Background
Invasive pulmonary aspergillosis (IPA) is mainly caused by
A. fumigatus and has a high morbidity and mortality in high-risk immunosuppressed patients [
1]. The incidence of IPA varies according to the underlying disease, being up to 24% in patients with acute leukemia, up to 10% in patients with allogeneic hematopoietic stem cell transplantation, and up to 7% in patients with lymphoid malignancies [
2]. Additionally, the mortality rate of IPA is reportedly as high as 36–75% in patients with hematological malignancies despite the application of antifungal agents such as triazoles and echinocandins [
3‐
5]. Thus, antifungal prophylaxis is currently recommended in high-risk immunosuppressed patients [
6,
7].
Currently, no reported study has investigated how to develop an optimal antifungal prophylactic dosage regimen, and so the most common practice in clinical is to apply the recommended dosage regimen for prophylaxis. Theoretically, an understanding of the pathogenesis of
A. fumigatus could provide clues for designing prophylactic antifungal regimen that are effective at preventing IPA. Inhalation is the primary route for acquiring
Aspergillus spores. Pulmonary epithelial cells are the first cells encountered by the inhaled spores, which then germinate and grow in these cells, followed by the development of serious IPA disease in immunosuppressed patients [
8]. Therefore, pulmonary epithelial cells can be viewed as the target of prophylaxis in IPA.
Voriconazole is a triazole exhibiting broad-spectrum antifungal activity against
Aspergillus species [
9], and is approved as the first-line therapy for invasive aspergillosis [
6,
10]. The Infectious Diseases Society of America recommends using voriconazole for prophylaxis against invasive aspergillosis in high-risk patients, such as patients with prolonged neutropenia, patients with graft-versus-host disease, and lung transplant patients (strong recommendation and moderate-quality evidence) [
6]. In addition, voriconazole is safe and effective for the secondary prophylaxis of systemic fungal infection in patients receiving allogeneic stem cell transplantation [
11]. It has been reported that the concentration of voriconazole in the pulmonary epithelial lining fluid (ELF) crucially influences the prevention of IPA [
12]. However, the ability of voriconazole penetrate the ELF varies widely in patients, with concentrations ranging from 0 to 83 mg/L being found in transplant patients receiving the recommended oral dosage regimen [
13,
14]. While the ELF is on the surface of the pulmonary epithelial cells [
15], the activity of voriconazole against
A. fumigatus in these cells is unclear, which hinders the ability to design an optimal prophylactic dosage regimen for voriconazole in immunosuppressed patients.
We established a model of infected human pulmonary epithelial cells caused by A. fumigatus conidia with the following aims: (1) to determine the cellular pharmacokinetics/pharmacodynamics (PK/PD) characteristics of voriconazole in pulmonary epithelial cells, and (2) to identify a strategy for designing prophylactic regimen of voriconazole that are effective at preventing IPA.
Discussion
Pulmonary epithelial cells invaded by
A. fumigatus conidia in human body is the first step of causing IPA, and IPA is still related to high rates of morbidity and mortality after the utilization of antifungal agents [
3‐
5]. Therefore, how to design the optimum prophylactic dosage regimen to prevent IPA is an intractable problem in clinical practice. Voriconazole has a strong activity against and it is vital to explore the intracellular activity of voriconazole against
A. fumigatus in pulmonary epithelial cells. This study built the cells model infected by
A. fumigatus to investigate, firstly, the PK/PD property of voriconazole in infected cells, and then use the Monte Carlo simulation and PK/PD breakpoint to design the prophylactic dosage regimen. This research provided a potential mechanism of voriconazole preventing IPA on a cellular level and provided a novel theoretical and experimental method for the designation of voriconazole prophylactic dosage regimen and the reduction of IPA morbidity.
Previous pharmacodynamics studies have used galactomannan (a major constituent of aspergillus cell walls) [
22] as a biomarker for assessing the antifungal activity against
A. fumigatus [
23‐
25]. The present study found a distinct relationship between the voriconazole concentration and the galactomannan index for the two
A. fumigatus strains (Fig.
1). In contrast to previous studies, [
23‐
25] we did not calculate the pharmacodynamics parameters based on the relationship between the voriconazole concentration and the galactomannan index. One of the limitations in semiquantitative assessment is that the content of galactomannan in the medium cannot precisely reflect the number of
A. fumigatus conidia in cells. Thus, the number of
A. fumigatus conidia in A549 cells was determined in this study by performing quantitative culturing in a subsequent step.
A. fumigatus conidia in A549 cells were exposed for 24 h to voriconazole over a wide range of concentrations to determine the overall pattern of intracellular activity. The data obtained in this study of voriconazole cellular pharmacodynamics properties have not been reported previously in detail. The E
max values of voriconazole for the AF293 and AF26 strains were 0.79 and 0.84 lg cfu, respectively, showing a decrease compared with the initial intracellular inoculum (Table
1), which meant that 78.20 and 85.57% of intracellular
A. fumigatus conidia were suppressed within 24 h for AF293 and AF26, respectively. At the infinitely low C
e, the number of intracellular
A. fumigatus conidia increased by 63.63 and 51.28% for AF293 and AF26, respectively. When the data were plotted against C
e, the C
s calculated using the sigmoid model was 0.52 mg/L for AF26 and 1.86 mg/L for AF293, respectively. A particularly interesting observation was how close the intracellular pharmacodynamics parameters of voriconazole against AF293 and AF26 became when the data were plotted against C
e/MIC, with C
s was 8.32-fold MIC for AF26 and 9.69-fold MIC for AF293, and E
50 was 32.93-fold MIC for AF26 and 36.71-fold MIC for AF293 (Table
1 and Fig.
2b). This result suggests that C
e and the inherent susceptibility of
A. fumigatus were the key factors influencing the voriconazole activity against
A. fumigatus in the intracellular milieu. In other words, the C
e/MIC ratio might be a crucial determinant of the voriconazole intracellular activity against
A. fumigatus.
Through the above analysis we were able to demonstrate that C
e/MIC might be the best predictor of the voriconazole intracellular antifungal effect. To our knowledge, the present study is the first to use infected A549 cells combined with an inhibitory effect sigmoid E
max model to study the PK/PD breakpoint. All of the data for the voriconazole intracellular activity against AF293 and AF26 are summarized in Fig.
3. The model showed that up to 84.01% of the initial intracellular inoculum was suppressed by voriconazole within 24 h (Fig.
3a). Given that 15.99% of the initial intracellular inoculum (about 4.4 lg cfu conidia) survived in the cells indicates that voriconazole could not completely suppress intracellular
A. fumigatus even at higher incubation concentrations. This phenomenon may be due to the saturation of voriconazole absorption and the low voriconazole concentration in A549 cells [
17,
26].
In the present study, we firstly found that the PK/PD breakpoint in the infected A549 model predicting the intracellular antifungal effect of voriconazole was C
e/MIC = 35.53, which was associated with a 50% suppression of intracellular
A. fumigatus (Fig.
3b). Previous studies have shown that PK/PD parameters of antimicrobial agents could be useful for designing dosage regimens. Thus, C
e/MIC = 35.53 was utilized as the PK/PD index for the subsequent Monte Carlo simulation to design the prophylactic regimen for voriconazole. For cases of IPA that are mostly caused by airborne
A. fumigatus, knowledge of the epidemiology of
A. fumigatus is pivotal and could be used to underpin prophylactic strategies. The value of cumulative fraction of response is an estimate of the proportion of a population achieving the target PK/PD index. The results of Monte Carlo simulation showed that the effective suppression of intracellular
A. fumigatus at least requires an oral dosage of 200 mg of voriconazole twice daily, which yielded a CFR value of 91.48% (Table
2). Actually, the Monte Carlo simulation result has been largely confirmed in clinical applications, because the dosage regimen has been widely demonstrated to be effective at preventing IPA [
27‐
31]. For example, an oral dosage of 200 mg of voriconazole twice daily has been described as an appropriate and effective prophylactic agent in children and adults with acute myeloid leukemia or myelodysplastic syndrome and in those undergoing hematopoietic stem cell transplantation or solid-organ transplantation [
32‐
35]. This study has also been demonstrated that the C
e/MIC breakpoint is a rational metric to use when designing dosage regimens for patients receiving voriconazole for prophylaxis.
The pulmonary ELF is on the surface of pulmonary epithelial cells and the concentration of an antifungal agent within the ELF is critical to the prophylaxis and treatment of the early stages of IPA [
12,
36]. Clinically, patients typically take 200 mg of voriconazole orally twice daily—which is the recommended dosage regimen on the package insert—during prophylaxis applications. However, there was a lot of sub-therapeutic concentrations among some patients taking the oral dosage of 200 mg twice daily. For example, the concentration was adequate (< 1.0 mg/L) in 45% of allogeneic hematopoietic stem cell transplantation recipients [
37]. Based on in vitro susceptibility data, the MIC
90 (MIC at which 90% of the strain was inhibited) for voriconazole against
A. fumigatus was 0.5 mg/L [
38] and the C
e/MIC value was 35.53. We can thus calculate that the target voriconazole concentration in ELF for prophylaxis is 17.77 mg/L. However, the actual voriconazole concentration in ELF is difficult to measure, and it is much easier to measure it in plasma. According to the mean value of R
ELF/plasma, we calculated that the target voriconazole plasma concentration was 1.55 mg/L. A prospective, observational study showed that voriconazole trough concentration higher than 1.5 mg/L was the most effective for prophylaxis [
39], which was close to the result of present study. Notably, when applying these results to clinical practice, we should characterize the local epidemiology of
A. fumigatus and monitor the concentration of voriconazole in ELF or plasma, which would ensure the C
e/MIC > 35.53 for voriconazole against most isolates of
A. fumigatus in patients receiving voriconazole prophylaxis. However, the relationship between C
e/MIC and clinical efficacy still needs to be verified in further clinical studies.
This study was subject to some major limitations. (1) The R
ELF/plasma value from the previous two studies may be somewhat unreliable due to the inclusion of only 20 samples; (2) currently the mechanism underlying how voriconazole is loaded into pulmonary epithelial cells remains unknown. The present study used extracellular voriconazole concentrations modelled on those reported in ELF fluid, whereas intracellular voriconazole may come from both ELF and plasma; (3) for the reliability of the results, additional experiments should be carried out in pulmonary epithelial cells different from A549. However, the method for building a cell model infected by
A. fumigatus in this study is based on the study of Wasylnka et al. [
16]., and the this model was proved to be reasonable and reliable in previous studies [
17,
26,
40,
41]. Therefore, this cells model could afford a reliable result for this study.