Prophylaxis with Isavuconazole or Posaconazole Protects Immunosuppressed Mice from Pulmonary Mucormycosis

ABSTRACT

We assessed prophylactic or continuous therapy of isavuconazole, posaconazole, or voriconazole in treating pulmonary murine mucormycosis. In the prophylaxis studies, only isavuconazole treatment resulted in significantly improved survival and lowered tissue fungal burden of immunosuppressed mice infected with Rhizopus delemar. In the continuous treatment studies, isavuconazole and posaconazole, but not voriconazole, equally prolonged survival time and lowered tissue fungal burden compared to placebo-treated mice. These results support the use of isavuconazole and posaconazole in prophylaxis treatment.

TEXT

Mucormycosis is a life-threatening infection that occurs predominantly in immunocompromised patients (e.g., diabetic ketoacidosis [DKA] or neutropenia) and is caused by the ubiquitous Mucorales, among which Rhizopus is the predominant cause of infection (1–3). Despite disfiguring surgical debridement and adjunctive antifungal therapy, the overall mortality of mucormycosis remains approximately 50% and can approach 100% in hematogenously disseminated disease and central nervous system (CNS) disease as well as in patients with prolonged neutropenia (2, 4–8). Clearly, new strategies to prevent and treat mucormycosis are urgently needed.

In the past 2 decades, mucormycosis has emerged as an important invasive fungal infection in hematologic malignancies, bone marrow transplant (BMT) and solid-organ transplant (SOT) (9, 10). These high-risk patients are often receiving prophylaxis with the azole voriconazole (VOR) or posaconazole (POS). Although VOR is used as a first-line therapy against aspergillosis, it is ineffective against Mucorales. Indeed, outbreaks of mucormycosis cases in patients who received VOR prophylaxis are frequently described (6, 11–14). In contrast, POS has activity against Mucorales in vitro, but breakthrough mucormycosis infections (especially with Rhizopus species) among patients who received POS prophylaxis have also been reported (15, 16).

Isavuconazole (ISA) has recently been approved by the U.S. Food and Drug Administration (FDA) and the European Commission for treating invasive mucormycosis. We have demonstrated the efficacy of ISA in treating mice infected with Rhizopus delemar (17). Hence, we wanted to assess the prophylactic activity of ISA compared to POS and VOR in protecting neutropenic mice from pulmonary mucormycosis.

Animal studies described here were approved by the IACUC of the Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, according to the NIH guidelines for animal housing and care (18). Male CD-1 mice (20 to 25 g from Envigo, Indianapolis, IN, USA) were used in this study. They were given irradiated feed and sterile water containing 50 mg/liter Baytril (enrofloxacin: Bayer, Leverkusen, Germany) ad libitum on day −3 and then switched to daily ceftazidime (5 mg/mouse) subcutaneous (s.c.) treatment starting day 0 through day +13, relative to infection with R. delemar 99-880 (a brain isolate obtained from the Fungus Testing Laboratory at the University of Texas Health Science Center at San Antonio). Neutropenia was induced by cyclophosphamide (200 mg/kg of body weight, administered intraperitoneally [i.p.]) and cortisone acetate (500 mg/kg, s.c.) on days −2, +3, and +8 relative to infection. The treatment regimens resulted in ∼16 days of leukopenia with the total white blood cell count dropping from ∼130,000/cm³ to almost no leukocytes detected as determined by using a Unopette system (Becton-Dickinson and Co.). Whenever mice were treated with VOR (Pfizer Pharmaceuticals), they were given 50% grapefruit juice in lieu of drinking water to inhibit metabolism of the drug by cytochrome P450 enzymes (19).

Pharmacokinetic (PK) studies were conducted using ISA (prodrug isavuconazonium sulfate from Astellas USA), POS (oral suspension from Merck & Co., Inc.), and VOR at 110, 30, and 40 mg/kg, respectively. The doses for ISA and POS were chosen because of prior demonstration of activity in murine pulmonary mucormycosis (20, 21). Drugs were given orally three times a day (t.i.d.) for ISA and POS and once daily for VOR for 4 consecutive days. Four hours following the last dose of each drug, the mice were sacrificed, and total blood was collected by cardiac puncture. An analytical method that uses ultraperformance liquid chromatography and single-quad mass spectrometry (UPLC/MS) for the determination of itraconazole and hydroxylitraconazole was adapted and validated for the measurement of ISA and POS (20, 22), while high-performance liquid chromatography (HPLC) was used to measure VOR levels. All three drugs demonstrated serum drug levels in excess of MIC values (defined as the lowest drug concentration that causes 100% growth inhibition) against R. delemar 99-880 (MIC values of 0.5, 0.78, and 8.0 μg/ml for ISA, POS, and VOR, respectively [17, 23]) (Fig. 1A).

FIG 1.

ISA prophylaxis protects immunosuppressed mice from R. delemar pulmonary infection. (A) Serum azole concentration of mice (six or seven mice in each group) treated with ISA or POS three times daily or VOR once daily for 4 days at the specified doses. Sera were collected 4 h after the last dose. *, P < 0.05 compared to the value for the placebo-treated group (no drug) by the Wilcoxon rank sum test. (B) Survival of mice (20 mice for the placebo group and 9 or 10 mice for all other groups) treated prophylactically (day −2 until day 0 prior to infection) with ISA, POS, or VOR and then infected with R. delemar (inhaled inoculum of 4.1 × 10³ per mouse). **, P = 0.01 compared to the value for the placebo-treated group by the log rank test. (C) Fungal burdens in lungs and brains (9 to 11 mice per arm) harvested on day +3 postinfection. Data are expressed as median ± interquartile range. ‡, P = 0.03 compared to the value for the placebo-treated group by the Wilcoxon rank sum test.

Next, the efficacy of each of the azoles given as prophylaxis or continuous treatment at the doses investigated in the PK studies was determined. For prophylaxis, treatment started on day −2 and continued until day 0, when the mice were infected 4 h after the last treatment. For continuous therapy, treatment started on day −2 and ended on day +2 relative to infection. Immunosuppressed mice were infected intratracheally (23) with a targeted inoculum of 2.5 × 10⁵ spores of R. delemar 99-880. The primary endpoint for efficacy was the time to moribundity of infected mice, while tissue fungal burden in lungs and brains harvested from mice sacrificed on day +3 served as the secondary endpoint. Tissue fungal burden was determined by quantitative PCR (qPCR) (24).

In the prophylaxis studies, only ISA prolonged survival time of mice with pulmonary mucormycosis, albeit this protection was modest with 21-day survival of ∼20% for ISA versus 0% for placebo treatment and a median survival time of 15 versus 9 days for ISA and placebo treatment, respectively (Fig. 1B). The improved survival in ISA-treated mice was accompanied by an ∼0.5-log-unit reduction in the lung, but not the brain, fungal burden compared to placebo-treated mice (Fig. 1C). With the continuous therapy, both ISA and POS resulted in 50% long-term survival of mice infected with R. delemar, whereas VOR did not improve survival versus placebo (Fig. 2A). Protection elicited by ISA or POS was further corroborated by ∼1-log-unit reduction in lung and brain fungal burden versus placebo-treated mice (Fig. 2B).

FIG 2.

ISA or POS continuous therapy prolongs survival time and reduces tissue fungal burden of immunosuppressed mice with pulmonary mucormycosis due to R. delemar. (A) Survival of mice (20 mice in the placebo group and 10 mice for all other groups) treated continuously (day −2 through day +2) with ISA, POS, or VOR. Mice were infected on day 0 with R. delemar (inhaled inoculum of 4.1 × 10³ per mouse). Values for ISA- or POS-treated mice that are significantly different (P < 0.05) from the value for mice treated with placebo by the log rank test are indicated by an asterisk. (B) Fungal burdens of lungs and brains (9 to 11 mice per arm) were harvested on day +3 postinfection. Data are expressed as median ± interquartile range. **, P < 0.05 compared to the value for the placebo-treated group by the Wilcoxon rank sum test.

Previously we have established that the protective activity of ISA is similar to that of liposomal amphotericin B in treating immunosuppressed mice with pulmonary mucormycosis caused by Rhizopus or Mucor species (17, 20). The efficacious animal data combined with a recently conducted single-arm open-label clinical trial of ISA in the treatment of mucormycosis (25) and case-control analysis resulted in the FDA and European Commission approving ISA for treating invasive mucormycosis. In the present study, we wanted to compare the activity of ISA to currently used azoles with respect to protection against mucormycosis in the prophylaxis setting. At the doses and treatment regimen tested, we established that ISA, POS, and VOR had serum drug levels that exceeded their MICs against R. delemar. At these doses, ISA prophylaxis was superior to POS prophylaxis in protecting mice from pulmonary mucormycosis, while both drugs were equally efficacious in continuous therapy. The difference in efficacy between ISA and POS when administered prophylactically could be due to the possible altered drug levels in the target organs of lungs and brains. For example, it is possible that higher ISA exposure may have been achieved in the lungs and brains after 3 days of dosing compared to that of POS. This assumption is supported by the equal efficacy of ISA and POS when administered in continuous therapy, which lasted for 5 days of treatment.

As expected, the activities of ISA and POS administered as continuous therapy exceeded their respective activities when given as prophylaxis, since the effective treatment continued after infection. Continuous therapy is more likely to be clinically relevant, since it represents treatment of a breakthrough infection while patients are on prophylaxis.

In contrast, VOR was not efficacious in protecting mice from mucormycosis despite the fact that serum VOR levels were threefold higher than the MIC of 8 μg/ml. Prophylaxis of BMT patients with VOR has been widely linked to breakthrough infections with mucormycosis (26, 27), and it is acceptable that VOR does not possess activity against Mucorales. In fact, it has been shown that treatment of Rhizopus with VOR prior to infecting mice or Drosophila increases the virulence of this mold (28). In summary, given their broad-spectrum activities, our findings support the use of ISA and POS in breakthrough infections in patients who are at high risk of contracting mucormycosis.