Biotechnol. Appl. Biochem. (2001) 33, 85–89 (Printed in Great Britain)
Erythrocyte-mediated delivery of dexamethasone in patients
with chronic obstructive pulmonary disease

Luigia Rossi*, Sonja Serafini*, Luigi Cenerini, Francesco Picardi, Leonardo Bigi,
Ivo Panzani
and Mauro Magnani*1
*Istituto di Chimica Biologica G. Fornaini ’, UniversitaZ degli Studi di Urbino, Via Saffi 2, 61029 Urbino (PU), Italy, †Ospedale Fraternita di S. Maria della Misericordia’, Via Bonconte da Montefeltro, 61029 Urbino (PU), Italy,and Di.De.Co. S.p.A., Via Statale 12 Nord, 41037 Mirandola (MO), Italy Human erythrocytes from ten patients with chronic
should be considered if a reversible component in COPD is obstructive pulmonary disease (COPD) were loaded
present. In fact, oral steroids appear to be beneficial for with increasing amounts of dexamethasone 21-phos-
acute exacerbations, giving faster impovement in FEV (i.e., phate and were re-infused into the original donors.
forced expiratory volume in 1 s) and fewer relapses [6].
Drug-loaded erythrocytes acted as circulating bio-
However, it is important to note that glucocorticoids reactors, converting the non-diffusible dexamethasone
have a short half-life (approx. 3 h), which make frequent 21-phosphate
administrations and high dosages necessary. Consequently, Pharmacokinetic analyses on these patients showed
this causes a dangerous peak in plasma concentration, toxic that a single administration of drug-loaded erythrocytes
side effects and poor patient compliance, especially during was able to maintain detectable dexamethasone con-
the chronic use of the drug. Moreover, when corticosteroids centrations in blood for up to seven days. This contin-
are administered by inhalation, only a low percentage of the uous release of dexamethasone was paralleled by the
drug (7 %) is able to reach the lungs, thus compromising the suspension of β -agonist and oral corticosteroid treat-
therapy. Therefore alternative strategies that are able to ments by all of the patients.Thus dexamethasone 21-
reduce the toxicity of the drugs, allow prolonged application phosphate-loaded erythrocytes are safe carriers for
intervals and use different kinds of administration could be of corticosteroid analogues and are a useful alternative to
clinical importance for the treatment of COPD.
frequent oral or inhaled drugs in elderly patients with
It is known that human erythrocytes, owing to the noteworthy ability of their membrane to be opened andresealed [7], provide an extraordinary vehicle for thedissemination of drugs in the circulation. Moreover, Introduction
engineered erythrocytes have been proposed as carriers andbioreactors suitable for use in the treatment of various Chronic obstructive pulmonary disease (COPD) is a major diseases [8–13]. Previous experiences in our laboratory [14] cause of morbidity and mortality in adults [1]. It includes have demonstrated that the non-diffusible pro-drug dexa- bronchitis (unspecified and chronic), emphysema, asthma methasone 21-phosphate can be entrapped in human and acute exacerbations of chronic bronchitis. In COPD, erythrocytes where it is slowly dephosphorylated to the inflammation of the airways is a pathogenic factor, inducing corresponding diffusible dexamethasone by erythrocyte- chronic sputum production, parenchyma destruction and a resident enzymes. Furthermore, a new apparatus called ‘ Red decrease in lung function as a result of airway obstruction.
Cell Loader ’ that allows the encapsulation of drugs in human Since glucocorticoid hormones are potent anti-inflammatory erythrocytes for clinical use is now available [15]. Thus, based drugs [2], they could act as efficient therapeutic agents for on the above mentioned considerations, we evaluated the the treatment of COPD. In fact, there are data that support potential use of autologous erythrocytes with encapsulated the use of systemic corticosteroids in the treatment of acute dexamethasone 21-phosphate as a slow delivery system exacerbations of COPD [3,4], although benefits deriving from long-term use of corticosteroids to manage chronicCOPD have not been demonstrated. Despite the lack ofevidence for this approach, inhaled corticosteroids are Key words : corticosteroids ; drug delivery ; erythrocytes.
widely used for the treatment of severe COPD, especially in Abbreviations used : COPD, chronic obstructive pulmonary disease ; FEV , the United States [5]. Moreover, the use of corticosteroids 1 To whom correspondence should be sent (e-mail magnani!bib.uniurb.it).
Materials and methods
solution, the addition of dexamethasone 21-phosphate andthe resealing of the lysed cells. This process was performed Materials
using the ‘ Red Cell Loader ’ apparatus previously described The blood-processing machine (Di.De.Co Compact A) and [15], and was completed in 2 h at room temperature under the red cell loader disposables were obtained from blood-banking conditions. Briefly, blood (50 ml) was col- Di.De.Co. S.p.A. (Mirandola, Italy). Dexamethasone 21- lected from COPD patients in syringes containing heparin as phosphate and the resealing solution, PIGPA.C (1.606 M anticoagulant and processed following the sequence of the KCl, 0.194 M NaCl, 33 mM NaH PO , 0.1 M inosine, operations described for the ‘ Red Cell Loader ’. To obtain 5 mM adenine, 20 mM ATP, 0.1 M glucose, 0.1 M pyruvate erythrocytes loaded with different amounts of dexa- and 4 mM MgCl ), were obtained from Mastelli (Imperia, methasone 21-phosphate, different amounts of a 40 mM Italy). Other solutions (washing and hypotonic) were as dexamethasone 21-phosphate (120 mOsM) solution (5– 20 ml) were added to the lysed cells. At the end of theloading procedure, dexamethasone 21-phosphate-loadederythrocytes were transferred to a final disposable bag and Patients
re-infused into the original donor. Before this step, the Ten patients with COPD [PO l 51.28±9.4 mmHg (mean± volume of the erythrocyte suspension was evaluated and a S.D.) ; normal value in 70-year-old patients is approx.
100 µl aliquot was used for dexamethasone 21-phosphate 75 mmHg ; FEV l 62.25±8.3 % compared with controls (mean±S.D.)] were enrolled in this clinical study. They weredivided into three groups : group A (patients 1–5), whoreceived a single administration of autologous erythro- Determination of the amount of dexamethasone 21-
cytes loaded with different amounts of dexamethasone 21- phosphate administered to COPD patients
phosphate ; group B (patients 6–8), who received two The amount of dexamethasone 21-phosphate administered administrations of dexamethasone 21-phosphate-loaded to COPD patients was determined by HPLC on boiled erythrocytes at 15 day intervals ; and group C (patients samples essentially as described in [15], with some modi- 9 and 10), who received a single administration of loaded fications. Briefly, 100 µl of the final erythrocyte suspension erythrocytes and were observed in order to evaluate the was diluted with 0.9 ml of doubly distilled water, boiled for pharmacokinetics of dexamethasone. In addition, the phar- 5 min and filtered through 0.22 µm-pore-size filters. HPLC macokinetics were also evaluated in patient 6 after the first determinations were performed with a 5 µm Res Elut dexamethasone 21-phosphate-loaded erythrocyte adminis- C -90A column (25 cmi4.6 mm internal diameter ; Varian, tration. All patients received β agonists (theophylline and Harbor City, CA, U.S.A.) protected by a guard column bromophylline) and oral or inhaled corticosteroids. Ad- (Pelliguard LC-18, 20 mmi4.6 mm internal diameter, 40 µm ministration of these drugs was suspended following particles). The mobile phase consisted of two eluents : buffer dexamethasone 21-phosphate administration until they A containing 10 mM KH PO (pH 5) ; and buffer B, which consisted of buffer A plus 70 % (v\v) acetonitrile (pH 5). The The following clinical parameters were evaluated : elution conditions were : 5 min at 100 % buffer A, increasing symptoms reported by patients (difficulty in breathing, to 100 % buffer B over 10 min and held for 8 min. The cough, quality of life, etc.) ; clinically evaluated symptoms gradient was returned to 100 % buffer A over 3 min and the (bronchospasm, bronchostenosis, etc.) ; and frequency of initial conditions were restored in 5 min. The flow rate was administration of β agonists and corticosteroids. All patients 0.9 ml\min and detection was performed at 239 nm. Ana- were monitored for 1 month after the last infusion of lyses were performed at room temperature and quantitative drug-loaded erythrocytes. The entire experimental pro- measurements were obtained by the injection of a standard cedure was approved by the Ethics Committee of the ‘ S.
of known concentration. The retention times under the Maria della Misericordia ’ Hospital of Urbino and was conditions used were 15.2 min for dexamethasone 21- performed in accordance with the guidelines given in the phosphate and 17.4 min for dexamethasone. The amount of Helsinki Declaration with informed written consent.
dexamethasone 21-phosphate (mg) administered was calcu-lated from the concentration of dexamethasone 21-phos-phate encapsulated (µmol\ml of erythrocyte suspension) Encapsulation of dexamethasone 21-phosphate in
and the volume of the erythrocyte suspension re-infused.
Dexamethasone 21-phosphate was selected as a non-
diffusible glucocorticoid analogue for encapsulation in
Extraction of dexamethasone from plasma samples
erythrocytes of COPD patients. The encapsulation pro- At different times after the infusion of drug-loaded cedure involves the lysis of erythrocytes in a hypotonic erythrocytes (time 0, and 1 and 7 days post-infusion) 10 ml Erythrocyte-mediated delivery of dexamethasone
of blood was drawn from COPD patients (group C) and
collected in heparin. After centrifugation at 2000 g, plasma
was separated from blood cells and dexamethasone was
extracted using diethyl ether. Briefly, 9 vol. of diethyl ether
were added to 1 vol. of plasma and incubated for 1 h at room
temperature with gentle mixing. Following centrifugation at
2000 g, the diethyl ether was separated, concentrated with
a Speed Vac concentrator (Savant Instruments, Hicksville,
NY, U.S.A.) and resuspended in acetonitrile (Sigma–Aldrich,
Milan, Italy). The amount of dexamethasone was quantified
by HPLC as described above. Recovery was determined by
adding a known amount of dexamethasone, and was found to
be 64.8±2.5 % (mean±S.D. ; n l 10).
Plasma levels of dexamethasone after administration of dexa- Dexamethasone 21-phosphate administrations using
methasone 21-phosphate-loaded erythrocytes the ‘ Red Cell Loader’
Dexamethasone concentrations were determined by HPLC on plasma Dexamethasone 21-phosphate was encapsulated in auto- ), 9 ($) and 10 (>) at time 0, and 1 and 7 days post- logous erythrocytes of COPD patients by the procedure infusion of dexamethasone 21-phosphate-loaded erythrocytes. The amounts of described in [15] using the specifically designed apparatus dexamethasone 21-phosphate administered were : 2.00 mg (patient 9) ;2.84 mg (patient 10) ; and 4.39 mg (patient 6, first infusion).
named ‘ Red Cell Loader ’. At the end of the procedure,65±9 ml of erythrocyte suspension at 13±2 haematocrit wasobtained with a cell recovery of 44±6 %. Given the fact thatsome of the erythrocytes are inevitably lost during the administrations of drug-loaded erythrocytes at 15 day washing steps and in the dead volume of the circuits, this cell intervals. These patients received on average 4.90 mg of recovery can be considered good. The amounts of dexa- dexamethasone 21-phosphate, a value (in two sequential methasone 21-phosphate administered to COPD patients administrations of dexamethasone) close to the highest are shown in Table 1. The patients enrolled in this study concentration administered to group A. Patients 9 and 10 were divided into three groups (A, B and C). The first group (group C) received a single drug administration (2.00 and (A) received one administration of dexamethasone 21- 2.84 mg of dexamethasone 21-phosphate respectively) and phosphate with doses ranging from 0.78–8.78 mg. Since no together with patient 6 (first infusion, 4.39 mg of dexa- toxic side effects were observed at any of the concentrations methasone 21-phosphate) were monitored for the deter- investigated and, moreover, clinical improvements were mination of dexamethasone in the circulation. The results obtained, the second group (B) underwent two sequential obtained (Figure 1), show that dexamethasone was still Dexamethasone 21-phosphate administered to COPD patients and the number of days passed without taking corticosteroids and β agonists The amount of dexamethasone 21-phosphate administered to patients was determined by HPLC on boiled samples as described in the Materials and methodssection. admin., administration.
Number of days without β agonists a This patient did not take any oral corticosteroid or β agonist for the entire period of observation after the first infusion.
present in the circulation 1 week post-infusion of dexa- chronically treated with steroids for at least 10 years. Thus methasone 21-phosphate-loaded erythrocytes.
the only possible monitoring was of the reported clinicalsymptoms and medical examinations.
The first group of patients was treated with increasing Clinical evidence
amounts of dexamethasone 21-phosphate to evaluate the The patients enrolled in the clinical trial were all suffering safety and efficacy of the procedure. Since no problems from severe COPD. All ten patients chronically used short- developed following erythrocyte infusion and overall clinical and\or long-acting β agonists and systemic, oral or inhaled benefits were obtained, a second group of patients received corticosteroids. Following the administration of dexa- two infusions of loaded erythrocytes at 2-week intervals. In methasone 21-phosphate-loaded erythrocytes, all patients this case, patients reported good health, which was borne suspended these therapies and were requested to record out above all by the fact that they never used steroids at least the time at which they felt the need to take these drugs.
up to 1 month post-erythrocyte infusion. The clinical benefits Patient 2 resumed inhaled corticosteroids 4 days post- observed were supported by the results of preliminary infusion. The other patients did not resume taking the drug pharmacokinetic studies of a third group of patients, which for at least 10 days post-infusion. It is worth noting that revealed the presence of dexamethasone in the circulation patients 6–8, who received two administrations of drug- up to 7 days post-infusion. Longer periods of time were not loaded erythrocytes at 15 day intervals, still did not need evaluated, but, given the results obtained (see Figure 1), we corticosteroids 1 month after the last infusion. During the can assume that low amounts of dexamethasone continue to same period, all patients were also monitored for the use of be present in the circulation for a longer time. Furthermore, β agonists. All patients avoided the use of β agonists for it is noteworthy that the best clinical benefits were obtained different periods of time (Table 1) and reported a decrease by administering, twice in a month, an amount of dexa- in coughing, breathing difficulties and brochospasms. Fur- methasone (3.7 mg on the average) that is much lower thermore, for all patients the physicians reported a reduction (approx. 25–50 times) than that usually administered in or absence of bronchostenosis and a reduction of dyspnoea.
therapeutic protocols (4 mg\2–3 times a day per patient).
Morover, in all patients (except patient 2) there was areduction in breathing difficulty, bronchospasm, broncho- Discussion
stenosis and dyspnoea, together with a remarkable reductionin the need to take β -agonists and corticosteroids.
In the present paper we report the first clinical study in We are confident that this procedure will be useful for which patients were treated with autologous erythrocytes the administration of drugs for which a slow and prolonged modified to act as bioreactors for the slow delivery of release in the circulation is needed. Further studies of drug corticosteroids in the circulation. In this study, patients delivery in vivo in human patients are in progress.
with COPD were treated with autologous erythrocytesloaded with the non-diffusible prodrug dexamethasone 21-phosphate. Once re-infused into patients, dexamethasone21-phosphate is slowly dephosphorylated by erythrocyte- Acknowledgments
resident enzymes (K l 11.1±0.6 mM ; V l 7.4±0.2 nmol\ h per mg of haemoglobin [14]) to dexamethasone, a We thank the patients and physicians of the ‘ S. Maria della corticosteroid able to reduce airway inflammation in Misericordia ’ Hospital of Urbino (Italy) for their invaluable collaboration. This work was partially supported by the Ten patients with COPD were enrolled in our clinical National Research Council. Target Project on Biotech- study. After the infusion of autologous dexamethasone nology. The ‘ Red Cell Loader ’ was provided by Di.De.Co.
21-phosphate-loaded erythrocytes, they were monitored essentially to evaluate clinical symptoms and the number ofdays that passed without taking their usual drugs (β -agonists and steroids). In fact, the parameter that most significantly References
provides evidence for lung function (FEV ) could not be determined, since all of the patients were unable to perform Higgins, M. W. and Thorn, T. (1990) in Clinical Epidemiology of the test correctly due to an advanced age and bad-general- Chronic Obstructive Pulmonary Disease (Hensley, M. J. and health status. We also considered other determinations to Saundres, N. A., eds.), pp. 23–43, Marcel Dekker, New York monitor possible side effects of corticosteroids, such as Haynes, R. C. (1990) in The Pharmacological Basis of assesment of ocular pressure and a computerized study of Therapeutics (Gilman, A. G., Rall, T. W., Nies, A. S. and Taylor, bone density, not significant, since all patients had been P., eds.), pp. 1431–1457, Pergamon Press, New York Erythrocyte-mediated delivery of dexamethasone
Albert, R. K., Martin, T. R. and Lewis, S. W. (1980) Ann. Intern.
Rossi, L., Bianchi, M. and Magnani, M. (1992) Biotechnol. Appl.
Murata, G. H., Gorby, M. S., Chick, T. W. and Halperin, A. K.
Magnani, M., Fazi, A., Mangani, F., Rossi, L. and Mancini, U.
(1993) Biotechnol. Appl. Biochem. 18, 217–226 American Thoracic Society. Standards for the diagnosis and Magnani, M., Giovine, M., Fraternale, A., Damonte, G., Rossi, L., care of patients with chronic obstructive pulmonary disease Scarfı', S., Benatti, U. and De Flora, A. (1995) Drug Delivery 2, (COPD) (1995) Am. J. Resp. Crit. Care Med. 152, S77–S120 Dompeling, E., van Schayck, C. P., van Grunsven, P. M., van Rossi, L., Brandi, G., Schiavano, G. F., Scarfı', S., Millo, E., Ilerwaarden, C. L., Akkermans, R., Molema, J., Folgering, H. and Damonte, G., Benatti, U., De Flora, A. and Magnani, M. (1999) van Weel, C. (1993) Ann. Intern. Med. 118, 770–778 AIDS Res. Hum. Retroviruses 15, 345–353 Hoffman, J. F. (1992) in The Use of Resealed Erythrocytes as D’Ascenzo, M., Antonelli, A., Chiarantini, L., Mancini, U. and Carriers and Bioreactors (Magnani, M. and De Loach, J. R., eds.), Magnani, M. (1997) in Erythrocytes as Drug Carriers in Medicine (Sprandel, U. and Way, J. L., eds.), pp. 81–88, Ropars, C., Teisseire, B., Avenard, M., Chassaigne, M., Hurel, C., Girot, R. and Nicolau, C. (1984) Ann. N.Y. Acad. Sci. 445, Magnani, M., Rossi, L., D’Ascenzo, M., Panzani, I., Bigi, L. and Zanella, A. (1998) Biotechnol. Appl. Biochem. 28, 1–6 De Flora, A., Zocchi, E., Guida, L., Polvani, C. and Benatti, U.
(1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3145–3149 Received 15 November 2000 ; accepted 4 December 2000

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