Efficacy of BAL5788, a Prodrug of Cephalosporin BAL9141, in a ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2004, p. 1105–1111 0066-4804/04/$08.00⫹0 DOI: 10.1128/AAC.48.4.1105–1111.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Vol. 48, No. 4

Efficacy of BAL5788, a Prodrug of Cephalosporin BAL9141, in a Mouse Model of Acute Pneumococcal Pneumonia E. Azoulay-Dupuis,1* J. P. Be´dos,1 J. Mohler,1 A. Schmitt-Hoffmann,2 M. Schleimer,2 and S. Shapiro2 INSERM, Faculte´ de Me´decine Xavier Bichat, Paris, France,1 and Basilea Pharmaceutica AG, Basel, Switzerland2 Received 19 June 2003/Returned for modification 3 September 2003/Accepted 19 December 2003

BAL5788 is a water-soluble prodrug of BAL9141, a new broad-spectrum cephalosporin with high levels of in vitro activity against methicillin- and vancomycin-resistant staphylococci and penicillin-resistant streptococci. In plasma BAL5788 is rapidly converted to BAL9141. We studied the activity of BAL5788 in a mouse model of acute pneumococcal pneumonia. Leukopenic female Swiss albino mice were challenged intratracheally with 107 CFU of clinical Streptococcus pneumoniae strains P-52181 (Pens Cros Ctxs), P-15986 (Penr Cros Ctxs), P-40422 (Penr Cror Ctxr), and P-40984 (Penr Cror Ctxr). Infected mice received subcutaneous (s.c.) injections of BAL5788 or ceftriaxone starting 3 h after pneumococcal challenge. Uninfected nonleukopenic mice received single s.c. doses of BAL5788 to determine the BAL9141 concentration-time profiles in serum and lungs. Untreated control mice died within 5 days postinfection. Ten-day cumulative survival rates for infected mice receiving BAL5788 (total daily doses of BAL9141 equivalents, 2.1 to 75 mg/kg of body weight) ranged from 57 to 100%, whereas with ceftriaxone (total daily doses, 10 to 400 mg/kg), the survival rates varied between 13 and 100%. In mice infected with P-15986, the survival rates achieved with BAL5788 (BAL9141 equivalent, 8.4 mg/kg) and those achieved with ceftriaxone (50 mg/kg) were significantly different (93 versus 13%; P < 0.0001) in favor of BAL5788; the outcomes of the trials with all other strains were not significantly different between the two antibiotics, but markedly lower doses of BAL5788 than ceftriaxone were required to obtain similar survival rates. Pharmacokinetic data showed that BAL9141 was effective against the four pneumococcal strains tested at very low values of the time above the MIC (T > MIC), which ranged from 9 to 18% of the dosing interval, whereas the values of T > MICs for ceftriaxone ranged from 30 to 50% of the dosing interval. Community-acquired pneumonia is the most common cause of death due to infection (3, 14, 15, 17, 22, 23), with Streptococcus pneumoniae remaining the leading pathogen as a cause of community-acquired pneumonia. The past decade has witnessed substantial increases in pneumococcal resistance to various classes of antibiotics (10). Although the recently developed broad-spectrum macrolides and fluoroquinolones have proven highly effective in combating pneumococcal infections, their widespread use on an outpatient basis will compromise their usefulness by promoting resistance. Moreover, the use of broad-spectrum cephalosporins, such as ceftriaxone and cefotaxime, which are characterized by higher levels in serum and MICs lower than those of amoxicillin, has been suggested for the treatment of pneumonia due to penicillin-resistant strains of S. pneumoniae for which penicillin MICs are ⬎2 ␮g/ml. However, the emergence of clinical isolates resistant to cephalosporins (MICs ⬎ 1 ␮g/ml) has been reported since 1992 (4, 9, 12, 19). BAL5788 (formerly known as Ro 65-5788) is a water-soluble prodrug of BAL9141 (formerly Ro 63-9141), a novel parenteral pyrrolidinone cephalosporin with a broad spectrum of activity against most clinically relevant gram-positive and gramnegative pathogens and high levels of in vitro activity against methicillin- and vancomycin-resistant staphylococci and penicillin-resistant streptococci (5, 11, 13). The superior in vivo efficacy of BAL5788 over that of amoxicillin-clavulanate and/or

vancomycin in experimental aortic-valve endocarditis due to methicillin-resistant Staphylococcus aureus or vancomycin-intermediate S. aureus has been demonstrated in rats and rabbits (8; L. A. Basuino, G. Madrigal, and H. F. Chambers, Abstr. 43rd Intersci. Conf. Antimicrob. Agents Chemother., abstr. B1090, 2003). The purpose of this study was to compare the in vivo activity of BAL5788 (BAL9141) with that of a “gold standard” cephalosporin, ceftriaxone (25), in a mouse model of acute pneumococcal pneumonia. Leukopenic mice were infected with poorly virulent penicillin- or cephalosporin-resistant pneumococcal strains, since all strains belonging to serotypes 6, 9, 14, 19, and 23 are naturally less virulent in mice (2, 6). Immunocompromised animals were challenged with a large inoculum to induce pneumonia. Infected mice were subsequently treated with either BAL5788 or ceftriaxone. Survival rates, bacterial clearance from lungs and blood, and pharmacokinetic parameters were determined for both cephalosporins; and the development of resistance to BAL9141 in vivo was assessed. (This work was presented in part at the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy [E. Azoulay-Dupuis, J. Mohler, J. P. Be´dos, A. Schmitt-Hoffmann, and S. Shapiro, Abstr. 42nd Intersci. Conf. Antimicrob. Agents Chemother., abstr. A-1522, 2002].)

* Corresponding author. Mailing address: INSERM EMI-U 9933, Faculte´ de Me´decine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France. Phone: (33) 1 44 85 61 53. Fax: (33) 1 44 85 61 47. E-mail: [email protected].

Antibiotics. The antibiotics used in this study were BAL9141 and its prodrug, BAL5788 (Basilea Pharmaceutica AG, Basel, Switzerland); amoxicillin sodium salt (Beecham Laboratories, Paris, France); cefotaxime (Glaxo Laboratories, Paris, France); ceftriaxone (Roche Laboratories, Neuilly-sur-Seine, France); and

MATERIALS AND METHODS

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penicillin G (Aventis Laboratories, Paris, France). The active drug, BAL9141, was used for in vitro studies of antibacterial activities, while the prodrug, BAL5788, was used for in vivo studies. Assessment of antibacterial activities in vitro. The MICs of BAL9141 were determined by the broth microdilution method (21) in Columbia medium supplemented with 5% (vol/vol) sheep blood. Minimal bactericidal concentrations (MBCs) were determined in accordance with NCCLS protocols (20). Animals. Female Swiss albino mice (body weight, 20 to 22 g) were obtained from Iffa-Credo Laboratoires, Les Oncins, France. Mice were rendered leukopenic by treating them with daily intraperitoneal (i.p.) injections of cyclophosphamide (150 mg/kg of body weight) for 3 days, beginning 4 days before pneumococcal challenge. On the day of challenge leukocyte counts in blood had fallen from approximately 7,000 cells/ml to a mean of 1,000 cells/ml. Under these conditions, the four poorly virulent pneumococcal strains fully express their pathogenicity and invasive properties. Induction of experimental pneumonia. Pneumonia was induced with a penicillin-susceptible strain, P-52181 (serotype 19), isolated from a blood culture; a penicillin-resistant strain, P-15986 (serotype 19), isolated from middle-ear fluid; and two highly penicillin-, ceftriaxone-, and cefotaxime-resistant strains, P-40422 (serotype 23F) and P-40984 (serotype 14), recovered from middle-ear fluid. Bacterial suspensions (50 ␮l) containing 107 CFU of S. pneumoniae were instilled intratracheally into the lower respiratory tracts of leukopenic mice by a procedure described elsewhere (1). Untreated leukopenic mice developed acute pneumonia, became bacteremic within 1 to 4 h after challenge, and died within 3 to 5 days (mortality peak, day 1 or 2), depending on the invasiveness of the strain. Survival studies. The doses of BAL5788 used in survival studies are expressed as milligrams of BAL9141 equivalents per kilogram of body weight. The individual doses of BAL9141, given twice daily for 3 days (3 ⫻ q12h), were 1.05 and 2.1 mg/kg (total daily doses, 2.1 and 4.2 mg/kg, respectively) for S. pneumoniae strain P-52181; 2.1, 4.2, and 8.4 mg/kg (total daily doses, 4.2, 8.4, and 16.8 mg/kg, respectively) for P-15986; and 17, 25, and 37.5 mg/kg for P-40422 and P-40984 (total daily doses, 51, 75, and 75 mg/kg, respectively), with the two lower doses given three times daily for 3 days (3 ⫻ q8h). The individual doses of ceftriaxone, which were given 3 ⫻ q12h, were 5 and 10 mg/kg (total daily doses, 10 and 20 mg/kg, respectively) in studies with P-52181; 25 and 50 mg/kg (total daily doses, 50 and 100 mg/kg, respectively) in studies with P-15986; 100 mg/kg (total daily dose, 200 mg/kg) in studies with P-40422; and 100 and 200 mg/kg (total daily doses, 200 and 400 mg/kg, respectively) in studies with P-40984. See Table 2 for a summary of the dosing schedules. Treatment with BAL5788 or ceftriaxone was started 3 h after pneumococcal challenge. Early treatment initiation was necessary because the immunocompromised mice developed pneumonia shortly after they received such a large bacterial inoculum (107 CFU, close to the bacterial burden of ⱖ108 CFU at the time of death). Cohorts of infected mice (approximately 15 animals per treatment group) received a total of six subcutaneous (s.c.) injections (3 ⫻ q12h) or nine s.c. injections (3 ⫻ q8h) of BAL5788 or six s.c. injections (3 ⫻ q12h) of ceftriaxone (see Table 2). Infected control mice received s.c. injections of isotonic saline. The death rates were recorded daily, and cumulative survival rates over a 10-day observation period were calculated. Bactericidal activity in vivo. (i) BAL5788. Twelve hours after the first and third doses and 5, 30, 72, or 96 h after the sixth dose, mice infected with P-15986 (n ⫽ 15) or P-40422 (n ⫽ 15) were killed by i.p. injection of sodium pentobarbital and exsanguinated by cardiac puncture. Blood samples were cultured, and the lungs from each mouse were removed and homogenized in saline (1 ml). Total CFU counts in whole-lung homogenates were determined by plating 10-fold serial dilutions onto Columbia agar supplemented with 5% (vol/vol) sheep blood. The results are expressed as the mean log10 CFU per lung for groups of three mice. (ii) Ceftriaxone. Total CFU counts in whole-lung homogenates from mice treated with ceftriaxone (100 mg/kg, 3 ⫻ q12h) were determined 8 h after the first and third doses and 2 or 26 h after the sixth dose. The results are expressed as the mean log10 CFU per lung for groups of three mice. Analysis of resistance development. Dilutions of lung homogenates from BAL5788-treated mice were spread on Columbia agar plates supplemented with 5% sheep blood and cultured for 18 h at 37°C. Individual colonies recovered from P-15986-infected mice treated with BAL5788 were selected, and the MICs of BAL9141 were determined. Determination of plasma and lung drug concentrations and pharmacokinetics of BAL9141. Uninfected immunocompetent mice received single s.c. doses of BAL5788 corresponding to 25 mg of BAL9141 per kg. Blood samples and lungs were collected from six animals each at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 h after dosing. Blood was treated with EDTA-heparin and centrifuged at 4°C, and the plasma was immediately frozen at ⫺80°C. The lungs were excised, placed in liquid nitrogen, and immediately transferred to a ⫺80°C freezer. To cover the range of doses evaluated in the present study, an additional study was performed

ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. In vitro activities of BAL9141 and comparators against pneumococcal strains Antibiotic

Penicillin G Amoxicillin Ceftriaxone Cefotaxime BAL9141

MIC (␮g/ml) or MIC/MBC (␮g/ml) P-52181

P-15986

P-40422

P-40984

0.03 0.03 0.03 0.03 0.008/0.06

4/8 2/4 0.5/1 0.5/1 0.25/0.5

2/4 2/4 4/4 4/4 1/2

8/8 4/8 8/16 8/16 1/2

with uninfected immunocompetent mice receiving single s.c. doses of BAL5788 corresponding to either 1 or 10 mg of BAL9141 per kg. Plasma samples were collected from three animals each at 0.08, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, and 7 h after dosing. Blood was processed as described above. Samples (20 ␮l) of mouse plasma were diluted with human plasma (80 ␮l), and an internal standard (Ro 67-0024; 50 ␮l of a 40-ng/ml solution) was added. After the pH was adjusted to 5.0 with citric acid, proteins were precipitated with perchloric acid (150 ␮l of a 20% [vol/vol] solution) and removed by centrifugation. To determine the lung BAL9141 concentrations, whole lungs were minced and homogenized in liquid nitrogen. Samples of lung homogenate (0.050 g, weighed accurately) were suspended in EDTA-treated human plasma (0.1 ml; pH 5), 50 ␮l of the aforementioned Ro 67-0024 solution was added, the mixture with the sample was vortexed, and the precipitated proteins were removed by centrifugation. Aliquots (20 ␮l) of the supernatant obtained were injected into a reversed-phase (RP), column-switching, high-performance liquid chromatography (HPLC) system operated in the back-flush mode at ambient temperature. Trapping was performed with an Oasis HLB trapping column (1.0 by 30.0 mm; Waters AG, Rupperswil, Switzerland) and then a Phenomenex Synergi Polar-RP ˚ ; Brechbu column (1.0 by 50.0 mm; particle size, 80 A ¨hler AG, Schlieren, Switzerland) for analytical separation. The mobile phase for trapping was H2Omethanol (95:5 [vol/vol]) containing 1% glacial acetic acid, which was used at a flow rate of 0.12 ml/min; the applied trapping time was 1 min. For RP-HPLC a binary linear gradient of eluent A (1% aqueous glacial acetic acid) and eluent B (methanol-acetonitrile [50:50; vol/vol] containing 1% glacial acetic acid) was applied. The mobile phase for RP-HPLC was delivered at a flow rate of 1 ml/min, beginning with 35% eluent B and ramping up to 95% eluent B within 2.75 min after valve switching. BAL9141 was detected by diverting the eluent into a triple quadrupole mass spectrometer (API365; ABI Sciex, Concord, Ontario, Canada) operating in the positive ionization SRM detection mode (m/z 5353308 for BAL9141, m/z 6093365 for Ro 67-0024). Under these conditions the retention time for BAL9141 was 3.1 min. The calibration range for BAL9141 was from 0.05 to 6.40 ␮g/ml; thus, the limits of quantification for BAL9141 after dilution fivefold were 0.25 ␮g/ml in plasma and 0.10 ␮g/g in lung tissue. Plasma samples with concentrations outside the linear range were analyzed undiluted or were diluted by factors of 40 or 100 to fit the unknown concentrations into the validated linearity range. The BAL9141 content (in nanograms per gram) in lung tissue was calculated by dividing the analytical result (in nanograms per milliliter) by the net weight of tissue homogenate (number of grams per 0.1 ml of plasma) and correcting the result by a factor of 10 (which acknowledges the different volumes taken for unknown samples and calibration standards). All unknown samples, calibration samples, and quality control (QC) samples were assayed in the same way. During analytical runs the concentrations in 12 QC samples (4 QC samples each with low, medium, and high concentrations) were determined. All QC samples were analyzed with a mean interassay precision and accuracy of 100% ⫾ 15%. Derived pharmacokinetic parameters (area under the concentration-time curve [AUC], terminal elimination half-life [t1/2]) were estimated by noncompartmental analysis with WinNonLin Pro (version 4.1) software (Pharsight Corp., Mountain View, Calif.). Due to the linearity of the pharmacokinetics of BAL9141 in plasma in both infected leukopenic mice and uninfected nonleukopenic mice (see below), the maximum residence time above the MIC (␦tMIC) and the time above the MIC (T ⬎ MIC) in plasma were estimated from the mean plasma concentration-time profile obtained with a dose of 25 mg/kg administered s.c. Plasma and lung ceftriaxone concentrations and pharmacokinetics of ceftriaxone. The plasma and lung ceftriaxone concentrations and the ␦tMIC and T ⬎ MIC of ceftriaxone were determined for infected leukopenic female Swiss albino mice by using data from a previous study (24) performed with an identical mouse strain, with identical pneumococcal strains, and by an identical infection protocol. Statistical analysis. Survival data were analyzed by the logrank (Mantel-Cox) test, for which P values ⱕ0.05 were regarded as statistically significant.

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TABLE 2. In vivo efficacies of BAL9141 and ceftriaxone against pneumococcal strains in a mouse pneumonia model according to 10-day cumulative survival rates BAL9141

Ceftriaxone

Strain

Dosage (mg/kg), dosing frequency

Total daily dose (mg/kg)

Survival rate (%)

Dosage (mg/kg), dosing frequency

Total daily dose (mg/kg)

Survival rate (%)

P-52181

1.05, 3 ⫻ q12h 2.1, 3 ⫻ q12h

2.1 4.2

91 100

5, 3 ⫻ q12h 10, 3 ⫻ q12h

10 20

87 100

P-15986

2.1, 3 ⫻ q12h 4.2, 3 ⫻ q12h 8.4, 3 ⫻ q12h

4.2 8.4 16.8

57 93 93

25, 3 ⫻ q12h 50, 3 ⫻ q12h

50 100

13a 75

P-40422

17, 3 ⫻ q8h 25, 3 ⫻ q8h 37.5, 3 ⫻ q12h

51 75 75

80 80 67

100, 3 ⫻ q12h

200

85

P-40984

17, 3 ⫻ q8h 25, 3 ⫻ q8h 37.5, 3 ⫻ q12h

51 75 75

73 80 73

100, 3 ⫻ q12h 200, 3 ⫻ q12h

200 400

54 83

a

P ⬍ 0.0001 (logrank [Mantel-Cox] test) versus the result for BAL9141 at 8.4 mg/kg.

RESULTS In vitro antipneumococcal activities. BAL9141 exhibited good antipneumococcal activity in vitro against all strains tested (Table 1). The MICs and MBCs of BAL9141 for ␤-lactam-resistant pneumococci ranged from 0.25 and 0.5 ␮g/ml, respectively, for P-15986 to 1.0 and 2.0 ␮g/ml, respectively, for P-40422 and P-40984 and were four- to eightfold lower than those of penicillin G, amoxicillin, ceftriaxone, and cefotaxime. Cumulative survival of mice receiving BAL5788 or ceftriaxone. In studies with P-52181 (Table 2; Fig. 1A), the untreated mice died within 2 days. For mice receiving BAL5788 at a total daily dose of BAL9141 equivalents of 2.1 or 4.2 mg/kg, the cumulative survival rates were 91 and 100%, respectively. Treatment with ceftriaxone at total daily doses of 10 or 20 mg/kg resulted in survival rates of 87 and 100%, respectively. While there was no significant difference in the cumulative survival rates among mice treated with the two cephalosporins, 100% survival was obtained at a fivefold lower total daily dose of BAL9141 than the dose of ceftriaxone used. In mice infected with P-15986, BAL5788 (total daily dose of BAL9141, 4.2 mg/kg) led to a 10-day cumulative survival rate of 57%, whereas larger daily doses (8.4 and 16.8 mg/kg) protected 93% of mice (Table 2; Fig. 1B). Treatment with ceftriaxone at total daily doses of 50 and 100 mg/kg was associated with survival rates of 13 and 75%, respectively (Table 2). The survival rates achieved with BAL5788 (total daily dose of BAL9141, 8.4 mg/kg) and ceftriaxone (total daily dose, 50 mg/kg) were significantly different (93 versus 13%; P ⬍ 0.0001) in favor of BAL9141, while the difference between BAL9141 (total daily dose, 8.4 mg/kg; survival rate, 93%) and ceftriaxone (total daily dose, 100 mg/kg; survival rate, 75%) did not reach statistical significance. Similar levels of protection against the Penr Cros Ctxs strain were obtained with a 10-fold lower dose of BAL9141. P-40422-infected mice receiving BAL5788 (total daily dose of BAL9141, 75 mg/kg given 3 ⫻ q12h) had a 10-day cumulative survival rate of 67%, while the same total daily dose (25 mg/kg given 3 ⫻ q8 h) or a total daily dose of 51 mg/kg (17 mg/kg given 3 ⫻ q8 h) protected 80% of the mice (Table 2; Fig.

1C). Treatment with ceftriaxone (total daily dose, 200 mg/kg given 3 ⫻ q12h) provided an 85% survival rate (Table 3). In mice infected with Penr Cror Ctxr strain P-40984 and treated with BAL5788 (total daily dose of BAL9141, 75 mg/kg given 3 ⫻ q12h), a 10-day survival rate of 73% was recorded, whereas the same total daily dose of 25 mg/kg given 3 ⫻ q8h or a total daily dose of 51 mg/kg (17 mg/kg given 3 ⫻ q8h) produced survival rates of 73 and 80%, respectively (Table 2). Treatment with ceftriaxone at total daily doses of 200 and 400 mg/kg administered 3 ⫻ q12h was associated with 10-day survival rates of 54 and 83%, respectively (Table 2; Fig. 1D). Both BAL9141 and ceftriaxone led to similar cumulative survival rates among mice infected with Penr Cror Ctxr strains, but the total daily dose of BAL9141 required to achieve this survival rate was four- to fivefold lower than that of ceftriaxone. Bacterial clearance from lungs. In control mice (n ⫽ 3) infected with strain P-15986, pneumococcal counts in the lungs increased from 7.0 log10 CFU/ml at 15 h postchallenge (p.c.) to 8.2 log10 CFU/ml at 70 h p.c., whereupon the mice died. At 39 h p.c., i.e., 12 h after the third dose of BAL5788 (BAL9141 equivalent dose, 4.2 mg/kg), the titers of P-15986 in the lungs of the mice were 4 orders of magnitude lower than those in the lungs of control mice. By 159 h, i.e., 5 h after the sixth dose of BAL5788, pneumococci could not be detected in blood (Table 3). In mice infected with strain P-40422, the pneumococcal titers in lungs 12 h after the third dose (39 h p.c.) of BAL5788 (BAL9141, 17 mg/kg) were 4 orders of magnitude lower than those in the lungs of untreated mice (Table 4). By the end of treatment with BAL5788, no pneumococci could be detected in lung tissues, and no regrowth occurred after the cessation of treatment. Blood cultures for all mice were negative for pneumococci by the end of treatment. Ceftriaxone (100 mg/kg given 3 ⫻ q12h) reduced the bacterial counts in the lungs of mice infected with strain P-40422 to undetectable levels, without regrowth (Table 5). The blood of ceftriaxone-treated mice was cleared of pneumococci by the end of treatment. Pharmacokinetics of BAL9141 and ceftriaxone in serum and lungs. In uninfected nonleukopenic mice, a single s.c. dose of

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FIG. 1. Activity of BAL9141 against pneumococcal strains. (A) Cumulative 10-day survival of mice challenged with Pens CroS Ctxs strain P-52181 and treated with BAL5788 3 ⫻ q12h (BAL9141 equivalents, 1.05 or 2.1 mg/kg). (B) Cumulative 10-day survival of mice challenged with Penr Cros Ctxs strain P-15986 and treated with BAL5788 3 ⫻ q12h (BAL9141 equivalents, 2.1, 4.2, or 8.4 mg/kg). (C) Cumulative 10-day survival of mice challenged with Penr Cror Ctxr strain P-40422 and treated with BAL5788 3 ⫻ q12h (BAL9141 equivalents, 17 or 25 mg/kg) or 3 ⫻ q8h (BAL9141 equivalents, 37.5 mg/kg). (D) Cumulative 10-day survival of mice challenged with Penr Cror Ctxr strain P-40984 and treated with BAL5788 3 ⫻ q12h (BAL9141 equivalents, 17 or 25 mg/kg) or 3 ⫻ q8h (BAL9141 equivalents, 37.5 mg/kg). Peni, penicillin; CRO, ceftriaxone; BAL, BAL5788.

BAL5788 (BAL9141 equivalent, 25 mg/kg) gave higher peak concentrations in serum than a single s.c. dose of ceftriaxone (50 mg/kg) (Fig. 2), whereas the AUC from 0 to 24 h (AUC0-24) for serum, t1/2, and ␦tMIC (calculated for Penr Cror

Ctxr strain P-40422) were much less for BAL9141 (25 mg/kg) than for ceftriaxone (50 mg/kg). The peak concentration of BAL9141 in the lungs after a single s.c. dose of BAL5788 (BAL9141 equivalent, 25 mg/kg) was much less than that after

TABLE 3. Pneumococcal clearance from lungs and blood of mice infected with Penr Cros Ctxs strain P-15986 by BAL5788 Mean S. pneumoniae titer (log10 CFU/ml of whole-lung homogenate [lowest and highest values]), no. of animals with positive blood culture/total no. of animals

Assessment time Actual timea

Time postchallenge (h)

Control mice (n ⫽ 3)

12 h after first dose 12 h after third dose 5 h after sixth dose 30 h after sixth dose 96 h after sixth dose

15 39 68 93 159

7.0 (6.5–7.7), 3/3 7.9 (7.1–8.4), 3/3 8.2 (8.0–8.5), 2/2 ⬎8c

a

The first dose was given 3 h after bacterial challenge. All three doses were administered 3 ⫻ q12h. Mortality rate, 100%. d Limit of detection, 1 log10 CFU/ml. b c

Mice treated with BAL9141 atb: 2.1 mg/kg; 3 q12h

4.2 mg/kg

8.4 mg/kg

6.1 (6.0–6.3), 2/3 5.0 (3.8–6.9), 2/3 4.6 (4.4–4.9), 1/3 3.8 (1.7–5.0), 1/2 ⬍1.0, 0/2

5.2 (4.5–6.4), 3/3 3.8 (2.8–4.5), 1/3 3.7 (3.3–4.2), 1/3 3.7 (1.7–4.9), 0/2 ⬍1.0, 0/2

5.0 (4.4–5.4), 3/3 5.7 (5.2–6.6), 3/3 3.5 (2.5–4.7), 0/3 ⬍1.0,d 0/3 ⬍1.0, 0/3

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TABLE 4. Pneumococcal clearance from lungs and blood of mice infected with Penr Cror Ctxr strain P-40422 by BAL5788 Mean S. pneumoniae titer (log10 CFU/ml of whole-lung homogenate [lowest and highest values]), no. of animals with positive blood culture/total no. of animals

Assessment time Actual timea


12 h after first dose 12 h after third dose 5 h after sixth dose 72 h after sixth dose 96 h after sixth dose

15 39 68 135 159

Mice treated with BAL9141 atb:

Control mice

7.3 (6.6–7.7), 3/3 7.7 (7.0–8.1), 3/3 7.9 (7.6–8.6), 3/3 ⬎8c

8.4 mg/kg

17 mg/kg

6.0 (4.7–7.3), 1/3 4.3 (3.0–5.0), 2/3 3.2 (2.4–3.7), 0/3 2.9,d 0/3 ⬍1.0, 0/3

7.2 (7.0–7.3), 2/3 3.9 (3.0–5.6), 1/3 0.0, 0/3 ⬍1.0,e 0/3 ⬍1.0, 0/3

a

The first dose was given 3 h after bacterial challenge. Both doses were administered 3 ⫻ q12h. Mortality rate, 100%. d Value for one mouse. e Limit of detection, 1 log10 CFU/ml. b c

a single dose of ceftriaxone (50 mg/kg). The t1/2 of BAL9141 in lung tissue was only marginally shorter than that of ceftriaxone, although BAL9141 had a much lower pulmonary AUC0-24 than ceftriaxone. For lung tissue, as for serum, the ␦tMIC was much lower for BAL9141 than for ceftriaxone (Table 6). For the four pneumococcal strains tested, very low values of T ⬎ MIC, ranging from 9 to 18% of the dosing interval, were adequate to combat pneumonia, whereas the corresponding values of T ⬎ MIC for ceftriaxone were 30 to 50% (Table 7). Development of endogenous resistance to BAL9141. No increase in the MIC of BAL9141 for pneumococcal isolates recovered from P-15986-infected mice treated with BAL5788 was observed (total daily dose of BAL9141, 2.1 mg/kg given 3 ⫻ q12h; data not shown). DISCUSSION

mococcal lung infection in the absence of a functional immune system. In earlier experiments conducted in these laboratories under identical conditions with the identical mouse strain and pneumococcal strains (18, 24), a sixfold higher dose of amoxicillin was required to achieve the same activity as that of ceftriaxone against Penr Cros Ctxs strain P-15986. Since doses of amoxicillin or cefotaxime two- to fourfold higher than those of ceftriaxone resulted in similar activities against Penr Cror Ctxr strains P-40422 and P-40984, it was expected that BAL9141 would be effective against Penr Cror Ctxr strains at much lower doses than either amoxicillin or cefotaxime. Our present observations are concordant with earlier findings with a mouse septicemia model (11), in which BAL9141 proved highly active against S. pneumoniae 23F-CTR (MIC, 1 ␮g/ml; 50% effective dose [ED50], 1.0 mg/kg), a strain with

BAL9141 displayed very good in vitro activity against highly penicillin- and cephalosporin-resistant S. pneumoniae strains, and these in vitro data agree with our in vivo findings. Although the survival rates achieved with BAL9141 and ceftriaxone for mice infected with Pens Cros Ctxs or Penr Cror Ctxr strains did not differ significantly, the doses of BAL9141 required to achieve survival were markedly lower than those of ceftriaxone. Thus, BAL9141 proved highly active in this stringent model of pneumonia, providing a good measure of the intrinsic activity of this cephalosporin for clearance of a pneu-

Compartment, cephalosporin, and dose (mg/kg)

TABLE 5. Time course of bacterial clearance from lungs and blood of mice infected with Penr Cror Ctxr strain P-40422 by ceftriaxone Mean S. pneumoniae titer (n ⫽ 3) (log10 CFU/ml of whole lung homogenate [no. of animals with positive blood culture/total no. of animals])

Assessment time

Actual timea


Control mice (n ⫽ 3)

Cefriaxoneb

8 h after first dose 8 h after third dose 2 h after sixth dose 26 h after sixth dose

11 35 65 89

7.52 ⫾ 0.27 (3/3) 8.23 ⫾ 0.33 (3/3) 8.93 ⫾ 0.30 (3/3) ⬎8d

5.70 ⫾ 0.38 (1/3) 3.69 ⫾ 0.25 (1/3) ⬍1c (0/3) ⬍1 (0/3)

a

The first dose was given 3 h after bacterial challenge. Ceftriaxone was administered at 100 mg/kg 3 ⫻ q12h. Limit of detection, 1 log10 CFU/ml. d Mortality rate, 100%. b c

TABLE 6. Pharmacokinetic parameters for BAL5788 and ceftriaxone in serum and lungs of Swiss mice after single s.c. injectionsa Cmax (␮g/ml)

t1/2 (h)

␦tMIC (h)

AUC0–24 (␮g 䡠 h/ml)

1.6 19.2 60.7

0.26 0.24 0.33

0.5 0.8 1.2

0.87 13.1 29.7

Ceftriaxone 50 100

46.5 91.5

1.41 1.35

4.0 4.6

Lung tissue BAL5788, 25

9.6

1.00

1.25

5.4

Ceftriaxone 50 100

36.7 44.3

1.12 1.68

4.0 4.0

79.5 59.6

Serum BAL5788 1 10 25

96.6 150.0

a Data are means for six mice (BAL5788) or three mice (ceftriaxone). The doses of BAL5788 are expressed as BAL9141 equivalents. Cmax, peak concentration; t1/2, terminal elimination half-life, calculated by least-squares regression of the log-linear terminal elimination phase; ␦tMIC, maximum residence time above MIC calculated for Penr Cror Ctxr strain P-40422 (BAL9141 and ceftriaxone, MICs, 1 and 4 ␮g/ml, respectively); AUC0–24, area under the curve (integral of the concentration over time), estimated by using the trapezoidal rule with noncompartmental analysis.

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ANTIMICROB. AGENTS CHEMOTHER.

FIG. 2. Mean concentration-time profiles of BAL9141 (25 mg/kg administered s.c. as BAL5788) in the sera and lungs of uninfected immunocompetent Swiss albino female mice (n ⫽ 6).

correlation between T ⬎ MIC and antibacterial activity (Andes and Craig, 40th ICAAC). BAL9141 was highly effective, despite a much lower ␦tMIC of BAL9141 in serum (calculated for Penr Cror Ctxr strain P-40422) compared with that of ceftriaxone. For the four pneumococcal strains tested, very low values of the T ⬎ MIC for BAL9141, ranging from 9 to 18% of the dosing interval, proved adequate to combat pneumonia in immunocompromised mice. This is especially noteworthy because mortality studies with animal models of pneumonia showed other that ␤-lactams were ineffective when the levels in serum exceeded the MIC for ⱕ20% of the dosing interval (7; Andes and Craig, 40th ICAAC). In contrast, the values of T ⬎ MIC for ceftriaxone were 30 to 50%. This range is in good agreement with the values of T ⬎ MIC (30 to 60%) reported to be necessary for activity (7; M. N. Dudley and P. G. Ambrose, Abstr. 42nd Intersci. Conf. Antimicrob. Agents Chemother., abstr. A-635, 2002). The differences between the effective T ⬎ MIC for BAL9141 and that for ceftriaxone might be explained, at least in part, by the difference in the extent of plasma protein binding of these cephalosporins (BAL9141 plasma protein binding, ⬍60% for all species, including hu-

reduced susceptibility to both ceftriaxone (MIC, 4 ␮g/ml; ED50, 8.8 mg/kg) and cefotaxime (MIC, 8 ␮g/ml; ED50, ⬎ 12 mg/kg). This has important clinical ramifications, since the worldwide development of resistant pneumococci is related to the spread of a few highly resistant clones, such as serotype 23F (16). BAL9141 exhibits time-dependent bactericidal activity (11; D. R. Andes and W. A. Craig, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1079, 2000). In the present study the pharmacokinetics of BAL9141 were determined in uninfected mice. This was warranted because earlier studies with a rat endocarditis model revealed that plasma BAL9141 concentrations in infected rats matched those anticipated from single-dose pharmacokinetic studies with uninfected rats (8). Pharmacokinetic studies with humans and several animal species, including female Swiss albino mice, indicated linear kinetics up to a dose of 360 mg/kg; and to date there have been no noticeable differences in pharmacokinetics according to the species tested. Therefore, the pharmacokinetic behavior of BAL9141 was reasonably presumed to be unaffected by pneumococcal infection. Earlier animal studies indicated a strong

TABLE 7. T ⬎ MICs for BAL9141 and ceftriaxone for activity against pneumococcal strains Strain (phenotype; BAL9141 MIC)

P-52181 P-15986 P-40422 P-40984

(Pens (Penr (Penr (Penr

Cros Cros Cror Cror

Ctxs; Ctxs; Ctxr; Ctxr;

0.008 ␮g/ml) 0.25 ␮g/ml) 1 ␮g/ml) 1 ␮g/ml)

BAL9141

Ceftriaxone

Active dose (mg/kg)

T ⬎ MIC (% of dosing interval)

Active dose (mg/kg)

T ⬎ MIC (% of dosing interval)

1.05, 3 ⫻ q12h 4.2, 3 ⫻ q12h 17, 3 ⫻ q8h 25, 3 ⫻ q8h

18 9 13 15

10, 3 ⫻ q12h 50, 3 ⫻ q12 100, 3 ⫻ q12 200, 3 ⫻ q12

⬎50 50 38 30

VOL. 48, 2004


mans; ceftriaxone plasma protein binding, ⬎80% for mice [18]). However, a strict comparison of the values of T ⬎ MIC of unbound BAL9141 and ceftriaxone was not possible due to the nonlinear plasma protein binding of ceftriaxone in mice over the range of the concentrations in plasma of the dose range evaluated (18). S. pneumoniae strain P-15986 was expected to be a sensitive gauge for the possible emergence of resistance to BAL9141 because of its cephalosporin-susceptible phenotype. The absence of the development of resistance to BAL9141 after the full treatment cycle among isolates recovered from mice infected with P-15986 is consistent with earlier findings from serial passage studies with Staphylococcus aureus and Staphylococcus epidermidis that resistance to BAL9141 does not emerge readily (11; S. Shapiro, unpublished observations). Although extrapolation of our present findings to the clinic is complicated by a number of experimental factors (use of immunocompromised animals, high inoculum, early treatment onset), studies of BAL5788 for the treatment of human pneumococcal pneumonia are clearly of interest, particularly in populations such as children under 2 years of age, elderly persons, and individuals infected with human immunodeficiency virus, whose immune status may be suboptimal. Toward this end, BAL5788 may prove to be a very valuable therapeutic option for the treatment of lung and possibly other infections due to pneumococcal strains resistant to conventional cephalosporins. ACKNOWLEDGMENT

9. 10.

11.

12. 13.

14. 15.

16.

17.

18.

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