Impact of Human Serum on Pharmacodynamic Action of Echinocandins against Aspergillus spp. : an in Vitro Study

Hussam Wahab Al-Humadi
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Keywords : growth inhibition, Aspergillus, galactomannan, caspofungin, micafungin, anidulafungin.
Medical Journal of Babylon  12:3 , 2015 doi:1812-156X-12-3
Published :18 November 2015

Abstract

Echinocandins represent a class of lipopeptide antifungal agents that possess a high protein binding capacity which could have an impact on their activity. This study was focusing on the role of human serum on the activities of echinocannins (caspofungin, micafungin, and anidulafungin) against Aspergillus species (A.fumigatus, A.flavus and A.terreus). The aspergillus growths have been assessed by measuring minimal effective concentrations of each (caspofungin (0.5-1 mg/L), micafungin (0.06-0.12 mg/L) and anidulafungin (0.03-0.09 mg/L). Broth microdilution method was used to study in vitro activities based on the Clinical and Laboratory Standards Institute documentation (CLSI M38-A2) by using standard medium (Roswell Park Memorial Institute (RPMI 1640)) with or without 50% human serum. Each drug was diluted ranging from 0.03-4 mg/L, inoculated with 2x104 Colony Forming Units/ml for each isolate, then incubated at 37?C for 10 hrs to A.fumigatus and A.flavus and 15 hrs to A.terreus. The fungal growth was assessed by measuring the galactomannan index for each concentration with ELISA (Platelia Aspergillus); percentage of fungal growth inhibition was measured from galactomannan index and analyzed as maximal growth inhibition (Emax) and drug concentration with 50% of growth inhibition (IC50). IC50 values of echinocandins were ranged from 0.03-0.06 mg/L without serum and 0.09-0.25 mg/L in presence of serum while ?max values were 50(44-56)% for A.fumigatus, 70(65-74)% for A.flavus and 65(63-66)% for A.terreus without serum and 94(92-96)% for A.fumigatus, 92(91-95)% for A.flavus and 80(75-83)% for A.terreus with human serum. The study concluded the effective concentrations of all echinocandins were increased concomitantly with more than 90% fungal growth inhibition with human serum.

Introduction

Echinocandins are regarded one of a new class of antifungal agents that is often used in the combination with other antifungal drugs for the treatment of invasive aspergillosis which is considered as a life threatening disease in prolonging and profound immunocompromised patients [1]. High protein binding capacity of these agents is changed their pharmacological activity. Several studies with antibiotics have demonstrated that the role of free or unbound drug is always available for antibiotic activity [2-5]. Moreover, numerous studies reported that pharmacodynamic effects of echinocandins have been increased in the presence of human serum not correlated with minimal inhibitory concentration (MIC) increases in concomitant with high protein binding capacity of echinocandins (ranging between 96-98%) [6-8]. The endpoints of susceptibility testing for the echinocandins are difficult to determine while the end point for other antifungal classes are easier by using of minimal inhibitory concentration methods [9]. However, a clear in vitro-in vivo clinical correlation is still lacking [10]. The fluctuation of drug concentrations on in vivo environment does not correlate well with in vitro study, where in vivo the drugs have interacted with the fungus in addition to the presence of human serum and tissues. Thus, in the presence of serum, the antifungal effects may be changed by using the same agent that were not seen in vitro without serum [11,12]. In addition, a clinical study found that the infections with A. fumigatus isolates with minimal effective concentrations (MECs) (0.125-8 µg/ml) in patients with haemopoitic stem cell transplantation receiving caspofungin prophylaxis leading to empirical treatment as breakthrough reported [13] while on in vivo study of an animal model with A. fumigatus infection suggested decreasing in susceptibility to caspofungin with high doses possibly by upregulation of the level of target enzyme [14]. This study investigated the in vitro pharmacodynamic changes of the antifungal activities of echinocandins (caspofungin, micafungin and anidulafungin) in presence of human serum against three clinical isolates Aspergillus species (A. fumigatus, A. flavus and A. terreus) were investigated.

Materials and methods

Antifungal Drugs:
  Caspofungin (Merck-USA), Micafungin (Astellas-USA) and Anidulafungin (Pfizer-USA) were prepared as stock solutions and dissolved in either dimethyl sulfoxide for anidulafungin or water for caspofungin and micafungin, and stored at -70oC until use. The Clinical and Laboratory Standards Institute (CLSI M38-A2) method, [15] with serial dilutions and the final concentration were prepared in double-strength test medium (Roswell Park Memorial Institute (RPMI 1640) medium buffered with morpholine propane sulfonic acid (MOPS) [0.165M], pH=7.0).
Isolates and Culture Media:
  The clinical isolates of each A.fumigatus, A.flavus and A. terreus were used. Inocula from 5-7 days-old cultures in Sabouraud dextrose agar were prepared in saline or human serum and adjusted twice with Neubauer counting chamber to get the final inoculum (2x104 Colony Forming Units (CFU)/ml).
The pharmacodynamic activities of the echinocandins were tested with a broth microdilution method[15] by using standard medium (RPMI 1640, 0.165 M [MOPS], pH=7.0) either with 50% (v/v) pooled human serum or without.
 
Human Serum Preparation:
  Whole human blood samples were centrifuged at 4,000 X g for 10 min, and the supernatant serum was taken and then incubated in a water bath at 56°C for 30 min with constant swirling. The serum sample was stored at -70°C that should be used within 7 to 10 days [16].
Susceptibility Testing:
 Tested plates were prepared in advance by dispensing 100 ?l of twofold serially diluted drugs ranging from 4 to 0.03 mg/L into 96-well flat-bottom microdilution plates and stored at -70oC. On day of testing, plates were thawed and inoculated with 2x104 CFU/ml in saline or human serum. The plates were incubated at 37?C for 10 hrs for A.fumigatus and A.flavus and 15 hrs for A. terreus [17].  The fungal growth was assessed by measuring the galactomannan index (GI) for each drug concentration with ELISA (Platelia Aspergillus; Bio-Rad-USA) [18] by taking 5 ?l from each well of microplate and diluted in 5 ml of normal saline (1:1000) then measured GI by ELISA. GI was inversely proportional with percentage (%) of fungal growth inhibition.
 
Statistical Analysis
   The % of fungal growth inhibition was analyzed by using nonlinear regression analysis with GraphPad Prism 5.0 Software. The sigmoidal slope of Emax model was described by drug concentration corresponding to 50% of growth inhibition (IC50).
The Emax and IC50 values were tested for each drug concentration and isolate without human serum and compared the results with those with human serum by plotting the log10 for each concentration of each echinocandins against the % of growth inhibition for each species of Aspergillus [Figure 1], [Figure 2], [Figure 3].

Results
IC50s values of the in vitro activities of three Echinocandins :
   The IC50 values of all three echinocandins were 0.05 (0.03-0.06) mg/L without serum and 0.21 (0.09-0.5) mg/L with human serum showing a significant mean increase [Table 1].  The addition of human serum increased the IC50 of echinocandins by 0.16 (0.09-0.19) mg/L for A.fumigatus [Figure 1], 0.24 (0.06-0.44) mg/L for A.flavus [Figure 2] and 0.1 (0.03-0.21) mg/L for A. terreus [Figure 3]. The largest increase in IC50s values was found with A.flavus and anidulafungin (8.0-fold) and the smaller with A. terreus and caspofungin (one-fold) [Table1].
?max Values of The In Vitro Activities of Three Echinocandins:
The ?max values of all three echinocandins were 50 (44-56)% for A.fumigatus [Figure 1], 70 (65-74)% for A.flavus [Figure 2] and 65(63-66)% for A. terreus [Figure 3] without serum and 94 (92-96) % for A.fumigatus [Figure 1], 92(91-95)% for A.flavus [Figure 2] and 80 (75-83)% for A. terreus [Figure 3] with serum [Table 2]. The addition of human serum increased the Emax of echinocandins by 44 (36-50)% for A.fumigatus [Figure 1], 22 (18-30)% for A.flavus [Figure 2] and 15 (9-21)% for A. terreus [Figure 3], resulting complete growth inhibition at concentrations 1-4 mg/L [Table 2]. The largest increase in Emax value was found with A.fumigatus and micafungin (1.6-fold) [Figure 1-B] while the smaller with A. terreus and anidulafungin (0.12-fold) [Figure 3-C].




Results

IC50s values of the in vitro activities of three Echinocandins : The IC50 values of all three echinocandins were 0.05 (0.03-0.06) mg/L without serum and 0.21 (0.09-0.5) mg/L with human serum showing a significant mean increase [Table 1]. The addition of human serum increased the IC50 of echinocandins by 0.16 (0.09-0.19) mg/L for A.fumigatus [Figure 1], 0.24 (0.06-0.44) mg/L for A.flavus [Figure 2] and 0.1 (0.03-0.21) mg/L for A. terreus [Figure 3]. The largest increase in IC50s values was found with A.flavus and anidulafungin (8.0-fold) and the smaller with A. terreus and caspofungin (one-fold) [Table1]. ?max Values of The In Vitro Activities of Three Echinocandins: The ?max values of all three echinocandins were 50 (44-56)% for A.fumigatus [Figure 1], 70 (65-74)% for A.flavus [Figure 2] and 65(63-66)% for A. terreus [Figure 3] without serum and 94 (92-96) % for A.fumigatus [Figure 1], 92(91-95)% for A.flavus [Figure 2] and 80 (75-83)% for A. terreus [Figure 3] with serum [Table 2]. The addition of human serum increased the Emax of echinocandins by 44 (36-50)% for A.fumigatus [Figure 1], 22 (18-30)% for A.flavus [Figure 2] and 15 (9-21)% for A. terreus [Figure 3], resulting complete growth inhibition at concentrations 1-4 mg/L [Table 2]. The largest increase in Emax value was found with A.fumigatus and micafungin (1.6-fold) [Figure 1-B] while the smaller with A. terreus and anidulafungin (0.12-fold) [Figure 3-C].

Discussions

Echinocandins are used as salvage therapy against invasive aspergillosis, but some recent studies suggested their effectiveness as a first line therapy in the treatment of the invasive Aspergillosis [19]. This increase in effectiveness may be due to synergy between antimicrobial factors present in serum and echinocandins. Serum has a direct effect on antimicrobial activity by altering their ability to interact with the target tissues; in addition, the inhibition of glucan synthase by echinocandins could be predicted by the percentage of their protein binding [8]. Indeed, the addition of the serum enhanced the echinocandins’ action to a 45%, 22% and 18% of their in vitro inhibitory activities against A.fumigatus, A.flavus and A. terreus respectively [Table 2] with concs. higher than the MEC and complete growth inhibition with no paradoxical effects; these results are supported by the results obtained by Odabasi et al [7] who tested echinocandins activities against only A.fumigatus with 50% enhancement in presence of serum. Other researchs showed that caspofungin has in vivo activity similar to that of anidulafungin, [20] while in vitro IC50 without serum indicated potency three times less than with serum [Table 1]. Moreover, animal studies showed that 5 mg/kg of caspofungin or micafungin resulted in similar in vivo outcomes (spleen and kidney fungal burden and survival) when caspofungin concentrations were two-times higher than those of micafungin in vitro [21]. Moreover, micafungin showed the greatest increase of Emax activity against only 4-fold increase in IC50 (0.03 versus 0.12 mg/L) in the presence of 50% human serum especially against A.fumigatus [Figure 1-B] [22] while anidulafungin exhibited the smallest increase in Emax activity against more than 8-fold differences (0.06 versus 0.25 mg/L) in the presence of 50% human serum, especially against A.flavus and A. terreus [Figure 2-C; Figure 3-C] [23]. Furthermore, the current study exhibited that the action of echinocandins was low in the presence of 50% human serum at lower near-MEC concentrations (<0.5 mg/L) in comparison with absence of human serum. This decrease of activity could be explained by the free-drug hypothesis, considering that echinocandins are highly protein-bound drugs.

Conclusions

The maximum activity of echinocandins was enhanced in presence of 50% human serum leading to the complete inhibition of fungal growth at concentrations ranging from 1 to 4 mg/L oppositely to less than 60% fungal growth inhibition without human serum with same concentration.

References

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