QUATERNARY BENZO[C]PHENAI\THRIDINB ALKALOIDS _ BIOLOGICALACTTVITIES

Intracellular Targets of Their Action and Application in Human and Veterinary Medicine

v. ŠnaÁNgK R.VESPALEC*, A.ŠEDo*, J. ULRICHoVÁ,AND J. VICARInstitute of Medical Chemistry,Palaclql Universiý,775 I5 Olomouc, Czech Republic,E-mail : vilim@tunw. upol. cz* Inst itute of Analytic al C hemistry,

Academy of Sciences of the Czech Republic,Veveří 97, 61 1 42 Brno*Laboratory of Cancer Cell Biolog,t,Institute of Biochemistry and Experimental Oncology,Charles Universiý,I28 53 Prague, Czech Republic

1. Introduction

Quarternary benzo[c]phenanthridine alkaloids (QBA) are a small class of compoundscommonly isolated from Caprifoliaceae, Fumariaceae, Meliacea, Papaveraceae andRutaceae plants. QBA belong to the elicitor-inducible secondary metabolites and areconsidered phytoalexines because of their antifungal and nematocidal activities.Quaternary benzo[c]phenanthÍidine alkaloids whose most studied representatives aÍesanguinarine (SA), chelerýhrine (CFIE), and fagaronine (FA) (Fig. 1) display a widespectrum of non-specific biological activities and affect basic molecular targets common tomammalian cells [1]. They are the subject oť sustained practical and research interestsbecause of their pronounced widespread physiological effects [2].

The plants containing SA and CF{E Sanguinaria canaderesls (Papaveraceae, bloodroot,rhizomes contain 4-7oÁ, roots about l.8% alkaloids), Chelidonium majus (Papaveraceae'celandine, roots contain 4.5% alkaloids) and Macleya cordata (Papaveraceae, aerial partscontain about 30Á alkaloids) were utilized in the practice of traditional medicine in NorthAmerica, Europe and China long before the isolation of the pure alkaloids. SA and CFIEdisplay antimicrobial, anti-inflammatory, adrenolyic, sympatholytic, and local anestheticeffects. The powdered rhizomes and roots of S. canadensis are the active components of theweight gain stimulant for farm animals SANGROVIT@. SANGUINARIA@, the extractfrom the rhizomes of ,S. canadensis, ffid SANGTIIRITRIN@, QBA fractions fuom M.cordata are used in toothpastes and mouthwashes as antiplaque agents, and the latter isapplied as antifungal and anti-inflammatory preparation in Russia. Nevertheless, the

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M.P. Schneider (ed.), Chemical Probes in Biology,245-254.O 2003 Kluwer Academic Publishers. Printed in the Netherlands.

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molecular basis of SA and CFIE pharmacological activities anďor toxic side effects remain

mysterious. In contrast to the beneficial effects of SA/CFIE, the epidemic dropsy syndrome

is a well-known toxic effect ascribed to QBA, which is associated with the consumption ofedible plant oils contaminated by the oil from Argemone mexicana seeds [3]. The seeds ofA. mexicana contun about 0.5yo of QBA [a].

R1

R2

SANGUINARINE R|+ť=oCHzo, ť+Ra:oCHzo' R5:H

FÍá'^Rtffi,tš^' řl:5i'"-9:ó?'t'-"o'Ť'ilť;t*

Figure 1. Structures of quaternary benzo[c]phenanthridine alkaloids

It has been reported that long-term use of oral products containing SANGUINARIA@appeaÍS to be associated with an increased incidence of leukoplakia of the maxillaryuirtiUut. t5l. Furthermore, SA elicited weak positive responses in the Salmonella

mutagenicity test after metabolic activation [6] and chelerýhrine induced respiration-

deficient mutants in Saccharomyces cereyisieae [7]. However, neither teratogenicity nor

increases in preneoplastic or neoplastic lesions have been shown in long-term bioassays

with a mixture of SA and CF{E in the rat [6].In contrast to SA/CFIE, FA has a different substitution pattern in the ring D. FA possesses

antileukemic activiry inhibits HIV-1 and HlV-2-reverse transcriptases and DNAtopoisomerases I and II [8]. FA is considered a potential antitumor drug.

One of the prerequisites for QBA activity is the presence of an iminium bond. In SA/CHEthis bond is susceptible to a nucleophilic attack and consequently plays a key role in the

inhibition of SH-proteins. Both alkaloids interconvert between the cationic vs. neutral form

(i.e. hydroxide adduct or pseudobase) (Fig. 2) displaýng sort of "Dr Jekyll and Mr Hyde''

duality.

+oH ----.->+

Figure 2. Quaternary ioďpseudobase equilibrium in benzo[c]phenanthridine alkaloids.

They penetrate across the cell membrane in the hydrophobic pseudobase form acting as

pro-drugs and convert into active cationic form once inside the cell. Unlike SA/CI#, FA,-due

to iis different ring substitution pattem, exists at physiological pH as a cation only, is a

weak electrophile and its iminium bond is not attacked by nucleophiles. SA/CFE react with

R1RI

R2R2

*-an,

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a biomacromolecule or subcellular structure by covalent bond formation, FA byelectrostatic interaction. Both of these interaction modes elicit dissimilar biologicalresponse.

In this paper, the following results of our investigation on QBA are presented: 1.

Identification of chemical forms of SA/CF{E that complex with mercapto nucleophiles, andthe determination of stability constants that characterize their binding with human serumalbumin; 2. Inhibitory effects of SA, CFIE and FA on DPP-IV-like enzymes isolated fromhuman blood plasma and human anďrut glioma cell lines; and 3. QBA role in the genesis ofepidemic dropsy syndrome.

2. Identification of chemical forms of SA/CHE that complex with mercaptonucleophileso and the determination of stability constants that characterwe theirbinding with human serum albumin

The red and yellow colors of SA and CFIE, respectively, are ascribed to their quaternarycations [9,10] (Fig. 2). zr-Electron densities on the nitrogen atom in position 5, and on thecarbon atom in position 6 in SA, are 1.660 and 0.646, respectively, according to quantum

chemistry calculations [11]. Thus, the double bond N(5) : C(6) of these quaternary cationsis polar and sensitive to attack by nucleophiles [11]. For hydroxide ions as nucleophiles, apH dependent equilibrium between the charged quaternary form and the uncharged, socalled pseudobase form (Fig. 2) is typical of benzo[c]phenanthridine alkaloids. Thisequilibrium may be formulďed as a reversible complexing of the heterocyc\ic cation, /and a hydroxide ion, Q* + OIf =- QOH or, more reasonably l7l, as acidobasicequilibrium Q. + HzO =- QOH + I{ with an equilib,rium constant Kn*:Fl

J [QOH]/[Q.J. In analogy to Bronsted acids, the pKpa denotes the pH at which theheterocyclic cation and pseudobase are present in equal concentrations [11]. Thealkanolamine structure given in Fig. 2 has been almost universally adopted becausequaternary hydroxides cannot exist U2]. pKp* Constants that characteize equilibriabetween charged and uncharged forms of SA and CřIE range between 7 anď 9 u3-15].Thus, at physiological pH7.4, both charged anduncharged forms of the alkaloids exist inaqueous solutions and in blood.SA and CFm reportedly interact with nucleophilic SH- groups of simple organiccompounds in a 1:1 ratio [16]. An analogous interaction is suggested with enzymes [16],and with human serum albumin [17] in which the one free SH-group is expected to be theinteraction point. The charged (iminium) forms of SA and CFIE have been indicated as theforms interacting chemically with nucleophiles including mercapto ones [16] ; the latterwas deduced from static photometric measurements. In contrast, the uncharged(pseudobase) form of SA was denoted as the form interacting with human serum albumin in

LI7I.Our electrophoretic investigation into the type of interaction between SA or CFIE andcysteine or mercaptoethanol evidenced that interaction of these alkaloids with bothmercapto compounds is not a chemical reaction but a complexing. The formed complexesbased on non-bonding intermolecular interactions are kinetically labile. Measurements at

various pH revealed that only uncharged forms of these alkaloids complex with simplemercapto compounds [18]. Cysteine and mercaptoethanol are electrophoretically unchargedin the pH range 5 - 7.4. Anionic migration of complexes formed by uncharged SA or CFIE

248

and mercaptoethanol at pH 7.4, and at high concentrations of mercaptoethanol in thebackground electrolýe was observed. This migration evidences that an anion, which maybe supplied only from the background electrolýe, participates in the formation of thecomplex between SA or CF{E and mercaptoethanol. The absence of an analogical effect inexperiments with cysteine may be explained by the participation of the negative charge ofcysteine in the complexing. The stability of complexes of SA and CFm withmercaptoethanol and cysteine depends on both buffer cation and buffer anion. Constantscorrected for the abundance of the uncharged form of these alkaloids therefore differmarkedly depending on the buffer composition. For example, stability of complexes of SAwith cysteine was from 8,400 to 18,000 Vmol, stability of complexes of CFm with cysteinewas from 21,500 to 30,800 l/mol in the buffers used [18]. The possible structure ofcomplexes of cysteine and mercaptoethanol with uncharged monomeric form of either SAor CHE, which is always present in some equilibrium concentration in water (in aqueousbackground electrolye), was explored and modeled using the software programHyperChem 2.0. The requirement of minimum total energy was applied. The twistedcysteine molecule, aligned by its carboxylic group to the vicinity of the N(5) - C(6) bond(Fig. 3) was the most probable result for each of the alkaloids. This supports the conclusionfrom experiments with mercaptoethanol that some participation of the negative charge isnecessary for the non-covalent binding of uncharged SA or CF{E with the mercapto group.If cysteine is the ligand, its carboxylic group may supply the necessary negative charge andthe resulting complex remains therefore outwardly uncharged. If the negatively chargedgroup is absent from the ligand, e.g., in mercaptoethanol, the necessary negative chargemay be supplied only from the solution. In this case, the resulting complex is negative. Thesimple 1 :1 interaction scheme 116,171, therefore, holds only for mercapto compoundsbearing negatively charged groups. Albumins evidently belong to such compounds.

Figure 3. Molecular modeling of the non-covalent interaction between cysteine and quatemary benzo[c]phenanthridine alkaloids.

Identical results have been obtained from the investigation of the complexing of SA andCFIE with human Serum ďbumin. Constants corrected for the abundance of the interactinguncharged form of these alkaloids are 332,000+38,400 and297,000+36,000 l/mol for SAand CFm, respectively. The value of stability constant for SA from electrophoreticexperiments agrees with the stability constant, K : 385,000 (or log K : 5.59) from staticexperiments [17].

R4

G<^-':/NH:*

249

3. Inhibitory effects of SA, CHB and FA on DPP-IV-like enzymes isolated fromhuman blood plasma and human and rat glioma cell lines

Proteinase inhibitors represent a very attractive and perspective topic in thepharmacological research and therapy. As we have shown [9], QBA are potent inhibitorsof DPP-IV-like en4rme activity in glioma cells. In fact, the total cellular DPP-N-likeenzpe activity represents the sum of hydrolyic activities of several separate molecularspecies [20] that play a critical role in the regulation of cell proliferation, differentiation,apoptosis and energy metabolism. Thus, we decided to study the effect of QBA onsubseparated DPP-IV-like enzyme activity bearing molecules in order to investigate thecomplexity of a possible mechanism of the effect of QBA alkaloids. To get a deeper insightinto the QBA biological activity, we should focus not only on their "direct" targets in thecell but also on possibly more complex pathways of their action. An example of such an"indirect" QBA effect is the interaction with enzymes involved in a multitude ofphysiological functions. We have been focusing on heterogeneous DPP-N-like activitybearing enzymes to investigate their inhibition by QBA as one possible pathway that couldhelp to explain the diverse QBA biological activities.Dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5) was originally believed to be the onlymembrane-bound enzyme specific for proline as the penultimate residue at the amino-terminus of the polypeptide chain. Many biologically active peptides contain anevolutionary conserved proline residue as a proteolysis-processing regulatory element and,therefore, proline-specific proteases could be seen as their important "check-points" [21].Thus, proteolýic activation and inactivation of such peptides was originally expected to bethe main physiological function of DPP-N. Subsequently, three general mechanisms ofDPP-N activity have been postulated: (i) Limited proteolysis, i.e. highly specificprocessing of biologically active peptides, leading to their functional activation orinactivation [22]. This mechanism has been shown to play a role in immune and endocrinesystem regulďions f2l,23f, diabetes mellitus pathogenesis [24] and HIV infection [21]; (ii)Cell-cell, cell-extracellular matrix and cell-virus contacts; DPP-IV was described to be acollagen- and fibronectin-binding protein 1251, a co-receptor for HIV-1 123f, and a homingfactor for organ-specific metastasizing of breast tumors [25]; (iii) Signal transduction; DPP-IV1CD26 is considered as a co-receptor transmitting specific signals through the plasmamembrane [26]. However, other molecules, even structurally non-homologous with theDPP-N but bearing corresponding enzpe activity, have been identified recently:

fibroblast activation protein cr, dipeptidyl peptidase IV-B, N-acetylated cr-linked acidicdipeptidase, quiescent cell proline dipeptidase/dipeptidyl peptidase II, attractin, anddipeptidyl peptidase 8 B0l. Comparing the structure and relatedness of moleculesassociated functionally or structurally to dipeptidyl peptidase IV led to a grouping classifiedas "DPP-IV activity- andlor structure homologues" (DASH). DASH were shown toparticipate in a broad ďray of complex processes like cell proliferation and differentiation

l2Tl,neoplastic fransformation [28 ] and apoptosis [26].In our experiment 1291, we found DPP-N-like enzyme activity associated with moleculesof about 320 kDa ("low-MW form") in C6 rat glioma cells, human U373 glioma cells,human U87 glioblastoma cells and with molecules of about 570 kDa ("high-MW form") inhuman U87 cells. In human plasm4 a predominant peak of DPP-N-like activity was foundfor molecules of about 440 kDA ("intermediate-MW form") and a minor peak of about 600kDa. Considering MW pH optima and inhibitory parameters of particular DPP-N-likeenrqe activity fractions, we assume that the high-MW, intermediate-MW and low-MW

2s0

forms represent attractin [30], DPP-IV1CD26 [31] and DPPS [32], respectively. All threeactivity fractions were inhibited by the QBA studied. The most potent inhibition wasobserved in DPP-8, resembling the low-MW form of DPP-IV-like enzyme activity. DPP8 isa ubiquitous soluble non-glycosylated serine protease, localized in the cýoplasmic (non-lysosomal) compartment, acting preferably at neutral pH [32]. Based on the structuralsimilarity with DPP-IV, DPPS was proposed to be involved in T cell activation andimmune function [32]. Functional studies dealing with DPPS have not been published so farand thus, biological impact of its inhibition by QBA remains fully speculative.Attractin was originally described to be an immunoregulatory protein, expressed andsecreted by activated T-cells, mediating their costimulation to recall antigen-drivenproliferation [33]. Later studies demonstrated its presence in other organ systems, where itis expected to participate in the regulation of pleiotropic phenotypic features includingtumor susceptibility, pigmentation and body weight [34]. At least some of these functionsmay be dependent on attractin enzyme activity. Thus, QBA-mediated attractin inhibitioncould result in a b'road array of functional effects, depending on the orgaÍl system affected.There is contradictory evidence whether attractin [33] or DPP-N/CD26 I35l represent themain source of serum DPP-IV-like enzyme activity. We observed two fractions of DPP-N-like enzyme activity in human plasm4 which supports the hypothesis of heterogeneity ofDPP-N there. Indeed, it is possible to speculate that their proportion could be dependent oneither individual or specific condition. Decreases in serum DPP-N-like enzyme activitywere observed to be inversely related to increases in severity of depression in patientsexposed to immunochemotherapy [36]. Interestingly, serum DPP-N-like activity is alsosignificantly decreased in patients with food intake disorders. Yet, it is worth mentioningthat glucagon-like peptide 1 and 2 are DPP-N substrates and, therefore, QBA mediatedinhibition of DPP-IV-like enzyme activity can influence intestinal motility and function aswell as to aÍfect the central body weight related effect of both hormones [37].Additionally, in all glioma cell lines, but not in human plasm4 we have found also a DPP-IV-like enzyme activity fraction with parameters (acidic pH optim4 low molecular weight,substrate preference) resembling quiescent cell proline peptidase/dipeptidyl peptidase II(unpublished results). Inhibition of quiescent cell proline peptidase (QPP) is believed to bea trigger of a specific apoptotic pathway in quiescent lymphocýes [38]. Unfortunately, wewere unable to detect arry inhibition of the abovementioned DPP-N-like enzyme activityby QBA. This could be due to QBA inactivity below pH 6, i.e. conditions optimal for QPPenrqe activity. Nonetheless, QPP could be speculated as a possible QBA target, at least insome cell systems.Inhibitors of DPP-IV-like activity bearing molecules are believed to be of significanttherapeutic impact in the treďment of HIV infection, diabetes mellitus, and as aÍI

immunosupressant in the transplantation surgery and autoimmune diseases, includingmultiple sclerosis. Even though valuable attempts have been made, there is still a lack ofcommercially available specific substrates and inhibitors of individual DPP-N-like enzymeactivity bearing molecules. On the other hand, from a functional point of view, an inhibitoritself does not need to be ultimately specific for a particular DASH molecule; its''specificitý' could be provided by cell/immediate environment specific expression paffernof these enzymes.To conclude, we propose that among numerous others, some of QBA biological effectscould be mediated by their interaction with the heterogeneous group of DASH. Moreover,a specific DASH expression pattern determines the quality of such alkaloid effects, whichcan be seemingly paradoxical in particular tissues and cell systems.

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4. QBA role in the genesis of epidemic dropsy syndrome

The objective of this study was to investigate whether a long-term oral administration ofQBA in feed generates adverse effects in swine. After Sarkar [39] isolated, identified andassessed the toxicity of SA and dihydrosanguinarine (dihydroSA) as the main alkaloids ofaÍgemone oil, other investigators have assumed that these alkaloids are responsible for theoral argemone oil toxicity and consequently responsible for the onset of epidemic dropsy.However SA cannot be considered the main toxic principle of argemone oil because thetoxicity data obtained with aÍgemone oil on one harrd and with pure SA or with definedfractions of benzo[c]phenanthridine alkaloids on the other hand diÍfer considerably: Sarkardescribed mortality in young albino rats in less than a week on feed containing 10-25 mg/kgof SA hydrochloride [39]. Oral LDso (rat) of argemone oil was determined as 1.1 ml/kg

[40]. At a 0.5oÁ SA/dihydroSA concentration this corresponds to a 5 mglkg dose forSA/dihydroSA. Other authors report a LD5s in rat for SA of 1658 mglkg [41]. There are nodata available on the oral toxicity of dihydroSA; however, this difference is too large to beexplained by the toxicity of dihydroSA in argemone oil. The parenteral toxicity ofdihydroSA in rats is two andahalftimes lower than that of SA [39]. A 400 mg/kg injectionof SA suspended in arachis oil was nontoxic in mice 1421. A daily administration of 3.5

mglkg SA and 10 ml/kg €rrgemone oil (i.e. 46 mýkg SA and dihydroSA) to young and oldrats over 225 anď 250 days, respectively' did not induce epidemic dropsy symptoms [42],(i.e. the toxicity was different by one order of magnitude from that reported by Satyavati

[a0]. In trials on monkeys it was determined that 2.0 gkg argemone oil (i.e. 10 mg/kg SAand dihydroSA) administered orally ťor 4 weeks induced the development of edema anderýhema along with distinct reddish angiomatous nodules over skin [43,44]. SA only at thedaily dose of 1.6 mglkg did not bring about epidemic dropsy [a5]. Only two studies exist ofthe effects of QBA in swine. Lal et aL la6l in a limited study observed a marked bodyweight gain after the feeding 5% argemone oil in mustard oil for 3 months. Australianauthors studied the tolerarrce of Argemone ochroleuca and A. mexicana seeďs in animalfeed |47]. Seeds of the Argemone species (- 20 mg of SA/dihydroSA/other quďernary anddehydrogenated benzo[c]phenanthridine alkaloids in I kg feed) elicited the first signs ofintoxication after 3-4 weeks (lower feed intake, lethargy, skin redness, mild diarrhea). At20Á proportion, the intoxication manifested after 5-7 days and after 2 weeks these animalshad to be removed from the experiment. However, toxicity was markedly changed byinadiation of ground seeds with direct sunlight for several days; pigs then to|erated 40Á

seeds in the ťeed. Sunlight inadiation of the seed-contaminated feed in the presence of air"destroys" most of the dihydroSA and dihydroCFIE [a7]. This might indicate that thesealkaloids rather than SA could be responsible for oral toxicity of A. mexicana. Thisobservation is consistent with Hakim's reference to recognized diminution of argemone oiltoxicity by heat, light, and ageingl42l.In our experiment [48], we administered two QBA doses to swine for three months: (i) alow dose (2 mgkg feed) conesponding to the content of QBA recommended for inclusionin commercial feed), atrd (ii) a dose fifty times higher (i.e. 100 mglkg feed), where theonset of adverse reactions could be predicted, based on literature data [3]. We achieved aplasma level of 0.1 1 pglml SA, which is the level comparable with that of a humanepidemic dropsy patient [49]. All animals remained in a very good health over the durationof the experiment and did not manifest any signs of toxicity described above. Histologicalexaminations of tissues and hematology did not reveal any toxicological damage. The onlysignificant deviations from the control group clinical chemistry were found for the activity

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of liver enzymes ALT, AST and GMT. Argemone oil feeding in rats caused an increase inthe AST activity in serum [50]; other authors described a significant loss of hepaticALT/AST activities while the activities were increased in the serum [51]. Dalvi reported an

increase in the ALT/AST activity following a single i.p. dose of 10 mg/kg SA in rat 1521.Previously, we found an in vitro itthibition of the ALT/AST activity (IDso 3.4.rc-6 M) in rďliver post-mitochondrial supernatant [16]. Our observation of a decreased ALT/AST in theplasma can be explained as an inhibition of the enzymes by the ca. lO-7 M concentration SApresent there, which is a magnitude sufficient to bring about inhibition [16].The intake of argemone oil (0.5 % of benzo[c]phenanthridine alkaloids) caused asignificant stimulation of endogenous lipid peroxidation t531. The serum level ofmalondialdehyde was found to be significantly increased (172 %) in 10 human (epidemicdropsy patients) following consumption of edible oils adulterated by argemone oil. Themalondialdehyde level was found to have positive correlation with the serum SA levels

[a9]. we did not observe significant diÍferences between control and experimental groups'i.e. we cannot confirm an increase in lipid peroxidation in the plasma of experimentalanimals with SA and CFIE levels of 0.11 and 0.024 pglml, respectively (a sanguinarinelevel comparable with that of an epidemic dropsy patient).In a 32P-postlabelling assay, we did not detect any SA/CFIE-derived DNA adducts in liversof pigs exposed to the alkaloids [48]. This indicates no genotoxicity of the compounds invivo.In conclusion, orrr experimentď animals, although fed doses of quaternarybenzo[c]phenanthridine alkaloids in the range of 5 mg/kg body weight, remained in goodhealth without any symptoms that could be associated to epidemic dropsy. Hence, theresults of this study support views of authors who do not consider benzo[c]phenanthridinealkaloids, particularly SA, being responsible for the disease. Attention should rather bedirected to other aÍgemone oil components when searching for the cause of epidemicdropsy outbreaks.

AcknowledgementFinancial support by the Ministry of Education (grant No. MSM 151100003) and GrantAgency of the Czech Republic (grant No. 20310210023) is gratefully acknowledged.

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