Review
Multiple non-psychiatric effects of phenothiazines: A review

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Abstract

The phenothiazine group of drugs has long been known as antipsychotic drugs and previously it was extensively used for the treatment of anxiety. Several pieces of evidence have shown that they interfere with a variety of cellular processes and in vitro can interact with biomolecules like DNA, proteins etc. Recent reports have also revealed some new properties like antimicrobial, antiprionic, anticancerous activities of certain members of the phenothiazine group. Appropriate clinical application of phenothiazines can be developed in the future after gaining a comprehensive knowledge about their function. Information relating to the toxic and beneficial effects of these drugs has been discussed in this review.

Introduction

The phenothiazine group of drugs is among the most widely prescribed psychotropic drugs in the world (Fourrier et al., 2000). Although best known and primarily used for antipsychotic effects, there are multiple other important issues, which are worth understanding and can potentially be used in clinical practice. Chemically, it is a sulfur-containing tricyclic organic compound with the formula S(C6H4)2NH. It has a side chain extending from the middle ring moiety (Fig. 1A). Moreover, it is related to the thiazine class of heterocyclic compounds. The group phenothiazine is divided into three subgroups that differ on the basis of nitrogen substitution, namely, the aliphatic compounds (bearing acyclic groups), the piperidines (bearing piperidine-derived groups), and the piperazines (bearing piperazine-derived substituents) (Fig. 1B and C). Examples of commonly used phenothiazines include: chlorpromazine (Fig. 1D), triflupromazine, trifluoperazine (Fig. 1E), fluphenazine, thioridazine, mesoridazine, perphenazine, prochlorperazine and promazine.

As mentioned earlier, phenothiazines are primarily used for clinical treatments as psychotropics for more than fifty years (Delay et al., 1952). Psychiatric application is principally based on the dopamine blocking at the D2 receptor (Snyder et al., 1974, Feinberg and Snyder, 1975, Sammut et al., 2007, Silverstone, 1990) and phenothiazines without D2 blocking are devoid of neuroleptic activity. However, they may interfere with other receptors and channels (Zarnowska and Mozrzymas, 2001). This is because of the fact that a large number of structural variations are introduced in the phenothiazine series and these deviations result into some pattern of important relationships between structure and activity. Hence apart from the usual psychotropic activity, the drugs under phenothiazine group show diverse biological activities including antibacterial, antiplasmid, antitumor and antihelmintic properties (Table 1).

The potency of this group of drugs activity may vary upon the level of substitutions. Molnar et al. (1993) proposed the involvement of different molecular orbitals for the expression of various biological activities by phenothiazines. As for example, the antiplasmid activity seemed to be intensified by Cl- or CF3-substitution at the 2C position of phenothiazines and altered by the side chain length and hydrophobicity. Triflupromazine, possessing a methyl-thio substituent at position 10 and a fluorine moiety at position 2, exhibits significant antibacterial activity. Thus the nature of substitutions has profound influence on the pharmacological actions of the drugs perhaps by changing the receptor specificity (Horn and Snyder, 1971) and future scope to design a new group of drugs with enhanced potency remains open.

Again different substituents on the phenothiazine ring determine the site of action by altering the distribution of the drug. Compounds with substitutions that increase their lipid solubility and facilitate their transport through the blood–brain barrier, are more potent. Their pharmacological nature depends on the electronic nature of the phenothiazine ring and involves electron donation in charge-transfer reactions (Silverstone and Turner, 1995). According to several reports (Ford et al., 1989, Zirkle and Kaiser, 1970), the most useful phenothiazine drugs have substituents at positions 2 and 10 (eg. chlorpromazine, trifluoperazine). The substitution at position 10 is mainly responsible for the psychotropic actions of the drugs and that at position 2 markedly influence the potency of the drugs. Highest activity is associated with an electron-withdrawing, lipophilic substituent (halogen) at position 2. Modifications in the length of the alkyl bridge and the type of amino side chain also influence the effectiveness of the drugs (Ford et al., 1989). An essential requirement for the psychotropic activity is the existence of a three-carbon chain between the nitrogen atom of the phenothiazine ring and the terminal nitrogen of the side chain (Fig. 1D (chlorpromazine, CPZ) and E (trifluoperazine)) (Zirkle and Kaiser, 1970). Such compounds also show antiemetic properties. Similarly phenothiazine compounds, containing a side chain at position 10 with two carbon atoms between the nitrogen atoms (eg. thioridazine), predominantly have antihistaminic properties instead of antischizophrenic activity (Zirkle and Kaiser, 1970). However, complexes, with a four-carbon bridge and a piperazinyl amine group rather than a noncyclic amino group, show increased activity against cellular proliferation and multi-drug resistance. Furthermore, the substitutions on the phenothiazine ring, that increased hydrophobicity, also lead to higher antiproliferative activities (Ford et al., 1989). Ratnakar et al. (1995) reported that the antitubercular activity (Table 1) conferred on the phenothiazines, especially trifluoperazine, was due to the presence of a methylpiperazin-yl-propyl group attached to the nitrogen atom at position 10 and the trifluoromethyl group at the second carbon atom. In continuation with these structure–activity relationships, it is found that the replacement of nitrogen in the phenothiazine ring with sulfur atoms decreases the antitumor activity (Azuine et al., 2004). According to Feinberg and Snyder (1975), the greater potency of drugs with the trifluoromethyl group rather than chlorine as a 2-substituent is due to the Van der Waal's forces of attraction. This attractive force can also explain for the enhanced activity of phenothiazines with piperazine instead of alkylamino side chains and the increased potency associated with hydroxy-ethyl-piperazines as contrasted to piperazine side chains. Current studies suggest that highly potent phenothiazine derivatives can be made by nitro substitution on the aromatic ring of phenothiazines (Motohashi et al., 1991). Thus considering all these aspects, the antimicrobial, antihelminthic, antiprionic, anticancerous activities of phenothiazines and non-psychiatric, non-dopamine blocking features of the drug should be evaluated.

It has already been stated that initially, phenothiazines are widely used as antidepressants. However, their multiple cellular effects confirm that they may not necessarily be confined to the central nervous system alone. For example, chlorpromazine possesses direct antibacterial activity (Molnar and Schneider, 1978). Chlorpromazine and trifluoperazine have in vitro activity against the pathogenic free-living amoebae like Naegleria fowleri, Acanthamoeba culbertsoni, and A. polyphaga (Schuster and Mandel, 1984). Though the mechanism of drug action is not very clear, it is suggested that the sensitivity is due to the phenothiazine-mediated antagonistic effect on calmodulin, the calcium regulatory protein present in amoeba. The lipophilic action of the drug on the plasma membrane may also be a reason for the suppression of amoeba. Thus, being a lipophilic compound, chlorpromazine can inhibit the growth of Leishmania donovani (Pearson et al., 1982) and Staphylococcus aureus (Ordway et al., 2002) in human macrophages, probably by concentrating in the tissues including the reticuloendothelial system, the site of leishmanial infection. It was observed that phenothiazines inhibit the replication of Mycobacterium tuberculosis and M. avium in cultured normal human macrophages (Crowle et al., 1992, Ratnakar and Murthy, 1992). Also, strains of Shigella spp., Vibrio cholerae and V. parahaemolyticus were reported to be sensitive to trifluoperazine (Mazumder et al., 2001) (Table 1). In addition, chlorpromazine has fungistatic and fungicidal activities against Candida albicans (Wood and Nugent, 1985) (Table 1). Here, chlorpromazine may have a direct effect on C. albicans membrane, similar to the postulated mechanism for the antibacterial properties (Hubbard et al., 1982). Further it may also interfere with the important cellular metabolism of Candida including calmodulin (Molnar and Schneider, 1978).

It is also worth mentioning that although phenothiazines, especially in large doses, are potentially toxic; the drugs have been given safely and successfully without noteworthy toxicity in the treatment of human Enterobius vermicularis infection, a common intestinal parasite (Most, 1943). Again for both Gram-positive and Gram-negative bacteria, the drugs are found to be equally sensitive, with the MIC varying between 25 and 100 μg/ml with most agents, which is well within the range of therapeutic tolerance (Dasgupta et al., 2008).

Considering intensive development in the studies on the new activities of phenothiazines, it seems necessary to organize and clarify the recent knowledge about the antiprionic activities of this group. The drugs, especially chlorpromazine, are known to pass the blood–brain barrier. Therefore currently the researchers are speculating that phenothiazine derivatives may have immense prospect in the treatment of Creutzfeldt–Jakob and other prion diseases leading to fatal neurodegenerative disorder (Korth et al., 2001). Similarly, Scrapie is an incurable neurological dysfunction that affects the nervous systems of sheep and goats. Scrapie agents can be inactivated by UV and ionizing radiation. Dees et al. (1985) suggested that this inactivation could further be increased by the addition of chlorpromazine as it penetrates the lipid bilayers of Scrapie agents and induces more single strand breaks in nucleic acids by irradiation. In another study (Korth et al., 2001), chlorpromazine was added to the hamster brain fractions enriched for membranes and the suspension was exposed to UV irradiation. It was noted that the prion titers were reduced by 100-folds in the irradiated samples containing the drug. In all these cases, the precise mechanism by which chlorpromazine inhibits the prion protein formation and possibly enhances clearance is still to be determined, although the main frame of action seems to be through its capability of crossing the blood–brain barrier.

Chlorpromazine has found potential use as a therapeutic agent in several other lethal neurodegenerative disorders like transmissible spongiform encephalopathies, which are associated with the misfolding of prion protein (Koster et al., 2003). It is reported that phenothiazine derivatives inhibit the formation of pathogenic prion proteins and may even enhance their clearance from infected cells (Korth et al., 2001). This group not only inhibits the conversion of soluble prion protein into the protease-resistant form (Dees et al., 1985, Roikhel et al., 1984, Doh-ura et al., 2000), but also hinders the formation of a disease-causing isoform of the normal host prion protein in cultured Scrapie-infected neuroblastoma (ScN2a) cells (Korth et al., 2001) (Table 1).

Section snippets

Interaction of phenothiazines with different biological macromolecules

One of the familiar approaches in understanding the mechanism of any drug action is to study its interaction with the functionally important biomolecules on which the drug may act. This can reveal the most potential target of the drug on the biological system. Apart from prion binding, phenothiazines are known to interact with different biological macromolecules like DNA, RNA, proteins etc. It is observed that phenothiazines, especially trifluoperazine, penetrate into human erythrocytes and

Phenothiazines at the cellular point

The interaction of phenothiazines to DNA finally reflects to cellular DNA replication. In vitro these drugs inhibit DNA synthesis in K-562 cells (Molnar et al., 1985), normal rat kidney cells (Lopez-Girona et al., 1992) and isolated nuclei of Meth A cells (Mizushima, et al., 1993). Going one step further, the fluorescence microscopic study with fibroblasts treated with chlorpromazine indicated that the drug is localized in the cytoplasm and nuclear membranes (Mizushima, et al., 1993). Examining

Phenothiazines and cancer

The successful non-surgical elimination of cancer cells ultimately involves apoptosis. Hence, based on the insights into the mechanisms involved in the regulation of apoptosis, new treatment options for cancer have been developed. Essentially, the induction of apoptosis by the cytotoxic drugs is not only confined to the malignant cells, normal cells are equally vulnerable. So there is a constant search for agents that specifically target tumor cells without perturbing normal tissue. In this

Use of bioinformatics with regard to phenothiazines

From our review, it is obvious that the age-old drug, phenothiazine, has a wide target inside the cell, and hence by the application of bioinformatics, a whole database can be prepared to ascertain the most promising and successful use of the drug. In this effort, recent experiments done by Borisy et al. (2003) and Ikediobi et al. (2008) have shown promising results.

After a thorough systematic screening of combinations of small molecules, Borisy et al. (2003) suggested that the phenothiazine

Acknowledgments

This work is supported by the Council for Scientific and Industrial Research, India [grant number 09/096(0530)/2K8 EMR-I] of Sudeshna Gangopadhyay under Professor Parimal Karmakar.

The authors declare that they have no conflicts of interest.

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