Influence of Molecular Level Interactions of Ofloxacin and Methylxanthines in Pharmacotherapy

Introduction

Concomitant administration of drugs has become a common practice and causes the pharmacokinetic drug-drug interactions, which often lead to an altered clinical response. Antacids delay the absorption of fluoroquinolones by complexation.¹ Caffeine and other methylxanthines are known to form complexes with sulfathiazole, benzocaine, and p-aminobenzoic acid.²

Theophylline is a drug of choice in the management of asthma. The chronic situation of asthma is sometimes associated with secondary infections. In such cases, the standard regimen in therapy includes theophylline and antibacterials such as fluoroquinolones. Simultaneous administration of theophylline and fluoroquinolones in patients and healthy subjects leads to increased theophylline concentration. This increase in concentration is due to decreased theophylline’s clearance and these changes can be of clinical significance.³ Theophylline and new fluoroquinolones are metabolized by hepatic cytochrome P₄₅₀ and only fluoroquinolones that form a 4-oxy metabolite inhibit theophylline clearance.⁴ Another hypothesis for fluoroquinolones to have a greater impact on theophylline clearances is due to their stereochemical similarity to theophylline. Therapeutic range of theophylline is 10-20 µg/ml; toxic effects often result when serum concentrations exceed 20 µg/ ml.⁵ Thus, avoiding of drug interactions is essential to overcome toxic effects. Concomitant oral administration of drugs leads to mutual interaction, thereby one may also alter the gastrointestinal absorption of the other. However, there is no evidence on the possible interaction of the quinolones and methylxanthines at absorption level.

Caffeine and theophylline were taken as model compounds for methylxanthine alkaloids. Ofloxacin, water soluble antimicrobial agent, rapidly absorbed by oral route, recommended for respiratory tract infections is taken as model for fluoroquinolone. Selected methylxanthines (caffeine and theophylline) in this study and ofloxacin have similar structure. Caffeine and theophylline are known to undergo self-association in solution. However, self-association of fluoroquinolones (ofloxacin) was not reported in the literature. As a corollary, it is conjectured here that it might be possible for fluoroquinolones to associate with methylxanthines molecules in the same way as these self-associate. Thus, in this study, suitability of application of complexation hypothesis to explore solutions for problems associated with drug-drug interactions is also investigated.

The objectives of the present investigation are (i) to identify the solubility phase interactions of methylxanthines with fluoroquinolone such as ofloxacin, (ii) to evaluate the in vitro diffusion and in situ absorption profiles of methylxanthines in presence of ofloxacin. Outcome of present investigation might be helpful in changing the dose and dosage regimen of theophylline which inturn helps to avoid its toxicity.

Materials and Methods

Ofloxacin was a gift sample from Dr. Reddy’s Laboratories Ltd, Hyderabad. Theophylline anhydrous and Caffeine anhydrous were obtained from Zydus Cadila Laboratories, Ahmedabad. Dialysis membrane was purchased from HiMedia Laboratories, Chennai. Silicones were procured from Reliance Petro Chemicals Ltd., Mumbai. Other chemicals used were of analytical grade.

UV analysis of ofloxacin

An UV scan of individual drug solutions of ofloxacin, caffeine and theophylline (10 µg/ml each) were taken between 200 - 400 nm to find out the l_max. Mixtures of ofloxacin (10 µg/ml) with methylxanthines (10 µg/ml each separately) were also scanned under UV region to check influence of drug interaction on absorption profiles at working wavelengths. Calibration curve of ofloxacin was plotted by using different concentrations ranging from 5 to 30 mg/ml.

Solubility studies of ofloxacin

The solubility method followed in this work was as described by Martin et al. and Subrahmanyam.^6,7 The stock solution of caffeine (0.04 M) was prepared and this solution was further diluted to get different solutions of concentration ranging from 0 to 4×10^-2 moles/litre. Different diluted caffeine solutions (20 ml) were taken in separate conical flasks.  Excess of ofloxacin (70 mg) was added to each flask. The flasks were shaken in a constant temperature reciprocating shaker water bath (Research & Test Equipments Ltd., Bangalore) at 100 strokes per minute and at 37^°C for 24 hours. The equilibrated solutions were then removed and filtered, the filtrate was diluted suitably with distilled water. There was no precipitation of ofloxacin on further dilutions with water. The concentration of ofloxacin was measured by UV spectrophotometer -1601 (Shimadzu Corporation, Japan) at 328 nm. Similar method was followed for the solubility studies of ofloxacin in presence of theophylline.

Preparation of solid complexes

A co-precipitation method was used to prepare solid complexes.^8,9 About 200 mg of ofloxacin was dispersed in acetone (20 ml) with the help of magnetic stirrer. Caffeine solution, 100 ml (0.04 M) was added drop by drop by a burette with continuous stirring. After completion of addition, acetone was evaporated on a constant temperature water bath (60^°C) for 20 minutes to get clear solution. This clear solution was kept in the deep freezer (4^°C for 12 hours) to precipitate the complex formed. The precipitate was collected by filtration and the residue was dried at room temperature for 36 hours in a desiccator. A similar method was used for the preparation of ofloxacin-theophylline complex.

characterization of Solid complexes

Determination of melting point

The melting points of individual components and solid complexes were determined in open capillaries. Melting points of substances having higher than 250^°C were determined in the same method but by using silicones, flash point 350^°C (Reliance Petro Chemicals Limited, Mumbai), as the medium of heating.

Fourier transformed infrared (FTIR) spectrometry

Individual spectra of methylxanthines, ofloxacin, and for their complexes were taken. The samples were intimately mixed with dry powdered potassium bromide. The powdered mixtures were taken in a diffuse reflectance sampler and the spectra recorded in the wavelength region of 400 to 4000 cm^-1 using a FTIR 1600 Perkin Elmer spectrophotometer (USA).

Differential scanning calorimetric analysis (DSC)

DSC analysis was performed using DSC-2C, Perkin Elmer, USA. Calorimetric measurements were made with empty cell (high purity alpha alumina discs of Duport company) as the reference. The instrument was calibrated using high purity indium metal as standard. The dynamic scans were taken in nitrogen atmosphere at the heating rate of 5^°C min^-1. The runs were made in triplicate.

HPLC analysis of methylxanthines and ofloxacin

A HPLC system (Shimadzu Corporation, Japan) equipped with LC–20AT Prominence LC Vi06 solvent delivery unit, Prominence auto sampler SIL-20A, Prominence degasser, Labsolutions-LC solutions software was used for the analysis.  A Phenomenex (C18) reverse phase stainless steel analytical column (250 x 4.6 mm), with 5 m particle size was employed for chromatographic separation. The mobile phase consisted of a filtered and degassed mixture of 87 volumes of orthophosphoric acid (0.025 M, previously adjusted pH to 3.0 + 0.1 with trichloroethanolamine) and 13 volumes of acetonitrile. The flow rate was maintained at 1.5 ml/ min. The injection volume for all the samples was 20 ml. Samples were detected using SPD-20A Prominence Vi04 UV-spectrophotometry detector at 278 nm.

In vitro transport studies

Drug solutions used for transport studies were (a) caffeine solution, 2 x 10^-3 M (b) theophylline solution, 2 x 10^-3 M (c) ofloxacin solution, 2 x 10^-3 M (d) mixture of caffeine solution, 4 x 10^-3 M and ofloxacin solution, 4 x 10^-3 M (e) mixture of theophylline solution, 4 x 10^-3 M and ofloxacin solution, 4 x 10^-3 M.

In vitro transport studies were performed using in house developed dialysis cell. Dialysis cell consisted of a hollow glass tube, one end of which covered tightly with dialysis membrane having pore size 2.4 nm, with molecular weight cut-off 12000. Membrane was soaked in distilled water for overnight before mounting in a dialysis cell. Dialysis cell containing drug solution under study (20 ml) was partially immersed in a beaker containing 100 ml of distilled water and fixed with help of stand. The level of liquids in dialysis cell and beaker were equal.  The solution in beaker was stirred at 100 rpm using magnetic stirrer. At fixed time intervals, 2 ml of the sample was withdrawn from the beaker and fresh distilled water was replaced to maintain constant volume. Samples withdrawn were suitably diluted and analyzed by HPLC method.

In situ intestinal absorption of drugs

Wistar albino rats of either sex weighing 200 to 275 g were selected for the study.  The animals were procured from JJM Medical College, Davangere and kept in the polypropylene cages with good ventilation. They were fed with standard diet (Lipton India Ltd., Bangalore) with water ad libtum. However, animals were fasted for 24 hours before the study and had free access to drinking water.

In situ rat gut technique reported by Swintosky and Elizabeta¹⁰ was followed. Rats were anaesthetized by an intraperitoneal injection of 60 mg kg^-1 of pentobarbitone sodium. Small incision was made at midline of abdomen and duodenum was located. Two plastic L-shaped cannula with a two way stop cock were inserted through small slits at the duodenal and ileal ends of the gut (at a length about 30 cm). The cannulas were secured by ligation with silk suture, and the gut segment was reinstated in abdominal cavity. Two hypodermic syringes (10 ml capacity) were connected to cannulas and were clamped up right.  Drug solution about 10 ml, sufficient to fill the gut segment was introduced into the intestine by means of one of the syringes.  Different drug solutions used in intestinal absorption studies were same as in case of transport studies mentioned above. At fixed time intervals, 2 ml of the sample was withdrawn from the intestine and fresh distilled water was replaced to maintain constant volume. Samples withdrawn were suitably diluted and analyzed by HPLC method as mentioned earlier. The entire study was conducted with prior permission from Institutional Animal Ethics Committee.

Statistical analysis

In vitro transport of methylxanthines in absence and in presence of ofloxacin and vice versa were compared by student ‘t’ test. In situ absorption data was also compared in similar way.

Results and Discussion

UV analysis of ofloxacin

UV scan of solutions of ofloxacin, caffeine, and mixture of solutions of ofloxacin and caffeine are reported in Figure 1. Ofloxacin and caffeine exhibited l_max of 328 nm and 273 nm, respectively. Caffeine did not show absorption at 328 nm, hence it did not interfere with the estimation of ofloxacin at the working wavelength. An UV spectrum (Figure 1) of mixture of ofloxacin and caffeine solutions did not show any changes in absorption profiles at 328 nm. A similar trend was observed in case of theophylline (273 nm) and its mixture with ofloxacin solution. This further confirmed the absence of interference of caffeine and theophylline for estimation of ofloxacin. Therefore, l_max 328 nm was selected for analysis of ofloxacin. Beer Lambert’s law obeyed in the concentration range of 5 to 30 mg/ml ofloxacin (R² = 0.9995). The regression equation, y = 0.037 x + 0.0097 was used for the calculation of ofloxacin in presence of caffeine and theophylline in case of solubility studies.

Solubility of ofloxacin in presence of caffeine at 37^°C

The determination of the stoichiometric ratio of donoracceptor and a quantitative expression of the complex stability are important in the study of complexes. Solubility method was used for investigating the complexation of p-amino benzoic acid and caffeine.⁷ The same method was used in the present investigation. Solubility studies indicated that as the concentration of caffeine increased from 0 to 40 × 10^-3 mol/litre, solubility of ofloxacin increased from 6.63 × 10^-3 to 9.91 × 10^-3 mol/litre. The equilibrium phase-solubility diagram of ofloxacin in presence of caffeine is shown in Figure 2. The increase in solubility of ofloxacin in presence of caffeine provided the prima facie evidence of molecular association of caffeine with ofloxacin. This type of association may be donor-acceptor or ion-pair of complexation. Saturation of complex solubility was not found, this inferred the absence of any higher forms of complexes at the working concentrations. These trends were different from those observed in case of PABA- caffeine⁶ and acetaminophen-methylxanthine complexes.

In the absence of plateau region in the solubility profile (Figure 2), it is difficult to estimate the donor-acceptor stoichiometric ratio of the substrate and ligand. In this situation, it is appropriate to assume the stoichiometric ratio of substrate to ligand as 1:1. The solubility data was subjected to regression analysis and regression equation found was S_OF = 0.0651 C_C + 6.6376 x 10^-3 (n = 10; R² = 0.9543). Where, S_OF and C_C are solubility/ concentrations of ofloxacin and caffeine expressed as mol/litre. As per regression equation, slope and intrinsic solubility (S_o) of ofloxacin were 0.0651 and 6.6376 x 10^-3 mol/litre, respectively. Equilibrium stability constant (K) of caffeine- ofloxacin complex was calculated using the following equation and was found as 10.5 litre/mol at 37^°C.

           K =Slope/S (1-slope)_{0   `}

A few equilibrium stability constants for caffeinedrug complexes reported in the literature were 15, 18, 40 and 48 litre/mol in case of aspirin, benzoic acid, salicylic acid, and p-amino benzoic acid, respectively.⁶ Comparison of stability constant of caffeine-ofloxacin (10.5 litre/mol) with the above-mentioned stability constants of caffeine-drug complexes indicated that the formed complex was sufficiently stable; though complex could not be isolated in this method. The pH of saturated solutions at different caffeine concentrations ranged from 7.64 to 7.89. According to Higuchi & Connors and Repta^11,12 classification of complex systems, ofloxacin caffeine complex belongs to class ‘type A’ and sub-class ‘A_L’ (linear relationship). ‘Type A’ complexes are soluble and do not form a precipitate regardless of the amount of ligand added. Also, ofloxacin-caffeine complex exhibits a linear relationship between St (solubility of ofloxacin) and L_t (concentration of caffeine). Hence, it belongs to sub class ‘A_L ’.

Characterization of complexes

Solid complexes of ofloxacin-methylxanthines were prepared using co-precipitation and solvent evaporation method. Since the solid complexes are water soluble, precipitation was carried out at low temperature (10° C).

Melting point

The melting points of ofloxacin, caffeine, and theophylline obtained were 252, 235, and 270° C, respectively. Melting points of complexes of ofloxacin with caffeine and theophylline were 293 and 273° C, respectively. Both complexes exhibited elevation of melting point compared to components. These results indicated the formation of complexes of methylxanthines with ofloxacin.

Fourier transformed infrared (FTIR) spectroscopic analysis

FTIR spectroscopy is helpful to confirm the identity of the drug and to detect the interaction of the drug with the other molecular compounds. FTIR spectra of ofloxacin, caffeine, and ofloxacin-caffeine complex are shown in Figure 4. FTIR spectrum of ofloxacin shows the characteristic peaks at 3420 cm^-1 (O-H, stretch) broad and 1771 cm^-1 (COOH, stretch). Caffeine shows the characteristic peaks at 1718 cm^-1 (C=O, carbonyl exocyclic) and 1645 cm^-1 (C=C, stretch, endocyclic). FTIR spectrum of ofloxacin-caffeine complex shows broadening of band at 3336 cm^-1 and changes in the sharpness of bands at 1700-1600 cm^-1 indicate the formation of complexes.

Similarly, FTIR spectrum of theophylline (Figure is not shown) shows the characteristic peaks at 3344 cm^-1 (NH, stretch) and 1716 cm^-1  (C=O, carbonyl, exocyclic). FTIR spectrum of ofloxacin-theophylline complex shows broadening of band at 3729 cm^-1 and changes in the sharpness of bands at 1700-1600 cm^-1 indicate the formation of complexes. These results confirm the formation of complexes between methylxanthines and ofloxacin.

Differential scanning calorimetric analysis (DSC)

DSC thermograms of ofloxacin, caffeine, and ofloxacincaffeine complex are shown in Figure 5. Individual thermograms of ofloxacin and caffeine showed their melting transition temperatures at 275 and 240° C, respectively. Ofloxacin-caffeine complex showed sharp melting transition temperatures at 230° C and sharp endotherm at 270° C. In thermogram of complex, melting transitions were slightly lowered compared to respective drugs. This observation indicated complex formation between ofloxacin and caffeine.

Theophylline showed (Figure is not shown) its melting transition temperature at 270° C. Thermogram of ofloxacin-theophylline complex showed melting transition temperature at 270° C. Because melting transition temperatures of individual drugs are close to each other, it is unable to confirm the formation of complex between ofloxacin and theophylline.

In vitro transport studies of drugs

A sensitive reverse phase HPLC with a UV detector was used for estimation of ofloxacin, caffeine and theophylline samples. The run time was approximately 45 min for each sample. Retention times of ofloxacin, caffeine, and theophylline were 6.8, 5.4, and 3.9 minutes, respectively. The standard curve having ofloxacin concentrations ranging from 58 to 290 mg/ml exhibited good linearity. The equation and correlation co-efficient of the calibration curve obtained from five points were y = 261.44 x and R² = 1.00 (n = 3). Similarly, caffeine calibration curve ranging from 78 to 390 mg/ml showed good linearity (y = 103.73x and R² = 1.00), whereas theophylline calibration samples ranged from 14.4 to 72 mg/ml showed good linearity (y = 1035.35 x and R2 = 1.00).

Transport of methylxanthines in presence of ofloxacin

Data obtained from in vitro transport studies was processed and cumulative percent of caffeine diffused in presence and absence of ofloxacin was plotted against time (Figure 6). A perusal to Figure 6 indicated that the transport of caffeine was low in presence of ofloxacin throughout the course of study. Thus, it reveals that caffeine was not readily available for transportation, i.e. it might be available as complex with ofloxacin. In order to appreciate the differences of cumulative percent of caffeine transported, regression approach was employed and attained values are reported in the Table 1. Theoretically, in vitro drug diffusion is expected to follow first order process, but correlation coefficients (R²) of caffeine alone obtained in the study indicates that caffeine diffusion followed zero order kinetics. However, caffeine transport in presence of ofloxacin followed first order diffusion (R² = 0.9978). In other words, the equilibrium was predictable between unbound caffeine molecules on both sides of the dialysis membrane.

Similar observations were attained in case of transport of theophylline in presence and absence of ofloxacin. Statistically significant differences were observed between transport of theophylline alone and in presence of ofloxacin (p < 0.05). However, there were no statistically significant differences observed between transport of caffeine alone and in presence of ofloxacin. Thus, statistical results highlighted the significant interactions between theophylline and ofloxacin to a large extent compared to caffeine and ofloxacin interactions. Theophylline was not readily available for transportation, in presence of ofloxacin due to its complex form. Theophylline transport in presence of ofloxacin followed first order diffusion (R² = 0.9859). From the transport constants (coefficient of x components, Table 1), it could be understood that the transport of theophylline (0.01 min^-1) is twice faster than that of the caffeine (0.0046 min^-1).

Transport of ofloxacin in presence of methylxanthines

In vitro transport studies indicated that the transport of ofloxacin was low in presence of methylxanthines throughout the course of study. Thus, it reveals that ofloxacin was not readily available for transportation, i.e. it might be available as complex with methylxanthines. From regression analysis, it is understood that transport of ofloxacin followed zero order kinetics (R² = 0.9106). However, in presence of caffeine and theophylline, the transport of ofloxacin followed first order (R² = 0.9988).

In situ absorption of drugs through rat gut

The absorption of caffeine and theophylline individually and in presence of ofloxacin was studied by rat gut method. Similarly, absorption of ofloxacin in presence and absence of methylxanthines was studied.

Absorption of methylxanthines in presence of ofloxacin

Data obtained from in situ absorption studies was processed and cumulative amount of caffeine absorbed in presence and absence of ofloxacin was plotted against time (Figure 7). A perusal to Figure 7 indicated that the absorption of caffeine was increased in the initial periods of study, in the presence of ofloxacin. Dissimilar caffeine absorption in presence of ofloxacin compared to caffeine alone was due to large molecular size of their complex formed. In order to appreciate the differences of absorption, regression approach was applied on absorption profile, rather than cumulative drug unabsorbed and attained values are reported in the Table 2. Correlation coefficients (R²) of caffeine alone obtained in the study helps to understand that caffeine absorption was found to follow first order kinetics. In other words, absorption was dependent on available concentration. However, in presence of ofloxacin, the caffeine absorption followed zero order kinetics. This showed the formation of complex between caffeine and ofloxacin. This formed complex acted as reservoir and continuously released the free caffeine molecules for absorption, i.e., absorption is independent of concentration.

The absorption of theophylline was decreased throughout the period of study, in the presence of ofloxacin (figure is not shown). Statistically significant differences were observed between absorption of theophylline alone and in presence of ofloxacin (p < 0.05). The possible reason for reduced theophylline absorption was due to large molecular size of complex molecules. Theophylline absorption alone and in presence of ofloxacin followed zero order kinetics (Table 2).

Absorption of ofloxacin in presence of methylxanthines

 Clear differences in the absorption of ofloxacin in absence and presence of caffeine were not found. (figure is not shown). Statistically significant differences were observed between absorption of ofloxacin alone and in presence of theophylline (p < 0.05). However, there were no statistically significant differences observed between absorption of ofloxacin alone and in presence of caffeine. Ofloxacin absorption alone followed first order kinetics, whereas in presence of caffeine followed zero order kinetics. The absorption of ofloxacin was enhanced in presence of theophylline. Ofloxacin absorption in presence of theophylline followed zero order kinetics. As explained above, complexes were formed between ofloxacin and methylxanthines, and acted as reservoir to exhibit zero order kinetics. Statistical results highlighted the significant interactions between ofloxacin and theophylline to a large extent compared to ofloxacin and caffeine interactions. The absorption rate constants were calculated from the slopes of the log percent drug unabsorbed versus time plots. Absorption rate constant of caffeine, theophylline and ofloxacin were 4.44, 5.25, and 3.70 h^-1 respectively. The absorption rate of theophylline was higher than that of the caffeine.

Conclusion

Solubility studies indicated that caffeine and theophylline had enhanced the solubility of ofloxacin. The increase in solubility of ofloxacin in presence of methylxanthines provided the prima facie evidence of molecular association of methylxanthines with ofloxacin. Stability constants determined for the complexes of methylxanthines with ofloxacin are in line with stable complexes found in literature and thus strengthen the evidence of formation of complex.

Solid complexes of ofloxacin-methylxanthines were prepared using co-precipitation and solvent evaporation method in spite of higher aqueous solubility of ofloxacin-methylxanthine complexes. FTIR and DSC results confirm the formation of complexes between methylxanthines and ofloxacin.

In vitro diffusion studies indicated that in presence of ofloxacin, methylxanthines’ transport is reduced due to formation of complex.  It means that methylxanthines were not readily available for transportation, as these may exist in complex form. Similarly, in presence of methylxanthines, transport of ofloxacin is also decreased. The transport of methylxanthines is concentration dependent i.e., followed first order. From the co-efficient of transport equations, it could be understood that the transport of theophylline is twice faster than that of the caffeine.

In situ rat gut absorption studies indicated that in presence of ofloxacin, the absorption of caffeine was almost unaltered, whereas theophylline absorption reduced compared to their individual absorptions. In presence of ofloxacin, the absorption of methylxanthines was found to follow zero order kinetics. This demonstrated the formation of complex. The complex acted as a reservoir and continuously released the free methylxanthine molecules. The absorption of ofloxacin was enhanced in presence of theophylline. Statistical results showed that theophylline had greater interaction with ofloxacin than interactions of caffeine with ofloxacin. It is also found that the absorption rate of theophylline through rat gut was higher than that of the caffeine.