Clinical Research and Regulatory Affairs, 2009; 26(4): 7383

RESEARCH ARTICLE

Comparison of effect of fasting and of five different diets on the bioavailability of single oral dose of clarithromycin 500mg extended release tablet
Sanjay J. Gurule1, Tausif Monif1, Priya Ranjan Prasad Verma 2, and Arshad H. Khuroo1
Ranbaxy Research Laboratories, Gurgaon, Haryana, India, and 2Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi, India
1

Abstract The objective of this cross-over bioavailability study on clarithromycin was to compare the bioavailability under fasting and five different diets, in 18 healthy adult male human volunteers using validated LC-MS/MS method. A single dose of clarithromycin 500mg extended release tablet was administered at six occasions: after overnight fasting, after two vegetarian diets (high fat and low fat), two non-vegetarian diets (high fat and low fat), and low fat vegetarian rice. Serial blood samples were collected up to 36h after dose. A statistically significant food effect was observed for all diets when compared to fasting treatment. Keywords: Clarithromycin; LC-MS/MS; bioavailability

Introduction
Fooddrug interactions can lead to alterations in the pharmacokinetic and pharmacodynamic profile of various drugs that may have clinical implications. Food may interact with co-administered drugs in various phases: (a) before and during gastrointestinal absorption; (b) during distribution; (c) during metabolism; and (d) during elimination. However, food has most effect during absorption and metabolism phases (1). There is considerable evidence to suggest that the absorption of various drugs is influenced by the presence of food in the gastrointestinal tract (2). The effects of food are not always predictable and can have clinically significant consequences. Some effects of food on the bioavailability of a drug includes: changes in gastric emptying, drug chelation, changes in the activity of drug metabolizing enzymes, changes in splanchnic blood flow and plasma protein binding, physical or chemical interaction of the meal with the drug product or drug substance, and changes in the

metabolic transformation of the drug by the gastrointestinal wall or liver (35). Clarithromycin is a semi-synthetic macrolide antibiotic containing a 14-membered lactone ring (6, 7) which has a unique principle metabolite (14-hydroxy clarithromycin) that has activity equal to or greater than that of the parent drug (810). Clarithromycin is active intracellularly, and its action is static or bactericidal, depending on the concentration and the organism. Similar to erythromycin and azithromycin, clarithromycin demonstrated activity against Mycobacterium avium complex (MAC) (1114). The activity of clarithromycin is enhanced by the formation of its active metabolite, 14-hydroxy clarithromycin, and by its extensive distribution into the tissues. Both parent and metabolite has been shown to inhibit the strains of Haemophilus influenzae in an additive and synergistic mode (15, 16). Clarithromycin extended-release tablets are used for the treatment of adults with mild-to- moderate infection caused by susceptible strains of the

Address for Correspondence: Sanjay J. Gurule, Ranbaxy Research Laboratories. Plot No. GP-5, Sector 18, HSIDC, Old DelhiGurgaon Road, Gurgaon 122 015, Haryana, India. E-mail: sanjay.gurule@ranbaxy.com (Received 20 April 2009; revised 11 July 2009; accepted 29 July 2009) ISSN 1060-1333 print/ISSN 1532-2521 online 2009 Informa UK Ltd DOI: 10.3109/10601330903252198 http://www.informahealthcare.com/crr

74 Sanjay J. Gurule etal.

designated microorganisms in the conditions stated below:

1. Acute maxillary sinusitis due to Haemophilus influenzae, Moraxella catarrhalis, or Streptococcus pneumonia; 2. Acute bacterial exacerbation of chronic bronchitis due to Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, or Streptococcus pneumonia; and 3. Community-acquired pneumonia due to Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, Streptococcus pneumoniae, Chlamydia pneumoniae (TWAR), or Mycoplasma pneumoniae (17).

The pharmacokinetics and tolerability of clarithromycin extended release (ER) have been discussed in several studies. Guay etal. (18) described the results of three studies of the steady-state pharmacokinetic profiles of clarithromycin and its active metabolite 14-hydroxy clarithromycin after multiple oral doses of clarithromycin 500mg ER tablets once daily, as well as the effect of administration in the fasting and fed regimen. They found that the bioavailability (AUC) of the ER tablet was 30% lower when administered under fasting compared with fed conditions. Gaete et al. (19) performed the comparative bioequivalence study on two formulations of clarithromycin 500-mg modified release tablets on 16 healthy male volunteers. The plasma clarithromycin concentrations were determined by microbiologic assay. The two products were found to be bioequivalent according to US Food and Drug Administration (FDA) guidelines. Cheng etal. (20) investigated the effect of grapefruit juice on inhibition of clarithromycin metabolism. Twelve healthy subjects were given water or grapefruit juice before and after a clarithromycin dose of 500mg in a randomized cross-over study. They found that administration of grapefruit juice increased the time to peak concentration of both clarithromycin and 14-hydroxy clarithromycin, but did not affect other pharmacokinetic parameters. In a 26 healthy volunteer study, Chu et al. (21) investigated that food intake before administration of clarithromycin immediate-release tablets increases the bioavailability of clarithromycin by ~ 25%. They also concluded that this increase was considered to have little or no clinical significance and immediate-release clarithromycin could be taken with or without food. Alkhalidi et al. (22) evaluated the pharmacokinetic parameters of clarithromycin extended 500mg tablet under fasting and fatty meal conditions in

38 Jordanian volunteers. The Cmax and AUC for test and reference formulation in fed condition were found to be increased, concluding the effect of food. Clarithromycin extended-release tablets provide lower and later steady-state peak plasma concentrations and equivalent 24-h AUCs for both clarithromycin and its active metabolite 14-hydroxy clarithromycin relative to an equal total daily dose of clarithromycin immediate-release tablets. It also provides extended absorption of clarithromycin from the gastrointestinal tract after oral administration. Although the extent of formation of 14-hydroxy clarithromycin after oral administration of clarithromycin extended release tablets (two 500-mg tablets once daily) was not affected by food, administration under fasting conditions was associated with an ~30% lower clarithromycin AUC relative to administration with food (18). Therefore, it is recommended that clarithromycin extended-release tablets should be administered with food (17). The objective of the present study was to assess the effect of different dietary status on the clarithromycin extended release 500mg tablet. India is a country largely inhabited by vegetarians and most non-vegetarians cannot afford to consume this diet more than once or twice a week. It will therefore be worthwhile to study the effect of isocaloric vegetarian and non-vegetarian diets (each with low and high fat content) on the oral bioavailability of clarithromycin extended release formulation. In this study,
1. Plasma concentrations were determined by rapid and sensitive LC-MS/MS method which is highly selective for clarithromycin and its active metabolite 14-hydroxy clarithromycin (23). 2. Complete validation as per regulatory requirements has been done before initiating the project. All parameters met acceptance limits. 3. Study was conducted with GCP and GLP compliance. 4. Almost all types of meals were taken into account to check drugfood interaction.

Experimental methods
Materials Clarithromycin and erythromycin were procured from USP. 14-hydroxy clarithromycin was supplied from analytical department of Ranbaxy Research Laboratory Gurgaon. Methanol and acetonitrile were of HPLC grade purchased from Sigma-Aldrich

Bioavailability of single oral dose of clarithromycin 75

(USA). Ammonium acetate was obtained from Fluka (Buchs, Switzerland). Water was purified using Milli-Q device (Millipore, Moscheim Cedex, France). Drug-free human plasma of healthy volunteers was obtained from Ranbaxy Clinical Pharmacology Unit (Majeedia, New Delhi, India). To ensure the safety during usage, all batches of plasma were screened for Hepatitis B and C, HIV (human immunodeficiency virus) 1 and 2, malaria, and syphilis. Human plasma batches, free of significant interference, were used to prepare calibration standards and quality control samples. Clinical study design The protocol was reviewed and approved by Jamia Hamdard Institution Review Board (IRB). All volunteers were healthy adult males who gave written consent before participating in the study. The protocol had pre-defined inclusion/exclusion criteria. The average ( SD) age and weight of volunteers was 27.25.9 years and 54.95.8kg, respectively (Table 1). Prior to dosing, each volunteer had undergone physical examination and laboratory tests of hematologic, hepatic, and renal functions. Only medically healthy

volunteers with clinically normal laboratory profiles were enrolled in the study. The study was designed as an open label, balanced, randomized six-treatment, six-period, sixsequence, single-dose, cross-over bioavailability study in 18 healthy, adult, male, human volunteers. None of the enrolled volunteers had history of allergy to clarithromycin and other macrolide antibiotics. Volunteers did not receive any medication during the study and 2 weeks prior to start of study. All the volunteers abstained from xanthine-containing food or beverages or alcohol products for 48h prior to dosing and during housing in each period as instructed. Volunteers were admitted in the clinical pharmacology unit from 12h before dose and were discharged 24h after dose during each period. After discharge ambulatory sample collection was done at 36h. A clinical nutritionist prepared five different study diets. The order of receiving treatments for each volunteer during the six periods of the study was determined according to SAS generated randomization schedule. Each volunteer received one 500mg clarithromycin extended release tablet (Ranbaxy Laboratories Ltd.) with 240mL of drinking water according to the following treatments on six different days separated by 5 days wash-out period.
Treatment A: A single oral dose of clarithromycin 500mg XL tablet under fasting conditions. Treatment B: A single oral dose of clarithromycin 500mg XL tablet within 30min of intake of a high-fat vegetarian diet. Treatment C: A single oral dose of clarithromycin 500mg XL tablet within 30min of intake of a high-fat non-vegetarian diet. Treatment D: A single oral dose of clarithromycin 500mg XL tablet within 30min of intake of a low-fat vegetarian diet. Treatment E: A single oral dose of clarithromycin 500mg XL tablet within 30min of intake of a low-fat non-vegetarian diet. Treatment F: A single oral dose of clarithromycin 500mg XL tablet within 30min of intake of a low-fat vegetarian rice diet. High-fat vegetarian diet (B) was composed of whole milk (200mL), cheese pakora (55g), one cheese toast, cheese (30g), butter (18g), green chatni (15g), and roasted peanuts (20g). High-fat non-vegetarian diet (C) was composed of chicken (60g), whole milk (240mL), hash brown potato 120 (g), two fried eggs, two bread slices, and butter (8g).

Table 1. Demographic details of study subjects. Parameter Statistics Age (years) n Mean SD CV Minimum Maximum Age category: n (%) 1825 2630 3135 > 35 Gender: n (%) Male Female Height (cm) n Mean SD CV Minimum Maximum Weight (kg) n Mean SD CV Minimum Maximum SD: Standard deviation; CV: Coefficient of variation.

n = 18 18 27.2 5.9 21.8 19 40 8 (44%) 5 (28%) 3 (17%) 2 (11%) 18 (100%) 0 18 165.1 5.0 3.0 157 176 18 54.9 5.8 10.6 46 66

76 Sanjay J. Gurule etal. Low-fat vegetarian diet (D) was composed of four bread slices, paneer (35g), dalia (250g), and fruit juice 240 (mL). Low-fat non-vegetarian diet (E) was composed of four toast slices, one boiled egg, boneless chicken (low fat) (30g), dalia (220g), fruit juice (240mL), and one banana. Low-fat vegetarian rice diet (F) was composed of cooked poha (300g), kheer and skimmed milk powder (300g), and one banana.

Pharmacokinetic methodology WinNonlin 5.0.1 pharsight was used for pharmacokinetic analysis of the data. The non-compartmental method was used to determine Cmax, Tmax, and AUC. The area under the curve to the last measurable concentration (AUC0t) was calculated by the linear trapezoidal rule. The area under the curve extrapolated to infinity (AUC0inf ) was calculated as AUC0t + Ct/Kel, where Ct is the last measurable concentration. The elimination rate constant (Kel) was obtained as the slope of the linear regression of the log transformed plasma concentration values vs time in the terminal phase. Terminal half life (t1/2) was calculated as 0.693/Kel. The time>MIC (time greater than minimum inhibitory concentration) and AUC>MIC at 0.125, 0.25, and 0.5g/mL were calculated. Bioavailability of clarithromycin and its metabolite 14-hydroxy clarithromycin under fasting conditions and with different diets were evaluated. Effect of fat (high-fat/ low-fat), effect of vegetarian and non-vegetarian diets, and effect of rice on the bioavailability of clarithromycin 500mg extended release tablet was evaluated. Statistical analysis Statistical analysis was performed on log transformed pharmacokinetic parameter Cmax and AUC. The log-transformed pharmacokinetic parameters were analyzed using a mixed effects ANOVA model using Type III sum of squares, with the main effects of sequence, period, and formulations as fixed effects and subjects nested within sequence as random effect. Each analysis of variance included calculation of least-squares means, the difference between the adjusted formulation means, and the standard error associated with the difference, and a p-value less than 0.05 (5% level of significance) was considered statistically significant for different source of variation. p-value was determined using student t-test for Cmax ; AUC0t and AUC0inf SAS version 9.1 was used as the software. Ratio of means was calculated using the LSM for log-transformed Cmax, AUC0t, and AUC0.

The blood samples were collected at pre-dose and at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 24, and 36h post-dose in each period. Plasma was separated from blood and plasma samples were stored below 15C until analysis. Tolerability Tolerability was assessed based on changes in vital signs (temperature, sitting blood pressure, and radial pulse), measured during volunteer admission, prior to dosing, and every 4h after administration of study drug in each period until discharge. In addition, a physician questioned volunteers about any adverse events occurring during the study, addressed them as necessary, and recorded them on the appropriate raw data forms. Product assays and in vitro dissolution testing Assays to determine the clarithromycin content of the formulation and in vitro dissolution studies were performed at Ranbaxy Research Laboratory. Dissolution was performed using the US Pharmacopeia (USP) method 2 (24) with paddles rotating at 100rpm. The temperature of dissolution media was maintained at 370.5C and samples were withdrawn at specified intervals up to 24h. The percentage of drug dissolved was calculated based on drug concentrations. Analysis of clarithromycin and 14-hydroxy clarithromycin in plasma The concentration of clarithromycin and 14- hydroxy clarithromycin were determined by a validated LC-MS/MS method. The method was validated in terms of selectivity, precision/accuracy, recovery, dilution integrity, matrix effect and stability studies (23).

Results
There were no significant protocol deviations during the study and no drug-related adverse event has been observed. Of the 18 healthy adult male volunteers originally enrolled in the study, three withdrew from

Bioavailability of single oral dose of clarithromycin 77

different periods of the study for personal reasons. There was no significant difference between volunteers in terms of demographic data. The data was analyzed on 15 volunteers who completed all six periods of the study. One set of calibration standards and two sets of quality control samples were used for analysis of each plasma volunteer samples. All batches passed the batch acceptance criteria. Figures 1 and 2 show the mean profile of clarithromycin and 14-hydroxy clarithromycin for single dose
Linear Plot of Mean Clarithromycin Concentration Versus Time 2000 1800 1600 1400 1200 1000 800 600 400 200 0 Clarithromycin Mean Concentration in Plasma (ng/mL) A B C D E F A Error B Error C Error D Error E Error F Error

of clarithromycin 500mg XL tablet from 15 healthy human male volunteers for all treatments. Tolerability Extended release tablet formulation of clarithromycin was well tolerated by all volunteers. No adverse effects were observed during the study, and no volunteer left the study with changes from baseline in vital signs or laboratory test results. Product assays and in vitro dissolution testing As per the FDA requirements for bioavailability and bioequivalence studies of orally administered drugs, the difference between drug content of test product and reference product should be<5% (25). In this study a single formulation was used and clarithromycin content was found to be 100.4%. Effect of food on the bioavailability of clarithromycin Refer to Tables 2 and 3 for mean SD (standard deviation) of the pharmacokinetic parameters viz Tmax, Cmax, AUC0t, AUC0inf, Kel, T1/2, mean residence time, apparent volume of distribution, and clearance after administration of clarithromycin 500mg XL tablet following fasting and five different diet treatments. The Tmax of clarithromycin in the fasting state was 8.534.71h. Tmax observed was 6.472.92h for high-fat vegetarian diet, 6.261.79h for high-fat non-vegetarian diet, 7.202.11h for low-fat vegetarian diet, 6.261.03h for low-fat non-vegetarian diet, and 6.333.24h for low-fat rice diet. The Tmax of 14-hydroxy clarithromycin in the fasting state was 7.003.25h. The Tmax was 7.732.28h for high-fat vegetarian diet, 8.131.92h for low-fat vegetarian diet, 7.731.98h for low-fat non-vegetarian diet, and 7.203.9h for low fat rice diet. The Tmax for high-fat non-vegetarian diet remained the same as fasting state. An increase in Cmax was observed with all diets (treatments B, C, D, E, and F) for clarithromycin and 14-hydroxy clarithromycin. The maximum increase in Cmax was observed with the high-fat vegetarian diet for both clarithromycin and 14-hydroxy clarithromycin (199% and 153% high when compared to fasting, respectively), and minimum increase was observed with the low fat rice diet. AUC0t also increased like Cmax with all diets for clarithromycin and 14-hydroxy clarithromycin, and

10

15

20 Time (hr)

25

30

35

40

Figure 1. Mean plasma concentration ( SD) time profile of clarithromycin after single oral dose of 500mg XL tablet (after fasting and five different diets) to healthy, human subjects (n=15). Treatment A (fasting), B (high-fat vegetarian), C (high-fat nonvegetarian), D (low-fat vegetarian), E (low-fat non-vegetarian), F (low-fat rice).

Linear Plot of Mean 14-Hydroxy Clarithromycin Concentration Versus Time 14-Hydroxy Clarithromycin Mean Concentration in Plasma (ng/mL) 700 600 500 400 300 200 100 0 0 5 10 15 20 Time (hr) 25 30 35 40 A B C D E F A Error B Error C Error D Error E Error F Error

Figure 2. Mean plasma concentration ( SD) time profile of 14-hydroxy clarithromycin after single oral dose of 500mg XL tablet (after fasting and five different diets) to healthy, human subjects (n=15). Treatment A (fasting), B (high-fat vegetarian), C (high-fat non-vegetarian), D (low-fat vegetarian), E (low-fat nonvegetarian), F (low-fat rice).

and clearance after

78 Sanjay J. Gurule etal.

Low-fat rice (F) 6.333.24 1,327.73456.95* 18,359.835,443.58 19,624.775,276.02 0.100.03 7.141.94 14.662.25 286.61129.12 27.247.43

Table 2. Mean SD (standard deviation) of the pharmacokinetic parameters viz Tmax, Cmax, AUC0t, AUC0inf, Kel, T1/2, MRT, apparent volume of distribution, administration of clarithromycin 500mg XL tablet following fasting and five different diet treatments (n=15) for clarithromycin. High-fat High-fat Low-fat Low-fat Fasting (A) vegetarian (B) non-vegetarian (C) vegetarian (D) non-vegetarian (E) Tmax (h) 8.534.71 6.472.92 6.261.79 7.202.11 6.261.03 983.73387.90 Cmax 1,958.65808.72* 1,888.23546.12* 1,797.71682.61* 1,903.31982.40* (ng/mL) AUC0t (h.ng/mL) 17,879.3210,264.70 21,322.467,593.70* 21,843.886,881.24* 22,037.829,446.55* 21,882.176,816.36* 20,371.1711,214.07 AUC0inf (h.ng/mL) 22,567.897,726.06* 23,011.876,538.90* 23,252.159,505.83* 23,125.296,508.92* 0.100.04 0.120.02 0.110.02 0.120.04 0.110.02 Kel (h1) 7.873.77 6.611.39 6.451.30 T1/2 (h) 5.960.88* 6.381.54* Mean residence time (h) 15.835.27 13.152.19 14.101.48 14.211.75 14.151.53 Apparent volume of 387.35324.40 210.1174.17 229.45101.37 221.8088.76 224.1697.87 distribution (L) Clearance (L/h) 37.1530.58 24.567.91 23.617.29 24.317.95 23.467.30 * p-value less than 0.05 indicates significant effect when compared to fasting treatment.

Table 3. Mean SD (standard deviation) of the pharmacokinetic parameters viz Tmax, Cmax, AUC0t, AUC0inf, Kel, T1/2, MRT, apparent volume of distribution and cearance after administration of clarithromycin 500mg XL tablet following fasting and five different diet treatments (n=15) for 14-hydroxy clarithromycin. High-fat High-fat Low-fat Low-fat Fasting (A) vegetarian (B) non-vegetarian (C) vegetarian (D) non-vegetarian (E) Low-fat rice (F) Tmax (h) 7.003.25 7.732.28 7.002.30 8.131.92 7.731.98 7.203.91 421.09127.28 447.50162.76 Cmax (ng/mL) 645.43178.65* 641.22161.72* 629.33119.48* 625.24129.90* 9,117.053,808.76 8,617.403,038.23 AUC0t (h.ng/mL) 10,604.212,687.07* 10,661.422,998.38* 10,661.562,721.22* 10,640.111,761.24* 11,607.835,113.51 11,832.062,799.98 12,074.863,699.03 11,960.523,047.78 11,834.272,015.23 9,859.243,657.20 AUC0inf (h.ng/mL) 0.060.03 0.080.02 0.080.02 0.080.02 0.080.02 0.070.02 Kel (h1) 13.076.37 10.513.18 T1/2 (h) 9.002.59* 9.843.08* 9.682.71* 9.481.85* Mean residence time (h) 22.669.25 17.903.40 19.013.49 18.993.31 18.771.92 19.474.28 Apparent volume of 927.76495.75 578.86224.59 607.96179.73 602.64170.89 591.56144.81 851.77374.52 distribution (L) Clearance (L/h) 55.5633.53 44.7711.97 44.5912.11 44.5412.64 43.609.06 57.0819.30 * p-value less than 0.05 indicates significant effect when compared to fasting treatment.

Bioavailability of single oral dose of clarithromycin 79

maximum increase was observed with the low-fat vegetarian diet (123% and 116% high when compared to fasting, respectively). There was no significant difference in values of AUC0inf when diets were compared with fasting for clarithromycin and 14-hydroxy clarithromycin. The maximum increase (114% high when compared with fasting) was observed with low-fat vegetarian diet for clarithromycin. Ratio of means (geometric means (LSM) based on log transformed data) and 90% confidence intervals for all the diet treatments vs fasting for Cmax, AUC0t, and AUC0inf are shown in Tables 4 and 5 for clarithromycin and 14-hydroxy clarithromycin, respectively. A decrease in half-life was observed when the drug was administered under non-fasting conditions. Half-life values observed under fasting condition for clarithromycin and 14-hydroxy clarithromycin was 7.873.77h and 13.076.37h, respectively, and maximum decrease (5.960.88 and 9.002.59, respectively) was observed with high-fat vegetarian diet. A significant effect was not observed for elimination rate constant under fasting and non-fasting conditions for both clarithromycin and 14-hydroxy clarithromycin. Effect of fat (low vs high) on the bioavailability of clarithromycin Cmax, AUC, and Tmax remain the same between high-fat vegetarian and non-vegetarian diets for

larithromycin and 14-hydroxy clarithromycin. There c was no significant change in half-life and Kel also. Effect of vegetarian and non-vegetarian diets on the bioavailability of clarithromycin There was no significant effect on the bioavailability when comparison is made between high-fat and low-fat vegetarian and non-vegetarian diets. A slight difference in values of Tmax, Kel, and half-life was observed from vegetarian to non-vegetarian diets for clarithromycin and 14-hydroxy clarithromycin. Statistical evaluation (treatment effect using least square means) Significant effect was observed for Cmax, AUC0t, and AUC0inf, and Cmax, AUC0t when fasting was compared against other diets for clarithromycin and 14-hydroxy clarithromycin, respectively. For Cmax, p-value less than 0.05 was observed (0.001 and<0.0001, respectively) between low-fat vegetarian diet and low-fat rice diet for both clarithromycin and 14-hydroxy clarithromycin. When the effect of low fat rice was compared against other diets, a significant effect was observed with all diets for Cmax for clarithromycin and Cmax, AUC0t, and AUC0inf for 14-hydroxy clarithromycin. p-values greater than 0.05 were observed when the effect of fat (high-fat and low-fat diets) and the effect of source (vegetarian and non-vegetarian diets) was evaluated, indicating an insignificant effect.

Table 4. Ratio of means (least square means (LSM) based on log transferred data) and 90% confidence intervals for all the diet treatments vs fasting for Cmax, AUC0t, and AUC0inf for clarithromycin. Cmax Treatment comparison Fasting (A) vs high-fat vegetarian (B) Fasting (A) vs high-fat non-vegetarian (C) Fasting (A) vs low-fat vegetarian (D) Fasting (A) vs low-fat non-vegetarian (E) Fasting (A) vs low-fat rice (F) Ratio (%) 195.05 198.89 183.59 194.77 137.62 CI (%) 178.09213.63 181.60217.83 167.63201.08 177.83213.32 125.65150.72 Ratio (%) 135.78 140.76 137.53 139.43 116.86 AUC0t CI (%) 122.60150.39 127.09155.90 124.17152.32 125.89154.43 105.51129.42 Ratio (%) 128.55 133.38 130.14 132.73 112.74 AUC0inf CI (%) 115.64142.89 119.99148.26 117.08144.66 119.40147.54 101.42125.32

Table 5. Ratio of means (least square means (LSM) based on log transferred data) and 90% confidence intervals for all the diet treatments vs fasting for Cmax, AUC0t, and AUC0inf for 14-hydroxy clarithromycin. Cmax Treatment comparison Fasting (A) vs high-fat vegetarian (B) Fasting (A) vs high-fat non-vegetarian (C) Fasting (A) vs low-fat vegetarian (D) Fasting (A) vs low-fat non-vegetarian (E) Fasting (A) vs low-fat rice (F) Ratio (%) 154.04 154.31 152.97 152.19 104.80 CI (%) 142.33166.72 142.58167.02 141.33165.56 140.61164.72 96.83113.43 AUC0t Ratio (%) 124.06 124.49 124.52 126.33 97.89 CI (%) 112.84136.39 113.23136.87 113.26136.90 114.91138.89 89.04107.62 Ratio (%) 110.77 111.65 110.92 111.77 88.96 AUC0inf CI (%) 100.51122.08 101.31123.05 100.65122.25 101.41123.17 80.7298.04

80 Sanjay J. Gurule etal.

Pharmacokineticpharmacodynamic (PK-PD) analysis Time>MIC and AUC>MIC was calculated for clarithromycin and 14-hydroxy clarithromycin at 0.125, 0.25, and 0.5g/mL (Tables 6 and 7). There is no significant effect on Time>MIC when different diets are compared with fasting; however, the diet treatments used in the study have a significant effect when AUC>MIC was compared with fasting for clarithromycin. The diet treatments used in the study have a significant effect when Time>MIC and AUC>MIC was compared with fasting for 14-hydroxy clarithromycin at 0.5g/mL.

Discussion
Interactions between food and drugs may inadvertently reduce or increase the drug effect. The majority of clinically important fooddrug interactions are caused by food-induced changes in the bioavailability of the drug. Since the bioavailability and

clinical effect of most drugs are correlated, the bioavailability is an important pharmacokinetic effect parameter (26). The type and size of a meal may have a marked effect on the nature of a drugfood interaction. Liquid meals, which are often used in an attempt to obtain mechanistic information, might have a totally different effect on drug absorption compared with solid meals, which are nonetheless more clinically relevant. The time interval between eating and medication will also affect the nature and extent of a drugfood interaction. Knowing that both the nature of the food and formulation of the drug play an important role in bioavailability, it becomes essential to precisely define their interaction (27). Alkhalidi etal. (22) reported the arithmetic mean (SD) Cmax for the test and reference formulation under fasting condition as 569.4 (189.3) ng/mL and 641.2 (202.0) ng/mL, respectively. The arithmetic mean AUC0t was 8602.9 (4105.1) and 8245.3 (4122.4) ngh/mL in the respective formulations. The arithmetic mean Tmax was 8.0 (5.6) and 6.1 (3.8) h. In the fed study, the Cmax and AUC of both formulations were significantly

Table 6. Time and AUC above MIC (0.125, 0.25, and 0.5g/mL) for clarithromycin. AUC>MIC AUC>MIC (0.25g/mL) in (0.125g/mL) in Time>MIC Time>MIC h.g/mL h.g/mL (0.25g/mL) in h Treatments (0.125g/mL) in h Fasting (A) 29.537.65 14.229.55 24.3210.01 10.878.69 High-fat 29.543.21 17.787.36 23.583.75 14.497.03 vegetarian (B) High-fat 30.633.36 18.036.37 24.794.66 14.585.96 non-vegetarian (C) Low-fat 29.684.12 18.359.13 24.304.71 15.008.75 vegetarian (D) Low-fat 30.191.92 18.146.48 24.163.17 14.776.21 non-vegetarian (E) Low-fat rice (F) 30.623.09 14.505.18 24.594.36 11.054.94 MIC: Minimum inhibitory concentration.

Time>MIC (0.5g/mL) in h 16.6510.35 15.743.69 16.885.02 16.354.81 16.243.84 15.854.86

AUC>MIC (0.5g/mL) in h.g/mL 5.746.61 9.636.29 9.464.94 9.997.83 9.835.48 6.054.26

Table 7. Time and AUC above MIC (0.125, 0.25, and 0.5g/mL) for 14-hydroxy clarithromycin. AUC>MIC AUC>MIC (0.25g/mL) in (0.125g/mL) in Time>MIC Time>MIC h.g/mL h.g/mL (0.25g/mL) in h Treatments (0.125g/mL) in h Fasting (A) 28.488.90 5.163.26 16.9611.23 2.212.29 High-fat 28.883.80 6.642.44 18.984.80 3.681.96 vegetarian (B) High-fat 28.923.45 6.652.76 19.275.68 3.652.20 non-vegetarian (C) Low-fat 28.804.30 6.692.42 19.615.22 3.691.91 vegetarian (D) Low-fat 29.202.50 6.581.68 19.393.71 3.571.35 non-vegetarian (E) Low-fat rice (F) 27.395.07 4.592.83 15.128.63 1.972.06 MIC: Minimum inhibitory concentration.

Time>MIC (0.5g/mL) in h 1.914.80 5.784.87 5.674.35 6.334.13 4.913.66 2.453.92

AUC>MIC (0.5g/mL) in h.g/mL 0.090.29 0.800.81 0.680.80 0.640.73 0.540.52 0.240.52

Bioavailability of single oral dose of clarithromycin 81

increased relative to the fasting study. The arithmetic mean Cmax of the two formulations was 1183.0 (637.5) and 1199.6 (496.3) ng/mL. The arithmetic mean AUC0t was 12,981.2 (7849.0) and 11,822.9 (5790.2) ngh/mL. The arithmetic mean Tmax was 5.7 (2.8) and 6.7 (2.5) h. In another study, Guay et al. (18) reported fasting Cmax and AUC as 2.330.70g/mL and 35.912.4g.h/mL and fed 3.911.04g/mL and 49.210.5g.h/mL, respectively, for clarithromycin. Fasting Cmax and AUC were 0.850.27g/mL and 14.23.7g.h/mL and fed 0.850.20g/mL and 14.63.1g.h/mL, respectively, for 14-hydroxy clarithromycin. There is no significant difference in Tmax values under fasting and non-fasting conditions for clarithromycin, whereas it increased in non-fasting conditions for 14-hydroxy clarithromycin. This indicates that food has a significant effect on the pharmacokinetic parameters of clarithromycin. Since a vast majority of the Indian population is vegetarian, it seemed prudent to further define the interaction with the administration of clarithromycin extended release tablet formulation along with equicaloric diets which varied both in content and source. Hence this study was planned to compare the effect of five different diets, viz. high-fat vegetarian diet, high-fat non-vegetarian diet, low-fat vegetarian diet, low-fat non-vegetarian, low-fat rice, and fasting state on the bioavailability of clarithromycin in normal human subjects. The fasting state served as reference for comparison. In this study, a 500mg oral dose of clarithromycin extended tablet under fasting conditions produced a Cmax of 983.73387.90ng/mL and 421.09127.28ng/mL for clarithromycin and 14-hydroxy clarithromycin, respectively. This value is in close agreement with Guay et al. (18) (Cmax 1160ng/mL and 425ng/mL for clarithromycin and 14-hydroxy clarithromycin, respectively, if extrapolated to a single dose). The Cmax value reported by Alkhalidi et al. (22) under fasting condition is low compared to this study for clarithromycin. In this study, the AUC0t values observed under fasting condition were 17,879.3210,264.70h.ng/mL and 9117.053808.76h.ng/mL for clarithromycin and 14-hydroxy clarithromycin, respectively. This value is in close agreement with Guay et al. (18). The AUC0t value reported by Alkhalidi etal. under fasting condition is 8602.9h.ng/mL for clarithromycin. Increases in Cmax and AUC values were observed in this study, when clarithromycin was given under non-fasting conditions compared to fasting condition. The increase in the values was not the same for all diets when compared with the fasting treatment. The maximum increase in Cmax was observed when clarithromycin was administered with a high-fat

vegetarian diet. The maximum increase in AUC was observed with low-fat vegetarian diet. The reason could be one of the meal components in these diets. In general, fatty meals will increase the extent of bioavailability of BCS class 2 compounds. This might be the probable cause of increasing the overall bioavailability of clarithromycin, being a BCS class 2 compound. This increased bioavailability with a concomitant high-fat meal could be due to inhibition of efflux transporters, such as P-gp, in the intestine (28). There was no significant difference in values of Tmax when diets were compared with fasting. p- values above 0.05 were observed when fasting Tmax was compared with other diets for both clarithromycin and 14-hydroxy clarithromycin, indicating an insignificant effect. Tmax values observed for both clarithromycin and 14-hydroxy clarithromycin are as per the reported literature (18, 22). In our study, the half-life under fasting condition was 7.873.77h and 13.076.37h for clarithromycin and 14hydroxy clarithromycin, respectively. A decrease in half-life was observed when the drug was administered under non-fasting conditions. The maximum decrease was observed with high-fat vegetarian diet. However, Alkhalidi et al. (22) reported an increase in half-life in non-fasting conditions compared to fasting. The confidence intervals of Cmax and AUC0t for log transformed data for treatment comparisons of fasting against all diets are not within 80125% for clarithromycin and 14-hydroxy clarithromycin, except for the low-fat vegetarian rice diet for 14-hydroxy clarithromycin. Thus, the results indicate a significant effect of food in Indian volunteers. PK-PD analysis Patterns of antimicrobial activity fall into one of the two major patterns: time-dependent killing (time>MIC) and concentration-dependent killing (AUC>MIC). Macrolide antibiotics (clarithromycin) fall under the time-dependent category (29). The relationship between time greater than MIC and resistance selection for time-dependent antimicrobials have not been well characterized. In the present study the antimicrobial activity was analyzed at three different concentrations levels, i.e. 0.125, 0.25, and 0.5g/mL. There is no significant effect on Time>MIC when different diets are compared with fasting. AUC>MIC under fasting conditions was 14.229.55, 10.878.69, and 5.746.61h.g/mL for clarithromycin and 5.163.26, 2.212.29, and 0.090.29h.g/mL for 14-hydroxy

82 Sanjay J. Gurule etal.

clarithromycin, respectively. All diets increased the AUC>MIC. The maximum increase was observed with the low-fat vegetarian diet (18.359.13, 15.008.75, and 9.997.83h.g/mL, respectively) for clarithromycin. The low-fat vegetarian diet increases AUC>MIC for 14-hydroxy clarithromycin at 0.125 and 0.25g/mL.

9.

10. 11.

Conclusion
In this study in healthy Indian subjects, a significant food effect was observed, as indicated by a significant increase in both the rate (Cmax) and extent of absorption (AUC) when fasting was compared with high-fat and low-fat vegetarian and non-vegetarian diets. Administration of clarithromycin 500mg extended release tablet under fasting condition was associated with lower rate and extent of absorption relative to administration with food. There was no significant effect on the bioavailability of clarithromycin when comparison is made between fat content (low and high) and source of food (vegetarian or nonvegetarian).

12.

13.

14.

15.

Acknowledgement
This paper is part of PhD thesis of Sanjay Gurule. The author thanks Ranbaxy research labs, Gurgaon, India, for carrying out this work. Declaration of interest: The authors report no conflict of interest.

16.

17. 18. 19.

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