Pulmonary Delivery of Low Molecular Weight Heparins Tianzhi Yang, 1 Fatima Mustafa, 1 Shuhua Bai, 1 and Fakhrul Ahsan 1,2 Received March 29, 2004; accepted July 26, 2004 Purpose. To investigate if pulmonary delivery of low molecular weight heparin (LMWH) formulated with tetradecyl--maltoside (TDM) or dimethyl--cyclodextrin (DMCD) could be a feasible alternative to subcutaneous injections for the treatment of pulmonary embolism. Methods. The pulmonary absorption of two LMWHs and unfraction- ated heparin formulated with TDM or DMCD was studied in cell culture and rodent model. The in vitro study was performed by mea- suring the transport of radiolabeled enoxaparin and mannitol across human bronchial epithelial cells (Calu-3) in the presence or absence of varying concentrations of TDM or DMCD. The changes in trans- epithelial electrical resistance (TEER) and enoxaparin metabolic sta- bility were also investigated using Calu-3 cells. In vivo absorption studies were performed by measuring plasma anti-factor Xa activity after pulmonary administration of enoxaparin, dalteparin, or unfrac- tionated heparin to anesthetized rats. Results. In vitro experiments conducted in Calu-3 cells suggest that the addition of TDM or DMCD to the apical chamber results in a significant increase in 3 H-enoxaparin and 14 C-mannitol permeability and a decrease in TEER across the Calu-3 cell monolayer. Enoxa- parin incubated in Calu-3 cell extracts was stable for 8 h. In vivo studies indicate that both TDM and DMCD enhance pulmonary absorption of LMWH. However, TDM was found to be more potent than DMCD in both in vitro transport and in vivo absorption stud- ies. Conclusions. TDM and DMCD enhance pulmonary absorption of LMWH both in vitro and in vivo, with TDM being more efficacious than DMCD. Both agents increase drug transport by acting mainly on the membrane rather than interacting with the drug. KEY WORDS: dimethyl--cyclodextrin; enoxaparin; low molecular weight heparins; permeability; pulmonary absorption; tetradecyl-- maltoside; transepithelial electrical resistance. INTRODUCTION Deep vein thrombosis affects 2 million Americans annu- ally, and an estimated 600,000 of these develop pulmonary embolism, a fatal complication that results in 200,000 deaths a year (1–3). Deep vein thrombosis and pulmonary embolism can occur in both healthy ambulatory individuals and bedrid- den hospitalized patients. A recent study has shown that one in 100 airplane travelers, with no previous history of clotting disorder, may develop deep vein thrombosis as a result of their lengthy flights (4). Of the thromboembolic disorders, pulmonary embolism is particularly important because in this acute thromboembolic episode, a thrombus originating from the circulation lodges in the pulmonary artery or one of its branches. As a result, death can occur within a few hours of onset due to partial or complete obstruction of pulmonary blood flow. Thus, rapid diagnosis and prompt treatment are critically important for the outcome of patients with pulmo- nary embolism (5). In recent years, because of improved pharmacokinetic profiles and reduced cost of therapy, low molecular weight heparins (LMWHs) have enjoyed success as an alternative to unfractionated heparin (UFH) in the treatment of deep vein thrombosis and pulmonary embolism (6,7). However, these new generation drugs are still delivered via the traditional routes of administration such as subcutaneous and intrave- nous routes. Administration of an anticoagulant drug directly to the pulmonary circulation would be ideal for the treatment of pulmonary embolism. A pulmonary formulation of LMWH will allow direct administration of the drug into the lungs, and consequently this formulation is likely to reduce the mortality from an attack of pulmonary embolism. Further, a portable noninvasive formulation would also be beneficial for both patients with thromboembolic disorders and healthy individuals who are susceptible to deep vein thrombosis. However, LMWHs do not get absorbed from the respi- ratory tract because of their excessive hydrophilicity and large surface charges. We have previously identified tetradecyl- maltoside as a nonionic surfactant that enhances nasal and pulmonary absorption of insulin (8,9). Similarly, dimethyl-- cyclodextrin (DMCD), a derivative of cyclodextrin with seven glucose units, has been studied extensively for its safety and efficacy as an absorption promoter for nasal and pulmo- nary delivery of peptide drugs (9–11). Recently, we have shown that both tetradecyl--maltoside (TDM) and DMCD enhance nasal absorption of enoxaparin, a low molecular weight heparin available in the U.S. market (12,13). Based on the efficacy of TDM and DMCD in enhancing nasal absorp- tion of LMWHs and peptide drugs, it is reasonable to assume that both of these agents can also increase pulmonary absorp- tion of LMWH. However, there is no experimental data in support of the assumption that TDM and DMCD enhance pulmonary absorption of LMWH, a polysaccharide with negative surface charges. On the other hand, because the lung provides a larger surface area and has a thin epithelial mem- brane, the bioavailability of a drug administered via the pul- monary route is expected to be much higher than that ob- tained via the nasal route. More importantly, clinical benefit offered by pulmonary LMWH in the treatment of pulmonary embolism could be of paramount significance because this delivery approach will permit to administer the drug directly at the site affected by the disease. This study tests the hypothesis that TDM and DMCD enhance pulmonary absorption of LMWH in vitro and in vivo. Enoxaparin and dalteparin, LMWHs that received wide- spread acceptation among the medical practitioners in the United States, have been used to evaluate the efficacy of TDM and DMCD in enhancing pulmonary absorption of LMWH. The mechanistic aspects of these absorption enhanc- ers have also been studied using human bronchial epithelial cells (Calu-3). 1 Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, USA. 2 To whom correspondence should be addressed. (e-mail: fakhrul. ahsan@ttuhsc.edu) Pharmaceutical Research, Vol. 21, No. 11, November 2004 (© 2004) Research Paper 2009 0724-8741/04/1100-2009/0 © 2004 Springer Science+Business Media, Inc.