The In vitro effects of Ranitidine hydrochloride on the Protein Binding of Naproxen Sodium in Aqueous Medium

Anusua Chowdhury1, Irfan Newaz Khan1, Md. Abdul Motaleb Bhuiyan1, Subrata Kumar Biswas2, Fouzia Siraji*1
1 Department of Pharmacy, USTC 2 Department of Pharmacy, Stamford University Bangladesh
1

Abstract: An in vitro study of protein binding has been carried out to observe the influence of Ranitidine hydrochloride on the protein binding of naproxen sodium by equilibrium dialysis method at 370C and at physiological pH (7.4). It has been found that Ranitidine hydrochloride slightly lowered the affinity and percentage of protein binding of naproxen sodium to bovine serum albumin. The scatchard plots were prepared to revel the number of binding sites and affinity for protein binding. It was seen that the highest percentage binding of naproxen sodium was 98% and the lowest was 92%. In the presence of Ranitidine hydrochloride, the highest and lowest value of percentage of protein binding was 89% and 92% respectively. It is thus inferred that Ranitidine hydrochloride or its complex with naproxen sodium can cause decrease in percentage of protein binding of naproxen sodium.Complexation of naproxen sodium with Ranitidine hydrochloride might, therefore, displace the drug from the plasma and the displaced drug may be redistributed , thus, increasing the free drug in plasma and tissue systems. This may change the pharmacokinetic properties of the drug and may affect the pharmacological and toxic effects. It is thus inferred that care and monitoring must be taken during combination therapy of naproxen sodium.

Keywords: Naproxen Sodium, Ranitidine hydrochloride, Protein binding, In vitro effects INTRODUCTION Naproxen is a non-steroidal anti-inflammatory drug (NSAID) commonly used for the reduction of moderate to severe pain, fever, inflammation and stiffness caused by conditions such as osteoarthritis, rheumatoid arthritis, psoriatic arthritis, gout, ankylosing spondylitis, menstrual cramps, tendinitis, bursitis, and the treatment of primary dysmenorrhea. It works by inhibiting both the COX-1 and COX-2 enzymes. Naproxen and naproxen sodium are marketed under various trade names including: Aleve, Anaprox, Antalgin, Miranax, Naprelan, Naprogesic, Naprosyn, Proxen, Synflex, Xenobid. . It works by reducing hormones that cause inflammation and pain in the body. The chemical name for naproxen sodium is 2-naphthaleneacetic acid, 6methoxy-a-methyl-sodium salt,Naproxen was first sold as the prescription drug Naprosyn in 1976; naproxen sodium was first sold under the trade name Anaprox in 1980 (Pocock). The Food and Drug Administration (FDA) approved naproxen sodium's use as an over-the-counter drug in 1994. naproxen binds very well to albumin and thus achieves a longer half-life in the blood than other drugs, lasting up to 12 hours per dose. Naproxen is also available as a sodium salt, naproxen sodium, which is more rapidly absorbed from the gastrointestinal tract. Ranitidine is a
* Author of Correspondence: Fouzia Siraji, Associate Professor, Department of Pharmacy, USTC

competitive, reversible inhibitor of the action of histamine at histamine H2-receptors, including receptors on gastric cells with a minimal effect on H1-receptors. It is one of the drugs of choice for the treatment of active duodenal ulcers, gastric ulcers, Zollinger-Ellison syndrome, gastroesophageal reflux disease, and erosive esophagi is [Louis et al, 2002] The drug has a short biological half-life of approximately 23 h, an absolute bioavailability of only 50%, and it is absorbed only in the initial part of the small intestine, Gramatt.1994, Lauriten 1990, Grant.1989. Plasma protein binding is one of the important and useful pharmacokinetics parameters of a drug. There are multiple binding sites on a protein molecule. The pharmacokinetic and pharmacodynamic behavior of a drug is governed by the strength of a complex formed with the protein. Drugs generally formed reversible complexes with the plasma proteins that as a reservoir releasing the free drug to the circulation. Free from (of the drug) shows pharmacological response is metabolized and excreted. Bound (to the plasma protein) drug is gradually released to maintain the equilibrium and thus the pharmacological response is maintained. The type and nature of the protein binding depend on the physicochemical properties of the drug molecules their concentratio9n, pH of the medium, number of available binding sites of the plasma proteins. protein binding is a limiting factor for drug effect. Simultaneous administration of two or more drugs into the systemic circulation can modify the affinity of the drug to bind with plasma protein and thus percentage of protein binding. Due to this modification, the combined therapy can change the volume of distribution, renal and hepatic clearance, and hence drug effect. This study was aimed to evaluate the influence of Ranitidine hydrochloride percentage of protein binding of naproxen sodium at physiological conditions (pH 7.4 and temperature 3710C).

Naproxen Sodium Mol. Wt.252.24 Mol. Formula: C14H13NaO3 MATERIALS AND METHODS

Ranitidine Hydrochloride Mol. Wt.350.87 Mol. Formula: C13H22N4O3 S.Hcl

Drugs and Chemicals. Naproxen Sodium and Ranitidine Hydrochloride were kind gifts from United Chemical and Pharmaceutical Ltd., Bovine serum Albumin (fraction V, 96-98%, Sigma) and semi permeable membrane (Medicell,England)and all other reagents were purchased from BDH (England).Phosphate buffers were prepared by following methods. Perrin, 1974 , Bates 1964 Apparatus. A digital pH meter ( Hanna, Portugal) was used to adjust the pH of the buffer solutions. A UV/VIS spectrophotometer (Thermo electrical company, England) was used for the measurement of absorbance of the unbound drugs present in the buffer compartment. A Dunbuff metabolic shaking incubator (Nickel, Electrical company, England) was used to shake the plasma drug mixtures for the attainment of equilibrium. Equilibrium dialysis. In this experiment, the membrane was activated by digestion

with 1.0 M NaHCO3 at 700C for four hours and washing with deionised water and immersing in 1/15 M (or, 0.067 M) phosphate buffer of pH 7.4. Activated membrane (4 ml capacity) were filled with solutions of protein with different concentration of drugs and their mixtures and immersed in fixed (60 ml ) of phosphate buffer and then shaken gently for six hours in a metabolic shaking incubator at (37010)C After completion of dialysis the absorbance of buffer (out side the membrane bags) was measured at 314 nm and the concentrations of bound and unbound drugs were found using a standard curve. Determination of the Lamda max (max) for Naproxen Sodium to be used in this work. A stock solution of Naproxen Sodium was prepared with deionised water and then working solutions were prepared by diluting with deionised water in all cases. Thus 5x10-5 M were prepared and their absorbance was measured to find out the max of Naproxen Sodium (Fig 1) and also for Ranitidine Hydrochloride. It was observed that for naproxen Sodium absorption maxima was occurred at 314 nm in aqueous solution.

Fig:1: Spectral Determination Preparation of standard curve. A standard curve was used for the spectrophotometric determination of concentration of drugs in the buffer chamber. A treated buffer was used for this purpose. This buffer was obtained by allowing the equilibrium dialysis of the permeable fragments of free plasma from the inside of the membrane bags to the buffer solution (0.067 M Phosphate Buffer, pH 7.4)

1.2 1 Absorbance 0.8 0.6 0.4 0.2 0 0 0.2 0.4 0.6 0.8 1 y = 0.9923x + 0.1366 R2 = 0.9548

concentrationx10-5M Fig 2. The Standard curve of naproxen sodium for equilibrium dialysis method. The data w as show n as mean + SEM

Calculation of percentage of protein binding. The percentage of protein binding (F) is given by F= [ (B-A) / B] x 100 Where A = Molar conc. Of free drug in buffer compartment B = Molar conc. of total drug in protein compartment Calculation of affinity constant and number of binding sites. The scatchard method Singlass.1997,Goldstein 1974,Scachard19496,10,11was used for this purpose and a curve was produced by plotting r/[A] versus r using the equation. r = ([B] - [A]) / [protein] Where r = the ratio between the molar concentration of the bound drug and the molar concentration of the protein. The intercept on the y axis representing nk, and the intersection on the x axis representing n and the slope of the line being k, where n = the number of binding sites on the protein available to bind drug molecule or its complex, k = affinity constant (or, binding force) for the binding of the drug or its complex. Statistical Analysis. The results were expressed as MeanSEM values. Differences in mean values between experimental data were analyzed, where necessary, by unpaired t test. A probability values less than 0.05 (p <0.05) was defined to be significant. RESULTS AND DISCUSSION The in vitro determination of percentage of protein binding of Naproxen Sodium and its 1:1 mixture of Ranitidine Hydrochloride has been conducted at room temperature and pH 7.4.The highest percentage of Naproxen Sodium binding with bovine serum albumin was found to be 98%. The percentage of protein binding of Naproxen Sodium has been found to decrease with increase concentration of Naproxen Sodium which attained a steady plateau state when the free drug concentration was around 5x10-5 m. This might be the saturation zone for the protein binding of Naproxen Sodium (Fig 3a). On the other hand, Ranitidine Hydrochloride was found significantly decrease (MeanSEM),* significant because p=0.01) the percentage of protein binding of Naproxen Sodium at the concentration range used but the attainment of steady plateau condition remained unchanged (Fig 3b).
100 98 96 % protein binding 94 92 90 88 86 84 0 5 10 15 conc. X 10-5 M Fig. 3. The protein binding of naproxen sodium and napr. raniti. Hydro. At pH 7.4 and temp. (3701 )C. The data are show n as mean + SEM.*indicates sig. change (p = 0.01) in protein binding

In other words, the complex formation of Ranitidine Hydrochloride with Naproxen Sodium can decrease unbound fraction in plasma at high conc.. This might the therapeutic response, toxic effect and pharmaceutical properties of Naproxen Sodium. These findings are similar with some findings. Amran 1999, 1998. It was also observed that all forms of the drugs (Naproxen Sodium and its 1:1 mixture with Ranitidine Hydrochloride) attained a steady plateau state at higher conc. This indicated the saturation of the sites of protein by the drug or its complex as was observed by other investigators. Arman,1999, Hossain 1991.The conc. of drug or the drug mixture were kept within (1 x 10-5 M to 9 x10-5M), while that of bovine serum albumin was kept at (1 x 10-5 )M so that protein binding sites were sufficient to bind all the drug molecules. It was found that nearly mole to mole binding at conc.(5 x 10-5 )M were 95% and 91% for Naproxen Sodium alone and Naproxen Sodium - Ranitidine Hydrochloride systems respectively. At this point the binding phenomenon reached nearly maximum values what is called saturation zone of binding. At low concs.(1 x 10-5 )M to 5 x10-5M) the percent of protein binding increased almost nearly (for complex) and at high conc. (6 x 10-5 to 8 x10-5 ) M the percent of protein binding did not increase sharply. It can therefore be inferred that complexation of Naproxen Sodium with the drug being studied can displace the drug from plasma. Since the drug displaced from the plasma protein will redistribute into its full potential volume of distribution, the conc. of free drug in plasma and tissues after redistribution might might be increased greatly and this might change the pharmacokinetics properties of the drug and thereby may affect its pharmacological and toxic effects. Arman,1999. It was found that there are at least two binding sites for naproxen sodium and its complexes with Ranitidine hydrochloride (fig 4 to 5 and table 1). The number of binding sites (n1) in class I was 31.86 and 32.15 for naproxen sodium and naproxen sodium -ranitidine hydrochloride. The value of affinity constant (k1) with this class have been found to be 17.1 and 0.29 respectively.
Table 1. The number of protein binding sites and the associated affinity constants of naproxen sodium and 1:1mixture with naproxen sodium and ranitidine hydrochloride.

Systems naproxen sodium naproxen sodium ranitidine hydrochloride.

Class I binding sites n1k1 k1 n1 106.88 17.1 31.86 9.24 0.29 32.15

Class II binding sites n2 k2 k2 n2 8.94 0.24 37.25 8.94 0.25 36.38

The number of binding sites (n1) in class II was 37.25 and 36.38 for naproxen sodium and naproxen sodium -ranitidine hydrochloride. The value of affinity constant (k1) with this class have been found to be 0.24 and 0.25 respectively.

150

100 r/Cx naproxen sodium

50

0 1 3 5 7 9 11 13 r Fig 4. The scatchard plot for protein binding capacity of naproxen sodium

15

10

r/cx

Naproxen - Raniti. hydrochloride 5

0 0 3 6 9 12 15 r Fig 5. The scatchard plot for protein binding capacity of naproxen-raniti.hydrochloride

It is obvious, from this result that the values of affinity constants for naproxen sodium is higher than that of its 1:1 complexes of naproxen sodium with Ranitidine hydrochloride at physiological temperature and pH conditions cause a decrease in values of affinity constants or binding forces .Due to this decrease in affinity of the drug to plasma protein binding, the volume of distribution of naproxen sodium may increase because affinity of a drug for protein binding is a limiting factor for the distribution of the drug Tillement, 1974. The values of affinity constants in class II binding sites revealed the same results,i.e.,increase in volume of distribution of naproxen sodium. As a result the intake of naproxen sodium as Ranitidine hydrochloride9concurent therapy) might increase both the hepatic and renal clearance of the drug as well as it may increase the half life of the drug. Hansten, 1989. CONCLUSION Poly pharmacy that is, prescribing many drugs at a time is a common practice in case of patient undergoing a major operation, hospitalized patient, and also injiatric patient.

Sometime co administration of more than two different classes of drugs may ensure effect that are neither safe nor effective but sometimes may be beneficial. It was observed that naproxen sodium and Ranitidine hydrochloride lowered the affinity of protein binding of naproxen sodium, and hence an increase in volume of distribution of naproxen sodium might be occurred. Therefore it can be inferred that care and monitoring should be practiced during administration of naproxen sodiumRanitidine hydrochloride complexes. REFERENCES 1. Pocock, Nicola (2006). "MHRA approves availability of OTC naproxen (Feminax Ultra)". NHS Press Release.. 2.http://www.bayer.ca/files/Aleve%20Release.July14.FINAL_.pdf 3. Louis st. Histamine H2 antagonists. In: Drug facts and comparisons. MO: Wolters Kluwer Co., 2002, 1192-1197. 4. Gramatt T., E. El Desoky, U. Klotz. Site-dependent small intestinal absorption of ranitidine Eur. J. Clin. Pharmacol, 1994, 46: 253-259. 5 Lauriten K.Clinical pharmacokinetics of drugs used in the treatment of gastrointestinal diseases. Clin. Pharmacokinet.,1990, 19: 94-125. 6. Grant. S Ranitidine: an updated review of its pharmacodynamicand pharmacokinetic properties and therapeutic usein peptic ulcer and other allied diseases. Drugs, 1989, 37: 801-887. 7. Singlass,E. 1987. protein binding of drugs, F.Hoffman La Roche & cio. Ltd.,Basic, Switzerland , 2nd edition, 17-32. 8. Perrin, D.D. and Boyd, D. 1974. Buffers for pH and metal ion control , Science papers back, New York, 44-64 9. Bates, 1964.Determination of pH Thoery and practice, Wily, New York, 50-67 10. Goldstein, A., Aronow, L. and Kalman, S.M. 1974, Priciple of drug action-the basis of pharmacology, 2nd edition, John wily and sons, New York, 47-52. 11. Scachard, G. 1949. The attraction of proteins for small molecules and ions, Ann NY Acad Sci, 660-673. 12. Arman, M.S., Zaman, M.R. and Hossain, M.A. 1999. Study protein binding of diltiazem in presence of nifedipine and kitotifen fumerate, Rajshahi Univ. Stud., PartB, 27, 45-54. 13. Arman, M.S., and Hossain, M.A. 1998. Study of protein binding of diltiazem in presence ofomeprazole and ranitidine, Z. Bio Sci, 6, 115-121. 14. Arman, M.S., and Hossain, M.A. 1999. Influence of ferrous sulphate on the protein binding of diltiazem in vitro, J. Bangladesh Acad Sci, 23(2), 125-131. 15. Hossain, M.A. and Kamal, A.K.M. 1991. protein binding of propanol in presence of theophyline, M pharm Thesis, Dept of pharmacy, University of Dhaka, Dhaka 16.Tillement. J.P., Zini, R. and Vassent, G. 1974. Binding of certain a15. cidic drugs to human albumin: theorical practical estimation of fundamental parameters, Euro J Clin Pharmacol. 7, 303-313. 17. Hansten, P.D. and Horn, J.R. 1989. Drug interactions-clinical significance of drug drug interactions, 6th edition, Philadelphia, 22-23.