Gamma scintigraphy imaging of murine invasive pulmonary aspergillosis with a 111In-labeled cyclic peptide

https://doi.org/10.1016/j.nucmedbio.2008.12.004Get rights and content

Abstract

Introduction

Invasive pulmonary aspergillosis (IPA) is a leading cause of infection-associated death in immunosuppressed patients. Early detection and early administration of antifungal therapy are critical factors in improving outcome for patients with IPA. Here, we evaluated the imaging properties of a 111In-labeled cyclic peptide targeted to Aspergillus fumigatus in an immunosuppressed murine model of IPA.

Methods

A cyclic peptide c(CGGRLGPFC)-NH2 was labeled with 111In by means of diethylenetriaminepentaacetic acid (DTPA). Two days after intranasal inoculation of 17.5×106 conidia of A. fumigatus, mice were injected 111In-DTPA-c(CGGRLGPFC)-NH2 intravenously. Biodistribution data were obtained at 2 h, and γ-images were acquired at 10 min and 2 h after radiotracer injection. Healthy mice were used as controls. In addition, a group of infected mice were co-injected with the radiotracer and unlabeled c(CGGRLGPFC)-NH2 to evaluate the inhibition of radiotracer's binding to infected lungs. Autoradiographs of lungs from infected and healthy mice were compared with corresponding photographs of transaxial sections of the lung tissues stained for A. fumigatus hyphae.

Results

The labeling efficiency was >98%, with specific radioactivity of up to 74 MBq/nmol peptide. Significantly higher uptake of 111In-DTPA-c(CGGRLGPFC)-NH2 was observed in the lungs of mice infected with A. fumigatus than in those of healthy mice (0.37±0.06 %ID/g vs. 0.14±0.02 %ID/g, P=.00044). Simultaneous injection with unlabeled peptide reduced radioactivity in the infected lungs by 41% (P=.0037). Increased radioactivity in the lungs of infected mice was visible in γ images at both 10 min and 2 h after radiotracer injection. Moreover, autoradiography confirmed radiotracer uptake in infected lungs, but not in the lungs of healthy mice or infected mice co-injected with unlabeled peptide.

Conclusions

γ-Imaging with 111In-DTPA-c(CGGRLGPFC)-NH2 clearly delineated experimental IPA in mice. Peptides directly targeting fungi therefore may be valuable agents for noninvasive detection of opportunistic mycoses.

Introduction

Invasive pulmonary aspergillosis (IPA) is an important cause of morbidity and mortality in immunocompromised patients [1], [2], [3], [4]. As the pathogenesis of IPA involves inhalation of airborne conidia by susceptible hosts, pneumonia is the most common clinical manifestation of IPA [5], [6]. In high-risk patients (e.g., those with acute leukemia or those who have received a bone marrow transplant), IPA is associated with a mortality rate of 42–83%, despite the administration of systemic antifungal therapy [7], [8]. The high mortality rate associated with IPA can be attributed to the immunosuppression of the affected patients, the typically late diagnosis and the suboptimal in vivo efficacy of antifungal agents against Aspergillus species. Thus, early detection and early administration of antifungal agents, i.e., when the tissue fungal burden is relatively low, may help improve the outcomes of patients with IPA. Because of the high mortality rate of Aspergillus infection, there is an urgent need to develop new strategies for the early diagnosis of IPA.

In cases of suspected pulmonary infection, computed tomography (CT) provides high-quality anatomic information, but in the absence of structural morphological changes in the lungs, it is difficult to diagnose a pulmonary infection in its early stages by CT alone. Other diagnostic methods, e.g., fungal culturing and non–culture-based methods such as serodiagnosis or polymerase chain reaction, may also be used [5], [9], [10], [11]. The sensitivity and specificity of these methods, however, remain suboptimal. Nuclear imaging techniques may be a valuable alternative in the diagnosis of IPA. Several nuclear imaging techniques have been evaluated for use in the diagnosis of fungal infections. In one approach, diagnosis is made based on structural and physiological changes in the lung induced by the invading microorganisms. An example of this approach is the injection and scanning of 67Ga-citrate, which binds to circulating transferrin and extravasates at the site of infection because of the increased vascular permeability [12]. Radiolabeled white blood cells (WBC) that migrate toward and infiltrate the inflammatory and infectious lesions have also been used for imaging fungal infections. WBC can be labeled either ex vivo [13], [14] or in vivo [15], [16]. In vivo labeling of WBC can be achieved using 99mTc-labeled antigranulocyte antibody tracers [17], [18]; 99mTc-labeled interleukin-8 (IL-8), which binds to IL-8 receptors [15]; or 111In-labeled leukotriene B4 (LTB4) antagonist, which targets LTB4 receptors [16]. However, because in vivo cell labeling depends on radiolabeled materials' attaching to receptors expressed on neutrophils, this approach might have limited utility in the diagnosis of IPA in severely neutropenic patients. [18F]Fluoro-2-deoxy-d-glucose (18F-FDG) accumulates avidly in metabolically active inflammatory cells [19]. Recent evidence suggests that 18F-FDG positron emission tomography (PET) imaging could be a useful tool in the diagnosis and management of opportunistic infections, including fungal infections in immunocompromised patients [20], [21]. However, diagnosis of fungal infection with 18F-FDG PET is not specific and is prone to errors (i.e., patients are often misdiagnosed as having a malignancy) [22].

An alternative approach for nuclear imaging of fungal infection, which may offer better detection specificity as compared to the methods described above, exploits the differences between fungi and normal host tissues or with bacteria. In this study, we investigated a 111In-labeled cyclic peptide that directly targets A. fumigatus in a clinically relevant model of IPA. The cyclic peptide c(CGGRLGPFC)-NH2 was identified through bacteriophage display technology and was found to bind in vitro to the surface of conidia and hyphae of A. fumigatus [23]. Radiolabeled peptides are promising nuclear imaging agents because of their pharmacokinetic properties, rapid binding and relatively low immunogenicity [24]. Our data suggest that 111In-labeled c(CGGRLGPFC)-NH2 can selectively accumulate in infected lungs; therefore it may facilitate diagnostic imaging of A. fumigatus infection.

Section snippets

Materials

All Nα-Fmoc amino acids, 1-hydroxybenzotriazole (HOBt), diisopropylcarbodiimide (DIC), triisopropylsilane (TIS) and Fmoc-Rink linker, were purchased from Novabiochem (San Diego, CA, USA). N,N-Diisopropylethylamine, trifluoroacetic acid (TFA), ethylenediaminetetraacetic acid (EDTA), cyclophosphamide and cortisone acetate were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). Aminobenzyl diethylenetriaminepentaacetic acid (DTPA-Bz-NH2) was obtained from Macrocyclics (Dallas, TX, USA).

Radiolabeling

DTPA-c(CGGRLGPFC)-NH2 was labeled with 111InCl3 in 0.1 M NaAC buffer at pH 5 (Fig. 1). Radiolabeling efficiency was 98.8±0.55%. Maximum specific activity was 8.5×1010 MBq/mol (74 MBq/μg). Fig. 2 shows the radiochromatograph and the UV/Vis chromatograph of 111In-DTPA-c(CGGRLGPFC)-NH2.

Stability of 111In-DTPA-c(CGGRLGPFC)-NH2

Negligible radioactivity was lost from 111In-DTPA-c(CGGRLGPFC)-NH2 after 4-h incubation in PBS containing either EDTA or human serum. Even after 24 h of incubation, greater than 97% of radioactivity was associated

Discussion

Our findings suggest that imaging of A. fumigatus in the lungs is feasible using 111In-DTPA-c(CGGRLGPFC)-NH2. c(CGGRLGPFC)-NH2 could be labeled with 111In through the DTPA chelator with high radiolabeling efficiency and high stability. Using an established murine model of IPA, we found that 111In-DTPA-c(CGGRLGPFC)-NH2 was selectively localized to lungs infected with A. fumigatus. The uptake of the radiotracer in the lungs of the infected mice was more than twice that in the lungs of the healthy

Conclusions

This study is the first to demonstrate the feasibility of specific nuclear imaging of fungal infection in a murine model of IPA using a radiolabeled peptide, c(CGGRLGPFC)-NH2, selected from a bacteriophage display library. The ability of this small cyclic peptide to accumulate in and delineate A. fumigatus colonies in the lungs emphasizes the specificity of its binding. Future work is needed to optimize its imaging properties through peptide mimetics approach, to determine the immunogenicity of

Acknowledgments

The authors thank Dr. Juri Gelovani for helpful discussion, and Lionel Santibañez for editing this manuscript.

References (35)

  • LatgeJ.P.

    Aspergillus fumigatus and aspergillosis

    Clin Microbiol Rev

    (1999)
  • SoubaniA.O. et al.

    Invasive pulmonary aspergillosis following bone marrow transplantation: risk factors and diagnostic aspect

    Haematologia (Budap)

    (2002)
  • EllisM.

    Invasive fungal infections; evolving challenges for diagnosis and therapeutics

    Mol Immunol

    (2001)
  • KhotP.D. et al.

    Development and optimization of quantitative pcr for the diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid

    BMC Infect Dis

    (2008)
  • TsanM.F.

    Mechanism of gallium-67 accumulation in inflammatory lesions

    J Nucl Med

    (1985)
  • PalestroC.J. et al.

    Combined labeled leukocyte and technetium-99 m sulfur colloid bone marrow imaging for diagnosing musculoskeletal infection

    Radiographics

    (2006)
  • PellegrinoD. et al.

    Inflammation and infection: imaging properties of 18F-FDG-labeled white blood cells versus 18f-fdg

    J Nucl Med

    (2005)
  • Cited by (19)

    View all citing articles on Scopus

    This research was supported in part by John S. Dunn Foundation.

    1

    On leave from the Department of Nuclear Medicine, Peking University School of Oncology, Beijing Cancer Hospital, Beijing 100142, China, and from the Isotope Department, China Institute of Atomic Energy, Beijing 102413, China.

    2

    These authors contributed equally to this work.

    View full text