1. INTRODUCTION:

Tamoxifen (2-[p-1, 2-diphenylbut-lenly) phenoxy ethyl dimethyl amine is an anticancer drug. A number of analytical methods for determination of tamoxifen viz. GLC, mass spectrometry, solid phase extractions, Capillary, electrophoratic separation^1,2 etc. have been reported in the literature.

However, formation of the Zn-tamoxifen complex and its characterizations by polarographic methods which are far better than the existing methods in the field as regards to their extra ordinary detection sensitivity, oligo determination capability, minimum detection limit, low cost, rapidity, accuracy, simplicity and non destructive nature, have not been used for the said purpose.

Several metals such as Fe, Co, Zn, Cd and Ni in trace amount are essential for health and for the maintenance of human biological system. The complexes of life essential metals play significant role in biological system, in which enzymes are known to be activated by metal ions³.

The advantages of complexes of Ni, Fe, Co, Zn etc. with different types of drugs have been discussed by number of workers^{4, 5}. In continuation of the work reported from our laboratory on the study of electrochemical and bioinoganic behavior of some drug and metal-drug complexes^6,7. The present paper describes the characterization of Zn (II) - tamoxifen complex using polarographic and spectral methods. Microbial study and pharmacological study of anticancer activity have been also reported in the paper.

2. EXPERIMENTAL:

2.1 Chemicals and Reagents:

All the chemicals used were Anala-R/BDH grade. The drug Tamoxifen (C₂₆H₂₉NO) was procured from Sigma Chemical Company, USA. Double distilled water and absolute ethanol were used as a solvent.

Polarographic measurements were made on an Elico (Hyderabad) pulse polarograph model CL-90, coupled with a X-Y polarocard model LR-108. The electrode system consisted of a dropping mercury electrode (DME) as a working electrode, a coiled platinum wire as an auxiliary electrode and saturated calomel electrode (SCE) as a reference electrode.

Experimental sets were prepared by keeping overall Zinc (metal ion) and potassium chloride (supporting electrolyte) concentration fixed at 1.0 mM and 1.0 mM respectively. The ligand concentration was varied from 0.0 mM to 5 mM. The pH of the test solution was adjusted to 7.0+0.1 using HCl/NaOH solution.

The amperometric titrations were performed on a manually operated set up, a polyflex galvanometer (sensitivity 8.1 x 10^-9 amp./div.) and an AJCO varnier potentiometer, a DME was used as an indicator electrode and a calomal electrode served as reference electrode. The capillary characteristics of a DME had a m^2/3 t ^1/6 = 2.13 mg^2/3 Sec^1/2 at 50 cm effective height of mercury column.

The pH of all the test solutions was measured on an Elico digital pH meter model LR-108.

Experimental sets, each having different but known amount of the drugs understudy were prepared in appropriate quantity of supporting electrolyte (potassium chloride) at pH 7.0+0.1were prepared and titrated separately against the standard solution of the titled Zn (II) ions whose pH has been adjusted to that of the titrate (7.0+0.1).

2.2 Synthesis Procedure of the solid complex:

Zinc chloride and Tamoxifen (drug) solutions were separately prepared in ethyl (40:60v/v) alcohol and were mixed in 1:1 molar ratio. The mixture was then refluxed in a round bottom flask for one-two hours. The residue complex was filtered and washed thoroughly to remove any unreacted material. The complex was dried at low temperature (40°C) and stored over P₄O₁₀.

The elemental, C, H, N and O analysis of the complex was done on a Heraeus Varlo Erba elemental analyser model-1108, at CDRI, Lucknow, whereas gravimetric method was used for the estimation of Zinc in the complex⁷.

The IR spectrum of the solid complex was recorded using KBr pallets on a perkin-Elmer IR spectrophotometer, model-379.

2.3 Biological Study of Zn (II) - Tamoxifen complex:

2.3.1 Microbial Study:

Raper's paper disc method¹² was followed for the microbial screening of Zn (II) - Tomoxifen complex against various bacteria viz. Pseudomonas mangiferae, Staphylococus aureus, Salmonella typhi and Vibreo colarae and fungi i.e. Trichothesium and Chrysosporium sp. sterlized filter paper discs (6mm) were dipped into the complex solution of 0.01M concentration. Prior to this, the bacteria and fungi were separately homogenized with nutrient agar and potatodextrose media (at 27-30°C) plated onto the sterilized Petri dishes. Dipped filter paper discs were placed on seeded medium. After 24 hour of incubation antimicrobial activities were recorded by measuring the inhibition zone against complex under study similar experiment was repeated with the control drug (tamoxifen).

The number of replicates in each case of three, percentage inhibition was calculated using the following formula –

% inhibition = (A-B)/A x 100

Where (A) represents the diameter of the inhibition zone for control (tamoxifen) and 'B' represents the diameter of the inhibition zone for sample tamoxifen complex.

2.3.2 Pharmacological Studies

In-vitro and in-vivo study of anticancer activity of prepared drug metal complex have been done by following procedure^13-15.

2.3.3 In-Vitro:

Mouse Sarcoma cell line-180, obtained from National Center of Cell Science, Pune, India, as a monolayer culture in Roux bottles (Corning Plastics, USA).

Cells Culture- The cells obtained was cultured in 5ml 24 well culture plate (corning plastics USA). The cells were seeded in 2x10 cells per well and 1.0ml of dulbecco's modified Eagles medium (DMEM) containing 10% (v/v) Foetal calf serum, penicillin 100/µg/ml and streptomycin 100µg/ml was added to each well. The cells were kept in incubator at 37⁰ C for 4h in 5% CO atmosphere and 95% humidity. The cells count was made on Neubaurs chamber (Fine Optic, Germany).

Three dilutions viz. 1µM, 10µM and 100µM/ ml of pure drug and its Zn complex was made and then the cells were treated as follows:

Column Complex	Free Drug	Column	Metal
A	1µM (1ml)	D	1µm (1ml)
B	10µM (1ml)	E	10µm(1ml)
C	100µM(1ml)	F	100µm(1ml)

After addition of the respective solutions, the culture plate was incubated at 37°C for 4 hours. Finally the cell counts were made as under. These are compared with the cell cultured in DMEM without treatment.

Cell Viability Counts:

Cell viability counts were made by Trypan blue dye exclusion test. Two drops of trypan blue were added to each cell culture wall and kept for 15 minutes. Now, a drop of culture was added to hemocytometer (Neubaurs Chamber) and the number of stained, non stained and total number of cells were counted, Then, the % inhibition was calculated using the equation.

No. of viable cells – No. of viable cells after treatment x100/

No. of viable cells without treatment

The experiment of each concentration of the drug and the complex was repeated thrice and statistical conclusions were drawn.

2.3.4 In-Vivo:

The comparative efficiency of pure and complex forms of Tamoxifen drug evaluated from the difference in response after treatment with two forms of drugs.

Animal model : Balb/c mice, weight 30-40 gm.

Tumor model : Sarcoma cell line-180

Drug : Tamoxifen and its Zn complex.

Cell growing in nutrient medium (DMEM) were obtained from NCCS, Pune. They were brought into single cell suspension by trypsinization (0.2% trypsin). The cell suspension was centrifuged to obtain concentrated suspension (1-2 x 10cell/ml). Approximately 10⁵ cells of tumor were injected on the dosal surface of the mouse and allowed to grow. Palpable size was reached by 6-8 days.

The time required to double the tumor volume (volume doubling time, VDT) from 100 to 200 mm was taken as a criterion to assess the antitumor efficiency of pure and complexed drug in S-180 tumor bearing mice. The treatment was started after tumor size reaches 100 ± 10mm³. Indicated dose (equivalent to 0.2 mg) of free drug and drug complex were injected intravenously and tumor growth was monitored. Tumor size was calculated by the formula 1/2 LW² where L-long diameter and W-short diameter of the tumor. The above in-vivo experiment was repeated on two other sets of mice groups.

3. RESULTS AND DISCUSSION:

3.1 Polographic behaviour of Tamoxifen with Zn (II):

In - 1.0 M KCl at pH 7.0+0.1 the Zn (II) and its complex with ligand under study were found to be reversibly reduced involving two electrons which was found to be diffusion controlled, which was evidenced by the plot Id vs Öh corr.

On gradual increase of the tamoxifen concentrations, the half wave potential of Zn (II) metal ion shifted to more negative value and the diffusion current also decreased thereby showing complex formation between Zn (II) with tamoxifen (fig.1).

Fig.1:Polarograms of Zn (II) (1mM) in 1M KCl supporting electrolyte at pH 7.0 ± 0.1 and A-without Tamoxifen; B-1 mM Tamoxifen; C, D and E-2, 4 and 5 mM Tamoxifen

Fig.2:Amperometric titration of (2mM/10ml) Tamoxifen (1mM/10ml Zn-II) solution

To study the composition and formation constant of the complex, plots of DE_½ (Shift in the E_½) i.e. DE_½ = (E_½)_c – (E_½)_s against log Cx (logarithm of the concentration of the ligand) were drawn. The plots were linear lines showing the formation of single complex species in solution. Lingane treatment⁸ of the observed polarographic data reveals 1:1 Metal : Tamoxifen comlex formation with log b = 5.1.

3.2 Amperometric determination of Tamoxifen with Zn (II):

Zn (II) gives a well defined polarographic wave in 1.0 M KCl at pH 7.0+0.1. The diffusion current was found proportional to the concentration of Zn (II). The tamoxifen drug does not produce any wave under the said experimental conditions. The platue potential for the polarographic wave of Zn (II) (-1.4v) vs Hg pool was applied for carrying out amperometric titration. On performing the amperometric titration of drug solution with standard solution of Zn (II) the current volume plots result in / shaped curves (Fig.2). The end point as located by graphical method revealed metal to drug ratio of 1:1 which is in agreement with the author's observations on the metal: ligand equilibria using polarographic method.

3.3 Characterization of Zn (II) Tamoxifen complex:

3.3.1 Elemental analysis:

The results of elemental analysis (Table-1) of the drug and it's complex with Zn(II) revealed 1:1 metal drug ratio in this complex, which supports author's findings using polarographic and amperometric method.

Table-1: Analytical data of Tamoxifen and its complex with Zn(II) Analysis/Calculated/(Found)

	Element	Tamoxifen	Zn(II) Tamoxifen Complex
1	Zn	-	23.52 (23.60)
2	C	39.61 (39.70)	30.28 (30.32)
3	H	5.4 (5.38)	4.22 (4.02)
4	N	46.2 (46.15)	35.24 (35.36)
5	O	8.79 (8.78)	6.74 (6.80)

Table – 2: Antimicrobial study of Tamoxifen Zn (II) complex

Sr. no.	Organism	Inhibition-zone (mm)
		Concentration of complex (per 10 ml)		Control metal Zn(II) 1.0 mM/10ml	Percentage change over control metal (A-B/A)x100	Control Drug (Y) 1.0 Mm/10ml	Percentage change over control drug (Y-B/Y)x100
		5mM	1.0mM(B)	Control metal Zn(II) 1.0 mM/10ml	Percentage change over control metal (A-B/A)x100	Control Drug (Y) 1.0 Mm/10ml	Percentage change over control drug (Y-B/Y)x100
1.	Bacterial
a.	Pseudomonas Mangiferae	5.8	12.8	57.0	77.54	12.2	0.00
b	Staphylococus Aureus	6.4	14.0	41.2	66.02	15.0	6.66
c	Salmonella typhi	8.4	15.3	53.1	71.18	22.0	30.45
d	Vibreo colarae	-	14.0	53.0	73.58	11.0	-27.27
2	Fungal
a	Trichothesium	8.0	15.0	40.2	62.68	-	-
B	Chryosposium sp.	7.0	17.1	39.0	56.15	-	-

3.3.2 IR Spectra:

Structurally important IR band of the tamoxifen drug and its Zn (II) complex which are particularly useful in assigning the position of metal ligand bonding is observed as a broad band at 1601-1640 cm^-1 due to C-N aliphatic vibrations⁹ in the IR spectrum of the drug, which is found to be affected in the spectrum of Zn(II) tamoxifen complex i.e. the broad band at 1600-1640 cm^-1 results in a single peaked band at 1590 cm^-1 with reduced intensity¹⁰. Th

e observations clearly indicate the involvement of the nitrogen of C-N group in complex formation¹¹. On the basis of above data a tentative structure of the Zn (II) - tamoxifen complex may be given as under –

3.4 Microbial study:

The results of antimicrobial activities of the Zn(II) - tamoxifen complex are shown in Table 2. A perusal of the data in table clearly shown that Zn - tamoxifen complex is found to be more toxic as compared to the control drug against above mentioned bacteria and fungi.

3.5 Pharmacological studies:

3.5.1 In vitro:

The results of in vitro experiments of pure drug and its complex are shown in Table 3.A perusal of the results show that Zinc-tamoxifen complex was found to be more effective than pure drug. The complex under study showed in increased inhibition against the S-180 tumor cells at all the test concentrations i.e. 1, 10, 100 μm/ml. The increased inhibition activity of the complex was 52.1+1.0%, 69.8+1.0% and 92.5+0.9% as against 36.4 + 1.0%, 54.7+0.6% and 78.6 + 0.8% shown by the drug, respectively. The statistical treatment of the observed inhibition data i.e. standard deviation and coefficient of variance which never exceeded 0.9 and 1.8% respectively, speaks the reliability of the observed inhibition data.

Table – 3: In vitro cytotoxicity of Tamoxifen and Zn(II)- Tamoxifen complex against S-180 tumor cells

Compound

Concentration μM/ml

% inhibition after 4h

Tamoxifen

1.0

10.0

100.0

36.4 ± 1.0(a) (b)

54.7 ± 2.6

78.6 ± 1.8

Zn (II)- Tamoxifen

Complex

1.0

10.0

100.0

52.1 ± 1.0

69.8 ± 1.6

92.5 ± 1.9

(a) Composite results of three experiments.

(b) Mean ± standard error at mean.

3.5.2 In vivo:

The results of the average of mice tumor against tamoxifen drug and its zinc complex under study are shown in Fig.3. The results indicated that the tumor volume was 0.05 cm² on the tumor cell injected mice without administering drug or complex after 20 days, percentage which was reduced to 0.035cm² on tumor injected mice who were also administered the tamoxifen drug. However, in case of Zn (II) tamoxifen administered mice (tumor cell injected) shows significant decrease in the tumor volume of 0.018cm² was observed. Thus it indicated the in vivo tumor inhibition power of the complex over the drug under study over the experiment time periods i.e. 20 days.

Fig.3:Effect of Tamoxifen and Zn(II) Tamoxifen complex on tumor volume.

A- Without drug

B- With Tamoxifen

C- With Zn(II)- Tamoxifen

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Received on 20.05.2010 Modified on 10.06.2010

Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 981-985