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The membrane perturbing potency of the highly effective anti-multidrug resistance (MDR) pyrimidine analog of fluphenazine (FPh-prm), has been studied using attenuated total reflectance Fourier-transfer infrared spectroscopy (FTIR-ATR). The temperature- and FPh-prm-dose-dependent evolutions of the infrared spectra of FPh-prm/sphingomyelin (SM) mixtures were analyzed using principal component analysis (PCA). It has been postulated that the distinct anti-MDR activity of FPh-prm could be related to its ability to affect the modification of SM membranes. A reduction in the temperature of the chain-melting phase transition was observed in FPh-prm-mixed SM membranes together with the loosing of the phase transition cooperativity. Increasing the temperature led to the trans to gauche isomerization of FPh-prm-rich lipid membranes, which resulted in the gradual release of FPh-prm from the lipid membrane to the water phase.

Fluphenazine (FPh) belongs to the phenothiazine family of compounds and exhibits a wide variety of biological effects, including antimutagenic, proapoptotic, antiproliferative and anti-multidrug resistance (MDR) activities. The ability of FPh to interact with lipid membranes can have a significant impact on its biological activities. However, the mechanisms involved in the interaction of FPh with lipid membranes are poorly understood. FTIR–ATR spectroscopy has been used in this study to visualize the interactions between FPh and a model lipid bilayer composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Subsequent interpretation of the temperature-dependent FTIR spectra obtained for FPh:DPPC systems containing different concentrations of FPh was efficiently supported by principal component analysis.

Phenothiazine compounds are known as effective inhibitors of a multidrug resistance (MDR) of tumor cells to chemotherapeutic agents. This group consists of many important substances used in human medicine such as antipsychotic drugs in the case of fluphenazine (FPh) or chlorpromazine (CPZ). Fluphenazine was on the World Health Organization (WHO) list of Essential Medicines of 2009, and its new pyrimidine analog (FPh-prm) presented in this work has been documented to have a high anti-MDR activity. In order to discover the character of alterations of the lipid bilayer structure caused by the presence of FPh-prm inside the lipid membrane, which is responsible for the essential increase of an anti-MDR activity of FPh-prm, microcalorimetric (differential scanning calorimetry), Laurdan fluorescence, (31)P nuclear magnetic resonance spectroscopy (NMR), and attenuated total reflectance Fourier transfer infrared spectroscopy (FTIR-ATR) were used for dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes mixed with a different concentration of amine analogue. It was stated that the formation of domains with different content of FPh-prm/DPPC can be a reason for the membrane-related mechanism of chemoprevention associated with the inhibition of the outward transport of anticancer drugs by the glycoprotein P (Pgp) in cancer cells by the pyrimidine analog of FPh. To our best knowledge, this report is the first to show the bilayer structure of domains formed by incomplete miscibility of fluphenazine-related compounds and phospholipid molecules. Our results provide a sound basis for the design of future modifications of anti-MDR drugs by providing very effective inhibitors of the pump activity of Pgp.

Wstep. Ceramid (cer) jest zazwyczaj produkowany na drodze hydrolizy sfingomieliny przez sfingomielinazy (SMase). Zbadano, ze aktywnośc chemoprewencyjna — propoptotyczna i chemouwrazliwiająca — piperazynowej pochodnej fenotiazyny (Pht), flufenazyny (FPh) i jej nowo syntezowanych analogow — związkow 1b i 3f — zalezy od stymulacji lizosomalnej, kwaśnej sfingomielinazy (aSMase), niezaleznej od Zn2+, jednego z enzymow uczestniczących w powsta­waniu endogennego cer. Innym waznym szlakiem tworzenia cer w komorce jest synteza de novo z udzialem syntetazy ceramidowej (CerS). Celem pracy byla ocena aktywności chemoprewencyjnej FPh i jej analogow: związkow 1b i 3f po nieodwracalnym zablokowaniu CerS. Material i metody. Aktywnośc chemoprewencyjną badanych związkow (10 μM, 2 godz.) oceniano w hodowlach limfocytow ludzkich uszkodzonych genotoksycznie przez inkubacje z benzo[ a ]pirenem (+B[ a ]P; 7,5 μM, 48 godz.) i po preinkubacji z selektywnym inhibitorem CerS — fumonizyną B1 (+FB1; 20 μM, 1,5 godz.). W ocenie efektow badanych związkow zastosowano metody: mikroskopii fluorescencyjnej, spektrofotometrii i spektrofluorymetrii. Istotnośc statystyczną uzyskanych rezultatow sprawdzono za pomocą testu t -Studenta. Wyniki. W hodowlach limfocytow w obecności inhibitora CerS (+B[ a ]P; +FB1) inkubacja hodowli z analogami 1b i 3f prowadzila do istotnie nizszego (p < 0,05) odsetka komorek w apoptozie i akumulacji rodaminy 123 (Rod-123) w po­rownaniu z hodowlą bez inhibitora CerS (+B[ a ]P; -FB1). W przypadku macierzystego związku FPh wplyw na hodowle limfocytow nie zalezal od obecności inhibitora CerS. Wnioski. Efekty proapoptotyczny i chemouwrazliwiający analogow 1b i 3f są uwarunkowane aktywacją CerS . Nato­miast dla macierzystej FPh wykazano, ze jej aktywnośc chemoprewencyjna nie zalezy od stymulacji szlaku syntezy de novo cer z udzialem CerS .

The effect of incorporating fluphenazine (FPh) into the dipalmitoylphosphatidylcholine (DPPC) multibilayers was studied by means of two-dimensional correlation spectroscopy (2DCOS) applied to attenuated total reflection (ATR) infrared spectra. DPPC is used as a model membrane that mimics the organization of lipids in biological membranes and their interaction with FPh. ATR-IR spectra for both DPPC dry film alone and the film doped with FPh were recorded as a function of temperature to provide information about the interaction between FPh molecules and DPPC lipid. The chain-melting phase-transition temperature changes are strictly correlated with the conformational order of the lipid hydrocarbon chains. To gain deeper insight into the accompanying spectral changes, we employed moving-window 2D correlation spectroscopy. Subdividing all the measurements from 10 to 90 °C into 20° subsets enables a detailed identification of spectral features induced by embedding FPh into DPPC multilayers. Moving-window analysis of the power spectra for the ν asym,sym CH 2 , δ s CH 2 , and δ r CH 2 vibrations provides evidence that FPh is embedded in the region between the bilayers, penetrating their hydrophilic part, which destabilizes the interchain interaction. Above 60 °C the FPh–DPPC system reaches the liquid crystalline phase with the well-established location of FPh. A further temperature increase to 90 °C has little effect on the intrachain conformational order and the packing character of the FPh–DPPC system in the liquid crystalline phase. In addition, FPh hinders the formation of large domains. Comparison of the moving-window analysis done by using slice spectra for DPPC and FPh-doped DPPC dry film for ν asym,sym CH 2 , νC O, and ν sym PO 2 - shows that the interaction between the DPPC and FPh molecules is accompanied by very distinct spectral changes located in a both lower and narrower temperature range than those observed in pure DPPC film.

Aim of the study: To evaluate the inhi -bitory effect of 17 new analogues of FPhon the Pgp transport function, by esti-mation of the rhodamine 123 (Rod-123)accumulation inside cultured lympho-cytes.Material and methods: Lymphocyte werecultured in the presence of a lectin (PHA;2%, v/v), incubated with benzo[ α]pyrene(B[α]P; 7.5 μM, 48 h) to induce genotox-ic damage and to increase Pgp expressionin the cells. Lymphocytes cultured with-out the tested compounds were consid-ered as controls. Results: It was established that 10 ana-logues of FPh, among 17 tested, signifi-cantly increased Rod-123 accumulation in lymphocytes at the concentration of10 μM. As compared to the control cul-tures the Pgp transport function was the most strongly inhibited by 1a, 1b, 1d,3f, 3h and 3i analogues (approximatelyby 25%).Conclusions: FPh analogues 1a, 1b, 1d, 3f,3h and 3i should be further studied aspromising candidates for adjuvant can-cer chemotherapeutics.Key words: fluphenazine analogues,MDR, P-glycoprotein, MDR modulators.

Phenothiazines belong to the oldest, synthetic antipsychotic drugs, which do not have their precursor in the world of natural compounds. Apart from their fundamental neuroleptic action connected with the dopaminergic receptors blockade, phenothiazine derivatives also exert diverse biological activities, which account for their cancer chemopreventive-effect, as: calmodulin- and protein kinase C inhibitory-actions, anti-proliferative effect, inhibition of P-glycoprotein transport function and reversion of multidrug resistance. According to literature data on relations between chemical structure of phenothiazines and their biological effects, the main directions for further chemical modifications have been established. They are provided and discussed in this review paper.

The influence of dihydrochloride fluphenazine (FPh) on the dipalmitoylphosphatidylcholine (DPPC) bilayer structure was investigated using ATR-IR and (31)P NMR methods. The ATR-IR results indicate an increase in conformational disorder in the hydrophobic part compared with pure DPPC liposomes and a decrease in temperature of the chain-melting phase transition in FPh/DPPC liposomes. These effects depended on the concentration of the drug in the DPPC bilayer. The dihydrochloride fluphenazine molecules form H-bonds with the proton-acceptor carbonyl groups of DPPC molecules. At a higher concentration of the drug, the lipid bilayer structure is destroyed, and an isotropic phase is observed using (31)P NMR spectroscopy. The interactions between FPh and the lipid bilayer have a crucial role in MDR (multidrug-resistant) activity of this drug. These results improve one possible strategy of cancer chemoprevention with FPh accompanied by fluidization and destabilization of the model lipid bilayer structure.

Fluphenazine (FPh) exhibited antimutagenic activity in lymphocyte cultures, markedly decreasing genotoxic effects of standard mutagenic agents present in cell cultures. However, the strong pharmacological activity of this neuroleptic drug, together with its serious side effects on the central nervous system, limits its use as an antimutagenic compound. In this paper we describe a route of chemical synthesis of FPh analogues that are more hydrophilic than the model compound, thus probably penetrate more weakly through the blood-brain barrier. These new analogues were tested for their antimutagenic and pro-apoptotic activities in human lymphocyte cultures, genotoxically damaged in vitro with benzo[a]pyrene [40 microM, 30 min] and subsequently cultured for 48 h in the presence of the tested compounds. The fluphenazine analogues enhanced apoptosis in genotoxically damaged lymphocytes more strongly than the model compound did. The increase of apoptotic cell frequency was the highest with compound 4a [2-(trifluoromethyl)-10-[3-(diethanolamino)-2-hydroxypropyl] phenothiazine]--a 35% higher effect than that of fluphenazine. The cytotoxicity of derivative 4a was the lowest among the tested compounds; it was 60% lower than that of fluphenazine. The antimutagenic effect of 4a was about 10% stronger than that of fluphenazine. Compound 4a also had the highest hydrophilicity of the new FPh analogues. Compound 4a was chosen for further study as a potentially usable antimutagen that would only weakly penetrate the central nervous system.

A series of 10 novel analogues of fluphenazine (FPh) were synthesized. Influence of the synthesized analogues of FPh on frequency of apoptosis and necrosis in cultures of human lymphocytes genotoxically damaged in vitro with benzo[a]pyrene (B[a]P; 7,5 microM, 48 h) was compared with the effect of FPh. Activity of the tested compounds was expressed by ED50 (pro-apoptotic activity) and TD50 (pro-necrotic effect, cytotoxicity). It was noticed that compounds 3-9 and 12 exerted a pro-apoptotic effect markedly stronger than that of FPh. Additionally, compounds 3, 9 and 10 exhibited the weakest influence on frequency of necrotic lymphocyte in cultures. 2D-QSAR analysis was done in order to find quantitative relationship between structures of the tested analogues and their pro-apoptotic activity or pro-necrotic effect in B[a]P-damaged cell cultures. Several statistically significant QSAR models were generated. Information obtained from 2D-QSAR study will be used in further design of analogues of FPh more active in cancer chemoprevention.

The membrane interactions can play a vital role in chemosensitizing activity of fluphenazine dihydrochloride, an inhibitor of P-glycoprotein transport function. In in vitro testings of the influence of fluphenazine dihydrochloride on model dipalmitoylphosphatidylcholine membranes and on human lymphocyte cultures, genotoxically damaged by benzo[a]pyrene, differential scanning calorimetry and rhodamine 123 retention test were applied. The results of both testings shown that the membrane interactions of fluphenazine dihydrochloride in model membrane structure and the chemosensitizing effect of this compound in examined human lymphocyte cultures, depend on its concentration in a sample.