Abstract
A dose-ranging study to investigate the in vivo effects of the presence of specific antibodies on the efficacy of β-lactam treatment of sepsis caused by Streptococcus pneumoniae (non-β-lactam-susceptible serotype 6B isolate) was performed with a BALB/c mouse model. Hyperimmune serum was obtained from mice immunized with the heat-inactivated strain. The rate of mortality was 100% in nontreated animals in the absence of specific antibodies. A single injection of a one-half or one-quarter dilution of hyperimmune serum produced 60 to 40% survival rates. In the absence of specific antibodies, the minimal effective doses of amoxicillin and cefotaxime that produced survival rates of 100 and 80% were 25 and 50 mg/kg of body weight (three times a day for up to six doses), respectively. These doses produced times that the levels in serum remained above the MIC (ΔT > MICs) ≈30% of the dosing interval. When specific antibodies were present (by administration of a one-half or one-quarter dilution of hyperimmune serum), the minimal effective doses of the antibiotics were 3.12 and 6.25 mg/kg (≈8 times lower), with the ΔT > MICs being approximately 3 and 5% of the dosing interval for amoxicillin and cefotaxime, respectively. This in vivo combined pharmacodynamic effect offers possibilities that can be used to address penicillin resistance.
Evidence shows that the successful outcome of infections caused by Streptococcus pneumoniae in humans depends on the humoral arm of the immune system and on treatment with an adequate antibiotic. Immunogenicity depends on the pneumococcal serotype (11). Evidence of the participation of immunogenicity in the outcome is based on the spontaneous resolution of fever in the absence of treatment at the time that capsular antibodies appear (15) and the increase in the severities of infections when immunoglobulin G2 (IgG2) (10) or C3 complement (4) deficiencies are present.
Colonization with S. pneumoniae is an immunizing event. In the absence of conditions that predispose an individual to infection, antibodies to the capsular polysaccharide of a colonizing organism are likely to appear before infection (15). The presence of anticapsular antibodies is regarded as a generally good, but not ideal, surrogate marker of immunity; the absence of such antibodies probably indicates a relative degree of susceptibility, even though many other factors contribute to protection against pneumococcal disease (15). In these circumstances, the appearance of pneumococcal sepsis indicates defective protection against pneumococcal invasion.
The administration of serum containing type-specific antibodies in the preantibiotic era was only moderately effective for the treatment of pneumococcal pneumonia (15) and was largely supplanted by the administration of antibiotics. Empirical antibiotic treatment should be chosen by consideration of data from susceptibility surveillance studies (1), antibiotic susceptibility profiles for isolates of a particular serotype (5, 6, 13), serotype distribution (5, 6, 13), and the disease being treated. For β-lactams, data from studies with animal models have demonstrated an excellent relationship between the survival rate and the duration of time that levels in serum exceed the MIC (ΔT > MIC), with very low survival rates detected when the ΔT > MIC is ≤20% of the dosing interval and 90 to 100% survival rates detected when the ΔT > MIC is ≥40% of the dosing interval (2). It is expected that β-lactams will be active against respiratory tract infections caused by S. pneumoniae when the MIC of penicillin for the infecting strain is up to 2 μg/ml, but an increase in the penicillin doses used for treatment is suggested (17). Nevertheless, the increase in antibiotic doses required to address the increase in penicillin resistance may have a limit with the available oral formulations of β-lactams since the National Committee for Clinical Laboratory Standards (NCCLS) (16) defines nonsusceptibility as an MIC ≥4 μg/ml for aminopenicillins and oral cephalosporins.
The dose-ranging study described here explored the efficacies of β-lactams in an experimental pneumococcal sepsis model in mice in which the animals were protected with different levels of specific antibodies before the pneumococcal challenge. A serotype 6B S. pneumoniae isolate was used as a representative of clinical isolates on the basis of data on its epidemiology (frequency of isolation) and susceptibility (the penicillin MIC for the isolate was similar to the MIC at which 90% of isolates tested are inhibited [MIC90]) (5, 6, 13). The antibiotics tested were amoxicillin and cefotaxime, representatives of the antibiotics commonly used for empirical therapy.
MATERIALS AND METHODS
The study was performed in accordance with the prevailing regulations regarding the care and use of laboratory animals in the European Community.
Infecting strain.
A serotype 6B S. pneumoniae strain (penicillin MIC and minimal bactericidal concentration [MBC], 2 and 4 μg/ml, respectively) was selected for the study on the basis of its resistance to β-lactams and its virulence in mice. The microorganism was grown until an absorbance of 0.3 (UV-1203UV-VIS spectrophotometer; Shimadzu, Tokyo, Japan) was obtained in Todd-Hewitt broth supplemented with 0.5% yeast extract (THYB; Difco, Detroit, Mich.), aliquoted, and stored at −70°C in 15% glycerol. These bacterial aliquots were used in all the following experiments.
In vitro studies.
MICs and MBCs were determined by a broth dilution method according to the procedures of NCCLS (16). Modal values for five separate determinations were considered.
Animals.
Eight- to 12-week-old female BALB/c mice weighing 19 to 22 g were used.
Determination of MLD and challenge dose.
Groups of 10 mice per dilution were inoculated intraperitoneally (i.p.) with different inocula ranging from 105 to 108 CFU/ml (spectrophotometrically measured) to determine the minimal dose that produced a 100% mortality rate over a 7-day follow-up period (i.e., the minimal lethal dose [MLD]). Bacteria in the logarithmic phase of growth in THYB were centrifuged, and the pellet was washed three times and resuspended in phosphate-buffered saline (PBS; pH 7.2) to reach 108 CFU/ml (spectrophotometrically measured). The inoculum was confirmed by culture of serial dilutions onto blood Mueller-Hinton agar incubated at 37°C in 5% CO2 and air. The number of dead mice was recorded daily. The MLD was determined from the results obtained in three independent experiments. Once the MLD had been determined, two times the MLD per milliliter was used as the infective inoculum (challenge dose).
Hyperimmune serum.
To obtain hyperimmune serum, bacteria in the logarithmic phase of growth were inactivated at 60°C for 1 h. The animals were inoculated weekly with 200 μl of the inactivated bacterial suspension (107 CFU/ml in PBS) by the i.p. route for 5 weeks. The animals were exsanguinated by cardiac puncture to obtain serum. Titers of specific IgG antibodies to capsular serotype 6B polysaccharide were determined for preimmune and immune sera by a standardized enzyme-linked immunosorbent assay (ELISA) protocol (workshop at the Centers for Disease Control and Prevention, Atlanta, Ga., 1996). Neutralization of antibodies to cell wall polysaccharide was carried out by adsorption with a commercial preparation of this antigen (C-Ps; Statens Serum Institute, Copenhagen, Denmark). For detection, horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad, Richmond, Calif.) was used.
Determination of protection by hyperimmune serum.
To determine the degree of protection offered by the immune serum, groups of five mice per dilution were inoculated i.p. with 200 μl of serial doubling dilutions of immune serum ranging from one-half to one-eighth. The animals included in the control group were injected with a placebo (apyrogenic sterile distilled water). After 1 h, the mice received one challenge dose of bacteria by the i.p. route. The animals were observed for 7 days. The experiment was repeated three times, with the mean value determined from the data.
Determination of the MAPDs of amoxicillin and cefotaxime.
To investigate the minimal antibiotic dose of amoxicillin and cefotaxime that produced a 100% survival rate in animals inoculated with the challenge dose over a 7-day follow-up period (i.e., the minimal antibiotic protective dose [MAPD]), the animals were inoculated i.p. with 200 μl of the challenge dose, and antibiotic treatment was started 1 h after bacterial inoculation. Groups of five animals per dose were treated three times a day for 48 h, and 100 μl was administered by the subcutaneous route in a dose-ranging study, with the doses ranging from 1.6 to 50 mg/kg of body weight. The animals included in the control group received placebo (apyrogenic sterile distilled water). The animals were observed, and the numbers of deaths were recorded for 7 days.
Determination of antibiotic protection in the presence of antibodies.
To investigate the in vivo combined effects of antibiotics and hyperimmune serum, groups of five animals were dosed with both hyperimmune serum and antibiotic by a checkerboard technique. The fractions of hyperimmune serum and the doses of antibiotics used were those individually tested for determination of the protection obtained with serum and MAPD, respectively. On each day of inoculation, a control group that received placebo was included. A single i.p. dose of hyperimmune serum was administered 1 h prior to i.p. inoculation of the bacterial challenge dose. Antibiotic treatment was initiated 1 h after the pneumococal challenge and was continued every 8 h, with a total of six subcutaneous doses being administered. The animals were observed, and the numbers of deaths were recorded for 7 days. The minimal antibiotic dose that produced a 100% animal survival rate in combination with hyperimmune serum over a 7-day follow-up period was determined. An antibiotic regimen was defined to be effective if it resulted in 100% survival when it was used with two serial dilutions of hyperimmune serum.
Determination of antibiotic concentrations in serum.
Amoxicillin and cefotaxime concentrations in serum were determined in healthy animals after administration of a single subcutaneous dose of the MAPD and after administration of the dose that produced 100% survival in the test with the combination therapy with two serial dilutions of hyperimmune serum. Blood samples were collected at 5, 15, and 30 min and 1, 2, 4, 6, and 8 h after dosing from groups of five animals per dose and per antibiotic. Concentrations were measured by bioassay with Micrococcus luteus ATCC 4698 for amoxicillin and Escherichia coli ATCC 25922 for cefotaxime as reference organisms. The bioassays were performed on 9-cm-diameter plates with 14 ml of antibiotic agar 2 (Difco) for amoxicillin and Mueller-Hinton agar for cefotaxime, with a final inoculum of 8 × 108 CFU/ml. Thirty-microliter aliquots of each sample were deposited into 6-mm-diameter wells in the inoculated plates, which were incubated at 36.5°C for 18 h. Standards containing from 0.012 to 1.6 μg/ml and from 0.4 to 50 μg/ml were prepared for amoxicillin and cefotaxime, respectively, in order to determine the assay regression line (standard curve) and to extrapolate antibiotic concentrations from the corresponding inhibition zone diameters.
Pharmacokinetic study.
Antibiotic concentration-time curves for each antibiotic were analyzed by a noncompartmental approach with the Win-Nonlin program (Pharsight, Mountain View, Calif.). The areas under the serum concentration-time curves (AUCs) from time zero to infinity (AUC0-∞) were calculated from the equation AUC0-480 + AUC480-∞ (where 480 indicates 480 min). The AUC0-480 values were calculated from plots of levels in serum versus time by using the trapezoidal rule. AUC480-∞ values were calculated from the expression C480/β, where C480 is the concentration at 480 min and β is the slope obtained from least-squares regression of the terminal elimination phase. β was calculated for each antibiotic by using the levels in serum at the last three sample times (18). The theoretical concentration at time zero (obtained by back extrapolation to the origin of the elimination regression line) was defined as the maximum concentration in serum (Cmax). ΔT > MIC was calculated graphically from the semilogarithmic plot of the concentration-time data.
Statistical analysis.
A Cox regression analysis was used to compare efficacy on the basis of the survival data obtained by using antibiotics, serum dilutions, and antibiotic doses as factors. In addition, the interactions between antibiotics and serum dilutions, antibiotics and antibiotic doses, and serum dilutions and antibiotic doses were included in the initial model (pharmacokinetic approach). A similar complementary analysis with antibiotic dose/MIC instead of the antibiotic dose as a factor was performed (pharmacodynamic approach). A forward-likelihood-ratio method was used. P values of ≤0.05 were considered significant. A hazard ratio of 1 was considered as a criterion for equal efficacy.
RESULTS
The titers of the immunoglobulin antibodies to the hyperimmune serum titrated in parallel with the corresponding preimmune serum to serotype 6B polysaccharide were 1:400, as determined by ELISA. Modal amoxicillin and cefotaxime MICs and MBCs for the infecting strain were 4 and 4 μg/ml and 2 and 4 μg/ml, respectively. The MLD was 5 × 107 CFU/ml, and the challenge dose was approximately 108 CFU/ml. Pretreatment with hyperimmune serum dilutions (from one-half to one-eighth) produced survival rates that ranged from 60 to 20%, with survival rates significantly (P < 0.001) related to the dilution administered.
With antibiotic therapy alone, statistically significant (P < 0.001) decreases in rates of mortality were obtained with increasing doses of both drugs. The minimal effective dose of amoxicillin was 25 mg/kg, whereas the maximum dose of cefotaxime tested (50 mg/kg) was not able to produce a survival rate of 100% but did produce one of 80%; the latter was considered the minimal effective dose without hyperimmune serum. Tables 1 to 3 show percent survivals with the different treatment regimens used. From a pharmacokinetic perspective, significantly (P = 0.011) higher levels of activity were found for cefotaxime at low doses (<5.5 mg/kg) and for amoxicillin at higher doses (>5.5 mg/kg). By the pharmacodynamic approach (using antibiotic dose/MIC as a factor), amoxicillin exhibited significantly (P = 0.0032) higher levels of activity than cefotaxime when the dose/MIC ratios were >1.12.
TABLE 1.
Percent survival of mice included in the control group (placebo) and mice protected with hyperimmune serum (diluted one-eighth, one-quarter, and one-half) over the 7-day follow-up period
| Serum dilution | % Survival on the following day of follow-up:
|
||||||
|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | |
| Control | 20 | 0 | 0 | 0 | 0 | 0 | 0 |
| One-eighth | 80 | 60 | 60 | 40 | 40 | 20 | 20 |
| One-quarter | 100 | 80 | 80 | 80 | 80 | 80 | 40 |
| One-half | 100 | 100 | 100 | 100 | 60 | 60 | 60 |
TABLE 3.
Percent survival of mice treated with different cefotaxime doses with and without hyperimmune serum (diluted one-eighth, one-quarter, and one-half) over the 7-day follow-up period
| Cefotaxime dose and serum dilution | % Survival on the following day of follow-up:
|
||||||
|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | |
| 1.6 mg/kg | |||||||
| No serum | 100 | 60 | 40 | 40 | 20 | 0 | 0 |
| One-eighth | 100 | 100 | 100 | 100 | 100 | 80 | 80 |
| One-quarter | 100 | 100 | 100 | 100 | 100 | 80 | 80 |
| One-half | 100 | 100 | 80 | 80 | 80 | 80 | 60 |
| 3.12 mg/kg | |||||||
| No serum | 60 | 40 | 40 | 20 | 20 | 0 | 0 |
| One-eighth | 80 | 80 | 80 | 80 | 60 | 60 | 40 |
| One-quarter | 100 | 100 | 100 | 100 | 100 | 80 | 60 |
| One-half | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| 6.25 mg/kg | |||||||
| No serum | 100 | 100 | 60 | 60 | 20 | 0 | 0 |
| One-eighth | 100 | 80 | 60 | 60 | 60 | 40 | 20 |
| One-quarter | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| One-half | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| 12.5 mg/kg | |||||||
| No serum | 100 | 100 | 40 | 0 | 0 | 0 | 0 |
| One-eighth | 100 | 100 | 100 | 80 | 80 | 60 | 40 |
| One-quarter | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| One-half | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| 25 mg/kg | |||||||
| No serum | 80 | 80 | 60 | 60 | 60 | 20 | 20 |
| One-eighth | 100 | 100 | 100 | 100 | 100 | 80 | 60 |
| One-quarter | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| One-half | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| 50 mg/kg | |||||||
| No serum | 100 | 100 | 100 | 100 | 100 | 80 | 80 |
| One-eighth | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| One-quarter | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| One-half | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
In the presence of specific antibodies, the minimal effective doses that produced 100% survival rates were 3.12 and 6.25 mg/kg for amoxicillin and cefotaxime, respectively, with one-half and one-quarter dilutions of hyperimmune serum.
The half-lives of high doses of amoxicillin and cefotaxime were 20.4 and 19.2 min, respectively. The respective Cmaxs and AUCs were 270.6 μg/ml and 7,657.7 μg·min/ml and 6.1 μg/ml and 191.3 μg·min/ml for the minimal effective dose of amoxicillin (25 mg/kg) without hyperimmune serum and for amoxicillin (3.12 mg/kg) and hyperimmune serum (one-quarter dilution). The respective Cmaxs and AUCs were 621.5 μg/ml and 16,483.8 μg·min/ml and 25.3 μg/ml and 368.1 μg·min/ml for the minimal effective dose of cefotaxime (50 mg/kg) without hyperimmune serum and for cefotaxime (6.25 mg/kg) with hyperimmune serum (one-quarter dilution). Table 4 shows the values of the pharmacodynamic parameters determined for the minimal protective doses of the antibiotics with and without immunoprotection (one-quarter dilution of serum).
TABLE 4.
Pharmacodynamic parameters of the minimal protective doses of antibiotics with and without immunoprotection
| Treatment | Minimal effective dose (mg/kg) | Cmax:MIC | ΔT > MIC (min [% dos- ing interval]) | AUC:MIC |
|---|---|---|---|---|
| Amoxicillin | 25 | 67.6 | 123 (25.6) | 1,914.4 |
| Amoxicillin + hyper- immune serum 1/4a | 3.12 | 1.5 | 13.6 (2.8) | 47.8 |
| Cefotaxime | 50 | 310.7 | 156 (32.5) | 8,241.9 |
| Cefotaxime + hyper- immune serum 1/4 | 6.25 | 12.6 | 26 (5.4) | 184.0 |
Immunoprotection consisted of a one-quarter dilution of serum (serum 1/4).
The presence of specific antibodies decreased the minimal effective doses of amoxicillin and cefotaxime 8 times; decreased the Cmax:MIC, ΔT > MIC, and AUC:MIC for amoxicillin 45, 9, and 40 times, respectively; and decreased the Cmax:MIC, ΔT > MIC, and AUC:MIC for cefotaxime 25, 6, and 44 times, respectively.
DISCUSSION
Several in vitro and ex vivo studies have suggested a combined effect of β-lactams and the immune system (8, 9, 14), via complement and opsonophagocytosis, because both β-lactams and the immune system act through or against the cell wall. The alteration of the cell surface by the β-lactam, which allows opsonization and phagocytosis (7, 19), overcomes the capsular impediment for recognition of C3b on the bacterial surface by polymorphonuclear cells. In a recently published study (3), the combination of immunoglobulins and an aminopenicillin was fully protective in an invasive pneumococcal pneumonia model of infection with a penicillin-susceptible serotype 3 strain in mice.
The only moderate effectiveness of administration of serum containing type-specific antibodies in the preantibiotic era was probably due, among other factors, to the following facts: (i) IgG2 is the predominant immunoglobulin isotype generated by the S. pneumoniae capsule in vivo, (ii) phagocytosis is limited to organisms coated with IgG1 and IgG3, and (iii) IgG2 is less capable of binding to C1q except when IgG2 is present at high local concentrations (10). In addition, serotype 6B, the most common serotype isolated from children with bacteremia (6), is poorly immunogenic (11). In any case, phagocytosis of isolates of this serotype may be enhanced indirectly by the cleavage of activated C3, which serves as a ligand for the type 3 receptor in phagocytic cells, as shown previously (11). This moderate effectiveness of serotherapy in humans can also be observed with respect to protection in this study in mice since the rate of mortality among nonprotected animals (100%) was reduced only to 40 to 80% when antibodies were present. These nonoptimal protection rates may be due to the fact that antibodies are not the direct effectors of bacterial clearance, in contrast to the situation with antibiotics. Bacterial clearance mediated by antibodies depends on the final membrane attack complex and the indirectly enhanced phagocytosis mentioned above, and these facts are a consequence of C3 cleavage and the subsequent amplification loop (4).
In terms of the antibiotics, a serotype 6B pneumococcus not susceptible to amoxicillin and cefotaxime, which MICs represent the MIC90s of both antibiotics for serotype 6 isolates in Spain (13), was used. Inoculation of untreated mice with this strain produced mortality rates of 100%, with decreases in mortality rates of 100 and 80% when the animals were treated with amoxicillin at 25 mg/kg and cefotaxime at 50 mg/kg, respectively. These antibiotic regimens achieved ΔT > MICs of approximately 30% of the dosing interval, a value close to that described for β-lactams by other investigators with different animal models of infection (2). The serum amoxicillin and cefotaxime concentrations obtained in mice after the administration of protective doses are much higher than those obtained in humans after the administration of standard doses. When antibiotic therapy was combined with immunoprotection (a single injection of hyperimmune serum at dilutions of one-half or one-quarter), the protective dose decreased eight times for amoxicillin (to 3.12 mg/kg) and cefotaxime (to 6.25 mg/kg). The levels obtained in the serum of mice after the administration of these doses are much lower than those obtained in the serum of humans after the administration of standard doses. From a pharmacodynamic point of view, this is of major concern since efficacy is obtained in the presence of antibodies with ΔT > MICs approximately 3 to 6% of the dosing interval.
In terms of immunoprotection, subtherapeutic doses of antibiotics increased the survival rates obtained by protection with hyperimmune serum from 60-40% to 100% (with one-half and one-quarter dilutions of serum, respectively). In terms of the antibiotic concentration, the presence of specific antibodies led to therapeutic efficacy with subinhibitory antibiotic concentrations. These facts are shown by the use of a highly restrictive endpoint: a decrease in the mortality rate from 100% (control groups) to 0%.
The combined effects of antibiotics and hyperimmune serum can be attributed to an increased rate of clearance of microorganisms after antibiotic treatment in preimmunized animals, as reported previously (J. Yuste, M. J. Giménez, I. Jado, A. Fenoll, L. Aguilar, and J. Casal, Letter, J. Antimicrob. Chemother. 48:594-595, 2001). This in vivo pharmacodynamic effect of the combination that decreases the ΔT > MIC needed for efficacy from 25.6 to 2.8% of the dosing interval for amoxicillin and from 32.5 to 5.4% of the dosing interval for cefotaxime in the presence of specific antibodies (induced by passive immunization) offers new insights into the design of therapeutic strategies or prophylactic strategies that will improve outcomes. For instance, serotypes could be evaluated for inclusion in vaccines by consideration not only of immunogenicity and prevalence data but also of susceptibility data. Further studies with other serotypes with different susceptibilities are needed to demonstrate this pharmacodynamic effect in an extended S. pneumoniae population. The development of immunoprotection and/or immunotherapy to overcome resistance may potentially provide new lifesaving strategies (12).
TABLE 2.
Percent survival of mice treated with different amoxicillin doses with and without hyperimmune serum (diluted one-eighth, one-quarter, and one-half) over the 7-day follow-up period
| Amoxicillin dose and serum dilution | % Survival on the following day of follow-up:
|
||||||
|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | |
| 1.6 mg/kg | |||||||
| No serum | 20 | 20 | 0 | 0 | 0 | 0 | 0 |
| One-eighth | 100 | 100 | 80 | 80 | 80 | 40 | 40 |
| One-quarter | 100 | 100 | 100 | 100 | 100 | 100 | 80 |
| One-half | 100 | 100 | 100 | 100 | 100 | 80 | 60 |
| 3.12 mg/kg | |||||||
| No serum | 100 | 80 | 40 | 20 | 20 | 20 | 0 |
| One-eighth | 100 | 60 | 60 | 40 | 20 | 20 | 0 |
| One-quarter | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| One-half | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| 6.25 mg/kg | |||||||
| No serum | 60 | 20 | 0 | 0 | 0 | 0 | 0 |
| One-eighth | 100 | 100 | 100 | 100 | 80 | 60 | 60 |
| One-quarter | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| One-half | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| 12.5 mg/kg | |||||||
| No serum | 100 | 100 | 100 | 100 | 60 | 0 | 0 |
| One-eighth | 100 | 100 | 100 | 100 | 100 | 80 | 80 |
| One-quarter | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| One-half | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| 25 mg/kg | |||||||
| No serum | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| One-eighth | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| One-quarter | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| One-half | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
Acknowledgments
We thank A. Carcas (Universidad Autónoma, Madrid, Spain) for pharmacokinetic analysis, A. Pedromingo and E. Letón for statistical analysis, and M. L. Gómez-Lus (Universidad Complutense, Madrid, Spain) for critical review of the manuscript. We also thank A. López-Pardo for help with the preparation of the manuscript.
This study was supported by European Funds for Regional Development and by funds from the Spanish National R&D Program (project 2FD 97-0554).
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