Antifungal Activity and Pharmacokinetics of Posaconazole (SCH 56592) in Treatment and Prevention of Experimental Invasive Pulmonary Aspergillosis: Correlation with Galactomannan Antigenemia

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,0066-4804/01/$04.0010 DOI: 10.1128/AAC.45.3.857–869.2001

Mar. 2001, p. 857–869 Vol. 45, No. 3

Antifungal Activity and Pharmacokinetics of Posaconazole(SCH 56592) in Treatment and Prevention of Experimental

Invasive Pulmonary Aspergillosis: Correlation withGalactomannan Antigenemia

RUTA PETRAITIENE,1 VIDMANTAS PETRAITIS,1 ANDREAS H. GROLL,1 TIN SEIN,1

STEPHEN PISCITELLI,2 MYRNA CANDELARIO,1 AIDA FIELD-RIDLEY,1

NILO AVILA,3 JOHN BACHER,4 AND THOMAS J. WALSH1*

Immunocompromised Host Section, Pediatric Oncology Branch, National Cancer Institute,1 PharmacokineticsResearch Laboratory, Pharmacy Department,2 and Department of Radiology,3 Warren Grant Magnuson

Clinical Center and Surgery Service, Veterinary Resources Program, Office of Research Services,4

National Institutes of Health, Bethesda, Maryland

Received 19 June 2000/Returned for modification 22 October 2000/Accepted 29 December 2000

The antifungal efficacy, safety, and pharmacokinetics of posaconazole (SCH 56592) (POC) were investigatedin treatment and prophylaxis of primary pulmonary aspergillosis due to Aspergillus fumigatus in persistentlyneutropenic rabbits. Antifungal therapy consisted of POC at 2, 6, and 20 mg/kg of body weight per os;itraconazole (ITC) at 2, 6, and 20 mg/kg per os; or amphotericin B (AMB) at 1 mg/kg intravenously. Rabbitstreated with POC showed a significant improvement in survival and significant reductions in pulmonaryinfarct scores, total lung weights, numbers of pulmonary CFU per gram, numbers of computerized-tomogra-phy-monitored pulmonary lesions, and levels of galactomannan antigenemia. AMB and POC had comparabletherapeutic efficacies by all parameters. By comparison, animals treated with ITC had no significant changesin outcome variables in comparison to those of untreated controls (UC). Rabbits receiving prophylactic POCat all dosages showed a significant reduction in infarct scores, total lung weights, and organism clearance fromlung tissue in comparison to results for UC (P < 0.01). There was dosage-dependent microbiological clearanceof A. fumigatus from lung tissue in response to POC. Serum creatinine levels were greater (P < 0.01) inAMB-treated animals than in UC and POC- or ITC-treated rabbits. There was no elevation of serum hepatictransaminase levels in POC- or ITC-treated rabbits. The pharmacokinetics of POC and ITC in plasmademonstrated dose dependency after multiple dosing. The 2-, 6-, and 20-mg/kg dosages of POC maintainedplasma drug levels above the MICs for the entire 24-h dosing interval. In summary, POC at >6 mg/kg/day peros generated sustained concentrations in plasma of >1 mg/ml that were as effective in the treatment andprevention of invasive pulmonary aspergillosis as AMB at 1 mg/kg/day and more effective than cyclodextrinITC at >6 mg/kg/day per os in persistently neutropenic rabbits.

Invasive pulmonary aspergillosis is an important cause ofinfectious morbidity and mortality in immunocompromised pa-tients, particularly in those with severe and prolonged neutro-penia as a consequence of cytotoxic chemotherapy for thetreatment of cancer (3, 10, 18, 36). Conventional amphotericinB (AMB), which binds to ergosterol and disrupts membraneintegrity, remains the mainstay of therapy for serious fungalinfections; however, its clinical utility may be thwarted by dose-limiting nephrotoxicity (2, 15, 42). There clearly is a great needfor safer yet effective antifungal compounds against pulmonaryaspergillosis, particularly in patients with persistent neutrope-nia and those undergoing bone marrow transplantation (18, 36,48).

Posaconazole (SCH 56592) (POC), a new triazole antifungalcompound, has a potent and broad spectrum of antifungalactivity (24, 37, 43). POC is structurally similar to the broad-

spectrum triazole compound itraconazole (ITC) (17). Themechanism of action of the antifungal triazoles is throughinhibition of cytochrome P-450-dependent 14a-sterol demeth-ylase, which results in inhibition of ergosterol biosynthesis (6).

In vitro studies have demonstrated the potent antifungalactivity of POC against Candida spp. (Candida albicans, Can-dida dubliniensis, Candida tropicalis, Candida parapsilosis, andCandida krusei), as well as Cryptococcus neoformans (4, 14, 26,38, 39, 40). Additional studies have demonstrated that POC ismore active than ITC and fluconazole against clinical isolatesof filamentous fungi such as Aspergillus spp., Fusarium spp.,Rhizopus spp., and Pseudallescheria boydii (11, 30, 34). POC isalso active against dimorphic fungi such as Blastomyces derma-titidis (45) and Histoplasma capsulatum (8). POC has also beenevaluated in animal models of cryptococcal meningitis (20, 38),disseminated aspergillosis (16, 23, 35), disseminated fusariosis(27), coccidioidomycosis (28), histoplasmosis (8), blastomyco-sis (45), and pheohyphomycosis (1).

Little is known, however, about the activity of POC againstprimary pulmonary aspergillosis in persistently neutropenichosts, where this compound may have an important role. Wetherefore investigated the antifungal efficacy, safety, and phar-

* Corresponding author. Mailing address: ImmunocompromisedHost Section, Pediatric Oncology Branch, National Cancer Institute,National Institutes of Health, Building 10, Rm. 13N240, Center Dr.,Bethesda, MD 20892. Phone: (301) 402-0023. Fax: (301) 402-0575.E-mail: [email protected].

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macokinetics in plasma of POC in the treatment and prophy-laxis of primary pulmonary aspergillosis in persistently neutro-penic rabbits. We further investigated the potential correlationbetween a therapeutic response to POC and galactomannanantigenemia.

MATERIALS AND METHODS

Animals. Healthy female New Zealand White rabbits weighing 2.6 to 3.7 kg(Hazleton, Deutschland, Pa.) at the time of inoculation were used in all exper-iments. Rabbits were individually housed and maintained with water and stan-dard rabbit feed ad libitum according to National Institutes of Health guidelinesfor animal care and in fulfillment of the criteria of the American Association forAccreditation of Laboratory Animal Care (32). A total of 102 rabbits were usedfor all experiments. Vascular access was established in each rabbit by the surgicalplacement of a silastic tunneled central venous catheter (50). The silastic cath-eter permitted nontraumatic venous access for repeated blood sampling for studyof biochemical and hematological parameters, study of pharmacokinetics inplasma, study of serum galactomannan, and administration of parenteral agents.Serum samples were drawn, when possible, from all rabbits at the initiation ofimmunosuppression, during the course of pulmonary aspergillosis, and beforedeath. Rabbits were euthanized by intravenous (i.v.) administration of sodiumpentobarbital injection (65 mg [1 ml]/kg of body weight; The Butler Company,Columbus, Ohio) at the end of each experiment, 24 h after administration of thelast dose of the drug.

Organism and inoculation. Aspergillus fumigatus (National Institutes of Healthisolate 4215) obtained from a fatal case of pulmonary aspergillosis was used in allexperiments. The MIC, determined by proposed NCCLS methods (12, 31), ofPOC (Schering-Plough Research Institute, Kenilworth, N.J.) was 0.125 mg/ml,that of ITC (Janssen Pharmaceutica NV, Beerse, Belgium) was 0.5 mg/ml, andthat of AMB deoxycholate (Bristol-Myers Squibb Company, Princeton, N.J.) was1.0 mg/ml. The minimum fungicidal concentration (MFC) of POC was 0.125mg/ml, that of ITC was 0.5 mg/ml, and that of AMB deoxycholate was 1.0 mg/ml.

Pulmonary aspergillosis was established as previously described (13). For eachexperiment, the A. fumigatus inoculum was prepared from a frozen isolate(stored at 270°C) that was subcultured onto Sabouraud dextrose slants (BBL,Cockeysville, Md.). Those slants were incubated for 24 h at 37°C and then keptat room temperature for 5 days before use. Conidia were harvested under alaminar airflow hood with a solution of 10 ml of 0.025% Tween 20 (FisherScientific, Fair Lawn, N.J.) in 0.9% NaCl (Quality Biological, Inc., Gaithersburg,Md.), transferred to a 50-ml conical tube, washed, and counted with a hemacy-tometer. The concentration was adjusted in order to give each rabbit a prede-termined inoculum of 108 conidia of A. fumigatus in a volume of 250 to 350 ml.The concentrations of the inocula were confirmed by serial dilutions cultured onSabouraud glucose agar (SGA).

Inoculation was performed on day 2 of the experiments under general anes-thesia. Each rabbit was anesthetized with 0.8 to 1.0 ml of a 2:1 (vol/vol) mixtureof i.v. administered ketamine (100 mg/ml) obtained as Ketaset (Phoenix Scien-tific, Inc., St. Joseph, Mo.) and xylazine (20 mg/ml; Animal Health, AgricultureDivision, Bayer Corp. Shawnee Mission, Kans.) obtained as Rompun. Oncesatisfactory anesthesia was obtained, a Flagg O straight-blade laryngoscope(Welch Allyn Inc., Skaneateles Falls, N.Y.) was inserted in the oral cavity untilthe vocal cords were clearly visualized. The A. fumigatus inoculum was thenadministered intratracheally with a tuberculin syringe attached to a 5 1/4-inc.16-gauge Teflon catheter (Becton Dickinson Infusion Therapy Systems Inc.,Sandy, Utah).

Immunosuppression and maintenance of neutropenia. To simulate the con-ditions of persistent neutropenia, therapy with cytarabine (Ara-C) (Cytosar-U;The Upjohn Company, Kalamazoo, Mich.) was initiated i.v. 1 day before theendotracheal inoculation of the animals. Profound and persistent neutropenia(granulocyte count of ,100/ml) was achieved by an initial course of 525 mg ofAra-C per m2 for five consecutive days. A maintenance dose of 484 mg of Ara-Cper m2 was administered for 4 additional days on days 8, 9, 13, and 14 of theexperiment. Concomitant thrombocytopenia ranged from 30,000 to 50,000/ml.Methylprednisolone (Abbott Laboratories, North Chicago, Il.) at 5 mg/kg ofbody weight was administered on days 1 and 2 of the experiment to inhibitmacrophage activity against conidia in order to facilitate establishment of infec-tion.

Ceftazidime (75 mg/kg given i.v. twice daily; Glaxo, Inc., Research TrianglePark, N.C.), gentamicin (5 mg/kg given i.v. every other day; Elkins-Sinn, Inc.,Cherry Hill, N.J.), and vancomycin (15 mg/kg given i.v. daily); Abbott Labora-tories) were administered from day 4 of immunosuppression until study com-

pletion to prevent opportunistic bacterial infections during neutropenia. In orderto prevent antibioticassociated diarrhea due to Clostridium spiriforme, all rabbitscontinuously received 50 mg of vancomycin per liter of drinking water.

Total leukocyte counts and the percentages of granulocytes were monitoredtwice weekly with a Coulter (Miami, Fla.) counter and by peripheral bloodsmears and differential counts, respectively.

Antifungal compounds. POC was provided by Schering-Plough Research In-stitute as standard powder. Drug stock solution (30 mg/ml) was prepared bydissolving the antifungal powder in a solution of distilled water and Tween 80(Fisher Scientific, Fair Lawn, N.J.). ITC was purchased as Sporanox (JanssenPharmaceutica NV) in a 10-mg/ml hydroxypropyl-b-cyclodextrin oral solution.AMB was purchased as Fungizone, an i.v. suspension (Bristol-Myers SquibbCompany), and was resuspended for use in sterile water according to the man-ufacturer’s instructions.

Treatment regimens. Treatment study groups were either untreated controls(UC) or animals treated with POC, ITC, or AMB. POC was administered per osat dosages of 2 mg/kg/day (POC2), 6 mg/kg/day (POC6), and 20 mg/kg/day(POC20). ITC was administered per os at dosages of 2 mg/kg/day (ITC2), 6mg/kg/day (ITC6), and 20 mg/kg/day (ITC20) as a 10mg/ml oral solution. AMBwas administered i.v. at 1 mg/kg of body weight per day (0.1 ml every 10 s).Antifungal therapy was initiated 24 h after endotracheal inoculation. Antifungaltherapy was continued throughout the course of the experiments for a maximumof 12 days in surviving rabbits.

Prophylactic regimen. In order to study the efficacy of POC for prevention ofaspergillosis in persistently neutropenic hosts, we compared this azole to ITC andno drug in the persistently neutropenic rabbit model for prophylaxis againstpulmonary aspergillosis. The prophylaxis experiments used the same methods asdescribed above with the following exceptions. Rabbits received the same dos-ages, POC2, 26, or 220 or ITC2, 26, or 220 administered for 4 days beforeendotracheal inoculation. On the day of inoculation, POC and ITC were admin-istered in the morning and the endotracheal inoculum was administered approx-imately 4 h later. POC and ITC were then continued for a maximum of 12 moredays after inoculation. In order to simulate the low initial tissue burden of A.fumigatus in the setting of antifungal prophylaxis, the administered inoculum was5 3 107 conidia. All other methods, including outcome variables, were identicalfor both treatment and prophylaxis experiments.

Outcome variables. A panel of outcome variables was used to assess antifungalefficacy. These variables consisted of survival, pulmonary infarct score, lungweight, microbiologic clearance (CL) from lung tissue (in log CFU per gram) andfrom bronchoalveolar lavage (BAL) specimens (log CFU per milliliter), com-puterized tomography (CT), galactomannan index (GMI), and pathology. Pul-monary infarct score, lung weight, and CT scan are measures of organism-mediated pulmonary injury.

Survival. The survival time in days postinoculation was recorded for eachrabbit in each group. Surviving rabbits were euthanized by sodium pentobarbitalanesthesia on the 13th day postinoculation.

Pulmonary lesion scores. The entire heart-lung block was carefully resected atautopsy. The heart was then dissected away from the lungs, leaving the tracheo-bronchial tree and lungs intact. The lungs were weighed and inspected by at leasttwo observers who were blinded to the treatment group and recorded hemor-rhagic infarct lesions (if any) in each individual lobe. The numbers of positivelobes were added, and the mean value of all positive lobes was calculated foreach treatment group. Hemorrhagic infarcts were dark-red consolidated lesionsthat corresponded histologically to coagulative necrosis and intra-alveolar hem-orrhage.

BAL. BAL was performed on each lung preparation by the instillation andsubsequent withdrawal of 10 ml of sterile normal saline two times into theclamped trachea with a sterile 12-ml syringe. The lavage was then centrifuged for10 min at 1,500 3 g. The supernatant was discarded, leaving the pellet, which wasthen resuspended in 2 ml of sterile normal saline. A 0.1-ml sample of this fluidand 0.1 ml of a dilution (1021) of this fluid were cultured on 5% SGA plates.

Histopathology. Pulmonary lesions were excised and fixed in 10% neutralbuffered formalin. Paraffin-embedded tissue sections were stained with periodicacid-Schiff and Gomori methenamine silver stains. Tissues were microscopicallyexamined for pulmonary injury and structural changes in Aspergillus hyphae.

Fungal cultures. Lung tissue from each rabbit was sampled and cultured by astandard excision of tissue from each lobe. Each fragment was weighed individ-ually, placed in a sterile polyethylene bag (Tekmar Corp., Cincinnati, Ohio), andhomogenized with sterile saline for 30 s per tissue sample (Stomacher 80; Tek-mar) (47). Lung homogenate dilutions (1021 and 1022) were prepared in sterilesaline. Aliquots (100 ml) from homogenates and homogenate dilutions wereplated onto SGA plates and incubated at 37°C for the first 24 h and then at roomtemperature for another 24 h. The number of CFU of A. fumigatus were counted

858 PETRAITIENE ET AL. ANTIMICROB. AGENTS CHEMOTHER.

and recorded for each lobe, and the log CFU per gram was calculated. A findingof one colony of A. fumigatus was considered positive.

CT. Serial CT of the lungs was performed during all experiments in order tomonitor the effects of antifungal therapy on organism-mediated pulmonary in-jury during the course of infection. Briefly, rabbits were sedated with ketamineand xylazine and then placed prone, head first, on the scanning couch. CT wasperformed with an ultrafast electron beam CT scanner (model C-100XL; Ima-tron, Oyster Point, Calif.), as previously described (49). Using the high-resolu-tion, table-incremented, volume acquisition mode, 3-mm-thick ultrafast CT scanswere performed every 4 s. A small scan circle and a 9-cm-diameter reconstruc-tion circle with a matrix of 512 by 512 were used, which resulted in a pixel sizeof less than 1 mm. Scan parameters were 130 kV and 630 mA, and scan durationwas 100 ms. In virtually all cases, 30 slices were sufficient to scan the entire thoraxof the rabbit. Images were photographed using lung windows with a level of 2600Hounsfield units (HU) and a width of 1,800 HU. The radiologic features ofinvasive pulmonary aspergillosis observed in this experimental system are similarto those reported for persistently neutropenic patients (7, 21, 25).

Galactomannan assay. Blood was collected every other day from each rabbitfor determination of serum galactomannan concentrations. Serum galactoman-nan concentrations we determined by the Platelia Aspergillus (Genetic Systems/Sanofi Diagnostic Pasteur, Redmond, Wash.) one-stage immunoenzymatic sand-

wich microplate assay method. The assay used the rat monoclonal antibodyEBA-2, which is directed against Aspergillus galactomannan. The monoclonalantibody is used to sensitize the wells of the microplate and to bind the antigen.Peroxidase-linked monoclonal rat antibody is used as the detector antibody.

Serum samples were heat treated in the presence of EDTA in order todissociate the immune complexes and to precipitate serum proteins. The treatedserum samples and conjugate were added to the wells coated with the monoclo-nal antibody and then incubated for 90 min at 37°C. A monoclonal antibody-galactomannan-monoclonal antibody–peroxidase complex was formed in thepresence of Aspergillus antigen. The strips were washed to remove any unboundmaterial. Next, we added the substrate solution, which reacted with the com-plexes bound to the well to form a blue color. The enzymatic reaction wasstopped by the addition of stopping solution (1.5 N sulfuric acid), which changedthe blue color to yellow. The optical absorbance values of specimens and controlswere determined with a microplate spectrophotometer equipped with 450- and620-nm-band-pass filters (Multiscan MMC/340; Titertek, Huntsville, Ala.).

Enzyme immunoassay data were expressed as serum GMIs plotted over time.The GMI for each test serum is equal to the optical density (OD) of the sampledivided by the threshold OD in serum. Sera with a GMI less than 1 wereconsidered to be negative. Sera with a GMI greater than 1.5 were considered tobe initially positive. Sera with GMIs between 1 and 1.5 were considered to be

FIG. 1. Comparative rates of survival of persistently neutropenic rabbits in treatment groups receiving POC, ITC, or AMB versus in the UCgroup. To gauge survival over 12 days (x axis) dosage groups for each antifungal compound were combined (P value determined by Mantel-Haenzsel chi-square test). The response of persistently neutropenic rabbits with primary pulmonary aspergillosis to antifungal therapy was alsomeasured by pulmonary infarct score, total lung weight, and pulmonary tissue concentration of residual organisms (log CFU/per gram) in UC (n 515); rabbits treated with POC2 (n 5 6), POC6 (n 5 6), and POC20 (n 5 6); rabbits treated with ITC2 (n 5 6), ITC6 (n 5 6), and ITC20 (n 55); and rabbits treated with AMB at 1 mg/kg/day (n 5 6). Values are given as means 6 SEM (p, P , 0.05; §, P , 0.01; ¶, P , 0.001) in comparisonto values for UC using an ANOVA test with Bonferroni’s correction for multiple comparisons.

VOL. 45, 2001 EFFICACY AND PHARMACOKINETICS OF POSACONAZOLE 859

indeterminate. Serial serum galactomannan levels were plotted over time as afunction of antifungal compound and time of initiation of study drug (treatmentversus prophylaxis).

Toxicity studies. A sample of blood was collected from each rabbit every otherday, starting from the first day after inoculation and continuing throughouttreatment. Plasma samples were stored in Sarsted (Newton, N.C.) tubes at270°C until all samples were processed simultaneously. Chemical determina-tions of potassium, aspartate aminotransferase (AST), alanine aminotransferase(ALT), serum creatinine, serum urea nitrogen, and total bilirubin concentrationswere performed on the penultimate sample drawn from each rabbit.

Pharmacokinetic experiments. The pharmacokinetics of POC and ITC inplasma were investigated in three infected animals per each dosage group. Timepoints for minimal plasma sampling were determined on the basis of full plasmaconcentration profiles from the same species (5, 22, 33) with the aid of the AdaptII computer program (9). Plasma sampling was performed 6 days after inocula-tion. Blood samples were drawn immediately prior to oral dosing and then at 1,4, 8, and 24 h postdosing. All samples were immediately centrifuged, and plasmawas stored at 280°C until assayed.

Concentrations of POC were determined after solid-phase extraction by liquidchromatography-tandem mass spectrometry at the Schering-Plough ResearchInstitute. The analytical procedure involved dilution of the samples in controlledplasma prior to extraction. The quantifiable range of the assay was 4 to 1,000ng/ml. Accuracies and intra- and interday variability (precision) were within615% and 620% at the lower limit of quantitation.

Concentrations of ITC were determined after protein precipitation by thereversed-phase high-performance liquid chromatographic method using saper-conazole as the internal standard (19). Ten-point standard curves were linear,with R2 values of $0.999. Accuracies were within 612%, and intra- and interdayvariability (precision) was ,6%. The lower limit of quantitation of the assay was25 ng/ml in plasma.

Pharmacokinetic parameters for POC and ITC were determined using com-partmental analysis. Experimental concentration in plasma data were fit to aone-compartment open model with first-order input, oral lag time, and first-order elimination from the central compartment using iterative weighted least-squares regression and the Adapt II computer program. Weighting was by max-imum a posteriori probability, and model selection was guided by Akaike’s

FIG. 2. Expression of galactomannan antigenemia in persistently neutropenic rabbits with pulmonary aspergillosis in the following treatmentgroups: UC, AMB-treated controls, and rabbits treated with POC (upper panel) or with ITC (lower panel). The differences between the antigenconcentration-time curves of UC versus those of rabbits treated with POC6 and POC20 are significant (P 5 0.05, ANOVA). Differences betweenantigen concentration-time curves of UC versus rabbits treated with ITC2, ITC6, and ITC20 are also significant (P , 0.001, ANOVA).


Information Criterion (51). The model fit the data with mean coefficients ofdetermination (r2) 6 standard errors of the mean (SEM) of 0.647 6 0.05 (POC)and 0.720 6 0.04 (ITC), respectively. Fitted parameters included total CL,volume of distribution (V), and elimination half-life (t1/2).

Statistical analysis. Comparisons between groups were performed by analysisof variance (ANOVA) with Bonferroni’s correction for multiple comparisons orby the Mann-Whitney U test, as appropriate. Kaplan-Meier survival plots wereanalyzed by the Mantel-Haenzsel chi-square test. All P values were two-sided,and a P value of ,0.05 was considered to be statistically significant. Values wereexpressed as means 6 SEM.

RESULTS

Antifungal therapy. There was a significant improvement inthe survival of rabbits treated with POC and AMB comparedto that of UC. Through the entire study, 13 of 18 rabbitstreated with POC, 5 of 6 rabbits (83%) treated with AMB, 1 of

18 rabbits (5.6%) treated with ITC, and none of the 15 UCsurvived (Fig. 1). Of the rabbits treated with POC2, only 1 of6 (16.7%) survived. By comparison, 100% (6 of 6) of therabbits treated with POC6 and 83% (5 of 6) of the rabbits inthe POC20 treatment group survived.

There also was a significant reduction in organism-mediatedtissue injury, as measured by pulmonary infarct score and totallung weight, in rabbits treated with POC and AMB. Rabbitstreated with POC6, POC20, and AMB had similarly significantlower mean infarct scores (P , 0.001, P , 0.001, P , 0.05,respectively); however, no differences in infarct scores werenoted in UC and rabbits treated with POC2, ITC2, ITC6, andITC20 (Fig. 1). The mean lung weights in rabbits treated withPOC20, POC6, and AMB were significantly reduced in com-parison to those of UC (P , 0.001); however, no differences in

FIG. 3. Expression of galactomannan antigenemia in persistently neutropenic rabbits with pulmonary aspergillosis in the prophylaxis group.Results for UC and rabbits treated with POC or with ITC are shown. The antigen concentration-time curves of rabbits treated with POC2, POC6,and POC20 are significantly different from those of UC (P , 0.001, ANOVA). The antigen concentration-time curve of ITC20-treated rabbits, butnot that of ITC2- or ITC6-treated rabbit is significantly different from that of UC (P , 0.05, ANOVA).


lung weight were observed between untreated animals andanimals treated with POC2, ITC2, ITC6, and ITC20 (Fig. 1).

There was a significant quantitative reduction in the growthof A. fumigatus in lung tissues from rabbits treated with POCand AMB in comparison to lung tissue from UC. Rabbitstreated with POC6, POC20 (P , 0.001), and AMB (P , 0.05)showed significant reductions in the number of pulmonaryCFU per gram (Fig. 1). In contrast, no difference in levels of A.fumigatus growth was observed between POC2- or ITC-treatedrabbits and UC.

There was a higher frequency of BAL cultures negative forA. fumigatus in rabbits treated with POC in all dosage groupsthan in untreated rabbits (16 of 18 [89%] versus 6 of 15 [40%]rabbits, respectively; P , 0.01). There also was a trend towardmore BAL cultures negative for A. fumigatus in ITC-treatedrabbits than in UC (13 of 17 [76%] versus 6 of 15 [40%] rabbits,respectively; P 5 0.07).

We investigated the expression of galactomannan in the seraof rabbits with pulmonary aspergillosis as a surrogate maker oftherapeutic response (Fig. 2 and 3). Expression of galactoman-nan was studied in rabbits receiving treatment and in rabbitsreceiving prophylaxis. The following groups were studied: UCand AMB-, POC2-, POC6-, POC20-, ITC2-, ITC6-, andITC20- treated animals. The GMI was positive ($ 1.5) in UC1 day after inoculation. Serial serum samples from UC andPOC2-treated rabbits showed progressive galactomannan an-tigenemia, correlating with progression of invasive pulmonaryaspergillosis. By comparison, serum GMI of rabbits treatedwith POC6 and POC20 demonstrated a significant decline

during therapy and correlated with low pulmonary log CFUper gram in those groups (P , 0.001, ANOVA). Serial serumGMI values of rabbits treated with AMB were negativethroughout treatment. Serum GMI remained negative afterday 6 in POC6- and POC20- treated rabbits. Serum GMI waspositive in ITC2 and ITC6 dosage groups, which also corre-lated with progression of invasive pulmonary aspergillosis andelevated tissue concentrations of A. fumigatus. Rabbits treatedwith ITC20 demonstrated a negative GMI. The responses ofgalactomannan antigenemia in rabbits treated with POC6,POC20, and ITC20 were comparable to that of rabbits treatedwith AMB.

Consistent with the reduction in organism-mediated pulmo-nary injury, ultrafast CT scans demonstrated resolution of pul-monary infiltrates in rabbits treated with POC (Fig. 4). Duringthe first 6 days of treatment, there was an increase in pulmo-nary infiltrates. Following day 6 of treatment, there was asubstantial reduction of infiltrates in rabbits receiving antifun-gal therapy. ITC-treated rabbits demonstrated a similar trendof resolution of pulmonary infiltrates during antifungal ther-apy.

Histopathological features of aspergillosis were studied inthe lungs of rabbits in all treatment groups. There was dosage-dependent damage of hyphal structures and clearance of theorganism from the lung tissue in animals treated with POC.Each panel in Fig. 5 demonstrates a representative section ofhyphal morphology in the POC dosage group. Organisms fromUC rabbits demonstrated typical dichotomously branchingseptate hyphae of A. fumigatus (Fig. 5A). Organisms in Fig. 5B

FIG. 4. Pulmonary infiltrate scores determined by image analysis of serial ultrafast CT scans of UC, POC-treated rabbits, and ITC-treatedrabbits. Pulmonary infiltrates increased during the first 6 days in UC and treated rabbits. The pulmonary infiltrate curve ends on day 6 due tomortality in the UC. Pulmonary infiltrates declined following day 6 in both the POC and ITC treatment groups. The ITC curve ends on day 8 dueto mortality.


from lung tissue of the rabbit treated with POC2 had short-ened hyphae. POC6- and POC20-treated rabbits (Fig. 5C andD) seldom revealed hyphal elements. The pulmonary his-topathological features of aspergillosis in POC6- and POC20-treated rabbits were similar to those observed in AMB-treated

rabbits. Damaged hyphal elements were observed at all dos-ages of ITC.

Antifungal prophylaxis. Prophylaxis was started 4 days be-fore endotracheal inoculation and continued throughout theexperiment. Significantly greater survival was achieved with

FIG. 5. Effects of POC, ITC, and AMB on hyphal structures and microbiological CL of A. fumigatus in rabbits with pulmonary aspergillosis.(A to D) Progressive reduction of hyphal elements in a representative section in the lungs from each dosage group. (A) UC; (B) POC2; (C) POC6;(D) POC20; (E) ITC20; (F) AMB at 1 mg/kg.


rabbits treated with POC than with UC and animals treatedwith ITC (P , 0.03) (Fig. 6). There was also a significantreduction in organism-mediated tissue injury in rabbits treatedwith POC, as measured by the mean pulmonary infarct scoreand mean lung weight, in comparison to that in UC. Rabbitstreated with POC6 and POC20 had no infarct lesions in thelungs, and animals treated with POC2 and ITC20 showed asignificant reduction in infarct score (P , 0.01). Rabbitstreated with POC at all dosages and ITC20 had significantlylower mean lung weights than those of UC (P , 0.01). Rabbitsreceiving prophylactic POC2, POC6, and POC20 had signifi-cant organism CL from the lung tissue in comparison to UC(P , 0.01). However, there was no significant CL from lungtissue in rabbits treated with ITC.

There was a higher frequency of negative BAL cultures forA. fumigatus in rabbits treated with POC in all prophylaxisgroups than in UC rabbits (17 of 18 [94%] versus 5 of 10 [50%]rabbits, respectively; P 5 0.01). However, there was no signif-

icant difference in BAL cultures between rabbits receivingprophylactic ITC and UC (13 of 18 [72%] versus 5 of 10 [50%]rabbits, respectively; P 5 0.41).

FIG. 6. Comparative rates of survival of persistently neutropenic rabbits receiving antifungal prophylaxis with POC or ITC versus that of UC.To gauge survival over 12 days (x axis) dosage groups for each antifungal compound were combined (P value determined by Mantel-Haenzselchi-square test). The response of persistently neutropenic rabbits with primary pulmonary aspergillosis to antifungal therapy was measured bypulmonary infarct score, total lung weight, and pulmonary tissue concentration of residual organisms (log CFU per gram) in UC (n 5 10); in rabbitstreated with POC2 (n 5 6), POC6 (n 5 6), and POC20 (n 5 6); and in rabbits treated with ITC2 (n 5 6), ITC6 (n 5 6), and ITC20 (n 5 6). Valuesare given as means 6 SEM (p, P , 0.05; §, P , 0.01; ¶, P , 0.001) in comparison to values for UC using an ANOVA test with Bonferroni’scorrection for multiple comparisons.

TABLE 1. Effects of POC, ITC, and AMB on concentrationsof serum creatinine, serum urea nitrogen, serum ALT, and

serum LAST in persistently neutropenic rabbitswith pulmonary aspergillosisa

Treatmentgroup (n)

Serumcreatinine

(mg/dl)

Serum ureanitrogen(mg/dl)

Serum ALT(U/liter)

Serum AST(U/liter)

UC (9) 0.68 6 0.05 21.7 6 2.3 33.2 6 4.8 36.8 6 10.8POC (6) 0.93 6 0.11 21.8 6 7.1 21.7 6 5.4 10.7 6 2.4ITC (6) 0.77 6 0.06 21.8 6 3.1 37.3 6 5.0 22.3 6 10.8AMB (6) 2.97 6 0.55** 99.5 6 14.3* 7.3 6 0.8 9.3 6 4.4

a All values are expressed as means 6 SEM. *, P , 0.05 (Kruskal-Wallisnonparametric ANOVA with Dunn’s correction for multiple comparisons); **,P , 0.001 (ANOVA with Bonferroni’s correction for multiple comparisons).


Serum GMI was positive in UC and ITC2- and ITC6-treatedrabbits by day 3 after inoculation, which correlated with in-creased tissue burden of A. fumigatus. By comparison, serumGMI was significantly reduced in POC2, POC6, POC20, andITC20 dosage groups (P , 0.001, ANOVA). These findingscorrelated with organism CL from the lungs expressed by logCFU per gram and dose-proportional drug concentrations inserum.

Safety. Rabbits treated with AMB had significant increasesin their mean levels of creatinine and urea nitrogen in serumcompared to those of POC- or ITC-treated rabbits or UC.There was no significant elevation of the concentrations ofhepatic transaminases in the sera of rabbits treated with POCor ITC. There were no differences in serum potassium andbilirubin concentrations for any of the treatment groups (Table1).

Pharmacokinetics of POC and ITC in plasma. Concentra-tion profiles of POC and ITC in the plasma of infected animalsreceiving treatment and prophylaxis regimens are depicted inFig. 7; the corresponding pharmacokinetic parameters are pre-sented in Tables 2 and 3, respectively.

Over the investigated dosage range, both compounds exhib-ited dose-dependent pharmacokinetics with a statistically sig-nificant decrease in CL and a significant increase in the elim-ination t1/2 with increasing dosages after multiple dosing over10 days (Table 3). Consistent with a dose-dependent disposi-tion of both drugs, there was a trend toward increases in thedose-normalized area under the concentration-time curvefrom 0 to 24 h (AUC0–24) with increasing dosages. Mean peaklevels of POC in plasma exceeded the corresponding MIC forthe test organism at all dosages and ranged from 170 to 5,176ng/ml in POC-treated rabbits. Mean peak levels of ITC in

FIG. 7. Concentration profiles of POC (A) and ITC (B) in plasma after multiple dosing over 6 days (treatment) and concentration profiles ofPOC (C) and ITC (D) in plasma after multiple dosing over 10 days (prophylaxis). F, 2 mg/kg/day; ■, 6 mg/kg/day; ‚, 20 mg/kg/day.


plasma exceeded the corresponding MIC at all dosages withthe exception of the 2-mg/kg dosage group and ranged from127 to 3,564 ng/ml (Tables 2 and 3). With the exception of thePOC2 dosage group and ITC2 dosage group, mean concentra-tions in plasma stayed above the MICs for the tested isolatethroughout most of the dosing interval. Both compounds ex-hibited a relatively large V, which is indicative of high proteinbinding and/or extensive distribution into tissues.

DISCUSSION

POC demonstrated potent dosage-dependent in vivo anti-fungal activity against A. fumigatus in persistently neutropenicrabbits with primary invasive pulmonary aspergillosis. The an-tifungal effects of orally administered POC6 were comparableto those of i.v. administered AMB at 1 mg/kg. The antifungalresponse was observed across all outcome parameters, includ-ing microbiologic burden (measured as log CFU per gram oflung tissue, serum galactomannan antigenemia, and histology),as well as organism-mediated pulmonary injury (measured aspulmonary hemorrhage score, lung weight, and CT scan). Thisantifungal efficacy of POC was superior to that of orally ad-

ministered cyclodextrin ITC at equal dosages. The antifungaleffect of POC was achieved without any detectable hepatotox-icity. ITC was well absorbed and achieved levels in plasmasimilar to those of POC6 and POC20. Concentrations of POC6and POC20 in plasma exceeded the MIC for A. fumigatusthroughout the 24-h dosing interval.

There was a significant reduction of microbiologic burden asmeasured by pulmonary log CFU per gram of A. fumigatus.Consistent with this antifungal effect was a parallel reductionof organisms observed histologically in tissue. Additionally,there appeared to be considerable disruption of fungal cell wallmorphology of residual organisms, suggesting azole-mediateddamage to residual hyphae.

The serum galactomannan levels also paralleled the micro-biologic decline in pulmonary tissue burden of A. fumigatus asa surrogate marker for a therapeutic response. The expressionof galactomannan in the sera of untreated neutropenic controlrabbits with pulmonary aspergillosis demonstrates patternssimilar to those observed in neutropenic patients initially di-agnosed with pulmonary aspergillosis (29, 41, 44, 46). As de-clining serum galactomannan levels reflected decreased micro-biologic tissue burden of A. fumigatus and a more favorable

TABLE 2. Pharmacokinetic parameters of POC (SCH 56592) and ITC in plasma after multiple dosing over 6 days (treatment)a

Drug dose(mg/kg QD) Cmax (ng/ml) Tmax (h) Cmin (ng/ml) AUC0–24 (ng z h/ml) V (liter/kg) CL (liter/kg z h) t1/2 (h)

POC2 183 6 58b 5.00 6 1.00c 82 6 13b 3211 6 657b 5.64 6 3.00d 0.476 6 0.240d 9.03 6 4.50d

POC6 940 6 142b 18.00 6 3.83c 454 6 54b 20,719 6 2,104b 3.45 6 0.47d 0.305 6 0.028d 7.76 6 0.35d

POC20 2,412 6 602b 18.37 6 3.37c 1,731 6 461b 56,153 6 12,633b 6.93 6 0.56d 0.558 6 0.055d 8.74 6 0.54d

ITC2 127 6 0.00e 4.00 6 0.00f 6 6 0.00g 2450 6 0.00g 15.3 6 0.00f 0.829 6 0.00f 12.79 6 0.00g

ITC6 711 6 234e 5.33 6 1.33f 229 6 60g 12,937 6 2,379g 9.79 6 1.65f 0.507 6 0.105f 13.69 6 1.67g

ITC20 3,564 6 0.195e 5.33 6 1.33f 2,338 6 0.446g 71,830 6 8,152g 9.17 6 0.90f 0.285 6 0.031f 22.72 6 2.82g

a Values are the means 6 SEM of results for three rabbits except for the ITC2 cohort (only one rabbit survived until the sampling). QD, once a day; Tmax, time tomaximum concentration of drug in serum; Cmin, minimum concentration of drug in serum.

b P # 0.005.c P # 0.05.d Not significant by ANOVA. P 5 0.058 by ANOVA for AUC per dose versus dose (linearity plot).e P # 0.001.f Not significant by ANOVA. P 5 0.1043 (not significant) by t test for AUC per dose versus dose (linearity plot). ANOVA comparing all three cohorts was not feasible

due to only one value in the ITC2 cohort.g P # 0.05 for the comparison of the ITC6 and ITC20 dosage level by t test (ANOVA comparing all three cohorts was not feasible due to only one value in the ITC2

cohort).

TABLE 3. Pharmacokinetic parameters of POC (SCH 56592) and ITC in plasma after multiple dosing over 10 days (prophylaxis)a

Drug dose(mg/kg QD) Cmax (ng/ml) Tmax (h) Cmin (ng/ml) AUC0–24 (ng z h/ml) V (liter/kg) CL (liter/kg z h) t1/2 (h)

POC2 170 6 44b 4.0 6 0.00c 73 6 5b 2990 6 537b 5.36 6 3.10c 0.719 6 0.132d 6.38 6 4.56c

POC6 1,528 6 322b 4.03 6 2.03c 990 6 145b 40,466 6 12,562b 3.57 6 1.12c 0.226 6 0.051d 7.86 6 1.43c

POC20 5,176 6 788b 5.30 6 2.67c 3360 6 563b 107,500 6 14,752b 2.89 6 0.92c 0.182 6 0.025d 10.75 6 1.48c

ITC2 314 6 263e 4.33 6 2.03 36 6 19e 2017 6 370e 15.88 6 1.30f 1.075 6 0.206f 10.73 6 1.48g

ITC6 992 6 030e 2.00 6 1.00 491 6 86e 18,650 6 2,660e 8.51 6 0.67f 0.341 6 0.053f 18.13 6 2.96g

ITC20 2,802 6 259e 5.33 6 1.33 2,061 6 332e 58,560 6 3,290e 11.30 6 0.84f 0.348 6 0.019f 22.73 6 2.47g

a Values are the means 6 SEM of results with three rabbits each. QD, once a day; Tmax, time to maximum concentration of drug in serum; Cmin, minimumconcentration of drug in serum.

b P , 0.001.c Not significant by ANOVA. P 5 0.0977 by ANOVA for AUC per dose versus dose (linearity plot).d P , 0.005.e P # 0.001.f P # 0.01.g P , 0.05 by ANOVA for comparison among all three cohorts; P , 0.01 by ANOVA for AUC per dose versus dose (linearity plot) among all three cohorts;

P 5 0.5965 for the comparison of the ITC6 and ITC20 dosage levels by t test.


experimental therapeutic response, serial GMI determinationswarrant further study as a surrogate marker of clinical antifun-gal efficacy.

The histopathological features of rabbits treated with POCdemonstrate progressive dose-dependent reduction of hyphalelements in lung tissue. In addition to quantitative reduction,there are marked structural changes in hyphal morphology.For example, hyphal structures in rabbits treated with POC6reveal marked distortion of fungal elements that include trun-cated and rounded hyphal fragments, as well as distendedchlamydospore-like remnants. These features are highly atyp-ical for A. fumigatus and may lead to misdiagnosis in a clinicalsetting in patients undergoing lung biopsy while receiving an-tifungal prophylaxis or therapy. Small sample sizes of biopsiesmay also miss foci of these distorted hyphal elements.

As the microbiologic tissue burden of A. fumigatus declines,pulmonary injury should also decrease. Indeed, pulmonary in-jury was significantly reduced at dosages of $6 mg/kg/day. Thereduction in organism-mediated pulmonary injury, evidencedby reduced total lung weight, mean pulmonary infarct score,and resolution of pulmonary infiltrates on CT scans, correlatedwith significant improvement in survival in comparison to thatof UC. This survival was comparable to that achieved withAMB.

The potent antifungal activity of oral POC against invasivepulmonary aspergillosis in persistently neutropenic rabbits wasdistinctive. One usually would not consider that an orally ad-ministered antifungal compound would attain this level of po-tent activity in a profoundly and persistently neutropenic host.That POC demonstrates fungicidal properties against A. fu-migatus at relatively low MICs of 0.125 mg/ml that were ex-ceeded in all dosage cohorts may explain in part this level ofactivity.

As an additional treatment control, oral ITC was investi-gated as the cyclodextrin solution. Cyclodextrin solution wasselected in order to optimize oral bioavailability. Despite ad-ministration of the oral cyclodextrin solution, levels of ITC inplasma above the MIC were not reliably achieved at the2-mg/kg dosage. When the ITC6 dosage was administered,concentrations in plasma were above the MIC throughout thedosing interval. However, despite these sustained levels, theITC6 dosage was less effective than the same dosage of POC.

This disparity in antifungal efficacy between POC and ITCmay be explained in part by differences in the potencies ofcompounds with similar molecular weights. The MICs of POCfor A. fumigatus have been reported as being relatively lowerthan those of ITC (12, 34). As the plasma concentration-timecurves and AUCs of POC and cyclodextrin ITC were similar,differences in efficacy between these antifungal triazoles couldnot be attributed to differences in bioavailability. This level ofactivity may be understood in terms of sustained levels inplasma being achieved above a relatively low MIC for A. fu-migatus. That the POC MIC and MFC were four times lowerthan those of ITC may contribute to the greater in vivo activityobserved in POC-treated rabbits. These lower MICs may beparticularly important in tissue sites of aspergillosis, wherepulmonary infarcts reduce the access of plasma antifungalcompound to the organism. Assuming levels of penetrationinto such infarcted sites to be similar for both triazoles, the

compound with lower MICs and MFCs will likely demonstrategreater in vivo antifungal activity.

The MICs and MFCs demonstrate a consistent fourfolddifference in in vitro potencies. Yet, the differences observed inour in vivo experiments suggest more potent activity than isreflected by the MICs and MFCs. This greater disparity be-tween the in vitro and in vivo potencies of POC was alsoobserved by Oakley et al. in a transiently neutropenic murinemodel of disseminated aspergillosis (35). Among the possibleexplanations for this difference are tissue penetration, rates ofmicrobicidal activity, and pharmacodynamics. The pharmaco-dynamics of POC and ITC against A. fumigatus are nonlinear(5; A. H. Groll, D. Mickiene, R. Petraitiene, V. Petraitis, T.Sein, J. Roach, K. Roth, S. C. Piscitelli, and T. J. Walsh, Abstr.40th Intersci. Conf. Antimicrob. Agents Chemother., p. 385,abstr. 1675, 2000). Perhaps the differences in antifungal activitybetween POC and ITC may be greater than a fourfold differ-ence in vivo as the result of the nonlinear in vivo pharmaco-dynamics. There are no apparent differences in levels of tissuepenetration of these two azoles reported at this point. Time-kill assays may prove to be helpful in understanding the dif-ferences in rates of kill of A. fumigatus.

Pharmacokinetic features of POC revealed that the maxi-mum concentration of the drug in serum (Cmax) greatly ex-ceeds that of the MIC at dosages of 6 and 20 mg/kg/day.Moreover, the relatively long t1/2 in plasma of 7 to 10 h alsomaintains existing plasma POC levels well above the MIC forA. fumigatus. Whether peak concentrations in plasma or expo-sure over time is the more critical pharmacodynamic parame-ter remains to be further investigated. Nevertheless, features ofboth properties were observed in these experiments and sug-gested a favorable pharmacokinetic profile of POC in the treat-ment of invasive pulmonary aspergillosis. Based upon thesefindings, as well as the MIC and MFC for the organism, asustained level of approximately 1,000 ng/ml is considered tobe the minimal highly active concentration in plasma.

Thus, POC6 per os generates sustained concentrations inplasma of $1 mg/ml that were as effective in treatment andprevention of invasive pulmonary aspergillosis as AMB at 1mg/kg/dag and more effective than cyclodextrin ITC6 per os inpersistently neutropenic rabbits. Given its potent antifungalactivity, excellent safety profile, and favorable pharmacokineticparameters, POC offers the potential for use as a prophylacticagent in the prevention of invasive pulmonary aspergillosis.These in vivo findings provide a foundation for understandingthe antifungal activity, safety, and pharmacokinetics of POC inpersistently neutropenic hosts and form a foundation for de-veloping clinical trials for the prevention and treatment ofinvasive pulmonary aspergillosis.

ACKNOWLEDGMENTS

We thank the staff of the Laboratory Animal Science Branch of theNational Cancer Institute and the staff of the Surgery Service of theOffice of Research Service for their excellent laboratory animal care.We also thank Jeremy Roach and Kenneth Roth for determining levelsof POC in plasma, Diana Mickiene for determining levels of ITC inplasma, Joanne Peter for determining MICs, and the staff of theDepartment of Radiology for CT imaging studies.

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