JAC Advance Access originally published online on February 7, 2006
Journal of Antimicrobial Chemotherapy 2006 57(4):732-740; doi:10.1093/jac/dkl015
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Caspofungin: antifungal activity in vitro, pharmacokinetics, and effects on fungal load and animal survival in neutropenic rats with invasive pulmonary aspergillosis
1 Department of Medical Microbiology & Infectious Diseases, Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands; 2 Department of Internal Medicine, Section Infectious Diseases, Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands; 3 Department of Hospital Pharmacy, Clinical Pharmacology Unit, Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands
* Corresponding author. Tel: +31-10-463-2174; Fax: +31-10-463-3875; E-mail: w.vanvianen{at}erasmusmc.nl
Received 13 October 2005; returned 6 December 2005; revised 20 December 2005; accepted 23 December 2005
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Objectives: Evaluation of the potential of caspofungin, in relation to pharmacokinetics, in order to optimize its use in the treatment of filamentous fungal infections.
Methods: The in vitro antifungal activity, pharmacokinetics and therapeutic efficacy of caspofungin versus amphotericin B was investigated in vitro as well as in a model of aerogenic Aspergillus fumigatus infection in neutropenic rats, using rat survival and decrease in fungal burden as parameters for therapeutic efficacy.
Results: In contrast to amphotericin B, caspofungin shows a concentration-dependent gradual decrease in fungal growth in vitro, which makes it difficult to perform visual readings of antifungal activity (CLSI guidelines). The quantitative XTT [2,3-bis(2-methoxy-4-nitro-5-[(sulphenylamino) carbonyl]-2H-tetrazolium-hydroxide] assay measuring a decrease in fungal metabolic activity seems more appropriate for caspofungin susceptibility testing. Using this assay, in vitro caspofungin was 4-fold less active than amphotericin B. In the infection model, therapy was started 16 h after fungal inoculation, and continued once daily for 10 days. Caspofungin was administered intraperitoneally at 1, 2, 3 or 4 mg/kg/day (CAS 1, 2, 3 or 4), amphotericin B at 1 mg/kg/day (AMB 1). Treatment with CAS 1 or AMB 1 provided modest prolongation of animal survival. The combination of caspofungin and amphotericin B did not show additive effects. Increasing the dosage of caspofungin to 2, 3 or 4 mg/kg/day resulted in a dose-dependent significant increase in efficacy. There was 100% survival among rats in the CAS 4 group, which was correlated with a significant decrease in fungal burden, based on the concentration of A. fumigatus galactomannan in serum and lung tissue and quantification of A. fumigatus DNA in lung tissue. Pharmacokinetic analysis suggested that the CAS 4 dose in rats produced drug exposure comparable to the human situation, visualized by similar 24 h AUC and trough concentrations.
Conclusions: The therapeutic efficacy of caspofungin is superior to amphotericin B, which seemed to be discrepant with their in vitro antifungal activity.
Keywords: amphotericin B , XTT assay , Aspergillus fumigatus , quantitative PCR , galactomannan
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Introduction |
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Patients receiving cancer chemotherapy or immunosuppressants are at risk of invasive pulmonary aspergillosis (IPA).1,2 The overall mortality rate for IPA remains dramatically high in populations of the most profoundly immunosuppressed patients.3,4 For many years amphotericin B (AMB) has been the drug of first choice. However, treatment with amphotericin B is often unsuccessful and its use is limited by dose-related nephrotoxicity.5,6 At present, voriconazole is being used as the drug of first choice.7 Nevertheless, voriconazole is not always effective or tolerated, so there is still a need to use other antifungal agents. Caspofungin (CAS) is the first representative of a new class of antifungal agents, the echinocandins, that inhibit 1,3-ß-D-glucansynthase. 1,3-ß-D-Glucan is a critical structural cell-wall component in many pathogenic fungi. As there is a lack of mammalian 1,3-ß-D-glucan, caspofungin shows a good safety profile with low toxicity.8
The predictive significance of in vitro data on susceptibility of Aspergillus spp. to antifungals for clinical efficacy remains challenging. One of the difficulties is the lack of accuracy and reproducibility of the susceptibility assays measuring fungal growth inhibition rate. In view of this there is a need for an objective and reproducible quantitative assay.9 Meletiadis et al. developed a quantitative assay to measure the antifungal activity of agents towards filamentous fungi in vitro, based on fungal dehydrogenase activity as a measure for the viable fungal mass.10 In the present study both this quantitative assay and the qualitative assay as described in the guidelines of the CLSI for other antifungal agents11 were used to assess in vitro antifungal activity.
Treatment with caspofungin produced a favourable response in patients with severe IPA.3,12 Nevertheless the activity of caspofungin with respect to decreasing fungal burden in blood and in infected tissues is not yet fully understood. A few studies in experimental models of aspergillosis have been performed to demonstrate the antifungal activity of caspofungin,13–18 with varying results with respect to the treatment outcome. In the present study the antifungal activity of caspofungin versus amphotericin B was investigated in vitro as well as in vivo in an inhalation model of Aspergillus fumigatus infection in neutropenic rats. This animal model is clinically relevant as aspergillus infection in patients is also through the respiratory route. Parameters of therapeutic response were animal survival and decrease in fungal burden in serum and infected lung tissue. For assessment of fungal burden, the formation of hyphae in the tissues after the inoculation of conidia makes traditional methods such as quantification of cfu unsuitable. A cluster of hyphae is often indistinguishable from a single-cell conidium when spread on agar, because both will usually yield one colony. In the present model Becker et al. demonstrated in untreated, infected rats that numbers of cfu in the infected left lung did not increase over time, despite progression of the fungal infection resulting in mortality of rats.19
Recently more sensitive assays for determining tissue A. fumigatus burden in animal models have been developed. Becker and co-workers used a quantitative galactomannan (GM) assay to measure fungal burden in the lungs of rats infected with A. fumigatus,20 and Bowman et al. developed a quantitative DNA assay to assess fungal burden in mice after inoculation with A. fumigatus.14 Both investigators demonstrated that these techniques are useful to monitor the progression of fungal infection as well as the efficacy of antifungal treatment. Both non culture-based assays as well as prolongation of rat survival were used in the present study to measure efficacy of antifungal treatment.
In the present study the therapeutic efficacy of caspofungin was related to its pharmacokinetic profile. Pharmacokinetic data are important to compare dosage schedules of clinical and animal studies. When pharmacokinetics in clinical and animal studies are comparable (pharmacokinetic equivalent), therapeutic efficacy in animal models might be relevant for clinical management.
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Organism
A clinical strain of A. fumigatus, originally isolated from an immunocompromised patient with IPA, was used in all experiments. To maintain the virulence of the strain, an isolate from the lungs of untreated control animals from each in vivo experiment was used in the next in vivo experiment.
In vitro antifungal susceptibility testing
In vitro antifungal susceptibility testing was performed in three ways. First, a broth macrodilution assay in tubes was performed (qualitative assay) according to the guidelines of the CLSI (M38-A document).11 A. fumigatus was cultured on Sabouraud glucose agar (Oxoid Ltd, Basingstoke, England) at 37°C for 4 days. A volume of 5 mL of sterile phosphate-buffered saline (PBS) containing 0.05% Tween 20 was transferred into the fully grown plate. Conidia were harvested by gently rotating the plate for 5 min. Next the conidia suspension was transferred into a sterile tube and heavy particles were allowed to settle for 4 min. Next the conidia suspension was transferred into a new sterile tube and the inoculum was standardized with a haemocytometer (Bürker Türk, Marienfeld, Germany) to a final concentration of 5 x 104 conidia/mL in RPMI 1640 medium (with L-glutamine but without bicarbonate) (Cambrex Bio Science, Verviers, Belgium) buffered to pH 7.0 with 0.165 M 3-N-morpholinopropanesulphonic acid (MOPS) (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) (assay medium). Quantification of viable inocula was performed by spreading serial dilutions on Sabouraud glucose agar. Colony-forming units were counted after 24 h of incubation at 37°C followed by 24 h of incubation at room temperature. Caspofungin was dissolved in water and serially diluted in assay medium to yield final concentrations of 0.06–128 mg/L. Amphotericin B was dissolved in dimethyl sulphoxide and serially diluted in assay medium to yield final concentrations of 0.03–32 mg/L. A drug-free growth control that contained 0.5% dimethyl sulphoxide in medium was included. After incubation for 48 h at 37°C, the degree of fungal growth was assessed visually and graded: score 0, optically clear or absence of growth; score 1, slight growth or 
25% of the growth control; score 2, prominent reduction in growth or 50% of the growth control; score 3, slight reduction in growth or 75% of the growth control; score 4, no reduction in growth. For amphotericin B the MIC was defined as the lowest drug concentration resulting in the absence of growth (score 0). For caspofungin a definition of MIC has not been described in the CLSI document. For this reason the definition for amphotericin B was followed.
Second, minimum effective concentration (MEC) values were determined for caspofungin according to Arikan et al.21 by microscopic examination. The lowest concentration of caspofungin causing abnormal hyphal growth with short, stubby and highly branched hyphae was defined as MEC.
Third, the viable fungal mass was assessed in a quantitative viability based assay as described by Meletiadis,10 with some modifications. In brief, in this assay the metabolic activity of the viable fungal mass was determined in terms of fungal dehydrogenase activity, converting XTT (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) into coloured formazan. XTT and menadione (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) were added to each tube in order to obtain final concentrations of XTT of 200 µg/mL and of menadione 4.3 µg /mL (25 µM). Menadione was first dissolved in acetone and then diluted 1/10 in PBS. Incubation was continued at 37°C for 2 h in the dark to allow conversion of XTT to its formazan derivative. While the XTT formazan product readily appears in solution, at caspofungin concentrations of 4 mg/L or higher a significant amount of retained intracellular product was visible in the hyphae probably through morphological changes of the hyphae.18,22,23 To solubilize the formazan product 300 µL of 100% dimethyl sulphoxide (Sigma-Aldrich Chemie) was added.24 The extinction of the supernatant was measured spectrophotometrically at 450 nm.
Infection model of invasive pulmonary aspergillosis (IPA)
The rat model of aerogenic left-sided IPA was used, as described previously19,20,25 with a few modifications. Specified pathogen-free female RP strain albino rats (breeding facilities of Erasmus MC, 18–25 weeks old, 200–250 g) were used. Profound neutropenia was induced by intraperitoneal administration of 75 mg/kg cyclophosphamide (Endoxan®, Baxter, Utrecht, The Netherlands) 5 days before fungal inoculation followed by a dose of 60 mg/kg 1 day before inoculation and 50 and 40 mg/kg, respectively on days 3 and 7 after inoculation. Neutrophil counts were assessed microscopically in a haemocytometer using Türk solution. For determination of neutrophil counts blood samples were taken on days –6, 0, 2, 5, 6, 9, 13, 15 and 23 (three animals per time point). The cyclophosphamide treatment protocol resulted in neutrophil counts of <0.1 x 109/L on the day of fungal inoculation, and rats remained neutropenic until day 10. From day 11 neutrophil numbers gradually rose.
To prevent bacterial superinfections, animals were given doses of 30 mg/kg teicoplanin (Targocid®, Aventis Pharma B.V., Hoevelaken, The Netherlands) intramuscularly (im) 4 and 5 days before fungal inoculation followed by doses of 15 mg/kg 1 day before inoculation, on the day of inoculation, and on day 3, 6, 8 and 10 after inoculation. In addition, rats received ciprofloxacin 660 mg/L and polymyxin B 100 mg/L in their drinking water throughout the experiment.
Fungal infection was established by intubation of the left main bronchus under general anaesthesia. A cannula was passed through the tube and the left lung was inoculated with 6 x 104 A. fumigatus conidia suspended in 20 µL of PBS. This is the minimal inoculum resulting in a lethal infection in untreated animals.
The experimental protocols adhered to the rules specified in the Dutch Animal Experimentation Act (1977) and the published Guidelines on the Protection of Experimental Animals by the Council of the EC (1986). The present protocols were approved by the Institutional Animal Care and Use Committee of the Erasmus MC Rotterdam.
Antifungal treatment
Treatment was started at 16 h after fungal inoculation, the time at which hyphal growth in the left lung was established, and was continued once daily for 10 days. Caspofungin (Cancidas®, Merck and Company, Rahway, NJ, USA) was diluted in saline and was given intraperitoneally. Amphotericin B (Fungizone®, Bristol-Myers Squibb B.V., Woerden, The Netherlands), was diluted in 5% dextrose and was given intravenously (iv) via the lateral tail vein. Treatment regimens included: caspofungin 1, 2, 3 or 4 mg/kg/day, amphotericin B 1 mg/kg/day [which is the maximum tolerated dose (MTD)], and caspofungin 1 mg/kg/day combined with amphotericin B 1 mg/kg/day. Control rats did not receive treatment, since previous studies showed that placebo treatment with either saline or 5% dextrose did not influence the course of infection and survival rate of rats.
Parameters for therapeutic activity
The survival rate of rats was monitored daily until day 21 after fungal inoculation. In addition, two non culture-based methods were used to quantify the fungal burden of the A. fumigatus-infected rats. The first method is the quantitative detection of GM, which is a fungal cell-wall polysaccharide that can be released by Aspergillus spp. during growth. The second method is the quantitative detection of A. fumigatus DNA by a real time PCR technique and calculation of conidial equivalents (CE) according to the procedure of Bowman et al.14
The GM concentrations in serum and left lung and the CE counts in left lung, were measured on days 1, 3 and 6, after fungal inoculation for the untreated control rats and on days 3, 6, 11 and 21 for the treated rats. At the indicated time intervals rats were euthanized. Blood was obtained for GM detection by puncture of the orbital plexus under CO2 anaesthesia. Then the left lung was dissected and stored at –80°C in pre-weighed WhirlPak bags (Fisher Scientific, Pittsburgh) until analysis. In rats that died before the indicated intervals organs were cultured to exclude bacterial superinfections. A tissue suspension was prepared by direct pressure in 3.6 mL of sterile saline per g of tissue.26 The tissue suspension was homogenized further according to the method of Bowman et al.14
Quantitative detection of GM
The antigen GM was quantified by a commercially available sandwich ELISA (Platelia Aspergillus, Bio-Rad, Marnes-la-Coquette, France). This assay was modified in our lab as described.20 The concentration of GM in positive test samples was expressed as nanograms of GM per mL of serum or per gram of tissue. The limit of detection of GM in a test sample was 1 ng/mL of serum (log GM = 0) and 3.6 ng/g of tissue (log GM = 0.56).
Quantitative detection of A. fumigatus DNA
DNA was extracted according to the method of Bowman et al.14 with some modifications.
Normalization for DNA recovery was performed by quantifying a non-murine, non-fungal DNA sequence on a plasmid which was added to all samples prior to homogenization.27 A plasmid bearing a 3 kb fragment containing the protein coding region of the Eimeria tenella PKG cDNA (Accession Number AF411961 [GenBank] ) was spiked into the saline added to organs in the Whirl-Pak bags. After homogenization and DNA isolation, samples were analysed by TaqMan® with the following primers and probe specific for the parasite gene sequence: (i) sense amplification primer: 5'-AGGGCTTTGCTGCACGAC-3', (ii) antisense amplification primer: 5'-TCCACCTCGGGACTGTTTG-3', (iii) hybridization probe: 5'- FAM-TGCTACTGTTGCAGACCGCCGCT-TAMRA-3'. PCRs were performed as for the A. fumigatus 18S rDNA target. TaqMan quantification of the PKG target sequence allowed for an estimate of the recovery of DNA in the experiment from the crude homogenate through the TaqMan reaction. This assessment of DNA recovery was made for each experimental sample. The percentage recovery of the PKG target sequence was used to estimate the recovery of tissue DNA from the sample. Accordingly, each TaqMan data point for the Af 18S rRNA gene target was normalized based on the recovery of DNA predicted by the PKG standard.
All qPCR results for samples from infected tissues were expressed as CE per gram equivalent of tissue.
The limit of detection of CE counts in a test sample was 96.83 CE/g tissue (log CE = 1.99).
Statistical analysis
Differences in rat survival rate were assessed by log rank test. Differences in quantitative parameters of fungal infection were assessed by Student's t-test.
Pharmacokinetics of antifungal agents
After administration to neutropenic rats of caspofungin 1 mg/kg, caspofungin 4 mg/kg or amphotericin B 1 mg/kg, serial blood samples were taken at 5 min, 1, 2, 3, 4, 6, 8, 12 and 24 h by retro-orbital puncture under CO2-anaesthesia from alternate groups of 3 rats. Blood samples were collected in heparinized tubes and after centrifugation the plasma was collected. Plasma concentrations of antifungal agents were assessed using a standard large plate agar diffusion procedure with diagnostic sensitivity test agar (Oxoid, Basingstoke, UK) and a Candida albicans strain (clinical isolate) as test organism. Samples of 200 µL were assayed. Twofold increasing standard concentrations of 0.25–4 mg/L were used (R2 = 0.998). The assay system for caspofungin as well as for amphotericin B was sensitive to 0.25 mg/L. The plasma-concentration profiles were fitted to a two-compartment pharmacokinetic model with the first-order absorption and elimination using WinNonlin software (Pharsight, Mountain View, CA, USA). The clearance (CL), volume of distribution at steady state (Vss), elimination half life (t1/2), and the area under the plasma concentration versus time curve (24 h AUC) were calculated.
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In vitro antifungal activity
The data for in vitro antifungal activity of caspofungin versus amphotericin B are shown in Table 1. Fungal exposure to caspofungin resulted in a concentration-dependent gradual reduction in fungal growth. In contrast, with amphotericin B a sharp endpoint between no reduction in growth and absence of growth was observed. As shown in Table 1 the MIC of amphotericin B determined visually according to the CLSI guidelines was 0.5 mg/L. The MIC of caspofungin according to the CLSI definition for amphotericin B was 8 mg/L. The MEC of caspofungin after microscopic inspection was 1 mg/L.
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At the MIC of caspofungin and amphotericin B, exhibiting visual absence of fungal growth, 16 and 2% fungal growth, respectively, was observed using the quantitative XTT assay. At concentrations of caspofungin 1 mg/L and amphotericin B 0.25 mg/L, which visually did not result in a reduction in fungal growth, a substantial decrease in viable fungal mass to 57 and 70%, respectively, was observed.
Effect of antifungal treatment on rat survival rate
Figure 1 shows the data for the therapeutic efficacy of caspofungin versus amphotericin B during the treatment period until day 11 when rats were persistently neutropenic (granulocyte counts <0.1 x 109/L) and the 10 day period after termination of treatment until day 21 when neutrophil numbers gradually rose.
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Untreated control rats all died between day 5 and day 10 after fungal inoculation. Administration of amphotericin B 1 mg/kg/day (AMB 1), significantly increased rat survival, up to 55% at the end of the treatment period (P < 0.0001). After termination of treatment, rat survival further decreased to 27% at day 21. The amphotericin B dosage could not be increased as the dose of 1 mg/kg is the MTD for amphotericin B in this animal model. Administration of caspofungin 1 mg/kg/day (CAS 1) had a similar therapeutic effect compared with AMB 1, with 60% rat survival at the end of the treatment period. However, in contrast to treatment with AMB 1, after termination of treatment with CAS 1 rat survival remained stable. The difference in rat survival rate after CAS 1 versus AMB 1 over the 21 day period was not significant (P = 0.24). The combination of AMB 1 and CAS 1 did not show an additive therapeutic effect compared with the drugs administered alone (P = 0.71 compared with CAS 1 and P = 0.10 compared with AMB 1). In view of the safety profile caspofungin could be administered at higher doses. Increase in dosage of caspofungin up to 2, 3 or 4 mg/kg/day resulted in an increased therapeutic activity, with 100% rat survival at the dose of 4 mg/kg/day (P = 0.02 compared with CAS 1).
Effect of antifungal treatment on fungal burden in rats
The GM concentrations in serum and tissues and the CE counts in tissues of surviving rats are presented in Figures 2
–4.
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Concentration GM in serum
As shown in Figure 2, in untreated control rats the mean log GM concentration in serum increased over time from undetectable at day 1 to 1.21 at day 6. All untreated control rats died before day 11. The mean log GM concentrations in serum at day 3 and day 6 of rats treated with AMB 1, CAS 1 or the combination AMB 1 + CAS 1 were not significantly different compared with the untreated controls. However in rats treated with CAS 4 the mean log GM concentration in serum was significantly decreased (P = 0.005) at day 6 compared with the untreated control rats and was undetectable in all rats at day 11 and day 21.
Concentration GM in left lung
As shown in Figure 3 in untreated control rats mean log GM concentration significantly increased over time from 2.18 ng/g at day 1 to 3.74 ng/g at day 6 (P = 0.002). From the start of treatment (16 h) in rats treated with AMB 1, CAS 1 or the combination AMB 1 + CAS 1 the mean log GM in the left lung significantly increased over time (P 
0.02). However, treatment with CAS 4 initially resulted in a significant increase in the mean log GM at day 3 and 6 (P = 0.002 and 0.006) followed by a gradual decrease at day 11 and 21 (P = 0.06 and 0.73). At day 11, treatment with CAS 4 produced a significant reduction of mean log GM compared with treatment with CAS 1 (P = 0.005) and the combination AMB 1 + CAS 1 (P = 0.0202), but not compared with treatment with AMB 1 (P = 0.1269). At day 21, treatment with CAS 4 resulted in a significant reduction in the mean log GM compared with all other treatment groups [AMB 1 (P = 0.0008), CAS 1 (P = 0.0017), combination AMB 1 + CAS 1 (P = 0.0212)].
CE counts in left lung
As shown in Figure 4, in untreated control rats the CE counts significantly increased over time from 5.67 CE/g at day 1 to 7.18 CE/g at day 6 (P = 0.002). In rats treated with AMB 1, CAS 1, CAS 4 or the combination AMB 1 + CAS 1 compared with the start of treatment (16 h) the CE counts of the left lung significantly increased at day 3, 6 and 11 for all treatment regimens (P 0.04) except for CAS 4, which resulted in a gradual decrease in CE counts in the left lung (P 
0.06). At day 11 in animals treated with CAS 4, the CE counts were significantly reduced compared with the CE counts in animals treated with AMB 1 (P = 0.0003), CAS 1 (P < 0.0001) and the combination AMB 1 + CAS 1 (P = 0.0001). Up to day 21 the CE counts decreased gradually in all treatment groups.
Pharmacokinetics of antifungal agents
The pharmacokinetic characteristics based on plasma concentrations after a single dose of amphotericin B or caspofungin to neutropenic rats are summarized in Table 2. Twenty-four hour AUC, Cmax and trough concentration were about four times lower in the CAS 1 group compared with the CAS 4 group and total clearance was similar in both groups which is indicative of linear pharmacokinetics. The CAS 4 and CAS 1 groups demonstrated 24 h plasma concentrations of 1.1 and 0.3 mg/L, respectively. Corresponding values for 24 h AUC were 91.8 and 20.1 mg·h/L. The concentration of amphotericin B in plasma 24 h after a single dose of 1 mg/kg was <0.25 mg/L. The 24 h AUC of AMB 1 was 12.2 mg·h/L.
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For filamentous fungi the predictive significance of in vitro antifungal susceptibility testing for in vivo antifungal efficacy remains unclear. Determination of MIC according to the CLSI guidelines11 particularly for agents that show a concentration-dependent gradual decrease in fungal growth is difficult and not accurate. Whereas amphotericin B shows a concentration-dependent sharp reduction in fungal growth, caspofungin shows a concentration-dependent gradual decrease in fungal growth. It is concluded that for caspofungin objectivity in visual reading of antifungal activity according to the CLSI guidelines11 and in microscopic assessment of abnormal hyphal growth21 is difficult to achieve. A quantitative assay to determine in vitro antifungal activity of caspofungin would be most helpful. The quantitative XTT assay measuring a decrease in viable fungal load in terms of mitochondrial dehydrogenase activity has recently been introduced.10. In the present study this assay was used to investigate the antifungal activity of caspofungin and amphotericin B. The concentrations of antifungal agent resulting in 50 and 90% inhibition of fungal growth, respectively, can be determined accurately using the quantitative XTT assay and can be used as an appropriate activity endpoint when comparing in vitro activity of antifungal agents of various classes. The present study shows that to obtain
50% inhibition of fungal growth, only a 4-fold difference in activity between caspofungin and amphotericin B was observed, whereas to obtain 90% inhibition of fungal growth as endpoint a 64-fold difference between both agents was seen. In the present study the therapeutic efficacy of caspofungin versus amphotericin B using different parameters was investigated in an inhalation model of IPA in neutropenic rats in relation to the pharmacokinetics of the agents. Amphotericin B at 1 mg/kg/day (MTD) and caspofungin at 1 mg/kg/day provided modest prolongation of survival. When caspofungin was administered at an increased dose of 4 mg/kg/day a significant improvement of rat survival was observed (100%). The therapeutic efficacy of caspofungin has also been investigated in other models of invasive aspergillosis induced by iv fungal inoculation in mice13 and guinea pigs,16 in a model of CNS aspergillosis in mice15 and in a model of pulmonary aspergillosis induced by intratracheal inoculation in rabbits.17 Similar to the present study Abruzzo observed an increased animal survival at increased dosages of caspofungin.13 Surprisingly, in the studies of Imai,15 Kirkpatrick16 and Petraitiene17 animal survival did not change with increasing dosages of caspofungin. Pharmacokinetic data which are important in the interpretation of results on therapeutic efficacy28 are not always available. The pharmacokinetic characteristics of caspofungin in rats observed in the present study were relatively similar to those previously reported by other investigators in rats,29,30 rabbits17 and in mice.18 In the present study 4 mg/kg caspofungin showed a 24 h AUC that is similar to the 24 h AUC achieved by 50 mg caspofungin dosage in man,31 which is the current maintenance dose in the clinical treatment of IPA. In addition, serum concentrations after a single dose of caspofungin 4 mg/kg to rats remained for 24 h above 1 µg/mL which was the target trough concentration in human pharmacokinetic studies.32 The 24 h AUC of AMB 1 was relatively similar compared with the 0.6 mg/kg dosage in men.33
In the study of Imai15 in mice and in the study of Kirkpatrick16 in guinea pigs pharmacokinetic data of caspofungin were not presented. In the study of Petraitiene17 in rabbits the 24 h AUC of caspofungin at the dose of 6 mg/kg/day was human pharmacokinetic equivalent. In that study a caspofungin dosage-dependent animal survival rate was not observed, in contrast to the present study. The discrepancy of the results of both studies cannot be explained.
In the present animal study, the combination of equal dosages of caspofungin and amphotericin B (1 mg/kg/day) did not show an additive effect. These data are in agreement with a study on combination therapy using caspofungin and Abelcet in mice.15 Arikan, however, supposed on the basis of in vitro observations that a combination of amphotericin B and caspofungin might be effective in infections due to Aspergillus spp.23 The caspofungin: amphotericin B dosage ratio in the combination investigated in the present study may not be optimal. In view of this, the benefit of combinations of caspofungin and amphotericin B at other dosages needs to be investigated in our animal model.
Besides the animal survival rate as overall parameter for therapeutic efficacy, the GM concentration in serum, the amount of A. fumigatus GM in infected lung tissue as well as the amount of A. fumigatus DNA (CE counts) in infected lung tissue were used to monitor the efficacy of treatment in the present study. The increased animal survival obtained after administration of caspofungin at the dose of 4 mg/kg/day was correlated with both decreased serum GM concentrations and decreased GM and CE counts in left lung tissue. It is assumed that the presence of GM in serum reflects active fungal multiplication in the infected left lung. It could be concluded that at the dose of 4 mg/kg/day caspofungin hyphal growth in the left lung was significantly inhibited. These data are in contrast with a study of pulmonary aspergillosis in caspofungin-treated neutropenic rabbits17 demonstrating increased serum GM levels, despite prolonged rabbit survival. In addition, histological examination in the rabbits suggests caspofungin dose-dependent hyphal damage. The discrepancy in results of both studies is difficult to explain. Nevertheless, the data of the present study are in agreement with the recent observation of Maertens, showing that trends in GM levels among 17 patients with invasive aspergillosis receiving caspofungin therapy, were correlated with both clinical and radiographic findings.4
The relatively high GM concentration and CE counts at day 21 in the treatment groups indicates that a substantial fungal burden is still present in the infected lung tissue in animals that are clinically cured. Whether this fungal burden represents viable A. fumigatus organisms is not known. Bowman observed in a model of invasive aspergillosis in mice treated with amphotericin B 0.5 mg/kg/day or caspofungin 1 mg/kg/day compared with untreated control animals a 4 log10 decrease in CE counts and in the same animals a 1 log10 decrease in the number of cfu counts.14 From this study it was concluded that the quantitative DNA assay is superior to traditional cfu determination. In the present study only at the CAS 4 dosage regimen a gradual decrease in both GM and CE counts in the infected left lung tissue was observed and both parameters were significantly reduced at the end of the study period compared with the other treatment groups including amphotericin B 1 mg/kg/day. In contrast to our data, Wiederhold observed in a model of pulmonary aspergillosis in neutropenic mice an increase in CE counts after treatment with caspofungin 4 mg/kg/day compared with caspofungin 1 mg/kg/day.18 In addition, 96 h animal survival was not different between these treatment schedules. The discrepancies between the data of Wiederhold and our data are difficult to explain. Possibly the relatively short 96 h study period plays a role. Pharmacokinetics of caspofungin in terms of clearance and 24 h AUC were relatively similar in both studies. In the present study, the decrease in CE counts with CAS 4 compared with CAS 1 was significantly correlated with both decreased GM concentrations and increased rat survival rate.
Summarizing, the present study shows that caspofungin administered at a dose of 4 mg/kg/day, which produces drug exposure comparable to the human situation, resulted in 100% survival in our animal model of IPA. The significant increase in animal survival correlated with a significant decrease in fungal burden in terms of A. fumigatus GM concentration in serum and amount of A. fumigatus GM and A. fumigatus DNA (CE counts) in left lung. A dosage-dependent efficacy of caspofungin was observed. Given this dosage-dependent efficacy and the excellent safety profile of caspofungin, it is worthwhile to investigate in a more advanced stage of aspergillosis in rats the therapeutic efficacy of caspofungin administered at further increased dosage. Such studies may contribute to optimization of dosage of caspofungin in patients. Investigation of the therapeutic efficacy of caspofungin in patients shows that dosing efficacy may be maximized.12
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None to declare.
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We thank Cameron Douglas and Joel Bowman for their valuable contributions in performing the quantitative DNA assays. This study was financially supported in part by Merck Research Laboratories, Rahway, NJ, USA.
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