Micafungin (FK463)

The echinocandins constitute a novel class of cyclic lipopeptides that inhibit the synthesis of 1,3-ß-glucan of the fungal cell wall (Kurtz and Douglas 1997). Three compounds have entered clinical development: Caspofungin (formerly MK-0991), FK463, and VER-002 (formerly LY-303366). These agents have very similar pharmacologic properties: A consistent spectrum of antifungal activity, no cross-resistance to existing antifungal agents, and lack of mechanism-based toxicity. They all possess favorable pharmacokinetic properties, but, at this point, are only available for parenteral administration (Groll et al., 1998). The following overview is limited to published data on FK463, which is being developed by Fujisawa Pharmaceutical Company, Inc.

 

Synthesis and mechanism of action

FK463 is a water-soluble echinocandin-like lipopeptide with a sulfate ester moiety in the cyclic hexapeptide nucleus. It is synthesized by chemical acylation of (5-(4-pentyl-oxyphenyl) isoxazol-3-yl) to the cyclic peptide nucleus FR179642, obtained by enzymatic cleavage of FR901379, a natural product of the fungus Coleophoma empedri (Tomishima et al., 1999).

FK463 inhibits 1,3-b-D-glucan synthase, an enzyme complex specific to fungi and essential for cell wall synthesis of many pathogenic fungi. Assessment of the effect of FK463 on 1,3-b-D-glucan synthase activity in membrane preparations of Candida albicans and Aspergillusfumigatus demonstrated significant inhibition at low concentrations with inhibition constants (Ki) of 0.21 µM for C.albicans and 0.016 µM for A.fumigatus . (Maki et al., 1998). Morphological changes observed in C.albicans after short term exposure to FK463 included abnormal swelling and irregular shape of the fungal cell, thinning of the fungal cell wall and aberrant budding, consistent with inhibition of cell wall synthesis (Nishiyama et al., 1999).

Antifungal activity in vitro

FK463 has potent and broadspectrum fungicidal activity against Candida spp., including C.albicans, C.dubliniensis, C.tropicalis, C.glabrata, C.krusei with somewhat higher MIC90 values for C.parapsilosis and C.guilliermondii (Table 1). Except for C.parapsilosis and C. guilliermondii, on a mg:mg basis, FK463 was generally more active than amphotericin B, fluconazole, and itraconazole (Maki et al., 1998; Mikamo et al., 1999; Tomishima et al., 1999; Tawara et al., 2000; Mueller et al. 2000; Ostrosky-Zeichner et al. 2000). Fluconazole-resistant clinical isolates of Candida showed no cross-resistance to FK463 (Maki et al., 1998; Tomishima et al., 1999). Negligible changes in MIC values after repeated exposure of C.albicans to subinhibitory concentrations of FK463 suggest a low potential for resistance induction (Maki et al., 1998). Of note, FK463 has shown virtually no in vitro activity against Cryptococcus neoformans and was inactive against Trichosporon cutaneum (Maki et al., 1998; Tomishima et al., 1999; Tawara et al., 2000).

FK463 has potent in vitro activity against Aspergillus spp. at lower concentrations than amphotericin B and itraconazole (Table 1). However, in contrast to the latter agents, FK463 is not fungicidal against these organisms (Maki et al., 1998; Tomishima et al., 1999; Tawara et al., 2000). FK463 was inactive in vitro (i.e., MIC values of ≥64µg/mL) against Fusarium spp., Pseudallescheria boydii, and Zygomyces ; moderate activity was found against dematiaceous fungi such as Cladosporium trichoides, Exophiala spp., and Fonsecaea pedrosi (MIC-values of 0.5 to 2.0 µg/mL) (Maki et al., 1998; Nakai et al., 1999; Tawara et al., 2000). In vitro activity against Histoplasma capsulatum, Blastomyces dermatidis, and Coccidioides immitis was limited to the mycelial forms of these organisms (Nakai et al. 1999). Combination of FK463 with amphotericin B was neither synergistic nor antagonistic in vitro against various clinicalAspergillus isolates (Petraitis et al., 1999; Stevens, 1999). Analysis of the in vitro activity of the combination of FK463 and nikkomycin Z against opportunistic moulds by a checkerboard macrodilution assay and vitality staining revealed synergy of these two cell wall-active agents against A.fumigatus, additive effects against Rhizopus oryzae, and indifference against A. flavus, A.terreus, A.niger and Fusarium solani (Chiou et al. 2000)

Table 1: Minimum Inhibitory Concentrations of FK463 against Clinical Yeast and Mould Isolates

Organism (no. of isolates)	MIC90 FK463 [µg/mL]	MIC90 FLCZ [µg/mL]	MIC90 ITCZ [µg/mL]	MIC90 AmB [µg/mL]
Candida albicans (37)	0.0156	0.5	0.0313	0.5
Candida albicans, FLCZ resistant (4)	0.0313	>64	>8	0.5
Candida tropicalis (20)	0.0313	8	0.5	0.125
Candida glabrata (20)	0.0156	64	8	1
Candida krusei (11)	0.125	64	1	1
Candida parapsilosis (17)	1	1	0.5	0.5
Candida guilliermondii (12)	2	4	1	0.25
Cryptococcus neoformans (5)	>64	8	0.5	0.5
Trichosporon cutaneum (5)	>64	16	0.5	32
Aspergillus fumigatus (29)	0.0156	>64	0.5	1
Aspergillus niger (15)	0.0078	>64	1	0.5
Aspergillus flavus (13)	0.0156	>64	0.5	1
Aspergillus terreus (7)	0.0078	>64	0.125	0.5

Antifungal susceptibility assays were performed according current NCCLS guidelines; for yeasts, the MICs of FK463 and AmB were defined as the lowest concentrations with no visible growth, and the MICs for FLCZ and ITCZ were defined as the lowest concentrations at which a prominent decrease in turbidity was observed. The MICs of all compounds tested for filamentous fungi were defined as the lowest concentrations at which a prominent decrease in turbidity compared with that for the growth control was observed.
FLCZ, fluconazole; ITCZ, itraconazole; AmB, amphotericin B. MIC90, 90st percentile for observed MIC values.
Table modified from Tawara et al. (2000).

Animal studies

Pharmacokinetics in laboratory animals:Single-dose pharmacokinetic studies of FK463 have been performed in mice, rats, and dogs after intravenous bolus administration of dosages of 0.32, 1, and 3.2 mg/kg (Suzuki et al., 1998), and in rabbits after administration of 0.5, 1 and 2 mg/kg (Groll et al. 2000). FK463 displayed linear pharmacokinetics over the investigated dosage range without fundamental inter-species differences in key pharmacokinetic parameters (Table 2). Plasma concentrations declined in a bi-exponential fashion with a half-life in the range of 3 to 6 hours. Total clearance ranged from 0.59 to 1.50 mL/min/kg, and the volume of distribution was 0.23 to 0.56 L/kg. In the rabbit, at the end of the initial distributive phase following the last of eight daily dosages of 0.5, 1, or 2 mg/kg, tissue concentrations ranged from 1.5 to 11 µg/g with highest concentrations noted in the lung, liver, spleen and kidney. FK463 was undetectable in CSF, but brain tissue concentrations exceeded the MIC90 and MEC90 values of most susceptible fungi (Groll et al., 2000). The serum protein binding exceeded 99% in all species. After bolus administration of 14C-FK463 into rats and dogs, approximately 15% of radioactivity was excreted in the urine with the remainder being excreted in the feces (Suzuki et al., 1998).

Table 2: Pharmacokinetic Parameters of FK463 in Animals and Humans

Species	Subjects [n]	Dosage	Route	Cmax [µg/mL]	AUC0-24hr µg/mLxhr]	VDss [L/kg]	CLt [mL/min/kg]	T1/2 [hr]
Male mice	3	0.32 mg/kg	IV bolus	~ 0.8	4.9	0.49	1.01	5.71
	3	1.0 mg/kg	IV bolus	~ 2	16.3	0.39	0.95	5.34
	3	3.2 mg/kg	IV bolus	~ 9	54.0	0.38	0.92	5.63
Male rats	3	0.32 mg/kg	IV bolus	~ 0.7	3.3	0.53	1.50	3.96
	3	1.0 mg/kg	IV bolus	~ 2	11.9	0.56	1.33	4.97
	3	3.0 mg/kg	IV bolus	~ 8	48.3	0.42	1.04	5.05
Female rabbits	3	0.5 mg/kg	IV bolus	7.62	5.66	0.30	0.09	2.97
	3	1.0 mg/kg	IV bolus	13.07	13.43	0.29	0.07	3.20
	3	2.0 mg/kg	IV bolus	16.08	21.79	0.34	0.08	3.04
Male dogs	3	0.32 mg/kg	IV bolus	~ 2	7.2 ±0.3	0.23 ±0.01	0.70 ±0.03	4.24 ±0.06
	3	1.0 mg/kg	IV bolus	~ 6	21.2 ±3.6	0.25 ±0.02	0.78 ±0.15	4.57 ±0.51
	3	3.0 mg/kg	IV bolus	~ 12	84.8 ±5.4	0.23 ±0.01	0.59 ±0.04	5.43 ±0.15
Male human volunteers	6	12.5 mg	2-hr infusion	0.94 ±0.04	17.11 ±1.22	0.242 ±0.024	0.197 ±0.021	15.2 ±0.9
	6	25 mg	2-hr infusion	1.86 ±0.31	32.63 ±4.18	0.239 ±0.023	0.201 ±0.025	14.8 ±1.2
	6	50 mg	2-hr infusion	3.36 ±0.27	60.93 ±7.32	0.237 ±0.021	0.192 ±0.022	15.2 ±0.5

Efficacy in animal models of invasive candidiasis:The antifungal activity of FK463, given intravenously in four daily dosages starting 1 hour after inoculation, against disseminated candidiasis has been studied in immunocompetent, transiently neutropenic, and corticosteroid-immunosuppressed mouse models (Matsumoto et al., 1999; Ikeda et al., 2000; Maesaki et al., 2000). In transiently neutropenic mice infected with C.albicans, treatment with FK463 resulted in a dose-dependent, statistically significant prolongation of survival. Against various Candida species, including fluconazole-resistant C.albicans, ED50 values of FK463 were 1.4 to 18 times higher than those of amphotericin B, but 9.6 to > 77 times lower than those of fluconazole. When survival of disseminated C.albicans infection was compared between normal, transiently neutropenic, and corticosteroid-immunosuppressed mice, efficacies of FK463 were reduced to 1/2.2 and 1/3.5, respectively, in the immunocompromised mice. Nevertheless, FK463 showed overall good activity in both types of immunocompromised mice with ED50 values of 0.28 to 0.45 mg/kg, and the expected reduction in efficacy was less than that observed for amphotericin B and fluconazole. Delay of the commencement of treatment to 24 hour post inoculation led to lesser reduction of the protective effect (to 1/1.3) in FK463-treated mice than in either amphotericin B- or fluconazole-treated animals (1/3.1 each). In neutropenic animals infected with C.albicans, 24 hours after administration of a single dose of FK463, a dose-dependent, statistically significant reduction in the residual fungal kidney burden was found that was comparable to amphotericin B; fluconazole merely had a suppressive effect at this timepoint. In model of subacute nonlethal disseminated candidiasis in persistently neutropenic rabbits, FK463, given at daily dosages ranging from 0.25 to 1 mg/kg starting 24 hours after inoculation, demonstrated dosage dependent clearance of C.albicans from tissues with complete elimination of the organism at the highest dosage level. Time-kill curves using the experimental strain from the infection model revealed time- and concentration-dependent fungicidal activity of the compound that correlated with the in vivo results (Petraitis et al. 2000). These results are similar to the results of a pharmacodynamic study of FK463 in a mouse thigh infection model that showed dose- and concentration dependent therapeutic efficacy against C.albicans infection of the soft tissues (Matsumoto et al. 2000).

Efficacy in animal models of invasive aspergillosis:Intravenous FK463, given once daily for four consecutive days starting 1 hour after inoculation, prolonged the survival of transiently neutropenic mice intravenously infected with Aspergillus fumigatus conidia in a dose-dependent manner; the efficacy of FK463 was about two times inferior to that of amphotericin B, with ED50s ranging from 0.25 to 0.50 mg/kg and 0.11 to 0.9 mg/kg, respectively [Ikeda et al., 2000] (Table 3). In mouse models of pulmonary aspergillosis (Wakai et al., 1998; Matsumoto et al., 2000], FK463, given for four days starting 1.5 hours after intranasal inoculation, significantly prolonged the survival of transiently neutropenic animals with ED50 values in the range of 0.26 to 0.51 mg/kg, which was comparable to amphotericin B (ED50, 0.26 to 0.46 mg/kg). In corticosteroid-immunosuppressed animals, a dose-dependent reduction in the residual burden of A.fumigatus in lung tissue was demonstrated when the drug was administered as continuous infusion with maintenance of plasma concentrations of 0.55 to 0.88 µg/mL or higher. In a rigorous persistently neutropenic rabbit model of invasive pulmonary aspergillosis, FK463, given at 0.5 to 2 mg/kg starting 24 hours after intratracheal inoculation, prolonged the survival and led to a significant decrease of organism-mediated pulmonary injury; the combination of FK463 (1mg/kg) with low-dose amphotericin B deoxycholate (0.1 mg/kg) or liposomal amphotericin B (0.5 mg/kg) was neither synergistic nor antagonistic (Petraitis et al., 1999; Petraitis et al. 2000). In less stringently immunocompromised mouse models of pulmonary aspergillosis, however, enhanced activity of the combination of FK463 and amphotericin B was noted using survival, residual fungal burden (Kohno et al. 2000) and histopathological evaluation of pulmonary infection (Nakajima et al. 2000) as endpoints of efficacy.

Table 3: Published Studies in Laboratory Animals Investigating the Efficacy of FK463 against Invasive Aspergillosis

Experimental Model	Endpoints of efficacy	Results
Transiently neutropenic (neutrophil recovery occurring at ≤ 6 days post infection) mouse model of pulmonary aspergillosis with start of treatment 1.5 hours after intranasal inoculation [Matsumoto et al. 2000]	Survival	FK463, given by the intravenous route once daily for 4 days, significantly prolonged the 15-day survival rate at dosages as low as 0.25mg/kg with an ED50 value comparable to that of intravenous amphotericin B (0.18-0.75 mg/kg/d vs. 0.24-0.67 mg/kg/d, respectively).
Corticosteroid-immunosuppressed (100 mg/kg hydrocortisone 1 day before and 1 day after infection) mouse model of pulmonary aspergillosis with start of treatment by continuous infusion after intranasal inoculation [Matsumoto et al. 2000]	Residual fungal burden in lung tissue	Significant reduction in the residual fungal tissue burden at day 5 under conditions of constant plasma levels of FK463 with steady/state plasma levels in the range of 0.55 to 0.80 µg/mL.
Transiently neutropenic (neutrophil recovery occurring at ≤ 6 days post infection) mouse model of disseminated aspergillosis with start of treatment 1 hour after intravenous inoculation [Ikeda et al. 2000]	Survival	FK463, given by the intravenous route once or twice daily for 4 days, significantly prolonged the 15-day survival rate at dosages ≥ 0.5 mg/kg. The efficacy of FK463 was about two times inferior to that of amphotericin B as measured by ED50 values (0.25-0.50 vs. 0.11-0.29 µg/mL, respectively).
Persistently neutropenic (no neutrophil recovery) rabbit model of invasive pulmonary aspergillosis with start of treatment 24 hours after intratracheal inoculation [Petraitis et al. 1999; Petraitis et al. 2000]	Survival, lung injury, residual fungal burden in lung tissue after therapy with FK463 alone and in combination with amphotericin B	Given alone at a dose of 0.5 to 2.0mg/kg, FK463 prolonged survival and significantly reduced the organism mediated tissue injury in comparison to untreated control animals. Combination of FK463 (1mg/kg IV) and either amphotericin B deoxycholate (0.1mg/kg IV) or liposomal amphotericin B (0.5mg/kg IV) once daily for a maximum duration of 10 days was neither synergistic nor antagonistic when compared with either agent alone.
Mouse-model of pulmonary aspergillosis (no further experimental details available) [Kohno et al. 2000]	Residual fungal tissue burden and survival after therapy with FK463 in combination with amphotericin B or therapy with either agent alone	Combination of FK463 and amphotericin B (0.5 mg/kg i.p. plus 0.5 mg/kg i.v.) enhanced survival and clearance of the organism from lung tissue as compared to either agent alone
Corticosteroid-immunosuppressed mouse model of pulmonary aspergillosis (no further experimental details available) [Nakajima et al. 2000]	Histopathological findings in infected lung tissue after therapy with FK463 in combination with amphotericin B or therapy with either agent alone	Combinations of i.v. FK463 and i.v. ampho-tericin B (1 plus 0.25 mg/kg and 2 plus 0.5 mg/kg respectively) enhanced antifungal effects as compared to treatment with either agent alone.

Efficacy against murine Pneumocystis carinii pneumonitis:FK463 was also highly effective for prevention of P.carinii pneumonitis in SCID mice leading to a complete absence of cyst forms of P.carinii and only minor injury to alveolar cells at the termination of the experiments (Ito et al., 1999). However, there was no effect on the trophozoite form as indicated by the detection of DNA-products of the organism in at least half of the animals.

Clinical studies

Phase I:The safety and pharmacokinetics of FK463 have been investigated in healthy adult male volunteers (Azuma et al., 1998) (Table 2). In the first part of the study, FK463 was administered as 2-hour intravenous infusion at dosages of 2.5, 5, 12.5, 25, or 50 mg to a total of 27 subjects. Plasma concentrations of FK496 after completion of the infusion declined in a biexponential pattern. The half-life ranged from 11.6 to 15.2 hours, total clearance from 0.192 to 0.225 mL/min/kg, and the volume of distribution from 0.215 to 0.242, respectively. A dose-proportional increase in Cmax and AUC0-24hr over the investigated dosage range was noted, indicating linear plasma pharmacokinetics of the drug. In the second part of the study, a cohort of 6 subjects received FK463 at a dosage of 25 mg once daily for a total of seven days. Modeling of the plasma data revealed a disposition that was best described by a 2-compartment open pharmacokinetic model. Steady state was achieved by day four after approximately 1.5 fold accumulation as determined by the AUC 0-24hr. The mean peak plasma level at day 7 was 2.46 ±0.27 µg/mL, AUC 0-24 29.6 ±4.6 µg/ml.h, half-life 14.6 ±1.5 hours, and total clearance 0.222 ±0.027 mL/min/kg, respectively. Less than 1% of drug was found in the urine in unchanged form (Azuma et al., 1998). At least six metabolites have been detected in both animals and humans to date; similar to caspofungin and VER-002, however, FK463 is not metabolized to a significant extent through the cytochrome P450 enzyme system (Groll et al., 1998). FK463 was well tolerated in both segments of this pharmacokinetic study (Azuma et al., 1998).

The safety and plasma pharmacokinetics of FK463 have been investigated in an ongoing phase I dose-escalation study in febrile neutropenic pediatric patients who received the compound for a mean of 8 days (range, 1 to 14 days) at dosages ranging from 0.5 to 3 mg/kg. Preliminary analysis of pharmacokinetic parameters at the 0.5 and 1.0mg/kg dosage level indicated linear disposition with a mean half life of 12 to 13 hours on day 1 and day 4 for 0.5mg/kg and on day 1 for 1.0 mg/kg; the half-life at day 4 for 1 mg/kg was variable resulting in a mean of 20.8 hours. The compound was well tolerated without evidence for differences in safety and efficacy compared to adults (Seibel et al., 2000).

A phase I, randomized, blinded, sequential group, dose-escalation tolerance study investigated FK463 plus fluconazole (400mg/day) vs. normal saline plus fluconazole 400 mg/day daily as prophylactic modality in adult patients receiving hematopoietic stem cell transplantation (Hiemenz et al., 1999). The study drug was started at the time of transplant and continued until neutrophil recovery. Seven dosage-levels of FK463 were studied: 12.5, 25, 50, 75, 100, 150, and 200 mg/day. At least 10 patients were enrolled per dosage group, with 8 individuals being randomized to the investigational regimen, and 2 to the control group. A total of 74 patients (investigational regimen: 62; control arm: 12) received at least one dose of study medication. The average length of dosing was 10 days (range, 1 to 27 days). FK463 was well tolerated; two patients (3.2 %) discontinued drug due to a possibly or probably related adverse event. No histamine-like reactions or infusion-related toxicity was noted. Overall, there were no changes from baseline to end of therapy in serum creatinine or liver function tests. Eighteen patients (29%) received alternate empirical antifungal therapy, and two patients developed fungal infections (3.2%). Pharmacokinetic investigations at day one and day 7 revealed dose-linearity and a half-life ranging from 11.3 to 13.9 hours at day 7.

Phase II:An open label, multicentre, dose de-escalation study has been completed to determine the safety and minimally effective dose of FK463 in HIV-infected patients with endoscopically documented Candida esophagitis (Pettengell et al., 1999). Doses of 50, 25, and 12.5 per day were evaluated. A total of 74 patients received FK463 as 1-hour infusion for a mean of 12 days (range, 1 to 21 days). In patients who received the drug for at least 8 days, resolution or improvement of clinical signs and symptoms was observed at end of therapy in 21/21 (100%) of patients receiving 50 mg, 18/20 (90%) receiving 25 mg, and 17/21 (81%) of patients receiving 12.5 mg. Three patients discontinued due to possible adverse events, and 1 discontinued due to lack of efficacy (12.5 mg). There was one serious adverse event reported (diarrhea and dehydration), that was assessed as possibly related to FK463. Infusion-related toxicity or reactions attributable to histamine release were not reported, and nephro- or hepatotoxicity was not observed.

Further dose escalation to a daily dosage of 75 and 100mg/kg and endpoint evaluation by endoscopy demonstrated consistent results with clearing or improvement of endoscopic lesions in 35/36 (97.2%) of patients least 10 days of therapy without safety issues (Pettengell et al. 2000)

Phase III:No phase III studies with FK463 have been completed and reported.

Conclusions

The preclinical and clinical data available to date strongly support the development of FK463 for treatment superficial and invasive Candida infections in immunocompetent, corticosteroid-immunosuppressed, and neutropenic patients. Based on its broad and fungicidal anti-Candida activity in neutropenic animals and the prolongation of survival in neutropenic animals in pulmonary and disseminated aspergillosis, the compound may also be useful for empirical antifungal therapy in persistently febrile neutropenic patients. FK463 and the other current echinocandins are only “fungistatic” against Aspergillus spp. Therefore, their role as single agent for treatment of proven invasive Aspergillus infections in the neutropenic host should be particularly carefully investigated. Due to the variable or lacking activity against other opportunistic moulds, these agents may not be suitable for preemptive therapy of clinically and radiographically suspected invasive mould infections. Nevertheless, FK463 and other echinocandins may prove useful for these indications in combination with agents that target the cell membrane, such as amphotericin B and the triazoles, and with future antifungal compounds.

The potential benefits of FK463 and other echinocandins for therapy of acute Pneumocystis carinii pneumonitis (Pcp) are difficult to predict because of the apparent specificity of these compounds for the cyst stage of the organism. However, while FK463 in intravenous form has limited practical applications for systemic Pcp-prophylaxis, aerosolized delivery of the compound might be useful for prevention of Pcp.

Andreas H. Groll, M.D., 
Senior Clinical Fellow, 
Pediatric Oncology Branch, 
National Cancer Institute, 
Bldg. 10, Rm. 13N240, 
10 Center Drive, 
Bethesda, MD 20891 / USA
Phone: (301) 435-3355, Fax: (301) 402-0575, Email: grolla@mail.nih.gov

Thomas J. Walsh (same location)

August 2001