The Immobilization of Gossypol Derivative on N-Polyvinylpyrrolidone

The conjugate of the gossypol derivative megosin (1) with N-polyvinylpyrrolidone named rometin (2) was synthesized. The effects of 1 and 2 on the stru...
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J. Med. Chem. 2009, 52, 4119–4125 4119 DOI: 10.1021/jm9002507

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The Immobilization of Gossypol Derivative on N-Polyvinylpyrrolidone Increases its Water Solubility and Modifies Membrane-Active Properties )

Maksim Ionov,†,§ Nataliya Gordiyenko,‡ Ewa Olchowik, Nina Baram,† Khairulla Zijaev,† Bakhtiyar Salakhutdinov,† Maria Bryszewska,§ and Maria Zamaraeva*, Institute of Bioorganic Chemistry, Academy of Sciences, Tashkent, Uzbekistan, ‡National Eye Institute, National Institutes of Health, Bethesda, Maryland, §Department of General Biophysics, University of Lodz, Lodz, Poland, and Department of Biophysics, University of Bialystok, Swierkowa 20C, 159-50 Bialystok, Poland )

†

Received February 27, 2009

The conjugate of the gossypol derivative megosin (1) with N-polyvinylpyrrolidone named rometin (2) was synthesized. The effects of 1 and 2 on the structure and permeability of human erythrocytes and rat liver mitochondria were compared. Compound 1 induced dose-dependent erythrocyte hemolysis and increased mitochondrial permeability, with concomitant changes in membrane structure as determined by ESR and fluorescence anisotropy methods. Immobilization of 1 on N-polyvinylpyrrolidone (compound 2) increased its water solubility and reduced the intensity of its effects on erythrocyte membrane integrity and mitochondrial permeability, which correlated with a decrease in the membranes structural changes induced by the compound. Although the same concentrations of free and N-polyvinylpyrrolidone bound 1 were used, far less 14C-labeled 1 was incorporated into the membranes from complex than free 1. The increase in water solubility and the reduction of membrane-active properties of 1 after immobilization on N-polyvinylpyrrolidone could explain our previous observation of the decreased toxicity of 1.

Introduction Gossypol is a natural polyphenolic pigment from cotton (Gossypium spp.) with a wide range of biological effects including antifertility,1-7 antiproliferative,8-13 antiviral, and immunomodulatory activities.14-17 The diversity of these pharmacological effects of gossypol and its derivatives is partly attributable to their membranotrophic properties. A study of the distribution of 14C-gossypol and its derivatives in subcellular fractions of rat hepatocytes showed that most of the label was located in the microsomes and mitochondria.18,19 Gossypol and its derivatives concentrate in the lipid bilayers of biological membranes as they possess high affinity for lipids and induce both structural and functional alterations.20-26 In particular, they make artificial and biological membranes permeable to mono- and divalent cations. The protonophoric activity of gossypol, uncoupling of mitochondria,7,27,28 may account for its antifertility4,5,7 and anticancerogenic10,11,29 effects. Gossypol also increases the intracellular Ca2þ concentration in cells of various types in the presence and absence of extracellular Ca2þ.30,31 These changes in Ca2þ homeostasis induced by gossypol and its derivatives can result in the activation of cellular functions (short-term effects) and may underlie their toxic effects (long-term effects). The development of efficient medicinal preparations of gossypol is hampered by its toxicity and insolubility in water. Gossypol is of a limited use as a male contraceptive and as a topical remedy for herpes. Currently, gossypol derivatives

with lower toxicity are being investigated with the aim of developing efficient medicinal preparations.32-34 One such gossypol derivative is disodium salt-[2,20 {[(7,70 ,8,80 -tetrahydro-1,10 ,6,60 -tetrahydroxy-5,50 -diisopropyl-3,30 -dimethyl-7,70 dioxo)-2,20 -dinaphthyl]-8,80 -methylenimino}-sulfonic acid (1, megosin).35 Compound 1 is of particular interest because of its high antiviral and interferon-inducing activities, suggesting a possibility of intresting medical applications.32 However, this drug, like other gossypol derivatives, has low solubility in water, making it difficult to use for clinical applications. Because the bioavailability of pharmacological preparations depends on their solubility in water, well-soluble derivatives could have superior pharmacokinetic properties and are expected be effective at lower therapeutic doses than poorly soluble ones. A useful approach to conferring water solubility on insoluble compounds is to modify them with natural or synthetic polymers, in particular with N-polyvinylpyrrolidone (PVPa). The aim of the present paper was to prepare a water-soluble conjugate of 1 with PVP (2, rometin) and to study how membrane-active properties of 1 change along with its immobilization on the polymer. We also studied the effects of both water-soluble and -insoluble forms of 1 on the structure and permeability of erythrocytes and mitochondria, which are used here as examples, respectively, of plasma and intracellular membranes.

*To whom correspondence should be addressed. Phone: þ:48 85 7457349. Fax: þ48 85 7457302. E-mail: [email protected].

a Abbreviations: PVP, polyvinylpyrrolidone; EDTA, ethylenediaminetetraacetic acid; RLM, rat liver mitochondria; DPH, 1,6-diphenyl1,3,5-hexatriene; ESR, electron spin resonance.

r 2009 American Chemical Society

Published on Web 06/25/2009

pubs.acs.org/jmc

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Figure 1. I, structure of 1; II, complex of 1 with N-polyvinylpyrrolidone (2); X, N-polyvinylpyrrolidone; Y, 1. Ratio X:Y = 9:1.

Results Chemical Synthesis. To prepare 2, compound 1 was first synthesized. Compound 1 belongs to the group of aliphatic imines of gossypol, where the sodium salt of β-aminoethylsulfuric acid substitutes for aldehyde groups and has low solubility in water.36 Then 1 was immobilized on N-polyvinylpyrrolidone (8000 MM) according to synthetic protocols presented in the Experimental Section. The identity and purity of 2 were determined by TLC analysis using Silufol thin layer plates. In addition, the percent content of the preparation was determined by spectrophotometry. Maximal yield of 2 was 88%,and purity of the compound was equal to or greater than 96%. The content of 1 in the compound 2 was 9 -10% (Figure 1). Effects of 1 and 2 on Hemolysis and Structural State of Erythrocytes Membranes. Erythrocytes are among the most sensitive cells for studying xenobiotics. We compared the effects of 1 and 2 on the integrity of erythrocyte membranes. Figure 2 shows the effects of these compounds on erythrocyte hemolysis. Compound 1 induced dose-dependent hemolysis over the range 20-100 μM, whereas 2 was only weakly hemolytic. Compound 2 at 50 μM induced on average 9(0.7% hemolysis, whereas the same concentration of 1 induced 68.0 ( 2.8% hemolysis. Thus, the hemolytic power of 2 was 7.5-fold less than for 1. The unequal hemolytic activity of the compounds may be related to differences in the interaction with the membrane lipid bilayer. To test this possibility,we examined the effects of 1 and 2 on erythrocyte membrane fluidity by measuring the fluorescence anisotropy values of 1,6-diphenyl-1,3,5-hexatriene (DPH) and determining the ratio (Ai/A0) between the fluorescence anisotropy of DPH in the presence (Ai) and absence (A0) of the compounds. The DPH fluorescence anisotropy values in the presence of increasing concentrations of 1and 2 are shown in Figure 3. These dose-dependent increases in fluorescence anisotropy as the result of the reduced rotational movements of the probe in the surrounding phospholipids were caused by the interactions of both compounds with the membrane and diminishing it fluidity. However, some differences in the effects of 1 and 2 should be noted. The fluorescence anisotropy increased linearly in the presence of 2 (0.05-0.75 μM), whereas in the presence of 1

Figure 2. Water-soluble 2 has a lower hemolytic effect than its water-insoluble precursor, 1. A 2% suspension of freshly isolated and washed rat erythrocytes in 140 mM NaCl, 10 mM Tris-citrate buffer, 1 mM EDTA, pH 7.5, was incubated for 30 min at 37 °C in the presence of the compounds tested. Concentration of 2 was equimolar with respect to that of 1 in the complex. The data are the means ( SEM, with n = 9. A representative experiment from three independent experiments is shown. For each individual concentration of the compounds the differences were considered to be significant if p < 0.05.

the dose-dependent curve leveled off at ∼0.4 μM. The Ai/A0 ratio increased far less markedly in the presence of 2 than in that of 1(Figure 3). Thus, at 0.4 μM, a 1.83 ( 0.006-fold enhancement of fluorescence anisotropy was observed for 1 (compared to a control without compound), whereas for 2, the corresponding value was 1.48(0.013. These data suggest that immobilization of 1 on PVP may decrease its interaction with erythrocyte membranes, thus diminishing its hemolytic action. Effects of 1and 2 on Structure and Permeability of Mitochondrial Membrane. Mitochondria are useful and sensitive systems for testing the pharmacological and toxic effects of various chemical compounds.37 In the present study, we examined the ability of gossypol derivatives to affect

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Figure 4. Influence of 2 and 1 on the permeability of nonenergized rat liver mitochondria. A, 20 μM 1; B, 50 μM 2. Concentration of 2 was equimolar with respect to that of 1 in the complex. Medium: 1, KNO3; 2, Ca(NO3)2; 3, Ba(NO3)2; 4, Mg(NO3)2. The protein concentration of mitochondria was 1.0 mg/mL. The traces represent recordings from five experiments.

Figure 3. Interaction of 1 or 2 with erythrocyte membranes reduces phospholipid mobility. The ratio (Ai/A0) of anisotropy signals in the presence (Ai) and absence (A0) of compounds was used to quantify the change in membrane activity. DPH was used at a final concentration of 10-6 M. The erythrocyte membrane protein concentration was 100 μg protein/mL. Concentration of 2 was equimolar with respect to that of 1 in the complex. The means ( SEM, with n = 8. A representative experiment from six independent experiments is shown. For each individual concentration of the compounds the differences were significant at p < 0.05.

mitochondrial membrane permeability by measuring the swelling of nonenergized mitochondria. To exclude the possible energy-dependent transport in these experiments, the incubation media were always supplemented with rotenone, inhibitor of mitochondrial respiratory chain I complex. The rates of passive mitochondrial swelling in a NO3containing medium in the presence of mono- and divalent cations, which were induced by 1 (A) and 2 (B) are shown in Figure 4. 1 at 20 μM increased the mitochondrial permeability to Kþ ions and to other ions in the order Kþ:Ca2þ: Ba2þ:Mg2þ =1.0:0.70:0.23:0.21. The compound 1 induced permeability of mitochondrial membrane to Kþ was taken as 1. The compound 2 at the same concentration (20 μM) did not alter mitochondrial permeability considerably; it induced an effect similar to that of 1 only at 50 μM. As in our erythrocyte hemolysis experiments, 2 had less affects than 1 on the mitochondrial membrane permeability because 2.5 times higher concentrations were required to achieve the same results. We also studied the influence of 1 and 2 on the structure of mitochondrial membranes by ESR, using the molecule depicted in Figure 5 as a spin probe. The spin probe partitions into the hydrophobic region of the lipid bilayer and changes in its spectral parameters allow for determination of the lipid hydrocarbon chain mobilities up to the terminal CH3 groups. The spectral parameter used was the rotational correlation time (τc), which reflects probe mobility in the bilayer. Figure 5 shows the dependence of τc on the concentrations of 1 and 2. Both compounds enhanced the τc values, indicating a decrease in the probe mobility and accordingly in the membrane fluidity. The change in τc was linear for 2 over the concentration range 0.025-0.3 μM, whereas the curve for 1 shows saturation at 0.15 μM. As we

Figure 5. Influence of 1 and 2 on the rotational correlation time (τc=value10-10 s) of the EPR probe in mitochondrial membranes. The EPR probe was used at a concentration of 10-6 M. Medium: 145 mM KCl, 10 mM Tris-HCl, pH 7.4. The protein concentration of mitochondria was 1.0 mg/mL. Concentration of 2 was equimolar with respect to that of 1 in the complex. The means ( SEM, with n=9. A representative experiment from three independent experiments is shown. For each individual concentration of the compounds the differences were significant at p < 0.05.

noticed with the erythrocyte membrane, the effect of 2 was weaker than that of 1 (Figure 5). The rotational correlation times of the probe were 22.54 ( 0.23  10-10 s and 32.13 ( 1.6  10-10 s for 0.15 μM 2 and 1, respectively. Our data demonstrate that the intensity of membrane effects of the compounds studied differed significantly in spite of the fact that the concentrations of free and PVPbound 1 were the same. These differences are assumed to result from the incomplete release of megosin from PVP over the time course of our study. Binding of Labeled Compounds to Membranes. Binding of 14 C-1 and 14C-2 to rat liver mitochondrial membranes was studied using a 50-fold excess of nonlabeled compounds to exclude the effects of unspecific binding. The compound 1 binding to the mitochondrial suspension showed saturation after 5 min of incubation (Figure 6) at 5882 ( 51 cpm, accounting for 88.2% of the total radioactivity, and there was no further significant change after 2 h. The compound 2 binding showed a different dynamics. After 5 min of

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Figure 6. Time-course of 14C-labeled 1 and 14C-labeled 1 þPVP (2) binding to mitochondrial membranes in the presence of nonlabeled compounds. Medium: 145 mM KCl, 10 mM Tris-HCl, pH 7.4. The protein concentration of mitochondria was 10 mg/mL. The labeled compounds were used at 10 nmol/mg protein and the nonlabeled at 500 nmol/mg protein. Concentration of 2 was equimolar with respect to that of 1 in the complex. The means ( SEM with n = 12. A representative experiment from four independent experiments is shown. Significance of the difference between 1 and 2: p < 0.05.

incubation, the amount of label bound was 3500 ( 38 cpm, accounting for 53.28% of the total radioactivity. The more prolonged incubation 2 with mitochondrial membranes led to a linear increase in the binding up to a maximal value at 2 h (5718 ( 10 cpm = 87.1% of total radioactivity). Discussion and Conclusions Development of new medicines and modification of the existing ones is driven by the need to enhance and to prolong their therapeutic effects, which are often related to the water solubility of compounds. Various carriers are used for preparation of medicines with prolonged action, including synthetic polymers, which meet such medical criteria as absence of side effects, as well as quick and nontoxic biodegradation and removal from the organism. Water-soluble low-molecular-weight fractions of PVP (MM 5000-15000) are polymers that satisfy these requirements. PVP can form complexes with various organic compounds in an aqueous solution. When a poorly soluble drug forms a complex with a PVP macromolecule, its solubility in water is increased and so is its stability in the solution and the duration of its action within the organism.38,39 In the present work, we described a method for preparing a water-soluble complex of 1 with PVP (compound 2) and the effects of the complex (formed) on its membrane-active properties. In this context, we compared the effects of 1 and 2 on the membrane integrity of human erythrocytes. The results showed that 1 induced dose-dependent hemolysis of erythrocytes, accompanied by an increase in the membrane rigidity. Immobilization of 1 on PVP reduced the intensity of its effects on erythrocyte integrity, which correlated with a lessening of the membrane structural changes induced by this drug. Known mechanisms by which erythrocyte hemolysis can be induced by drugs include formation of nonspecific channels and pores in the membranes or also perturbation of the structural order of the membrane, which depends on the interactions between the compounds and membrane proteins

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or lipids. When exogenous compounds interact with erythrocyte integral membrane proteins, new interactions with the cytoskeleton as well as an increase in membrane rigidity and hemolysis can be induced. External ligands can also interact with membrane lipids, thus inducing changes in the hydrophobic part of the membrane, particularly a disturbance in the orientation of the lipid hydrocarbon chains. This can also lead to erythrocyte swelling and subsequently to hemolysis.40 We demonstrated that 1 caused change in structural order in the hydrophobic part of the erythrocyte membrane. Previously we observed this effect of 1 in liposomes.41 Therefore, we hypothesize that 1 induced erythrocyte hemolysis via interaction with membrane lipids. We also studied changes in the interaction of PVP-immobilized 1 with mitochondrial membranes, and we chose the same test used for erythrocytes: a passive permeability. Compound 1 increased the permeability of inner mitochondrial membrane for selected cations studied but not for sucrose. It was previously shown that gossypol and its derivatives make artificial and biological membranes more permeable to monoand divalent cations, which demonstrates a low specific ionophoric activity of the compounds.7,27,28 It may be assumed that the mitochondrial swelling we observed in the presence of the tested compounds can be attributable to their ionophoric activity. Incorporation of hydrophobic molecules of 1 into the mitochondrial membranes led to a change in the lipid packing and consequently to decrease in the membrane fluidity. The more 1 molecules are incorporated into the membrane, the lower fluidity as well as the greater permeability are detected. Immobilization of 1 on PVP reduced the intensity of its effects on the mitochondria. The differences in the effects of 1 and 2 on the mitochondria as well as on the erythrocytes are apparently connected to the smaller amount of 1 incorporated into the membranes when it is released from the PVP complex. This was demonstrated by our experiments on the binding of the radioactively labeled compounds to the mitochondrial membranes. We found out that significantly fewer labels were incorporated into the membranes when the 14C-1-PVP complex was used instead of pure 14C-1. Moreover, the incorporation of the label from the complex was time-dependent. We suggest that this is connected with a gradual release of 1 from the complex. Two mechanisms of action of immobilized organic compounds by polymers are known: (1) If the chemical bond between a polymer and a drug is weak in the complex, it is gradually split and the drug exerts its effect in the free form. (2) If the chemical (covalent) bond between a polymer and a drug is strong, the drug exerts its effect together with the polymer molecule.38 According to IR spectroscopy data, hydrogen bonds involving OH- and NH- groups participate in the forming of 1PVP complex and there are van der Waals interactions between polar groups of PVP and dSO2 groups of 1.42 These bonds are weak, so we suggest that megosin is released gradually from the polymer molecule and exerts its effect in its free form. Our investigation of the structural parameters of erythrocytes and mitochondrial membranes in the presence of 2 confirmed this assumption. If the whole complex had been incorporated into the mitochondrial or erythrocyte membranes, we would have observed a greater diminution of membrane mobility in comparison with free 1. However, we found an opposite effect. The polarity of the compound is of

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the greatest importance in this process. Experiments with the fluorescent probe AHC located on the surface of the lipid bilayer showed that 2 displaced the probe from the membrane to a lesser extent than 1. This indirectly indicates a decreased affinity of 2 for lipids.41 Apparently, the hydrophobic parts of the 1 molecule in the complex with PVP (2) are screened by the polymer molecules, decreasing the affinity of 1 for lipids and consequently reducing the rate at which it accumulates in the membranes. Cellular effects of drugs depend on the extent to which they associate with the cells. A sharp accumulation of surfaceactive substances in a membrane can lead to a loss of the cell stability. In the case of megosin conjugated with PVP, a gradual release of the active component from the polymer carrier may explain its attenuated effect on the membranes. It is well-known that the toxicity of a substance is largely determined by its concentration. A drug may be nontoxic and exert a positive effect at low concentrations but may be toxic at high concentrations. Many compounds including gossypol and its derivatives have a narrow therapeutic dose range, and the exceeding these doses leads to toxic effects of the compounds. Also, of course, prolonged presence of low concentrations of active substances at the organism level is the most favorable option. In pharmacokinetic experiments, the circulation of 14C 2 was prolonged in comparison with free 1.19 Under in vivo conditions, polymeric complexes can circulate in the blood for a long period, gradually releasing an active component from the complex. This may result in a more prolonged therapeutic effect and a decrease in total toxicity. Experimental Section Chemicals. EGTA, EDTA, rotenone, TRIS, and DPH were purchased from Sigma (St. Louis, MO). Spin probe 1-[20 ,20 ,60 ,6tetramethyl-1-N-oxo-piperidinyl]-2-phenylethin used in the present study was generously provided by Dr. Shapiro (Russia). All other reagents, of the highest purity available, were purchased from Reakhim (Mikhailovsk, Russia) and POCH (Poland). Animals. Experiments were carried out on male Wistar rats (180-200 g). Wistar rats were bought from the Medical Academy, Bialystok. Permission for rats breeding is N no. 1/2006 from 19.07.2006. All protocols on animals were approved by Local Ethical Committee for Animal Studies in Bialystok, Poland, N 2006/02. Synthesis of 1 and 14C Labeled 1. Compound 1, disodium salt bis-[2,20 {[(7,70 ,8,80 -tetrahydro-1,10 ,6,60 -tetrahydroxy-5,50 -diisopropyl-3,30 -dimethyl-7,70 -dioxo)-2,20 -dinaphtyl]-8,80 -methylenimino}-sulfonic acid] was obtained as described previously.35,36 14 C labeled 1 was synthesized according to Sumin.19 Preparation and Characteristics of Compound 2. To produce 2, we added an aqueous solution of N-polyvinylpyrrolidone (8000 MM, 45 g) to an acetone-water (3:1) mixture containing 5 g of 1. The resulting mixture was agitated for 2 h at 28 °C and then filtered. The acetone was distilled from the filtrate, and the remaining aqueous solution was lyophilized. The residue was a yellow powder (44 g). Compound 2, the water-soluble complex of 1 with N-polyvinylpyrrolidone (8000 MM), was obtained (Figure 1). Maximal yield of 2 was 88%. The identity and purity of the compound was checked by TCL on Silufol plates developed in acetone:diethyl ether:water (6.0:0.5:0.5) in comparison with the standard specimen, Rf = 0.72. In addition, percent content of the preparation was determined by spectrophotometry in comparison with the content of the standard specimen. The purity of 2 was equal to or greater than 96% .The compound conforms with spectral and other characteristics presented in the patent N 2123.36 Tmelt = 185-187 °C, UV

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absorbance (acetone:water = 3:1, C = 0.002%) over 245-385 nm, λmax = 380 nm. IR (KBr)νmax 1690, 3300, 3500 cm-1. By spectrophotometry, the 1 content in the complex with N-polyvinylpyrrolidone was 9.0-9.2%. The compound 2 was readily soluble in water, ethanol, and DMSO. 14C-labeled 2 was obtained using 14C-labeled 1 according to the above-described method. Determination of Erythrocyte Hemolysis. The destructive effects on membranes were determined using erythrocyte hemolysis as a test. A 2% suspension of freshly isolated erythrocytes in 140 mM NaCl, 10 mM Tris-citrate buffer, 1 mM EDTA, pH 7.5, was incubated for 30 min at 37 °C in the presence of 1 or 2. Samples were spun at 1000g and the OD540 of the supernatant was measured. The OD540 corresponding to 100% hemolysis was determined by lysing the erythrocytes with distilled water. Isolation of Liver Mitochondria. Mitochondria were isolated from rat liver by differential centrifugation.43 Isolation medium A contained 250 mM sucrose, 5 mM Tris-HCl, 0.5 mM EDTA, pH 7.4. Immediately after decapitation, the liver was excised, chopped, and homogenized in 40 mL medium A with a PotterElvehjem homogenizer. The homogenate was centrifuged at 800g for 7 min. The supernatant was separated and recentrifuged at 6000g for 15 min. The resulting pellet was resuspended with EDTA-free isolation medium B and centrifuged at 6000g for 15 min. The final pellet was resuspended again in medium B at approximately 50 mg/mL of protein (measured by the Biuret method),44 stored on ice, and used within a few hours. All procedures were performed at 0-4 °C. Determination of the Permeability of Mitochondrial Membranes to Cations. The passive permeability of mitochondrial membranes to ions was measured by following the energyindependent swelling in iso-osmotic nitrate solutions as described.45 Salt concentrations were: 120 mM for NH4þ and Kþ, 80 mM for Mg2þ and Ba2þ, and 40 mM for Ca2þ. The Ca2þ solution also contained 120 mM sucrose. All solutions were buffered with Tris-HNO3 to pH 7.4. To exclude the possibility of energy-dependent transport in these experiments, all the incubation media were supplemented with rotenone (0.33 μg/mL). Measurements were performed at 25 °C in 3 mL glass cuvettes. The final concentration of mitochondrial protein was 1.0 mg/ mL. The suspension was continuously agitated with a magnetic stirrer. Swelling was observed as a decrease in absorbance at 520 nm, using an LMF-2 photometer (LOMO, Leningrad, Russia). Preparation of Red Blood Cell Membranes. Blood samples from healthy volunteers (n=12) were anticoagulated with 3.2% citrate and centrifuged at 4 °C at 5000g for 5 min to collect erythrocytes. The plasma and buffy layer containing leukocytes and platelets were removed, and the erythrocytes were washed three times with isotonic buffer. The erythrocytes were then hemolyzed with Tris-HCl buffer (pH 8.0) containing 1 mM EDTA, and the mixture was centrifuged at 15000g (4 °C) for 15 min. The supernatant was discarded, and the process was repeated 3-4 times. The erythrocyte membranes were suspended in PBS (pH 7.4) to a concentration of 100 μg membrane protein/mL PBS and were stored for up to 3 days pending experiments. Measurement of Erythrocyte Membrane Fluidity. Membrane fluidity was measured by the steady-state fluorescence anisotropy of the probe 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into erythrocyte membranes. An erythrocyte membrane suspension was diluted with cold PBS (8.1 mM Na2HPO4, 0.15 mM NaCl, 1.9 mM NaH2PO4, pH 7.4) to 0.05% hematocrit and then incubated at 37 °C for 2 min with gossypol derivatives at final concentrations of 0.05-0.75 μM, followed by incubation in the dark with DPH at room temperature for 15 min. The probe was used at a final concentration of 1 μmol/L and fluorescence was measured with an LS-50B luminescence spectrofluorimeter (Perkin-Elmer, Great Britain). The excitation

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and emission wavelengths were 348 and 426 nm.46 Fluorescence anisotropy was calculated from the equation given in ref 47. Measurements of Mitochondrial Membranes Fluidity by Electronic Spin Resonance Method (ESR). Mitochondria (1 mg/mL) suspended in 145 mM KCl, 10 mM tris-HCl, pH 7,4 were supplemented with spin probe 1-[20 ,20 ,60 ,6-tetramethyl-1-Noxo-piperidinyl]-2-phenylethin as an ethanol solution. A spin probe structure is given on Figure 5. Sufficient spin label in ethanol solution was added to the suspended mitochondria to produce a final concentration of 10-6 M. The aqueous suspension of spin labeled mitochondria (control) and labeled mitochondria incubated 2 min with tested compounds were placed in a 0.1 mm diameter glass tube in the ESR spectrometer resonator, and the ESR spectra were recorded. ESR spectra were registered on a Bruker spectrometer (Germany) at amplitude of modulation no greater than 1 Gs and resonator power no greater than 20 mW. ESR measurements were performed at 25 °C. The rotational correlation time τc was calculated according to the formula given in ref 48. Measurement of Binding of Gossypol Derivatives by Radioisotope Labels. Binding of gossypol derivatives to mitochondrial membranes was determined using nitrocellulose filters (pore size 1.5 nm, Millipore, USA). Briefly, 2 mL of a mitochondrial suspension (10 mg protein/mL) containing 14C-1 or 14C-2 were placed in 5 mL quartz vials. Stock solutions of labeled compounds were 20 mM. Specific activity was 9.45 mCi/mmol. The labeled compounds were used at 10 nmol/mg protein and the nonlabeled at 500 nmol/mg protein. Following incubation (5, 30, 60, or 120 min) with constant stirring at 22 °C temperature, the samples were passed through the filters using vacuum suction. The filters were rinsed twice with water to remove the free radiolabeled compounds and dried at room temperature for 24 h. The filters were transferred to appropriate vials with 5 mL scintillation medium having the following composition: PPO, 5.0 g; POPOP, 0.125 g; toluene, 66.7 mL; Triton X-100, 333.3 mL and used to determine the radioactivity in a scintillation counter (Beta-1, Russia). Protein content was determined using Biuret reagent.41 Statistics. Data were generated from three independent experiments (biological repeats), using a minimum replication from 3-4 different samples (technical repeats). Data are presented as the mean ( SEM. Comparisons between two compounds were made using unpaired, two-tailed Student’s t-test. For each individual concentration, the differences were considered to be significant if p < 0.05.

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