Research Paper

Comparative bioavailability of three oral formulations of sustained release theophylline in healthy human subjects
N. Parvez, T. Ahmed, T. Monif,* N. Saha,* P. L. Sharma

ABSTRACT Department of Pharmaceutical Medicine, Faculty of Pharmacy, Jamia Hamdard, Hamdard University, New Delhi 110062, India. *Department of Clinical Pharmacology and Pharmacokinetics, Ranbaxy Research Laboratories, Gurgaon 122001, India.
Received: 5.5.2003 Revised: 18.8.2003 Accepted: 24.8.2003

Correspondence to:
P. L. Sharma E-mail: mailtaus@yahoo.com

Objective: To determine the bioequivalence of two marketed test formulations (A, B) as compared to a reference formulation (R) of slow release theophylline in healthy volunteers. Material and Methods: The study was conducted as an open label, balanced, randomized, threetreatment, three-period, three-sequence single-dose crossover study to determine the bioequivalence of Phylobid 200 mg SR tablets (A) and Theobid 200 mg SR tablets (B) as compared to Theostan CR 200 mg capsule (R) under fasting conditions. A group of 12 healthy, adult, male human subjects participated in this study. The bioavailability was compared using pharmacokinetic parameters Cmax, Tmax, AUC0-t, and AUC0-. Moreover, the 90% confidence interval (CI) for the ratio of logarithmic transformed Cmax, AUC0-t and AUC0- was also used to determine bioequivalence. A washout period of seven days was kept between each study period. Serial blood samples were collected at 0, 0.5, 2, 3, 4, 6, 8, 10, 12, 15, 18, 21 and 24 h during each study. Results: The 90% CI for the log transformed data forCmax, AUC0-t, and AUC0- for both the test products fell outside the prescribed limits of bioequivalence for narrow therapeutic index drugs i.e. 90-111%. The T/R (test/reference) ratio of product A was quite close to the prescribed limits of bioequivalence (95-105%), while for product B the T/R ratio was not satisfactory. Conclusions: None of the test products of theophylline were bioequivalent to the reference product. The finding is of special significance since substitution of one brand of theophylline, a drug with a narrow therapeutic index, with another brand may result in sub-therapeutic response in patients. KEY WORDS: Bioequivalence, theophylline.

Introduction Systemic drugs administered orally or parenterally must reach the general circulation in their pharmacologically active form to be distributed throughout the body and to exert therapeutic effect. The intensity of the therapeutic actions of many drugs correlate well with the concentration of the drug in the biological fluid.1 The rate of absorption is therapeutically important with single doses of drugs, especially in case of narrow therapeutic index drugs,2 where relatively small changes in the concentration can lead to marked changes in pharmacodynamic response. Theophylline is a narrow therapeutic index drug and exhibits dose-dependent pharmacokinetics.3 It is commonly used for the control and management of acute asthma and acute exacerbations of chronic obstructive lung disease.4 Although most of the available oral preparations are well tolerated, the

desired plasma theophylline concentrations of 10-20 g/ml are difficult to achieve.5 Theophylline is a drug with a short half-life, so rapid release theophylline preparations are required to be administered in multiple daily doses, which may lead to poor patient compliance and hence inadequate therapeutic response. In order to overcome these problems, sustained release (SR) preparations of theophylline have been developed and marketed. A number of sustained release preparations of the drug are commercially available on the Indian market. These slow release products are formulated in various ways that decrease the rate of disintegration and dissolution of the drug, thus affecting the bioavailability.6 The medical profession has realized the problem of wide variations in the therapeutic effectiveness of various brands of oral formulations containing the same active ingredient in equal amounts. Once patients are stabilized on one slow release preparation, they should not be

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changed to another product without retitration of dosage with careful monitoring of plasma theophylline levels, as it is a narrow therapeutic index drug to achieve optimum therapeutic levels with minimum toxicity.4 Most of the bioequivalence studies on which the claims of bioequivalence to innovator product do not use confidence intervals (CI). Determination of CI is a current regulatory requirement of DCGI (Drug Controller General of India) and also of FDA7 (http://www.fda.gov/gov/cder/guidance/index.htm; April, 2003) to document bioequivalence. Thus, the only way to verify these claims is to do a comparative bioequivalence study with the innovator drug formulation, using confidence intervals. Hence, the present study was undertaken to compare the bioavailability of three brands of 200 mg sustained release theophylline in healthy, adult, male, human subjects under fasting conditions. Materials and Methods Products evaluated
Reference (R)

ceive any medication during the two weeks period prior to the start of the study. They were instructed during screening not to take any prescription and OTC medications subsequently until the completion of the study. All the subjects abstained from any xanthine-containing food or beverages or alcoholic products for 48 h prior to dosing and throughout the sampling schedule during each period. Smokers and alcoholics were excluded from the study. Subjects were admitted and housed in the Clinical Pharmacology Unit from 12 h before dose and were discharged 24 h after dose during each period. A total of 42 blood samples (5 ml each) were collected pre dose and at 0.5, 1.0, 2, 3, 4, 6, 8, 10, 12, 15, 18, 21 and 24 h post dose through an indwelling cannula placed in a forearm vein during each period. After collection, the blood samples were centrifuged to separate the plasma. All plasma samples were stored at -700C until analysis. Ethical considerations The study was conducted at Ranbaxy Clinical Pharmacology Unit (Majeedia Hospital, New Delhi) according to a protocol approved by the Jamia Hamdard Institutional Review Board. This research was carried out in accordance with the clinical research guidelines defined in the U.S. 21 CFR part 312.20 and the principles enunciated in the Declaration of Helsinki (South Africa 1996). All the subjects provided written informed consent before entering the study. Bioanalytical method Theophylline was assayed by high performance liquid chromatography (HPLC) according to the method described in a previous study,8 but with some modifications. The sample preparation process was accomplished by protein precipitation using acetonitrile. The supernatant was chromatographed on an ODS spherisorb column (25 cm X 4.6 mm ID) with a particle size of 10 m using the mobile phase consisting of anhydrous sodium acetate buffer and acetonitrile (92:8 v/v). The pH of the mobile phase was adjusted to 5.0 with 10% glacial acetic acid. The analysis was carried out at a wavelength of 273 nm. Signals from the detector were captured in a computer, and processed by using Millennium 32 software. The method was validated by following international guidelines.9 The assay was found to be selective, accurate and precise with a linear range of 0.625 g/ml to 20.0 g/ml. The within batch accuracy ranged from 92.0 to 105.5% while the total accuracy of the assay ranged from 94.32% to 101.96%. The total precision of the method ranges from 5.51 to 8.52% while the within batch precision of the method ranges from 1.7 to 13.25%. The stability of the spiked plasma samples was determined during two freeze thaw cycles. Replicate numbers of low and high quality control samples were analyzed for the first and second freeze thaw cycles. The stability in the first and second freeze thaw cycles was 93.22 to 108.7% and 96.06 to 99.33% respectively. Hence theophylline was stable for two freeze thaw cycles. Pharmacokinetic analysis The various pharmacokinetic parameters were calculated using WinNonlin software Version 1.5.10 The maximum plasma concentration (Cmax) and the time to reach Cmax (Tmax) were de-

Theostan CR-200 mg capsule manufactured by Sidmak Labs. Ltd.,Gujrat, Batch No. T-00600, Mfd. Nov. 2000, Exp. Oct. 2002. This was taken as reference as it was the first entrant in the Indian market.
Test (T)

(A) Phylobid 200 mg SR tablets manufactured by Tridoss India Ltd., Aurangabad, Batch No. 11026, Mfd. Dec. 2001, Exp. Nov. 2002. (B) Theobid 200 mg SR tablets manufactured by Osaka Pharma Ltd., Satara, Batch No. S10255, Mfd. Sep. 2001, Exp. Aug. 04. Study design The study was conducted as an open label, balanced, randomized, three-treatment, three-period, three-sequence single-dose crossover study in 12 healthy adult, male, human subjects, under fasting conditions, comparing the bioequivalence of the two test products (A, B) with the reference formulation (R). Each subject received one 200 mg theophylline formulation with 240 ml of drinking water after an overnight fast during each of the study day. The order of receiving the different treatments for each subject during the three periods of the study was determined according to SASgenerated randomization schedule. There was a washout period of seven days between each study period. Study subjects Twelve healthy, adult, male subjects participated in the study. The mean+SD age and weight of the subjects was 22.66+2.74 years (range 19-26) and 57.75+9.15 kg (range 46-76) respectively. All were in good health as evidenced by the medical histories, complete physical examination and routine laboratory tests performed within 28 days prior to the commencement of the study. None had history of any allergy to theophylline and related compounds. Subjects did not re30
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termined by visual inspection of the individual plasma-concentration time profiles. Terminal elimination rate constant (Kel) was calculated from a semi-log plot of the plasma concentration versus time curve. This parameter was calculated by linear least square regression analysis using the last three (or more) non-zero plasma concentrations. The terminal halflife was calculated by the formula 0.693/Kel. The area under the concentration-time curve (AUC) was calculated by the linear trapezoidal method. Statistical analysis All statistical analyses were carried out using SAS software version 6.12 (SAS Institute Inc. Cary NC, USA). The AUC0-t, AUC0- and Cmax values were log converted before analysis. Ratio of mean (in percentage) was calculated using the least square mean (LSM) for log transformed AUC0-t, AUC0- and Cmax values. Ratios of mean were expressed as a percentage of the LSM for the respective treatment comparisons. The 90% CI of AUC0-t, AUC0- and Cmax for the ratio of test and reference was determined to assess the bioequivalence between different products using the equivalence interval of 0.80 and 1.25. The USFDA7 recommend the limit of 80-125% for AUC and 90111% for Cmax for narrow therapeutic index (NTI) drugs. The Indian regulatory guidelines also recommend the same limits for NTI drugs. The intrasubject variability was determined using WinNonlin software Version 1.510 for the pharmacokinetic parameters AUC 0-t, AUC 0- and C max. Analysis of variance (ANOVA) was also performed and a statistically significant difference was considered at P<0.05. Results There were no significant protocol deviations and all the 12 subjects completed the study. The various pharmacokinetic

parameters after the administration of theophylline 200 mg sustained release formulation manufactured by different pharmaceutical companies are shown in Table 1. The mean plasma theophylline concentration versus time profile for all the treatments is shown in Figure 1. There was no period, sequence and formulation effect as indicated by the P values for different treatments. The T/R (test/reference) ratios for the pharmacokinetic parameters AUC0-t, AUC0- and Cmax were satisfactory for product A (Phylobid) and were 100.44%, 104.18%, and 89.78% respectively. For product B (Theobid) the ratios for these parameters were 61.67%, 52.67%, and 61.10%. However, when 90% CI was applied for the log-transformed pharmacokinetic parameters, the upper and lower confidence limits for both

Concentration (mg/ml)

0
0

10

15

20

25

30

-1

Time (hrs)

Figure 1: Mean plasma concentration (+SD) Vs time profile of theophylline in 12 subjects

Table 1
Mean + SD and log transformed pharmacokinetic parameters of three formulations of theophylline in 12 healthy human subjects
Formulation type
Reference (R) GM CV (%) Test A GM CV (%) Test B GM CV (%) Ratio of LSM A/R(%) B/R(%) 90% CI A/R(%) B/R(%)

Cmax (g/ml)
4.07 + 1.30 3.85 34.12 3.92 + 0.83 3.83 33.92 2.61 + 1.18 33.92 55.33 100.44 61.67 73.05-138.54 44.78-84.93

Tmax (h)
5.00 + 1.04 26.83 48.38 4.33 + 1.07 27.64 46.41 5.33 + 1.30 46.41 91.85 101.25 52.67 61.28-177.10 30.98-89.53

AUC0-t (g.h/ml)
30.85 + 12.83 41.31 42.16 29.70 + 9.71 37.09 46.96 18.70 + 13.68 46.96 68.23 87.09 61.10 63.22-127.51 42.47-87.91

AUC0- (g.h/ml)
45.03 + 17.42

T1/2 (h)
6.43 + 2.83

Kel (h-1)
0.13 + 0.06

38.61 + 10.33

5.10 + 2.12

0.16 + 0.08

28.70 + 14.49

5.73 + 2.21

0.13 + 0.04

GM-Geometric mean, LSM-Least square mean, CI-Confidence interval, n=12

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the products fell outside the prescribed limits of bioequivalence for narrow therapeutic index drugs i.e. 90-111% for log-transformed data for Cmax and AUC. The intrasubject variability (ISV) for the log-transformed pharmacokinetic parameters was quite high (For Test A: ISV for Cmax, AUC0-t, and AUC0- 33.78, 22.68 and 14.10; Test B: 53.14, 28.63 and 15.79 respectively). Discussion This study demonstrates that both the test products of theophylline were not bioequivalent to the reference product. The T/R ratios of product A were quite close to the prescribed limits of bioequivalence (95%-105%). The bioavailability of product B was very poor indeed. The desired ratio of AUC0-t/ AUC0- (80% or more) is low in this study, this could be due to the fact that in present study the choice of limit of quantitation (LOQ) is high and should have been preferably much lower. In a study carried out by Bhandari,11 the bioequivalence of two brands of SR theophylline was studied in 12 subjects. A single dose of 400 mg SR theophylline was administered. The Cmax of the reference formulation was 8.56+0.50 g/ml at 11.00+1.28 h. The Cmax of the test formulation was 9.95+0.68 g/ml at 10.83+1.28 h. The AUC0-24 was 140.57+8.19 g.h/ ml for the reference formulation and for the test formulation it was 165.80+9.83 g.h/ml. The AUC0- for the reference formulation was 483.65+193.20 g.h/ml and for the test formulation it was 374.77+66.54 g.h/ml respectively. They concluded that both the preparations resulted in comparable mean plasma theophylline levels throughout 24 h and were bioequivalent with respect to the T/R ratios. However, the confidence inter vals, the acid test for demonstrating bioequivalence12 were not applied. Another crucial drawback of this study was that only one time point was sampled after the Cmax and adequate sampling (at least 3 blood samples) was not done during the elimination phase of the drug, but surprisingly all the pharmacokinetic parameters i.e. AUC0- Cmax, t1/2 and Kel were calculated. In an earlier bioavailability study carried out on 8 healthy adult volunteers, three solid and one (reference) liquid theophylline products were studied.13 In yet another study14 conducted on 12 healthy human volunteers, two SR theophylline preparations were studied. In both these studies, confidence intervals were not used to document bioequivalence. It is thus clear that most of the bioequivalence studies carried out on theophylline did not use confidence intervals while concluding bioequivalence. Theophylline being a narrow therapeutic index drug, it is legally incumbent on the manufacturers to ensure the quality of the marketed formulations, since a small change in the systemic concentrations may lead to a marked change in the pharmacodynamic response, with undesirable therapeutic consequences. Thus, asthmatic patients whose therapeutic response to theophylline, in approved doses, is sub-optimal may not necessarily be drug-resistant. The sub-optimal response may be due to achievement of sub-therapeutic plasma levels due to poor oral bioavailability of some of the marketed theophylline formulations. In the present study, the bioavailability of the test product A was 89.78% with respect to the reference and the
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bioavailability of the test product B was 61.1 % with respect to the reference. So the product A i.e. Phylobid has almost comparable bioavailability to the reference product i.e. Theostan CR and can safely be substituted in case of non-availability of the reference product, but the same cannot be recommended for the product B i.e. Theobid. The results obtained in this study indicate that various formulations of theophylline (a narrow therapeutic index drug) marketed by different pharmaceutical companies in India may differ significantly in their bioavailability. The physicians prescribing these products, unsuspectingly presume that the quality of different formulations if not exactly the same, is nearly the same. The resultant sub-therapeutic response in many patients may, in fact be due to significantly poor bioavailability from the particular formulation prescribed. Since most practicing physicians in India do not have access to therapeutic drug monitoring, it is the legal as well as moral duty of the manufacturers of marketed formulation of theophylline to ensure bioequivalence with the innovators formulation, not only at the time of obtaining Regulatory Marketing Approval but also for subsequent batches of the formulations marketed by them over the years. Acknowledgements
This research was supported by a grant provided by Ranbaxy Research Laboratories, Gurgaon, India. The authors would like to thank the staff of the Ranbaxy Clinical Pharmacology Unit and Department of Clinical Pharmacology and Pharmacokinetics for their support to conduct this study and analysis of clinical samples. The authors are also grateful to Late Janab Hakeem Abdul Hameed for providing excellent facilities at the university.

References
1. Koch-Wesser J. Serum drug concentrations as therapeutic guides. N Engl J Med 1982;287:227-31. 2. Benet LZ, Goyan JE. Bioequivalence and narrow therapeutic index drugs. Pharmacotherapy 1995;15:433-40. 3. Dahlquist R, Billing B, Miners JO, Birkett DJ. Nonlinear metabolic disposition of theophylline. Ther Drug Monit 1984;6:290-7. 4. Markham A, Faulds D. Theophylline A review of its potential steroid sparing effects in asthma. Drugs 1998;56:1081-91. 5. Flink JN, Levy MB, Conard E. Study of two controlled release theophylline preparations. Am J Med 1990;25:58-61. 6. Weinberger M, Hendeles L, Brighley L. The relation of product formulation to absorption of oral theophylline. N Engl J Med 1978;299:852. 7. Centre for Drug Evaluation and Research, U.S. Department of Health and Human Services Food and Drug Administration: Guidance for Industry: Bioavailability and Bioequivalence Studies for Orally Administered Drug Products-General Considerations. 2003. 8. Subramaniam S, Garg SK, Dhand R, Malik SK, Sharma PL. Measurement of plasma theophylline concentration by HPLC. Bull P.G.I. 1986;20:20-5. 9. Shah VP, Midha KK, Dighe S, McGilveray IJ, Skelly JP, Yacobi A, et al. Conference Report: Analytical Method Validation: Bioavailability, Bioequivalence and Pharmacokinetic studies. Pharm Res 1992;9:588-92. 10. Win Nonlin, Professional Edition, version 1.5, Pharsight Corp., 800 West Elcamino Real, Suite 200, Mountain View, CA 94040. 11. Bhandari P. Bioequivalence of two brands of sustained release theophylline. J Asso Phys India 1999;47:461. 12. Centre for Drug Evaluation and Research, U.S. Department of Health and Human Services Food and Drug Administration: Guidance for Industry: Statistical Approaches to Establishing Bioequivalence-General Considerations. 2001.

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13. Regazzi MB, Rondanelli R, Vidale E, Cristiani D. Bioavaiability of sustained release theophylline formulations. Int J Clin Pharmacol Ther Toxicol 1983;21:245-51.

14. Gulati R, Roy V, Sachdeva A, Tripathi CD, Gupta U, Bapna JS. Bioequivalence of two brands of sustained release theophylline preparations. J Asso Phys India 1997;45:780-2.

AW ORATIONS 2004 IPS AWARDS & ORATIONS 2004

Indian Pharmacological Society honors distinguished pharmacologists for their significant contributions through various Awards and Orations. To encourage research in pharmacology, IPS also gives away various prizes for the published papers in addition to prizes awarded for presentations during the Annual Conference. As per the decision taken during the Executive meeting held at Ahmedabad, nominations for various orations, awards and prizes must reach the General Secretary, IPS, (except for P.P. Suryakumari Prize where the Chief Editor is the convener) on or before 30th April 2004. The nominations or a copy of the same may also be sent to the General Secretary who in the capacity of Chief Executive Officer of the society will forward it to the concerned convener and monitor the procedure. NAME OF THE AWARD & ITS AREA 1. Prof. Naranjan S. Dhalla Award for life time contributions in the field of Cardiovascular sciences Col. Ram Nath Chopra Oration Dr. B.N. Ghosh Oration Dr. Govind Achari Oration Dr. Saket B. Pandey Oration CDRI Oration in Biomedical / Life Sciences Drug Discovery Research Dr. S. Lahiri Oration in Neuro/Psychopharmacology@ Prof. P.K. Das Award for Young Scientist working in Cardiovascular Pharmacology REQUIREMENTS 1. 2. 3. 4. 1. 2. 1. 2. 1. 2. 1. 2. 1. 2. 1. 2. 1. 2. 3. Bio-data in the prescribed format along with Annexures One page write up on contributions Copies of some selected publications Nomination by Distinguished Scientists Bio-data in the prescribed format Nomination by the IPS members Bio-data in prescribed format Nomination by the IPS members Bio-data in the prescribed format Nomination by the IPS members Bio-data in the prescribed format Nomination by the IPS members Bio-data in the prescribed format Nomination by the IPS members Bio-data in the prescribed format Nomination by the IPS members Bio-data in the prescribed format Nomination by HOD/ member Reprint of imp. Papers 4 copies of the paper published in 2002-2003 5 copies of the paper published during last 5 years (1999-2003). Last date : 31st March 2004 4 copies of the paper published during 2002-2003

2. 3. 4. 5. 6. 7 8.

B. Uvnas Prize for the best research paper published in any journal in autacoids & endogenous substances 10. P.P. Suryakumari Prize for the best research paper published in diabetes mellitus and other endocrine and metabolic disorders* 11. Sharma Prize for the best research paper published in Cardiovascular pharmacology * This should reach the Chief Editor, IJP. @ Subject to receipt of the FDR money from the donor.

9.

Prof. Ramesh K. Goyal General Secretary, IPS.

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