Laboratory of Experimental Infection, Infectious Diseases Service and Microbiology Service, IDIBELL, Hospital Universitari de Bellvitge, Barcelona, Spain
Received 30 June 2004; returned 23 August 2004; revised 1 October 2004; accepted 1 October 2004
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: In vitro killing curves of teicoplanin, with and without ceftriaxone, were performed. Groups of eight animals per treatment were inoculated with a cephalosporin-resistant pneumococcal strain (penicillin MIC, 4 mg/L; ceftriaxone MIC, 2 mg/L; teicoplanin MIC, 0.03 mg/L) and treated over a 26 h period. Teicoplanin was administered at a dose of 15 mg/kg, alone and in combination with ceftriaxone at 100 mg/kg with or without dexamethasone at 0.25 mg/kg. CSF samples were collected at different time-points, and bacterial titres, white blood cell counts, lactate and protein concentrations and bacteriostatic/bactericidal titres were determined. Blood and CSF teicoplanin pharmacokinetic and pharmacodynamic parameters were determined.
Results: Teicoplanin alone promoted a decrease in bacterial counts at 6 h of 2.66 log cfu/mL and was bactericidal at 24 h, without therapeutic failures. Similar good results were obtained when dexamethasone was used simultaneously, in spite of the penetration of teicoplanin into the CSF being significantly reduced, from 2.31% to 0.71%. Teicoplanin and ceftriaxone combinations were synergic in vitro, but not in the meningitis model.
Conclusions: Teicoplanin alone was very effective in this model of cephalosporin-resistant pneumococcal meningitis. The use of concomitant dexamethasone resulted in lower CSF teicoplanin levels, but not in therapeutic failures. The combination of teicoplanin plus ceftriaxone and dexamethasone might be a good alternative for the empirical therapy of pneumococcal meningitis. Additional data should confirm our experiments, in advance of clinical trials to assess efficacy in humans.
Keywords: glycopeptides , bacterial meningitis , dexamethasone , Streptococcus pneumoniae
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Teicoplanin is a glycopeptide antibiotic, obtained in 1978 by fermentation from Actinoplanes teichomyceticus. It is less active than vancomycin against staphylococci, but equal or more active against streptococci; specifically, it presents excellent in vitro activity against S. pneumoniae strains, usually with MICs four or five dilutions under those of vancomycin and similar to those of oritavancin. The pharmacokinetics of teicoplanin allow its administration in a single daily dose, and the drug exhibits a lower propensity than vancomycin to cause side effects.1519
Clinical experience in the treatment of meningeal infections with teicoplanin is very limited,2022 and non-existent in the particular case of pneumococcal meningitis. In this work, we sought to evaluate the efficacy of teicoplanin, alone and in combination with ceftriaxone, in a rabbit model of cephalosporin-resistant pneumococcal meningitis, and to assess the effects of dexamethasone on the CSF antibiotic levels and inflammatory parameters.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The S. pneumoniae strain HUB2349, which had been isolated from a patient with meningitis, was used in the study. MICs and MBCs of penicillin, cefotaxime, ceftriaxone, vancomycin and teicoplanin were determined by the macrodilution method in cation-adjusted MuellerHinton broth supplemented with 3%5% of horse lysed blood according to NCCLS guidelines.23 MICs/MBCs were as follows: penicillin, 4/4 mg/L; cefotaxime and ceftriaxone, 2/4 mg/L; vancomycin, 0.25/0.50 mg/L; and teicoplanin, 0.03/0.25 mg/L.
In vitro killing curves
Killing curves were performed with glass tubes containing a final volume of 10 mL of cation-adjusted MuellerHinton broth with 5% of horse lysed blood. The final bacterial inoculum was 5 x 105 cfu/mL. Concentrations of 32 x MIC, 8 x MIC, 2 x MIC, 1 x MIC and 1/2 x MIC of teicoplanin, concentrations of 2 x MIC, 1 x MIC, 1/2 x MIC and 1/4 x MIC of ceftriaxone, and several associations of both antibiotics with one of them at subinhibitory concentration, were studied. Bacterial titres were determined at 0, 6 and 24 h of incubation by serial dilution of samples that were plated on agar containing 5% sheep blood. No carryover effect was observed. The detection limit was 1 log10 cfu/mL. Synergy was defined as the effect of a drug combination >2-log killing over the most active drug alone with one of the drugs at subinhibitory concentration. The bactericidal effect was defined as a decrease 3 log cfu/mL of the initial inoculum.
Inoculum preparation
The microorganism was grown overnight on blood-agar plates (BAP) and bacterial suspensions were prepared. The inoculum was made by suspending the colonies in brainheart infusion broth, adjusting the turbidity to the equivalent of a 0.5 McFarland standard (108 cfu/mL) and making 10-fold dilutions in saline in order to obtain a concentration of 106 cfu/mL. For each experiment, the inoculum size was determined by plating 0.1 mL on BAP, with subsequent counting of the colonies after 2448 h of incubation at 35°C.
Pharmacokinetics and pharmacodynamics
Several pharmacokinetic (PK) and pharmacodynamic (PD) parameters of teicoplanin and teicoplanin + dexamethasone were determined in serum and CSF: maximal achievable concentration, area under the curve calculated by linear trapezoidal rule, percentage of CSF penetration, time above the MIC and Cmax/MIC. Two groups of rabbits (n=10 each) were intracisternally inoculated with the study strain. Eighteen hours later, during a period of 24 h, the rabbits were treated with 15 mg/kg/day of intravenous (iv) teicoplanin or teicoplanin + dexamethasone (0.25 mg/24 h, divided in two doses). CSF and serum samples were taken at 1, 2, 5, 8, 10, 12 and 24 h of therapy, and were used to determine teicoplanin concentrations by fluorescence polarized immunoassay using a TDX analyzer calibrated to achieve a minimal detectable concentration of 0.03 mg/L. CSF cultures were also performed to determine bacterial concentration at each time-point. PK and PD parameters were obtained by a computer-assisted method (PK functions for Microsoft Excel; J. I. Usansky, A. Desai and D. Tang-Liu, Department of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, CA 92606, USA) after determination of antibiotic concentration at the different time points.
Meningitis rabbit model
The rabbit model of meningitis was performed according to an established protocol24 and was approved by the Ethical Committee for Animal Experiments at the University of Barcelona (Campus de Bellvitge). For all experiments, rabbits (2 kg female New Zealand white) were challenged in groups of eight animals per group. On the first day of the experiment, they were anaesthetized intramuscularly with ketamine 35 mg/kg (Ketolar; Parke-Davis, El Prat de Llobregat, Spain) and xylazine 5 mg/kg (Rompun; AG Bayer, Leverkusen, Germany). A dental acrylic helmet was fixed to the calvaria and the animals were returned to their cages. Twenty-four hours later, the rabbits were anaesthetized again by the same method and placed in a stereotaxic frame. A 25 gauge spinal needle was introduced in the cisterna magna and 200 µL of CSF was withdrawn as a sterility control. Meningitis was then induced by instillation of 200 µL of a bacterial suspension adjusted to 106 cfu/mL of the studied strain into the subarachnoid space (see inoculum preparation above). Eighteen hours later, the rabbits were anaesthetized subcutaneously with urethane 1.75 g/kg (ethyl-carbamate; Sigma Chemical Company, St Louis, MI, USA) and phenobarbital 5 mg/kg (Penthotal sodico; Abbott Laboratories, Madrid, Spain). Blood cultures were taken at this time to detect secondary bacteraemia; 0.1 mL of blood collected from the central ear vein was suspended in 5 mL of trypticase soy broth and incubated overnight at 37°C. A baseline 200 µL CSF sample was taken. Then an iv dose of 0.25 mg of dexamethasone (Fortecortin; Merck, Mollet del Vallés, Barcelona, Spain) or saline (Suero fisiológico Braun; Braun S.A. Rubí, Barcelona, Spain) was administered, and 10 min later, antibiotic therapy was started with one of the following therapy schedules: teicoplanin, teicoplanin + dexamethasone, teicoplanin + ceftriaxone, teicoplanin + ceftriaxone + dexamethasone. There was also a control groupinfected but not treated. Antibiotic doses were as follows: teicoplanin 15 mg/kg/day (Targocid; Aventis Pharma, Alcorcon, Madrid, Spain) and ceftriaxone 100 mg/kg/day (Rocefalin; Roche, Madrid, Spain), all in a single dose. The total dose of dexamethasone was 0.5 g/24 h, divided every 12 h over a 48 h period (four doses). The teicoplanin dosage of 15 mg/kg/day was chosen in order to have good serum levels based on previous experience with rabbit models.25 Ceftriaxone and dexamethasone doses were the same as used in previous experiments.26 CSF samples were taken at 0, 2, 6, 24, 26 and 48 h of therapy. Brain oedema, expressed as g of water/100 g of dry weight, was determined after sacrifice of the rabbits with an overdose of phenobarbital at 48 h. Therapeutic failure was defined as an increase in bacterial concentration of at least 1 log cfu/mL compared with a previous count. A therapy was considered bactericidal when a reduction of 3 log cfu/mL was achieved.
Sample processing
CSF bacterial titres, white blood cells (WBC), protein and lactate concentrations, and bacteriostatic/cidal activity were determined. For colony counts, direct cultures and serial 10-fold dilutions were performed (the detection limit by this method was 102 cfu/mL and a value of 1.9 log cfu/mL was assigned to the first sterile culture and of 0 to the subsequent ones). To avoid carryover antimicrobial agent interference, the sample was placed onto the plate in a single streak down the centre, allowed to be absorbed into the agar until the plate surface appeared dry and then the inoculum was spread over the plate.27 For WBC counts, 10 µL of each sample was diluted 1:1 with Turk solution (acetic acid and Methylene Blue prepared in-house) and read with a Neubauer camera. After centrifugation, CSF was stored at 80°C in order to determine the rest of the parameters.
CSF protein concentration was determined by the Bradford method (Bio-Rad Protein Assay), and lactate CSF concentration by Lactate PAP (bioMérieux, France).
CSF bactericidal activities were performed by a microdilution method28 with cation-adjusted MuellerHinton broth (Difco Laboratories, Detroit, MI, USA) with 2%5% lysed horse blood.
Statistical analysis
Comparisons among different therapy groups were, respectively, performed by ANOVA multiple-comparison test or KruskallWallis test (inflammatory activity). A P value of <0.05 was considered significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Teicoplanin alone was bactericidal at 24 h at a concentration of 8 x MIC and higher, equivalent to 0.25 mg/L. Ceftriaxone was also bactericidal at 24 h at a concentration of 1 x MIC, equivalent to 2 mg/L. A combination of 1/2 x MIC of both (subinhibitory concentrations) showed a synergic effect at 6 h and a bactericidal effect at 24 h. Combinations of teicoplanin 1 x MIC + ceftriaxone 1/2 x MIC, ceftriaxone 1/2 x MIC + teicoplanin 2 x MIC and ceftriaxone 1/4 x MIC + teicoplanin 2 x MIC were also synergic at 6 h and bactericidal at 24 h. Variations in log cfu/mL at 6 and 24 h are shown in Table 1.
|
The different parameters are shown in Table 2. Peak and trough CSF teicoplanin concentration with the concomitant use of dexamethasone was statistically significantly lower than with teicoplanin alone. Accordingly, CSF penetration decreased from 2.31% to 0.71%. PD parameters such as t > MIC and Cmax/MIC were also lower when using concomitant dexamethasone.
|
Eighteen hours after the inoculation, 100% of the animals presented positive blood cultures, suggesting that this is a highly invasive pneumococcal strain.
In vivo activity of the antimicrobial regimens
Table 3 shows the in vivo bacterial reduction in CSF at 6 and 24 h with the different therapeutic schedules. All therapies were bactericidal and all CSF samples under the level of detection at 24 h of the experiment. No therapeutic failures were observed in any of the combinations. Teicoplanin alone showed good behaviour, indifferent to the concomitant administration of dexamethasone (2.66 log cfu/mL for teicoplanin versus 2.75 log cfu/mL for teicoplanin + dexamethasone at 6 h; not significant). The addition of ceftriaxone did not significantly improve the activity of teicoplanin with or without dexamethasone (2.05 log cfu/mL for teicoplanin + ceftriaxone and 2.14 log cfu/mL for teicoplanin + ceftriaxone + dexamethasone at 6 h; not significant).
|
Bacteriostatic and bactericidal titres are shown in Table 4. Peak bactericidal activity (2 and 26 h) ranged between 1:4 (teicoplanin, teicoplanin + dexamethasone) and 1:32 (teicoplanin + ceftriaxone). Trough bactericidal activity (24 and 48 h) ranged between <1:2 (teicoplanin, teicoplanin + dexamethasone, teicoplanin + ceftriaxone + dexamethasone) and 1:8 (teicoplanin + ceftriaxone). Overall, the combination of teicoplanin + ceftriaxone gave higher bacteriostatic/bactericidal activities, whereas the addition of dexamethasone produced a decrease in the bacteriostatic/bactericidal titres.
|
The median of CSF WBC and the arithmetic means of lactate and protein concentrations at 2, 6 and 24 h are shown in Table 5. No statistically significant differences were observed in the cytochemical activity promoted by the different antibiotic therapies. The effect of dexamethasone was studied, bearing in mind the percentages of variation of the different parameters at a determined time-point compared with 0 h. The teicoplanin ± dexamethasone group did not present significant differences in terms of variation of WBC, lactate concentration or protein concentration in CSF. However, the combination of teicoplanin + ceftriaxone presented a higher inflammatory activity than the combination plus dexamethasone, regarding WBC concentration at 2 h and protein concentration at 24 h. Brain oedema was in the range 379401 g of water/100 g of dry weight, without significant differences between groups.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The conserved efficacy of teicoplanin in the presence of dexamethasone might be of special interest in the clinical setting. If additional studies confirm our results, the possibility of using teicoplanin instead of vancomycin as empirical therapy could be considered and assessed in clinical trials. Currently, more experience is needed before recommending the concomitant use of dexamethasone and teicoplanin alone to treat patients.
The addition of ceftriaxone to teicoplanin did not represent a significant improvement in bacterial decrease, despite the synergy shown in the in vitro studies at most concentrations tested. A synergic effect of ceftriaxone and vancomycin against resistant strains has been found in an experimental model of pneumococcal meningitis,12 but we have been unable to demonstrate this effect in any of the experiments performed with the different glycopeptides.14,26 However, the combination produced higher bactericidal titres (1:32) than teicoplanin alone (1:4). As with oritavancin, it is possible that the rapid decrease produced by teicoplanin in the bacterial concentration prevented the manifestation of a synergic effect. The use of ceftriaxone evoked some degree of enhanced inflammatory activity (increase in protein concentration) that was down-modulated by dexamethasone.
The possibility of meningitis cases resulting from S. pneumoniae, with very high resistance to third-generation cephalosporins, is of special interest following the promising results with this glycopeptide: empirical therapy with the combination cephalosporinglycopeptide would result, in fact, in monotherapy.
The experience with teicoplanin in bacterial meningitis is very limited. The administration of 400 mg to four patients resulted in a CSF concentration of 1 mg/L in one case and of less than 0.5 mg/L in the other three cases. With a dose of 1200 mg/day, the CSF levels achieved were 1.52 mg/L.31
The dose of 15 mg/kg used in our experiments achieved a serum teicoplanin concentration 7075 mg/L in the rabbits, similar to that achieved with the usual dose of 400 mg in humans;32
however, to treat severe infections higher doses (around 800 mg/day) have been used in humans and are known to be safe. A loading dose of teicoplanin would be of interest to treat pneumococcal meningitis in humans, in order to increase levels enhancing Cmax/CMB ratios in the first hours of therapy.
The high degree of protein binding of teicoplanin, 90% in humans and rabbits, seems not to be a problem in terms of efficacy, as occurs with other antibiotics used in meningitis, such as ceftriaxone.
In conclusion, teicoplanin alone was very effective in this model of penicillin and cephalosporin pneumococcal meningitis. The use of concomitant dexamethasone resulted in lower CSF teicoplanin levels but not in therapeutic failures. Combinations of teicoplanin and ceftriaxone were synergic in vitro, but did not produce any significant improvement in bacterial decrease. In the clinical setting, the combination of teicoplanin plus ceftriaxone and dexamethasone might be a good alternative for the empirical therapy of pneumococcal meningitis. Additional data should confirm our experiments before performing clinical trials to assess this efficacy in humans.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2
.
Doern, G. V., Heilmann, K. P., Huynh, H. K. et al. (2001). Antimicrobial resistance among clinical isolates of Streptococcus pneumoniae in the United States during 19992000, including a comparison of resistance rates since 19941995. Antimicrobial Agents and Chemotherapy 45, 17219.
3
.
Whitney, C. G., Farley, M. M., Hadler, J. et al. (2000). Increasing prevalence of multidrug-resistant Streptococcus pneumoniae in the United States. New England Journal of Medicine 343, 191724.
4 . Cottagnoud, P. H. & Tauber, M. G. (2004). New therapies for pneumococcal meningitis. Expert Opinion on Investigational Drugs 13, 393401.[CrossRef][ISI][Medline]
5
.
McMaster, P., McIntyre, P., Gilmour, R. et al. (2002). The emergence of resistant pneumococcal meningitis-implictions for empiric therapy. Archives of Disease in Childhood 87, 20710.
6
.
Kaplan, S. L. & Mason, E. O. (1998). Management of infections due to antibiotic-resistant Streptococcus pneumoniae. Clinical Microbiology Reviews 11, 62844.
7 . Viladrich, P. F., Cabellos, C., Pallares, R. et al. (1996). High doses of cefotaxime in treatment of adult meningitis due to Streptococcus pneumoniae with decreased susceptibilities to broad-spectrum cephalosporins. Antimicrobial Agents and Chemotherapy 40, 21820.[Abstract]
8 . Bradley, J. S. & Connor, J. D. (1991). Ceftriaxone failure in meningitis caused by Streptococcus pneumoniae with reduced susceptibility to ß-lactam antibiotics. Pediatric Infectious Diseases Journal 10, 8713.[ISI][Medline]
9 . Catalan, M. J., Fernandez, J. M., Vazquez, A. et al. (1994). Failure of cefotaxime in the treatment of meningitis due to relatively resistant Streptococcus pneumoniae. Clinical Infectious Diseases 18, 7669.[ISI][Medline]
10 . Viladrich, P. F., Gudiol, F., Liñares, J. et al. (1991). Evaluation of vancomycin for therapy of adult pneumococcal meningitis. Antimicrobial Agents and Chemotherapy 35, 246772.[ISI][Medline]
11
.
Ahmed, A., Jafri, H., Lutsar, I. et al. (1999). Pharmacodynamics of vancomycin for the treatment of experimental penicillin- and cephalosporin-resistant pneumococcal meningitis. Antimicrobial Agents and Chemotherapy 43, 87681.
12 . Friedland, I. R., Paris, M., Ehrett, S. et al. (1993). Evaluation of antimicrobial regimens for treatment of experimental penicillin- and cephalosporin-resistant pneumococcal meningitis. Antimicrobial Agents and Chemotherapy 37, 16306.[Abstract]
13 . Tunkel, A. R. & Scheld, W. M. (2002). Treatment of bacterial meningitis. Current Infectious Disease Reports 4, 716.[Medline]
14
.
Cabellos, C., Fernandez, A., Maiques, J. M. et al. (2003). Experimental study of LY333328 (oritavancin), alone and in combination, in therapy of cephalosporin-resistant pneumococcal meningitis. Antimicrobial Agents and Chemotherapy 47, 190711.
15 . Knudsen, J. D., Fuursted, K., Espersen, F. et al. (1997). Activities of vancomycin and teicoplanin against penicillin-resistant pneumococci in vitro and in vivo and correlation to pharmacokinetic parameters in the mouse peritonitis model. Antimicrobial Agents and Chemotherapy 41, 19105.[Abstract]
16
.
Knudsen, J. D., Fuursted, K., Raber, S. et al. (2000). Pharmacodynamics of glycopeptides in the mouse peritonitis model of Streptococcus pneumoniae or Staphylococcus aureus infection. Antimicrobial Agents and Chemotherapy 44, 124754.
17 . Lazzarini, L., Tramarin, A., Bragagnolo, L. et al. (2002). Three-times weekly teicoplanin in the outpatient treatment of acute methicillin-resistant staphylococcal osteomyelitis: a pilot study. Journal of Chemotherapy 14, 715.[ISI][Medline]
18 . LeFrock, J. & Ristuccia, A. (1999). Teicoplanin in the treatment of bone and joint infections: an open study. Journal of Infection and Chemotherapy 5, 329.[CrossRef][Medline]
19 . Wood, M. J. (2000). Comparative safety of teicoplanin and vancomycin. Journal of Chemotherapy 5, 215.
20 . Jourdan, C., Convert, J., Peloux, A. et al. (1996). Adequate intrathecal diffusion of teicoplanin after failure of vancomycin, administered in continuous infusion in three cases of shunt associated meningitis. Pathologie Biologie (Paris) 44, 38992.
21 . Kralinsky, K., Lako, J., Dluholucky, S. et al. (1999). Nosocomial staphylococcal meningitis in neonates successfully treated with intraventricular teicoplanin. Chemotherapy 45, 3134.[CrossRef][ISI][Medline]
22 . Fanos, V. & Dall'Agnola, A. (1999). Antibiotics in neonatal infections: a review. Drugs 58, 40527.[ISI][Medline]
23 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.
24 . Dacey, M. G. & Sande, M. A. (1974). Effect of probenecid on cerebrospinal fluid concentrations of penicillin and cephalosporin derivates. Antimicrobial Agents and Chemotherapy 57, 43741.
25 . Chambers, H. F. & Kennedy, S. (1990). Effects of dosage, peak and trough concentrations in serum, protein binding, and bactericidal rate on efficacy of teicoplanin in a rabbit model of endocarditis. Antimicrobial Agents and Chemotherapy 34, 5104.[ISI][Medline]
26 . Cabellos, C., Martínez-Lacasa, J., Martos, A. et al. (1995). Influence of dexamethasone on efficacy of ceftriaxone and vancomycin therapy in experimental pneumococcal meningitis. Antimicrobial Agents and Chemotherapy 39, 215860.[Abstract]
27 . Chapin-Robertson, K. & Edberg, S. C. (1991). Measurement of antibiotics in human body fluids: techniques and significance. In Antibiotics in Laboratory Medicine (Lorian, V., Ed.), pp. 295366. Williams and Wilkins, New York, NY, USA.
28 . Knapp, C. & Moody, J. A. (1992). Test to assess bactericidal activity, sec 5.16 Clinical Microbiology Procedures Handbook (Isenberg, H. D., Ed.), p. 12. American Society for Microbiology, Washington, DC, USA, sec 5.16, sec 5.16.
29 . Paris, M., Hickey, S. M., Usher, M. I. et al. (1994). Effect of dexamethasone on therapy of experimental penicillin- and cephalosporin-resistant pneumococcal meningitis. Antimicrobial Agents and Chemotherapy 38, 13204.[Abstract]
30
.
Martínez-Lacasa, J., Cabellos, C., Martos, A. et al. (2002). Experimental study of the efficacy of vancomycin, rifampicin and dexamethasone in the therapy of pneumococcal meningitis. Journal of Antimicrobial Chemotherapy 49, 50713.
31 . Cruciani, M., Navarra, A., Di Perri, G. et al. (1992). Evaluation of intraventricular teicoplanin for the treatment of neurological shunt infections. Clinical Infectious Diseases 15, 2859.[ISI][Medline]
32 . Martin, C., Bourget, P., Alaya, M. et al. (1997). Teicoplanin in cardiac surgery: intraoperative pharmacokinetics and concentrations in cardiac and mediastinal tissues. Antimicrobial Agents and Chemotherapy 41, 11505.[Abstract]
|