Anticancer pentamethinium salt is a potent photosensitizer inducing mitochondrial disintegration and apoptosis upon red light illumination.

Despite progress in the development and application of novel therapeutic agents, cancer remains a major cause of death worldwide. Therefore, there is a need for new approaches to increase therapeutic options and efficiency. The metabolism of cancer cells differs from that of non-malignant cells and their mitochondria show altered activities that can be utilized as a target for drug development. Salt 1 is a low-molecular weight heterocyclic compound of the polymethine class that accumulates in the mitochondria of cancer cells and selectively disrupts their metabolism. Salt 1 leads to a non-apoptotic form of cell death in vitro that is associated with an autophagic cellular response and eventual metabolic collapse, and inhibits human tumor xenograft growth in vivo without apparent toxicity for normal cells. As a pentamethinium compound, salt 1 exhibits intrinsic fluorescence and is a candidate for photosensitization after excitation by appropriate wavelengths of light. Herein, we report that salt 1 is a potent photosensitizer, which generates a photodynamic effect and provides enhanced cytotoxicity compared to salt 1 without light exposure. Importantly, photosensitization is optimally induced by red light, which is used clinically for photosensitization and penetrates further into tissues than lower wavelengths. Cancer cells treated with non-cytotoxic doses of salt 1 and subsequently exposed to 630 nm light show severely damaged mitochondria, manifested by reduced mitochondrial membrane potential and disintegration of the mitochondrial tubular network. As a consequence, cancer cells lose their proliferative potential and die via apoptosis in the presence of light. These findings indicate that salt 1 is a promising photosensitizer with potential to be combined with 630 nm light to strengthen its efficacy in cancer therapy.

Interactions of miltefosine with erythrocyte membrane proteins compared to those of ionic surfactants.

For miltefosine (MIL), a zwitterionic alkylphospholipid approved for leishmaniasis treatment, the mechanism of action is not well established. Electron paramagnetic resonance (EPR) spectroscopy has indicated that the interaction of MIL with membrane proteins has similarities to that of ionic surfactants. A general concern about leishmanicides is their high hemolytic potential, so we decided to compare the interactions of MIL and three ionic surfactants with the erythrocyte membrane. Measurements with two different spin labels indicated that the surfactants sodium dodecyl sulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS, zwitterionic) as well as MIL increase the dynamics of erythrocyte membrane proteins in a concentration-dependent manner. SDS produced the smallest increases in protein dynamics and was also the least hemolytic for measurements in PBS and in whole blood. Spin label EPR measurements performed directly in the blood plasma detected increased albumin stiffness caused by 2.5 mM SDS due to electrostatic/hydrophobic interactions. For 10 mM concentrations of the compounds, the EPR spectra showed a fraction of albumin with greater mobility and another with the same as that of the untreated plasma. The zwitterionic compounds MIL and HPS did not present significant differences in this study.

A simple spectroscopic method for studying erythrocyte ghost resealing.

A novel spectroscopic method is described for following the kinetics of resealing of hemolysed erythrocyte ghosts. The procedure is based on the broadening of the EPR spectrum of nitroxyl radicals by paramagnetic ions. The method is used to study the effect of Ca2+, Mg2+ and dimethonium ion on the kinetics of resealing.

Shape and volume changes in rat erythrocytes induced by surface-active alkyltrimethylammonium salts and sodium dodecyl sulphate.

Surface-active alkyltrimethylammonium salts (C12, C14 and C16) and sodium dodecyl sulphate (SDS) caused shape alterations and a volume increase in rat erythrocytes. The alkyltrimethylammonium salts caused echinocytic shapes at both prelytic and lytic concentrations during the first minutes of incubation at 37 degrees C but as the incubation proceeded some of the echinocytes were transformed into stomatocytes. This transformation developed through the normal discocyte shape and it occurred only above certain concentrations. With C14 and C16 the concentration at which stomatocytic shapes appeared coincided with those at which the volume increase began. With the C12 homologue stomatocytic shapes did not appear until lytic concentrations were reached, whereas the volume increase began at prelytic concentrations. SDS caused only echinocytic shapes at 37 degrees C and these appeared at prelytic concentrations, whereas the volume increase was associated with lytic concentrations. When erythrocytes crenated by SDS were cooled to room temperature they were transformed into stomatocytes and discocytes. Our results indicate that (a) even though ionic surfactants induce both swelling and shape alterations in erythrocytes these two changes are not necessarily connected, and that (b) the different shapes induced by cationic and anionic surfactants cannot be due to differences in the distribution of the surfactant molecules within the lipid bilayer of the erythrocyte membrane alone.

Ionic control of the size of the vesicle matrix of beige mouse mast cells.

Isolated matrices of the giant secretory vesicles of mast cells of the beige mouse were reliably produced by the osmotic lysis of isolated vesicles. These matrices maintained their form, and their sizes were easily measured using Nomarski optics. The size of the matrix depended on the ionic composition of the bathing solution. The physiologically relevant ions, histamine and serotonin, contracted the matrix. Multivalent cations condensed the matrix relative to univalents. Ag+, acid pH (below 5), and basic pH (above 9) expanded the matrix. In the presence of 10 mM histamine, lowering the pH from 9 to 5 contracted the matrix more than can be attributed to the pH-dependent matrix contraction in zero histamine. The nontitratable organic cation, dimethonium, contracts the matrix with little effect of pH in the range of 5-9. These results suggest that histamine acts as a matrix contractor in the divalent form. The dose-response (contraction) relation for histamine was gradual from micromolar to 316 mM (millimolar) histamine. Experiments with mixtures of histamine and sodium show antagonistic effects on the matrix but are inconsistent with either a model where ions compete for identical sites or a parallel model where ions interact with separate independent sites. In vigorous histamine washoff experiments, the half time for vesicle expansion in 10(-4) M pH buffer was approximately 4 s; in isotonic NaCl solution, it was 0.5 s. When 1 M histamine was presented to closely apposed matrices, fusion resulted. The matrix material returned to its initial shape after being mechanically deformed with a glass probe. These results suggest that the matrix size is controlled by its ion exchange properties. The matrix expansion can quantitatively account for the vesicular size increase observed upon exocytosis (as a postfusional event) and the osmotic nonideality of intact vesicles. The mechanical expansion is probably significant in the widening of the exocytotic pore and the dispersal of the vesicular contents.

Effects of surface-active alkyltrimethylammonium salts on concanavalin A-mediated agglutination in Acanthamoeba castellanii.

Pretreatment of Acanthamoeba castellanii (Neff strain) with sublytic concentrations of surface-active alkyltrimethylammonium salts (C12, C14, C16) enhanced ConA-mediated agglutination of the amoebae. Treatment with the surfactants alone did not affect the "spontaneous" agglutination of the amoebae. Electron microscopic (SEM and TEM) examinations of amoebae treated with sublytic concentrations of the surfactants did not reveal any significant alterations in cell shape or in cell surface morphology in treated cells. The binding of [3H]ConA to the amoebae was not affected by pretreatment with sublytic concentrations of the surfactants. It is suggested that the increase in ConA-mediated agglutination in surfactant-treated amoebae may be due to a fluidizing effect of the surfactants on plasma membrane of the amoebae. Part of the results of this work has previously been published in Eur. J. Cell Biol. 22, 210 (1980). (Abstract M 624).

Actions of a series of bisquaternary compounds on nicotinic acetylcholine receptors in insects: ligand binding and electrophysiological studies.

A series of bisquaternary ammoniums, with chain lengths of between 4-12 carbon atoms (C4-C12), have been tested for their ability to block acetylcholine-induced responses in the fast coxal depressor motor neurone (Df) of the cockroach (Periplaneta americana) and to displace [125I]alpha-bungarotoxin from membrane preparations of the CNS of the cockroach. The physiological studies showed that tetramethonium was inactive, whereas hexa-, octa- and dodecamethonium showed an enhanced ability to block acetylcholine-induced responses as the chain length increased. Decamethonium resulted in a slight increase in acetylcholine-induced depolarizations. Ligand binding studies showed that the ability of the compounds to inhibit the specific binding of [125I]alpha-bungarotoxin increased with size from C4-C12. The results show that neuronal nicotinic receptors in insects differ in aspects of their pharmacology from both the major subclasses of nicotinic receptors of vertebrates.

The channel-blocking action of methonium compounds on rat submandibular ganglion cells.

The effects of drugs of the polymethylene bis-trimethylammonium (methonium) series on the characteristics of the synaptic currents (e.s.cs) recorded from voltage-clamped rat submandibular ganglion cells have been studied. The drugs studied were from C4 to C10 (decamethonium). All of the drugs except C4 shortened the initial decay phase of the e.s.c.; C9 and C10 produced an additional slowly decaying component. These effects were interpreted in terms of an open channel block mechanism, the calculated rate constants for association with the open channel at -80 mV being fairly similar (5.9 X 10(6) to 18.1 X 10(6)M-1S-1) for all of the compounds except C4, which had no effect on the e.s.c. decay. All of the compounds produced use-dependent block when tested with short trains of stimuli at 10 Hz, or with trains of ionophoretic pulses of acetylcholine, consistent with their channel blocking property. Tubocurarine had a similar effect, but not trimetaphan or mecamylamine. Recovery from use-dependent block with short chain methonium compounds, up to C8, was very slow in the absence of agonist, being incomplete even after several minutes. With C9 or C10 or tubocurarine, recovery from use-dependent block was complete within a few seconds. With C6 recovery in the absence of agonist was unaffected by membrane potential, but could be accelerated by applying acetylcholine with the cell depolarized to -40 mV. This persistent block was ascribed to the ability of the blocking molecule to become trapped by closure of the channel. With C9 and C10 it is assumed that their presence inhibits channel closure, so they can escape without the help of agonist. When use-dependent block is avoided by leaving the ganglion unstimulated during equilibration with the blocking drug, the first e.s.c. elicited shows no appreciable reduction of amplitude, though with C6, C7 or C8 subsequent responses elicited at 0.1 Hz become progressively more blocked. Even at 1 mM, C6 does not prevent acetylcholine from opening ionic channels. It is concluded that all of the effects on e.s.c. amplitude can be interpreted in terms of channel block, there being no evidence of any receptor blocking action.

Interaction of competitive antagonists: the anti-curare action of hexamethonium and other antagonists at the skeletal neuromuscular junction.

1. In the rat isolated diaphragm preparation hexamethonium and other low potency competitive antagonists of acetylcholine (ACh), including gallamine and hyoscine butylbromide, reverse block by the potent antagonists tubocurarine, pancuronium and alcuronium. 2. In the presence of tubocurarine, hexamethonium increases the amplitude of the end-plate potential without increasing the quantal content. It enhances the response to ACh applied iontophoretically to the end-plate but does not enhance the response to ACh applied in the bath. 3. The anti-curare effect of hexamethonium is abolished in the diaphragm of the rat, guinea-pig and mouse by inhibitors of acetylcholinesterase. The effect is not observed in the indirectly stimulated toad sartorius muscle. 4. The effect is explained if tubocurarine does not dissociate appreciably in the time taken for ACh to achieve high occupancy of receptors, so that a fraction of receptors is completely excluded from occupation by ACh. Equilibration with hexamethonium reduces the fraction excluded by tubocurarine and the transmitter now competes with hexamethonium for more receptors and produces a larger response. 5. On the basis of this explanation the half-time for dissociation of tubocurarine must be about 1 millisecond. It follows that tubocurarine does not act competitively with ACh at synapses when transmitter action is sufficiently brief, and that its binding to the receptor is probably diffusion-limited.

Structural requirements of compounds to inhibit pulmonary diamine accumulation.

The diamine, putrescine, is accumulated into slices of rat lung by a temperature and energy dependent process similar to that responsible for the uptake of cadaverine, the polyamines spermidine and spermine, and the herbicide paraquat. Structure-activity studies using monoamines and diaminoalkanes, amino acids and guanidino compounds, have shown that in order to inhibit the pulmonary accumulation of putrescine, chemicals should possess at least one but preferably two nitrogen-containing cationic groups. In the series of alpha, w-diaminoalkanes studied, the inhibitory potential increased with increasing chain length, reaching a plateau at 1,7-diaminoheptane. These observations together with the fact that putrescine is a good substrate for the uptake system (Km 15 microM, Vmax 704 nmoles/g wet wt/hr) suggest that effective inhibitors require at least four methylene groups between their cationic centres and that diamines with more methylene groups may fold to give this separation. With both the monoamines and the alpha, w-diaminoalkanes, changes in the free energies of interaction suggest that the observed increases in inhibitory potential with increasing chain length are due to increased hydrophobic bonding, which is a consequence of the addition of methylene groups to the alkyl chain. Furthermore, the ability of compounds to inhibit putrescine uptake appears to be related to their propensity to bind with the appropriate site for putrescine. Steric hindrance of this ionic interaction by the quaternisation of the cationic centres of the inhibitors with methyl groups, results in a total loss of measurable inhibitory activity. Also, the introduction of anionic carboxyl groups into inhibitors result in a loss of inhibitory potential, probably due to ionic repulsion. The antileukaemic drug, methylglyoxal-bis-guanylhydrazone (MeGAG), and its congeners, were some of the most potent inhibitors of putrescine uptake studied. Our findings suggest similarities between the uptake system for putrescine into the lung with other uptake systems described for MeGAG and certain polyamines.

In search of new neuromuscular blocking drugs with quick onset and short duration of effect.

In experiments using cats the neuromuscular blocking effect of bis-trimethylammonium derivatives having a non-depolarizing mode of action had an onset of action shorter than that of their triethylammonium analogs. The difference in equieffective doses of neuromuscular blocking agents administered intravenously or intraarterially was less in the case of compounds with rigid molecules than that with flexible molecules. Results indicate that a more considerable portion of flexible substance absorbs on the 'site of loss' in blood vessels than for rigid compounds. This suggests that rigid molecules promote a faster achievement of effective concentration of a neuromuscular blocking agent at synapses. Proceeding from these data a bis-trimethylammonium derivative with a rigid molecule, IEM-1213, was synthesized and studied in cats.

Characterization of the antimuscarinic effect of heptane-1,7-bis-(dimethyl-3'-phthalimidopropyl ammonium bromide).

The effect of heptane-1,7-bis-(dimethyl-3'-phthalimidopropyl ammonium bromide) (C7/3'-phthalimidopropyl), an alkane bisquaternary compound with muscarinic receptor blocking activity was studied in guinea-pig atria and ileal longitudinal muscle. C7/3'-phthalimidopropyl was a more potent inhibitor of atrial muscarinic receptors, the cardioselectivity being ca. 32-fold. Previous studies in guinea-pig atria have shown that its antimuscarinic effect was of an allosteric nature. In ileal longitudinal muscle C7/3'-phthalimidopropyl (3 to 100 microM) appeared to behave in a competitive manner towards carbachol but the combination of atropine or homatropine with C7/3'-phthalimidopropyl produced a supra-additive inhibitory effect on the responses to carbachol. In both atria and ileal longitudinal muscle homogenates, C7/3'-phthalimidopropyl also slowed the dissociation rate of [3H]QNB suggesting an allosteric mechanism. In binding studies using either [3H]QNB or [3H]oxo-M, C7/3'-phthalimidopropyl recognized two binding sites in atria and ileum. In both tissues, C7/3'-phthalimidopropyl bound with high affinity (ca. 30-70 nM) to 60-85% of the sites and with low affinity (ca. 1-9 microM) to the remaining sites. Correlation of these affinity constants with the dissociation constants obtained in functional studies in the two tissues is discussed.

Interactions of agonists with an allosteric antagonist at muscarinic acetylcholine M2 receptors.

The interaction of heptane-1,7-bis(dimethyl-3'-phthalimidopropylammonium bromide) (C7/3'-phth), with several agonists, was investigated at the muscarinic M2 receptor in guinea-pig left atria. C7/3'-phth shifted concentration-response curves for the agonists, carbachol, oxotremorine-M and (+)-cis-dioxolane, to the right in a parallel fashion. Arunlakshana-Schild regressions of the data yielded slopes significantly different to unity, suggesting non-competitive antagonism. Non-linear regression analysis, using an equation based on allosteric modulation, gave quantitative estimates of co-operativity (alpha values) and the dissociation constant of C7/3'-phth (KB). In all cases, the KB estimates for C7/3'-phth were not significantly different. Increasing the carbachol contact time 10-fold did not significantly influence the KB or the alpha value obtained with C7/3'-phth. Changing from Krebs to Tyrode solution did not significantly alter the KB for C7/3'-phth, although alpha values obtained were consistently lower in Tyrode solution, suggesting that the allosteric action may be sensitive to buffer composition. A 4-fold higher degree of negative, heterotropic co-operativity between C7/3'-phth and agonists than between C7/3'-phth and competitive antagonists was also found.

Characterization of the muscarine receptor subtype on sympathetic nerve endings in the rat caudal artery.

The prejunctional muscarine receptor on sympathetic nerves in the rat caudal artery was characterized using several selective antagonists. The inhibitory response of carbachol on vasoconstriction elicited by sympathetic nerve stimulation was antagonized by benzhexol (trihexyphenidyl; pKB 7.1), heptane-1,7-bis(dimethyl-3'-phthalimidopropyl ammonium bromide) (C7/3-phth; pKB 6.5) and hexahydrosiladifenidol (HHSiD; apparent pKB 6.0). These pKB values suggest that the receptor most closely resembles the muscarine M2 receptor subtype rather than the muscarine M1, M3 or M4 receptor subtypes.

An angiotensin-independent, hypotension-induced, sodium appetite in the rat.

We have investigated the role of peripheral angiotensin II in the generation of hypotension (without hypovolaemia)-induced salt intake in rats treated with an IV infusion of the quaternary ammonium peripheral ganglion blocker Penthonium. At a dose of 15.4 mg/ml this compound induces a significant decrease in blood pressure from the beginning of the infusion. Intake of 3% NaCl was significantly increased only on the first day (1.7 +/- 0.3 ml, n = 7, p < 0.01 vs. the day before) and this induced salt appetite was not altered (2.3 +/- 0.9 ml, n = 7, p < 0.05 vs. the day before) by intracerebroventricular administration of a nonpeptide AII-type 1 receptor specific antagonist, Losartan, at a dose known to block AII-induced drinking (250 ng). We conclude from these results that AII liberated in response to the hypovolaemia is probably not responsible for the subsequent induced intake of NaCl which may be the result of a direct barosensitive input to the salt appetite centers of the brain.

Bioelectrical homeostasis as a component of acupuncture mechanism.

Low frequency electrical current and super-high frequency electromagnetic field were applied to acupuncture points of stomach meridian in dogs. The stimulation effect on Bioelectrical potentials of 5 acupuncture points of stomach, spleen, liver, kidney, small intestine meridians and non-acupuncture skin zones was studied in conditions of blocked autonomic ganglia or neuro-muscular junctions of the dog. The influence of ganglioblockading and myorelaxating drugs on Bioelectrical potentials of acupuncture points was also researched. The results are discussed from the neurohumoural and bioelectrical hypotheses points of view. The conclusion that both mechanisms of acupuncture supplement each other is drawn. The principle of bioelectrical homeostasis as a component of acupuncture mechanism is proposed. Bioelectrical homeostasis along with other kinds of homeostasis forms a system of first level homeostats which is united into second level homeostat by the autonomic nervous system.

[Effect of dodeconium on carbohydrate metabolism]. / Vliianie dodetsoniia na uglevodnyi obmen.

It is shown in experiments on albino rats that dodeconium in therapeutic doses stimulates the glycolytic processes and inhibits the aerobic oxidation of glucose in the pentose phosphate cycle as well as the final stages of gluconeogenesis. Such an action of dodeconim leads to hypoglycemia and normalizes many indices of carbohydrate metabolism in alloxane diabetes.

[Role of the autonomic nervous system in the differentiated response of pancreatic enzyme secretion]. / O roli vegetativnoi nervnoi sistemy v differentsirovannoi reaktsii pankreaticheskoi fermentovydeleniia

In chronic studies on fistular dogs, the basal secretion of pancreas and secretion stimulated by the duodenal perfusion of acid (pH 1.5) solutions of albumen and its poly-peptide hydrolysate was studied. Perfusion of the hydrolysate stimulated the pancreatic release of enzyme more than albumen itself. Pentamin, atropin, and ergotal suppressed basal secretion and secretion stimulated by the duodenal perfusion with the above solutions.

[A nondepolarizing muscle relaxant with rapidly developing and short-term action]. / Nedepoliarizuiushchii miorelaksant s bystro razvivaiushchimsia i kratkovremennym deistviem.

The pharmacological properties of the new nondepolarizing myorelaxant IEM-1213 and of its mixture with tercuronium were studied in experiments on anesthetized cats. Intravenous infusion of a blocking dose of IEM-1213 did not cause a change in the level of arterial pressure and blockade of the sympathetic ganglia but induced blockade of the heart muscarine receptors. The effect of IEM-1213 develops more rapidly and lasts for a shorter time than that of dithylin. Intravenous infusion of a mixture of IEM-1213 and tercuronium constituting 35 and 60% of the blocking dose of the former and, respectively, 35 and 20% of the blocking dose of the latter causes an effect similar in the time of its development to that of intravenous infusion of a total dose of IEM-1213 alone.

[The conformational aspects of the interaction of the cholinesterases of Pacific Ocean squid and vertebrates with derivatives of polymethylene bis(trimethylammonium)]. / Konformatsionnye aspekty vzaimodeistviia kholinésteraz tikhookeanskogo kal'mara i nekotorykh pozvonochnykh s proizvodnymi polimetilenbis(trimetilammoniia).

Conformational properties of a series of polymethylenebis (trimethylammonium) derivatives [formula: see text] (n = 4-10), cholinesterase reversible inhibitors, were studied by molecular mechanics method. Conformation-activity relationships between these inhibitors and human erythrocyte acetylcholinesterase, horse plasma butyrylcholinesterase, cholinesterases from the brain of the frog Rana temporaria and from visual ganglia of the squid Todarodes pacificus were investigated by correlational methods. Differences in mechanisms of antienzyme actions were determined.