those of the high-molecular-weight dextran sulfate (8000) or curdlan sulfate (79000). The objective of this study was to estimate the inhibitory effects of sulfated alkyl oligosaccharides (Fig. 1) on the replication of several HIV strains in vitro and to
272
H. Nakashima et al.
Fig. 1. Structure of sulfated alkyl oligosaccharides. (a) Sulfated alkyl malto-oligosaccharide; (b) sulfated alkyllaminari-oligosaccharide.
120
establish their anti-binding/fusion properties and primary mechanism of action. We also investigated whether alkyl oligosaccharides administered to animals can be delivered systemically in a form effective against HIV for a prolonged period of time.
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Anti-HIVactivities of sulfated alkyl oligosaccharides The anti-human immunodeficiency virus type-1 (HIV-1) activities of sulfated laminarioligosaccharide (0-5) and OL-110 were assessed by the protection of the HIV-1induced cytopathic effect (CPE) and inhibition of virusspecific antigen expression in MT-4 cells in vitro (Fig. 2). While untreated HIV-Hnfected MT-4 cells did not remain viable after virus infection, both 0-5 and OL-11 0 showed cell-protective activity against virus-induced CPE in a dose-dependent manner. The potency of OL-110, which has a terminal long alkyl chain, was almost 10 times greater than that of 0-5; the median inhibitory concentrations (ECso) of OL-110 and 0-5 were 0.2 and 1.8 J..lM, respectively. Several sulfated alkyl oligosaccharides characterized by different lengths of sugar and of alkyl chains, different linkage type and different terminal alkyl groups were evaluated in MT-4 cells and human peripheral blood mononuclear cells (PBMC) (Table 1). The ECso values of LDL-1, OL-110, LTE-1 and LTR-2 [all had a ~(1...-73) linkage and a terminal alkyl chain of n-C1 2H2 S ; sugar chain lengths were 10,5,4 and 3, respectively] in HIV-1-infected MT-4 cells
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.a The ECso is the concentration required to achieve 50% inhibition of the HIV-1-induced cytopathic effect in MT-4 cells as determined by the MTT method. Abbreviations: D-5, D-Iaminari-pentaose sulfate; DL-110, n-dodecyl ~-D-Iaminari-pentaoside sulfate; LTE-1, n-dodecyl /3-D-laminari-tetraoside sulfate; LTR. 2, n-dodecyl ~-D-Iaminari-trioside sulfate; LDL-1, n-dodecyl ~-D-Iaminari-decadoside sulfate; MAL-l, n-dodecyl ~-D-malto-pentaoside sulfate; MAL-2, ndodecyl ~-D-isomalto-pentaoside sulfate; DL-10, n-decyl ~-D-Iaminari-pentaoside sulfate; LDP-1, n-docosyl ~-D-Iaminari-pentaoside sulfate; DL-24, 4-n-octyloxyphenyl /3-D-laminari-pentaoside sulfate; DL-16, dl-u tocopheryl ~-D-Iaminari-pentaoside sulfate, and DLPM-9, 4-t-octylphenyl ~-D-Iaminari penta oside sulfate, b EC so in PBMC is calculated as the 50% reduction of RT activity of the culture supernatant. Please note that these data were generated using HIV-1 LA 1• C Cytotoxicity (CC so), the concentration required to reduce the viability of mock-infected MT-4 cells by 50% after 5 days of incubation in the presence of the compound. d CC so in PBMC is determined by ["Hl-thymidine incorporation, except for AZT for which cell counts were used. Data represent the average values for at least duplicate experiments.
were 0.04, 0.2, 0.9 and 44.1 JlM, respectively. The results suggest that the anti-HIV activities of these sulfated alkyl oligosaccharides depended on the length of the sugar chain. However, the compound with five sugar chains had good anti-HIV-1 activity. The EC5 0 for DL-110, LTE-1, and LTR-2 in HIV-1-infected PBMC were 0.2,0.9 and 7.2 11M, respectively. The EC5 0 values of DL-10, DL-110 and LDP-1 [which had a ~(1-73) linkage and a sugar chain length of 5, but terminal alkyl chains of n-C10H 2 1 and nC1 2H2 5 , n-C2 2H4 5 , respectively] in HIV-1-infected MT-4 cells were 12.9, 0.2, and 0.3 11M, respectively. This suggests that the anti-HIV activity of this group depends on the length of the terminal alkyl chain, but n-C12H 2 5 showed strong activity. Comparison of the EC5 0 values of DL-24, DL-16 and DL-110 [which had a P(1-73) linkage and a sugar chain length of 5, but whose aglycons consisted of n-octyloxyphenyl, dl o-tocopheryl and an nC12H2 5 alkyl chain, respectively], demonstrated almost equipotent anti-HIV activities. However, the cytotoxicity of DL-16 was greater than that of DL-24 or DL-110. EC5 0 values of MAL-1 and MAL-2 [which had five sugar chains with a terminal n-C12H 25 alkyl, but a linkage of u(1 -7 4) and u(1 -7 6), respectively] in HIV-Hnfected MT-4 cells
were 0.2 and 0.6 11M, respectively (Table 1). Anti-HIV activity of DL-110 and LDL-1 was observed with several HIV-1 strains and HIV-2 Ro D in MT-4 cells and human peripheral macrophages (Table 2). Irrespective of the criteria used to assess anti-HIV activity (I.e., inhibition of viral cytopathic effect, antigen expression and p24 production), both DL-110 and LDL-1 inhibited HIV-1, including an 3'-azido-3'-deoxythymidine (AZT)-resistant strain. No cytotoxicity was observed with any of these compounds when tested up to 100 11M (Table 1). These results indicate that sulfated alkyl oligosaccharides consisting of five sugar chains and an n-C12 H25 alkyl chain have potent and selective anti-HIV activity in vitro.
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To elucidate the mechanism of action of sulfated alkyl oligosaccharides, DL-110 was evaluated to determine whether it inhibited the binding of HIV-1 particles to MT-4 cells, as assessed by laser flow cytometry analysis. DL-110 inhibited HIV-1 binding by 95% at 9.5 11M. This inhibitory activity was comparable to that of dextran sulfate (data not shown).
© 1995 Blackwell Science ltd, Antiviral Chemistry & Chemotherapy, 6, 271-280
274
H. Nakashima et al.
Table 2. Antiviral activity spectra of sulfated alkyl oligosaccharides and 2',3'-dideoxynucleoside analogues
Virus and strain
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MTT p24 IF IF
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24.8 0.13 12.9 12.2
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0.18
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18.6
Antiviral activity (EC so, 11M)"
a ECso is calculated on the basis of the inhibition of the HIV-induced cytopathic effect or HIV antigen expression in MT-4 cells, or the reduction of p24 antigen in the culture supernatant of human peripheral macro phages. Data represent mean values for at least three separate experiments. b Monocyte/macrophage-tropic HIV-1 strain. c AZT-resistant HIV-1 strain.
Inhibition of syncytium formation
Co-cultures of persistently HIV-infected MOLT-4 cells (MOLT-4/HIVlIIs ) and uninfected MOLT-4 cells were used. Dextran sulfate and DL-110 effectively inhibited syncytium formation; both their ECso values were -0.8 !!M (6.4 and 2.1 !!g ml-\ respectively) (Fig.3). Since DL-110 and DLPM-9 (for full chemical names, see Table 1) are charged molecules, it was important to determine whether they interacted with the CD4+ receptor on lymphocytes. It appears that DL-110 and DLPM-9 did not markedly (35.2 and 15.6%, respectively at 50 !!M) affect the binding of anti-CD4 antibody to CD4+CEM cells (data not shown). In contrast, HPA-23, used as a positive control, strongly interfered with CD4+ binding at 100 !!M (80.9% inhibition). In an assay of fusion between CEM and TF228.1.16 cells, DL-110 and DLPM-9 had potent activities at a concentration of 1 !!M; their ECso values
were 0.05 and 0.25 !!M, respectively. Dextran sulfate (8000) and suramin, which were used as positive controls, inhibited fusion with ECso values of 0.34 and 11.5 '!!M, respectively; DL-110 was almost 1 order of magnitude more potent than dextran sulfate (8000) under similar assay conditions. Effect of serum concentration on in vitro an ti-Hl V activity of DL-110
The anti-HIV activity of DL-110 was not altered by pretreatment with human or animal serum (Table 3). As expected, the antiviral activities of AZT, 2',3'-dideoxyadenosine (ddA) and 2',3'-dideoxyinosine (ddl) were also not significantly affected by pretreatment of FCS (Tables 2 and 3). The anti-HIV activities of DL-110, ddl and dex-
Table 3. Anti-HIV-1 activity of DL-11 0 pretreated with various mammalian sera in MT-4 cells
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