pharmacokinetics of cefazolin administered as a new drug delivery system in healthy volunteers

BIOPHARMACEUTICS & DRUG DISPOSITION, VOL. 9,377-388 (1988)

PHARMACOKINETICS OF CEFAZOLIN ADMINISTERED AS A NEW DRUG DELIVERY

SYSTEM IN HEALTHY VOLUNTEERS

J. M. LANAO, M. T. VICENTE AND A. DOMINGUEZ-GIL

Departamento de Farmacia y Technologia FarmacPutica, Facultad de Farmacia, Universidad de Salamanca, Salamanca, Spain

ABSTRACT A study was made of the pharmacokinetic behaviour in plasma and urine of cefazolin in seven healthy volunteers following parenteral administration of sodium cefazolin (1250 mg) and a sustained release formulation containing sodium cefazolin: cefazolin- dibenzylamine (1:4) at a total dose of 1250 mg, both formulations being administered over 1 week by the i.m. route. Cefazolin concentrations in plasma and urine were determined by a HPLC technique. Kinetic analysis of the experimental results was performed using an open single-compartment kinetic model and a sustained release model for the drug administered as standard formulation and the sustained release formulation, respectively. The results obtained point to significant variations in the pharmacokinetic profile of the drug when administered in the DDS.

The time of cefazolin at levels greater than 1 pg ml-1 was 16.03 f 2 5 1 and 47.76 f 14.18 h-1 after administration of the standard formulation and the DDS, respectively. The urinary excretion rate curves also show the existence of sustained drug levels in the urine following administration of the DDS. The renal clearance of the drug did not show statistically significant differences between the two formulations administered. The process of release of cefazolin from the cefazolin-dibenzylamine complex proved to be a first order kinetic process. The release constant of the antibiotic was calculated according to three different methods : the Wagner-Nelson method; the statistical moments method; and the fitting of the plasma levels curves to a compartmental model considering release and absorption. The values obtained for this constant ranges from 0.026 f 0.02h-’ calculated with the method of statistical moments to 0.094 f 0.03 h-1 as calculated by the equation derived for the plasma fitting of cefazolin administered as DDS.

KEY WORDS Cefazolin Drug delivery system Pharmacokinetics

INTRODUCTION

In recent years drug delivery systems have undergone considerable advances, allowing modifications in dosage schemes to be made in the case of many drugs.

In the field of the beta-lactam antibiotics the protocol followed in obtaining a sustained release effect is dual: the obtaining of structural derivatives, with

0142-2782/ 88 / 04377-12 $06.00 8 1988 by John Wiley & Sons, Ltd.

Received 10 April 1987

378 J. M. LANAO, M. T. VICENTE AND A. DOMINGUEZ-GIL

alterations in certain pharmacokinetic processes, especially at elimination level, such as the case of ceftriaxone, cefonicid, etc.,’” and the formation of fairly insoluble derivatives in which the release process is modified thus inducing a decrease in the absorption rate of the antibiotic with alterations in the pharmacokinetic profile and hence giving rise to sustained-type kinetic^.^

Cefazolin is a beta-lactam antibiotic widely used in clinical practice and is active against Gram (+) microorganism^.^ The speed of its elimination process implies reduced dosage intervals with a view to maintaining the serum levels of the drug within the therapeutic range. This situation has led to the development of several drug delivery systems for this antibiotic.

The aim of the present study was to gain insight into the pharmacokinetic profile of cefazolin administered in a new sustained drug delivery system developed from a fairly water-insoluble derivative of the drug; the cefazolin- dibenzylamine complex.

MATERIAL AND METHODS

Products Standard formulation (SF): sodium cefazolin in 1250 mg vials. Drug Delivery System (DDS): cefazo1in:cefazolin-dibenzylamine ( 1 :4) in

1250 mg vials. The dibenzylamine salt of cefazolin is very slightly soluble. Both preparations were supplied by Antibioticos S. A. (Madrid, Spain).

Volunteers The pharmacokinetic study was performed in seven healthy volunteers with

an age range between 23 and 30 years (25.86 ? 2.48) and a weight range of

The study was approved by the Ministry of Health and by the Ethical Committee of the University Clinical Hospital of Salamanca. The volunteers were informed as to the purpose of the study and gave written consent to participate. They were also subjected to a clinical examination both before starting and after completing the trial.

Administration and sampling Each of the volunteers received a single dose of the standard formulation

intramuscularly. After 1 week they were given a single dose of the DDS through the same route.

Blood and urine samples were collected for both formulations at the following times:

Blood: 15, 30,60, and 90 min: 2, 3 ,4 ,6 ,9 , 12, and 24h*. Urine: 1, 2, 4, 6, 9, 12, and 24h*.

46-73 kg (58.14 ?r 10.35).

*Only in the case of the DDS.

CEFAZOLIN 379

Blood samples (2ml) were collected in heparinized tubes by direct venipuncture of a forearm vein; plasma was separated by centrifugation and frozen at -2OO for later analysis.

Urine samples were collected at the same time as those of blood, measuring the volume collected and separating an aliquot of 10 ml which was frozen until later assay.

Analytical technique Determination of plasma cefazolin concentrations was performed by an

HPLC technique. To 1 ml of plasma was added 0.1 mi of adeproteinizing solution prepared with 9.5 ml of methanol, 0.5 ml of trichloracetic acid to which 10 mg of barbital were added as internal standard. After shaking the mixture it was centrifuged at 5000 rev min-' for 3 min. The supernatant fluid warused to determine the antibiotic following injection of 50 pl into the chromatograph.

The apparatus employed was a Varian 5000 chromatograph equipped with a UV detector with a variable wavelength detector. p-Bondapak C-18 reverse phase columns were employed. The chromatographic conditions were as follows:

Stationary phase: reverse phase (RP-18). Mobile phase: I / 150 M phosphate buffer, pHz7.4, Methanol (78/22). Wavelength 254 nm. Flow rate 1 ml min-'.

The retention times established for cefazolin and barbital were 6-1 and 9.6 min, respectively.

The determination of cefazolin in urine was carried out by the above method after dilution of the samples (150) in phosphate buffer, pHz7.4, and injection of the samples directly into the chromatograph.

For analysis of the problem samples, calibration curves of cefazolin were prepared in plasma and phosphate buffer, pH 7.4, with concentration values ranging between 2.5 and 50 pg ml-'. The sensitivity limit was 2 pg ml-' and the variation coefficient was less than 15 per cent.

Pharmacokinetic analysis Cefazolin administered as standard formulation. The plasma levels of

cefazolin administered as SF followed a single-compartment open kinetic model with first order absorption and elimination kinetics.5 The choice of this model from among a series of possibilities was based on the following criteria: a sum of the weighted squares of the residual values; standard deviation of the parameters evaluated by the covariance matrix from a Gauss-Newton non- linear regression algorithm: and according to Akaike's ~r i ter ion.~

The fitting of the experimental data to the proposed model was performed using a non-linear regression program based on the Nelder-Mead algorithm which searches for initial estimates and the Gauss-Newton algorithm for the final optimization of the parameters.*


From the experimental values of cefazolin in human plasma the following potential relationship between the variance of the data (a2) and the mean plasma concentration of cefazolin (c) was defined by the following equation:

According to this expression, in the fitting of the data by non-linear regression we used a statistical weight equivalent to the reciprocal of the square of the observed plasma concentration.

With this model it was possible to determine the elimination constant (k,) of cefazolin and the apparent distribution volume ( V& considering for such calculations a bioavailability of 100 per cent for the antibiotic when administered by the i.m. route.4

Determination of the absorption constant (ka) was performed according to the Wagner-Nelson method.’

Other parameters determined were the maximum plasma concentration (C,,,), the time that Cma, is reached ( tmax) , plasma clearance (Cl,), and the plasma elimination half-life (t s).

Renal clearance of cefazolin (Cl,) was calculated from the urinary excretion rate (V) according to the expression:

where C, is the plasma cefazolin concentration.

Figure I. Compartmental model for the disposition kinetics of cefazolin administered as the drug delivery system

Cefazolin administered as the drug delivery system. To characterize the release and absorption kinetics of cefazolin administered as the DDS a compartmental model whose scheme is shown in Figure 1 was used:

Compartment I represents the dose of cefazoline present in the DDS as the cefazolindibenzylamine complex whose fraction with respect to the total dose of cefazolin was defined as Fa.

CEFAZOLIN 381

Compartment 2 represents the drug released from the complex remaining at the site of administration. Compartment 3 represents the drug present in the DDS as sodium cefazolin whose fraction was defined as Fb, considering only kinetic absorption processes for this fraction of the dose. Compartment 4 represents the human body in which cefazolin is absorbed and eliminated according to the single-compartment model referred to above.

The processes of release, absorption, and elimination were considered as first order kinetic processes after previous selection of the model among the other possible candidates using the above mentioned criteria. The kinetic release process of cefazolin from the cefazolin-dibenzylamine complex was characterized by the constant kr.

The mass transfer in the model considered can be formulated mathematically according to the following systems of differential equations:

(3) - - kr XI dXI

dt Compartment 1 --

(4) - kr XI - ka X2 dX2 dt Compartment 2 - -

dX3 ( 5 ) Compartment 3 7 = - ka X3

dX4 Compartment 4 - - - ka ( X 2 + X3 ) - ke X4 dt (6)

The solving of this system by the use of Laplace transforms yields the following integrated equations that permit the characterization of the variation on the levels of the drug at the site of absorption ( X ) and in the organism (X4): Site of absorption:

-kr t X = ( Da- kr + a) Ckat + ( Da - Da rkk,' kr e kr - ka (7)

(where Da and Db are the dose of cefazolin present in the DDS as cefazolin- dibenzylamine and sodium cefazolin, respectively). Organism:

x 4 = ( Dakakr e-k t Dakakr ka e-ka t+ (ka-kr) (ke-kr) + ( (kr-ka) (ke-ka) (ka-ke)

1 (8) Da ka kr + Db ka -ke t

+ ( (kr-ka) (ke-ka) (ka-ke) )


Equations (7) and (8) may be expressed in the form of the fraction of the dose (fl of cefazolin remaining at the absorption site, in the first case, and in the form of the plasma cefazolin concentration in the second, obtaining the following equations:

-kr I kr ) e -ka t F=(Fa- kr + Fb) e +(Fa - Fa- (kr-ka) (kr-ka)

1 Db ka -ket +- Da ka kr

+ ( (kr-ka) (ke-ka) (ka-ke)

(9)

Fractions Fa and Fb were considered in all cases to be constant and equivalent to 0.8 for the cefazolin dose present as the cefazolin-dibenzylamine complex and 0.2 for the dose of drug present as sodium cefazolin in the DDS.

The cefazolin remaining at the absorption site as a function of time was calculated in the form of a fraction (fl from the plasma cefazolin concentrations obtained after administration of the DDS by application of the Wagner-Nelson method, using for each volunteer the value of the elimination constant (Ke) previously determined after administration of SF. Previous statistical comparison between the values of renal clearance determined in all the volunteers by the two treatments, using a paired test, permits one to rule out the existence of intrasubject variations in the elimination constant of cefazolin.

The data corresponding to the fraction of drug remaining at the absorption site and to the concentrations of cefazolin in plasma were fitted to equations (9) and (10) and allowed us to deduce the value of the release constant (Kr) of the antibiotic from the dibenzylamine salt of cefazolin.

At the same time, the release constant of cefazolin from the dibenzylamine salt was determined from the plasma concentration data of cefazolin obtained in each volunteer after administration of both formulations using a model- independent technique based on the statistical moment theory. ''

The mean residence time (MRT) of cefazolin in plasma was calculated for each of the two formulations from the following expression:

ot C, dt

o C,dt (1 1)

M R T = J m 7 J

Mean residence time was defined as MRTa for the time calculated after administration of the DDS and as MRTb for the time calculated after administration of SF.

CEFAZOLIN 383

The mean residence time of the process of release of' cefazolin from the cefazolin-dibenzylamine complex (MRT,) was calculated as follows:

1 also considering that Kr = m, Statistical analysis

In view of the reduced number of samples studied (n=7), the statistical comparison of the different experimental results was performed by use of the non-parametric Wilcoxon rank sum test for independent samples and the signed rank test for dependent or paired samples. The latter proved to be very useful for the study of possible intrasubject variations."

Parallel to the study of statistical significance carried out simultaneously with all the methods employed in the determination of the release constant, a one way ANOVA variance test was conducted."

RESULTS

Figure 2 shows the mean plasma levels of cefazolin obtained in the seven volunteers following administration as the SF and as the DDS.

24 *' HOURS 4 8 12 16

Figure 2. Mean plasma levels of cefazolin following the administration of SF and DDS


Table 1. Pharmacokinetic parameters of cefazolin obtained after administration of SF calculated with a one-compartment model

Cmax tman v d Clp (% D)*$ c1r (1 ih) 5 Kt Vol. (h-) (h- ) $h (pglml) (h) (1) U / h )

no. 1 0.979 0.309 no. 2 2.308 0.404 no. 3 0.420 0.522 no. 4 1.302 0.289 no. 5 1.024 0.246 no. 6 3.594 0.274 no. 7 5.007 0.269

X 2.090 0.330 un 1.539 0.090

~ n - 1 1.663 0.098

2.246 1.716 1.328 2.394 2.815 2.529 2.580 2.229 0.486 0.525

75.704 53.998 91.152 85.820 46.450

102.235 84.274 77.090 18.638 20.132

0.779 0.826 1.842 1.314 1.995 0.2 17 0.435 1.058 0.630 0.680

12.98 1 16.695 5.246 9.964

16.465 11.519 13.196 12.279 3.640 3.930

4.004 6,695 2.736 2.884 4.054 3.156 3.544 3.867 1.250 1.350

74.510 65.640 8 1 .OOO 48.610 75.440 66.370

100~000 73-081 14.610 15.781

2-983 4.395 2.216 1.402 3.058 2.095 3,544 2.813 0.925 0.999 -

Table I shows the pharmacokinetic parameters of cefazolin administered as the SF characterized according to a single-compartment open kinetic model.

The results obtained show that in the absence of release processes cefazolin is absorbed and eliminated rapidly, obtaining for the standard formulation mean values of the absorption and elimination constants of 2.090 f 1.663 h-' and 0-330 f 0-098h-', respectively. These values are similar to those reported by other authors following i.m. administration of the antibiotic." The maximum plasma concentration had a mean value of 77.09 f 20.13 pg ml-', in some cases being lower than the values of the maximum concentrations observed experimentally, which is attributable to the kinetic model employed.

Following administration of the DDS the plasma levels of cefazolin show a significant alteration patent in a decrease in the maximum plasma levels, which proved to be statistically significant (p = 0.0005), and an increase in the time at which cefazolin levels were greater than 1 pg ml.-' This time had a mean value of 16.030 f 2.5 1 1 h after administration of the SF i.m. up to 47.758 f 14.184 h after administration in the DDS, the difference being statistically significant (p = 0-006).

The time at which the maximum concentration is reached, however, did not have any significant differences between the two formulations; this is attributable to the absorption of the compound with immediate release present in the DDS which guarantees therapeutic levels of the antibiotic at times similar to those seen with the conventional formulation.

Figure 3 shows the mean urinary excretion rate curves of cefazolin administered in both kinds of formulation. A strong parallelism may be seen between the plots of these curves compared with the homologous curves relating to the plasma levels. Regarding the urinary excretion process the DDS guarantees urinary cefazolin levels for prolonged periods.

The renal clearance of the drug had a mean value of 2.813 f 0.999 Ih-' following administration of the drug as SF and of 3.269 f 1.194 1 h-' when

CEFAZOLIN 385

- 200 c . - F W I- a

0 a

a 150 z

I- W

U X w

> 1oc LT 4 f a 3

50

20 24 HOURS

4 8 12 16

Figure 3. Mean excretion rate of cefazolin following administration of SF and DDS

1

Q8

0 W m a S: 0.6 m a

f w 0.4 d

z 3

2

W U

z 0 a L 0.2 LT LL

4 8 12 16 24 HOURS

4 8 12 16 20 24 HOURS

Figure 4. Fraction of cefazolin remaining at the absorption site as a function of time for both formulations


administered as the DDS. Application of a paired test to this pharmacokinetic parameter shows that there are no statistically significant differences (p = 0.375) and hence no intraindividual variations in the renal elimination of the antibiotic administered by both formulations.

Figure 4 shows the mean fraction of cefazolin remaining at the absorption site calculated by the Wagner-Nelson method for both formulations. In the figure it is possible to observe the biexponential nature of the plot when the DDS is administered, reflecting the existence of two simultaneous kinetic processes at the site of administration; release and absorption.

Table 2 shows the value obtained for the release constant of cefazolin from the cefazolin-dibenzylamine complex calculated from the fraction of drug remaining at the absorption site using equation (9); from the plasma levels of cefazolin administered as the DDS by application of equation (lo), and from the theory of statistical moments and according to equation (13). The table also shows the value obtained for the first order constant characterized from the slope of the terminal phase of the plasma levels curve obtained following administration of the DDS. One sees that the release constant shows a higher mean value when it is calculated by the statistical moments and a lower value when it is calculated according to equation (lo), the mean value being lower than 0.1 h-’ for all the methods used.

DISCUSSION

When administered as the SF, the absorption and elimination of cefazolin are fast; it rapidly reaches therapeutic plasma levels but only maintains these within the therapeutic range for relatively short periods of time which in clinical practice obliges the physician to use reduced dosage intervals (6 and 8 h).

Table 2. Release constant of cefazolin from the DDS formulation caluclated using: (a) Wagner-Nelson method, (b) statistical moments (c) slope of terminal phase, (d) using the equation

(10) no. K,(a) Kdb) K(c) Kdd) 1 0.102 0.136 0.090 0060 2 0.062 0.067 0.054 0-030 3 0.067 0.084 0061 6 4 0.074 0.111 0073 0.038 5 0.043 0035 0.031 0.018 6 0.063 0.104 0075 0.021 7 0.065 0-124 0.096 0.009 X 0.068 0-094 0.069 0.025 an-1 0.017 0.035 0.022 0.020

CEFAZOLIN 387

The results obtained also confirm the notion that although renal excretion is the major elimination route for this drug, there are other routes that account for 27.25 per cent of the overall elimination of the antibiotic.

Administration of the drug in the form of the DDS leads to significant modifications in the pharmacokinetic profile of the drug, mainly producing a prolongation of its plasma levels that is controlled by the release process of cefazolin from the cefazolin-dibenzylamine complex.

The presence of 20 per cent of sodium cefazolin in the DDS means that therapeutic levels of the drug are reached rapidly, as may be seen in the experimental results. From the therapeutic point of view the results obtained allow one to affirm that the DDS assayed permits modifications in the conventional dosage scheme in that the dosage intervals can be extended to 24h with this system, although this latter aspect would require experimental confirmation at clinical and pharmacokinetic level in multiple dosage regimens using this new dosage form.

The use of compartmental analysis to characterize the release process of cephazoline from the DDS using the fraction remaining at the site of absorption calculated from the Wagner-Nelson method, on one hand, and from the plasma levels curve of cefazolin after administration of the DDS on the other, allows one to infer the following:

1. Release of the antibiotic from the cefazoline-dibenzylamine complex follows a first order kinetic process. 2. The values obtained for the release constant with both calculation methods proved to be significantly lower (p 3.10-5) than the value obtained for the absorption and elimination constant when the drug was administered in conventional form to the same healthy volunteers. The difference in magnitude between the three constants clearly shows that the release process is the limiting kinetic process and hence is responsible for the prolonged drug plasma levels observed.

The value obtained for the release constant implies that the release half-life of the antibiotic has mean values greater than 7h. This allows one to predict the efficiency of the DDS with respect to the release phase and to affirm that over minimum periods of 24 h this formulation, when administered through the i.m. route, will guarantee a sustained action of the drug.

The results suggest that the apparent rate constant calculated from the slope of the terminal phase of the plasma levels curve after administration of the DDS, although release of the drug is maintained, should reflect the value of the release constant of cefazolin, in view of the fact that it may be considered as the limiting kinetic process. Table 2 confirms such a hypothesis; similar results were obtained for the release constant by the different methods with the exception of the apparent rate constant obtained from the plasma levels curve.


The limitations of the Compartmental models in the characterization of certain kinetic proce~ses '~ imply the need to use model-independent techniques that will corroborate the results obtained by compartmental analysis.

The application of statistical moments to the plasma kinetics of cefazolin obtained according to both formulations validates the applicability of compartmental models and in particular the Wagner-Nelson method, in the determination of release constants, as seen in the data offered in Table 2.

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Pharmacol. Ther., 30,587 (1981). 3. V. H-L. Lee and J. R. Robinson, in Sustained and Controlled Release Drug Delivery Systems,

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pharmacokinetics of cefazolin administered as a new drug delivery system in healthy volunteers

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