Antimicrobial Agents and Chemotherapy, October 2004, p . 3959-3967, Vol . 48, No . 10

PLD-118 (formerly BAY-10-8888) is a novel antifungal compound and a synthetic derivative of the naturally occurring ß-amino acid cispentacin (Fig . 1) . The mechanism of action of PLD-118 is thought to arise from the inhibition of isoleucyl-tRNA synthetase, resulting in the inhibition of protein synthesis and of fungal cell growth (16, 32, 33) . In vitro studies have demonstrated antifungal activity of PLD-118 against Candida albicans strains, including azole-resistant strains (A . Hasenoehrl, M . Skerlev, N . Marsic, and W . Schoenfeld, Abstr . 41st Intersci . Conf . Antimicrob . Agents Chemother., abstr . 2143, 2001) and Candida spp . other than C . albicans (W . Schoenfeld, Abstr . 42nd Intersci . Conf . Antimicrob . Agents Chemother., abstr . 812, 2002) . In vivo studies also demonstrated antifungal activity of PLD-118 in animal models of disseminated candidiasis (W . Schoenfeld, J . Mittendorf, A . Schmidt, and U . Geschke, Abstr . 41st Intersci . Conf . Antimicrob . Agents Chemother., abstr . 2144, 2001; A . Hasenohrl, N . Marsic, and K . Orescovic, Abstr . 43rd Intersci . Conf . Antimicrob . Agents Chemother., abstr . 1229, 2003) .

 
Little is known, however, about the activity of PLD-118 against OPEC in immunocompromised hosts . We therefore investigated the safety and antifungal efficacy of PLD-118 in an immunosuppressed-rabbit model of fluconazole-resistant OPEC .

MICs. The inoculum was prepared by selecting three to five colonies from 24-h cultures of C . albicans grown on Sabouraud glucose agar (SGA) plates . Colonies were suspended in sterile normal saline, and the density of the fungal suspension was adjusted to an optical density at 600 nm of 0.85, which corresponds to approximately 10⁷ CFU/ml (18) . From that suspension, a 10^–4 dilution was made in yeast nitrogen broth-1% glucose medium (YNB; K-D Medical, Inc., Columbia, Md.; 0.67% yeast nitrogen base [Difco], 1% glucose [pH 7.0]) . This inoculum was further adjusted to prepare a suspension of C . albicans in yeast nitrogen glucose (YNG) medium to yield a final inoculum concentration of 10³ CFU/ml . The exact inoculum used was confirmed by quantitative culture on SGA plates . The MICs were determined as the lowest concentrations in which a prominent decrease in turbidity was observed after incubation at 37°C for 24 h . The MICs of PLD-118 for the four isolates of fluconazole-resistant C . albicans were 0.5 (isolates 96 and 105) and 1 µg/ml (isolates 80 and 126) .

Time-kill assays. To characterize the in vitro pharmacodynamics and potential fungicidal activity of PLD-118 (Pliva Pharmaceutical Industry, Inc., Zagreb, Croatia), time-kill assays were performed in the same fashion for all four C . albicans isolates, with fluconazole (Roerig-Pfizer, New York, N.Y.) used as a treatment control . Isolates treated with PLD-118 (4, 8, 16, and 64 µg/ml) or fluconazole (128 µg/ml) and untreated growth controls in YNB medium were studied . The inoculum for the time-kill assays was prepared by subculturing each isolate twice for 48 h at 37°C on SGA, inoculating three to five colonies into a starter broth of 50 ml of Sabouraud glucose broth (SGB; K-D Medical), and incubating overnight in a shaking water bath at 37°C . The isolates were washed three times with 0.9% saline (Quality Biological, Inc., Gaithersburg, Md.), resuspended, and counted by hemacytometer .

Concentrations of 10⁴ to 10⁵ CFU of C . albicans/ml were used for time-kill assays for PLD-118 and fluconazole . An aliquot of 300 µl of each dilution was placed in the wells of a 24-well flat-bottom plate (Costar, Cambridge, Mass.); there was a separate well for each time point . The growth suspensions were sampled at predetermined time points (0, 2, 4, 6, 12, and 24 h following the addition of antifungal), and a 100-µl aliquot was removed from each culture vial and plated in dilutions of 10^–2, 10^–3, and 10^–4 onto one SGA plate per aliquot . The colonies were counted after 48 h of incubation at 37°C, and the calculated CFU per milliliter were plotted for each time point . The lower limit of quantitation for the time-kill assay was 10 CFU/ml . Time-kill assays for all concentrations were performed in triplicate .

Animals. Fifty-eight female New Zealand White rabbits (Hazleton Research Products, Inc., Denver, Pa.) weighing 2.1 to 3.1 kg at the time of inoculation were used in all experiments . All rabbits were monitored under humane care and use in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and according to National Institutes of Health guidelines for animal care in fulfillment of Guidelines of the National Research Council (19) and approved by the Animal Care and Use Committee of the National Cancer Institute . Rabbits were individually housed and maintained with water and standard rabbit feed ad libitum . Vascular access was established in each rabbit by the surgical placement of a silastic tunneled central venous catheter as previously described (30) . The silastic catheter permitted nontraumatic venous access for administration of parenteral agents and for repeated blood sampling for study of biochemical and hematological parameters and plasma pharmacokinetics . Serum samples were drawn from all rabbits at prespecified intervals throughout each experiment . Rabbits were euthanized by intravenous (i.v.) administration of pentobarbital (65 mg of pentobarbital sodium/kg of body weight) (euthanasia solution; Schering-Plough Animal Health Corp., Union, N.J.) at the end of each experiment, 12 h after administration of PLD-118 and fluconazole and 24 h after administration of desoxycholate amphotericin B (DAMB) .

Oral inoculation. Organisms from stock isolates were stored in skim milk at –80°C . Cells from these suspensions were streaked onto SGA plates and incubated at 37°C for 24 h and then maintained at 4°C . Five discrete colonies were then inoculated into 50 ml of Emmon's modified Sabouraud broth (pH 7) in a 250-ml Erlenmeyer flask and incubated at 37°C for 16 h on a shaking incubator at 80 oscillations per min . The Candida suspension was centrifuged at 3,000 x g for 10 min and washed three times with sterile 0.9% saline . Candida blastoconidia were counted with a hemacytometer and diluted to the desired concentration . An inoculum of 2 x 10⁸ blastoconidia was suspended in 1.5 ml of 0.9% saline, and the suspension was administered per os from day 1 through 7 of the experiment . There were no interisolate differences in the capacity to establish oropharyngeal or esophageal infection .

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

Antifungal compounds and treatment groups. Therapy was initiated on day 8 of the experiment following per os inoculation and continued throughout the course of the experiments for 7 days . PLD-118 was provided as a powder for parenteral administration by Pliva Pharmaceutical Industry, Inc . PLD-118 was dissolved in 0.9% saline and administered i.v . at dosages of 2 (PLD4 group), 5 (PLD10 group), 12.5 (PLD25 group), and 25 (PLD50 group) mg/kg twice daily (BID) . DAMB (Fungizone; Apothecon Bristol-Myers Squibb Company, Princeton, N.J.) at 0.5 mg/kg/day was administered as slow i.v . infusion (0.1 ml every 10 s) . Fluconazole was administered at 1 mg/kg twice daily i.v . All compounds were initiated 24 h after the last inoculation on day 7 of the experiment .

Outcome variables. The rabbit model of fluconazole-resistant OPEC provides a strong in vitro-in vivo correlation of therapeutic response to fluconazole (28) . This system permits quantitative assessment of therapeutic response along clinically relevant sites of the upper alimentary tract: tongue, oropharynx, esophagus (proximal, middle, and distal), stomach, and duodenum . The tongue, oropharynx, and esophagus were resected en bloc postmortem . Segments of duodenum and stomach were resected separately . Antifungal efficacy was assessed by microbiologic clearance of C . albicans from tissue . Sections of tongue, oropharynx, esophagus (proximal, middle, and distal), stomach, and duodenum were cultured by excision of a representative region . Each fragment was weighed and then homogenized in sterile reinforced polyethylene bags (Tekmar Corp., Cincinnati, Ohio) (29) . Each tissue homogenate was serially diluted 100-fold from 10 to 10^–4 in sterile 0.9% saline . An aliquot of 100 µl of undiluted homogenate and of each dilution was separately plated onto Emmon's modified SGA containing chloramphenicol and gentamicin . Culture plates were incubated at 37°C for 24 h, after which CFU were counted and the number of CFU per gram of tissue was calculated for each organ . Carryover of the drug is controlled by serial dilution and by streaking a small-volume (100-µl) aliquot onto a large volume of agar (1 full agar plate per 100-µl aliquot) . The method was sensitive enough to detect 10 CFU/g . The culture-negative plates were counted as 0 CFU/g . Data from all four isolates used in experiments were graphed as the means ± standard errors of the means (SEM) of log₁₀ (CFU per gram) .

Histopathology. Representative sections of the tongue were prepared for histological studies . Tissue specimens were excised and fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and then stained with periodic acid-Schiff and Grocott-Gomori methenamine-silver stain .

Pharmacokinetic studies. The plasma pharmacokinetics of PLD-118 in six infected animals each per dosage cohort were investigated . Plasma sampling was performed on day 6 of antifungal therapy . Blood samples, collected in heparinized syringes, were drawn immediately after dosing and then at 0.17, 0.5, 1, 2, 4, 6, and 12 h postdosing . Plasma was immediately separated by centrifugation . Saliva was sampled 2 h following the seventh dose . Salivation was induced with 0.5 mg of pilocarpine (catalog no . 45H05181i; Sigma) . Plasma and saliva samples were stored at –80°C until assay .

Concentrations of PLD-118 in plasma and saliva were determined after protein precipitation with methanol (2:5, vol/vol) and derivatization with o-phthaldialdehyde (OPA) by reversed-phase high-performance liquid chromatography (HPLC) (5) . Sample preparation was performed by adding 500 µl of methanol to 200 µl of matrix, vortexing, and centrifuging for 10 min at 8,000 x g . An aliquot of 100 µl of the supernatant was used to fill autosampler vials . One hundred microliters of fresh OPA solution was added from a reservoir and mixed for 1 min before 30 µl of the resulting solution was injected onto the HPLC column . For preparation of the OPA reagent, 50 mg of phthaldialdehyde was dissolved in 1 ml of ethanol in a 25-ml volumetric flask and the flask was filled up to the mark with 0.1 M borate buffer (pH 9) . Twenty microliters of 2-mercaptoethanol was then added . The mobile phase consisted of acetonitrile-methanol-0.01 M phosphate buffer (3.50:1.25:5.25 [vol/vol/vol]), and the flow rate was 1.1 ml/min . Separation was achieved with a C₁₈ analytical column (Nucleosil; 125 by 4.6 mm [internal diameter]; particle size, 5 µm; Thomson Liquid Chromatography) maintained at 40°C . PLD-118 was detected fluorescently at an excitation of 340 nm and emission of 430 nm . Quantitation was based on the peak-height concentration response of the reference standard, prepared in either normal rabbit plasma (plasma) or 0.9% saline (saliva) . Ten-point standard curves (range of concentrations, 0.05 to 10 µg/ml) were linear, with r² values greater then 0.994 . Samples containing concentrations exceeding the upper limit of the standard curve were assayed after dilution with the mobile phase subsequent to determination of over-curve concentration response linearity . The lower limit of quantitation (LLQ) was 0.1 µg/ml in plasma . Accuracies were within 14%, and intra- and interday variability (precision) was <12%; at LLQ, accuracy and precision were within 11 and 12%, respectively .

Pharmacokinetic parameters for PLD-118 were determined by model-independent analysis . The following pharmacokinetic parameters were determined: maximum concentrations in plasma (C_max); concentrations at 12 h after dosing (C_min); the area under the plasma concentration-time curve from 0 to 12 h (AUC_0-12), calculated by trapezoidal estimation; the area under the plasma concentration-time curve from 0 to 24 h (AUC_0-24), calculated by multiplying the AUC_0-12 by 2; and dose linearity, determined by comparison of the mean dose-normalized calculated AUC_0-24 . Plasma clearance (CL), apparent volume of distribution at steady state (VD), and half-life (t_1/2) were calculated from standard equations (26) . Statistical comparisons across dosage cohorts were made by analysis of variance (ANOVA) .

Toxicity studies. Chemical determinations of serum potassium, aspartyl aminotransaminase (AST), alanine aminotransaminase (ALT), serum creatinine, alkaline phosphatase, and total bilirubin were performed on the penultimate sample drawn from each rabbit .

Statistical analysis. Comparisons between groups were performed by ANOVA with Dunn's correction for multiple comparisons or the Mann-Whitney U test, as appropriate . All P values were two sided, and a P value of <0.05 was considered to be significant . Values are expressed as means ± standard errors of the means (SEM) .

 
Antifungal therapy. PLD-118 demonstrated a significant dosage-dependent antifungal effect in treatment of fluconazole-resistant OPEC . Rabbits treated with PLD-118 showed a significant dosage-dependent reduction of organisms in the tongue and oropharynx at 10, 25, and 50 mg/kg in comparison to untreated controls (P < 0.001) (Fig . 3 and 4) . There was also a significant reduction of C . albicans in the tongue and oropharynx (P < 0.001 and P < 0.01, respectively) from rabbits treated with DAMB in comparison to untreated controls, while rabbits treated with fluconazole showed no reduction in CFU per gram in these tissues .

 
Rabbits treated with PLD-118 also showed significant reduction of the concentration of C . albicans in esophagus tissue (Fig . 5) . In comparison to untreated controls, rabbits treated with PLD-118 at 10, 25, and 50 mg/kg showed a significant reduction in organism burden from proximal (P 0.05), middle (P 0.05), and distal (P 0.01) segments of the esophagus . There was no significant reduction in tissue burden in esophageal tissue of rabbits treated with PLD-118 at 4 mg/kg and fluconazole in comparison to untreated control animals .

 
There also was a reduction in C . albicans from the stomach and duodenum in rabbits treated with PLD-118 (Fig . 6) . Rabbits treated with fluconazole showed no reduction .

 
Histopathology. The organisms in tissue of PLD-118-treated animals demonstrated morphological changes in hyphal and pseudohyphal structures in comparison to those of untreated controls (Fig . 7A and B) . Organisms in PLD-118-treated rabbits demonstrated disrupted and shortened hyphal structures (C and D) . The long slender hyphae and pseudohyphae observed in tissues of untreated controls were seldom observed in treated animals .

 
Pharmacokinetics. The observed plasma concentration-versus-time profiles of PLD-118 following administration of 2, 5, 12.5, and 25 mg/kg BID are depicted in Fig . 8, and the corresponding pharmacokinetic parameters are listed in Table 1 .

 
Dosages of 2, 5, 12.5, and 25 mg/kg BID resulted in escalating peak plasma levels that ranged from 31.99 ± 3.19 to 98.19 ± 5.53 µg/ml, exceeding the level at which the experimental isolates manifest fungicidal or inhibitory activity (16 µg/ml) in time-kill assays . Plasma PLD-118 concentrations decreased, with a mean elimination ß t_1/2 of 1.62 to 4.03 h . The compound exhibited a VD that approximated the VD of total body water and that was independent of the dosage . Across the investigated dosage range, PLD-118 demonstrated nonlinear plasma pharmacokinetics, as evidenced by a dose-dependent increase in plasma CL and a decrease in the dose-normalized AUC_0-12 and AUC_0-24, respectively . Concentrations of PLD-118 in induced saliva at 2 h following the seventh dose ranged from 0.040 ± 0.01 to 0.143 ± 0.02 µg/ml .

Safety. Rabbits treated with PLD-118, fluconazole, or DAMB had no significant increase or decrease in levels of serum creatinine, serum potassium, ALT, or serum bilirubin in comparison to untreated controls . On the other hand, there was a trend toward an increase in serum AST of rabbits treated with PLD-118 at 10, 25, and 50 mg/kg/day and with fluconazole (Table 2) . However, this trend did not achieve statistical significance .

 
PLD-118 demonstrated significant antifungal activity against fluconazole-resistant OPEC due to C . albicans . The in vivo efficacy of PLD-118 was dosage-dependent and correlated with the in vitro concentration-dependent effect of PLD-118 against fluconazole-resistant C . albicans . Fluconazole had no effect in this rabbit model of fluconazole-resistant OPEC .

This model of OPEC reflects the profound impairment of mucosal immunity encountered in severely immunocompromised hosts, such as HIV-infected patients, corticosteroid-treated patients, and recipients of organ or marrow transplants . High-dose corticosteroids induce a marked suppression of mucosal immunity in rabbits (24) . Specifically, the mucosa-associated lymphoid tissue (MALT) in corticosteroid-treated rabbits reveals a profound depletion of lymphoid follicles and severe loss of B lymphocytes . The dome epithelium of MALT is also compromised, as evidenced by apoptosis of M cells and loss of intraepithelial lymphocytes . These immune impairments compromise surface immunoglobulin production and immunoregulation of mucosal host defenses against opportunistic pathogens . Such mucosal immunosuppression results in a higher tissue burden of C . albicans than that which occurs in normal animals . The refractoriness of this infection to fluconazole resembles that of azole-resistant candidiasis in humans (6, 7, 22, 23) .

The microbiological outcome variables in this model permit quantitative analysis of dosage-dependent antifungal responses in several sites of the upper alimentary track . Evaluation of different sites in the upper alimentary track is important, as the antifungal effect of different concentrations of antifungal agents in saliva and tissues may be site dependent . For example, one may observe patients with fluconazole-resistant OPEC who achieve a response in the oral cavity but who continue to have persistent esophageal disease .

Cispentacin, a cyclic ß-amino acid isolated from Bacillus cereus and Streptomyces setonii, was found by Ziegelbauer and colleagues to have potent anti-Candida activity (32, 33) . After cyclic ß-amino acid derivatives with superior efficacy were identified, PLD-118 (former BAY 10-8888) was chosen for further development . PLD-118 is actively accumulated by amino acid permeases within the cell . PLD-118 inhibits isoleucyl-tRNA synthetase, resulting in the disruption of fungal protein biosynthesis and cell growth . This novel mechanism of action should not be affected by azole resistance mechanisms .

PLD-118 demonstrated antifungal activity in time-kill assays . Concentration-dependent antifungal activity was observed at 24 h . However, at earlier time points of 2, 4, and 6 h, there was substantially less concentration-dependent antifungal activity across the range of concentrations of PLD-118 . Some regrowth was observed after 24 h . Reculture of organisms from wells containing PLD-118 with regrown C . albicans at 24 h revealed no changes in MICs, suggesting that deterioration of drug in vitro may account for this effect . The time-kill effects of PLD-118 are consistent with those observed in Candida-infected tissues of the upper alimentary track .

The PLD-118 dosage of 10 mg/kg BID was active in clearing C . albicans from the tongue, oropharynx, esophagus, stomach, and duodenum . The different tissue sites of tongue, oropharynx, and esophagus appear to have similar dose-dependent patterns of response to PLD-118 . The dose-proportional tissue PLD-118 concentrations correlated with the dose-dependent in vitro microbiological response .

As little is known about the pharmacokinetics and pharmacodynamics in treatment of esophageal candidiasis, we investigated the plasma, esophageal, and salivary concentrations of PLD-118 . The concentrations of PLD-118 demonstrated a nonlinear dosage-dependent increase in plasma . Preliminary studies indicated that the kidneys are the likely route of excretion of PLD-118 (W . Schoenfeld, Abstr . 41st Intersci . Conf . Antimicrob . Agents Chemother., abstr . 11, 2001; W . Schoenfeld and M . Parnham, Abstr . 41st Intersci . Conf . Antimicrob . Agents Chemother., abstr . 2145, 2001) . This nonlinear rise in AUC with increasing dosages suggests induction of another clearance pathway . These findings are consistent with clearance by glomerular filtration and inducible tubular secretion .

Substantially greater levels of PLD-118 were found in esophageal tissue than in saliva . The relatively higher concentrations of PLD-118 in esophageal tissue suggest that the tissue route may be the dominant kinetic mechanism of antifungal efficacy in treatment of OPEC in comparison to the relatively low salivary concentrations at the lumen .

Dosages of PLD-118 needed to eradicate C . albicans from esophageal tissue appear to be higher then those required to eliminate C . albicans from deep tissue, including liver, spleen, and kidney . Using the same quantitative culture techniques, we found from our preliminary data that 12.5 mg/kg BID achieved complete eradication of C . albicans from these deep tissues (V . Petraitis, R . Petraitiene, A . A . Sarafandi, A . M . Kelaher, T . Sein, D . Mickiene, A . H . Groll, J . Bacher, and T . J . Walsh, Abstr . 43rd Intersci . Conf . Antimicrob . Agents Chemother., abstr . M-369, 2003) . By comparison a dosage of 25 mg/kg BID did not fully eliminate C . albicans in esophageal candidiasis . This disparity of response between esophageal and deep visceral candidiasis also is found in echinocandins . For example, the dosages of anidulafungin sufficient to eradicate C . albicans from deep visceral tissue were lower than those required to eradicate it from esophageal tissue (20, 21) .

Although higher dosages of PLD-118 were associated with increased elevation of serum AST, these elevations were not statistically significant . Notably, the serum AST elevations in PLD-118-treated rabbits were comparable to those treated with fluconazole . Other parameters, including ALT and serum bilirubin, were not appreciably elevated compared to controls . Nonetheless, careful monitoring of plasma biochemical parameters in patients enrolled into clinical trials of PLD-118 is warranted .

In summary, these findings demonstrate proof of principle that the cispentacin compound PLD-118 is active in treatment of experimental fluconazole-resistant OPEC, thus laying the foundation for potential prospective clinical trials of this infection .

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What Is Bioassay?, What Is Activated Sludge?, What Is Dna?, What Is Bioreactor?, What Is MIC?, i, Bacterium, n, Microbes, e, Bacteriology, i, Microbiology, r, Microorganism, o, Pseudomonas, a, Vibriosis, c, Bacteriophages, a, Gram positive, e, Haemophilus, c, Escherichia coli, s, Salmonella typhimurium, e, Pseudomonas, o, Streptococci, a, Escherichia coli, s, Streptococci, e, Enterobacteriacea, s, Pseudomonas, e, Staphylococcus, e, Enterobacteriacea, n, Multidrug resistant, e, Bacillus subtilis, n, Salmonella typhimurium, e, Lactobacillus, n, Escherichia coli, s, Suspension cells




 

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Last modified: May 25, 2005