Macrolide pharmacokinetics and dose scheduling of roxithromycin

DIAGN MICROB1OLINFECT DIS 1992;15:71S-76S

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Macrolide Pharmacokinetics and Dose Scheduling of Roxithromycin Odd G. Nilsen, Trond Aamo, Kolbjt~rn Zahlsen, and Per Svarva

The 150- and 300-mg single-dose pharmacokinetics of roxithromycin were investigated in 12 healthy subjects in a crossover study. Serum concentrations were determined by highperformance liquid chromatography (HPLC) and microbiologic assay (MA). Peak serum levels as measured by HPLC were 6.7 +- 2.6 (150 mg) and 11.0 +- 2.2 Izg/ml (300 mg) and did not differ significantly from the values obtained by MA. Mean serum roxithromycin levels 12 hr after the 150-mg dose and 24 hr after the 300-mg dose were 2.50 and 2.55 i~g/ml, re-

spectively. HPLC analysis of a comparable macrolide, clarithromycin, showed peak serum levels of 1.2 +- 0.6 and 2.3 +0.6 I~g/ml after oral dosing with 250 and 500 mg in the same subjects. The 14-0H metabolite reached a level that was 50% and 40%, respectively, of that of the parent compound. Roxithromycin showed a prolonged elimination half-life compared with clarithromycin and its 14-0H metabolite. Mean values of 14.6, 3.5, and 5.5 hr, respectively, indicate the need for less frequent dosing of roxithromycin.

INTRODUCTION

the 150- and 300-mg single-dose pharmacokinetics of roxithromycin, and thus evaluate the suitability of a 300-mg once-a-day dosing schedule of roxithromycin as compared with 150 mg twice daily. A subsidiary aim was to compare the pharrnacokinetics of roxithromycin with those of clarithromycin and its active 14-OH metabolite.

The search for erythromycin derivatives with improved antibacterial and/or pharmacokinetic properties has led to the synthesis of several new agents. Among the new macrolide antibodies, roxithromycin demonstrates a characteristic pharmacokinetic profile with good absorption, favorable tissue and intracellular distribution, prolonged elimination halflife (tt), and low-accumulation and interaction profiles (Nilsen, 1987; Bergogne-Berezin, 1987; Tremblay et al., 1988; Carlier et al., 1989; Young et al., 1989). Clinical experience with roxithromycin has demonstrated good efficacy and tolerability with 150 mg twice daily for - 1 0 days (Young et al., 1989). From a pharmacokinetic point of view, however, a macrolide, such as roxithromycin, with favorable absorption and tolerance profiles and a prolonged elimination half-life, should be considered for a oncea-day dosing schedule. The present study was undertaken to compare From the Department of Pharmacologyand Toxicology (O.G.N., T.A., K.Z.) and Department of Microbiology(P.S.), Faculty of Medicine, Universityof Trondheim, Trondheim, Norway. Additional copies of this supplement are availablefrom Roussel UCLAF, DomaineThdrapeutiqueAntibiothdrapie,35 Boulevardde~ h~valides,75007Paris, France. © 1992ElsevierSciencePublishingCo., Inc. 655 Avenue of the .&aericas, New York, NY 10010 0732-8893/92/$5.00

MATERIALS AND METHODS Participants Twelve healthy men aged from 22 to 37 years (mean, 31.2 -+ 4.7 years) participated in the study. All showed normal clinical chemical and hematologic values before, during, and after the study. No hard exercise or blood donation was allowed 4 weeks prior to or during the study. No alcohol was allowed during the study, and no adverse effects related to any of the treatment drugs were observed.

Study Design The participants were randomly divided into groups of equal size and received the drugs in a Latin square fashion with a 1-week washout betweer~ treatments. All treatments were given at 08:00 hours after an

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O.G. Nilsen et al.

overnight fast. The study drug was taken will a full glass of water. A standardized hospital breakfast was given 4 hr after dosing. No further restrictions were made on the intake of food. Blood (8 ml) was collected from an indwelling catheter at the following points after dosing: 0 hr Cvefc;redosing), 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5, 3, 4, 6, 8, 12, 24, 30, and 36 hr. Blood was protected from light and allowed to clot at room temperature for 0.5 hr before centrifugation at 1100 g. Serum was divided for high-performance liquid chromatography (HPLC) and microbiologic assay (MA), and frezen immediately at - 20°C. No sample was stored for more than 8 weeks before analysis.

4.6 mm i.d.) protected by a 20-mm Supelguard C18 precolumn. The mobile phase consisted of acetonitrile-methanol-water (5:2:3) containing 4.0 g of ammonium acetate. The mobile phase was filtered through 0.22-p~m filters (Millipore) before use. Detection was performed at an oxidation potential of + 0.89 V. The retention times for roxithromycin and internal standard were 3.5 and 7.5 min, respectively, at a mobile phase flow r~te of 1.0 ml/min. Roxithromycin was quantified by the peak height ratio from a calibration curve covering the range from 1 to 20 p,g/ml. The calibration samples were frozen as -20°C, thawed at room temperature, and extracted identically with the serum samples. The detection limit was 0.05 p,g/ml.

Microbiologic Assay

Internal standard [50 ~tl (erythromycin A-6-O-methyloxime)] was added to 0.5 ml of serum, dissolved in 1:1 acetonitrile-water (15 p,g/ml), 0.2 sodium carbonate, and 3 ml of ethyl acetate-hexane (1:1 vol/vol). After vortexing for 1 min, the solution was centrifuged at 800 g for 5 min. The organic layer ~.,as transferred to a clean test tube and evaporated to dryness under a stream of nitrogen (40°C). The residue was dissolved in 0.15 ml of the mobile phase by ultrasonication for 1 min, transferred to autosampler (Shimadzu SIL 6A) vials with 0.2-ml glass inserts, and capped with screw caps with Teflon septa.

Extraction Procedure for Clarithromycin An agar-weil diffusion technique 0ailing et al., 1972) was used with the test organism Bacillus subtilis ATCC 66,33. This was poured as a spore solution onto 14cm agar plates to a confluent growth. The culture medium consisted of Bacto Antibiotic no. 1 (Difco Laboratories, Detroit, MI) with 1.5% agar adjusted to pH 8.5. As standards, stock solutions of both antibiotics were diluted in phosphate buffer, pH 8, to final concentrations of 0.5, 1.0, 2.0, and 4.0 ~tl/ml. The standard solutions were deposited in 4-mm wells on each plate. The samples were, if necessary, diluted in serum and deposited in duplicate in a volume of 25 p,l. After 0.5 hr prediffusion, the plates were incubated overnight. The detection limit of the assay for both macrolides was 0.5 p,g/ml.

HPLC Methodology Extraction Procedurefor Roxithromycin Internal standard [50 p,l (RU 29767)] was added to 0.5 mi of serum, and dissolved in methanol (20 p,g/ml) and 2.5 m] hexane-isoamyl alcohol (95:5 vol/vol). After vortex mixing for 20 s, the solution was centrifuged at 1500 g for 5 min. The organic phase (2 ml) was transferred to a new test tube. After evaporation under nitrogen (40°C), the residue was dissolved in 0.2 ml of the mobile phase and vortexed for another 20 s. Ultrasonication was performed for 1 min, followed by further centrifugation at 1500 g for 5 rain. The final solution was decanted into autosampler (Shimadzu SIL 6A) vials with 0.2-ml glass inserts and capped with screw caps with Teflon septa.

Separation and Detection. Quantitao_'on of roxithromycin was performed on a Shimadzu LC 9A pump equipped with an ESA Coulochem 5100A electrochemical detector with a 5010 analytic cell and a Shimadzu CR 3A integrator. A 50-ttl sample was separated on a Supelcosil 5-p,m C18 column (50 mm x

Separation and Detection. Clarithromycin and its 14OH metabolite were analyzed with a Shimadzu LC 9A pump equipFed with an ESA Coulochem 5100A electrochemical detector with a 5120 analytical cell and a Shimadzu CR 3A integrator. A 75-p,1 sample was separated on a Supelcosil 5-p,m C8 column (150 x 4.6 mm i.d.) protected by a Supelguard C8 precolumn. The mobile phase consisted of 0.025 M acetic acid, 46% acetonitrile, and 10% methanol, adjusted to pH 6.8 with sodium hydroxide. The mobile phase was filtered through 0.22-1~m filters (Millipore) before use. Detection was performed at an oxidation potential of 0.78 V, and the retention times for the 14-OH metabolite, clarithromycin, and internal standard were 11, 17, and 25 min, respectively, at a mobile phase flow rate of 1.0 ml/min. Clarithromycin and the 14-OH metabolite were quantified by peak height ratios from calibration curves covering the range from 0.1 to 3 p,g/ml. Calibration samples were frozen at -20°C, thawed at room temperature, and extracted identically with the serum samples. The detection limit was 0.05 p~g/ml for both compounds. Calculation of Pharmacokinetic Parameters Model independent pharmacokinetic parameters were calculated. The elimination half-life was determined

Roxithromycin, Pharmacokinetics, and Dosing

from the linear part (13phase) of the semilogarithmic serum concent.~ation-time plot, computerized from the least-square regression line with equal weight on each point. ]'h(: area under the curve (AUC) from zero to infinity was calculated by the trapezoid rule using each experimental point up to the detection limit of the compounds in serum, C~, with addition of the remaining area, C~ - T~/ln2. The time, t . . . . taken to reach the maximum serum concentration, C. . . . was derived from the experimental values.

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Statistics

Statistical significance was determined by Student's t-test for paired values, and a p of 0.05 was used for comparing means. Results are presented as means -+ SD. Test T r e a t m e n t S u p p l y Roxithromycin tablets, 150 mg (lot 82) and 300 mg (lot CR22731-145), were supplied by Roussel Uclaf (Paris) together with roxithromycin dry powder for analysis (lot 25). aarithromycin tablets, 250 mg (lot 33065TF), were supplied by Abbott AB (Stockholm, Sweden) together with clarithromycin (lot 37-022VC) and 14OH clarithromycin (lot 110-434AX) dry powder for analysis. Ethics The investigation was performed in compliance with the Declaration of Helsinki (18th World Medical Assembly, 1964), revised 35th Wortd Medical Assembly, Venice, Italy, 1983. The study was approved by the Ethical Committee in Health Region W, Trondheim, and by the Medicine Control Authority, Oslo, Norway.

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FIGURE 1 Serum concentration-time prof'des of roxithromycin 300 mg (0--0) and 150 mg (O--O) and of clarithromycin 500 mg (O--O) and 14-OH metabolite (o----o) in healthy subjects after single oral dosing (I-1PLCassay). metabolite and clarithromycin were measured concurrently using the microbiologic assay, they were closer to those of roxithromycin, as showu in Figure 2, but were still lower. The parameters in Table 1 show that the clarithromycin 14-OH metabolite has a longer half-life than does the parent compound, and that it increases with dose. The peak metabolite concentration is -50% that of the parent compound. The serum concentration of roxithromycin 12 hr after dosing is - 2 0 times higher than that of clarithromycin, when calculated per milligram substance administered. Table 2 shows a good correlation between the HPLC and MA results for roxithromycin in serum, and a 25% overestimation of clarithromycin by MA compared with HPLC. 100

RESULTS The serum concentration-time profiles given in Figures I and 2 were constructed from data from all 12 =lJbiecfs at each point shown. Any points where two or more subjects were lost due to serum concentraLions below the limit of detection of the as,~ay were excluded from the figures. However, all data presented in Tables 1 and 2 are based on individual calculations, including all experimental values obtained for each subject. Figure 1 demonstrates significantly higher serum concentrations of roxithromycin after single dosing with 300 and 15 mg than of cla~-ithromycin and its 14-OH metabolite after a single 500-rag dose of clarithromycin. When serum levels of the active 14-OH

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FIGURE 2 Serum concentration-time profiles of roxithromycin 300 mg (0---0) and 150 mg (O---O) and of clarithromycin 500 mg (O-O) in healthy subjects after single oral dosing (microbiologic assay).

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TABLE 1

Single Oral-Dose Pharmacok/netic Parameters for Roxithromycin and Clarithromycin in Healthy Subjects (HPLC Assay) Roxithromycin (rag)

Pamme~r

150

C,,~ (p.g/ml) t,,~ (hr) C12hr (p,g/ml) C241~ (,.g/ml) hr2 (hr) AUC (ixg/ml x hr)

6.68 ± 2.54 ± 2.59 ± 1.35 ± 12.97 ± 107.3 ±

Clarithromycin (rag)

300 2.58 1.42 1.34 0.83 5.69 52.9

11.02 ± 2.25 ± 4.95 ± 2.76 ± 16.24 ± 214.7 ±

2.19 1.62 1.67 1.15 7.76 82.5

250

500

250

500

1.20 ± 0.56 1.57 ± 1.14 0.18 ± 0.13 ND 3.17 ± 0.85 7.6 ± 4.2

2.25 ± 0.63 2.54 ± 1.57 0.62 ± 0.35 ND 3.82 ± 1.33 19.7 ± 7.9

0.62 ± 0.19 2.46 ± 1.26 0.16 ± 0.09 ND 4.60 ± 1.94 5.7 ± 2.3

0.90 ± 0.33 2.56 ± 1.14 0.38 ± 0.18 ND 6.43 ± 2.31 10.2 ± 4.0

ND, not detectable, detection limit 0.05 Ixg/ml.

TABLE 2

Single Oral-Dose Pharmacokinetic Parameters for Roxithromycin and Clarithromycin in Healthy Subjects (Microbiologic Assay) Roxithromycin (rag)

Parameter Cm~ (p,g/ml) t,,~ (hr) Cl2hr (ixglml)

C24hr (p.g/ml) tlrz (hr) AUC (l~g/ml x hr)

Ciarithromycin (rag)

150

300

250

500

6.93 --: 3.43 2.52 ± 1.26 2.37 "" 1.a_l 1.33 ± 0.85 11.28 ± 5.74 94.9 --. 50.1

12.45 -- 3.54 2.42 ± 1.49 4.74 ± 1.77 2.29 ± 0.90 14.16 ± 11.89 189.4 +_ 70.5

1.33 _+ 0.45 1.98 ± 0.88 ND ND 4.47 ± 1.89 9.4 +- 3.4

2.80 ± 0.83 2.27 ± 0.52 0.79 ± 0.24 ND 5.42 ± 1.97 24.0 ~: 6.5

l%rD,not detectable, detection limit 0.5 ~.g/ml.

TABLE 3

Comparative Pharmacokinetic Parameters of Roxithromycin in Healthy Subjects After a Single Oral Dosing with 150 a n d 300 m g

Dose (mg) tlrz Oar) C ~ (ILg/ml) C1~, (l~g/ml) C24t~(p.g/ml) 150 150 150

13.0 8.3 10.5

Mean 300 300 300 300 Mean

6.7 6.6 7.9

2.6 1.8 2.3

10.6

7.1

2.2

16.2 10.9 11.9 11.2

11.0 9.7 10.8 9.7

2.8 1.2 1.5 1.8

12.5

10.3

1.8

PI, present investigation.

Clarithromycin 14-OH Metabolite (rag)

Reference PI Puri and Lassman (1987) Tremblay et al. (1988)

PI Puri and Lassman (1987) Tremblay et al. (1988) Kisicki (1985)

Roxithromycin, Pharmacokinetics, and Dosing

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FIGURE 3 Serum concentration-time profiles of roxithromycin 150 mg twice daily (C)--C))and 300 mg once daily (O----t) in healthy subjects at steady state on day 11 of repeated dosing. Data adapted from Purl and Lassman (1987). Table 3 summarizes some pharmacokinetic parameters of roxithromycin in healthy subjects from different single-dose pharmacokinetic studies with 150 and 300 mg. A similar trend is observed in all studies, a slight but not significant increase in Tt with increasing dose, a nonlinear increase in Cm~,, and similar serum concentrations 12 hr after the 150-mg dose and 24 hr after the 300-mg dose. Figure 3 shows the steady-state serum concentration-time profiles of roxithromycin on day 11 (Purl and Lassman, 1987) in two different dosage regimens: 150 mg twice daily and 300 mg once daily. Maximum serum concentrations were 9.3 +- 1.4 i~g/ml (150 mg b.i.d.) and 10.9 ± i.4 Fg/ral (300 mg daily), demonstrating a nonlinear increase in (=maxalso at steady-state levels. The 12-hr serum concentration at steady state after 150 mg twice daily (trough level) was 3.6 ± 1.3 p~g/ml, whereas the 24-hr serum concentration after 300 mg daily (trough level) was 2.2 _+ 0.64 ~g/ml.

DISCUSSION The single-dose pharmacokinetic parameters obtained in this study for roxithromycin and clarithromycin are in the same range as reported by others (Kisicki, 1985; Purl and Lassman, 1987; Tremblay et al., 1988; Chu et al., 1990). However, only a few reports are available on the pharmacokinetics of 300 mg roxithromycin. Table I shows a higher clarithromycin 14-OH metabolite ratio with the 500-m~ single dose than with the 250-mg dose with respect to Cm~, C12hr, and AUC, showing decreased conversion of clarithro-

mycin to its 14-OH metabolite at the higher dose. This indicates that the elimination of clarithromycin is saturable, which is not evident from the slight nonsignificant increase in its elimination half-life. Saturable elimination also applies to the 14-OH metabolite, given the significant increase in the elimination half-life with increasing dose. Even though all parameters calculated for the 14-OH metabolite must be considered as apparent, due to the similar serum concentrations of the parent compound and metabolite, an increased accumulation of both compounds can be suspected on repeated dosing, especially in the elderly and in patients with liver failure. The longer elimination half-life of roxithromycin compared with those of clarithromycin and its 14OH metabolite indicates that less frequent dosing is required to maintain adequate steady-state serum concentrations. As roxithromycin does not seem to have active metabolites, the dosing frequency of roxithromycin can be determined from the pharmacokinetk ~ of the parent compound alone. Single dosing with roxithromycin in the present study produced serum concentrations of 2.4 and 2.6 pJ/ml 12 hr after the 150-mg dose (MA and HPLC, respectively) and 2.3 and 2.8 p.gl/ml 24 hr after the 300-mg dose. This strongly suggests that trough serum levels of roxithromycin at steady state far exceed the minimum concentration required for antibacterial activity, with either dosage regimen, 150 mg twice daily or 300 mg once daily. Our single-dose data are similar to those of other studies (Kisicki, 1985; Purl and Lassman, 1987; Tremblay et al., 1988). Repeated dosing with 150 mg twice daily and 300 mg once daily for 11 days in healthy subjec~.s confirms the suitability of both dosage regimens. It demonstrates f,~,'ther the impact of the nonlinear pharmacokinetics of roxithromycin in that C ~ , and trough serum levels at steady state increased less on both dosage regimens than expected from the single-dose pharmacokinetic data. The nonlinear pharmacokinetics are also evident in Table 3, which shows a nonlinear increase in C , ~ with the increase in single dose from 150 to 300 mg. The reason for the nonlinear pharmacokinetics is thought to be saturation of roxithromycin binding to a~-acid glycoprotein in serum within the therapeutic range (Zini et al., 1988). One consequence is that roxithromycin apparently accumulates on repeated dosing to a lesser extent than expected from its elimination half-life. This has also been shown to be true in old age, debilitation, and even renal failure (though the elimination halflife does increase somewhat). Others have also discussed the nonlinear pharmacokinetics of roxithromycin (Nilsen, 1987; Tremblay et al., 1988). On the basis of the data presented here and elsewhere on the pharmacokinetics of roxithromycin, it

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can be concluded that repeated dosing schedules of roxithromycin of 150 mg twice daily or 300 mg once daily will both produce serum concentrations at steady state well above the minimum concentration re-

O.G. Nilsen et al.

quired for antibacterial activity. Furthermore, this level will be reached with the first dose using either regimen.

REFERENCES Bergogne-Berezin E (1987) Tissue distribution of roxithromycin. J Antimicrob Chemother 20(Suppl B):113-120. Carlier MB, Zenebergh A, Tulkens PM (1989) Cellular uptake and subcellular distribution of roxithromycin and erythromycin in phagocytic cells. ! Antimicrob CIlemother 20(Suppl B):47-56. Chu SY, Wilson DS, Eason C, Deaton RL, Cavanaugh J, Sonders RC (1990) Single and multi-dose pharmacokinetics of clarithromycin [abst 759]. In 30th ICAAC Meeting, Atlanta, GA, p 212. Jailing B, Malmborg A, Lindman A (1972) Evaluation of a micromethod for determination of antibiotic concentrations in plasma. Eur J Clin Pharmacol 4:150-156. Kisicki JC (1985) Single dose pharmacokinetics of RU 965 in young and elderly men. Roussel on file, protocol 110.

Nilsen OG (1987) Comparative pharmacokinetics of macrolides. J Antimicrob Chemother 20(Suppl B):81-88. Purl SK, Lassman HB (1987) Roxithromycin: a pharmacokinetic review of a macrolide. ] Antimicrob Chemother 20(Suppl B):89-100. Tremblay D, Jaeger H, Fourtillan JB, Manuel C (1988) Pharmacokinetics of three single doses (150, 300 ann-. 450 mg) of roxithromycin in young volunteers. Br ] Clin Pract 42(Suppl 55):49-50. Young RA, Gonzales JP, Sorkin EM (1989) Roxithromycin: a review of its antibacterial activity, pharmacokinetic properties and clinical efficacy. Drugs 37:8-41. Zini R, Fournet MP, Barre J, Tremblay D, Tillement JP (1988) In vitro study of roxithromycin binding to serum proteins and erythrocytes in humans. Br J Clin Pract 43(Suppl 55):55-56.