Indian Journal of Chemistry Vol. 398, September 2000, pp.680 - 687

Hydrophobic interactions of phenoxazine MDR modulators with bovine serum albumin

G B Eregowda, B C Channu, S Jagadeesh, H N Kalpana, Ravi Hegde, P J Houghtont & K N Thimmaiah*

Department of Studies in Chemistry, Manasagangotri , University of Mysore, Mysore 570 006, India tDepartment of Molecular Pharmacology, St. lude Children's Research Hospital , Memphis, Tennessee-38 105 . USA

Received 22 July 1999; accepted (revised) 20 october 1999

The binding of 1O-[3'-(N-piperidino)propyl]-2-trifluoromethylphenoxazine 1, 1O-[3'-(I3-hydroxyethylpiperazino)­propyl]-2-trifluoromethylphenoxazine 2, IO-[4'-(N-diethylamino)butyl]-2-trifluoromethylphenoxazine 3, 10-[4'-(N-piper­idino~butyI 1 -2-trifluoromethylphenoxazine 4 and 10-[4'-(N-diethylamino)butyl]-2-chlorophenoxazine 5 to bovine serum al­bumin (BSA) has been measured by gel filtration and equilibrium dialysis methods. The binding of these modulators to al­bumin has been characterized by the following parameters: percentage of bound drug (13), the association constant (K,), the apparent binding constant (k) and the free energy (f1F") . In addition, the displacing activity of hydroxyzine and acetylsali­cylic acid on the binding of phenoxazine to albumin has been examined. The binding of phenoxazine derivatives to serum transporter protein, BSA., is correlated with their partition coefficients. The results of the displacing experiments reveal that the phenoxazine benzene rings and the tertiary amines attached to the side chain of the phenoxazine moiety are bound to a hydrophobic area on the albumin molecule.

The binding of drugs to albumin has been investigated in numerous papers because of their pharmacokinetic significance l

. But, the drug-albumin complex may be considered as a model for gaining general fundamental insights into drug-protein binding. General rules of protein binding gained from this model could apply at least partially to the drug­receptor complex provided that the receptor has a protein structure. The determination of albumin binding of several structurally related compounds is a valuable tool for identifying the groups of a drug molecule which are involved in binding and for characterizing the binding forces concerned with the interaction of drugs with protein.

The binding of analogous phenothiazine derivatives to BSA has been studied l. Although, most of the authors obtained total binding constants of the same order of magnitude, the number of binding sites varied considerably. It has been found2 that the number of binding sites on BSA for promazine and chlorpromazine changed with the concentration of drugs, higher numbers being obtained at higher drug concentrations. The results suggested that phenothiazine derivatives ale bound by hydrophobic interaction with the aromatic amino acids of the BSA molecule and that under the influence of high drug concentrations, the number of avai lable sites increased by swelling and unfolding of the BSA

molecules in solution. Glasser and Krieglstein3

correlated the LoglOP of some phenothiazine drugs and related compounds with their log (~/C() values, 'W and 'C(' being the fractions of bound drug and free drug respectively. A fairly good linear correlation (r = 0.969) for five lO-dimethylaminopropyl derivati ves of phenothiazine was obtained. When other drugs were included the correlation deteriorated .

Compounds of pharmacologi.cal interest have been found among phenoxazine derivatives and they have been claimed to be nervous system depressants in particular with sedative, antiepileptic, tranquil ising activit/,5, spasmolytic activit/, antitubercular activit/ and anthelmintic activit/,. Multidrug resistance (MDR) has become a major obstacle for the clinical treatment of cancer as well as microbial diseases. A variety of small molecules capable of modulating MDR have been prepared and examined. While a number of pharmacological agents have been shown to reverse MDR in vitro, there remains a need to identify more potent, more specific and less toxic chemosensitizers for clinical use. In an attempt to search for more potent and less toxic chemosensitizers, Thimmaiah et al. 9 have reported that the parent phenoxazine potentiated the uptake of vincristine (VCR) and vinblastine (VLB) in MDR GCicl and KBChR -8-5 cells to a greater extent than verapamil. In a subsequent studylO.l l, twenty N lO_

EREGOWDA el al.: BINDING OF PHENOXAZINES TO BOVINE SERUM ALBUMIN 681

substituted phenoxazines were synthesised and examined for their ability to enhance the uptake of VLB and VCR in GC3/cl and KBChR -8-5 cells. Recently, Thimmaiah et a/. l2 have demonstrated that 2-chlorophenoxazines were able to partially reverse VLB resistance in MDR colon carcinoma cell line GCjcl and completely reversed the 86-fold VLB resistance in the MDRI overexpressing breast carcinoma cell line BC 19/3. The same agents could only partially sensitize BC 19/3 cells to taxol and doxorubicin suggesting that the chlorophenoxazines show some specificity for modulating VLB resistance. The results revealed that substitution on the phenoxazine ring at position NIO was associated with an increase in antiproliferative and anti-MDR activities. Nevertheless, the exact mechanism of pharmacological action of phenoxazines remains unknown. Since these molecules are found to be useful as potential anti-MDR agents, studies have been undertaken to elucidate the nature of interactions of phenoxazine derivatives namely, 10-[3'-(N-piper­idino)propyl]-2-trifluoromethylphenoxazine 1, 10-[3'­(6 - hydroxyethylpiperazino)propyl] - 2 - trifluoro­methylphenoxazine 2, 1O-[4'-(N-diethylamino)butyl]-2-trifluoromethylphenoxazine 3, 10-[ 4'-(N-piper­idino)butyl]-2-trifluoromethylphenoxazine 4 and 10-[4'-(N-diethylamino)butyl]-2-chlorophenoxazine 5 (ref. Table I) with serum transporter protein, BSA.

Results and Discussion

Evaluation of binding parameters

The binding of MDR modulators (1-5) to BSA based on the dialysis experiment was characterized by the parameters namely, percentage of bound modulator (~), t.he association constant (K), the apparent binding constant (k), and the free energy (I1JfJ) (Table II). The symbols, dimensions and methods of analysis of the values used to characterize the protein binding and the hydrophobic character of the modulators are summarised in Table III. The results of gel filtration experiment revealed that the bound phenoxazine modulator moves with the velocity of BSA. When experiments were performed in series for one substance, only the fractions after the protein zone have been assayed for phenoxazine modulator.

The effect of concentrations of modulators 1-5 on the binding to BSA was studied by dialysis experiments (Table IV). In these studies, the concentrations of the modulator were varied in the range 0.1- 5 x 1O-4M keeping the protein concentration constant (1 %) (ref. Figure 1). Examination of the data revealed that the binding increased with the increasing concentration of phenoxazine at low modulator/protein ratios. The amount of free drug remains the same inspite of the

Table I--Names, molecular structutre, molecular weight, loglO P values and pKa values of the phenoxazine derivatives.

r?'p0y.::p ~N~

I X R

Compd R X MW loglOP pKa

1 -CH2-CH2-CH2-N~ -CFJ 412 1.70 3.60 8.30

r-'\ 4.45 2 . -CH2-CHr CH2-N N-CH2-CH2-OH -CFJ 494 1.46 '-----/ 8.30

/CH2-CH3

3 CH2-CH2-CH2-CH2_N -CFJ 414 1.58 5.25

"- 8.60 CH2-CH3

4 -CH2-CH2-CH2-CH 2_<~ -CFJ 426 1.60 3.55 8.05

~ CH2-CH3

5 -CHr CH2-CH2-CH2_

-CI 37R 5.40

"-CH2-CH 3 2.40

9.10

682 INDIAN J CHEM, SEC B, SEPTEMBER 2000

Table II-Binding of phenoxazine derivatives 1-5 to bovine serum albumin

Phenoxazine % of bound Regression Apparent binding Association constant Free energy 10g IO (~/a) deri vati ves drug (~)* coefficient (mt constant (k)· K"1[104 x Mil ~ps

1 63.45 0.978 1.7360 3.85 6177 0.2395

2 53.40 0.979 J.l460 2.30 5876 0.0591

3 34.70 0.988 0.5333 0.80 5258 -0.2475

4 46.10 0.980 0.8553 1.65 5682 -0.1678

5 77.70 0.971 3.471 6.95 6523 0.5420

• ~ is the percentage of bound drug in a 1% BSA solution wi th a total concentration c= 10·4M of phenoxazine modulator.

+ ro, the regression coefficient and ·k, the apparent binding constant, were obtained from Figure 2, see also Table I .

• K I , is the association conslant obtained from the scatchard plot (Figure 3).

sl'lF" is the free binding energy calculated from -R71nK I .

Table JII-Symbol, dimensions and methods of analysis of the parameters used

Parameter Symbol Dimension Method of analysis

Total concentration of phenoxazine deli vati ve c M By weight, colorimetry

Concentration of free phenoxazine derivative Cr M Gelfiltration , dialysis

Concentration of bound phenoxazine deri va- Cb M cb=c-cr tive

Concentration of albumin Co gl lOOmL By weight, biuret method

Percentage of free phenoxazine derivative a % Gel filtration, dialysis

Percentage of bound phenoxazine derivative ~ % ~=IOO-a

Specific binding capacity r MlM r = cJca in moles

Regression coefficient m Fig. 3

Apparent binding constant k ( 10·5Mlm k=cJcr (Fig. 3)

Association constant KI 104 M·I Scatchard plot (Fig. 4)

Free binding energy I'll'" CalIM I'll'" = -RT InKI

Partition coefficient P M/M Partition between II -oclonal and buffer solution, pH 7.4

Table IV-Statistical data for the binding of varying concentrations of phenoxazine derivatives with bovine serum albumin

Phenoxazine Equation of the regression line

derivative in the double logarthemic sys-

tern

1 y=0.0288+0.978 x

2 y=0.0198+0.98 x

3 y=-0.00539+0.98 x

4 y=O.O 1655+0.98 x

5 y=0.02 16+0.97 x

* For compounds 1-5, r was found to be significant

fact that the concentration of the modulator was further increased (Figure 1), suggest ing higher number of binding sites on BSA. A similar observation was made in the case of binding of phenothiazines to BSA I. The apparent binding constant 'k' and. regression coefficient 'm' were

No. of single Coue1ation

experiments (n) coefficient (rl"

26 0.9986

26 0.9991

26 0.99954

26 0.99933

26 09984

calculated by plotting concentration of free­phenoxazine modulator versus concentration of bound phenoxazine modulator (Figure 2). In order to calculate the total binding constant, K\ , sc hatchard plot for the binding of the phenoxazine modulator to BSA was done (Figure 3). Comparison of the k and

EREGOWDA el al.: BIND ING OF PHENOXAZINES TO BOVIN E SERUM ALBUM IN 683

70

... ......... 010·· .. • .. • .. 60

CD 0 0 ::J g .... ..-0-0-0 0 Q 0 e 0 a e

"C 50 Cb 0 • • • • Cb •••• .... ~

• - • -0 40 • • • • • • • • 0 ~

, . Q) 30 • CD

S • t: . • , . . • • • D - • • • Q) 20 • +1 u .... . .A 2 Q)

a.. 03 10

-4 ·5

0

0,0 0,5 1,0 1,5 2.0 2,5 3,0 3,5 4,0

Total concentratlon'c'(10~ M)

.!,:urt' I- Binding or varying co nce ntrations of the phenoxazi ne modulators to bovi ne se rum albumin. Ordinate : Percentage of free phe­)xazine derivative. Ahsc i s~ a : lO tal concentration of phenoxazine derivative ( I O·.J M). Binding measure ments were carri ed out in a I '7r SA so lution (I' ll 6.9. 22"C. incu hated at 37"C for 6 hr) . Each poin t represe nts the mean value or two expe riments .

~ "I 0 .... Cl

2 10 '0 '0 C ::J 0 .c ..... a D

(.)

~c 0

rtI .!:; C III (.)

C a U

0 ,1

0,1 10 100

Concentrat l on-crof free drug ( 10~ M)

Figure 2-Binding capacity or a I '7< album in sol ut ion for phenoxazine derivat ives . Ordin ate: Concentration 'Ct: of bound phenoxazille deri vative ( 10 5M ). Ahscissa : conce nl ration or free phe noxazine deri vative ( I 0.5 M) . Bindi ng measurements were carried out in a 1'7r BSA so lution (p ll 6.9. 22"C. incubated at 37"C fo r 6 hr). Each [loint rep re~e nts a sin gle ex peri ment. Thi s plot is pe rformed in order to obtain the binding constant s 'Ill' and 'k' . Sec also Tahlt, II . For , tati stical data sec Table IV.

684 INDIAN 1 CHEM, SEC B, SEPTEMBER 2000

• Q ....

a

7

6

5

0

~ 0 0 0

)( " . 3

0 0

0 0

0 .1 .2 ...3 -4 05

• • • •

.. . • • 2 ~ A ~" •• ~.r---~.--~.~---r--4---____ -& ______ ~ .... ' Z • * • • A

o+---------+---------~--------~--------~--------~--0.0 1,0 2.0 3,0 ".0 5,0

r Figure 3--Scalchard plot of binding of phenoxazine deri vatives to bovine serum albumin . Ordinate: rlcr in (l0·4M-I). c( = molar' concen­tration of free phenoxazine derivative in the albumin solution . Abscissa: r = number of moles of phenoxazine per moie of albumin . All measurements were made in a I % albumin so lution (PH = 6.9, 22°C). Each point represents the mean value of two single experiments. For total binding constant K, see Table II .

KJ values within the compounds examined, showed that the modulators bind to BSA in the order: 5>1>4 >3>2, indicating that phenoxazine containing -Cl in position C-2 has greater affinity to protein than those phenoxazines containing -CF3 in position C-2. The extent of binding of these modulators to BSA was further supported by l1F" values (Table II) .

Relationship between bovine serum albumin binding and hy­drophobic character of phenoxazine modulators

Several authors have demonstrated a correlation between the hydrophobic character and protein binding of low molecular weight substances I 3

-1?

These results suggest that hydrophobic interactions play an important role in protein binding to organic compounds. However, a good correlation between protein binding and pal1ition coefficients can be shown only for substances of structurally related groups. Hence, not only hydrophobic interactions can be present in protein binding of organic molecules, but also other binding mechanisms such as ionic binding, hydrogen binding or steric effects, etc. must be also invol ved 's.

The hydrophobic character of phenoxazine modulators (loglOP values) were determined (Table I) to look for a possible quantitative correlation between the lipid solubility of these compounds and their protein binding ability. Analysis of the relationship

0.6

0.5

0.4

0,3

__ 0,2

o C. 0,1 ~

co

~ °t-~--------~~------~'------~I ~ U - ~ , t

.1 ~,3

Figure 4-Relation betwecn albumin binding and partition cocl~ lici ent s or phcnoxazi ne derivati ves. Ordinate: log (pIa), p = per­ccnt phcnoxazinc derivative bound, a = pcrc1!nt frcc . Abscissa : Log lO 1', I' = partition coeffic ient between l1-oetanol and buffer so lution . Thc equation of the rcgrt:ss ion linc: Log lO pIa = 2.412 + 1.4971 Log IO P.

between lipid solubility of modulators used in this study and the fraction, log (Bfa), where 'B' is the percentage of phenoxazine bound and 'a' is the percentage of free phenoxazine modulator showed a good correlation (Figure 4). (Bfa) function is

preferred for thi s type of correlation because it is directly analogous to the organic solvent-buffer partition coefficient. The following equation 1 was

EREGOWDA e/ at.: BINDING OF PHENOXAZINES TO BOVINE SERUM ALBUMIN 685

arrived at. from the log (~/a) and 10gIOP data for five phenoxazine derivatives substituted in position C-2 of the phenoxazine nucleus .

'Iog (~/a) = -2.412 + 1.4971 10gIOP ...... (1) (correlation coefficient, r = 0.9576).

For the five phenoxazine modulators, hydrophobic ity decreased in the order: 5> 1 > 4 > 3 > 2. This rank order is the same as for the binding efficiency of these modulators with BSA.

Displacement of phenoxazine modulators from their bovine serum albumin binding sites

Simple aromatic substances like benzoic acid or aniline are able to displace phenothiazines from their binding site on the albumin molecule? In order to understand the BSA binding mOIetIes of the phenoxazine modulators, 1 - 5, the authors have studied the displace ment experiments by dialysi s method using hydroxyzi ne and acetylsalicylic acid . In these experiments, the binding of the phenoxazine modul ators to BSA was determined afte r the di splac ing agent, hydroxyzi ne or acetyl salicylic ac id was added to the incubation mi xture (Table V) .

The partic ipation of the side chain in the total binding of the phenoxazine derivati ves is indicated in the di splacing experiments with hydroxyzine (Figure 5). Hydroxyzine. which like modul ator 2, possesses a piperazine ring, di spl aces 2 but not 5 and 3 from its BSA binding si tes. Thi s mi ght be ex pl ained by supposi ng that the a liphatic side chain between N IO of phenoxazi ne nucle us and N-atom of s ide chain teJ1iary amine is not in a position to contribute to the binding of the phenoxazi ne modulators used . Aga inst that N, N-diethylamino group in the a liphatic side chain may lie sufficient ly c lose to the surface of the BSA molecu le to intensify binding of the phenoxazine mo lecul e by hydrophobic inte raction .

Acety lsalicylic ac id competes with the benzene rings of the phenothiazine ring system for binding to BSA I. S ince acetyl sa licy lic ac id competes for

phenothiazine ring system for binding to BSA, the author has examined the effect of acetylsalicylic ac id as a displacing agent to know whether benzene rings of phenoxazine modulator is involved in binding \0

BSA. The displacing experimental data are shown in Figur e 6. Examjnation of the data from Figure 6 has highlighted that acetyl sa licylic acid d isplaces phenoxazine modulator from the BSA binding site . In summary, the results of the investigation suggest that possibly phenoxazines are bound to albumin by hydrophobic interactions.

Experimental Section

The synthesis of 2-trifluoromethy l-N 10-subst ituted phenoxazines 1-4 and 10-[4'-N-(diethylamino)butyIJ-2-c hlorophenoxazine 5 are descri bed e l sewhereI 9.~o The structural formulas of the compounds are gi ven in Table I. The compounds were separated and purified by column chromatography and c haracterized by UV. IR 1 · 11C , H and ' NMR and mass spec tra l ana lyses. The spectral data are consistent with the structures.

Acetylsalicylic ac id , bovine se rum a lbum in and hydroxyzine were purchased from Sig ma Chemica ls Company, USA. All o ther che mjcals were o f reagent grade. All bi nding measure ments were made in the presence of 0.02M phosphate bu ffe r, pH 6.9 conta ining 0.15M NaCl and 1mM sod ium thiosulphate . Just be fore protei n binding of a drug was de te rmined, the pH of the sample solution was measured and where necessary adjusted to pH 6.9 with O. IN HCI or O.IN NaOH.

Separation of phenoxazine-albumin complex by gel-filtration experiment

The binding of phenoxazine deriva ti ves to albumin was studied wi th the aid of ge l fi ltrati on experiments. Gel filtrati on experiments were performed on a 20 x 1.3 c m column of sephadexR G-SO fine (Pharmac ia) at 22°C, equilibrated with standard buffe r solution. the flow rate be ing maintained at 25 mL/hr. 20mL of the

Tahl~ V--Sta ti slical data for the di splacem en t of phenoxazine d eriva tives from their albumin bind ing sites by hydroxyzine

Compd No . Equation of the regression line No. of single experiments (n) Correlati on coefficient ( r )*

I + Hydrox yzi ne y=36.55+0.60x 20 0 .9147

4 + Hydroxyzi ne y=46.6+0. 15x 20 0 .8209

2 + Hydroxyzine y=65.3+0.225x 20 09225

3 + Hydrox yzine Y=53.9+0x 20 0 . 1130

5 + Hydroxyzine y=22 .3+0.025x 20 0 .1592

* For co mpou nds 1.2 and 4 the value of r was signili cant and for compounds 3 and 5, r was insignificant.

686 INDIAN J CHEM , SEC B, SEPTEMB ER 2000

10

., 70

C N .. >( 10 0 c !! ~ 50 ., • ., .: 0

'0 ~

-tl ., 30 C1

5 c .,

20 u "-I)) c..

10

0

O.D

• .. • •

• •

5,0

.

•

10.0

• •

•

o

.

15.0

'aT .2 .3 • 4 .5 -

-

• o

•

20.0 25.0

Hydroxyzine (10-4 M)

Figure 5--lnfluence of hydroxyzine on the binding of phenoxazine deri vati ves to bovine serum albumin . Ordinate: Percentage of free phenoxazine deri vative in the albumin solution. Abscissa: total concentration of hydroxyzine (lO,4M) . All measurements were made in a I % al bumin solut ion containi ng 1O,4M phenoxazine derivati ve and varying concentration on hydroxyzine. Each point represents the mean value of two single experiments. For stati sti cal evalua tion see Table V.

eo

II 70 c: .. .. " ID 0 c: II .I:

... •

• • . • -Q. !IO II II .: • '0 «> . , 0 , II :10 Cl

~ o o

c: II 20 ~ II e-

10

0.0 5.0 10.0 15.0 20.0 :15.0

Acetylsalicylic acid (10" M)

Figure 6--Displaccment of phenoxazinc derivati ves by acetylsalicy lic acid from binding to bovine serum albumin . Ordinate: Percentage of free phenoxazine deri vat ive. Abscissa: total concentration of acetylsali cylic acid ( 1O,4M) . All measurements were made in a I % albu­mi n so lution containing one of the phenoxazine derivatives ( I O,4M) and varying concentrati ons of acetylsalicyli c acid. Each point repre­sents the mean value of two single ex periments.

BSA solution (l %) containing l.Ox 10'4 M phenoxazine modulator, after incubated at 37°C for 6 hr, was loaded on to the column and washed with standard buffer. The effluent from the column was collected into fractions of 3mL. 1.5mL of each fraction was used to deterrrune the protein

concentration and remaining 1.5mL for the estimation of phenoxazine modulator. The albumin content was determined by 'biuret' method and phenoxazine modulator estimated after extracting with n-heptane colorimetrically in 50% sulphuric acid containing 10 mg% FeCI).

EREGOWDA et at.: BINDING OF PHENOXAZINES TO BOVINE SERUM ALBUMIN 687

Study of interaction of phenoxazines with BSA by equilibrium dialysis method

Sample solution (20mL) containing BSA (I %) and one of the modulators 1 - 5 in the concentration range 0.1- 5 x 1O-4M was taken in a 50mL centrifuge tube and incubated at 37°C for 6 hr in a shaking water-bath incubator. For each of the four dialysis tubings (3/4"

diameter), 5 mL of the above reaction mixture was pipetted out. After closing, the dialysis tubing was immersed in standard buffer solution taken in a measuring jar. The samples were dialysed for 12 hr at 22°e. At the end of the dialysis experiment, the free phenoxazine modulator from the buffer medium was extracted into n-heptane. After evaporating the organic layer, the phenoxazine concentration was measured colorimetrically using 4mL of 50% sulphuric acid containing 10mg % FeCI).

Displacement of phenoxazine modulator from BSA binding site by equilibrium dialysis

The displacement of phenoxazines by hydroxyzine or acetylsalicylic acid was studied by means of equilibrium dialysis . In these experiments, the binding of the phenoxazine modulator to BSA was determined after the displacing agents, hydroxyzine or acetylsalicylic acid were added to the incubation mixture.

Measurement of lipophilicity

The relative Iipophilicity of each of the compound used in this study, was assessed using an adaptation of the method of Zamora et al 21

. This method involves measuring the partitioning of modulator between 1-octanol and PBS (PH 7.2). HPLC grade I-octanol was pre-saturated with aqueous phase buffer and converselylbuffered aqueous phase was pre-saturated with HPLC grade I-octanol before use. The modulator was dissolved in aqueous phase buffer / octanol at a final concentration of I x 1O-4M, an equal volume of I-octanollbuffer was added and the tubes were then continuously inverted for 15 min (experiments carried out over time intervals ranging from 5 to 60 min confirmed that equilibration was reached within 15 min) . The final concentration of modulator in both aqueous and octanol fractions was assessed by measuring the UV absorbance of these experimental fractions . The partition coefficient, P,

was determined by dividing the concentration of modulator in the l-octanol phase by the concentration in the aqueous phase. LoglOP was used as a measure of lipophilicity.

Acknowledgements

This work was partially supported by the Department of Science and Technology (DST), Government of India. One of the authors (GBE) thanks the Mysore University for providing the University Postgraduate Junior Research Fellowship.

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