New approach for accurate simulation of human pharmacokinetics in an in vitro pharmacodynamic model: application to ciprofloxacin
Boubakar B. Baa,*,
Arnaud Bernarda,
Athanassios Iliadisb,
Claudine Quentinc,
Dominique Ducinta,
Renaud Etiennea,
Mathieu Fourtillana,
Isabelle Maachi-Guillota and
Marie-Claude Sauxa
a Laboratoire de Pharmacocinétique et de Pharmacie Clinique, Faculté de Pharmacie, Université de Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux cedex;
b Laboratoire de Pharmacocinétique et de Toxicocinétique, Faculté de Pharmacie, 27 Boulevard Jean Moulin, 13385 Marseille cedex 5;
c Laboratoire de Microbiologie, Faculté de Pharmacie, Université de Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux cedex, France
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Abstract
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An in vitro pharmacodynamic model using a disposable dialyser unit and computer-controlled devices was developed. Feedback control of peristaltic pump flow rates is used to maintain constant flow rates, thus avoiding the problem of the modification of the physical properties of the tubing that generally occurs. Fast equilibrium is obtained with capillaries, which allows simulation of the same kinetic profile in the central and the peripheral compartments. Thus, more accurate simulation of plasma, extracapillary fluid or whole tissue kinetics can be performed. Our model was validated by simulation of a 30 min infusion of a 200 mg dose, and of an oral administration of a 500 mg dose of ciprofloxacin.
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Introduction
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Most in vitro pharmacodynamic models use mainly the continuous dilution technique to reproduce drug kinetics. This technique needs pumps that maintain constant flow rates. Since volumetric pumps are expensive and require a long sterilization procedure, roller pumps are commonly used; however, it is difficult to maintain absolutely constant flow rates over several days with these.1 Indeed, overheating of peristaltic tubing by friction of pump rollers modifies the physical properties of the plastic, leading to variations in the flow rate, and therefore, the central compartment volume during long-term experiments. Consequently, the resulting exponential variation of drug concentrations does not completely fit the reference human pharmacokinetic profile. In addition, several in vitro kinetic models based on serum concentrations of antibiotics are based only on human first order elimination kinetics by setting the pumps' flow rate to simulate the elimination half-life. Additional pumps and flasks are required for oral and intramuscular administration simulation,1 making the system bulky and difficult to control.
We have developed a new model in which inconsistency in peristaltic pump flow rates is prevented by their computer control via flowmeters with an RS 232 interface. Computerization of pump speed control enables simulation of a multiexponential pharmacokinetic profile. Both intravenous infusion and oral administration were performed using the same computer-controlled syringe pump. This model was validated by determining the ciprofloxacin plasma concentration versus time profiles, following an intravenous 200 mg dose and an oral 500 mg dose.
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Materials and methods
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Description of the model
The model (see Figure
) derives from the two-compartment kinetic model with artificial capillary units proposed by Blaser et al.2 and was designed to expose bacteria to changing antibiotic concentrations, without dilution of the bacterial inoculum together with the antibiotic. The central compartment (CCp) consisted of a 1 L thermostatable flask with a magnetic stirrer (Wheaton, Polylabo, Strasbourg, France) containing 500 mL broth, tubing and the lumina of the capillaries within a disposable dialyser unit (F40S, Fresenius, Sèvres, France). The peripheral compartment (PCp) consisted of the extracapillary space of the dialyser unit plus tubing. A computer-controlled peristaltic pump (Minipuls 3M/HF, Gilson Medical Electronics, Villiers le Bel, France) provided a fast equilibrium of antibiotic concentration between the CCp and the PCp in order to simulate blood levels also in the PCp. Broth flows into and out of the CCp were checked by the mean of two liquid flowmeters with an RS 232 interface (Phase Separations France, Saint Quentin en Yvelines, France). The dialyser unit was placed in an incubator at 37°C.

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Figure. Schematic illustration of the in vitro pharmacokinetic model and device connections [, broth or antibiotic solution flow;---, RS 232 cable;--, Gilson serial input/output channel (GSIOC) cable].
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Antibiotic and culture medium
Ciprofloxacin was obtained as reference powder (85.9% free base) and as marketed infusion solution (CIFLOX 200 mg/100 mL) from Bayer Pharma (Puteaux, France). All experiments were performed using MuellerHinton broth (A.D.L., Tresses, France).
Determination of antibiotic concentration
A validated high performance liquid chromatographic (HPLC) method3 was used. Briefly, it consisted of an isocratic HPLC technique with column switching and direct injection of broth. Fluorescence detection allowed a quantification limit of 0.078 mg/L with a 0.040 mL sample size. The standard curves were linear between 0.078 and 1.25 mg/L. Intraday and interday coefficients of variation between 0.078 and 10 mg/L ranged from 0.34 to 1.04% and from 2.09 to 7.07%, respectively.
Data analysis
The software PHARMACOKIN4 was used to perform compartmental analysis of designed drug concentration data. The goodness of fit for each concentrationtime curve vas evaluated both by the correlation coefficient between experimental and software-calculated data, and the objective function F, which represents the sum of weighted squared deviations. The following parameters were determined: peak concentration (Cmax), concentration at 12 h (C12), time to reach the peak (tmax), distribution half-life (t1/2
), elimination half-life (t1/2ß), area under the curve from 0 to 12 h (AUC012), area under the curve from 0 to
(AUC0
), absorption rate constant (Ka) and the lag time (tlag).
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Results and discussion
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The concentrationtime curve of ciprofloxacin showed a good reproducibility of sequential experiments and comparable values for CCp, PCp and human reference data. Indeed, pharmacokinetic parameters from simulated curves (Table
) ranged within the confidence intervals of the means of human data.5,6
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Table. Mean pharmacokinetic parameters (± s.d.) for the central compartment (CCp) and the peripheral compartment (PCp) after simulation of a 30 min infusion of a 200 mg dose and a 500 mg oral dose of ciprofloxacin, and the human corresponding reference data
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Several improvements were afforded in the proposed in vitro model to allow accurate simulation of the human concentrationtime profile. Indeed, computer control of flowmeters and pumps avoided flow rate inconsistency. The flowmeters were checked each second by the computer and the information was recorded by the program. An average flow rate was then calculated from five consecutive values and compared with the theoretical flow rate. Finally, an adjustment could be calculated and the speed of the different pumps was brought up to date to fit the required value. PC-assisted feedback control based on the detection of the liquid level in the CCp was proposed by Ledergerber et al.,7 but the vigorous agitation necessary for drug homogenization in this compartment made this technique difficult. The rapid rate of drug equilibration across dialyser membranes observed with the present system (<5 min; data not shown) allowed for similar drug concentrationtime profiles to be obtained in the CCp and the PCp. Therefore, appropriate adjustment of experimental conditions was the same for the two compartments. This allowed systemic kinetics and tissue infection site concentrationtime profiles to be simulated accurately using the corresponding human data. Indeed, as the majority of infections are located in peripheral tissues, it is considered more appropriate to study the process of drug penetration of the target site rather than assessing serum pharmacokinetics.8 Since it is believed that most ordinary infections occur in the interstitial fluid, levels of antibiotics in the extracapillary fluid (ECF) are presumably more relevant than those in whole tissues. Considering the case of ciprofloxacin, the concentrations in human lung tissue are about five times higher than the corresponding serum concentrations.9 In contrast, interstitial free drug concentrations (determined by microdialysis in skeletal muscle) are significantly lower than corresponding total serum drug concentrations; the interstitium-to-serum concentration ratios range from 0.55 to 0.73.8 In general, data obtained in the model peripheral compartment were distantly related to human ECF free drug or whole tissue levels such as those obtained by Dudley et al.,10 which overestimated ECF free drug concentrations and underestimated lung whole tissue levels. Our model, based on direct simulation of the infection site concentrationtime profile, may produce more accurate data. Additional devices used for oral administration simulation were replaced by a computer-controlled syringe pump, also used for infusion simulation, facilitating computer control of administration processes.
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Acknowledgments
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We thank V. Philip for providing biomedical devices, V. Marquais for technical assistance, and G. Etchetto and the Haut Leveque Sterilization Service staff for their help.
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Notes
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* Corresponding author. Tel: +33-5-57-57-17-36; Fax: +33-5-56-93-04-07; E-mail: boubakar.ba{at}phcocin.u-bordeaux2.fr 
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References
|
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1
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Blaser, J. & Zinner, S. H. (1987). In vitro models for the study of antibiotic activities. Progress in Drug Research 31, 34981.[Medline]
2
.
Blaser, J., Stone, B. B. & Zinner, S. H. (1985). Two compartment kinetic model with multiple artificial capillary units. Journal of Antimicrobial Chemotherapy 15, Suppl. A, 1317.[ISI][Medline]
3
.
Ba, B. B., Ducint, D., Fourtillan, M. & Saux, M. C. (1998). Fully automated high-performance liquid chromatography of ciprofloxacin with direct injection of plasma and MuellerHinton broth for pharmacokinetic/pharmacodynamic studies. Journal of Chromatography, Biomedical Sciences and Applications B714, 31724.
4
.
Kister, G., Bres, J. & Cassanas, G. (1998). PHARMACOKIN: a software for pharmacokinetic teaching. In Program and Abstracts of the Second Scientific Meeting, Grenoble, France, 1998. Abstract 13, p. 14. Association of Pharmacy Faculties Pharmacologists, Paris, France.
5
.
Drusano, G. L., Plaisance, K. I., Forrest, A. & Standiford, H. C. (1986). Dose ranging study and constant infusion evaluation of ciprofloxacin. Antimicrobial Agents and Chemotherapy 30, 4403.[ISI][Medline]
6
.
Crump, B., Wise, R. & Dent, J. (1983). Pharmacokinetics and tissue penetration of ciprofloxacin. Antimicrobial Agents and Chemotherapy 24, 7846.[ISI][Medline]
7
.
Ledergerber, B., Blaser, J. & Lüthy, R. (1985). Computercontrolled in-vitro simulation of multiple dosing regimens. Journal of Antimicrobial Chemotherapy 15, Suppl. A, 16973.[ISI][Medline]
8
.
Brunner, M., Hollenstein, U., Delacher, S., Jäger, D., Schmid, R., Lackner, E. et al. (1999). Distribution and antimicrobial activity of ciprofloxacin in human soft tissues. Antimicrobial Agents and Chemotherapy 43, 13079.[Abstract/Free Full Text]
9
.
Caruso, E., Castro, J. M., Chamoles, N., Galimberti, R., Jorge, L. & Rohwedder, R. (1988). Penetration of ciprofloxacin into human lung tissue after oral and i.v. dosing. In Ciprofloxacin (Percival, A. & Nicoletti G., Eds), pp. 6570. Sixth Mediterranean Congress of Chemotherapy, Taormina, Italy, 1988. Schwer Verlag, Stuttgart.
10
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Dudley, M. N., Mandler, H. D., Gilbert, D., Ericson, J., Mayer, K. H. & Zinner, S. H. (1987). Pharmacokinetics and pharmacodynamics of intravenous ciprofloxacin. Studies in vivo and in an in vitro dynamic model. American Journal of Medicine 82, Suppl. 4A, 3638.[ISI][Medline]
Received 2 August 2000;
returned 2 October 2000; revised 23 October 2000;
accepted 6 November 2000