1 INTRODUCTION:

Amantadine was discovered in petroleum and name is derived from Greek name adamantinos (relating to steel or diamond), due to its diamond-like structure [1]. Tricyclic amines have a great potential in the treatment and prevention of influenza A of which the most significant is amantadine (Figure 1). The Amantadine was first in this class to be clinically approved in 1966 agent against Asian influenza and ultimately received approval for the treatment of Influenza virus A in adults. In 1969 the drug was also discovered by accident to help reduce symptoms of Parkinson's disease [2].

The common thing in tricyclic amines antiviral drugs are the cage like basic structure Adamantane (C₁₀H₁₆) is a colorless, crystalline compound with a camphor-like odor with a formula it is a cycloalkane and also the simplest diamondoid. First time adamantane was discovered in petroleum in 1933 [3].

Figure 1

Angiotensin Converting Enzyme Inhibitors:

Angiotensin converting enzyme (ACE) inhibitors plays a physiological role in the renin-angiotensin system to increase blood pressure, which removes a dipeptide from the C terminus of angiotensin I to form angiotensin II, a potent vasoconstrictor and inactivates the vasodilator nonpeptide bradykinin [4]. An important consideration affecting the use of aspirin in diabetic patients is its interaction with ACE-inhibitors, [5] ibuprofen also interacted with ACE-inhibitors [6].

Cephalosporins:

Presently cephalosporins antibiotics are classified as first, second, third and fourth generation. Individually cephalosporins differ in their pharmacokinetics properties, especially plasma protein binding and half-life, but the structural bases for these differences are not obvious [7, 8]. The most common adverse reactions are allergy and hypersensitivity [9], which are identical to those caused by the penicillins, and this may be related to the shared β-lactam structure of both groups of antibiotics [10].

H₁-receptor Antagonists

The second-generation antihistamines are more similar pharmacologically than structurally; Most of these compounds produce prolong antihistaminic effects due to the slow dissociation from H₁ receptors and the formation of active metabolites with similar receptor binding profile [11]. There are number of drug interactions reported as the effect of the antifungal drug ketoconazole, which blocks hepatic first-pass biotransformation of ebastine and terfenadine leading to increased cardiotoxicity of terfenadine in man, on the ECG effects of terfenadine and ebastine in animal model [12].

H₂-receptor Antagonists

Histamine H₂-receptor antagonists are potent inhibitors of all phase of gastric acid secretion, they do so by binding to H₂- receptors on the parietal cells of stomach and are analogs of histamine, contain bulky side chain in place of ethylamine moiety. Burimamide and cimetidine are early members of this group. Cimetidine contains the imidazole ring of histamine, which is replaced by furan in ranitidine or a thiazole in famotidine and nizatidine. This group is more hydrophilic than H₁antagonists [13]. Cimetidine altered steady-state kinetics of oral theophylline, slowing theophylline clearance 25%. H₂-receptor antagonists exhibited decline in bioavailability when ingested with antacid.

Non-Steroidal Anti-inflammtaory Drugs (NSAIDs)

NSAIDs are inhibitor of the COX enzymes that catalyze the conversion of aracidonic acid to various eicosanoids including thromboxane and prostaglandins [14, 15]. Conversely, prostacyclin synthesis, which is largely mediated by COX-2 activity in macrovascular endothelial cells, counteracts these effects resulting in inhibition of platelet aggregation, vasodilation and antiproliferative effects [16].

Piroxicam and ibuprofen markedly blunt the effects of antihypertensive drugs while acetaminophen is almost inert [17, 18]. NSAIDs also interacted with antibiotics [19].

Quinolones

Fluoroquinolones as a class are generally fairly insoluble in water, modern quinolone agents are zwitter ionic in character, due to the presence of both a carboxylic acid and a basic amine; pKa values for these functional groups are 5.5-6.3 for the carboxylic acid and 7.6-9.3 for the distal amino group [20]. Fluoroquinolones class is involved in a number of clinically important drug interactions, which exhibit little inter-patient variability [21, 22].

The mechanism of interaction of amantadine with these drugs either occupying p-p* transitions or by forming charge transfer complexes (CTC), not very sturdy covalently bonded molecules were formed but due to the favorable temperature, solvent conditions and concentrations they are described as charge transfer complexes.

2 EXPERIMENTAL:

i Materials

Amantadine was purchased from Sigma-Aldrich, captopril from Zafa Pharmaceutical Laboratories (Pvt) Ltd, hydrochlorothiazide from Novartis Pharma, (Pvt) Ltd and lisinopril from Atco Laboratories Ltd. Cefpirome from Aventis Pharma (Pakistan) Ltd, cephradine Bosch Pharma, ceftriaxone from Macter Pharmaceutical, ceftazidime from Glaxo Smith Kline Pakistan Ltd. Levocetirizine and fexofenadine were UCB Farchim SA, Switzerland and Getz Pharma Pakistan (Pvt) Ltd respectively. Cimetidine from Glaxo Smith Kline Pakistan Ltd, famotidine from Getz Pharma Pakistan (Pvt) Ltd and ranitidine were obtained from Zafa Pharmaceutical Laboratories (Pvt) Ltd. NSAIDs used were diclofenac sodium from Novartis Pharma, (Pvt) Ltd., ibuprofen from Abbott Laboratories (Pvt) Ltd, mefenamic acid were obtained from Parke-Davis & Co Ltd, meloxicam from Ali Gohar Pharmaceuticals (Pvt.) Ltd and naproxen from Roche Pharmaceutical. Ciprofloxacin from Brookes Pharma, levofloxacin and ofloxacin from Aventis Pharma (Pakistan) Ltd, moxifloxacin Bayer Pakistan (Pvt) Ltd, norfloxacin from Novartis Pharmaceutical and sparfloxacin from Abbott Laboratories (Pvt) Ltd.

ii Reagents

Methanol (TEDIA^®, USA), hydrochloric acid, sodium hydroxide from Merck, Darmstadt, Germany. All other chemicals used were of analytical grade. Deionized water used throughout the work was freshly prepared daily.

iii Equipments and glassware

UV-Visible double beam spectrophotometer (Shimadzu 1601), 1 cm rectangular quartz cells, ground glass distillation assembly, (Quickfit), de-ionizer (Stedec CSW -300), water distillation unit (GFL type 2001/2, No. 10793600G), melting point apparatus (Gallenkamp), All the glassware’s were of Pyrex A grade, washed with chromic acid followed by thorough washing with water and finally rinsed with double distilled or deionized water, which was freshly prepared in the laboratory.

iv Dissolution test apparatus

The dissolution equipment was Compact Dissolution Test Apparatus-Type PTWS 310 manufactured by Pharmatest according to USP/EP [23, 24]. The assembly consisted of a covered vessel made of glass, a motor, a metallic drive shaft and a cylindrical basket. The vessel is partially immersed in a suitable water bath which permitted holding the temperature inside the vessel at 37±0.58 during the test and keeping the bath fluid in constant, smooth motion. The assembly was placed in an environment free of significant motion, agitation, or vibration except due to the smoothly rotating stirring element.

Table 1 Reference Standards of H₁andH₂-receptor antagonists in simulated gastric juice

M x 10^-5	Cetri-zine	M x 10^-5	Fexo-fenadine	M x10^-4	Cimitidine	M x10^-4	Raniti-dine	M x10^-5	Famotidine
1	0.164	0.5	0.081	1	0.2	1	0.178	0.3	0.379
2	0.321	1	0.515	2	0.365	2	0.382	0.4	0.498
3	0.472	1.5	0.728	3	0.591	3	0.579	0.5	0.673
4	0.654	2	0.90	4	0.785	4	0.778	0.6	0.778
5	0.791	2.5	1.079	5	0.979	5	0.964	0.7	0.899
6	0.977	3	1.175	6	1.174	6	1.191	0.8	1.033
7	1.131	3.5	1.398	7	1.371	7	1.365	0.9	1.159
8	1.32	4	1.476	8	1.567	8	1.567	1	1.424
9	1.496	4.5	1.539	9	1.755	9	1.768	1.1	1.523
10	1.633	5.5	1.583	10	1.923	10	1.959	1.2	1.686
e (M^-1cm^-1)	16320	e (M^-1cm^-1)	43770	e (M^-1cm^-1)	19457	e (M^-1cm^-1)	19303	e (M^-1cm^-1)	12848

Table 2 Reference Standers of Ciprofloxacin, Norfloxacin, levofloxacin and Moxifloxacin in simulated gastric juice

M x10^-5	Cipro-floxacin	Mx10^-5	Nor-floxacin	M x10^-4	Levo-floxacin	Mx10^-5	Moxi-floxacin	M x10^-5
1.2	0.514	0.50	0.311	0.5	0.170	0.5	0.102	0.178
1.4	0.585	0.75	0.475	1.0	0.32	1.0	0.183	0.382
1.6	0.650	1.00	0.621	1.5	0.476	1.5	0.270	0.579
1.8	0.736	1.25	0.760	2.0	0.63	2.0	0.357	0.778
2.0	0.821	1.50	0.920	2.5	0.801	2.5	0.455	0.964
2.2	0.884	1.75	1.041	3.0	0.959	3.0	0.548	1.191
2.4	0.958	2.00	1.185	3.5	1.116	3.5	0.632	1.365
2.6	1.047	2.25	1.299	4.0	1.266	4.0	0.724	1.567
2.8	1.116	2.50	1.611	4.5	1.434	4.5	0.815	1.768
3	1.194	2.75	1.797	5.0	1.586	5.0	0.911	1.959
e (M^-1cm^-1)	41000	e	61000	e (M^-1cm^-1)	32030	e	18320	19303

Table 3 Reference standards of ofloxacin, sparfloxacin, cefpirome, cephradine and lisinopril in simulated gastric juice

M x10^-5	Ofloxacin	M x 10^-5	Spar-floxacin	Mx 10^-3	Cef-pirome	Mx10^-3	Cephra-dine	Mx10^-3	Ceftazidime	Mx10^-3	Lisino-pril
1.0	0.106	4.4	0.683	0.01	0.141	0.01	0.21	0.01	0.017	0.01	0.162
1.5	0.157	4.6	0.704	0.02	0.311	0.02	0.4	0.02	0.033	0.02	0.324
2.0	0.216	4.8	0.732	0.03	0.439	0.03	0.63	0.03	0.05	0.03	0.481
2.5	0.263	5.0	0.791	0.04	0.616	0.04	0.84	0.04	0.063	0.04	0.643
3.0	0.331	5.2	0.825	0.05	0.831	0.05	1.08	0.05	0.08	0.05	0.789
3.5	0.385	5.4	0.837	0.06	0.981	0.06	1.29	0.06	0.096	0.06	0.956
4.0	0.421	5.6	0.857	0.07	1.157	0.07	1.43	0.07	0.114	0.07	1.103
4.5	0.461	5.8	0.900	0.08	1.318	0.08	1.69	0.08	0.131	0.08	1.251
5.0	0.528	6.0	0.916	0.09	1.524	0.09	1.89	0.09	0.145	0.09	1.409
5.5	-	6.2	1.004	0.1	1.673	0.1	2.17	0.1	0.163	0.1	1.571
e (M^-1cm^-1)	10660	e	10660	e	18590	e	15500	e	1650	e	15745

Table 4 Reference standards of Diclofenac sodium, Meloxicam, Mefanamic Acid, Ibuprofen and Captopril in simulated gastric juice

M x10^-5	Diclofenac sodium	Mx10^-5	Meloxi-cam	M x10^-5	Mefanamic Acid	Conc. Mx10^-4	Ibuprofen	M x10^-5	Captopril
1.2	0.825	1	0.016	1	0.078	2.0	0.336	0.05	0.487
1.4	0.922	2	0.039	2	0.119	2.5	0.422	0.06	0.567
1.6	1.017	3	0.056	3	0.157	3.0	0.535	0.07	0.640
1.8	1.115	4	0.089	4	0.185	3.5	0.638	0.08	0.713
2.0	1.206	5	0.114	5	0.223	4.0	0.748	0.09	0.814
2.2	1.294	6	0.133	6	0.253	4.5	0.846	0.1	0.900
2.4	1.294	7	0.155	7	0.285	5.0	0.965	0.11	0.962
2.6	1.384	8	0.181	8	0.309	5.5	1.076	0.12	1.058
2.8	1.569	9	0.204	9	0.35	6.0	1.165	0.13	1.139
3	1.642	10	0.22	10	0.387	6.5	1.261	0.14	1.209
e (M^-1cm^-1)	9000	e (M^-1cm^-1)	2000	e (M^-1cm^-1)	5000	e	19570	e	8771

The apparatus permitted observation of the specimen and stirring element during the test. The vessel had a capacity of 1 L and conformed to the USP performance test and the procedure statement [23]. The shaft was positioned so that its axis is not more than 2 mm at any point from the vertical axis of the vessel and rotated smoothly without significant wobble. A speed-regulating device was used that allows the shaft rotation speed to be selected and maintained at 100±0.5 rpm.

3 RESULT AND DISCUSSION:

i Validation of dissolution apparatus

Due to the nature of the test method, quality by design is an important qualification aspect for in-vitro dissolution test equipment. Geometrical and dimensional accuracy and precision, any irregularities such as vibration or undesired agitation by mechanical imperfection were avoided. Besides, the specification of the apparatus and qualification of dissolution equipment was considered as a critical parameter, e.g. temperature of test medium, rotation speed, flow rate and volume, sampling; it was monitored periodically during the work. Apparatus suitability test with calibrator prednisone and salicylic acid tablets was used to ensure the performance of apparatus [24].

The method selected for our studies is simple, accurate and quick. Fexofenadine showed maxima at 218 nm in all mediums, cetirizine at 211 nm, cimetidine at 216 nm, ranitidine 313 nm and famotidine 266 nm in simulated gastric juice. The drugs selected from cephalosporins were cefpirome it exhibits absorption at 271nm, cephradine at 260 nm, ceftazidime at 262 nm and ceftriaxone at 227 nm. The drugs selected from quinolones were ciprofloxacin exhibit strong absorption in the ultraviolet region at 270-277, 314-324 and 328-334 nm, norfloxacin at 273 nm, levofloxacin at 294 nm, moxifloxacin at 290 nm, ofloxacin at 300 nm and sparfloxacin at 298 nm in simulated gastric juice. Captopril showed its strong absorption at 203 nm, lisinopril at 206 nm and hydrochlorothiazide at 212 nm in simulated gastric juice. The drugs selected from NSAIDs were mefanamic acid exhibited its UV maxima at 285 nm, diclofenac sodium at 271, meloxicam at 362 nm, ibuprofen at 226 nm and naproxen sodium at 240 nm in simulated gastric juice. Measurement of these absorption bands has been employed for the assay of these drugs, which followed Beer’s Lambert law in the concentration range studied. Molar absorptivity values were calculated for each medium the slight deviation may arrive due to the change in the medium by changing the buffer the minimum wavelength selected as most favorable for the research was 206 nm. Reference standards for different drugs with their epsilon values are showed in Table 1-4; Figure 3a.

ii Interaction with Amantadine

Drug interaction studies of amantadine were carried out at 37°C in simulated gastric juice as it was insoluble at all other pH levels found in human body. Prior to these interactions studies the assay of all the drugs were carried out according to their Pharmacopoeial methods. This was followed by dissolution studies of each formulation. Conforming to the availability of these drugs (99±1%) according to Pharmacopoeial standards drug interactions of amantadine were carried out with representative examples from different classes of drugs, i.e. ACE inhibitors, cephalosporins, H₁ and H₂ receptor antagonists, NSAIDs and quinolones.

iii Procedure for drug interaction studies

Drug interaction studies of amantadine were carried out on a Dissolution Test Apparatus as described above. There have been number of other drug interaction studies reported by this method [25, 26].

In each set of experiment, amantadine solution 10 mL (0.5 mM) was added to the dissolution medium at zero time while 10 mL (0.5mM) of each interacting drug was added to the dissolution medium separately in each individual set of experiment. Aliquots were withdrawn at fifteen minutes interval and assayed for interacting drugs contents and graphs were plotted between drug concentration versus time which showed drug status during and at the end of the experiments.

The in vitro availability of all drugs was found to be markedly retarded in the presence of amantadine. The results of availability H₁andH₂-receptor antagonists in presence of amantadine are given in Table 5 and are plotted in Figure 3.1and 3.2. These results reflect the possible complexation of these drugs with amantadine.

Amantadine cephalosporin drug interactions have not been directly studied. However, several drug interactions have been reported involving renal clearance of drugs [27]. In our studies of interaction of amantadine with cephalosporin’s (Table 6; Figure 3.3), the availability of cephalosporins were decreased to a considerable extent. As both of these types of drugs has number of lone pairs of electrons and hence have centre of reactivities. These electron clouds easily overlap with each other to form charge transfer complexes. Such types of charge transfer complexes although having low dissociation energy (~ 3-5 Kcal^-mole) result in high molecular weight complexes (than parents) and therefore may not be detected merely by the uses of UV spectrophotometry and hence other tools are to be implemented.

In line with the literature, the drug interactions of amantadine with ACE inhibitors and Hydrochlorothiazide decreased the availabilities of the later drugs (Table 7; Figure 3.4).

Co-administration of quinine or quinidine with amantadine has been shown to reduce the renal clearance of amantadine by about 30 % [28]. In dogs with osteoarthritic pain refractory to an NSAID, physical activity was improved by the addition of amantadine. Amantadine might be a useful adjunct therapy for the clinical management of canine osteoarthritic pain [29]. In our studies, the results of availability of NSAIDs and quinolones in presence of amantadine are given in Table 8 and are plotted in Figure 3.5 and in Table 9 plotted in Figure 3.6 respectively.

4 CONCLUSION:

The in vitro interactions studies of H₁andH₂-receptor antagonists, cephalosporins, ACE-inhibitors, hydrochlorothiazide, NSAIDs and quinolones with amantadine show that % availability of all these drugs were affected either increased or decreased in simulated gastric juice at 37˚C, which illustrate possible complex formation due to p-p* transitions or by forming charge transfer complexes (CTC), in that way changes occurred in the molecular weight due to donor acceptor equilibrium incorporation.

A + B AB

Where,

A = electron pair acceptor

B = electron pair Donor

Table 5 In vitro availabilities of H₁andH₂-receptor antagonists in presence of amantadine at 37˚C in simulated gastric juice

S. No	Time (min)	Fexofenadine	Cetirizine	Ranitidine	Cimetidine	Famotidine
S. No	Time (min)	% availability
1	0	100.00	100.00	100	100	100
2	15	90.44	88.95	86.04	91.67	72.67
3	30	52.90	81.40	59.36	57.87	62.73
4	45	58.36	62.79	49.54	52.08	54.35
5	60	20.48	40.41	30.06	32.41	23.91

Table 6 In vitro availabilities of cephalosporins in presence of amantadine at 37˚C in simulated gastric juice

S. No	Time (min)	Cefpirome	Cephradine	Ceftazidime	Ceftriaxone
S. No	Time (min)	% availability
1	0	100.00	100.00	100.00	100
2	15	86.03	74.53	96.76	95.21
3	30	59.45	61.05	86.33	89.00
4	45	39.45	59.55	75.18	72.10
5	60	15.62	34.46	68.35	65.23

Table 7 In vitro availabilities of ACE-inhibitors and hydrochlorothiazide in presence of amantadine at 37˚C in simulated gastric juice

S. No	Time (min)	Captopril	Lisinopril	Hydrochlorothiazide
S. No	Time (min)	% availability
1	0	100	100	100
2	15	88.62	80.69	82.54
3	30	78.44	73.76	76.59
4	45	72.46	53.47	82.54
5	60	44.31	28.71	22.22

Table 8 In vitro availabilities of NSAIDs in presence of amantadine at 37˚C in simulated gastric juice

S. No	Time (min)	Mefenamic acid	Diclofenac sodium	Meloxicam	Ibuprofen	Naproxen sodium
S. No	Time (min)	% availability
1	0	100	100	100	100	100
2	15	86.80	82.44	91.43	86.31	89.15
3	30	78.26	71.84	73.05	80.13	82.45
4	45	64.57	57.12	59.19	68.43	71.13
5	60	49.60	56.49	45.02	56.07	64.43

Table 9 In vitro availabilities of quinolones in presence of amantadine at 37˚C in simulated gastric juice

S. No	Time (min)	Ciprofloxacin	Norfloxacin	Levofloxacin	Moxifloxacin	Ofloxacin	Sparfloxacin
S. No	Time (min)	% availability
1	0	100	100	100	100	100	100
2	15	90.91	91.34	80.51	90.3	84.08	82.64
3	30	89.26	88.98	78.97	78.73	77.61	73.58
4	45	77.69	77.17	71.28	71.64	70.65	62.64
5	60	21.49	22.05	52.82	60.07	53.23	35.47

Reference Standard of cimetidine, famotidine, ranitidine and enoxacin in buffer of various pH at 37˚C

Figure 3(a)

iv Dissolution test apparatus

23 The USP Performance Test and the Dissolution Procedure Statement. October 24, 2006. http://www.usp.org/ USPNF/notices/uspPerformanceTestStatement.html