Introduction

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During the last decades it has been shown that pharmacokinetic parameters of small molecular weight drugs scale across species as a function of body weight, using a power function of the general form: P = a W^b, where P is the pharmacokinetic parameter, W the body weight, a the allometric coefficient, and b the allometric exponent. After log-transformation the power function can be written as a linear equation: log P = log a + b log W, where log a is the intercept and b the slope.

Allometric scaling of small molecular weight xenobiotics has been applied to compounds excreted by physical processes such as glomerular filtration of unchanged drug (Sawada et al., 1984; Mordenti, 1985), but also to metabolized compounds if suitable measures are taken to correct for interspecies differences in metabolic clearance (Ings, 1990; Lave et al., 1996). Pharmacokinetic parameters of nonmetabolized compounds usually scale with body weight according to power functions with a certain allometric exponent, depending on the pharmacokinetic parameter (Mordenti, 1986). Clearance tends to scale with an exponent of 0.6 to 0.8, whereas volumes of distribution and half-lives scale with exponents of approximately 0.8 to 1.0 and 0.2 to 0.4, respectively. It has recently been shown that the principles of allometric scaling established for nonmetabolized small molecular weight compounds can also be applied to therapeutic proteins covering a molecular weight range from 6 to 98 kDa (Mordenti et al., 1991; McCarthy et al., 1993).

Lenercept is a recombinant fusion protein consisting of the extracellular domain of two human p55 tumor necrosis factor (TNF)¹ receptors and the hinge as well as the constant domain C2 and C3 sequences of the human immunoglobulin G1 (IgG1) heavy chain (Ashkenazi et al., 1991). Lenercept, previously referred to also as Ro 45-2081 or TNF receptor immunoadhesin, binds TNF with high affinity and a high kinetic stability of the resulting complex (Evans et al., 1994). Its therapeutic benefits derive from its capability of neutralizing TNF activity under disease conditions such as severe sepsis and septic shock, which are characterized and mediated by an overproduction of TNF. The protective effect of lenercept in severe sepsis and septic shock has been demonstrated in laboratory animals (Lesslauer et al., 1991; Van Zee et al., 1996) and, recently, in clinical trials (Abraham et al., 1997). Lenercept is purified from eukaryotic cell expression and is present as a disulfide-bonded homodimer with a molecular weight of about 120 kDa and eight potential asparagine-N-linked glycosylation sites. In the course of the development of lenercept, its pharmacokinetics were characterized in several animal species. This allowed for both better planning of toxicology trials and for predicting the pharmacokinetics in humans by allometric scaling, in order to help in the design of first clinical trials.

In this paper we describe the allometric scaling of lenercept. A first allometric scaling for lenercept was performed prospectively to help in the design of first clinical protocols. This prediction was based on preliminary pharmacokinetic data in several animal species. Here we communicate results from a refined, retrospective scaling, which was performed when additional data on animal pharmacokinetics became available. The results of the experiments showed that the pharmacokinetics of lenercept do not follow the described scales of allometry; rather, the drug shows similar pharmacokinetic behavior across species from rat to human, i.e., both clearance and volume of distribution scale with a slope factor close to 1.

	Materials and Methods

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Test Material. Lenercept was made by Genentech, Inc. (South San Francisco, CA). It was produced in Chinese hamster ovary cells and purified by ion-exchange and affinity chromatography, as previously described (Ashkenazi et al., 1991). The test substance was formulated as an aqueous solution at a concentration of 5 mg/ml. This formulation was used as such for i.v. bolus administration to rats and rabbits, or was further diluted for administration by i.v. short infusion to cynomolgus monkeys and dogs.

Animal Experiments. Lenercept was administered i.v. either by bolus administration or short infusion. Short infusion was chosen for those animal species (cynomolgus monkey, dog) that had not been exposed to lenercept before the described experiments.

Drug Assay Method. Lenercept was analyzed by means of an enzyme-linked immunological and biological binding assay. In brief, in a single step lenercept is bound to a microtiter plate coated with mouse monoclonal antibodies against the human sTNFR-55 portion of lenercept (clone htr-20), and at the same time the free TNF binding sites of lenercept are labeled with its ligand TNF-, which is coupled to horseradish peroxidase. Nonbound material is removed by washing and the quantity of peroxidase bound to the microtiter plate is measured enzymatically. Measurement range was 0 to 20 ng/ml lenercept, with a detection limit of 0.2 ng/ml. Biological samples were analyzed after appropriate dilution with a suitable buffer solution.

Determination of Antibodies against Lenercept. For the determination of neutralizing antibodies against lenercept a modified sandwich-type assay was used. Different dilutions of serum or plasma were coincubated with 10 ng/ml lenercept in microtiter plates coated with noninhibitory mouse monoclonal antibodies against the human sTNFR-55 portion of lenercept (clone htr-20), then a TNF--peroxidase conjugate was added to label still active lenercept. After washing the plates the amount of bound peroxidase was determined enzymatically. In the presence of antibodies the measurable amount of lenercept was less than the 10 ng/ml added. The difference between this 10 ng/ml and the measured concentration gave the amount of lenercept that was neutralized by antibodies. Antibody levels are reported as the concentration of lenercept that can be neutralized.

Pharmacokinetic Analysis. Pharmacokinetic parameters were calculated by noncompartmental methods, using the computer program TOPFIT 2.0 (Heinzel et al., 1993). For rats, parameters were determined from composite plasma concentration data. The area under the plasma or serum concentration-time curve (AUC) was calculated using the linear trapezoidal rule and extrapolated to time infinity. However, both AUC and area under the first moment curve (AUMC) were estimated by extrapolation from the apparent linear portion of the semilogarithmic plot before the change in the kinetics due to the onset of the immune response against the test substance (for rationale, see Discussion). The apparent half-life (T_1/2) was calculated using the equation T_1/2 = ln 2/. The apparent rate constant  was determined from the linear portion of the log plasma or serum concentration-time curve before the onset of the immune response. Total clearance (Cl) was calculated as Dose/AUC. The volume of distribution at steady state (V_ss) was calculated as Dose × AUMC/(AUC)². The initial volume of distribution (V_i) was calculated as Dose/concentration of the first sample taken after administration.

Allometric Scaling. The mean pharmacokinetic parameters (Cl, V_ss, and T_1/2) for lenercept in laboratory animals were log-transformed and correlated with the log-transformed mean body weights (W) using the log-transformed allometric equations of the general form: log P = log a + b log W, where P is the pharmacokinetic parameter, W the body weight, a the allometric coefficient, and b the allometric exponent. The values of allometric coefficients a and exponents b were estimated by linear least-squares regression. The allometric equations obtained were used to calculate the respective pharmacokinetic parameter for a 70-kg human.

	Results

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Pharmacokinetic Experiments. Characteristic individual plasma or serum concentration-time curves of lenercept and antibodies against lenercept following i.v. administration of lenercept to rabbits, cynomolgus monkeys and dogs are shown in Fig. 1. Composite plasma concentration-time curves in rats are depicted in Fig. 2. All derived pharmacokinetic parameters are presented in Table 1.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Representative individual plasma or serum concentrations of lenercept (closed symbols) and antibodies against lenercept (open symbols) following a single i.v. dose to (A) rabbit (5 mg/kg), (B) cynomolgus monkey (5 mg/kg), and (C) dog (0.1 mg/kg). For the cynomolgus monkey, determined antibodies are not shown (single determination made at day 19 postdose).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Composite individual plasma concentrations of lenercept and antibodies against lenercept following a single i.v. dose to rats at dose levels of 0.2 or 5 mg/kg. n = 4/dose level; sampling from two rats/time point/dose level except for last time point.

Allometric Scaling. Table 2 shows the results of the linear least-squares fitting of log Cl, log V_ss, and log T_1/2 versus log W data in animals, including the predicted and observed values in humans.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Allometric scaling of lenercept and observed values in humans. A, clearance; B, volume of distribution at steady state; C, half-life (including data in mice). Closed symbols, observed values in animals with regression line for prediction; open symbols, observed values in humans (Kneer et al., 1996).

	Discussion

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Preclinical studies with human proteins are often accompanied by an immune response against the protein, leading to the formation of antibodies (Working, 1992). Due to the human amino acid sequence and structure of lenercept, an immune response against the test substance was fully expected in laboratory animals. Indeed, in all species tested, except in the rat at 5 mg/kg, an immune response was observed usually within 6 to 10 days after single dosing, as evident from antibody analysis and a rapid drop in the plasma concentration-time curves. The apparent absence of an immune response in the rat at 5 mg/kg probably reflects the interindividual variability of the immune response rather than a species difference. Thus, in other pharmacokinetic experiments with lenercept in the rat at this dose level an immune response was observed (data not shown).

The rapid drop in the plasma concentration-time curves caused by the immune response was excluded from the pharmacokinetic analysis. This exclusion was justified because the immune response in nonhomologous species bears no relevance to humans, so that pharmacokinetic parameters from an analysis including the immune response cannot be extrapolated to humans. However, the onset of this immune response meant that the pharmacokinetics could only be followed for about one to two half-lives, leading to some imprecision in the values estimated for the parameters. This is especially true for V_ss because the contribution of the extrapolated portion to the AUMC was very high. Furthermore, an elimination from the central compartment had to be assumed for the estimation of V_ss by noncompartmental methods.

Nevertheless, the pharmacokinetics of lenercept could be characterized in laboratory animals. In all species tested, lenercept was cleared very slowly, at rates far below physiological flow rates like liver or kidney plasma flow. The estimated values for V_ss were small, about twofold the plasma volume, indicating a very limited distribution of lenercept to tissues. The extremely low clearance was reflected in long apparent half-lives ranging from 3.4 days in the rabbit to 6.5 days in the rat.

Despite the difficulties in the pharmacokinetic assessment of lenercept caused by the immune response, allometric scaling of its pharmacokinetic parameters was reasonably predictive of the human pharmacokinetics (see Table 2). The greatest deviation of the predicted values from the observed ones was found for clearance, where the predicted value was twice as high as the observed one. The allometric equations turned out to be different from the ones reported for other proteins (Mordenti et al., 1991), the allometric scalings of which tend to follow the principles established for nonmetabolized small molecular weight compounds. Thus, for rCD4, CD4-IgG, rhGHm, rt-PA and relaxin the exponents in the allometric equation for Cl and V_ss vary from 0.65 to 0.84 and 0.84 to 1.02, respectively. By contrast, with lenercept the allometric exponents for both Cl and V_ss were close to 1.0. Consequently, the allometric exponent for T_1/2 was close to 0, i.e., a similar half-life is seen in all species regardless of weight. The deviations of the predicted human parameters from the observed ones may be partially explained by the discussed shortcomings of the pharmacokinetic assessment in laboratory animals. Due to the immune response, the terminal phases of most concentration-time curves could not be well characterized. Thus, some Cl values might be overestimated, while some T_1/2 values were underestimated. It is noteworthy that the longest period available for pharmacokinetic assessment (13 days in the rat) was associated with the longest estimated T_1/2 (6.5 days), which was also closest to the observed T_1/2 in humans. In all other species, only 6 to 10 days postdose were available for pharmacokinetic assessment before the onset of the immune response.

Possible reasons for the uncommon allometric scaling of lenercept's clearance may include its extremely slow clearance, which represents only a marginal fraction of physiological flow rates to liver or kidney in all species studied. Accordingly, the allometric scaling of lenercept pharmacokinetics does not follow the allometric scaling of these flow rates; this is in contrast to results reported for smaller, more rapidly cleared proteins such as rt-PA (Mordenti et al., 1991). Other possible explanations for the uncommon allometric scaling may include potential specific clearance pathways of lenercept, suggested by its molecular structure. Due to its high molecular weight above the glomerular filtration threshold of about 70 kDa (Knauf et al., 1988) and its high number of glycosylation sites, lenercept is probably cleared, at least partially, via glycoprotein receptors. In addition, Fc-receptor mediated clearance pathways appear to be possible due the Fc-portion of lenercept. Interspecies differences of these clearance routes, however, are largely unknown for low intrinsic clearance proteins. Therefore, a full explanation for the unusual allometric scaling of lenercept cannot be provided yet. However, future examples of proteins with such allometric scaling properties may enhance the understanding, for which therapeutic proteins such extrapolation behavior can be expected.

Overall, the results from this study demonstrate an uncommon allometric scaling of the pharmacokinetics of lenercept, in that it shows similar half-lives from mice to humans. Despite an immune response against this all-human protein in laboratory animals the pharmacokinetic data in animals were found to be predictive for humans, given that the more rapid immune-modulated clearance in animals could be identified and excluded from the pharmacokinetic evaluation.