Pathophysiological Effects of Synthetic Derivatives of Polymeric Alkylpyridinium Salts from the Marine Sponge, Reniera sarai

Abstract

Polymeric 3-alkylpyridinium salts (poly-APS) are among the most studied natural bioactive compounds extracted from the marine sponge, Reniera sarai. They exhibit a wide range of biological activities, and the most prominent among them are the anti-acetylcholinesterase and membrane-damaging activity. Due to their membrane activity, sAPS can induce the lysis of various cells and cell lines and inhibit the growth of bacteria and fungi. Because of their bioactivity, poly-APS are possible candidates for use in the fields of medicine, pharmacy and industry. Due to the small amounts of naturally occurring poly-APS, methods for the synthesis of analogues have been developed. They differ in chemical properties, such as the degree of polymerization, the length of the alkyl chains (from three to 12 carbon atoms) and in the counter ions present in their structures. Such structurally defined analogues with different chemical properties and degrees of polymerization possess different levels of biological activity. We review the current knowledge of the biological activity and toxicity of synthetic poly-APS analogues, with particular emphasis on the mechanisms of their physiological and pharmacological effects and, in particular, the mechanisms of toxicity of two analogues, APS12-2 and APS3, in vivo and in vitro.

Full text

Mar. Drugs 2014, 12, 2408-2421; doi:10.3390/md12052408
marine drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Review
Pathophysiological Effects of Synthetic Derivatives of Polymeric
Alkylpyridinium Salts from the Marine Sponge, Reniera sarai
Marjana Grandič 1 and Robert Frangež 2,*
1 Institute for Hygiene and Pathology of Animal Nutrition, Veterinary Faculty, University of
Ljubljana, Cesta v Mestni log 47, Ljubljana 1000, Slovenia; E-Mail: marjana.grandic@vf.uni-lj.si
2 Institute of Physiology, Pharmacology and Toxicology, Veterinary Faculty, University of Ljubljana,
Gerbičeva 60, Ljubljana 1000, Slovenia
* Author to whom correspondence should be addressed; E-Mail: robert.frangez@vf.uni-lj.si;
Tel.: +386-1-477-91-31.
Received: 17 March 2014; in revised form: 4 April 2014 / Accepted: 4 April 2014 /
Published: 30 April 2014
Abstract: Polymeric 3-alkylpyridinium salts (poly-APS) are among the most studied
natural bioactive compounds extracted from the marine sponge, Reniera sarai. They
exhibit a wide range of biological activities, and the most prominent among them are the
anti-acetylcholinesterase and membrane-damaging activity. Due to their membrane
activity, sAPS can induce the lysis of various cells and cell lines and inhibit the growth of
bacteria and fungi. Because of their bioactivity, poly-APS are possible candidates for use
in the fields of medicine, pharmacy and industry. Due to the small amounts of naturally
occurring poly-APS, methods for the synthesis of analogues have been developed. They
differ in chemical properties, such as the degree of polymerization, the length of the alkyl
chains (from three to 12 carbon atoms) and in the counter ions present in their structures.
Such structurally defined analogues with different chemical properties and degrees of
polymerization possess different levels of biological activity. We review the current
knowledge of the biological activity and toxicity of synthetic poly-APS analogues, with
particular emphasis on the mechanisms of their physiological and pharmacological effects
and, in particular, the mechanisms of toxicity of two analogues, APS12-2 and APS3,
in vivo and in vitro.
Keywords: alkylpyridinium compounds; APS12-2; APS3; cardiotoxicity; hemolysis;
nicotinic acetylcholine receptors; neuromuscular junction; mouse; rat; synthesis
OPEN ACCESS

Mar. Drugs 2014, 12 2409
1. Introduction
Polymeric 3-alkylpyridinium salts (poly-APS) are one of more than 80 biologically active
compounds found in several marine sponges of the order, Haplosclerida [1–4]. They have been
isolated from crude extracts of the Mediterranean marine sponge, Reniera sarai. Poly-APS have been
reported to comprise two polymers with molecular weights of 5520 and 18,900 Da, corresponding to
29 and 99–100 covalently, head-to-tail linked N-butyl-3-butyl pyridinium monomers [5]. In water
solutions, they form larger supramolecular aggregates [5–7]. However, recent analyses have indicated
that poly-APS are composed of one monomeric species only, with a molecular weight of
5520 Da [8].
Poly-APS are water-soluble compounds with high degrees of association and a broad spectrum of
interesting biological activities [4,6]. These include hemolytic, cytolytic and cytotoxic activities [6],
antifouling [9,10] and antimicrobial properties, including antibacterial [11] and anti-algal activities [12].
Poly-APS are also very potent, irreversible acetylcholinesterase (AChE) inhibitors [13–15]. Due to
their ability to induce transient pore formation in biological membranes [16,17], poly-APS have been
used for stable transfection of various mammalian cells with heterologous DNA and, thus, have a
potential in gene therapy [18–20]. Moreover, poly-APS exert selective cytotoxicity against non-small
cell lung cancer (NSCLC) cells, which are the most common form of lung cancer, and express
α7-nicotinic receptors [21–23]. Cytotoxic concentrations of poly-APS are in the nanomolar range
(0.36–0.86 nM) [23] and are much lower than the calculated concentrations in blood plasma inducing
toxic and lethal effects after intravenous (i.v.) compound application. Toxic effects on mammals,
arising from poly-APS interference with the cholinergic system, have been observed following
administration of low doses (0.7 mg/kg) of poly-APS. At higher doses, these effects were masked by
the more pronounced lethal activity of the compound related to hemolysis and platelet aggregation.
The half-lethal dose (LD50) of poly-APS in rats has been estimated to be 2.7 mg/kg ([24], reviewed
in [25]). Poly-APS have recently been shown, at a 1 μM concentration, to diminish endothelium-dependent
relaxation of isolated rat thoracic aorta and to significantly decrease coronary flow in the heart [26].
Such biological effects of natural poly-APS and their possible application in the fields of industry
(as components of environmentally friendly antifouling paints) and medicine (as new anti-cholinergic,
transfection and chemotherapeutic agents) have led to the synthesis of several 3-alkylpyridinium
analogues (sAPS) with different degrees of polymerization and different lengths of the constituent
alkyl chains [27–29]. The synthesis of structurally well-defined analogues with different chemical
properties and degrees of polymerization has enabled the regulation of the biological activities
of sAPS.
The aim of this review is to summarize current knowledge on the biological activities and toxicity
of sAPS, with particular emphasis on mechanisms of toxicity of two synthetic analogues, APS12-2 and
APS3, in vivo and in vitro.
2. Synthetic Analogues of Polymeric Alkylpyridinium Salts
Their interesting biological effects, their potential use in the pharmaceutical and chemical
industries, coupled with the insufficient quantities of natural poly-APS, have contributed to the

Mar. Drugs 2014, 12 2410
development of new methods for synthesizing poly-APS analogues. This could enable the commercial
production of sAPS with modified characteristics. In 2004, Mancini and colleagues synthesized dimers
and tetramers of 3-alkylpyridinium salts [27]. In 2010, Houssen and colleagues reported a new
protocol enabling synthesis of larger polymers that possess greater biological activities [28]. To
determine how the structure of sAPS influences the biological activities, several sAPS, with various
lengths of the alkyl chain, numbers of pyridinium rings and with different counter ions (bromide or
chloride), have been synthesized.
Figure 1. Synthesis of poly-(1,3-alkylpyridinium) salts. Reagents and conditions: for
R = alkyl chain: (i) HBr, toluene, reflux overnight followed by neutralization to yield
products with X = Br; thionyl chloride, dichloromethane, room temperature to yield
products with X = Cl; (ii) reflux in acetonitrile or methanol (in the presence of a small
amount of KCl for monomeric chloride), followed by microwave irradiation at 130 °C for
the time length stated for each compound under the experimental section. Adapted from
Zovko et al. [29], with permission from © 2012 Elsevier Ltd.

Mar. Drugs 2014, 12 2411
sAPS Synthesis
A method that enables simple, rapid and affordable synthesis of highly purified alkylpyridinium
compounds with a high degree of polymerization was developed [28,29]. Monomers were prepared
according to a small modification of the method described by Davies-Coleman in 1993 [30]. Pyridyl
alcohol was produced by coupling bromo-alcohol with 3-picoline. Bromide monomers were produced
by neutralization of the alcohol treated with hydrogen bromide, while chloride monomers were
produced by reacting the substrate with thionyl chloride. The monomers were further oligomerized in
the presence of acetonitrile and methanol. Polymers were then formed using microwave-assisted
polymerization. Their length depended on the time of irradiation [28,29]. Interestingly, the critical
micelle concentration of selected sAPS (APS7, APS8 and APS12-2) was found to be above 1 mg/mL [31],
e.g., considerably higher than that determined for natural poly-APS [5].
The chemical synthesis of poly-(1,3-alkylpyridinium) salts is shown in Figure 1.
The method is quick, safe, economical, eco-friendly and enables the production of large amounts of
product [32]. Several sAPS have been produced with various degrees of polymerization, different
cations and different lengths of the alkyl chain. Some analogues are mixtures of polymers with
different degrees of polymerization. The basic chemical properties of the most studied sAPS are
presented in Table 1.
Table 1. Basic chemical properties of polymeric 3-alkylpyridinium salts (poly-APS) and
their synthetic analogues.
Compound
No. of Alkyl
C-atoms
No. of Polymers
and Molar Ratio
Molecular Weight
(kDa)
Degree of
Polymerization
Counter Ion
Reference
Poly-APS
8
1
5.52
29
Cl−
[6]
APS3
3
2 (9:1)
1.46 (1.2/3.8)
10 and 32
Cl−
[29]
APS7
7
2 (2:1)
2.33 (1.4/4.2)
8 and 24
Cl−
[29]
APS8
8
1
11.9
63
Br−
[28]
APS12
12
1
12.5
51
Br−
[28]
APS12-2
12
1
14.7
60
Br−
[28]
3. Biological Activities of sAPS
3.1. Hemolytic and Antimicrobial Activity
Like natural poly-APS, the synthetic analogues (sAPS) have structures similar to those of cationic
detergents [33]. The hemolytic activity for both is directly proportional to the length of the alkyl chain
and the degree of polymerization [34,35]. The hemolytic activity of analogues with low molecular
weights is very low or negligible [28,29,31]. The nature of the counter ion does not influence the
hemolytic activity [29]. The electrophysiological effects of mono-, di- and tetra-meric sAPS [27] were
evaluated also on cultured hippocampal neurons [17]. Here, again, low-molecular sAPS were found to
be much weaker pore formers than the natural poly-APS, indicating that the polymerization degree and
the subsequent formation of the supermolecular structure are crucial for the observed membrane activity.
sAPS possess antimicrobial properties and have proven to be more effective against Gram-positive
(S. aureus) than Gram-negative bacteria (E. coli). The latter are more resistant to sAPS action,

Mar. Drugs 2014, 12 2412
probably due to the additional lipopolysaccharide layer on the cells [27,29]. Their antibacterial activity
increases with the increasing number of positive charges and the length of the alkyl chain. sAPS with a
bromide counter ion are more active than sAPS with a chloride counter ion [11,29]. Interestingly, all
sAPS, except APS3, which is the smallest, have higher antibacterial activities than natural poly-APS [29].
Compared with structurally similar compounds, like cetylpyridinium chloride (CPC), which has
minimal inhibitory concentrations (MIC) for S. aureus and E. coli of <1.47 μM and 470 μM, sAPS are
quite effective, their antibacterial activity against E. coli being greater (MICAPS-12-2 = 34.01 μM) and
against S. aureus being comparable (MICAPS-12-2 = 6.8 μM) to that of CPC [29].
sAPS also inhibit the growth of pathogenic fungi; the length of the alkyl chain and the degree of
polymerization are important. APS12-2, the analogue with the longest alkyl chain and the highest
degree of polymerization, has the highest antifungal activity [29]. The effectiveness of several sAPS
has been compared with that of some standard antifungal drugs. The antifungal activity of analogues
APS12-2 and APS3 was similar to that of miconazole, while other antifungal drugs were ten to a
hundred times more effective than sAPS [29]. sAPS, especially those with longer alkyl chains, are also
effective against saprophytic fungi. The oxygen atom in the alkyl chain of APS8 significantly
decreases its effectiveness. However, APS12-3 is appropriate as a biocide for protecting wood against
the fungus, Gloeophyllum trabeum [29]. Finally, sAPS oligomers and polymers have the ability to
effectively inhibit the settling of the marine barnacle, Amphibalanus amphitrite, larvae and are thus
interesting as antifouling agents [36,37].
3.2. Effects of sAPS on Acetylcholinesterase
The most prominent biological activity attributed to sAPS is probably the inhibition of AChE, the
enzyme in the nervous system synapses that hydrolyses the neurotransmitter, acetylcholine (ACh).
Hydrolysis of ACh takes place at the bottom of a 2 nm-deep enzyme active site gorge, where the
anionic site responsible for choline recognition and the catalytic site with its active serine are located.
At the rim of the gorge, there is another binding site for the substrate and other ligands, called the
peripheral anionic site [38]. This is also the binding site for natural poly-APS. The first
non-competitive binding of poly-APS is followed by several successive phases ending in the
irreversible inhibition of the enzyme, which is due to the aggregation and precipitation
of AChE [13,14].
Unlike the natural poly-APS, the time-course of AChE inhibition by sAPS12 and APS12-2 is linear,
showing the reversibility of inhibition [28]. This compounds act as noncompetitive AChE inhibitors,
by binding to the peripheral anionic site and preventing the binding of ACh inside the enzyme gorge. It
is assumed that binding takes place at this site, because the size of the synthetic analogues is too great
to allow entry to the enzyme gorge, as these sAPS are very potent AChE inhibitors, acting in
picomolar concentrations [28]. They could be used in medicine as drugs for treating conditions in
which ACh secretion is reduced, i.e., Alzheimer’s disease, myasthenia gravis and eye glaucoma [39].
3.3. Antitumor Activity of sAPS
Recent studies with synthetic analogue APS8 have shown that it is a potent inhibitor of α7-nicotinic
receptors, at concentrations of less than 1 nM [40]. Since this concentration is lower than the inhibition

Mar. Drugs 2014, 12 2413
constant for AChE (1.88 nM), APS8 activity is probably due to the inhibition of receptors and not
AChE. APS8 inhibits the growth of various cancer cell lines, like A549 and SKMES-1, but is not toxic
for normal fibroblasts [40]. Moreover, using flow cytometry and differential staining, it was found that
APS8 triggers the apoptosis of cancer cells in a concentration-dependent manner [40]. This effect may
be due to the antagonistic effect of APS8 on α7-nicotinic receptors, which are particularly abundant in
various tumor cells of the respiratory tract in contrast to non-cancer cells [40]. The apoptosis caused by
APS8 involves both intrinsic and extrinsic pathways and is activated by cell stress. In the extrinsic
pathway, the death receptors are involved and are activated after binding certain ligands. A number of
reactions are triggered, ultimately leading to apoptosis [40]. The results suggest that APS8 or similar
compounds could be considered as promising compounds for antitumor drugs development for some
types of lung cancer [40].
The basic biological activities of the most studied sAPS are summarized in Table 2.
Table 2. Biological activities of poly-APS and their synthetic analogues.
Compound
AChE Inhibition—Ki (nM) *
Hemolysis (s−1 at 500 nM) **
IC50 for NSCLC (μM) ***
Poly-APS
irreversible inhibition
0.05
4.41
APS3
85
0
3000
APS7
10
0.1
480
APS8
1.875
2.6
478
APS12-2
0.036
5.0
470
NSCLC, non-small cell lung cancer; * [28]; ** [31]; *** [41].
4. Toxicity of APS12-2 and APS3
In view of the possible use of sAPS in medicine and the pharmacy setting, it was essential to
evaluate their effects on mammals and to explore the mechanisms of their toxicity. APS12-2 and APS3
are the most studied sAPS. They were chosen for research due to the different mechanisms of their
toxicity and their different chemical properties, which could account for their physiological,
toxicological and pharmacologic activities.
APS12-2 is an analogue with a higher degree of polymerization and a longer alkyl chain, bearing a
bromide counter ion. It is strongly hemolytic and acts as a non-competitive AChE inhibitor. APS3 is
smaller and shorter, with a chloride counter ion. It is non-hemolytic and acts as a competitive AChE
inhibitor. In vivo and in vitro experiments have provided significant data on the possible adverse
effects of APS12-2 and APS3 on the vital functions of mammalian organisms, related to their effects
on organ systems, organs, tissues and cells, as well as on the molecular level, as described below.
4.1. In Vivo Effects of APS12-2 and APS3
Before performing in vivo experiments, the median lethal dose for both sAPS analogues was
estimated in Balb/c mice. Different doses of APS12-2 and APS3 were administered intravenously to
male Wistar rats. Blood pressure, respiratory activity and electrocardiograms (ECG) were monitored.
At the end of each experiment, vital organs were removed for histological analysis. The estimated
median lethal doses for APS12-2 and APS3 in mice were 11.5 and 7.25 mg/kg [8,42]. Compared to

Mar. Drugs 2014, 12 2414
natural poly-APS, with an estimated LD50 in rats of 2.7 mg/kg [24], the toxicity of APS12-2 and APS3
is low. In in vivo experiments, it was found that rats are more sensitive to both analogues than
mice [8,42].
Sublethal effects of APS12-2 in vivo were determined in rats (sublethal doses of four and
5.5 mg/kg) to provide more understanding of the mechanistic specificity of this APS. i.v. application
leading to mild transient bradycardia similar to that described for poly-APS above, but in this case, the
heart rate gradually recovered. Arterial blood pressure (aBP) decreased significantly immediately
following application. This was followed by a transient increase, then finally, a gradual return to the
basal value. The bradycardia produced by the anticholinergic activity of the compounds, the
hyperkalemia or the lung reflexes may be responsible for the reduction in aBP. The subsequent
increase in aBP could be the consequence of a compensatory increased sympathetic tone as a response
to the hypotension or the direct or indirect effect of the substance on peripheral blood vessel resistance.
The fact that no increase in heart beat frequency was observed during the period of transient
hypertension supports this view. Sublethal doses of APS12-2 also caused significant elevation of blood
potassium levels, which could be an important cause of the cardiorespiratory toxicity of APS12-2 [8].
In rats, the death caused by a lethal dose (11.5 mg/kg) of APS12-2 was due to cardiorespiratory
arrest [8]. Since the latter can be produced at plasma potassium concentrations above 10 mM [43–45],
the cardiotoxic effects of APS12-2 may be related to its hemolytic activity and hyperkalemia
(10.44 ± 0.44 mM) [8,31]. Respiratory arrest could be produced by the stimulation of juxtapulmonary
capillary receptors in lung parenchyma [46,47]. These receptors are mechano-sensitive and are
therefore activated by conditions, like pulmonary edema, congestion or pulmonary microembolism [48].
This could be the mechanism of respiratory arrest produced by lethal doses of APS12-2. This
explanation is supported by histopathological findings of acute lesions observed in the pulmonary
vessels of rats, the lysis of aggregated erythrocytes within their lumina and pulmonary edema [8].
APS3 was not lethal in experimental rats at doses up to 20 mg/kg and at cumulative doses up to
60 mg/kg. Only transient changes in blood pressure were observed. The serum potassium level was, as
expected, not significantly altered, due to the absence of APS3 hemolytic activity [42]. In vivo
experiments with APS3 further confirmed the putative role of hyperkalemia in the cardiotoxic activity
of APS12-2.
The effects of APS12-2 and APS3 on in vivo measured parameters are summarized in Table 3.
In vivo experiments with APS 12-2 on mice, injected (2.2 μg/kg) intramuscularly at the base of the
tail, showed that it decreased the compound muscle action potential (CMAP) [49]. Similar time- and
dose-dependent reversible effects on CMAP amplitude were observed in mice after administration of
APS3 at sublethal doses (0.3–3 mg/kg). Administration by i.v. of cumulative doses of APS3 (up to
60 mg/kg) in rats produced dose-dependent inhibition of nerve-evoked muscle contraction with an ID50
of 37.25 mg/kg. Since APS3 is a water-soluble substance composed of two relatively small polymers
in a molar ratio 9:1,with molecular weights (m.w.) of 1.2 and 3.8 kDa, in contrast to APS12-2
(m.w. 17.7 kDa), it can pass the slit-pore in muscle capillary membranes, reach the postsynaptic
membrane of the neuromuscular junction and cause neuromuscular block. The relative permeability of
skeletal muscle capillary pores to substances with molecular weights of 342 and 5000 Da is
0.4 and 0.2.

Mar. Drugs 2014, 12 2415
Table 3. The effects of APS12-2 and APS3 on significant parameters in rats.
Measured Parameters
APS12-2 *
APS3 **
LD50 (mice)
11.5 mg/kg
7.25 mg/kg
ECG (rats)
 bradycardia
 second degree atrioventricular block
 Ventricular extrasystoles
 Transient tachycardia
Arterial blood pressure
Steep decrease immediately after application
First a decrease, then an increase above
base-line value
Breathing
Respiratory arrest soon after application
No effect
Biochemical parameters
Statistically significant increase in K+ level
(10.44 ± 0.44 mM)
Statistically significant increase in K+ level
(5.66 ± 0.37 mM)
Muscle contraction
No effect up to 8.6 mg/kg
ID50 = 37.25 mg/kg
LD50, half-lethal dose; ID50, median inhibitory dose; ECG, electrocardiography; * [8]; ** [42].
In contrast to APS3, APS12-2 (at 11.5 mg/kg) produced cardiorespiratory arrest, due to its
hemolytic activity, associated with hyperkalemia [8,42]. A possible in vivo effect of APS12-2 on
skeletal muscle contraction could therefore not be observed in vivo, since APS 12-2 produces cardiac
arrest and the death of experimental animals due to its hemolytic activity and hyperkalemia at much
lower doses (11.5 mg/kg), as expected for a skeletal muscle contraction block (i.e., as shown by the
calculated non-hemolytic median inhibitory dose (ID50) of 37.25 mg/kg for APS3-induced skeletal
muscle contraction in vivo). In addition, the relative permeability of skeletal muscle capillary pores to
substances with an m.w. of approximately 17 kDa (close to that of APS12-2) is ten times lower
(at 0.03) than in APS12-2, so that the diffusion of APS12-2 is expected to be much slower.
4.2. In Vitro Physiological and Pharmacological Effects of APS12-2 and APS3
Based on the structure and anti-AChE activities of APS12-2 and APS3 (both are quaternary
ammonium compounds), effects on neuro-muscular transmission were expected. sAPS are structurally
related to quaternary ammonium compounds, like physostigmine, bis(7)-tacrine and BW284c51, some
of which have dual effects and, in a concentration-dependent manner, inhibit either AChE or nicotinic
acetylcholine receptors (nAChR) [50–53]. The effects on neuro-muscular transmission were revealed
by experiments with both analogues on neuromuscular preparation [42,49]. APS12-2 and APS3 block
nerve-evoked isometric muscle contraction in a concentration-dependent manner [42,49]. To determine
their molecular mechanisms of action, the microelectrode technique on mouse hemidiaphragm
preparations was applied in order to study the effects of APS12-2 and APS3 on skeletal muscle fiber
resting membrane potential (RP), miniature endplate potential (MEPP) and evoked endplate
potential (EPP). The direct influence of sAPS analogues on nAChRs expressed on Xenopus oocytes
was also studied. Both analogues decreased the amplitude of EPPs and MEPPs in a
concentration-dependent manner, indicating that their action may be on nAChRs [42,49]. To confirm
the possibility of the direct effects of APS12-2 and APS3 on muscle-type nAChRs at the
neuromuscular junction, experiments were performed on Xenopus laevis oocytes into which Torpedo
(α2β1γδ) muscle-type nAChRs had been incorporated. It was proven that APS12-2 (IC50 = 0.0005 μM)

Mar. Drugs 2014, 12 2416
and APS3 (IC50 = 0.19 μM) effectively block the acetylcholine-evoked current through the
muscle-type nAChRs expressed in oocyte membranes, due to nAChRs inhibition [42,49].
In order to study the effects of APS12-2 and APS3, to better establish the mechanisms of their
cardiovascular effects and to provide more data on mechanism specificity, experiments were
performed on isolated porcine coronary vessels. In contrast to APS3, which displayed no effect,
APS12-2 induced the contraction of coronary ring preparations in a concentration-dependent manner
(at 1.36 to 13.60 μM). Lanthanum chloride, a non-selective cation channel blocker [54,55], and
verapamil, a selective antagonist of L-type voltage-dependent calcium channels [56], completely
abolished the contraction of coronary rings induced by APS12-2. This indicates that, due to increased
Ca2+ influx through the voltage-gated Ca2+ channels, APS12-2 induces vascular smooth muscle
contraction in a concentration-dependent manner. These results show, for the first time, that APS12-2
induces a concentration-dependent contraction of coronary ring preparations. Coronary vasoconstriction, as
well as hyperkalemia, may contribute to the cardiotoxic effects of APS12-2. It is notable that the
maximal final concentration of APS12-2 (13.60 μM) that produces a significant increase in coronary
ring tension in vitro is comparable to the maximal concentration of APS12-2 in blood plasma in vivo
following the administration of one LD50, which produced arrhythmia and cardiorespiratory
arrest [57].
The effects of APS12-2 and APS3 on the in vitro measured parameters are summarized in Table 4.
Table 4. Physiological and pharmacological effects of APS12-2 and APS3 in vitro.
Measured Parameters
APS12-2 *
APS3 **
Effect
IC50
Effect
IC50
Skeletal muscle
contraction
Nerve-evoked
stimulation
Inhibition
0.74 μM
Inhibition
20.3 μM
Direct stimulation
No effect up to
2.72 μM
N/A
No effect up to
20.55 μM
N/A
Pharmacological effect
atropine
No effect up to 80 μM
N/A
No effect up to
80 μM
N/A
neostigmine
No effect up to 1 μM
N/A
No effect up to
1 μM
N/A
3,4-DAP
Stops muscle contraction
blockade
(300 μM)
N/A
Stops muscle contraction
blockade
(300 μM)
N/A
Effect on
RP
No effect up to 3.40 μM
N/A
No effect up to
68.49 μM
N/A
MEPP
Amplitude decrease,
MEPP disappear above
0.68 μM
N/A
Amplitude decrease,
MEPP disappear above
6.85 μM
N/A
EPP
Amplitude decrease
0.36 μM
Amplitude decrease
7.28 μM
nAChRs inhibition
Inhibition
0.0005 μM
Inhibition
0.19 μM
Effect on coronary rings ***
Contraction
(4.1–13.6 μM)
N/A
No effect up to
137 μM
N/A
N/A, Not applicable; 3,4-DAP, 3,4-diaminopyridine; RP, resting membrane potential; MEPP, miniature endplate potential; EPP,
endplate potential; nAChRs, nicotinic acetylcholine receptors; * [49]; ** [42]; *** [57].

Mar. Drugs 2014, 12 2417
Their hypotensive action, hemolytic activity (of some compounds) and cytotoxic activity may limit
the use of these substances as anti-tumor therapeutics and anti-cholinergic drugs. These effects are
expressed in vitro at very low concentrations of APS12-2. However, relatively high doses of the tested
compounds have to be used to see these effects in vivo, which makes these compounds suitable for
preclinical testing. In conclusion, the in vivo toxicity of APS3 is probably the result of the reversible
antagonistic action of the compound on nAChRs on motor endplates, as shown in in vivo and in vitro
experiments. On the other hand, the toxicity of hemolytically active APS12-2 is probably related to the
high blood potassium levels and cardiac arrest or to its direct functional effects (mechanical
dysfunction) of APS12-2 on the heart conduction system. This remains to be proven. The coronary
vasoconstriction produced by APS12-2 constitutes an important mechanism that can contribute to the
cardiotoxicity of APS12-2. In general, the toxicity of tested sAPS is relatively low, when compared to
that of natural poly-APS. The sAPS, in particular those that are non-hemolytic, are of interest for
preclinical testing as novel lung tumor chemotherapeutics.
5. Conclusion
Synthetic APS exert a wide range of interesting biological activities that can vary according to their
structure. It was shown that some of them inhibit the growth of lung cancer cells lines, either by
inducing apoptosis or by inhibiting cell division. The putative underlying mechanism might be the
block of the cholinergic system, which is physiologically important for lung cancer cells homeostasis.
Therefore, sAPS could be suitable especially as a new class of chemotherapeutic drugs for treating
non-small cell lung cancer. In recent studies, it was shown that sAPS have low toxicity that encourages
their further investigation and testing as anticancer drugs. The antitumor effects of one of sAPS
(APS8) are currently being preclinically evaluated on a lung carcinoma rodent model and show some
very encouraging results. Synthetic APS could also find their use as agents allowing the stable
transfection of cells, which could lead to their potential applications in medicine and cell biology.
Finally, due to their ability to inhibit the settlement of marine organisms to submerged surfaces, they
could be potentially used as active components of antifouling paints.
Acknowledgements
The authors acknowledge the Slovenian Research Agency (research program P4-0053(RF) and
P4-0092 (MG)) for their financial support, Kristina Sepčić for helpful comments on the manuscript and
Roger Pain for the critical reading of the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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