Ann Thorac Surg 1995;59:277-282
© 1995 The Society of Thoracic Surgeons
Thalidomide as Replacement for Steroids in Immunosuppression After Lung Transplantation
Kay Uthoff, MD,
Kenton J. Zehr, MD,
Paul B. Gaudin, MD,
Pankaj Kumar, BMBCh,
Peter W. Cho, MD,
Georgia Vogelsang, MD,
Ralph H. Hruban, MD,
William A. Baumgartner, MD,
R. Scott Stuart, MD
Division of Cardiac Surgery and Departments of Pathology and Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland
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Abstract
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Steroids have been implicated in postoperative complications after lung transplantation: infections, delayed wound healing, and poor bronchial anastomotic healing. Thalidomide (
-phthalimidoglutarimide), a sedative drug with known immunomodulatory properties, was used to replace corticosteroids after canine lung transplantation. Fifteen mongrel dogs underwent single-lung transplantation: group I (n = 5) received cyclosporin A (20 mg/kg twice a day), azathioprine (2.5 mg/kg once a day), and thalidomide (50 mg/kg twice a day). Group II (n = 5) received standard immunosuppression of cyclosporin A (20 mg/kg twice a day), azathioprine (2.5 mg/kg once a day), and prednisone (2 mg/kg once a day), and group III (n = 5) received cyclosporin A (10 mg/kg twice a day), azathioprine (2.5 mg/kg once a day), and thalidomide (50 mg/kg twice a day). Open lung biopsy and bronchoscopy were performed weekly until sacrifice on day 28. Serum thalidomide and cyclosporin A levels were followed up weekly. Group I showed essentially no rejection until week 2 and minimal rejection (grade 1) until day 28. Group II had moderate rejection (grade 2) of the graft at all time points. Group III animals had moderate to severe rejection (grades 3 to 4) after 21 days (p < 0.05 for group I versus groups II and III). The number of clinically evident episodes of pneumonia was also significantly lower in group I than in groups II and III (p < 0.05). We conclude that thalidomide appears to replace corticosteroids effectively in early postoperative immunosuppression after lung transplantation and is associated with a decreased incidence of pneumonia. It was not efficacious in combination with low-dose cyclosporin A. This drug may have a significant impact after clinical lung transplantation by reducing steroid-associated complications.
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Introduction
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L ung transplantation is well-established therapy for end stage pulmonary disease. Standard immunosuppressive therapy has consisted of cyclosporin A, azathioprine, and corticosteroids. The use of corticosteroids during the postoperative course is associated with well-known early and late complications [1–5]. Clinical studies have tried to replace or withdraw corticosteroids in the traditional immunosuppression therapy with varying success. Thalidomide was introduced into experimental and clinical immunosuppression trials as a possible replacement for steroids. Thalidomide is a sedative and antiemetic drug with known T-cell directed immunomodulatory properties. Clinical application of the drug has proved beneficial in the treatment of erythema nodosum leprosum [6, 7], diseases with autoimmune characteristics [8], and graft-versus-host disease in bone marrow transplant patients [9–11]. Thalidomide has been used successfully with low-dose cyclosporin A in experimental cardiac transplantation [12, 13]. It has failed to suppress the immune response as a single agent after renal transplantation [14]. We sought to replace steroids with thalidomide in postoperative triple immunosuppression therapy and to evaluate the effectiveness with low-dose cyclosporin A after canine single-lung transplantation.
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Material and Methods
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Surgical Procedure
Conditioned mongrel dogs (21 to 24 kg) were used as donors (n = 15) and recipients (n = 15) and randomized to three treatment groups. Donor and recipient animals were premedicated with intravenous cefamandol (500 mg) and anesthetized with intravenous pentobarbital (25 mg/kg). All surgical procedures were performed under sterile conditions. After intubation, donor organ harvest was performed through a median sternotomy after full intravenous heparinization (300 U/kg). The main pulmonary artery was flushed with cold Euro-Collins solution (4°C). The donor organ was bench prepared and transferred immediately for implantation into the simultaneously prepared recipient. The lung was kept cold by topical 4°C Ringer's lactate solution. The recipient's left pneumonectomy was performed through a left posterolateral thoracotomy. Heparinization (300 U/kg) was done before cross-clamping the pulmonary artery. The pulmonary artery anastomosis was done with a running 5-0 polypropylene suture. The donor bronchus was trimmed to the second cartilage proximal to the division of the lobular bronchi and was telescoped into the donor bronchus using a running 4-0 polypropylene suture. The venous anastomosis was performed along an incision made to combine the orifices of the recipient pulmonary veins with the left atrial cuff of the donor lung. The edge of the anastomosis was everted to prevent left atrial thrombosis. Air was carefully removed from the graft by reverse reperfusion. All dogs were weaned from the ventilator and extubated several hours postoperatively.
Immunosuppression and Postoperative Management
Postoperative immunosuppression was initiated by intravenous administration of cyclosporin A (3 to 7 mg/kg every 12 hours). Maintenance immunosuppression was started 12 hours after extubation. Thalidomide was prepared as suspension in vegetable oil and given orally. Cyclosporin A (a gift from Sandoz Inc, Pharmaceutical Division, East Hanover, NY) was given orally. Group I (n = 5) received modified triple immunosuppression with oral cyclosporin A (20 mg/kg twice a day), azathioprine (2.5 mg/kg once a day), and thalidomide (50 mg/kg twice a day). Group II received modified triple immunosuppression comprising oral cyclosporin A (20 mg/kg twice a day), azathioprine (2.5 mg/kg once a day), and prednisolone (2 mg/kg once a day). Group III received low-dose oral cyclosporin A (10 mg/kg twice a day) and thalidomide (50 mg/kg twice a day). Immunosuppression was maintained until day 28. On postoperative days 7, 14, 21, and 28 open lung biopsies were performed under general anesthesia. All specimens were taken from the left lower lobe. The biopsy specimens were prepared with hematoxylin and eosin staining technique and evaluated in a blinded fashion by two cardiac pathologists. Histologic grading of acute rejection was done according to the working formulation of the Lung Rejection Study Group [15]:
- Acute rejection
- Grade 0—No significant abnormality
- Grade 1—Minimal acute rejection
- With evidence of bronchiolar inflammation
- Without evidence of bronchiolar inflammation
- With large airway inflammation
- No bronchioles are present
Grade 2—Mild acute rejection
Grade 3—Moderate acute rejection
Grade 4—Severe acute rejection
Active airway damage without scarring
Lymphocytic bronchitis
Lymphocytic bronchiolitis
Bronchoscopy was performed on days 0, 7, 14, 21 and 28. Trachea and main bronchi were cleared of secretions and blood clots. Bronchial anastomoses were inspected and stenoses more than 50% noted. Blood was obtained on days 0, 1, 7, 14, 21, and 28 for white blood cell count, transaminases, and serum creatinine levels, serum cyclosporin A levels were obtained weekly and measured by radioimmunoassay. The enteral absorption and pharmacokinetics of thalidomide was investigated in a single normal dog to compare the dose/response curve with previously published data. Serum levels were determined by high-performance liquid chromatography. All dogs were examined daily by two independent observers. At onset of symptoms of pneumonia (coughing, fever, rales, or decreased activity) intravenous antibiotics (gentamicin, chloramphenicol, and cefoxitin) were administered for 10 days. All animals were sacrificed at day 28. Heart and lungs were removed en bloc and submitted to pathology for gross inspection. Any significant stenoses of the bronchial tree were noted. All anastomoses were examined histologically.
Statistical Analysis
Comparison between histologic grading, cyclosporin A levels, and blood chemistry between the three groups were made by analysis of variance using Neumann-Keul's multiple comparisons test. Comparisons of incidence of pneumonia, wound dehiscence and bronchial stenosis were made using 
2 analysis. Statistical significance was accorded to p values less than 0.05. Data are presented as mean ± standard error of the mean.
Animal Care
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).
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Results
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Cyclosporin A Levels
Cyclosporin A trough levels of the group II dogs were therapeutic (231 to 295 ng/mL) throughout the postoperative course. Group I dogs had slightly increased cyclosporin A levels (287 to 342 ng/mL) compared with group II, although not statistical significant. Group III had lower cyclosporin A levels (198 to 231 ng/mL) than group II, particularly at week 3 (Fig 1
)..
Thalidomide Pharmacokinetics
To show adequate enteral absorption of thalidomide, we administered a single oral dose of thalidomide (50 mg/kg) to a 21-kg mongrel dog. Serum peak level (17.3 nmol/mL) was observed 5 hours after the oral dose. Serum half-life was approximately 3 hours. Dosing was done in the absence of other drugs.
Blood Chemistry
Renal and hepatic function were unimpaired (Table 1). Levels of liver transaminases and creatinine remained normal in all dogs throughout the postoperative period.
Bronchoscopy
Bronchoscopic evaluation revealed initial postoperative stenoses of the left upper lobe bronchus in 4 of 5 group I animals. These stenoses were attributed to technique; otherwise, all bronchial anastomoses were patent in group I throughout the follow-up period. There were no immediate stenoses in group II, but two late left upper lobe stenoses were observed. Group III had one immediate left upper lobe stenosis, and left upper lobe stenoses developed in 2 dogs at week 3 (Table 2) All airway stenoses were confirmed by gross inspection after sacrifice. Anastomotic microabscesses were observed in 1 animal in each group.
Bronchopneumonia and Wound Healing Disturbances
The major postoperative complications were wound dehiscences at the thoracotomy site and bronchopneumonia. Partial wound dehiscence occurred in no dogs group I, 3 of 5 dogs in group II, and in 1 in group III (see Table 2
). Bronchopneumonia, requiring intravenous antibiotics were observed in 1 dog in group I, 5 dogs in group II, and 5 dogs in group III (p < 0.05, group I versus groups II and III). At sacrifice, there was microscopic evidence of poststenotic left upper lobe bronchopneumonia in 4 group I dogs, whereas only 1 dog of the same group had additional left lower lobe bronchopneumonia. This was the only symptomatic dog (see Table 2).
Rejection
GROUP I.
All group I dogs survived the follow-up period. Two of 10 biopsy specimens taken on weeks 1 and 2 showed mild acute rejection (grade 2) (Fig 2). Only 1 of the 2 dogs with mild acute rejection had accompanying small airway inflammation. The third biopsy (day 21) revealed grade 1 rejection in 3 of 5 dogs. The median histologic grade of rejection remained stable during the last 2 weeks. Overall, there was minimal evidence of rejection within the first 2 weeks with an increase to grade 1 in weeks 3 and 4 (Fig 3). There was no evidence of lymphocytic bronchitis or bronchiolitis in this group.
GROUP II.
Three dogs had grade 2 rejection over the 4-Week course (Table 3). One animal had stable moderate rejection (grade 3) with additional small airway inflammation over the postoperative course (see Figs 2, 3). The remaining animal had no histological evidence of rejection.
GROUP III.
Despite successful prior experiences with combination therapy of low-dose cyclosporin a and thalidomide in a rat heart transplantation model, this drug regimen had the lowest therapeutic effect after single lung transplantation in this study. The histologlc Picture at 1 week was dominated by features of mild to moderate acute rejection (Grades 2 to 3), bonchiolar inflammation was noted in 3 of 5 biopsy specimens (see Fig 2). At week 2, 4 of 5 dogs had grade 3 rejection with accompanying bronchiolar inflammation in 3 animals. One dog had grade 2 rejection. There were two postoperative deaths at week 3 after biopsy. Neither dog could be weaned from the ventilator. Of the week 3 biopsies, two showed grade 4 rejection. Alveolar damage was present. One of these 2 dogs had necrotizing vasculitis and frank organ failure. Additional arterial thrombi were noted in the vascular bed of this animal. One animal had a mild form of acute graft rejection (grade 1). In the remaining two biopsies acute rejection ranged from grade 3 to grade 4. One of the grade 3 to 4 biopsy specimens was taken from the area of an old biopsy site, which we excluded from histologic evaluation. In summary group III dogs showed mild to moderate rejection (grades 2 to 3) after 7 days progressing to grade 3 to 4 during the follow-up period (see Fig 3). The 3 surviving dogs had associated large airway (cartilaginous bronchial) inflammation.
Group I had significantly less rejection than groups II and group III (p < 0.05). Group II rejected significantly less than group III (p < 0.05). The development of early chronic rejection was seen twice in group II and twice in group III.
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Comment
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The majority of postoperative complications after lung transplantation are related to the immunosuppressive drugs administered. Particularly problematic are those associated with steroids. Early morbidities are anastomotic dehiscence and increased incidence of pulmonary infections. Chronic use can lead to systemic hypertension, diabetes mellitus, growth retardation in children, osteoporosis, and aseptic necrosis. Alternative approaches in postoperative immunosuppression therapy after organ transplantation have been suggested. One approach to increase host tolerance has been to administer a synergistically acting compound or the additional application of biological treatments in combination with subtherapeutic doses of cyclosporin A [16]. Elimination of steroids has been emphasized [17]. Thalidomide is a sedative compound with known immunosuppressive properties. It was widely used in the early 1960s but was withdrawn from use because of its teratogenic side effects. Its subsequent use has been limited to the treatment of a few autoimmune disorders [6–8]. In the late 1980s an experimental study showed efficacy in the treatment of graft-versus-host disease in rat bone marrow transplantation [9, 10]. These results led to clinical application for treatment and prevention of graft-versus-host disease after human bone marrow transplantation [11]. The most effective dose/response relation has been determined [18, 19]. The precise mechanism of action has not been illicited. There is evidence to suggest that thalidomide has some inhibitory effect on polymorphonuclear leukocytes and monocytes [20]. More important is its effect on T-lymphocytes, and its synergy with cyclosporin A and FK506 has been investigated [21–23]. Thalidomide inhibited T-lymphocyte proliferation in mitogenic and allogeneic assays in a dose-dependent manner and showed additive effects in combination with low-dose cyclosporin A. This was corroborated in vivo, where a combination of thalidomide and low-dose cyclosporin A suppressed the immune response in rat heart transplants as effectively as cyclosporin A and steroids [12].
The main goal of this study was to show the efficacy of thalidomide as replacement for steroids in the postoperative course after lung transplantation. This study provides evidence for this within the limited time frame of 1 month. In fact the thalidomide group showed significantly less rejection than the standard immunosuppression group. Because previous studies showed efficacy of thalidomide (50 mg/kg) in combination with low-dose cyclosporin A (10 mg/kg) [12, 18, 19], we used this regimen in the third group. We were unable to accomplish satisfactory immunosuppression with similar dosages. In fact, this group consistently had the most severe rejection with parenchymal necroses after 4 weeks despite relatively smaller differences in cyclosporin A levels. However, week 3 levels were markedly lower in group III. This was the time when acute rejection was most severe. We observed arterial endothelialitis in both the low-dose cyclosporin A and the standard immunosuppression groups. This finding is thought to be a precursor to chronic vascular rejection.
The issues of bronchial complications and bronchopneumonia were complicated in this study. The numbers are too small to provide firm conclusions but do support that the standard immune suppression and low-dose cyclosporin A groups were associated with more infectious complications. In each, 5 animals received intravenous antibiotic treatment for pneumonia. Bronchial dehiscences were not observed in any dog. This may be due to the fact that none of the animals were pretreated with steroids. The relation between steroid treatment and the occurrence of bronchial complications has been controversial [4, 24]. We observed bronchial stenoses occurring within the postoperative period limited to the standard immunosuppression group and the low-dose cyclosporin A/thalidomide group. The thalidomide study group did not have progressive bronchial stenoses, although 4 dogs had early left upper lobe stenoses attributed to technique. These stenoses had minimal influence on the clinical course. The upper lobe in the canine species is much smaller and the decreased function did not compromise respiratory status. These stenoses were not associated with infectious complications. All biopsy specimens were taken from the lower lobe; therefore, these data were not affected. The remaining progressive stenoses noted may have contributed to higher rates of bronchopneumonia seen primarily in the standard immunosuppression group. Further studies are necessary to determine the effect of this regimen on bronchial healing.
No behavioral or physiologic side effects of thalidomide were noted. Based on canine pharmacokinetics we administered thalidomide twice daily. We observed none of the hepatotoxic side effects during the 4-week course seen in previous studies (Table 4).
This study demonstrates that use of thalidomide after single-lung transplantation was associated with decreased acute rejection, decreased episodes of pulmonary infections, and a trend toward lower incidence of bronchial stenoses when compared with standard immunosuppressive therapy. This suggests that thalidomide may be efficacious in a similar role as steroids in standard triple immunotherapy in single-lung transplantation.
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Footnotes
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Presented at the Poster Session at the Thirtieth Annual Meeting of The Society of Thoracic Surgeon, New Orleans, LA, Jan 31–Feb 2, 1994.
Address reprint requests to Dr Stuart, Division of Cardiac Surgery, The Johns Hopkins Hospital, 600 N Wolfe St, Blalock 618, Baltimore, MD 21287-4618.
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Abstract
Introduction
