dosage-dependent antifungal efficacy of v-echinocandin (ly303366) against experimental...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,0066-4804/01/$04.0010 DOI: 10.1128/AAC.45.2.471–479.2001

Feb. 2001, p. 471–479 Vol. 45, No. 2

Dosage-Dependent Antifungal Efficacy of V-Echinocandin(LY303366) against Experimental Fluconazole-Resistant

Oropharyngeal and Esophageal CandidiasisVIDMANTAS PETRAITIS,1 RUTA PETRAITIENE,1 ANDREAS H. GROLL,1 TIN SEIN,1

ROBERT L. SCHAUFELE,1 CARON A. LYMAN,1 ANDREA FRANCESCONI,1 JOHN BACHER,2

STEPHEN C. PISCITELLI,3 AND THOMAS J. WALSH1*

Immunocompromised Host Section, Pediatric Oncology Branch, National Cancer Institute,1 Surgery Service, VeterinaryResources Program, Office of Research Services,2 and Pharmacokinetics Research Laboratory, Pharmacy Department,

Warren Grant Magnuson Clinical Center,3 National Institutes of Health, Bethesda, Maryland

Received 23 March 2000/Returned for modification 30 August 2000/Accepted 9 November 2000

V-echinocandin (VER-002; LY303366) is a semisynthetic derivative of echinocandin B and a potent inhibitorof fungal (1, 3)-b-D-glucan synthase. We studied the antifungal efficacy, the concentrations in saliva and tissue,and the safety of VER-002 at escalating dosages against experimental oropharyngeal and esophageal candi-diasis caused by fluconazole-resistant Candida albicans in immunocompromised rabbits. Study groups con-sisted of untreated controls, animals treated with VER-002 at 1, 2.5, and 5 mg/kg of body weight/day intrave-nously (i.v.), animals treated with fluconazole at 2 mg/kg/day i.v., or animals treated with amphotericin B at0.3 mg/kg/day. VER-002-treated animals showed a significant dosage-dependent clearance of C. albicans fromthe tongue, oropharynx, esophagus, stomach, and duodenum in comparison to that for untreated controls.VER-002 also was superior to amphotericin B and fluconazole in clearing the organism from all sites studied.These in vivo findings are consistent with the results of in vitro time-kill assays, which demonstrated thatVER-002 has concentration-dependent fungicidal activity. Esophageal tissue VER-002 concentrations weredosage proportional and exceeded the MIC at all dosages. Echinocandin concentrations in saliva were greaterthan or equal to the MICs at all dosages. There was no elevation of serum hepatic transaminase, alkalinephosphatase, bilirubin, potassium, or creatinine levels in VER-002-treated rabbits. In summary, the echino-candin VER-002 was well tolerated, penetrated the esophagus and salivary glands, and demonstrated dosage-dependent antifungal activity against fluconazole-resistant esophageal candidiasis in immunocompromisedrabbits.

Esophageal candidiasis is one of the most common oppor-tunistic fungal infections in immunocompromised patients, in-cluding human immunodeficiency virus (HIV)-positive pa-tients and those who are immunosuppressed as a result ofunderlying diseases or medications (2, 35). A number of agentshave been used to treat esophageal candidiasis, including nys-tatin, miconazole, ketoconazole, fluconazole, itraconazole, andamphotericin B. Fluconazole is frequently selected for sys-temic therapy because it is well tolerated and has excellent oralbioavailability. During the past several years, however, therehave been increasing reports of fluconazole-resistant oropha-ryngeal and esophageal candidiasis (OPEC) (27). The emer-gence of fluconazole-resistant OPEC has heightened the needfor development of new antifungal compounds with novel tar-gets.

The echinocandins are semisynthetic lipopeptides with po-tent and broad-spectrum antifungal activity which act by inhib-iting the synthesis of (1,3)-b-D-glucan, leading to cell wall dam-age and ultimately cell death (6, 7, 10, 16, 17). This novel modeof action and potent antifungal activity in vitro have led to the

design of several new echinocandin compounds for potentialclinical development.

V-echinocandin (VER-002; LY303366) is a semisyntheticechinocandin B derivative (9; I. Rajman, K. Desante, B.Hatcher, J. Hemingway, R. Lachno, S. Brooks, and M. Turik,Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother,abstr. F-74, p. 158, 1997) which demonstrates potent and non-cross-resistant antifungal activity against Candida albicans,Candida tropicalis, Candida glabrata, and other non-C. albicansspecies (8, 13, 14, 15, 24, 26, 29). Little is known, however,about the activity of VER-002 is treatment for fluconazole-resistant OPEC. We therefore investigated the concentrationsin the esophageus and saliva the safety, and the antifungalefficacy of VER-002 in an immunosuppressed rabbit model offluconazole-resistant OPEC.

MATERIALS AND METHODS

Organisms and MICs. Two isolates (isolates NIH 105 and NIH 126) offluconazole-resistant C. albicans were used for all experiments. Each isolate wasobtained from an HIV-infected child who had fluconazole-resistant OPEC andwho was monitored at the Pediatric Oncology Branch of the National CancerInstitute. MICs were determined by National Committee for Clinical LaboratoryStandards reference methods as described previously (23). The MICs of flucon-azole (Roerig-Pfizer, New York, N.Y.) and VER-002 (Eli Lilly & Company,Indianapolis, Ind.) were determined in morpholinepropanesulfonic acid (MOPS)-buffered RPMI 1640 (BioWhittaker, Walkersville, Md.), and the MICs of am-photericin B (Bristol-Myers Squibb Company, Princeton, N.J.), purchased asFungizone, were measured in antibiotic medium 3 (AM3; National Institutes of

* 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].

471

Health Media Department, Bethesda, Md.). The MIC was defined as the con-centration that resulted in complete optical clearance of fungal growth. TheMICs for the isolates studied in these experiments are shown in Table 1.

Time-kill assay. In order to characterize the in vitro pharmacodynamics andpotential fungicidal activities of VER-002 and amphotericin B, time-kill assayswere performed against C. albicans (NIH 105 and NIH 126). Three concentra-tions of VER-002 and amphotericin B (0.01, 0.1, and 0.25 mg/ml) were studied.These concentrations span the range of MICs of the two compounds. Theinoculum for the time-kill assay was prepared by growing the isolate for 48 h at37°C on Sabouraud glucose agar (SGA), inoculating colonies into a starter brothof 50 ml of Sabouraud glucose broth, and incubating the colonies in the broth for2 h in a shaking water bath at 37°C. One milliliter of this suspension wastransferred into 50 ml of fresh AM3 broth in each of four 250-ml Erienmeyerflasks, and the flasks were incubated at 37°C for 16 h in a shaking water bath inorder to generate logarithmic phase growth. The suspension was centrifuged,washed three times, adjusted for concentration with a hemacytometer, and in-oculated into 250-ml Erlenmeyer flasks containing 50 ml of AM3 broth alone(untreated growth control) and AM3 plus antifungal compound. The final base-line (time zero) concentration of approximately 3.0 3 105 CFU/ml was confirmedby quantitative culture. These flasks were incubated simultaneously in a shakingwater bath at 37°C. The growth suspensions were sampled at time zero and at 2,4, 6, and 24 h, and 100-ml aliquots were plated in dilutions of 1022, 1023, and1024 onto one SGA plate/per aliquot. The colonies were counted after 48 h ofincubation at 37°C, and the calculated number of CFU/per milliliter was plottedfor each time point. The lower limit of quantitation for the time-kill assay was 10CFU/ml. Time-kill assays for all concentrations were performed in triplicate.

Animals. Sixty-three female New Zealand White rabbits (Hazleton, Deutsch-land, Pa.) weighing 2.5 to 3.5 kg at the time of inoculation were used in allexperiments. Rabbits were individually housed and were maintained according toNational Institutes of Health guidelines for animal care and in fulfillment ofAmerican Association for Accreditation of Laboratory Animal Care standards(5). Vascular access was established in each rabbit by the surgical placement ofa tunneled silastic central venous catheter (30). The silastic catheter permittednontraumatic venous access for repeated blood sampling for study of biochem-ical safety parameters and pharmacokinetics in plasma, as well as administrationof parenteral agents. Rabbits were euthanatized at the end of each experiment byinjection of an intravenous (i.v.) bolus of pentobarbital (65 mg [1 ml]/kg of bodyweight; Veterinary Laboratories, Inc., Lenexa, Kans.).

Oral inoculation. Organisms from stock isolates were stored in skim milk at270°C. Cells from these suspensions were streaked onto SGA plates, and theplates were incubated at 37°C for 24 h and then maintained at 4°C. Five discretecolonies were then inoculated into 50 ml of Emmon’s modified Sabouraud broth(pH 7) in a 250-ml Erlenmeyer flask, and the flask was incubated at 37°C for 16 hon a shaking incubator at 80 oscillations per minute. The Candida suspensionwas centrifuged at 3,000 3 g for 10 min and was washed three times with sterilenormal saline (Quality Biological, Inc., Gaithersburg, Md.). Candida blasto-conidia were counted with a hemacytometer and diluted to the desired concen-tration. An inoculum of 2 3 108 blastoconidia was suspended in 1.5 ml of normalsaline, and the suspension was administered per os from day 1 through day 7 ofthe experiment. There were no interisolate differences in the capacity to establishoropharyngeal or esophageal infection.

Immunosuppression and antibiotics. Methylprednisolone (Abbott Laborato-ries, North Chicago, Ill.), at 5 mg/kg of body weight was administered from day1 to day 14 of the experiment for suppression of mucosal cellular immunity.Gentamicin (Elkins-Sinn, Inc., Cherry Hill, N.J.) at 40 mg/liter and vancomycin(Abbott Laboratories) at 50 mg/liter were administered in the drinking waterstarting on day 1 and continuing through day 14 in order to reduce mucosalbacterial colonization competitive with C. albicans.

Antifungal compounds and treatment groups. Therapy was initiated on day 8of the experiment following per os inoculation and was continued throughout the

course of the experiments for 7 days. Eli Lilly & Company provided VER-002 asa 10-mg/ml solution for parenteral administration. VER-002 was administeredi.v. at dosages of 1 mg/kg/day (VER1) (n 5 9), 2.5 mg/kg/day (VER2.5) (n 5 9),and 5 mg/kg/day (VER5) (n 5 9). The initial solution of 10 mg/ml was dilutedwith sterile 0.9% NaCl to a concentration of 1 mg/ml for the 1-mg/kg dosage andto a concentration of 2 mg/ml for the 2.5-mg/kg dosage. VER5 was administeredin an initial solution of 10 mg/ml. Amphotericin B at 0.3 mg/kg/day (n 5 9) wasadministered as a slow i.v. infusion (0.1 ml every 10) in order to assess thelow-dose regimen of amphotericin B which is used in the management of esoph-ageal candidiasis (22). Fluconazole was administered at 1 mg/kg (n 5 9) twicedaily i.v. Administration of compounds was initiated 24 h after the last inocula-tion on day 7 of the experiment. The untreated control group consisted ofsaline-treated infected rabbits that did not receive antifungal treatment (n 5 18).

Outcome variables. The rabbit model of fluconazole-resistant OPEC providesa strong in vitro-in vivo correlation of the therapeutic response to fluconazole(31). This system permits quantitative assessment of therapeutic response alongclinically relevant sites of the upper alimentary tract: tongue, oropharynx, esoph-agus (proximal, middle, and distal), stomach, and duodenum. The tongue, oro-pharynx, and esophagus were resected en bloc postmortem. Segments of duo-denum and stomach were resected separately. Antifungal efficacy was assessed bymicrobiologic clearance of C. albicans from tissue. Sections of the tongue, oro-pharynx, esophagus (proximal, middle, and distal), stomach, and duodenum werecultured by excision of a representative region. Each fragment was weighed andthen homogenized in sterile reinforced polyethylene bags (Tekmar Corp., Cin-cinnati, Ohio) (34). Each tissue homogenate was serially diluted 100-fold from 10to 1024 in sterile 0.9% saline. A 0.1-ml quantity of undiluted homogenate and ofeach dilution was separately plated onto Emmon’s modified SGA containingchloramphenicol and gentamicin. Culture plates were incubated at 37°C for 24 h,after which the members of CFU were counted and the number of CFU pergram of tissue was calculated for each organ. Previous studies with serial dilu-tions demonstrated that the method was sensitive to detection of $10 CFU/g.The culture-negative plates were counted as having 0 CFU/g. Data were graphedas the mean 6 standard error of the mean (SEM) log10 (CFU per gram).

Pharmacokinetic and pharmacodynamic studies. Drug concentrations inplasma, saliva, and esophageal tissue were determined for all treated animals,and the relationships between drug levels and residual fungal burden wereinvestigated for VER-002-treated rabbits. Plasma and saliva were sampled fromeach rabbit 2 h after administration of the seventh dose and after induction ofsalivation with 0.5 mg of pilocarpine (catalog no. 45H05181i Sigma), and esoph-ageal tissue was obtained 24 h after administration of the seventh dose atautopsy. All samples were stored at 280°C until assay.

The concentrations of VER-002 were determined after solid-phase extractionby reversed-phase high-performance liquid chromatography (HPLC) as de-scribed previously (25). Esophageal tissue was homogenized prior to extractionin ice-cold normal saline (1:10 [wt/wt]) with using a tissue homogenizer (Tiz-zumizer; Tekmar Corp.), and drug concentrations in tissue were calculated for1 g of tissue. External standards and quality controls were prepared separatelyfor all matrices by spiking pooled normal rabbit serum, saliva, and esophagealhomogenate with VER-002 and the internal standard. Eight-point standardcurves were linear, with R2 values of $0.993. Accuracies were within 65%, andintra-and interday variabilities (precisions) were ,7%. The lower limit of quan-titation of the assay was 20 ng/ml in plasma and saliva, respectively, and 200 ng/gin tissue.

Concentrations of fluconazole were determined after solid-phase extraction byreversed-phase HPLC (12) and detection at 260 nm. Esophageal tissue washomogenized prior to extraction in ice-cold normal saline (1:2 [wt/wt]), and drugconcentrations were calculated for 1 g of tissue. External standards and qualitycontrols were similarly prepared by spiking pooled normal rabbit serum, saliva,and esophageal homogenate with fluconazole. Eight-point standard curves werelinear, with R2 values of $0.997. Accuracies were within 67%, and intra- andinterday variabilities (precisions) were ,8%. The lower limit of quantitation ofthe assay was 0.5 mg/ml in plasma and saliva, respectively, and 1.5 mg/g in tissue.

Concentrations of amphotericin B were determined after liquid extraction byreversed-phase HPLC (3) and detection at 382 nm. Esophageal tissue was ho-mogenized and extracted in ice-cold methanol (1:3 [wt/wt]), and drug concen-trations were calculated for 1 g of tissue. External standards and quality controlswere similarly prepared by spiking pooled normal rabbit serum, saliva, andesophageal homogenate with amphotericin B. Eight-point standard curves werelinear, with R2 values of $0.999. Accuracies were within 66.5%, and intra- andinterday variabilities (precisions) were ,12%. The lower limit of quantitation ofthe assay was 40 ng/ml in plasma and saliva, respectively, and 160 ng/g in tissue.

The relationships between concentration data and the residual fungal burdenin tissue were determined by pharmacodynamic modeling. Experimental con-

TABLE 1. MICs for isolates from rabbits with experimentalfluconazole-resistant OPEC

C. albicans isolateMIC (mg/ml)

Fluconazolea VER-002a Amphotericin Bb

NIH 105 .64 0.015 0.05NIH 126 .64 0.004 0.05

a MOPS-buffered RPMI 1640.b AM3.

472 PETRAITIS ET AL. ANTIMICROB. AGENTS CHEMOTHER.

centration-effect data were fit to an inhibitory effect sigmoidal maximum effect(Emax) model by iterative uniformly weighted nonlinear least-squares regressionwith the WinNonlin computer program (Scientific Consulting, Lexington, Ky.).

Histopathology. Representative sections of the tongue were prepared forhistological studies. Tissue specimens were excised and fixed in 10% neutralbuffered formalin, embedded in paraffin, sectioned, and then stained with peri-odic acid-Schiff and Gomori methenamine silver stains.

Toxicity studies. Chemical determinations of serum potassium, aspartyl ami-notransaminase, alanine aminotransaminase, serum creatinine, alkaline phos-phatase, and total bilirubin levels were performed with the penultimate sampledrawn from each rabbit.

Statistical analysis. Comparisons between groups were performed by theKruskal-Wallis nonparametric analysis of variance test with Dunn’s correction

for multiple comparisons. All P values were two sided, and a P value of ,0.05was considered significant.

RESULTS

Time-kill assay. Time-kill curves demonstrated the concen-tration-dependent fungicidal activities of VER-002 and am-photericin B (Fig. 1). At 4 to 6 h, $99.9% killing was achievedwith VER-002 and amphotericin B at 0.1 and 0.25 mg/ml. Thekilling was sustained, and regrowth was not seen at 24 h atVER-002 concentrations of 0.1 and 0.25 mg/ml.

FIG. 1. Time-kill assay of VER-002 and amphotericin B against C. albicans in antibiotic medium 3. Concentrations of VER-002 (VER) andamphotericin B (AmB) at 0.01, 0.1, and 0.25 mg/ml were studied in relation to a growth control. Data plotted are the mean 6 SEM from threeseparate experiments for each growth curve, including the control and amphotericin B at 0.25 mg/ml. As the SEM was small for several time points,the error bars may not always be apparent in the time-kill curves.

VOL. 45, 2001 ANTIFUNGAL EFFICACY OF V-ECHINOCANDIN 473

Antifungal therapy. VER-002 showed a significant dosage-dependent antifungal effect as treatment for fluconazole-resis-tant OPEC. Rabbits treated with VER1, VER2.5, and VER5showed significant dosage-dependent organism clearance fromthe tongue in comparison to the organism clearance for theuntreated controls (P , 0.01) (Fig. 2). There was also a sig-nificant reduction of C. albicans the tongues of rabbits treatedwith amphotericin B in comparison to that for untreated con-trols (P , 0.001), while rabbits treated with fluconazole showedno reduction in the number of CFU per gram in this tissue.

There was a significant reduction in C. albicans growth in theoropharynx from rabbits treated with VER1, VER2.5, andVER5 in comparison to the reductions for untreated controlsand fluconazole-treated rabbits (P , 0.01) (Fig. 2). Rabbitstreated with VER2.5 and VER5 mg/kg had significantly lowerorganism burdens in the oropharynx than rabbits treated withamphotericin B (P , 0.01).

Rabbits treated with VER-002 also showed a significant

reduction of the concentration of C. albicans in the esophagus.There was complete clearance of organisms from all threesegments of the esophagus in rabbits treated with VER5 (Fig.2). In comparison to untreated controls, rabbits treated withVER2.5 showed a significant reduction in organism growthfrom the proximal middle (P , 0.001) and distal (P , 0.001)segments of the esophagus. There was no significant reductionin tissue burden in the esophageal tissue of rabbits treated withVER1, amphotericin B, and fluconazole in comparison to thatfor untreated control animals.

There also was significant organism clearance from thestomach and duodenum in rabbits treated with VER-002. Rab-bits treated with VER2.5 and VER5 had no detectable C.albicans in the stomach or duodenum (Fig. 2). Rabbits receiv-ing VER1 showed significant reductions in organism growthfrom the duodenum in comparison to that for untreated con-trols (P , 0.05), while rabbits treated with amphotericin B andfluconazole showed no reductions.

FIG. 2. Response of experimental OPEC caused by fluconazole-resistant C. albicans in immunocompromised rabbits to antifungal therapymeasured by mean log (CFU per gram) concentration of organism in the tongue, oropharynx, esophagus, stomach, and duodenum in untreatedcontrols (control) (n 5 18), rabbits treated with VER1 (n 5 9), VER2.5 (n 5 9), and VER5 (n 5 9), rabbits treated with amphotericin B at 0.3mg/kg/day (AmB) (n 5 9), and rabbits treated with fluconazole at 1 mg/kg/day (FLU) (n 5 9). Values are given as mean 6 SEM. p, P , 0.05; †,P , 0.01; and ¶, P , 0.001, in comparison to untreated controls by the Kruskal-Wallis nonparametric analysis of variance test with Dunn’scorrection for multiple comparisons.


Concentrations of antifungal compounds in plasma, saliva,and esophageal tissue. The concentrations of VER-002, flu-conazole, and amphotericin B in esophageal tissue, saliva, andplasma at steady-state are depicted in Table 2. VER-002 ex-hibited dosage-proportional increases in concentrations at allthree sites. Mean trough concentrations in esophageal tissueranged from 0.564 to 2.402 mg/g and exceeded the MICs forthe infecting isolates by more than 30-fold. Potentially thera-peutic VER-002 concentrations, which were greater than orequal to the MICs, were also found in saliva. The mean levelsin plasma 2 h after dosing ranged from 1.653 to 7.426 mg/ml.Fluconazole achieved concentrations in esophageal tissue andplasma that were subtherapeutic (below the MICs). The con-centrations of amphotericin B in esophageal tissue were belowthe MICs for the C. albicans isolates. In comparison to VER-002, both fluconazole and amphotericin B were undetectablein saliva.

Concentration-effect relationships. The pharmacodynamicrelationships between the concentrations of VER-002 versusthe residual fungal burden in tissue are depicted in Table 3 andFig. 3. The pharmacodynamic model was consistent with thelinear pharmacokinetic and the linear dosage-response rela-tionship of VER-002 in the infection model. The mean coef-ficient of determination between observed and estimated val-ues (r) was 0.884 (range, 0.8397 to 0.9302) (Table 3) for therelationships between drug concentrations in plasma, saliva,and esophageal tissue versus residual fungal burden in tissue inthe oropharynx and esophagus. Relationships between concen-trations in plasma and saliva and residual fungal burden in

stomach and duodenum were not as strong (r 5 0.5371 to0.7324; data not shown).

Histopathology. These histologic studies demonstrated aclear dose-dependent correlation between microscopic reduc-tion of organisms in tissues and quantitative reduction of fun-gal burden in tissue (log CFU per gram). There was completehistologic eradication of the organisms with VER5. As shownin Fig. 4, there also were dose-dependent morphologicalchanges from hyphae and pseudohyphae in untreated controls(Fig. 4A) to an appearance of yeast-like structures in VER2.5-treated rabbits (Fig. 4B). Amphotericin B- and fluconazole-treated rabbits had no significant changes (Fig. 4C and D) incell wall morphology.

Safety. Rabbits treated with VER-002, fluconazole, and am-photericin B and untreated controls had no detectable increaseor decrease in serum creatinine, serum potassium, aspartylamino transaminase alanine aminotransaminase alkaline phos-phatase, or total bilirubin levels (Table 4).

DISCUSSION

The semisynthetic echinocandin VER-002 demonstratedsignificant in vitro and in vivo activity against fluconazole-resistant OPEC due to C. albicans. The in vivo efficacy ofVER-002 was dosage dependent and correlated with the invitro fungicidal concentration-dependent effect of VER-002against fluconazole-resistant C. albicans. The in vitro concen-tration-dependent fungicidal effects of VER-002 were similarto the fungicidal concentration-dependent effects of ampho-

TABLE 2. Concentrations of VER-002, fluconazole, and amphotericin B in esophageal tissue, saliva, and plasma after repeat dosing over 7days

Treatment groupaConcn (mg/g or mg/ml)b

Esophagus Saliva Plasma

VER1 0.564 6 0.149 0.017 6 0.007 1.653 6 0.365VER2.5 1.276 6 0.273 0.024 6 0.011 3.623 6 0.882VER5 2.402 6 0.401 0.046 6 0.022 7.426 6 1.616Fluconazole 3.577 6 0.787 Not detectable 1.357 6 0.129Amphotericin B 0.020 6 0.007 Not detectable 0.995 6 0.056

a There were nine animals in each group.b All values are expressed as means 6 SEMs. P was ,0.001 by analysis of variance across the three VER-002 dosage groups. The lower limits of quantitation in plasma

and saliva were 0.020 mg/ml for VER-002, 0.5 mg/ml for fluconazole, and 0.040 mg/ml for deoxycholate amphotericin B; the lower of limits of quantitation in tissueswere 0.200 for VER-002, 1.5 mg/g for fluconazole, and 0.160 mg/g for amphotericin B.

TABLE 3. Pharmacodynamic relationships between concentrations of VER-002 versus residual fungal burden and goodness of fit of themodel equationa

Pharmacodynamicrelationship

Emax[log (CFU/g)]

EC50(mg/ml or mg/g)b Gamma

Correlation (C) betweenobserved and

predicted values

C in plasma/log (CFU/g) for oropharynx 5.22 6 0.17 2.69 6 0.22 2.11 6 0.33 0.9297C in plasma/log (CFU/g) for esophagus 4.87 6 0.24 2.20 6 0.28 2.08 6 0.52 0.8781C in esophagus/log (CFU/g) for esophagus 4.84 6 0.19 0.81 6 0.07 2.80 6 0.55 0.9302C in saliva/log (CFU/g) for oropharynx 5.20 6 0.25 0.01 6 0.00 1.62 6 0.44 0.8464C in saliva/log (CFU/g) for esophagus 4.89 6 0.28 0.01 6 0.00 1.83 6 0.60 0.8397

a All values are expressed as the mean 6 standard deviation. The equation for the model follows the Emax function, where E 5 Emax z {(1) 2 [CGamma/(CGamma 1EC50

Gamma)]}, where E is the effect, Emax is the maximum effect, C is the concentration, EC50y is the 50% effective concentration, and Gamma is the slope in the centralpart of the curve.

b Units are micrograms per milliliter for plasma and saliva; units are micrograms per gram for esophageal tissue.


tericin B. Fluconazole had no effect in this rabbit model offluconazole-resistant OPEC.

The in vitro time-kill antifungal activities of VER-002 ob-served in this study are consistent with those observed by Ernstand colleagues (8) and Klepser and colleagues (14). However,the fungicidal activity of the echinocandin observed in thisstudy was more potent than that observed against most of theisolates studied by those investigators. The use of AM3 in ourassays may account for this difference. Emst et al. (8) andKlepser et al. (14) indicate that RPMI 1640 is probably not arepresentative medium for assessment of the in vitro antifungalactivities of echinocandins. On the basis of their subsequentstudies, those investigators further recommend that AM3 maybe a more suitable medium.

The model of OPEC described here reflects the profoundimpairment of mucosal immunity encountered in severely im-munocompromised hosts, such as HIV-infected patients, cor-ticosteroid-treated patients, and recipients of organ or bonemarrow transplants. High-dose corticosteroids induce a pro-found impairment of mucosal immunity in rabbits (28). Spe-cifically, the mucosa-associated lymphoid tissue in corticoste-roid-treated rabbits reveals a profound depletion of lymphoidfollicles and severe loss of B lymphocytes. The dome epithe-lium of mucosa-associated lymphoid tissue is also compro-mised, as evidenced by apoptosis of M cells and loss of intra-epithelial lymphocytes. These immune impairments compromisesurface immunoglobulin production and immunoregulation ofmucosal host defenses against opportunistic pathogens. Suchmucosal immunosuppression results in a burden of C. albicansin tissue that is higher than that in healthy animals. The re-fractoriness of this infection to fluconazole resembles that ofazole-resistant candidiasis in humans (31).

The microbiological outcome variables in this model permitquantitative analysis of dosage-dependent antifungal responsesin several sites of the upper alimentary track. Evaluation ofdifferent sites in the upper alimentary track is important, as theantifungal effect of different concentrations of antifungalagents in saliva and tissues may be site dependent. For exam-ple, one may observe patients with fluconazole-resistant OPECwho achieve a response in the oral cavity but who continue tohave persistent esophageal disease.

The echinocandin VER-002 demonstrated concentration-dependent fungicidal activity in time-kill assays. As our pre-liminary findings demonstrated more potent activities of echi-nocandin and amphotericin B and a wider distribution of MICsfor C. albicans in AM3 than in RPMI 1640, the MIC assays andtime-kill assays were performed in AM3 throughout the study.The concentration-dependent effects of the echinocandin wereconsistent with those observed in tissues of the upper alimen-tary track. Both echinocandin and amphotericin B demon-strated similar profiles on time-kill curves, reflecting the con-centration-dependent fungicidal properties of these compounds.Each concentration (0.01, 0.10, and 0.25 mg/ml) of VER-002and amphotericin B with antifungal activity demonstrated asimilar level of microbicidal activity. The concentration of 0.25mg of amphotericin B per ml caused a more rapid decline inthe number of organisms in the initial part of incubation.However, no significant differences in killing between VER-002 and amphotericin B were detected at 24 h. Although themechanisms of action of amphotericin B and echinocandinsare clearly different, with the former causing direct disruptionof cell membrane integrity and the latter causing inhibition ofcell wall biosynthesis, both mechanisms lead to a lethal effectagainst C. albicans. These concentration-dependent fungicidalproperties of the echinocandin molecule demonstrated in thein vitro time-kill assays were reflected in the dosage-dependentand concentration-dependent fungicidal activities in thetongue, oropharynx, and esophagus in experimental flucon-azole-resistant OPEC.

VER5 was most active in clearing C. albicans from theesophagus, stomach, and duodenum. The effects in the tongueand oropharynx also were greatest for VER5, with which a$104-fold reduction in the level of C. albicans was achieved.The different tissue sites (the tongue, oropharynx, and esoph-

FIG. 3. Concentration-effect relationship for VER-002 in the treat-ment of experimental OPEC. The relationship follows an inhibitoryeffect sigmoidal Emax model with no effect at a concentration of 0 andcomplete inhibition at a concentration extrapolated to infinity. De-picted are the fitted curve and the scatter of the observed values: (A)Residual fungal burden in esophageal tissue versus near-peak concen-trations of VER-002 in plasma; (B) residual fungal burden in esoph-ageal tissue versus concentrations of VER-002 in esophageal tissue24 h after dosing. The coefficients of determination between observedand predicted data (r) were 0.8781 and 0.9302, respectively. Note thesteep negative slope over a small concentration range, indicating thepresence of a narrow threshold concentration for antifungal efficacy.


agus) appeared to have similar patterns of response to VER-002, amphotericin B, and fluconazole. The gastric and duode-nal tissues demonstrated complete clearance of C. albicanswith VER2.5 in comparison to the clearance seen for the othertissues, which were cleared with VER5. This greater responsemay be related to the lower burden of C. albicans at these sitescompared to those in the tongue, oropharynx, and esophagus.

As little is known about the pharmacokinetics and pharma-codynamics of amphotericin B, fluconazole, and the echino-candin as treatment for esophageal candidiasis, we investigatedthe concentrations of these three compounds in plasma, tissue,

and saliva. The concentrations of echinocandin demonstrateda dosage-proportional increase in the esophagus, saliva, andplasma. The concentrations in esophageal tissue exceeded theMIC for C. albicans by at least 37-fold when VER1 was usedand by 160-fold when VER5 was used. By comparison, theconcentrations of the echinocandin in saliva exceeded the MICfor C. albicans by as little as one- to threefold. These datasuggest that the relatively large molecule of the echinocandinis not excreted well into saliva, perhaps due to barriers oftransport from the capillaries through the basement membraneand into the epithelial cells of the salivary glands. Restrictionsto interepithelial penetration by tight junctions may furtherlimit transport of the echinocandin molecule into saliva. Bycomparison, the echinocandin molecule appears to penetraterelatively well into the capillary bed of esophageal tissue,where the barriers to penetration present in the salivary glandsare not applicable. The linear dosage-proportional plasma andtissue pharmacokinetic properties of VER-002 contrast withthose of its predecessor, cilofungin (LY121019) (11). The ear-lier echinocandin demonstrated nonlinear saturation plasmaand tissue pharmacokinetics (19). The tissue cilofungin con-centrations correlated with the antifungal response in a patternsimilar to that observed for VER-002 (33).

FIG. 4. Antifungal effect on microscopic morphology of the cell structure of fluconazole-resistant C. albicans in glossal tissue of rabbits treatedwith VER2.5, amphotericin B at 0.3 mg/kg/day, and fluconazole at 2 mg/kg/day. (A) Untreated controls; (B) VER2.5; (C) amphotericin B; (D)fluconazole. Gomori methenamine silver stain magnification, 3428 was used. (original magnification, 3630). (A and B) Transition frompredominantly hyphae and pseudohyphae in untreated controls to predominance of yeast-like structures in rabbits treated with VER2.5. Rabbitstreated with amphotericin B and fluconazole had no significant changes (C and D) in cell wall morphology.

TABLE 4. Effects of VER-002, amphotericin B, and fluconazole onserum creatinine and potassium levels

Treatmentgroup

Serum creatinine level(mg/d)a

Serum potassium level(mmol/liter)a

Control (n 5 15) 0.43 6 0.04 4.60 6 0.15VER-002 (n 5 27) 0.47 6 0.02 4.40 6 0.09Amphotericin B (n 5 9) 0.50 6 0.10 4.13 6 0.07Fluconazole (n 5 9) 0.42 6 0.04 4.20 6 0.20

a All values are expressed as means 6 SEMs.b Mean serum creatinine and potasium levels for all dosage groups.


Rabbits were treated with amphotericin B (0.3 mg/kg/day) inthe well-known low-dose regimen, which has been advocatedfor management of esophageal candidiasis (22). The dosagesof amphotericin B used in rabbits have pharmacokinetic pro-files that are similar to those for the dosages used in humans.Comparison of the data for amphotericin B between rabbitsand humans for the 1.0-mg/kg dosage level (1, 18) revealsvalues that are very similar to those for humans for the maxi-mum concentration of drug in plasma (Cmax) and the areaunder the concentration-time curve from 0 to 24 h (AUC0–24)(4.7 versus 2.9 mg/ml and 31 versus 36 mg z h/ml, respectively),suggesting no fundamental differences in the level of exposureto amphotericin B between rabbits and humans. Further cor-roboration of these observations is a detailed analysis of con-ventional amphotericin B and amphotericin B lipid complex inrabbits (32). The pharmacokinetics of amphotericin B in thethree-compartment model in rabbits are similar to those inhumans across a broad dosage range. Thus, the results ob-tained for the key pharmacokinetic parameters (Cmax andAUC0–24) indicate that the effect of the dosage of 0.3 mg/kg/day in rabbit is likely to be similar to that in humans.

In previous studies performed in our laboratory, fluconazoleat 1 mg/kg twice daily maintained concentrations in plasmaabove the MICs for fluconazole-sensitive isolates (#0.125 mg/ml) throughout the dosing interval (31). Rabbits infected withfluconazole-sensitive isolates and treated with fluconazole hadsignificant reductions in the levels of organisms in quantitativecultures of oral mucosal samples (P , 0.001) and burdens of C.albicans in the tongue, soft palate, and esophagus (P , 0.001)(31). As the half-life of fluconazole in the rabbit is shorter (thehalf-life is approximately 12 h in rabbits) than that in adulthumans but similar to that in children (20) and since exposureover time has been shown to be important for antifungal effi-cacy (21), a twice-daily dosing regimen was used.

The ratio of the concentration in esophageal tissue to that inplasma of 0.02 for amphotericin B is substantially lower thanthe ratio of 0.3 achieved with echinocandin. Nevertheless, theconcentration of amphotericin B in esophageal tissue is ap-proximately four times higher than the MIC for C. albicans.This concentration appears to be sufficiently active to achievean approximate 10-fold or 1-log reduction of Candida levels intissue. Higher concentrations of amphotericin B would likelylead to greater fungicidal activity and to more complete clear-ance from tissue. These findings suggest that low-dose ampho-tericin B may not achieve complete microbiological eradica-tion or result in a clinical response in profoundly compromisedpatients with esophageal candidiasis due to the relatively lowconcentrations of amphotericin B in tissue. To our knowledge,the study described here is the first in vivo investigation of thepharmacokinetics and pharmacodynamics of low-dose ampho-tericin B for the treatment of esophageal candidiasis. Furtherstudies of the dosage-response relationships of amphotericin Bin this model of experimental esophageal candidiasis may helpprovide an understanding of the correct dosing strategies forimmunocompromised patients with esophageal candidiasis.

The clinical and microbiological responses in our immuno-compromised patients with fluconazole-resistant OPEC havebeen variable, prompting us to use higher amphotericin Bdosages (0.5 to 0.6 mg/kg/day) (4). This lack of response may

reflect profoundly impaired mucosal immunity as well as rela-tively low levels of amphotericin B in esophageal tissue.

The tissue fluconazole concentration of 3.6 mg/ml and theplasma fluconazole concentration of 1.36 mg/ml are substan-tially lower than the MICs (.64 mg/ml). These findings furtherdemonstrate a relatively high ratio of the concentration intissue to that in plasma (approximately 3:1). The excellentresponse achieved with fluconazole against esophageal candi-diasis caused by susceptible organisms may be related to itsfavorable esophageal penetration (31).

The concentrations of an antimicrobial compound in tissueare the sum of the concentrations in the intravascular, theintestitial, and the intracellular tissue compartments. However,it is not clear which of these compartments is important as atarget tissue for invasive fungal infections. The pseudohyphaeand hyphae of Candida spp., for example, invade cells, bloodvessels, and interstitial compartments of tissues. Furthermore,the pathogenic hallmark of tissue-invasive fungal infections istissue necrosis and inflammation, in which different equilibriaof drug concentrations are likely to prevail, suggesting thatsampling of the entire tissue homogenate may be more reflec-tive of the pathogenesis of invasive mycoses.

Our data show a similar concentration-response relationshipof the concentrations of VER-002 in tissue and the levels ofVER-002 in plasma with the residual fungal burden. This sim-ilarity further validates the relationship between plasma phar-macokinetic parameters as a surrogate for the concentrationsof echinocandins in the alimentary tract. Whether these phar-macokinetic relationships are valid for other tissue sites such asthe central nervous system remain to be explored.

In summary, this study demonstrates that the echinocandinVER-002 exerts concentration-dependent and dosage-depen-dent fungicidal activity against experimental fluconazole-resis-tant OPEC. The new group of echinocandins, as exemplified byVER-002, represents a potentially important therapeutic ad-vance in the management of esophageal candidiasis and war-rants further investigation in clinical trials of treatments forthis infection.

ACKNOWLEDGMENTS

We are grateful to Myrna Candelario and Aida Field-Ridley forassistance in the laboratory animal facility, to Joanne Peter for per-forming MIC determinations, to Diana Mickiene for performingHPLC, and to Jeniffer Rabb for expert secretarial assistance in prep-aration of the manuscript.

REFERENCES

1. Amantea, M. A., R. A. Bowden, A. Forrest, P. K. Working, M. S. Newman,and R. D. Mamelok. 1995. Population pharmacokinetics and renal function-sparing effects of amphotericin B colloidal dispersion in patients receivingbone marrow transplants. Antimicrob. Agents Chemother. 39:2042–2047.

2. Barbaro, G., G. Barbarini, W. Calderon, B. Grisorio, P. Alcini, and G. DiLorenzo. 1996. Fluconazole versus itraconazole for Candida esophagitis inacquired immunodeficiency syndrome. Gastroenterology 111:1169–1177.

3. Brassinne, C., C. Laduron, A. Coune, J. P. Sculier, C. Hollaert, N. Collette,and F. Meunier. 1987. High-performance liquid chromatographic determi-nation of amphotericin B in human serum. J. Chromatogr. 7:401–407.

4. Chiou, C. C., A. H. Groll, C. E. Gonzalez, D. Callender, D. Venzon, P. A.Pizzo, L. Wood, and T. J. Walsh. 2000. Esophageal candidiasis in pediatricacquired immunodeficiency syndrome: clinical manifestations and risk fac-tors. Pediatr. Infect. Dis. J. 19:729–734.

5. Committee on the Care and Use of Laboratory Animals of the Institute ofLaboratory Animal Resources, Commission on Life Sciences, National Re-search Council. 1996. Guide for the care and use of laboratory animals.National Academy Press, Washington, D.C.


6. Debono, M., and R. S. Gordee. 1994. Antibiotics that inhibit fungal cell walldevelopment. Annu. Rev. Microbiol. 48:471–497.

7. Debono, M., W. W. Turner, L. LaGrandeur, F. J. Burkhardt, J. S. Nissen,K. K. Nichols, M. J. Rodriguez, M. J. Zweifel, D. J. Zeckner, R. S. Gordee,J. Tang, and T. R. Parr, Jr. 1995. Semisynthetic chemical modification of theantifungal lipopeptide echinocandin B (ECB): structure-activity studies ofthe lipophilic and geometric parameters of polyarylated acyl analogs ofECB. J. Med. Chem. 38:3271–3281.

8. Ernst, M. E., M. E. Klepser, E. J. Wolfe, and A. Pfaller. 1996. Antifungaldynamics of LY303366, an investigational echinocandin B analog, againstCandida spp. Diagn. Microbiol. Infect. Dis. 26:125–131.

9. Fromtling, R. A. 1994. LY303366. Drugs Future 19:338–342.10. Georgopapadakou, N. H., and J. S. Tkacz. 1995. The fungal cell wall as a

drug target. Trends Microbiol. 3:98–104.11. Gordee, R. S., D. J. Zeckner, L. C. Howard, W. E. Alborn, and M. DeBono.

1988. Anti-Candida activity and toxicology of LY121019, a novel semisyn-thetic polypeptide antifungal antibiotic. Ann. N. Y. Acad. Sci. 544:294–309.

12. Inagaki, K., J. Takagi, E. Lor, M. P. Okamoto, and M. A. Gill. 1992.Determination of fluconazole in human serum by solid-phase extraction andreversed-phase high-performance liquid chromatography. Ther. Drug Monit.14:306–311.

13. Karlowsky, J. A., G. A. J. Harding, S. A. Zelenitsky, D. J. Hoban, A. Kabani,T. V. Balko, M. Turik, and G. G. Zhanel. 1997. In vitro kill curves of a newsemisynthetic echinocandin, LY-303366, against fluconazole-sensitive and-resistant Candida species. Antimicrob. Agents Chemother. 41:2576–2578.

14. Klepser, M. E., E. J. Ernst, M. E. Ernst, S. A. Messer, and M. A. Pfaller.1998. Evaluation of endpoints for antifungal susceptibility determinationswith LY303366. Antimicrob. Agents Chemother. 42:1387–1391.

15. Krishnarao, T. V., and N. J. Galgiani. 1997. Comparison of the in vitroactivities of the echinocandin LY303366, the pneumocandin MK-0991, andfluconazole against Candida species and Cryptococcus neoformans. Antimi-crob. Agents Chemother. 41:1957–1960.

16. Kurtz, M. B. 1997. New antifungal drug targets: a vision for future. ASMNews 64:31–39.

17. Kurtz, M. B., and C. M. Douglas. 1997. Lipopeptide inhibitors of fungalglucan synthase. J. Med. Vet. Mycol. 35:79–86.

18. Lee, J. W., M. A. Amantea, P. A. Francis, E. E. Navarro, J. Bacher, P. A.Pizzo, and T. J. Walsh. 1994. Pharmacokinetics and safety of a unilamellarliposomal formulation of amphotericin B (AmBisome) in rabbits. Antimi-crob. Agents Chemother. 38:713–718.

19. Lee, J. W., P. Kelly, J. Lecciones, D. Coleman, R. Gordee, P. A. Pizzo, andT. J. Walsh. 1990. Cilofungin (LY121019) shows non-linear plasma pharma-cokinetics and tissue penetration in rabbits. Antimicrob. Agents Chemother.34:2240–2245.

20. Lee, J. W., N. T. Seibel, M. A. Amantea, P. Whitcomb, P. A. Pizzo, and T. J.Walsh. 1992. Safety, tolerance, and pharmacokinetics of fluconazole in chil-dren with neoplastic diseases. J. Pediatr. 120:987–993.

21. Louie, A., P. Banerjee, G. L. Drusano, M. Shayegani, and M. H. Miller. 1999.Interaction between fluconazole and amphotericin B in mice with systemicinfection due to fluconazole-susceptible or -resistant strains of Candida al-bicans. Antimicrob. Agents Chemother. 43:2841–2847.

22. Medoff, G., W. E. Dismukes, R. H. Meade, and J. M. Moses. 1972. A newtherapeutic approach to Candida infections. A preliminary report. Arch.Intern. Med. 130:241–245.

23. National Committee for Clinical Laboratory Standards. 1997. Referencemethod for broth dilution antifungal susceptibility testing of yeasts. Tenta-tive standard. NCCLS document M27-A. National Committee for ClinicalLaboratory Standards, Wayne, Pa.

24. Petraitiene, R., V. Petraitis, A. H. Groll, M. Candelario, T. Sein, A. Bell, C.A. Lyman, C. L. McMillian, J. Bacher, and T. J. Walsh. 1999. Antifungalactivity of LY303366, a novel echinocandin B, in experimental disseminatedcandidiasis in rabbits. Antimicrob. Agents Chemother. 43:2148–2155.

25. Petraitis, V., R. Petraitiene, A. H. Groll, A. Bell, D. P. Callender, T. Sein,R. L. Schaufele, C. L. McMillian, J. Bacher, and T. J. Walsh. 1998. Anti-fungal efficacy, safety, and single-dose pharmacokinetics of LY303366, anovel echinocandin B, in experimental pulmonary aspergillosis in persis-tently neutropenic rabbits. Antimicrob. Agents Chemother. 42:2898–2905.

26. Pfaller, M. A., S. A. Messer, and S. Coffman. 1997. In vitro susceptibilities ofclinical yeast isolates to a new echinocandin derivative, LY303366, and otherantifungal agents. Antimicrob. Agents Chemother. 41:763–766.

27. Rex, J. H., M. G. Rinaldi, and M. A. Pfaller. 1995. Resistance of Candidaspecies to fluconazole. Antimicrob. Agents Chemother. 39:1–8.

28. Roy, M. J., and T. J. Walsh. 1992. Histopathological and immunohistochem-ical changes in gut-associated lymphoid tissues following treatment of rabbitswith dexamethasone. Lab. Investig. 64:437–443.

29. Uzun, O., S. Kocagoz, Y. Cetinkaya, S. Arikan, S. Unal. 1997. In vitro activityof a new echinocandin, LY303366, compared with those of amphotericin Band fluconazole against clinical yeast isolates. Antimicrob. Agents Che-mother. 41:1156– 1157.

30. Walsh, T. J., J. Bacher, and P. A. Pizzo. 1988. Chronic silastic central venouscatheterization for induction, maintenance, and support of persistent gran-ulocytopenia in rabbits. Lab. Anim. Med. 38:467–470.

31. Walsh, T. J., C. E. Gonzalez, S. Piscitelli, J. D. Bacher, J. Peter, R. Torres,D. Shetti, V. Katsov, K. Kligys, and C. A. Lyman. 2000. Correlation betweenin vitro and in vivo antifungal activity in experimental fluconazole-resistantoropharyngeal and esophageal candidiasis. J. Clin. Microbiol. 38:2369–2373.

32. Walsh, T. J., A. J. Jackson, J. W. Lee, M. Amantea, T. Sein, J. Bacher, andL. Zech. 2000. Dose-dependent pharmacokinetics of amphotericin B lipidcomplex in rabbits. Antimicrob Agents Chemother. 44:2068–2076.

33. Walsh, T. J., J. W. Lee, P. Kelly, J. Bacher, J. Lecclones, V. Thomas, C.Lyman, D. Coleman, R. Gordee, and P. A. Pizzo. 1991. The antifungal effectsof the non-linear pharmacokinetics of cilofungin, a 1,3-b-glucan synthaseinhibitor, during continuous vs. intermittent infusion of cilofungin in treat-ment of experimental disseminated candidiasis. Antimicrob. Agents Che-mother. 35:1321–1328.

34. Walsh, T. J., C. McEntee, and D. M. Dixon. 1987. Tissue homogenizationwith sterile reinforced polyethylene bags for quantitative culture of Candidaalbicans. J. Clin. Microbiol. 25:931–932.

35. Wilcox, C. M., R. F. Straub, L. N. Alexander, and W. S. Clark. 1996. Etiologyof esophageal disease in human immunodeficiency virus-infected patientswho fail antifungal therapy. Am. J. Med. 101:599–604.


dosage-dependent antifungal efficacy of v-echinocandin (ly303366) against experimental...

Documents