Public Interest Note

On Putative Critical Role of Cell-Surface Polyanions as Broad-Spectrum Anti-Pathogens

Indications from the literature and laboratory notes on the anti-pathogen including the anti-HIV
activities of polyanionic substances

D. Grant, Turriff UK

Summary
The early years of intense research activities which had been undertaken in an attempt to quickly
identify useful anti- human immunodeficiency virus (HIV) drugs which might have helped to combat
AIDS, had identified numerous classes of polyanionic substances which could inhibit the replication of
this virus which exhibited additional broad spectrum anti-pathogen activities. Examples of such
substances include sulphated polysaccharides, lignin derivatives and inorganic polyphosphates.
Further research is now suggested to be required to optimise the purification and formulation such
polyanionic preparations in order to maximise their anti-pathogen activities. It is e.g. of interest to
determine if inorganic silicon and phosphorus containing molecules commonly found (e.g. in some
incalcated form) to be attached to the naturally occurring polyanionic polysaccharides can beneficially
influence their in vivo anti-pathogen anti-cell surface adhesion activities.
It is pointed out that since the sulphated polysaccharide carrageenan which has recently failed to
demonstrate an expected high level of anti-HIV barrier gel effectiveness in clinical trials can enter
tissues where it can behave to act as an inflammatory agent which can also directly promotes HIV
infection, a circumstance which however also suggests that the effectiveness of such gels might be
greatly improved by the incorporation of suitable anti-inflammatory (e.g., anti-tumour necrosis factor
alpha [TNFα ]) agents in their formulation.

Introduction

Owing to the growing resistance of pathological organisms to the currently used antibiotics there is an
urgent need to properly identify novel types of microbiocides.
It is now suggested that the exogenous administration of therapeutic agents which can upgrade the
natural cell surface polyanionic barriers -to-adsorptiona and host cell entry of numerous types of
pathological agents might be a useful concept which can be adapted to this task.

The administration of heparinoids (which mimic the ubiquitous animal cell surface protective system of
heparan sulphate family of sulphated polysaccharides) might by signalling for the upgrading of natural
polyanionic defence mechanisms, allow for novel, more highly effective methods not only of
combating drug-resistant bacteria but also of combating all kinds of infections.

Polyanionic Tissue Protective Systems which are

Putatively Adaptable for AIDS Therapy
A number of polyanionic systems seem e.g. to be potentially adaptable to potentially provide anti-
AIDS therapeutic agents. The type of polyanionic molecules which seemed to have promise for
combating the primary HIV viral as well as the secondary opportunistic infections associated with
AIDS contain sulphonate, sulphate half-ester (e.g. the products obtained e.g. by sulphation of plant or
algal polysaccharides using pyridine/chlorosulphonic acid b) phosphonate or phosphate anionic groups
which showed potent in vitro antiviral, including anti-HIV activities. Schwarz PNAS 1973 70 1608-
12) reported that such polysaccharides always seem to occur naturally in association with inorganic
silicon (but the amounts of this and other inorganic elements present can be greatly diminished by
conventional purification processes [this phenomenon seems to have been, at least partly, responsible
for the lack of consistency in the studies of the antiviral activities of polyanionic polysaccharides]).
The presence of such inorganic cofactors could, it is now pointed out, also have contributed directly to
the observed antiviral activities of such polysaccharide extracts since silicate nanoparticles (the most
likely form present in natural polysaccharides) can exhibit antiviral properties per se (cf. e.g. Rahman
et al., Naturwiss 2009 96 31-8). Such nanoparticles (e.g. the former Monsanto product Santocell) can
also act as effective insecticides.
Evidence that the inorganic phosphates which apparently can co-puify with heparin-like molecules and
can putatively greatly modify their biochemical activities, might also contribute to the antiviral
activities of these polysaccharides comes from the report (Lorenz et al. J Acquire Immune Defic Syndr
Hum Retrovir 1997 14 110-8) that purely inorganic phosphate polyanionic molecules can, acting alone,
both inhibit HIV proliferation and syncytium formation in vitro.
This suggests that the exogenous administration of inorganic elements might also in suitable form,
improve the natural anti-HIV activities of the cell surface polyanionic polysaccharides molecules
which occur at all adherent animal cell surfaces.
Highly sulphated semisynthetic polymers could, it might be predicated, be enabled to act more
effectively act as microbiocides in animal tissues under conditions where they can more closely mimic
fragments of heparan sulphate polysaccharides which are natural messenger molecules which impact
on the cellular immune system. This fragmentation process is accomplished by heparanase and by
nitric oxide metabolite scission. The latter action, which is capable of being regulated by exogenous
drug administration (e.g. by nitric oxide boosters) which may conceivably boost the antiviral potency
of the natural antiviral polysaccharide systems.
An analogous argument can be applied to manner that the high molecular weight cells surface
polyphosphates might behave in vivo.
The full evaluation of the in vivo potential of sulphated polysaccharides and other polyanionic
substances as antiviral agents of possible use for the treatment of AIDS victims has however, not to
been pursued to any rational conclusion since more effective anti HIV drugs became available before
this task had been completed and furthermore the potency of many of these substances had been
indicated in preliminary tests to be considerably reduced in the presence of blood serum. The
sulphated polysaccharide heparinoid types of possible antiviral drugs a further major perceived
problem since some of them exhibited undesirable in vivo blood anticoagulant activities. Nevertheless,
it should be noted that, towards the end of this preliminary antiviral substance evaluation period, a type
of low molecular weight heparin, was reported, on the basis of the favorable results of a small scale
clinical trial, to offer potentially useful novel therapeutic benefits to AIDS victims (cf., Howell Int J
Clin Lab Res 1996 26 124-31). This is likely to, at least partly be because such heparin can both
inhibit viral replication as well as combat the numerous opportunistic infections which are associated
with AIDS.

It should be noted that cell surfaces throughout biota are associated with both inorganic and organic
polyanions, all of which might be capable of inhibiting viral activities. The incidence of viral infections
may at least, it is now suggested, could at least in part be dependent on the occurrence of deficiencies
in this and other possible antiviral systems especially the anionic polysaccharides.
The most ubiquitous inorganic polyanion system which occurs widely throughout biota is believed to
be inorganic polyphosphate (cf. e.g. Brown & Kornberg PNAS 2004 101 16085-7). Kornberg had
earlier suggested that this polymer was of importance in the first stages of the evolution of life.
It should be noted that both short and long chain polyphosphates can also demonstrate moderately
strong anti- HIV activity in vitro (cf., Lorenz et al., J Aquire Immune Defic Syndr Hum Retrovirus
1997 14 (2) 110-8 {these inorganic polyanions can apparently inhibit both viral replication and
syncytium formation which is a phenomenon of cell fusion which is thought to promote the spread of
HIVviruses in vivo}. This suggests that the inorganic polyphosphate molecules and their adducts
which occur at numerous types of cell walls in humans might also be enabled under some
circumstances to contribute to the natural in vivo antiviral defense system. Is this of relevance to how
humans become infected with HIV? Could the efficiency of that the putative cell surface antiviral
systems might allow for an improvement of their effectiveness via therapeutic intervention?

It should be further noted that at least a portion of the natural cell surface inorganic polyphosphate is
incalcated at cell walls in calcium poly-β -hydroxy butryate (cf. Reusch Proc Soc Exp Biol Med 191
377-81). Perhaps this interaction by inhibiting the availability and access to inorganic polyphosphate
molecules can inhibit the antiviral action of the polyphosphate system.
Inorganic polyphosphates may also, co-exist in vivo together with the anti-adhesive antiviral
glycosaminoglycans. This hypothesis is in accord with the paradoxical ability of the animal cell
surface heparin/heparan sulphate systems to be able to bind both cations and anions. A well-
established example is the co-occurrence of inorganic silicate and phosphate with heparin (the
unidentified phosphate containing molecule has previously often assumed to be monomeric phosphate
but may conceivably include some ‘antiviral polyphosphate’. [It should also be noted that the presence
of such molecules in pharmaceutical heparin could dependent on the method of purification which
seem to vary between manufacturers]. A possible antiviral scenario is the release of such
polyphosphate following nitrosative degradation of the polysaccharide. A further possibility is that
such interaction might be critically dependent on the chemical nature of the inorganic counterion
cocktail present. as this nonenzymic nitrosative scission process is known to require inorganic
cofactors such as Cu and Zn. {Such inorganic cofactors can also impact on interphase water structure
putatively involved in viral fusion processes}.
(N.b. it has been reported that the anti-HIV activity of a sulphated polysaccharides can strongly depend
on the chemical nature of such inorganic counterions has been confirmed by studies of the anti-HIV
potency of a Spirulina species derived sulphated polysaccharide anionic antiviral agent which was
found to be highly dependent on the chemical nature of the counterions present (Lee Chem Pharm
Bull 2001 49 108-111). Some of these may not be easily replaceable as would be the case for simple
salt formation. It may be possible therefore to adjust the counter-ion in other experimental sulphated
polysaccharides so to achieve optimal antiviral and anti-bacterial effectiveness.

It should be noted that in vitro studies of polyanions having a similar chemical structure to abundant
naturally occurring cell surface polyanions (or which can mimic their chemical behaviour) have been
shown to be highly effective inhibitors of the pathological activities of misfolded prions, enveloped
viruses, bacteria, fungi, protozoa, toxic crystals and fibrils.
The polyanionic broad spectrum anti-pathogen antiviral drugs, although showing early promise, were
apparently eventually not put into wide clinical use (a possible exception seems to be ι + κ
carrageenan which has reported, cf. Ghosh loc. cit. which reports the clinical use of these sulphated
polysaccharides as topical anti-HIV protective agents); other anti-retroviral therapeutic methods which
had, by 1988 (cf. e.g. Richman Infect Dis Clin N Amer 1998 2 397-407) found favor (and later more
successfully employed in multi-synergistic drug protocols) are generally believed to have achieved an
acceptable “cure” for the AIDS epidemic although this seems strictly not to be a accurate assumption.
It may yet be the case that the sulphated polysaccharide and analogous substances, because they offer a
wide spectrum anti-pathogen activity suitable for combating the wide range of opportunistic infections
which are characteristic of AIDS and can also inhibit mutated HIV viruses, could offer major future
therapeutic benefits (cf. Luscher-Matti 2000, Antivir Chem Chemother 11(4) 249-59).
A possible beneficial synergistic interaction might also be achievable with a combination anti AIDS
therapy containing more than one of the known anti-HIV anionic systems which include polysulphated,
polysulphonated and polycarboxylated polymers as well as polyphosphates which have been identified
by in vitro experimentation to inhibit HIV viral activities.

(Cf., also vide infra, old literature anti-HIV sulphated polysaccharides and related substances
excerpted from a personal research historical database).

Footnote a
A possible rationalization of the ability of various classes of polyanions to achieve broad spectrum anti-
pathogen protection effects is that such polyanions are able to mimic the heparan sulphate-based tissue
protection including antiviral mechanisms which apparent exist in all multicellular animals involving
the ability of heparin like molecules (heparinoids) both to block the entry of viruses and bacteria which
enter host cells to host cells through heparan sulphate receptors but also to upregulate the biosynthesis
of such heparan sulphate cf., e.g. Pinal et al. Thromb Res 1004 74 143) containing cell surface barriers.
A further corollary to this idea is that appropriate drugs for inclusion in any antiviral combination
therapy might consider the inclusion of substances which boost the degree of sulphation of the cell
surface heparan sulphates a modification which also seems to augment their barrier and tissue
protection potencies; such heparan sulphate boosters include ascorbate, retinoic acid as well as
exogenousously administered heparin.

Footnote b
Cf. web.scribd.com/doc/34916461/Why-is-PPS-Such-an-Effective-Anti-NvCJD-Agent-Revised
Appropriate researches have apparently not yet been performed to fully analyse the extracts of algae, plants etc., so
as to fully quantify their natural inorganic cofactor profiles (cf. b-1) or, e.g., to confirm if preliminary indications
that in vitro anti-HIV activities of sulphated polysaccharides can be (perhaps greatly) increased by using an
appropriate salt (counterion) form of the potential antiviral sulphated polysaccharide and if an augmentation of the
broad spectrum anti-pathogen potency might also be achievable for in vitro and in vivo health benefits by altering
the inorganic cofactor composition of polyanionic molecules which have demonstrated potent in vitro anti-HIV
and other anti-pathogen potency.
b-1
Cf. web.scribd.com/doc/34178257/WRL1873-With-Fig
and
web.scribd.com/doc/26994439/Publication-2-Web

Cf. also documents in which a hypothesis the ‘Ascorbate and Nitric Oxide Control of Heparan Sulphate
was presented by D Grant in 2000 at
web.ukonline.co.uk/dg4/ (outline of the hypothesis);
web.ukonline.co.uk/dg5/ (appendix)
web.ukonline.co.uk/dg8/ (references)

Cf. also publications of WF Long et al. listed at web.abdn.ac.uk/~bch118/publications2003march.doc

which includes most of the formal research publications of the University of Aberdeen former
polysaccharide laboratory (which had been headed by Professor WF Long with Dr FB Williamson).

Old Literature Collection – Reappraisal

SUGGESTED STARTING POINTS FOR THE EVALUATION OF ROLES OF INORGANIC
COFACTORS FOR THE OPTIMISATION OF ANTI-VIRAL & ANTI-PATHOGEN
ACTIVITIES OF ANIONIC POLYSACCHARIDES Etc.
Some at least of these kinds of anti-pathogen polyanionic molecules (e.g. identified in the following
reference list) are now believed to occur naturally in the form of multi-inorganic element complexes
(“metallomic” matrices). The ability of these polyanions to sequester multiple inorganic cofactors
could be highly relevant to the mechanism of their anti-pathogen potencies. A possible practical
problem which has emerged is that different extraction and purification procedures can dramatically
alter the inorganic cofactors which co-purified with these molecules.

The references listed below, which could form the basis of inorganic (multielement) biochemical
researches, were obtained from information database collected to ca. 1993 by D. Grant et al. [to which
a few more recent references had been added]. [This work had been commenced as part of a
multidisciplinary antiviral research program conducted at Marischal College Aberdeen UK, which had
attempted to assess the relative (anti-HIV) efficiency of numerous substances including various
sulphated polysaccharides. Other research programmes in these labororatories had shown by spark
source mass spectrometry that heparin-like molecules were seawater like multielement matrices
[cf. e.g. Grant et al. Biochem J 1987 244 143-9 ). These data can provide a convenient starting point for
via internet update searches to evaluate the current status of sulphated polysaccharide research].
The relevance of the anti-viral activities of sulphated polysaccharides is thought to be that these agents
can directly and indirectly reinforce the natural multi-pathogen effects of heparin/heparan sulphate an
endogenous tissue protection system which can potentially protect against all manner of pathogens and
hence is uniquely relevant to the possible harnessing of such properties to a combined drug system to
combat opportunistic infections which are a hallmark of AIDS. It should be noted that sulphated
polysaccharides seem to be able to successfully inhibit the various mutated forms of HIV ( the presence
of strains of HIV presents a bottleneck to the development of an effective vaccine from the prevention
of AIDS).

[*]][Papers which had been identified as a possible sources of information which could be of value for
the preparation of new anti-HIV-1polysaccharides].

PRIMARY PAPERS =P

P
Baba M Pauwels R Balzarini J Arnout J Desmyter J De Clercq E
Mechanism of inhibitory effect of dextran sulfate and heparin on replication of human
immunodeficiency virus in vitro
PNAS USA 1988 85 6132-6
[It was reported that dextran sulphate (mol. wt. 5000) and heparin (mol. wt. 15000) completely
protected MT-4 cells against HIV-1 –induced cytopathogenicity at 25 µ g/ml with EC50 values of 9.1
and 7.0 µ g/ml respectively, there being no observed toxicity to the host cells (at 625 µ g/m)[these
polysaccharides were also effective against HIV-2, herpes simplex I and II and some other enveloped
viruses]; the activities of different heparins seemed to correlate with the anticoagulant activities; these
polysaccharides both inhibited revere transcriptase and the adsorption to host cells, the latter process
was indicated to be the principal mechanism (a direct viral inactivation by these polysaccharide or
interference with the CD4 antigen receptor was not indicated); a predicted problem of creating
unacceptably high anticoagulant activity in patients was indicated for heparin but not for dextran
sulphate AIDS therapy]. [Mitsuya H et al., had simultaneously reported (Science 1988 240 646-649)
similar findings].

P
Ehlers B Diringer H
J Gen Virol 1984 65 1325-1330

P
De Clercq D
Cancer Lett 1970 8 9
Arzneim-Forsch/Drug Res 1985 35 (no.6) 1007=8
Cf. J Med Chem 1986 29 1561-9
Cf. Eur Pat Appln 0293826 31/05/88
Cf. Antiviral Res 1989 12 1-20
Cf. Microbiologica 1990 13 165-78

P
De Prost D Katlama C Pialouc G Karsenty-Mathonnet F Wolff M (1987)
Heparin-like anticoagulant associated with AIDS
Thrombosis Haemostasis 57 239
[Acquired heparin-like anticoagulant previously found with leukemia, systemic mastocytosis and
plasma cell myeloma was also found in a patient with AIDS]
(who indicated 0.32 and 0.52 IU heparin/ml by
calcium thrombin time and anti-Xa activity, respectively)
{This poses the question: does the animal organism instigate a heparin-like anti-HIV response?}.

*Iriki Y Suzuki T Nisizawa K Miwa T

Xylan of siphonaceous green algae
Nature 1960 187 (4731) 82-83
[The authors had reported that each of the main cell walls of Bryopsis maxima, Caulerpa anceps,
Halimeda cuneata collected on the Pacific coast not far from Tokyo and
Chlorodesmis formosana from Okinawa consisted, not of cellulose as had previously been thought
likely, but of xylan in which the xylose residues were linked by a 1,3 bond; the resistance towards
periodate oxidation of such xylans other than end groups allowed the degree of polymerisation to be
estimated as 45-67; the xylans obtained by Iriki et al. seemed also to contain ca. 9% glucose residues
Which had suggested that the cell wall xylans were actually glucoxylans (cf., however, Fukushi Y &
Maeda M, later described the preparation of a glucose-free xylan from the cell wall of Bryopsis
maxima Botanic Marina 1986 XXIX 387-90)
Iriki et al. had noted the previous work of
Mackie IM and Percival EE, J Chem Soc 1959 1159
which had established the presence of β -1,3 linkages in xylan from Caulerpa filiformis cell walls].
{This type of polysaccharide is suitable for chlorosulphonic acid/pyridien sulphation a procedure
which allows the formation of pentosan polysulfates}.

Cf. Doctor VM Sauls V

Isolation and anticoagulant properties of a new sulfated xylan: comparison with heparin and a sodium
pentosan polysulfate (SP54)
Thrombosis Research 1983 30 573-8
[This paper described the preparation of sulphated larch wood xylan which differed from SP54
(it’s mol wt was higher and its anticoagulant activity also higher; the relevance of this paper is that it
indicates that a range of xylan sulphates with potential antiviral potency which differ from that of SP54
are to be expected to exist; cf. also web.scribd.com/doc/34916461)].
P
Mitsuya H Popovic M Yarchoan R Matsushita S Gallo RC Broder S
Suramin protection of T cells in vitro against infectivity and cytopathic effect of HTLV-III
Science 1984 226 172-4
[Suramin had been reported in 1979 by de Clercq loc. cit. to inhibit the reverse transcriptases of a
number of retroviruses {this dyestuff had previously been used therapeutically by intravenous injection
to combat Rhodesian trypanosomiasis and onchocerciasis, it was known to persist in the bloodstream
for sufficiently long periods at high enough concentrations to enable it to inhibit HTLV-III (HIV)};
known toxic side effects included renal damage and possibly shock and coma, however].
Broder S et al., Lancet I 1986 575-580 reported the use of suramin in AIDS patients.
Cf. Mitsuya H et al. Science 1988 240 646-649 who reported studies og heparin and dextran sulphate.
.

*Oliva EM Cirelli AF De Lederkremer RM

Chemical composition of the cell wall of the tree fungus Cyttaria harioti Fischer
Exper Mycol 1986 10 150-156
[Alkaline treatment gave a major soluble β (1-3) glucan (deduced on the basis of resistance to
oxidation) and an insoluble structurally related substance, which in total accounted for 89% of the cell
wall preparation which in turn was 67% of the fungus dry weight;
The authors had noted that the monsaccharides present in the cell walls of C. harioti Fischer (glucose,
mannose and glucosamine) occurred in extremely variable proportions in cell walls of almost all fungi.
This makes these polysaccharides of potential interest for the preparation of anti-HIV polysaccharides].

P
*Sydow G Klocking H-P
Effect of pentosan polysulfate (SP54) on the reverse transcriptase activity of several retroviruses
Biomed Biochim Acta 1987 46 6 527-530
[The in vitro anti reverse transcriptase activity of SP54 (from Arzneimittel Munich the molecular
weight of which was ca. 4700) against HIV/HTVL-1 was reported to be reduced from 100, 31 to 1
activity units when SP54 was increased from 0, 1 to 2µ g/ml respectively (with A204 cells from a
human rhabdomyosarcoma) infected with 7 retroviruses including HIV {type C retrovirus HTLV-I
obtained from R.C. Gallo (for which suramin HPA-23 and phenolic polymers, had previously been
reported in the literature)}; 6 of the 7 virus reverse transcriptases were observed to be inhibited by
SP54. The authors had noted that SP54 had previously been reported like other polyanions to inhibit
herpes viruses and by Ehlers B & Diringer H loc. cit to inhibit scrapie pathogenicity in mice;
It should be noted the only the reverse transccriptase which was resistant to SP54 was in BLV virus
differed from HIV I (etc?) in having a lower optimum temperature for activity 25 vs 37C. Perhaps this
might suggest some role of water structure modulation in SP54 anti-retroviral activity].

→
Internet search (27/7/10) starting from De Prost et al above did not reveal any carry-on interest in this
subject but eventually accessed the following papers which give insight into the continued interest in
the anti-viral activities especially the anti-HIV-1 activities of sulphated polysaccharides including
heparin and heparinoids.

Hayashi K Hayashi T Kodima I

A natural sulfated polysaccharide calcium spirulan isolated from Spirulina platensis:
in vitro and ex vivo evaluation of anti-herpes simplex and anti-human immunodefiecincy virus
activities
AIDS Res Hum Retroviruses 1996 12 1463-7

Harrop HA Rider CC
Heparin and its derivatives bind to HIV-1 recombinant envelope glycoprotein rather than to
recombinant HIV-1 receptor CD4
Glycobiology 1998 8 (2) 131-7
[These authors showed that heparin differed from dextran sulphate in the apparent mechanism of its
anti-HIV-1 activity; whereas dextran sulphate targeted CD4, heparin targeted a conserved highly basic
amino acid sequence in the V3 loop of gp120 of HIV-1; this was confirmed by a correlation between
the antiviral activities and the binding activity of modified heparin for this V3 loop].
Stone AL
Glycoconjug J 1998 15 (7) 7-12
[A study of sulphated xylans and their fractions as anti-HIV-1 agents]

Lee LB et al.,
Antiviral activities against HSV-1, HCMV and HIV-1 of rhamnan sulfate from Monostroma latissima
Planta Med 1999 65 439-41

Schaeffer DJ Krylov VS
Anti-HIV activity of extracts and compounds from algae and cyanobacteria
Ecotoxicol Environ Sci 2000 45 208-27

Vives RR et al.
Heparan sulfate targets the HIV-1 envelope glycoprotein gp120 coreceptor binding site
J Biol Chem 2005 280 21353-7

Matsuhiro B et al.
Structural and antiviral activity of a sulfated galactan from the red seaweed Schizymenia binderi
(Gigartinales Rhodophyta)
Carbohydr Res 2005 340 2392-402

Terada M et al.
Polysulfated sialic acid derivatives as anti-human immunodeficiency virus
Biomed Pharmacother 2005 59 (8) 423-429

Chattopadhyay K et al.,
Galactan sulfate of Grateloupia indica. Isolation structural validation and antiviral activity
Phytochem 2007 68 1428-35

Mandel P et al.
Xylans from Scinaia hatei: structural features, sulfation and anti-HSV activity
Int J Biol Macromol 2008 69 243

Cassolato JE et al.
Chemical structure and antiviral activity of the sulfated heterorhamnan isolated from the green seaweed
Gayrali oxysperma
Carbohydr Res 2008 343 3085-95

Trinchero J et al.
Anti-HIV activity of fucoidans extracted from the brown seaweed Adenocystis utriculans
Phytother Res 2009 23 707-12

Ghosh T et al.
Focus on antivirally active sulfated polysaccharides: From structure activity analysis to clinical
evaluation
Glycobiology 2009 19 (1) 2-15
[Anti HIV-1 sulphated carbohydrates have a high selectivity index and very high antiviral potency
(up to 0.01µ g/ml activity) and retain high activity against mutated forms of HIV which are resistant
to nucleoside analogues.
The anti-HIV sulphated polysaccharides show no toxicity to host cells up to 1-2.5 mg/ml.
The reason for the effectiveness of such substances evidently derives from the circumstance that
HIV-1 appears first to bind to heparan sulphate of target cells through a high affinity
selective interaction with coat gp120 followed by a second lower affinity interaction with the
conserved chemokine co-receptor region of gp120.
Sulphate half-ester anionic groups are essentially required for activity whereas carboxyl groups were
thought generally not to promote antiviral activity. In addition to the well-documented degree of
sulphation dependence the specific positions of these sulphate half-ester groups appear to be important
for antiviral activity. Thus the antiviral activity may be especially structurally specified via
heparin(oid)6-O-SO3- groups.
Usually the antiviral activity correlates with molecular weight but may have a minimum size for
activity and above a limiting molecular weight no further increase in activity may occur.
That the nature of the inorganic countercations associated with the sulphated polysaccharides might
also apply to anti-HIV activity could be suggested by the finding that a marked diminution of antiviral
activity against herpes virus of different ‘salt forms’ of a sulphated Spirulina polysaccharide varied in
the order of activity Ca>Na>>K>Ag>Cd (Lee et al cited by Ghosh et al., also Lee et al., Chem
Pharm Bull 2001 49 108-110).
An algal (ι , κ carrageenan) sulphated polysaccharide [(Carraguard ® ) barrier gel for vaginal
application before or after sexual intercourse] was apparently, when Gosh et al. published this article,
the Population Council’s lead microbiocide for topical vaginal application. A double blind (2 x 3000
subject) clinical trial was however statistically inconclusive (Lancet 2008 372 1977) perhaps because
the gels had been used in only about half the occasions intended (nevertheless even under these
conditions the Carraguard ® group showed a lower rate of HIV infection (3.3/100 women years,
compared to 3.8/100 for the placebo). The modest reduction (13.2%) in infection which attributable to
this algal sulphated polysaccharide-based barrier gel should however be compared with the results of
another similar clinical trial of plant-derived sulphated cellulose-based gel which had to be stopped
since it was found that this sulphated polysaccharide microbiocide actually significantly increased the
rate of HIV infection in the treated group [cf. Tao et al., AIDS Res Hum Retrovir 2008 24 925]). This
might indicate that plant-derived sulphated polysaccharide microbiocides barrier gels are intrinsically
less effective than algal derived microbiocides for the inhibition of viral infections in humans. A
corollary might be that better barrier gel performance might be anticipated by use of animal-sulphated-
polysaccharide-based microbiocide barrier gels for topical application in humans.
Furthermore since carrageenan can cause inflammation in wounds (involving e.g. induction of
cytokines such as TNFa (which has been reported to upregulate the expression of HIV) it is
suggested that the inclusion of inhibitors of such inflammatory cytokines (cf. Gong D et al., Phytother
Res 2009 23 645-50) might enable more effective anti-HIV carrageenan-based barrier gels to be
formulated.
[The pro-inflammatory effects of carrageenan have been much studied in a rat model
cf., e.g. Ruch ACC et al., Br J Pharmacol 2006 146 688-95].

{The Ghosh et al., review article which seeks to find correlations which might indicate the modus
operandi of the anti viral including anti-HIV-1 activity of numerous reported investigations into
heparin and semisynthetc heparinoids derived from a variety of e.g. algal species seems to draw
conclusions similar to those indicated by Grant and coworkers, including those listed in
web.abdn.ac.uk/~bch118/publications2003march.doc) cf. also
web. scribd.com/doc/34916461/Why-is-PPS-Such-an-Effective-Anti-NvCJD-Agent-Revised;
It should be noted that endogenous heparin with anti-HIV-1 activity naturally occurs in some
individuals and varies with diet, including e.g. cholesterol status and might even be induced as a result
of viral infection remains an intriguing prospect}.

Cf. Ghosh P et al.

In vitro anti-herpetic activity of sulfated polysaccharide fractions from Caulerpa recemosa
Phytochemistry 2004 65 3151-7
[11 natural sulphated polysaccharides from 10 green algae and 4 synthetic sulphated xylans prepared
from the β -(1,3)-xylan of C. Brachypus showed potent anti-Herpes simplex virus type 1 (HSV-1)
activities with IC50 values of 0.38-8-8.5µ g/ml without toxicity to host cells and also with low blood
anticoagulant activity properties].

{Useful internet searches to identify natural polysaccharides for future inorganic biochemical
researches can start from search terms “xylans from green algae” (cf. Irike et al loc. cit.)

1992 Marishal Collage DG File 2 Refs

The modification of tissue protecting cell surface anionic polysaccharides and polyphosphates may
have promise for augmenting the in vivo inhibition effects of such molecules for numerous kinds of
pathological insult including by viruses (e.g. virus HIV) and bacteria .
A possible starting off point for future researches aimed at find novel broad specificity anti-pathogens
is now suggested to be potentially provided by the now largely abandoned research work on the anti-
pathogen actions of polyanions which had been conducted during the early days of the AIDS epidemic.
This work which had been carried out in numerous institutions including in the University of Aberdeen
UK, had identified a wide range of possible therapeutic agents which might have been enabled, when
suitably formulated, to combat this disease.

A collection of reprints from the Aberdeen work includes the following papers which have now been
selected as being useful starting-off points for future research.

AMINOSUGAR DERIVATIVES
Karpas A Fleet GW Dwek RA Petursson S Namgoong SK Ramsden NG Jacob GS Rademacher
TW
Aminosugar derivatives as potential anti-human immunodeficiency virus agents
PNAS USA 1988 85 9229-33

[A role of glycosylation of gp120 in the spread of HIV virus was suggested since purified
(glycosylated) gp120 inhibited cell fusion (aggregation) but this was not the case for unglycosylated
gp120 (this protein was apparently also immunologically indistinguishable from glycosylated gp120).
The antiviral effects of castanospermine (1,6,7,8 tetrahydroxy octahydroindolizine) and
deoxynojirimycin (DNJ) were thought to arise because of the effect of these substances on
glycosylation effects altering the N-linked oligosaccharides on the viral envelope glycoproteins where
a major proportion of the molecular weight is due to N- and O- glycosylation.
It had been reported that HIV gp120 glycosylation could be subject to an alteration by castanospermine
or DNJ, a process which might be adaptable for designing an anti-HIV therapy.
It should be noted that this report centered on the possibly anti-HIV activity of sugar-like substances
which containing N atoms in 6 or 5 membered rings. Of such ‘aminosugar’ derivatives 47 were
screened for anti-HIV activity, 5 of these compounds were identified with potentially useful anti- HIV
activities at concentrations which were not toxic to host cells. These included 1,4-dideoxy-1,4-imino-L-
arabinitol (LAB) and N-(5-carboxymethyl-1-pentyl)-1,5-imino-L-fucitol (LFT) which inhibited the
cytopathic effect (giant cell, syncytia formation etc.) of HIV and yield of infectious virus but only
partially reduced viral titers. Perhaps of greatest interest was N-butyldeoxynorjirimycin (BuDNJ)
which was found to reduce the yield of HIV by 105 and cause an eventual elimination of HIV viruses
from the cell culture studied; {it was also reported that N-methyldexynojirimycin (MeDNJ) and N-
ethyldeoxynojirimycin (EtDNJ) also reduced the yield of infectious HIV by 104 and 103 respectively}.

N.b. the HIV-1 gp120 envelope glycoprotein contains 24 potential N-glycosylation sites and half its
weight is sugar-derived].

{Paper added later to 1993 photocopy file

CHITIN SULPHATES
Nishimura S-I Kai H Shinada K Yoshida T Tokyra S Kuita K Nakashima H Yamamoto N Uryu T
Regioselective synthesis of sulfated polysaccharides: specific anti-HIV-1 activity of novel chitin
sulfates
Carbohydr Res 1998 306 427-33
[This 1998 paper noted the continued requirement for the more rational design of anti AIDS drugs
repeating argument,s which had been expressed in papers some ten years previous to this study, of the
inappropriate nature of AZT for use in AIDS therapies; furthermore it seems that these authors
thought that in 1998 the anti-AIDS effectiveness of linear β -(1-3) linked glucopyranose backbone
curdlan sulphate (which strongly inhibited AIDS virus at 3.3 µ g/ml) could be regarded as one of the
most potent and practical antiretroviral polysaccharide reagents. The authors described how
selective sulphation of chitin/ chitosan at O-2 and/or O-3 affords potent antiretroviral agents showing
a much higher inhibitory effects on the infection of AIDS virus in vitro than that by the previously
known 6-O-sulphated chitin derivatives. {Previous work had shown that N-carboxymethylchitosan,
N,O-sulphate, a heparin-like polysaccharides derived from N-carboxymethylchitosan by a random
suphation procedure had been found to inhibit HIV-1 replication and viral binding with CD4. A
preparation which completely inhibited the infection of AIDS virus to T lymphocytes at 0.28µ g/ml was
described. The specific interaction with gp120 which was evidently required for this activity which
depended significantly on the sites of sulphation rather than on the overall degree of sulphation]}.

CARRAGEENAN
Nakashima H Kido Y Kobayashi N Motoki Y Neushul M Yamamoto N
Purification and characterization of an avian myeloblastosis and human immunodeficiency virus
reverse transcriptase inhibitor, sulfated polysaccharides extracted from sea algae
Antimicrob Agents Chemother 1987 31 1524-8
[A highly active antiviral agent had mol. wt. ca. 2,000,000 and i.r. spectrum and hydrolysis products
consistent with it consisting of a member of the λ -carrageenan family.
The reverse transcriptase activity of this polysaccharide studied with AMV was similar to that of κ
carrageenan and ι carrageenan being about a half of that of λ carageeenan but ca. thirty times more
effective than heparin, 180 times more effective than dermatan sulfate, ca. 500 times more effective
than heparan sulphate and ca.103 times more effective than chondroitin sulphate (keratan sulphate and
non-sulphated alginic acid, chondroitin and hyaluronic acid lacked any detectable activity].

Cf. Neushul M
Antiviral carbohydrates from marine red algae
Hydrobiolgia 1990 204/205 99-104
[The natural ‘heparin-like-substances’ (e.g. carrageenans) obtainable from red algae or fractions thereof
seemed to offer a low-cost broad-spectrum antiviral including anti-HIV agent].

Suspected Polysaccharide anti-HIV agent ex marine red alga:

Nakashima H Kido Y Kobayashi N Motoki Y Neushul M Yamamoto N

Antiretroviral activity in a marine red alga: reverse transcriptase inhibition by an aqueous extract of
Schizymenia pacifica
J Cancer Res Clin Oncol 1987 113 413-6
[A highly effective inhibitor of reverse transcriptase was extracted from the marine red alga].

Cf. also
Ehresmann DW Deig EF Hatch MT Di Salvo LH Vedros NA
Antiviral substances from California marine algae
J Phycol 1977 13 37-40
[Anti-herpes virus (and other anti-viral) substances (probably polysaccharides perhaps sulphated
polysaccharides) were extracted from ten Rhodophyta species].

CARRAGEENAN
& DEXTRAN SULPHATE, PENTOSAN POLYSULFATE, FUCOIDAN)
Baba M Snoeck R Pauwels R De Clercq E
Sulfated polysaccharides are potent and selective inhibitors of various enveloped viruses, including
herpes simplex virus, cytomegalovirus vesicular stomatitis virus, and human immunodeficiency virus
Antimicrob Agents Chemother 1988 32 1742-5
[(When, two decades previously it had been discovered that various polyanionic substances were
potent antiviral agents, the supposed non-specific nature of this action had caused the biomedical
scientific interest in such substances as possible in vivo therapuetic agents to become muted, but this
situation dramatically changed at the start of the AIDS epidemic since it became evident that the
anti-HIV activities of dextran sulphate, heparin and other polysulphated substances seemed to offer
easily available potentially useful therapeutic agents for this disease. {It should be noted that inhibitory
activity was not observed for adenovirus Coxsackie virus, poliovirus parainfluenza virus and reovirus
was observed with the tested substances}
Six molecular types of sulphated polysaccharides (dextran sulphate heparin, pentosan polysulphate,
fucoidan, κ -carrageenan and λ -carrageenan were confirmed to possess potentially useful antiviral
properties and this confirmed previous indications that sulphated polysaccharides can inhibit a variety
of DNA and RNA viruses, which suggested the general rule that sulphated polysaccharides are potent
and selective inhibitors of enveloped viruses (an exception to this rule, however, was parainfluenza
virus). Dextran sulphate, it was indicated, can be especially potent as an inhibitor of both human
cytomegalovirus (CMV) as well as of HIV-1 with EC50 values of 0.5µ g/ml and was not inhibitory
(toxic) to the host cell (HEL or MT-4) at 400 µ g/ml. Dextran sulphate apparently acted by blocking
the binding of HIV-1 herpes simplex virus-1 (HSV-1), herpes simplex virus-2 HSV-2, to host cells and
was similarly moderately inhibitory of vaccinia virus (VSV)]. Dextran sulphates of mol. wts. greater
than a critical 104 value showed complex relationships with the antiviral activity which approximated to
the existence of a similar antiviral potency above the critical mol. wt. value. (The authors studied the
effect of dextran sulphate mol. wt. for the inhibition of vaccinia virus, HSV-1, (thymidine kinase-
deficient (TK- ) HSV-1,
HSV-2 , CMV, Sindbis virus, HIV-1 and vesicular stomatitis virus (VSV)].
Cf., Baba M De Clercq E
Sulfated polymers as inhibitors of HIV replication
Design of anti-AIDS drugs. Ed, E de Clercq Elsevier Amsterdam…,1990, p. 85-101
{This paper includes data presented above plus additional information on the antiviral activities of
pentosan polysulphate which is especially active against HIV-2 (but heparin was less active); this
report also discussed the antiviral effects of sulphated substances suggested by literature reports which
included clinical trails of dextran sulpahte which had failed to show any effect of oral administered
dextran sulpahte against AIDS; a problem with intravenous administartion of pentosan polysuphate
was the occurrence of side effects; an on-going problem with sulphated polysaccharides is the
possibility that all sulphated polysaccharides that have useful anti-HIV activities also might cause
unacceptable blood-anticoagulant-related side-effects}.

CHONDROITIN POLYSULPHATE
Jurkiewicz E Panse P Jentsch K-D Hartmann H Hunsmann G
In vitro anti-HIV-1 activity of chondroitin polysulfate
AIDS 1989 3 423-7
[Chondroitin sulphate of mol. wt. 9000 (CPS9000, manufactured by Luitpold-Werk Munich) was
found to be a highly effective anti-HIV (HTLV-IIIB strain grown in Jurkta cells] agent as measured for
viral replication and anti-tanscriptase activity. It was more active than other chondroitin sulphates (A
and C of mol. wts. 32000), dextran sulphate, surmain and zidovudine (which it was noted, in contrast to
the polyanionic antiviral substances, was toxic to the host cells) in MT-4 cell culture assay.
These authors reported that CPS900 was considerably more active against HIV than suramin, heparin,
dextran sulphate and pentosan polysuphate].

CURDULLAN SULPHATE (see Lentinan sulphate)

DEXTRAN SULPHATE
Bagasra O Lischner HW
Activity of dextran sulfate and other polyanionic polysaccharides against human immunodeficiency
virus
J Infect Dis 1988 158 1084-7
[Dextran sulphate and other polyanions had been discovered to block syncytia formation (a possible
indicator of in vivo spreading potency) between HIV-producing cells and CD4-bearing cells. This
paper reported the effect of different mol. wt. dextran sulphates for the action it being indicated that the
effect increased with increasing mol. wt. It should be noted that AZT does not block syncytium
formation].

Hartman NR Johns DG Mitsuya H

Pharmacokinetic analysis of dextran sulfate in rats as pertains to its usefulness for therapy of HIV
infection
AIDS Res Hum Retroviruses 1990 6 805-11
[This study of the uptake and excretion of using radiolabelled dextran sulphate by male Sprague
Dawley rats showed that oral administration was ineffective as an possibly anti-HIV agent as only
small amounts of very low molecular weight (anti-HIV inactive) dextran sulphate reached the blood
and urine. Like heparin and pentosan polysulphate, dextran sulphate appeared mainly to be cleared by
the reticuloendothelial system with a minor percentage excreted in the urine. Following intravenous
administration a biexponentail elimination of dextran sulphate label occurred with first phase of half
life ca. 20 min (undegraded material) and a second phase of half life 480 min of slightly degraded
material.
Separate experiments which tested the in vitro anti HIV activities of a range of dextran sulpahte
obtained from Ueno Fine Chemicals showed that anti-HIV activity was absent when mol. wt. was less
than 2300, was decreased at 4500 but fully active at 5600 and 7800 but this anti-HIV activity
was reduced from 5-10µ g/ml to >50µ g/ml in the presence of 85% fresh human serum].

Ueno R Kuno S
Dextran sulphate, a potent anti-HIV agent in vitro having synergism with zidovudine
The Lancet 1987 June 13 1379
Dextran sulfate had been in use as an anti-lipaemic agent in Japan for many years.
Dextran sulphate (mol wt. 7999-8000 S cont 17-20% showed at >5µ g/ml potent in vitro antiviral
activities against HIVand therein acted synergistically with AZT 10nmol/L].

Cf. Nakashima H Yoshida O Baba M De Clercq E Yamamoto N

Anti-HIV activity of dextran sulphate as determined under different experimental conditions
Antiviral Res 1089 11 233-46
[The anti-HIV activity of dextran sulphate was highly dependent on its sulphate conteny and evidently
exerted its activity via the inhibition of viral binding to target cells].

Cf. also under Carageenan

FUCHSIN ACID
Baba M Schols D Pauwels T Balzarini J De Clercq E
Fuchsin acid selectively inhibits human immunodeficiency virus (HIV) replication in vitro
0006-291X/88 Academic Press Inc 1404
Fuchsin acid a sulphonated anionic dye (cf suramin) [approximatly structurally related to
aurintricarboxylic acid) is a selective inhibitor of HIV-1 and HIV-2 in vitro being more active and less
toxic than suramin (with EC50 was 16µ g/ml for HUT-78 antigen expression inhibition; the HIV-1
was obtained from the culyure supernatant of a HUT-78 cell line persistently infected with HTLV-IIIB)

FUCOIDAN
Sugawara I Itoh W Kimura S Mori S Shimada K
Further characterization of sulfated homopolysaccharides as anti-HIV agents
Experientia 1989 45 996-998
[Fucoidan and dextran sulpahte showed potnet anti-HIV activities in cells from AIDS patients and were
suggested as being suitable for combination therapy together with AZT].
Cf. also under Carrageenan

GLYCYRRHIZIN SULFATE etc.

Tochikura TS Nakashima H Tanabe A Yamamoto N
Human immunodeficiency virus (HIV)-induced cell fusion: quantification and its application for the
simple and rapid screening of anti-HIV substances in vitro
Virology 1988 164 542-6
[This paper reported a method of evaluating anti-HIV drugs via the inhibition of syncytia formation in
the co-culture system using Molt-4 and its HIV producing cell Molt-4/HIVHTLV-IIIB .
Substances which offered potent antiviral effectiveness included glycyrrhizin sulfate, dextran sulfate
and PSK a protein-bound polysaccharide extracted from Basidiomycetes].

Tochikura TS Nakashima H Yamamoto N

Antiviral agents with activity against human retroviruses
J Acquired Immune Deficiency Syndrome 1989 2 441-7
[Glycyrrhizin sulphate was compared with lentinan sulphate, dextran sulphate, a non sulphated
polysaccharide PSK for the inhibition of HIV-1, HIV-2 and HTLV-1.
A list of substances which included glycerrhizin sulphate etc. which had been shown to demonstrate
anti-HTLV-IIIB activity efficiently suppressed cell-free infection of other HIV-1 strains including
LAV, ARV and YU-6 and the GH strain of HIV-2. The YU-6 strain was, however,
uncharacteristically less effectively inhibited].

Cf. Nakashima H Matsui T Yoshida O Isowa Y Kido Y Motoki Y Ito M Shigeta S Mori T
Yamamoto N
A new anti-human immunodeficiency virus substance, glycyrrhizin sulfate; endowment of glycyrrhizin
with reverse transcriptase-inhibitory activity by chemical modification
Jpn J Cancer Res (Gann) 1987 78 767-71
[Sulphate residues play a key role in the inhibition process].

HEPARIN
Cardin AD Taylor DL Krstenansky JL Tyms AS Jackson RL
A heparin which binds to the envelope glycoprotein gp120 inhibits human immunodeficiency virus
replication
Trans Assoc Am Physicians 1989 102 101-9. Cf. Clinical Research 1989 37 (2)
[It was demonstrated that residues 307-330 of gp120 of HIV (RP-135) contains basic amino acids
typical of the heparin-binding domains of other proteins.
The heparin binding domain of RP-135 showed a sequence similarity to that of vitronectin cf.
HIV BH10 of HTLV-III gp120 RP-135 NNTRKS I R I QRGPG RAFV T I GKI
Vitronectin HRNRKGYRS QRGHS RGRNQNSRR
Ca. 2% of a starting heparin bound to a RP-135 affinity column and at ED50 0.3-0.4 µ g/ml
demonstrated ca. 8 times higher anti-HIV activity than unfractionated heparin or dextran sulphate (mol.
wt. 500,000) ; a disadvantage of the fractionated heparin for potential AIDS therapy was that it showed
high anticoagulant activity than did unfractionated heparin].

Cf.
Ito M Baba M Sato A Pauwels R De Clercq E Shigeta S
Inhibitory effect of dextran sulfate and heparin on the replication of human immunodeficiency virus
(HIV) in vitro
Antiviral Res 1987 7 361-7
[Dextrans sulphate and heparin effected a 50% reduction in the cytopathogenicity of HIV for MT-4
cells at a concentration of 4.7 and 7.5 µ g/ml respectively].

LENTINAN SULPHATE
CURDULAN SULPHATE
Yoshida C Nakashima H Yoshida T Kaneko Y Yamamoto I Matsuzaki K Uryu T Yamamoato N
Sulfation of the immunomodulating polysaccharide lentinan: a novel strategy for antivirals to human
immunodeficiency virus (HIV)
Biochem Pharmacol 1988 37 (15) 2889-91
[The authors discuss their previous findings that a chondroitin sulphate from a sea algal extract from
Schizymenia pacifica inhibits HIV viral adsorption and inhibits reverse transcriptase activity and noted
that other anti-HIV active substances such as Evans blue and suramin were sulphated compounds.
Curdulan galactose sulphate, curdulan arabinaose sulphate and lentinan sulphate (sulphated compounds
of D-galactosyl curdulan, oligo-D-arabinosyl curdulan and lentinan respectively) strongly inhibited
reverse transcriptase (of purified avian myeloblastosis virus (AMV) at 2.5, 1 and 0.3µ g/ml
respectively; inhibited viral antigen synthesis and HIV-cytopathogenicity at 10µ g/ml; all the studied
sulphated substances also inhibited syncytia formation (of MOLT-4 and MOLT-4/HIVHTLV-IIIB ) at
concentrations of 10-100µ g/ml; the substances also blocked cell fusion reactions of MOLT-4 cells.
The reason for choosing the studied polysaccharides in the first place was that the parent compounds
had been known to exhibit immunostimulatory effects useful for anti-tumour therapy. It was reported
that the sulphated compounds studied as anti-HIV agents however abolished such antitumour activity
as studied in transplanted allogenic or syngenic tumour models (the greatest non-sulphated parent
substances anti-tumour activity was for lentinan (a fungal branched (1-3)-β -D-glucan); this was
reduced during the sulphation procedure attributable to the formation of sulphate (half-ester) groups.

Hatanaka K Yoshida T Uryu T Yoshida O Nakashima H Yamamoto N Mimura T Kaneko Y

Synthesis of an inhibitor of human immunodeficiency virus infection
Jpn J Cancer Res 1989 80 95-8
[Lentinan sulphate with a sulphur content greater than 13.9%, with more than 1.7 sulphate half ester
groups/glucose unit, effectively prevented HIV-induced cytopathic effects at concentrations of
>3.3µ g/ml indicating that this type of preparation was amongst the most effective anti-HIV agents of
any known sulphated polysaccharide type. It was noted that the anti-HIV sulphated lentinans inhibited
HIV infection at concentrations 300 fold lower concentrations than were required for the inhibition of
the growth of the same kind of cells without HIV infection. Lesser amounts of sulphation or with more
than 50% of Na+ countercations replaced by pyridinium produced dramatically altered (ineffective) anti
HIV activities. {This could suggest an important role for polysaccharide hydration in the anti-HIV
activity}. The synthesis of sulphated derivatives could apparently also be negatively affected by poor
solubility. An advantagen of lentinan sulphate for anti-HIV therapy was indicated to be the greater
activity and lesser potential in vivo antigentic reaction than was the case for dextran sulphate].

LICHEN POLYSACCHARIDE SULPHATE

Hirabayashi K Iwata S Ito M Shigeta S Narui T Mori T Shibata S
Inhibitory effect of a lichen polysaccharide sulfate, GE-3-S on the replication of human
immunodeficiency virus (HIV) in vitro
Chem Pharm Bull 1989 37 2410-2
[Sulphation of GE-3, a partially acetylated β (1-6) glucan of the lichen Umbilicaria esculenta inhibited
HIV proliferation and ssyncytium formato, inhibiting plaque formation by 50% aat 19.5µ g/ml; this
sulphated polysaccharide had no direct effect on the reverse transcriptase of HIV].

LIGNIN DERIVATIVES
Suzukii H Okubo A Yamazaki S Suzuki K Mitsuya H Toda S
Inhibition of the infectivity and cytopathic effect of human immunodeficiency virus by water-soluble
lignin in an extract of the culture medium of Lentinus edodes mycelia (LEM)
Biochem Biophys Res Commun 1989 160 367-373
[It was believed that a polycarboxylated lignin derivative was the active ingredient in the mixture of
different polymeric substances (which included polysaccharides) present in LEM was the principle
anti-HIV agent completely inhibiting viral proliferation at >10µ g/ml. The potency of such
heterogeneous extract mixtures may conceivably derive from synergism between the different possible
antiviral molecules present].

MANNAN SULPHATE
Ito M Baba M Hirabayashi K Matsumoto T Suzuki M Sukuki S Shigeta S De Clercq E
In vitro activity of mannan sulfate, a novel sulfated polysaccharide, against human immunodeficincy
virus type I and other enveloped viruses
Eur J Clin Microbiol Dis 1989 8 171-3
[Anti HIV-1 activities were studied in Molt-4 and MT-4 cells where activities were EC50 1.5 and 9.3
µ g/ml respectively, low toxicities to host cells were found; useful antiviral activities against herpes
simplex types 1 and 2 vaccinia virus and vesicular stomatitis virus were also indicated].

PENTOSAN SULPHATE
Anand R Nayyar S Galvin TA Merril CR Bigelow LB
Sodium pentosan polysulfate (PPS), an anti-HIV agent also exhibits synergism with AZT,
lymphoproliferative activity and virus enhancement
AIDS Res Hum Retroviruses 1990 6 (5) 679-689
[The HIV-1 isolate used in this work was LAV-1BRU as well as HIV-1BR NA4 and HIV-1451.
The studies persulphated polysaccharides caused transient viral enhancement observed at 1.0µ g/ml or
lower.
The antiviral effects of PPS dominated however at 5.0µ g/ml. The viral enhancing effect was absent in
the presence of AZT with which PPS demonstrated marked anti-HIV synergism.
It was concluded that combined AZT PPS therapy might be therapeutically useful for AIDS therapy
design].

A press release (ca.1989) from the Max-Planck-Gesellscchaft Munchen

(“Hemmstoff gegen AIDS in klinischer Prufung”)
[Clinical trial of pentosan polysulfate as a therpeutic agent for AIDS]
(Polysulfatierte Polysaccharide study was planned; the note mentioned Prof Molling (Max –Planck-
Institut fur molekulare Genetik Berlin as being involved.
Pentosan polysulfate had showed an exceptionally high HIV anti reverse transcriptase activity
Cf.
Wagner W-H
Hoe/Bay 942-a new compound with activity against the AIDS virus
Arzneim-Forsch/Drug Res 1989 39 112-3
Hoe/Bay 946 pentosan sulphate an xylan polysulphate containing 1.8 sulphate half ester groups/
monomer and molecular weight of 6000, lacked host cell toxicity and showed a high in vitro
antireverse transcriptase activity (ID50 2µ g/ml) and a high ability to inhibit syncytia formation and
also performed well in experiments in mice (who received daily doses of 12.5-50 mg/kg during 10
days) with virus infection-induced splenomegaly. This drug was reported to have been identified by
major joint research of Bayer AG and Hoechst AG conducted during the 1980s in response to the
perceived urgent need for more effective AIDS therapeutic agents than e.g. AZT and promising effects
against AIDS seemed to have been confirmed by a 4 month trial of HIV-infected individuals.
This pentosan polysulphate seemed to offer a highly promising treatment for AIDS but in order to
confirm this, it was thought to require human trials lasting e.g. five years of continuous treatment.

Cf., De Clercq E
New acquisitions in the development of anti-HIV agents
Antiviral Res 1998 12 1-20
[This in depth review includes a comprehensive historical perspective which indicates that sulphated
polysaccharides are therapeutically advantageous for AIDS therapy].

Cf. Baba M Schols D Pauwels R Nakashima H De Clercq E

Sulfated polysaccharides as potent inhibitors of HIV-induced syncytium formation: a new strategy
towards AIDS chemotherapy
J Acq Immune Deficiency Syndrome 1990 3 493-9
[The possibility that the syncytium formation which is inhibitable by sulphated polysaccharides is a
key event in the etiology of AIDS is discussed. It can be suggested that a sulpahted polysaccharide
based therapy for AIDS could be of value. It should be noted that as studied by these authors, pentosan
polysulfate, dextran sulfate and various other sulfated polysaccharides but not heparin inhibited this
cell fusion process and hence protected the target CD4+ cells against destruction by the killer HIV-1
infected cells. AZT did not inhibit this process. The requirements for the inhibition of giant cell
formation seemed to be more stringent than those for the inhibition of virus replication.

The authors further suggested that the anti-HIV anti syncytium formation ability of polyphosphates and
polyphosphonates be evaluated.
(This was later accomplished and the anti-HIV (including antisyncytium forming activity) of these
substances has been confirmed (cf. Lorenz, loc. cit., cf., Introduction)].

Cf. also under Carrageenan

PRUNELLIN
Tabba HD Chang RS Smith KM
Isolation, purification and partial characterization of prunellin, an anti-HIV component from aqueous
extracts of Prunella vulgaris
Antiviral Res 1989 11 263-74
[The authors obtained what appeared to be a sulphated polysaccharide of very high anti-HIV activity in
vitro from an aqueous extract of the Chinese medicinal herb Prunella vulgaris].

PUSTULAN (partly acetylated) (LICHEN POLYSACCHARIDE) SULPHATE

Hirabayashi K Iwata S Ito M Shigeta S Narui T Mori T Shibata S
Inhibitiory effect of a lichen polysaccharide sulfate, GE-3-S, on the replciation of human
immunodeficiency virus (HIV) in vitro
Chem Pharm Bull 1989 37 (9) 2410-2
[A product designated as GE-3-S obtained by sulphation of GE-3 a partially acetylated β (1-6) glucan
of the lichen Umbilicaria esculenta inhibited the cytopathic effect of HIV and suppressed the HIV-
antigen expression in Molt-4 (clone 8) cells, suppressed syncytia in HIV-infected Molt-4 cells and
inhibited HIV-induced plaque formation by 50% at 19.5µ g/ml. It was noted that GE-3-S had no direct
effect on the reverse transcriptase activity of HIV. The parent polysaccharide was ineffective as an
anti-HIV agent which indicated an essential role for sulphate half ester groups (however other (pr.)
sulphated lichen derived polysaccharides studied by the authors , PC-3S, pachyman – and lichenan-S
were not effective anti-HIV agents)].
SCHIZOPHYLLAN SULPHATE
Itoh W Sugawara I Kimura S Tabata K Hirata A Kojima T Mori S Shimada K
Immunopharmacological study of sulfated schizophyllan (SPG) I. Its action as a mitogen and anti-HIV
agent
Int J Immunopharmac 1990 12 225-33
Schizophyllans, members of the class of β -1,3 glucans possessing immunomodulating activities which
were believed to be useful for the treatment of uterine cervical cancer; such polysaccharides when
sulphated to high S contents showed potent anti-HIV effects indicating their possibly usefulness as
anti-AIDS drugs; the authors studied effects in Molt-4 clone no. 8 or MT-4 cells infected with HIV as
well as on lymphocytes from AIDS patients. The S content was the most important determinant of the
anti-HIV effect.
The higher (but not the lower) molecular weight sulphated schizophillans studied showed no toxicity to
host cells at 5-2000µ g/ml; at 100-500µ g/ml showed mitogenic activites of murine spleen cells
similar to dextran sulphates. Non-sulphated schizophillans lacked this property. Sulphated
schizophyllans with S > 3.7% inhibited viral proliferation; the lower molecular weight schizophyllans
studied were effective at 20µ g/ml being more effective than dextran sulphate and seemed to be the
preferred form for achieving useful anti-AIDS therapy. Test with HIV-infected lymphocytes from
AIDS patients confirmed that sulphated schizophyllans significantly blocked the growth of the HIV-
infected lymphocytes. Comparison of the anti-HIV activities of linear vs. branched chain
polysaccharides suggested that the structure of the main polysaccharide chain (and not the structure of
side chains) is the principal determinant of the observed antiviral activities.

Cf., Mizumoto K Sugawara I Ito W Kodama T Hayami M Mori S

Sulfated homopolysaccharides with immunomodulating activities are more potent anti-HTLV-III
agents than sulfated heteropolysaccharides
Japan J Exp Med 1988 58 3 145-51
[Wheras the authors had found that the ‘sulphated heteropolysaccharides’ heparin and heparan sulphate
had little anti-HTL-III in their assay methods, ‘sulphated hompolysaccharides’ such as fucoidan,
dextran sulphate (of three different molecular weights) cellulose sulphate and κ -carrageenan showed
potent anti HTLV-III (HIV) effects; the latter were also intrinsic immunomodulators]

Added Later
SPIRULAN SULPHATE
Effects of structural modification of calcium spirulan, a sulfated polysaccharide from
Spirulina Platensis on antiviral activity
J.B. Lee et al.,
Chem Pharm Bull 2001 49 108-110
{Different salt forms of this sulphated polysaccharide which is active against HIV showed markedly
different activities and selectivities for anti herpes activites; multivalent counterions, except calcium,
tended to greatly reduce the activity}.

SURAMIN
Suramin Analogues [These sulphonated dyestuffs can be considered as HEPARIN ANALOGUES]
De Clercq E
Inhibitors of Reverse Transcriptase Replication
Autoreferat vom 290. Kolloquium des Paul-Ehlrich-Instiyutes, des Georg-Speyer-Hauses und des
Ferdinand-Blum-Instituts zu Frankfurt/Main am 28 Marz 1985
Arzniem-Forsch/Drug Res 1985 35 (1) No.6
Known inhibitors of reverse transcriptase were discussed.
With an ID50 of 1µ g/ml for the reverse transcriptase activity of Moloney murine leukemia virus,
suramin ranked amongst the most potent RT inhibitors; equally potent as suramin were ethidium
bromide and some diarylamididine and imidazoline derivatives and more potent than suramin were
(ms2A)n with ID50 0.15 µ g/ml and (fl2A)n with ID50 0.02µ g/ml (the most effective anti-RT agent
known at that time).
The inhibition of RT by suramin was competitive with the template primer (A)n-oligo(dT) suggesting
that suramin interacts with the binding site of RT for which activity the sulphonate group was finding
that several polysulphonic acid dyestuffs which are structurally related to suramin are also RT
inhibitors (these included Guinea Green B (ID50 3.65µ g/ml), Trypan Blue (ID501.91µ g/ml), Procian
Blue HB (ID50 1.49µ g/ml, Aniline Blue (ID50 1.18 µ g/ml) , Congo Red (ID50 0.64µ g/ml),
Chicago Sky Blue 6B (ID50 0.60 µ g/ml), Direct Yellow 50 (ID50 0.47µ g/ml) and Evans Blue (ID50
0.33µ g/ml) as compared with test conditions where suramin showed ID50 1.91µ g/ml).
These compounds also were inhibitors of cellular transformation of C3H mouse embryo cells by
Moloney sarcoma virus; for this effect a series of 5-substituted 2/-deoxyuridines behaved in a
correlated fashion with their inhibitory efficiency for vaccina virus replication.

SULPHATED POLYVINYL ALCOHOL (PVAS)

SULPHATED COPOLYMERs of ACRYLIC ACID WITH VINYL ALCOHOL (PAVAS)
Baba M Schols D De Clercq E Pauwels R Nagy M Gyorgyi-Edelenyi J Low M Gorog S
Novel sulfated polymers as highly potent and selective inhibitors if human immunodeficiency virus
replication and giant cell formation
Antimicrob Agents Chemother 1990 34 (1) 134-8
[The studied polymers were highly effective inhibitors of HIV-1 and HIV-2 in vitro.
They completely inhibited HIV-1 cytopathogenicity in MT-4 cells and HIV-1 antigen expression in
CEM cells at 0.8µ g/ml; similar results were apparent for HIV-2; in contrast to AZT they suppressed
syncytium formation at 4µ g/ml a concentration which was thought to be highly achievable in human
plasma (which is also more efficient than dextran sulphate which for which 100µ g/ml was required a
value which was thought highly unlikely to be achievable in human plasma). It was believed that the
modus operandi of the studied substances was the inhibition of virus adsorption by the cells.
It is evident that these substances are analogues of sulphated polysaccharides and that the precise
nature of the backbone skeleton is not required. The presence of sufficiently abundant and correctly
arranged sulphate half-ester groups is however apparently mandatory. This could be further suggested
to be evidence that the water structure generated by such linear persulphated polymers may also be
critical to their antiviral actions]
Cf. also
De Clercq E
Review Selective Virus Inhibitors
Microbiologica 1990 13 165-78.
[In p.179 “Suranim had been recognized as a potent inhibitor of retrovirus-associated reverse
transcriptase (by the author in 1979) which led Mitsuya et al. (1984) to evaluate its inhibitory effect on
the infectivity of HIV in vitro. The compound proved inhibitory to the cytopathic effect of HIV at drug
concentrations which were not toxic to the host cells. Suramin was also the first anti-HIV agent found
to be capable of inhibiting HIV replication in AIDS patients (Broder et al., 1985). However, a short-
term (6-week) treatment course with suramin did not lead to an either clinical or immunological
improvement (Broder et al., 1985) and, upon, prolonged treatment, suramin proved rather toxic
(Kaplan et al., 1987) so that its use as single-agent therapy for AIDS is not recommended. Shortly
after suramin 3/-azide-2/,3/dideoxythymidien (azidothymidine ,AxddThd, AZT) was discovered as a
selective anti-HIV agent (Mitsuya et al., 1985), and, as AZT appeared to be superior to suramin in both
potency and selectivity, it was promptly introduced in the clinic (Yarochoan et al., 1986). A short-term
(6-week) treatment of AIDS patients with AZT led to clinical, virological and immunological
improvement and, consequently, AZT was submitted to a double-blind, placebo –controlled trial,
which, in turn, indicated that following a 24-week treatment period with AZT, there was a significant
decrease (delay) in mortality and reduction in the frequency of opportunistic infections (Fischl et al,
1987). However, the long term administration of AZT is associated with severe toxicity, in particular
bone marrow suppression (anemia, leukopenia) (Richman et al., 1987) and the initial immunological
improvement , i.e. increase in CD4+ cells after the first few weeks of AZT therapy, CD4+ cell counts
have returned to their pretreatment levels and further decline despite continued AZT treatment….
(cf. p.172-3) In addition to ATA (aurintricarboxylic acid), sulfated polysaccharides also specifically
interfere with the virus adsorption process. This group contains a number of compounds i.e. heparin,
dextran sulfate, pentosan polysulfate, λ , κ , ι − carrageenans, mannan sulfate, lentinan sulfate
“supersulfated” chondroitin sulfate, sulfated bacterial glycosaminoglycan, periodate-treated heparin,
sulfated polyvinylalchohol (PVAS) and sulfated co-polymers of acrylic acid with vinyl-alcohol
(PVAS) that inhibit HIV replication in vitro at concentrations that are far below the cytotoxicity
threshold (Dr Clercq, 1989.., Baba et al., 1990….While exquisitely selective as inhibitors of HIV
replication in vitro , it is as yet unclear whether these compounds are also effective against HIV
replication in vivo. The sole clinical study that has so far been conducted with dextran sulfate (Abrams
et al. 1989) did not reveal much benefit, but this trial dextran sulfate was administered orally, and this
is not the ideal route for compounds that are assumed to be poorly absorbed when given orally……A
rather unique feature of the sulfated polysaccharides (and sulfated polymers in general) is their ability
to interfere with giant cell (syncytium) formation, resulting from the fusion between HIV-infected cells
and uninfected cells. Like virus attachment to the cells, this fusion process depends on a specific
interaction between the viral gp120 (expressed by the HIV-infected cell) and the CD4 receptor of the
uninfected cell. The role of syncytium formation in the pathogenesis of AIDS has not been
unequivocally established…..the most potent inhibitors of syncytium formation are PVAS and
PAVAS”].

Cf. Parish CR Low L Warren HS Cunningham AL

A polyanion binding site on the CD4 molecule. Proximity to the HIV-gp120 binding region
J Immunol 1990 145 1188-95
[Dextran sulphate mol. wt. 500000, polyvinyl sulphate and polyanethole sulfonate were especially
effective types of sulphated polyanions for blocking the binding of 11 of 12 studied anti CD4
antibodies to the putative identified polyanion binding site; it was indicated however that while some
sulphated polysaccharides such as dextran sulpahte may inhibit HIV by this mechanism, some other
mechanisms evidently operate with pentosan polysulphate and heparin since these sulphated
polysaccharides failed to show the studied CD4 reactivity].

TUMOR NECROSIS FACTOR alpha

Tumor necrosis factor antagonizes inhibitory effect of azidothymidine on human immunodeficiency
virus (HIV) replication in vitro
Ito M Baba M Mori S Hirabayashi K Sato A Shigeta S De Clercq E
Biochem Biophys Res Commun 1990 166 1095-101
[Tumor necrosis factor alpha (TNFα ) a typical cytokine, which had been considered as a component
of combination therapy for AIDS was reported to strongly antagonize the inhibitory effect of
azidothymidine (AZT) on human immunodeficiency virus (HIV) replication in vitro MOLT-4 cell
cultures. TNFα also, but less strongly, blocked the HIV antiviral activity of 2/ , 3/-dideoxycytidine
(DDC) but, perhaps highly significantly for possible therapeutic implications, had very little effect on
the anti-HIV activity of dextran sulphate. This indicated the unsuitability of TNFα as a component of
AZT or similar substance mixed cytokine (TNFa) anti-HIV therapy. TNFα can completely abolish
the antiviral action of AZT. A consideration of the possible mechanism suggested a role of TNFα in
increasing the in giant cell (syncytia) formation following HIV infection was centrally involved. A
further relevant consideration was that granulocyte-macrophage colony stimulating factor (GM-CSF)
could also, like TNFα, markely upregulate the expression of HIV-1. The effect of TNFα was
indicated not to be due to altered phosphorylation (rate) of AZT (which occurs during the metabolism
of this drug)].

Cf. Plotz FB et al.

Artif Organs 1992 16 336; Chem Abs 117 198455c
[Heparin coating provided complete protection from TNF formation].

Suggestions for Future Research

It is suggested that future research should seek to determine if different salt forms of the naturally
occurring putatively tissue protecting cell surface anionic polysaccharides and polyphosphates can
augment the inhibition by such molecules of numerous kinds of pathological insult including by viruses
(e.g. HIV variants) and bacteria which have become resistant to conventional antibiotics.

The modulation of inorganic biochemical aspects of anionic polysaccharides might generate specific
non viral pathogen potencies which include broad-spectrum antibacterial actions which conceivably
might be harnessed to combat bacterial “superbug” infections.

Improved performance for topical vaginal application for the prevention of HIV infection by sulphated
polysaccharide (e.g. carrageenan)-based barrier gels formulated may be achieved by the incorporation
of anti-inflammatory (especially anti-TNFα agents).