CHAPTER 4 EXPERIMENTAL INVESTIGATIONS. L-histidine, L-alanine, L-tyrosine and glycine. (ii) Synthesis of Mefenamic acid with amino acids such as

Transcription

1 75 CHAPTER 4 EXPERIMENTAL INVESTIGATINS 4.1 METHDLGY The detailed methodology adopted for the present research is furnished as follows: 1. Synthesis of prodrugs (i) Synthesis of Aceclofenac with amino acids such as L-histidine, L-alanine, L-tyrosine and glycine (ii) Synthesis of Mefenamic acid with amino acids such as L-histidine, L-tryptophan, L-tyrosine and glycine (iii) Synthesis of Dexibuprofen with amino acids such as L-tryptophan, L-phenylalanine, glycine and L-tyrosine 2. Characterization of drugs and prodrugs (i) Thin layer chromatography (ii) Melting point determination (iii) Molecular weight determination (iv) Solubility studies (v) Elemental analysis (vi) Spectral analysis by UV, IR, 1 HNMR, 13 C NMR and mass spectroscopy (vii) Partition coefficient determination in octanol/acidic buffer (ph 1.2) and in octanol/phosphate buffer (ph 7.4) (viii) Protein binding studies in phosphate buffer saline (ph 7.4) 3. Hydrolysis study of synthesized prodrugs (i) in simulated gastric fluid (ph 1.2)

2 76 (ii) in simulated intestinal fluid (ph 7.4) (iii) in rat fecal matter (ph 7.4) (iv) in 80 % human plasma (ph 7.4) 4. Pharmacological screening of drugs and prodrugs (i) Anti inflammatory activity (ii) Analgesic activity (iii) Ulcerogenic activity (iv) Histopathological studies 4.2 MATERIALS AND INSTRUMENTS The amino acids were obtained from M/s Hi-Media Ltd., Mumbai, India and drugs aceclofenac, mefenamic acid and dexibuprofen were obtained as gift sample from Alkem Laboratories, Mumbai, India. ther reagents and solvents were of analytical grade. The melting points were recorded by melting point determination apparatus (Sigma Instrument, Mumbai, India) and are uncorrected. The elemental analysis was performed in CDRI, Lucknow using Carlo-Erba Model 1108 Analyzer (Italy). The characterization of drugs and anticipated structures of synthesized prodrugs were confirmed by various spectral analyses such as UV, IR, 1 H NMR, 13 C NMR and mass spectroscopy. The infrared spectra were recorded on IR spectrophotometer (Shimadzu 8201 PC) in KBr phase, Indian Institute of Chemical Biology, Kolkata. 1 H NMR and 13 C NMR spectra were recorded in NMR spectrophotometer (Bruker DRX 300, USA) in Indian Institute of Chemical Biology, Kolkata and Analytical R and D, Arabindo Lab, Hyderabad. The samples were made in D2 and

3 77 DMS-D6 using tetra methyl silane (TMS) as internal standard and are given in δ (ppm) scale. The standard abbreviations s, d, t, q, m, dd, dt and brs refers to singlet, doublet, triplet, quartet, multiplet, doublet of a doublet, doublet of a triplet and broad singlet respectively. Mass spectra were recorded in mass spectrophotometer (Jeol SX-102 (FAB), Japan), Indian Institute of Chemical Biology, Kolkata. The hydrolysis data and drug content determination were performed by UV spectrophotometer. The solution of prodrug was prepared in a concentration of 10 μg/ml and the absorbance was measured in the UV spectrophotometer (Shimadzu, Japan) at a range of nm. Determination of physicochemical properties and the pharmacological evaluation were carried out in Department of Pharmaceutical Chemistry and Department of Pharmacology, Sree Vidyanikethan College of Pharmacy, Tirupati. The histopathological studies were carried out in Department of Pathology, Sri Venkateswara Veterinary University, Tirupati, India. 4.3 SYNTHESIS F PRDRUGS The synthesis of amide prodrugs was carried out in three steps Step 1: Synthesis of acid chlorides of selected drugs Step 2: Synthesis of amino acid ester hydrochlorides Step 3: Acylation of amino acid ester with acid chloride using Schotten Baumann technique

4 Synthesis of Amide Prodrugs of Aceclofenac Aceclofenac is 2-[(2, 6-dichloro phenyl amino) phenyl] acetyloxy acetic acid. The amino acids such as L-histidine, L-alanine, L-tyrosine and glycine were selected to synthesize amide prodrugs of AC. The synthesis was carried out in the following steps: Step 1: Synthesis of aceclofenac acid chloride g (0.05 M) of aceclofenac was dissolved in minimum amount of chloroform and freshly distilled thionyl chloride (0.05 M, 6 ml) was added slowly to it. The mixture was refluxed for 15 h at 60-70ºC with continuous stirring on magnetic stirrer. The viscous liquid was immediately poured on petridish and was vacuum dried to give yellow coloured crude aceclofenac acid chloride. Step 2: Synthesis of methyl ester hydrochlorides of amino acids Freshly distilled (0.05 M, 6 ml) of thionyl chloride was slowly added to methanol (100 ml) with cooling and amino acid (0.1 M) was added to it. The quantity of amino acids used for conjugation with aceclofenac is shown in Table 4.1. The mixture was refluxed for 6-8 h at 60-70ºC with continuous stirring on magnetic stirrer. Excess thionyl chloride and solvent was removed under reduced pressure giving crude amino acid methyl ester hydrochloride. It was treated with 20 ml portion of cold ether at 0ºC until the excess of dimethyl sulphate was removed. The resulting solid product was collected and dried under vacuum. It was recrystallized from hot methanol by slow addition of ml ether followed by cooling at 0ºC. The crystals were collected on next day and washed twice with ether-methanol mixture (5:1) followed by pure ether

5 79 and dried under vacuum to give pure amino acid methyl ester hydrochloride. Table 4.1 Quantity of amino acid used for conjugation with aceclofenac Sl. No. Amino acid Strength (M) Quantity (g/1000 ml) 1 L-histidine L-alanine L-tyrosine Glycine Step 3: Acylation of amino acid ester with aceclofenac acid chloride The step 3 involved the acylation of amino acid ester with aceclofenac acid chloride and is carried out using Schotten Baumann technique. Ice cold, aqueous sodium hydroxide solution (5 %) was taken in 250 ml beaker and methyl ester of amino acid (0.05 M) was added to it. The quantity of various methyl ester of amino acids used for conjugation with amino acid are given in Table 4.2. The reaction mixture was mechanically stirred for 30 min at room temperature, after which the beaker was transferred to an ice bath kept on mechanical stirrer, maintaining the temperature at 10ºC. Aceclofenac acid chloride (0.01 M, 3.72 g) was added in small portions with continuous stirring for 7-8 h. The solid that separated out was filtered using vacuum pump and dried. The crude conjugate was recrystallized from methanol to obtain amide prodrugs of aceclofenac.

6 80 Table 4.2 Quantity of methyl ester of amino acid used for conjugation with aceclofenac Strength Quantity Sl. No. Amino acid ester (M) (g/1000 ml) 1 Methyl ester of histidine Methyl ester of alanine Methyl ester of tyrosine Methyl ester of glycine The aceclofenac acid chloride (1) was conjugated with methyl ester of histidine (2) to synthesize histidine conjugated aceclofenac (AC1). The schematic representation of synthesis of AC1 is shown in Scheme 4.1. The same procedure was followed to synthesize alanine conjugated aceclofenac (AC2), tyrosine conjugated aceclofenac (AC3) and glycine conjugated aceclofenac (AC4).

7 81 Step I: Synthesis of aceclof enac acid chloride C CH 2 CH NH + SCl 2 Cl Cl Cl NH C Cl CH 2 CCl 1 Step II: Synthesis of methyl ester of histidine NH 2 CH SCl 2 2 CH CH CH 2 NH 2 Cl CH CCH 3 HN N CH 3 H HN N 2 Step III: Acylation of aceclof enac acid chloride w ith methyl ester of histidine C CH 2 CCl NH 2 Cl Cl NH 1 Cl + HN 2 N CH 2 CH NH C CCH 3 CH 2 C NaH H 2 C 2-8 C C H N NH CH N CCH 3 Cl Cl AC 1 Scheme 4.1 Synthesis of histidine conjugated aceclofenac Synthesis of Amide Prodrugs of Mefenamic Acid Mefenamic acid is 2-[(2, 3-dimethyl phenyl) amino] benzoic acid. The amino acids such as L-histidine, L-tryptophan, L-tyrosine and glycine are chosen for conjugation with mefenamic acid. The steps involved in the synthesis of amide prodrugs of mefenamic acid are explained as follows.

8 82 Step 1: Synthesis of mefenamic acid chloride 12 g (0.05 M) of mefenamic acid was dissolved in minimum amount of chloroform and freshly distilled thionyl chloride (0.05 M, 6 ml) was added slowly to it. The mixture was refluxed for 15 h at 60-70ºC with continuous stirring on magnetic stirrer. The viscous liquid was immediately poured on petridish and was vacuum dried to give yellow coloured crude mefenamic acid chloride. Step 2: Synthesis of methyl ester hydrochlorides of amino acids The synthesis method is the same as in step 2 of section The quantity of amino acids used for conjugation with mefenamic acid is shown in Table 4.3. Table 4.3 Quantity of amino acid used for conjugation with mefenamic acid Sl. No. Amino acid Strength (M) Quantity (g/1000 ml) 1 L-histidine L-tryptophan L-tyrosine Glycine Step 3: Acylation of amino acid ester with mefenamic acid chloride The procedure is the same as explained in step 3 of section The quantity of various methyl ester of amino acids used for conjugation with mefenamic acid chloride (0.01 M, 2.60 g) are given in Table 4.4.

9 83 Table 4.4 Quantity of methyl ester of amino acid used for conjugation with mefenamic acid Strength Quantity Sl. No. Amino acid ester (M) (g/1000 ml) 1 Methyl ester of histidine Methyl ester of tryptophan Methyl ester of tyrosine Methyl ester of glycine The mefenamic acid chloride (1) was conjugated with methyl ester of histidine (2) to synthesize histidine conjugated mefenamic acid (MA1). The schematic representation of synthesis of MA1 is shown in Scheme 4.2. The same procedure was followed to synthesize tryptophan conjugated mefenamic acid (MA2), tyrosine conjugated mefenamic acid (MA3) and glycine conjugated mefenamic acid (MA4).

10 84 Step I: Synthesis of mefenamic acid chloride CH CH 3 CCl CH 3 NH CH 3 NH CH 3 + SCl 2 + CHCl 3 1 Step II: Snthesis of methyl ester of histidine CH 2 CH CH SCl2 NH 2 CH NH 3 Cl CH CCH 3 HN N CH 3 H HN N 2 Step III: Acylation of mef enamic acid chloride w ith methyl ester of histidine CCl CH 3 NH CH 3 + CH 2 NH 2 Cl CH CCH 3 HN N 1 2 CH 3 NH H 3 C NH H 2 C C CH N CCH 3 NaH 2-8 C H N 0 MA 1 Scheme 4.2 Synthesis of histidine conjugated mefenamic acid Synthesis of Amide Prodrugs of Dexibuprofen Dexibuprofen is (2S)-2-[4-(2-methylpropyl) phenyl] propanoic acid. The amino acids chosen for conjugation with dexibuprofen are L-tryptophan, L-phenylalanine, glycine and L-tyrosine. Following steps were involved in the synthesis of amide prodrugs of dexibuprofen:

11 85 Step 1: Synthesis of dexibuprofen acid chloride Dexibuprofen of gm (0.05 M) was dissolved in minimum amount of chloroform and freshly distilled thionyl chloride (0.05 M, 6 ml) was added slowly to it. The mixture was refluxed at 60-70ºC with continuous stirring on magnetic stirrer. The viscous liquid was immediately poured on petridish and was vacuum dried to give yellow coloured crude dexibuprofen acid chloride. Step 2: Synthesis of methyl ester hydrochlorides of amino acids. The procedure is same as that of step 2 of section The quantity of amino acids used for conjugation with dexibuprofen is shown in Table 4.5. Table 4.5 Quantity of amino acid used for conjugation with dexibuprofen Sl. No. Amino acid Strength (M) Quantity (g/1000 ml) 1 L-tryptophan L-phenylalanine Glycine L-tyrosine Step 3: Acylation of amino acid ester with dexibuprofen acid chloride The procedure was the same as that of step 3 of section The quantity of various methyl ester of amino acids used for conjugation with dexibuprofen acid chloride (0.01 M, 2.23 g) is given in Table 4.6.

12 86 Table 4.6 Quantity of methyl ester of amino acid used for conjugation with dexibuprofen Sl. No. Amino acid ester Strength (M) Quantity (g/1000 ml) 1 Methyl ester of tryptophan Methyl ester of phenylalanine Methyl ester of glycine Methyl ester of tyrosine The dexibuprofen acid chloride (1) was conjugated with methyl ester of tryptophan (2) to synthesize tryptophan conjugated dexibuprofen (Dex 1). The schematic representation of synthesis of Dex 1 is shown in Scheme 4.3. The same procedure was followed to synthesize phenylalanine conjugated dexibuprofen (Dex 2), glycine conjugated dexibuprofen (Dex 3) and tyrosine conjugated dexibuprofen (Dex 4).

13 87 Step I: Synthesis of dexibuprof en acid chloride H 3 C CH 3 + SCl 2 H H 3 C CH 3 Cl CH 3 1 CH 3 Step II: Synthesis of methyl ester of L-tryptophan N H CH 2 CH NH 2 CH SCl 2 CH 3 H N H 2 CH 2 CH NH 3 + Cl CCH 3 Step III: Acylation of dexibuprof en acid chloride w ith methyl ester of tryptophan H 3 C CH 3 Cl + CH C H 3 CH 3 N H CH 2 CH NH 3 + Cl CH 3 CCH 3 NH Dex 1 CH CCH 3 NaH Scheme 4.3 Synthesis of tryptophan conjugated dexibuprofen 4.4 CHARACTERIZATIN F DRUGS AND PRDRUGS CH C A study of the physicochemical properties of drug molecule is a prerequisite for product formulation and often leads to a better understanding of the inter relationship between molecular structure and drug action 186. In general, in order for a drug to exert its biologic effect, it must be soluble in and transported by the body fluids, traverse the required biologic membrane barriers, escape widespread distribution to unwanted areas, penetrate in adequate concentration to the sites of action and interact in a specific fashion causing an alteration of cellular function. All these events depend upon the physicochemical and electronic parameters of the drug. Therefore it is N H

14 88 essential to study the physicochemical parameters of the synthesized derivatives to understand its biopharmaceutical behaviour. Various physicochemical properties viz., solubility, dissolution, partition coefficient, ionization, hydrogen bonding, chelation, redox potential, surface-activity and drug receptor interactions are useful in drug action. Prodrugs are reversible derivative of various drugs of pharmaceutical importance which get converted to the active therapeutic agent either by the ph of the buffer system of the GIT fluid or by the enzyme system of the body, Therefore, inspite of the physicochemical consideration, it is equally essential to understand the mechanism of prodrug to drug conversion because it is the drug that would produce biologic response. Whether the prodrug will act in the designed manner or not, can only be confirmed by the study of the kinetics and mechanism of hydrolysis of synthesized compound in GIT fluids, colonic fluid and in plasma. The various physicochemical properties such as purity, melting point, molecular weight, solubility, elemental and spectral analyses, partition coefficient and protein binding of AC, MA and Dex and their prodrugs were determined. The procedures for the same are explained in the following section 187, Thin Layer Chromatography 189 The purity of aceclofenac, mefenamic acid and their prodrugs were monitored by thin layer chromatography (TLC) on pre coated silica G plates using iodine vapour as detecting agent. The solvent system used for AC was toluene: 2-propanol: ammonia (4:4:0.4) and that for

15 89 MA was acetone: chloroform: acetic acid (3:2:1). The purity of dexibuprofen and its prodrugs were checked by TLC on pre-coated silica GF252 plates using iodine vapor as detecting agent with n-hexane: ethyl acetate: glacial acetic acid (7.5:2.5:0.5) as mobile phase Melting Point Determination Melting point of drugs and prodrugs were determined by capillary fusion method using melting point determination apparatus (Sigma Instrument, Mumbai, India) and are uncorrected Molecular Weight Determination 190 Mass spectrum of drugs and prodrugs were recorded to prove the exact mass or M/Z ratio, with field dissorption technique in magnetic sector of mass spectrometer, with an applied voltage of 70 ev for the fragmentation of molecular ions. Field dissorption technique in mass spectroscopy was specially used for evaluation of peptidal type drugs. It was carried out in mass spectrophotometer (Jeol SX-102 (FAB), Japan), Indian Institute of Chemical Biology, Kolkata Solubility Studies 191 Solubility can be defined quantitatively as the concentration of the solute in a saturated solution at a certain temperature. Approximately 5 mg of drug was dissolved in 5 ml of each solvent at 37 ± 1ºC in glass test tubes. The solvents used were 0.1 N NaH, 0.1 N HCl, 0.1 N KH, methanol, ether, chloroform, acetone, and water. Test tubes were gently shaken and solubility was observed. In case of any observed insoluble fractions, the known amount of solvent was further

16 90 added to ascertain the solubility of the compound. The same procedure was repeated for AC, MA and Dex as well as their prodrugs Elemental Analysis The elemental analysis of prodrugs of AC, MA and Dex were performed in Central Drug Research Institute, Lucknow, India, using Carlo-Erba Model 1108 Analyzer. It was carried out to find the percentage of C, H and N in the prodrugs Spectral Analysis The characterization of drugs and anticipated structures of their prodrugs were confirmed by various spectral analyses using IR, 1 H NMR, 13 C NMR and mass spectroscopy 192, 193. The infrared spectra were recorded on IR spectrophotometer (Shimadzu 8201 PC) in KBr phase, Indian Institute of Chemical Biology (IICB), Kolkata. 1 H NMR and 13 C NMR spectra were recorded in NMR spectrophotometer (Bruker DRX 300, USA) in IICB, Kolkata and Analytical R and D, Arabindo Lab, Hyderabad. Mass spectra were recorded in mass spectrophotometer (Jeol SX-102 (FAB), Japan), IICB, Kolkata UV Absorption Studies 194 The solution of AC, MA and Dex and their prodrugs were prepared in a concentration of 10 to 20 μg/ml and the absorbance was measured with the Shimadzu UV spectrophotometer at a range of nm Preparation of Calibration Curve of Aceclofenac (a) In Methanol A standard solution containing 1 mg/ml of aceclofenac was prepared in methanol by dissolving 50 mg of pure aceclofenac in 50 ml

17 91 of methanol. From this solution, working standard solutions of concentrations 0 to 20 µg/ml of aceclofenac was prepared by dilution with methanol. The absorbance of the solutions was measured at 275 nm against reagent blank. Calibration curve was prepared. (b) In Various Simulated Fluids The calibration curve of aceclofenac was prepared using phosphate buffer (ph 7.4), SGF (ph 1.2), SIF (ph 7.4) and 80 % human plasma (ph 7.4). An accurately weighed amount of aceclofenac equivalent to 100 mg was dissolved in small volume of methanol, in a 100 ml volumetric flask and the volume was adjusted to 100 ml with phosphate buffer (ph 7.4) and further dilutions were made with 7.4 ph phosphate buffer. The same procedure was repeated using SGF (ph 1.2), SIF (ph 7.4) and 80 % human plasma (ph 7.4). A series of standard solution containing 2 to 20 µg/ml of aceclofenac were prepared and absorbance was measured at 275 nm against reagent blank. All spectral absorbance measurements were made on Shimadzu UV spectrophotometer. (c) Determination of Aceclofenac Content An accurately weighed amount of aceclofenac was dissolved in small volume of methanol and further diluted with methanol. The content of aceclofenac was determined spectrophotometrically at 275 nm using Shimadzu UV spectrophotometer.

18 Preparation of Calibration Curve of Mefenamic Acid (a) In Methanol A standard solution containing 1 mg/ml of mefenamic acid was prepared in methanol by dissolving 50 mg of pure mefenamic acid in 50 ml of methanol. From this solution, working standard solutions of concentrations 0 to 20 µg/ml of mefenamic acid was prepared by dilution with methanol. The absorbance of the solutions was measured at 230 nm against reagent blank. Calibration curve was prepared. (b) In Various Simulated Fluids The calibration curve of mefenamic acid was prepared using phosphate buffer (ph 7.4), SGF (ph 1.2), SIF (ph 7.4) and 80 % human plasma (ph 7.4). Accurately weighed 100 mg of mefenamic acid in a 100 ml volumetric flask was dissolved in SGF and the volume was made upto 100 ml with SGF (ph 1.2) to get a final concentration of 5 g/ml. The aliquots of 0.5, 1.0, 1.5, 2.0 and 2.5 ml were diluted to 10 ml with SGF in a series of 10 ml volumetric flask. Thus the solutions with a concentration range of 5 μg/ml to 25 μg/ml were prepared. The same procedure was repeated using phosphate buffer (ph 7.4), SIF (ph 7.4) and 80 % human plasma (ph 7.4). Absorbance of each solution was measured against blank at 230 nm by UV spectrophotometer. The drug solutions obeyed Beer-Lambert s law in the concentration range 5 μg/ml to 25 μg/ml.

19 93 (c) Determination of Mefenamic Acid Content An accurately weighed amount of mefenamic acid was dissolved in small volume of methanol and further diluted with methanol. The content of mefenamic acid was determined spectrophotometrically at 230 nm using Shimadzu UV spectrophotometer Preparation of Calibration Curve of Dexibuprofen (a) In Methanol A standard solution containing 1 mg/ml of dexibuprofen was prepared in methanol by dissolving 50 mg of pure dexibuprofen in 50 ml of methanol. From this solution, working standard solutions of concentrations 0 to 20 µg/ml of dexibuprofen was prepared by dilution with methanol. The absorbance of the solutions was measured at 223 nm against reagent blank. Calibration curve was prepared. (b) In Various Simulated Fluids The calibration curve of dexibuprofen was prepared using phosphate buffer (ph 7.4), SGF (ph 1.2), SIF (ph 7.4) and 80 % human plasma (ph 7.4). Accurately weighed 100 mg of dexibuprofen in a 100 ml volumetric flask was dissolved in SGF (ph 1.2) and the volume was made upto 100 ml with SGF to get a final concentration of 5 g/ml. The aliquotes of 0.5, 1.0, 1.5, 2.0 and 2.5 ml were diluted to 10 ml with SGF in a series of 10 ml volumetric flask. Thus the solutions with a concentration range of 5 μg/ml to 25 μg/ml were prepared. The same procedure was repeated using phosphate buffer (ph 7.4), SIF (ph 7.4) and 80 % human plasma (ph 7.4). Absorbance of each solution was measured against blank at 223 nm by UV spectrophotometer. The drug

20 94 solutions obeyed Beer-Lambert s law in the concentration range 5 μg/ml to 25 μg/ml. (c) Determination of Dexibuprofen Content An accurately weighed amount of dexibuprofen was dissolved in small volume of methanol and further diluted with methanol. The content of dexibuprofen was determined spectrophotometrically at 223 nm using Shimadzu UV spectrophotometer Determination of Partition Coefficient Partition coefficient is defined as the ratio of distribution of the solute between two immiscible solvents, basically between aqueous and non aqueous phases. It is indicative of drug s lipophilicity and its ability to cross the biological membrane. If an excess of compound is added to a mixture of two immiscible liquids it will distribute itself in between the two immiscible phases so that each becomes saturated. If the substance is added to the mixture of immiscible solvents in an amount insufficient to saturate the solutions, it will become distributed between the two layers in a definite concentration ratio. If C1 and C2 are the equilibrium concentrations of the substance in solvent 1 and solvent 2, the equilibrium expressions becomes K=C1/C2 in which the equilibrium constant K is known as the distribution coefficient or partition coefficient. Knowledge of partition coefficient is important, as it is useful in several areas of pharmaceutical interest including preservation of oil-water systems, drug action at non specific sites, absorption and distribution of drugs

21 95 throughout the body and screening for some biological properties The partition coefficient of drugs and prodrugs were determined by shake flask method between n-octanol saturated with acidic buffer (ph 1.2) and n-octanol saturated with phosphate buffer (ph 7.4). A 20 mg of drug was weighed and dissolved in 20 ml octanol. This solution was divided in two parts and to each part was added l0 ml acidic buffer (ph 1.2) and phosphate buffer (ph 7.4) separately. The contents were thoroughly shaken for 24 h at room temperature followed by its transfer to a separating funnel. The octanol layer was dried under high vacuum and the residue obtained was again dissolved in methanol (l0 ml). 2 ml of this solution was further diluted to 100 ml with methanol. From this solution an aliquot of 25 ml was withdrawn and was mixed with 4.5 ml solution of acidic buffer (ph 1.2) or phosphate buffer (ph 7.4). Volume was finally made to l00 ml by addition of methanol. The resulting solution of each drug and prodrugs of AC, MA and Dex was analyzed and the absorbance of the solutions was measured spectrophotometrically at 275 nm, 230 nm and 223 nm respectively Protein Binding Studies Protein binding studies are important due to the interaction between proteins and drugs in various biochemical and pharmacological processes. Most drugs are bound to some extent by plasma proteins such as albumin, globulin, glycoprotein or lipoproteins. Such type of binding can influence the disposition of drug in the body in terms of distribution and elimination as well as affect the

22 96 pharmacodynamic properties of a drug 197. Also, the drug protein interaction in the blood stream hinders the action, metabolism and excretion of drug 198. It has been reported that the unbound drug exerts its pharmacological action, while bound drug may provide the reservoir, which is converted to the active free form as needed to maintain a constant blood level over extended period of time. A number of methods are used to determine the amount of drug bound to protein. They are ultra filtration, equilibrium dialysis, electrophoresis, nuclear magnetic resonance and gel filtration. In the present study, protein binding of drugs and prodrugs of AC, MA and Dex were carried out by equilibrium dialysis method in a phosphate buffered saline (PBS) (ph 7.4). A solution of synthesized prodrug (10 mg/ml) was made in phosphate buffered saline (PBS, ph 7.4). A 100 ml of this solution was taken in a beaker. The cellophane membrane (molecular weight cut off in the range of Da obtained from Hi-Media, India) was first washed with distilled water and then with buffer solution (ph 7.4). The opening end of dialysis tube was tied; the dialysis tube containing (6 %) egg albumin was dipped into the drug solution and covered. The whole assembly was placed on a magnetic stirrer and switched at low revolutions per minute. The temperature was maintained at 37 ± 0.5ºC. After every 1 h, 1 ml of the PBS containing drug solution was replaced with fresh 1 ml of PBS. Withdrawn sample was diluted further with 1 ml phosphate buffer and the concentration of prodrug was estimated using spectrophotometer.

23 97 Preparation of phosphate buffer (ph 7.4): l.38 g of disodium hydrogen phosphate, 0.19 g of potassium dihydrogen phosphate and 8 g of sodium chloride were dissolved in sufficient water to produce l000 ml. ph was adjusted to 7.4 immediately before use. 4.5 HYDRLYSIS STUDY F SYNTHESIZED PRDRUGS Prodrug, bio-reversible derivative of drug, has to elicit the pharmacological activity but eliminate the undesirable side effects of the drug. Therefore it is utmost necessary for a prodrug to hydrolyze rapidly either due to ph change or due to enzymes present in the body fluids. Study of kinetics and its mechanism are performed to ascertain whether the prodrug will act in the designed manner or not In vitro Hydrolysis Study In vitro hydrolysis of compounds in simulated fluids will help to predict the amount of drug that would be available at the site of action. It involves two types of mechanism, acidic and basic, which are explained as follows 199, Acid Catalyzed Hydrolysis R C NHR' Fast + H R C NH 2 R + H 2 Slow H 2 + Fast R C H 2 + R'NH 2 R C NH 2 R' + HH + R C + R'NH 3 + Fig 4.4 Mechanism of acid catalyzed hydrolysis of amides

24 98 Acid hydrolysis of amides requires that the substituent should exert only weak polar effects, but when suitably situated they should exert strong steric effects (Fig 4.4) Base Catalyzed Hydrolysis Nucleophillic attack by the hydroxide ion occurs in base catalyzed hydrolysis and is shown in Fig R C NHR' + H Slow R C NHR H Fast R C + R'NH 2 R C H+ R'NH Fig 4.5 Mechanism of base catalyzed hydrolysis of amides Hydrolysis Rate Determination of Prodrugs The hydrolytic studies of prodrugs of AC, MA and Dex were carried out in simulated gastric fluid (SGF) at ph 1.2, simulated intestinal fluid (SIF) at ph 7.4, rat fecal matter at ph 7.4 and 80 % human plasma at ph 7.4 maintained at a temperature of 37 ± 0.5ºC In Simulated Gastric Fluid (ph 1.2) Preparation of SGF g of sodium chloride and 3.2 g of pepsin were dissolved in 7 ml of hydrochloric acid and sufficient water was added to make up to 1000 ml. ph was adjusted to 1.2. Procedure: In vitro hydrolysis studies of synthesized prodrugs were carried out in SGF at ph 1.2 (USP 1970). A solution of 10 mg of prodrug was prepared in 90 ml of SGF (ph 1.2). An aliquot of 15 ml of

25 99 this solution was withdrawn repeatedly and kept in test tubes maintained at 37 ± 0.5ºC. At a definite interval of time (0.5, 1, 2 up to 8 h), an aliquot was withdrawn from different test tubes and was transferred to micro centrifuge tubes followed by addition of methanol to make up the volume. The tubes were placed in freezing mixture in order to arrest further hydrolysis, followed by vortexing at high speed for 5 min. After vortexing, the tubes were centrifuged at high speed (3000 rpm) for 5 min. A 5 ml of clear supernatant obtained from each tube was measured on UV spectrophotometer for the amount of free drug released after the hydrolysis of AC, MA and Dex in SGF at 275, 230 and 223 nm respectively In Simulated Intestinal Fluid (ph 7.4) Preparation of SIF (ph 7.4) g of monobasic potassium phosphate was dissolved in 250 ml of water, followed by the addition of 190 ml of 0.2 N sodium hydroxide and 400 ml of water. A 10 g of pancreatin was added and mixed. Resultant solution was adjusted to ph 7.4 with 0.2 N sodium hydroxide. Water was added to make up the volume to 1000 ml. Procedure: Same as the procedure explained in section In Rat Fecal Matter (ph 7.4) 202 Male albino rats weighing g and maintained on a normal diet were used for the study. Six rats were asphyxiated using carbon dioxide. The prodrug was dissolved in phosphate buffer (ph 7.4) so that final concentration of solution was 250 µg/ml. Fresh fecal material of rats were weighed (about 1 g) and placed in different sets of test tubes.

26 100 To each test tube, 1 ml of the prodrug solution was added and diluted to 5 ml with phosphate buffer (50 µg/ml). The sets of test tubes were incubated at 37 ± 0.5ºC for different interval of time (0.5, 1, 2 up to 8 h). For analysis, the free drug was extracted with 5 ml of methanol and estimated on UV spectrophotometer In 80 % Human Plasma (ph 7.4) 203, 204 A solution of 10 mg of prodrug was prepared in methanol (2 ml) and was added to 98 ml of 80 % human plasma (ph 7.4 prepared by mixing 80 portion of plasma and 20 portion of phosphate buffer at ph 7.4). An aliquot of 15 ml of this solution was withdrawn and kept in test tubes maintained at 37 ± 0.5ºC. At definite interval of time (0-8 h) an aliquot of 22 ml was withdrawn and mixed with 0.5 % ZnS4 solution. Samples were centrifuged at 6000 rpm for 10 minutes and a clear supernatant solution was analyzed spectrophotometrically Kinetics of Drug Hydrolysis The in vitro hydrolysis of prodrugs was followed by the determination of the order of reaction and half life The kinetics of hydrolysis was monitored by increase of free drug concentration with time. The velocity with which a reaction or a process occurs is called as its rate 215. Drug A drug B 4.1 da The rate of forward reaction is expressed as. The negative dt sign indicates that the concentration or drugs decreases with time t. As

27 101 the reaction proceeds, the concentration of drug B increases and the db rate of reaction can be expressed as. dt The manner in which the concentrations of drug influence the rate of reaction or process is called as the order of reaction. If C is the concentration of drug A, the rate of decrease in C of drug A as it is changed to B can be described by a general expression as a function of time t. dc dt n KC 4.2 where K = rate constant, n = order of reaction If n = 0, it is a zero order reaction and if n = 1, it is a first order reaction. Zero rder Kinetics If n=0, equation 4.2 becomes dc dt K C K0 where K0 = zero order rate constant From equation 4.3, the zero order process can be defined as the one in which the reaction is independent of the concentration of drug undergoing reaction i.e., the rate of reaction cannot be increased further by increasing the concentration of reactants. Rearrangement of equation 4.3 yields dc K0dt 4.4

28 102 Integration of equation 4.4 gives C 0 0 C K t or C 0 0 C K t 4.5 where C0 = concentration of drug at t = 0 and C = concentration of drug yet to undergo reaction at time t. Zero rder Half Life The half life (t½) is defined as the time period required for the concentration of drug to decrease by one-half. When t = t½, C= C0/2 and equation 4.5 becomes C 0 2 C K t Solving 4.6, C 0 t K 0 First rder Kinetics If n = 1, equation 4.2 becomes dc dt where K = first order rate constant KC 4.8 From equation 4.8, it is clear that a first order process is the one whose rate is directly proportional to the concentration of drug undergoing reaction. It is because of such proportionality between rate of reaction and the concentration of drug that a first order process is said to follow linear kinetics 216. Rearrangement of equation 4.8 yields dc Kdt 4.9 C

29 103 Integration of equation 4.9 gives Equation 4.10 can be written as ln C ln C Kt 4.10 o C Kt C 0 e 4.11 where e = natural log base. Since ln = log, equation 4.11 can be written as Kt LogC LogC If x = the amount of prodrug hydrolyzed and C0 = the initial concentration of prodrug, then concentration of prodrug at time t, C = C0-x and equation 4.12 can be rearranged as K C0 log t C x First rder Half Life Substituting the value of C C 0 2 at t 1 2 in equation 4.12 and solving it yields t K 4.6 PHARMACLGICAL SCREENING F DRUGS AND PRDRUGS Drugs as well as the synthesized prodrugs were evaluated for anti inflammatory activity, analgesic activity, ulcerogenicity and histopathology. A comparative study between pharmacological activities of parent drug and their prodrugs were performed.

30 Experimental Animals Wistar albino rats were randomly divided into six groups each of 6 rats, including a control and a standard group. The selected animals were housed in acrylic cages at standard environmental conditions at 25 ± 2ºC, relative humidity of 45 to 55 %, in a well ventilated room maintained at 12: 12 h light: dark cycle, fed with standard rodent diet and water ad libitum. All the animals were acclimatized for a week before experiment. All animal experiments were carried out according to the guidelines of the Committee for the Purpose of Control of Experiments on Animals and approval of the Institutional Animal Ethics Committee (Reg. No. 930/a/06/CPCSEA), Sree Vidyanikethan College of Pharmacy, Tirupati, India was obtained Anti Inflammatory Activity Inflammation is the response of the tissue to an infection or irritation by a foreign substance. Protein denaturation is one of the well documented causes of inflammation 217. Production of auto antigens in certain rheumatic diseases may be due to in vitro protein denaturation. Anti inflammatory agents helps in removing the signs of inflammation like pain, tenderness, swelling, vasodilation and leukocyte infiltration by acting on various systems responsible for inflammation such as plasmin clotting, arachidonic acid or complimentary systems 218, 219. They are said to inhibit the formation of inflammation mediators like histamine, serotonins, prostaglandin and kinins. Activity of the phosphorylase is inhibited which deprives the inflamed tissue of much needed metabolic energy in the form of ATP.

31 Screening Methods Various methods are reported for screening anti inflammatory activity of a drug Carrageenan induced hind paw oedema method, 5-hydroxy tryptamin induced hind paw oedema method, formalin induced hind paw oedema method, hyaluronidase hind paw oedema method, Histamine induced hind paw oedema method and turpentine oil induced arthritis in knee joint are some of the methods adopted for the evaluation of acute inflammation. Carraeenan granuloma pouch technique and cotton pellet granuloma are used for the evaluation of subacute inflammation. Miscellaneous methods include formaldehyde induced arthiritis and immunological methods Experimental Method In the present study, the anti inflammatory activity of drugs and prodrugs were determined by hind paw oedema method using carrageenan (0.1 ml, 1 % w/v) as phlogistic agent 224. Wistar albino rats ( g) were divided into six groups, each comprising of six animals, including a control and a standard group. The initial volume of right hind paw of rat was measured by plethysmometer without administration of drug. A 1 % sodium carboxy methyl cellulose (CMC) suspension containing drug (100 mg) was prepared and a volume of this suspension containing an equivalent dose (AC-100 mg/kg/body wt, MA-50 mg/kg/body wt, Dex-20 mg/kg/body wt) was administered orally to the standard groups. Similarly equivalent quantity of each prodrug was administered to the test groups. After 30 min of administration of the drug or prodrugs, carrageenan solution in normal

32 106 saline was injected into the planter surface of right hind paw of each animal. The volume of swelling of right hind paw of each rat was measured after 0.5, 1, 2, 4 and 6 h. The mean increase in the volume of the right hind paw of rats was compared with control and standard. The percent inhibition of paw oedema was calculated as Percentage inhibition = (1-Vt/Vc) x where Vt - mean relative change in paw oedema volume in test group, Vc mean relative change in paw oedema volume in control group Analgesic Activity Pain is not easily or satisfactorily defined, and therefore it is often interpreted as the suffering that results from the perception of painful stimuli. Pain signifies that the structural or functional integrity of the body has been impaired. Generally, pain is classified clinically into two distinct types, acute and chronic. Either type may be mild or severe. Acute pain is temporary, having an instantaneous onset e.g. renal, colic, trauma and headache. n the other hand chronic pain is continuous and gradual in onset so that the exact time of onset is uncertain e.g. rheumatoid arthritis and pain of malignancy. Analgesia denotes the relief of pain but with simultaneous retention of consciousness. Although pain in rheumatoid arthritis and in angina pectoris can be controlled by the use of corticosteroid and nitroglycerine respectively, these agents are not considered as analgesics. Thus, analgesic covers only those agents which when administered systematically provide non-specific relief from pain without loss of consciousness.

33 Screening Methods The methods to test the drugs for relieving the pain vary with the type of stimuli used. Various methods have been applied using chemical, mechanical, electrical and thermal stimuli to determine the analgesia produced by a test drug Tail clip method, hot plate method, tail flick method, tail immersion test and writhing induced by sodium chloride (4 %) solution are some of the methods Experimental Method The analgesic activity of the drugs and prodrugs was determined by thermal stimulus using tail flick method 228, 229. Analgesiometer was used for the determination of pain threshold of albino mice. Cold water was circulated through the water jackets of the instrument to avoid heating of the area around the hot wire. Mice (25-30 g) were divided into six groups, each comprising six animals, including a control and standard group. The mice were placed in a holder through which the tail of the animal protruded out. The normal reaction time, i.e., the time taken to flick the tail was noted at 0.5, 1, 2, 3 and 4 h after the treatment and cut-off time was 9 s. The animals, which showed significant (above 9 s) delayed response, were rejected. 1 % sodium carboxy methyl cellulose (CMC) suspension containing drug (100 mg) was prepared and a volume of this suspension containing an equivalent dose (AC-100 mg/kg/body wt, MA-50 mg/kg/body wt, Dex-20 mg/kg/body wt) was administered orally to the standard groups. Similarly equivalent quantity of prodrugs was administered to the test groups. Percentage analgesia was calculated by the formula given as

34 108 Percentage Analgesic activity = [(T2-T1)/ (Tc-T1)] x where T1 - reaction time (s) before prodrug administration, T2 - reaction time (s) after prodrug administration and Tc - cutoff time (s) Ulcerogenic Activity Peptic ulcer constitutes a major disease that affects the human gastrointestinal tract. The common clinical features of peptic ulcers are hyperacid secretion and ulcer formation in the stomach and duodenal part of the intestine. Acute gastric ulcers can be produced in laboratory animals by the use of corrosive substances such as alcohol, NSAIDs, surgical manipulation or by subjecting the animals to acute stressful conditions. While, NSAID s are effective in the management of pain and inflammation in a large number of conditions, it is now established that these are associated with the development of upper GI damage, including lesions, ulcers and life threatening perforations and haemorrhage. The importance of the direct contact effect in GIT toxicity of NSAID was examined in rats and the study suggested that direct tissue contact of NSAIDs plays an important role in the production of GIT lesions. The use of prodrugs to temporarily mask the acidic group of NSAIDs has been postulated as an approach to decrease GIT toxicity. Ulcerogenic index of the compounds further ensures whether the synthesized compounds are less irritating to gastric mucosa than the parent NSA1Ds or not. Hence, it is necessary to investigate the ulcerogenic activity of the prodrugs 230.

35 Experimental Method Gastrointestinal toxicity of the drugs and prodrugs was measured and compared with the parent drug by measuring mean ulcer index 231. Wistar albino rats ( g) were divided into six groups, each comprising six animals, including a control and standard group. The control group was administered orally by 2 % acacia suspension. Test compounds and standard were administered orally (at 10 times higher dose) as a suspension with 2 % acacia daily for 5 days. The rats were fasted after the administration of last dose, thereafter they were sacrificed by decapitation and the stomach was removed, opened and washed with distilled water. The lesions on the gastric mucosa were counted by visual examination using a binocular magnifier. Ulcers greater than 0.5 mm were recorded. The mean ulcer index (UI) was calculated by severity of gastric mucosal lesions which are graded as grade 1: less than1 mm erosions, grade 2: 1-2 mm erosions and grade 3: more than 2 mm erosions. The UI was calculated as UI = [1 (number of lesions of grade 1) + 2 (number of lesions of grade 2) + 3 (number of lesions of grade 3)]/ Histopathological Studies The stomach gastric mucosa is divided arbitrarily, into three regions: the cardia, the corpus (fundus), and the antrum. In contrast to surface mucous cells that are continuous over the entire stomach surface, regional differences are mainly due to the composition of gastric tubular glands within each compartment. Tubular glands within the gastric corpus have four cell types: parietal (oxyntic) cells that

36 110 secrete acid and the intrinsic factor necessary for vitamin B12 reabsorption, chief cells responsible for pepsinogen production, mucous neck cells and endocrine cells [gastrin producing cells (G cells) and enterochromaffin-like cells (ECL cells)]. The pyloric glands are more coiled, branched and tubular glands that are predominantly mucous (i.e. few parietal cells, chief cells are usually absent) with endocrine cells [G cells and somatostatin-producing cells (D cells)]. Endocrine cells, particularly G cells, are more frequent in the antrum than in the corpus 232. The gastric cardia has branched, tubular glands that are primarily mucous and oxyntic type glands. Individual glands are separated from one another by a thin stroma of loose connective tissue (the lamina propria) that has fibroblasts, endothelial cells and few, if any, chronic inflammatory cells (lymphocytes, plasma cells and macrophages). In the gastric corpus, as the long glands in the stomach are lined by prominent cells, it may be difficult to see the lamina propria, except just under the surface mucous cells Experimental Method The histopathological studies 234, 235 of stomach of rats were carried out using haemotoxylin and eosin stain at Pathology Department, Sri Venkateswara Veterinary University, Tirupati. Wistar albino rats ( g) were divided into six groups, each comprising six animals, including a control and standard group. The stomach tissues were removed from the rats and fixed in 10 % normal saline for at least 48 h. These were then processed routinely and the tissues were embedded in paraffin wax. Histological sections were cut at 5-6 μm and stained with

37 111 routine haematoxylin and eosin. These were then examined by a consultant histopathologist. The lesions observed were assessed for the following mucosal atrophy, the presence of inflammatory cells in the wall, oesinophils, lymphocytes and plasma cells. Photomicrographs of representative lesions at various magnifications were taken on Zeiss optical microscope (Germany), Stemi 2000-C, with a resolution of 10 x 45 X, attached with trinocular camera. Statistical Analysis Statistical analysis of the pharmacological activity of the synthesized prodrugs on animals was evaluated using a one-way analysis of variance (ANVA). Student s t-test was applied for expressing the significance and the experimental data are expressed as mean ± SD (standard deviation).