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Ann Thorac Surg 1999;68:2044-2048
© 1999 The Society of Thoracic Surgeons
a Department of Cardiothoracic and Vascular Surgery, University of Texas-Houston, Houston, TX, USA
b Departments of Medicine, Surgery, Microbiology, Immunology, Molecular Physiology, and Biophysics, Baylor College of Medicine, Houston, Texas, USA
Address reprint requests to Dr Letsou, Department of Cardiothoracic and Vascular Surgery, The University of Texas-Houston Medical School, 6431 Fannin St, Suite 1.220, Houston, TX 77030
Presented at the Forty-fifth Annual Meeting of the Southern Thoracic Surgical Association, Orlando, FL, Nov 12–14, 1998.
Abstract
Background. The results of pulmonary transplantation are compromised by acute and chronic rejection. We hypothesized that a liposomal form of aerosolized cyclosporine A (CsA) would be selectively deposited and concentrated in the lungs. The theoretical advantage of this therapy is selective pulmonary immunosuppression with prolonged utilization.
Methods. Eighteen dogs were endotracheally intubated; aerosolized liposomal CsA was administered for 15 min. CsA levels were measured in whole blood, lung, trachea, heart, kidney, liver, and spleen at various times after treatment.
Results. The lung rapidly absorbs aerosolized liposomal CsA; other organs have much lower concentrations. The retention of pulmonary CsA delivered by liposome aerosol is approximately 120 min in this model.
Conclusions. Aerosolized liposomal CsA is selectively deposited and concentrated in the lungs; other organs absorb less CsA.
Pulmonary transplantation is a widely accepted technique for the treatment of end-stage pulmonary disease. However, long-term results are not optimal; improved methods of immunosuppression and new therapeutic agents are constantly under investigation.
Another possibility for improving the clinical results of pulmonary transplantation is the application of currently used medications via an aerosolized route. Experimental work from the University of Pittsburgh in an animal model using an aerosolized form of cyclosporine dissolved in ethanol administered via a nebulizer limited acute early rejection [1]. Clinical investigation of a similar aerosolized form of cyclosporine dissolved in ethanol by Keenan and colleagues showed some effectiveness in the treatment of refractory acute graft rejection [2]. Aerosolized ethanolic cyclosporine therapy can ameliorate histologic rejection and improve pulmonary function [3]. Further studies using aerosolized cyclosporine in propylene glycol reported deposited dose-related attenuation of acute lung rejection [4]. However, administration of aerosolized medications dissolved in solvents such as ethanol or propylene glycol may not be the best method for pulmonary absorption, and they are often poorly tolerated in humans. This form of therapy may not be optimally suited for long-term management of allograft rejection.
We investigated the pharmacokinetics of cyclosporine A dilauroylphosphatidylcholine (CsA-DLPC), a liposomal form of cyclosporine developed in our laboratories. Liposomal formulations of medications have higher absorption in the lungs and are well tolerated clinically. They also have other advantages including aqueous compatibility, facilitated intracellular delivery (particularly to alveolar macrophages and lymphocytes), and sustained release [5]. The reduced toxicities of aerosolized liposomal cyclosporine suggest that this formulation would be preferable for the chronic treatment of pulmonary rejection [6]. Because such liposomal preparations have not been used in pulmonary transplant recipients, we performed a feasibility study to determine the deposition patterns and organ distribution of CsA-DLPC before clinical trials.
Material and methods
Preparation of CsA-DLPC liposomes
CsA and 12:0 dilauroylphosphatidylcholine (DLPC) are dissolved in t-butanol at 37°C at an optimal ratio. For CsA-DLPC formulations, the optimal ratio is 1:7.5 by weight. The drug liposomes are lyophilized overnight to remove the t-butanol. Multi-lamellar (MLV) liposomes are produced by adding 5 mL of ultrapure water above the desired final CsA concentration of 5 mg/mL. The mixture is incubated for 15 minutes at room temperature with intermittent mixing to produce MLV liposomes [7]. For these studies, CsA was obtained from Chemwerth Chemical Co. All lipids were obtained from Avanti Polar Lipids (Alabaster, AL).
Drug liposome aerosols
The CsA-DLPC liposome aerosols for this study were generated using an Aerotech II nebulizer (ATII) (CIS-USA, Bedford, MA). The ATII is a high-output, efficient nebulizer demonstrated to produce liposome aerosols in the optimal size range of 1 to 2 µm mass median aerodynamic diameter (MMAD) for peripheral lung delivery [8]. A source of dry air was delivered to the nebulizer and its internal drying air intake was regulated via a flow meter at 10 L/min.
Drug liposome aerosol particle size distribution
Aerodynamic particle sizing of the drug liposome aerosols was measured using an Andersen I ACFM nonviable ambient particle sizing sampler (Andersen Instruments Inc, Atlanta, GA) equipped with an artificial throat as a simulator of the human respiratory system [9]. After determination of the drug concentrations for each stage by HPLC, the MMAD and geometric standard deviation (GSD) of the drug liposomes were computer calculated by a log probability plot. The MMAD and GSD were determined by the liposomal drug content distributed within the array of droplets comprising the aerosol.
Pulmonary delivery of CsA-DLPC aerosol
Mongrel dogs (20 to 50 kg; obtained from a local supplier) were utilized in these experiments. All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH Publication 85-23, revised 1985). Eighteen animals were anesthetized using pentobarbital (20 mg/kg) intravenously and halothane (0.5% to 1.5%) as inhalational anesthetic. CsA-DLPC liposomes (25 mg CsA per vial for each 15-minute treatment) and aerosols were generated at 10 L/m with an Aerotech II nebulizer connected via a T-tube (with one-way valves distal to the outlet port) into an in-line aerosol reservoir connected to the respirator. The CsA-DLPC liposome aerosol was mixed with 21% oxygen and delivered through an endotracheal tube. The ventilator was adjusted to deliver 4 L/m with 500-mL tidal volume and 12 beats per minute. Dogs were treated for 15 minutes. Blood samples were obtained at time 0, at the end of the 15-minute aerosol treatment, and at the time of death. Tissue samples of peripheral lung, central lung, heart, kidney, spleen, and liver were obtained for drug analysis. In transplant recipient dogs, only lung tissues were analyzed.
CsA analysis by HPLC
The CsA in DLPC liposomal formulations (to determine CsA drug content and liposome association) in aerosol samples was determined by high-performance liquid chromatography (HPLC). Samples for parallel CsA analysis were dissolved directly in methanol. CsA was measured using a Supelcosil LC-1 (5.0 cm x 4.6 mm, 5 µm) column (Supelco, Bellefonte, PA) at 75°C with acetonitrile/methanol/water (50:20:30 v/v/v) mobile phase [9]. Peaks were detected at 214 nm using the multi-wavelength ultraviolet detector and quantified with the Millennium 2010 Chromatography Manager (Waters, Millford, MA). The limits of detection were 10 ng CsA.
Solid-phase extraction and drug analysis of lung and other tissues
After inhalation, animals were killed under Halothane anesthesia or after removal of the heart and lungs for transplantation. Ten micrograms of cyclosporine D (CsD; Sandoz Research Institute, East Hanover, NJ) (10 µL of a 1-mg/mL stock solution) was added to the weighed tissues samples as an internal standard, and the tissues were homogenized for 2 min in a Waring blender [9]. The homogenized slurry was then mixed with an equal volume of 98% acetonitrile: 2% methanol solution, mixed thoroughly and centrifuged (1000g, 20 minutes) to obtain a clear supernatant. An equal volume of sterile water was added to dilute the tissue extraction. Meanwhile, Sep-Pak Plus C18 cartridges for solid-phase extraction (Waters) were prepared for use. These columns were activated with 5 mL of sterile water before layering the extracted supernatant onto the column. The sample was applied slowly to the column and all fluid was allowed to drain from the resin bed. The column was washed again with 5 mL of methanol and 0.5 mL of sterile water. The eluted material was evaporated and then reconstituted in CsA HPLC mobile phase, described above. These samples were then analyzed by HPLC to determine the concentration and extraction efficiency of the CsA. Data were expressed as micrograms CsA/gram tissue or whole blood analyzed.
Results
The aerosol particle size distribution of nebulized CsA-DLPC liposomes is shown in Figure 1. The median aerodynamic diameter is 1.6 µm. This size range is optimal for penetration into the lung periphery upon inhalation [10].
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Despite major advances in the development of newer, more potent, and more selective immunosuppressive agents, rejection remains a significant obstacle to long-term pulmonary allograft survival. The development of renal toxicity after prolonged systemic administration of cyclosporine limits its use in the treatment of pulmonary rejection. Furthermore, cyclosporine may not efficiently penetrate the pulmonary tissues when administered intravenously or orally [11].
Experimental work performed by Mitruka and colleagues at the University of Pittsburgh [1] has documented that the administration of cyclosporine dissolved in ethanol and administered as an aerosol prevents acute pulmonary allograft rejection in rats at significantly lower doses than systemic therapy alone. The aerosolized dose was as much as 80% lower than a concomitantly administered systemic dose in second group of control animals.
Keenan and colleagues reported similar data in 1992 [2]. An aerosolized preparation of CsA dissolved in ethanol was administered at 180 mg/m3 with a mean particle size of 0.7 µm, and estimated pulmonary depositions of CsA were 0.98 to 3.6 mg/kg/day. Blot’s group also documented similar findings [12]. They used nebulized CsA dissolved in ethanol in a rat model and documented effective pulmonary delivery of cyclosporine.
Keenan and colleagues documented the efficacy of an aerosolized cyclosporine dissolved in ethanol for acute and chronic refractory pulmonary rejection 9 of 12 patients as well as an associated suppression of cytokines interleukin-6 and interferon-
[13].
A criticism of these studies is that although they document the presence of cyclosporine in the lungs after aerosol administration, they do not clearly establish any benefit over intravenous administration. Ideally, aerosolized cyclosporine administration offers potential advantages such as minimizing renal exposure. However, such benefits have yet to be proven. A blinded, randomized study has yet to be performed in humans. Consequently, the therapy (using an ethanol or propylene glycol formulation) has yet to be widely accepted in humans. Long-term utilization (as desired for optimal immunosuppression) is also precluded.
The liposomal form of aerosolized cyclosporine offers the same theoretical advantages as the ethanol form: renal sparing, ease of administration, and application directly to the target organ. Liposomal CsA offers the potential advantages of rapid exchange across lipid membranes (a characteristic of all liposomal compounds) and the possibility of increased therapeutic activity at lower dosing regimens.
Aerosolized cyclosporine may be present on the surface of the alveoli but not absorbed into the pulmonary interstitium. Preliminary in vitro work has demonstrated the efficacy of liposomal formulations of CsA-DLPC [9]. Our results demonstrate that liposomal CsA-DLPC particle size is similar to that of aerosolized CsA dissolved in ethanol, and of small enough size for rapid distribution through the central and peripheral pulmonary alveoli. The mean size is 1.6 µm. Particles of this size readily migrate into the alveoli when administered via nebulizer or endotracheal tube; particles from 0.5 to 3.0 µm migrate into the lung after nebulization, whereas particles greater than 1.5 µm deposit in the mouth and are swallowed [5].
Our study documents the liposomal form of aerosolized CsA to be effectively and selectively absorbed into the lungs. Heart and spleen are not sites of cyclosporine concentration. The kidneys and spleen begin to demonstrate higher levels of cyclosporine after 3 hours but demonstrate lower concentrations at shorter times after aerosol administration. Our data demonstrate early effective accumulation of cyclosporine in central and peripheral lung with an initial sparing of the other organs such as a liver, kidney, and spleen. This early renal sparing is of possible therapeutic importance in allowing increased delivery of CsA to the target organ, the lung, without the required blood trough levels of 200 to 800 ng/mL necessary with oral or intravenous cyclosporine.
There is a late accumulation of cyclosporine in the renal parenchyma without any increased levels being detected in the lung when aerosolized CsA-DLPC is given for 15 minutes. This late accumulation is of concern as it may indicate that aerosol administration of CsA will have less of a renal sparing effect than anticipated. It may also explain why clinical trials of aerosolized CsA delivered in ethanol-based solution have not had the dramatic effects in improvement of rejection that might be expected. Low serum levels of cyclosporine reflect the rapid exchange of liposomal preparations across bilayer membranes [14]. Our study does not purport to show effective pulmonary immunosuppression but merely the fact that liposomal aerosolized cyclosporine is rapidly accumulated in and/or on the lung.
In conclusion, our study demonstrates that the deposition and organ distribution of a liposomal preparation of CsA delivered to the lung via aerosol are quite favorable for pulmonary transplantation. Our study does not document the effectiveness of this form of aerosolized CsA as an immunosuppressant but merely its favorable distribution profile after aerosol delivery. Late accumulation of aerosolized cyclosporine in the kidneys is of interest in that it may reflect an intrinsic affinity of the kidneys for cyclosporine with consequent implications for renal damage. The rapid accumulation of cyclosporine in the lungs is an important observation that provides a new means for relatively selective pulmonary immunosuppression. Further work in documenting the actual clinical effectiveness of this immunosuppressive technique needs to be done in animal lung transplant models.
Acknowledgments
This work was supported by the Clayton Foundation for Research, Houston, TX, and Biotime, Inc., Berkeley, CA.
References
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