Delivery of therapeutic levels of heparin and low-molecular-weight heparin through a pulmonary route.

Although heparin and low-molecular-weight heparins (LMWH) have been widely used clinically as anticoagulants, their broader use has been limited by the lack of noninvasive delivery methods for this class of molecules. In this study, we demonstrate an efficient, rapid, and reproducible delivery system for heparin through the lungs that is not confined to particles of a certain geometric or aerodynamic diameter. Importantly, blood levels after intrapulmonary administration of either heparin or LMWH were comparable to that of s.c. administration but are characterized by a more rapid onset of action (t(1/2) = 40 min vs. 2.5 h, respectively). Furthermore, we show in animal models, that inhaled heparin species efficiently inhibit diseases such as thrombosis and emphysema, and that the repetitive inhalation of formulated LMWH results in no observable toxicity from the delivery of reproducible systemic levels of heparin or LMWH.

Endothelial cells preparing to die by apoptosis initiate a program of transcriptome and glycome regulation.

The protein-based changes that underlie the cell biology of apoptosis have been extensively studied. In contrast, mRNA- and polysaccharide-based changes have received relatively little attention. We have combined transcriptome and glycome analyses to show that apoptotic endothelial cell cultures undergo programmed changes to RNA transcript abundance and cell surface polysaccharide profiles. Although a few of the transcriptome changes were protective, most appeared to prepare cells for apoptosis by decreasing the reception and transduction of pro-survival signals, increasing pro-death signals, increasing abundance of apoptotic machinery, inhibiting cellular proliferation, recruiting phagocytes to regions of cell death, and promoting phagocytosis. Additional transcriptomal changes appeared to alter the synthesis and modification of cell surface glycosaminoglycans. The resultant reduced abundance of sulphated cell surface glycosaminoglycans may further promote cell death by inhibiting the presentation of extracellular matrix-tethered survival factors to their receptors on dying cells. We propose that the transcriptome and glycome regulation presented here synergize with previously described protein-based changes to guide the apoptotic program.

Rational design of low-molecular weight heparins with improved in vivo activity.

Heparin and low-molecular weight heparins (LMWHs), complex, sulfated polysaccharides isolated from endogenous sources, are potent modulators of hemostasis. Heparin and LMWHs interact with multiple components of the coagulation cascade to inhibit the clotting process. Pharmaceutical preparations of these complex polysaccharides, typically isolated from porcine intestinal mucosa, are heterogeneous in length and composition and, hence, highly polydisperse. Because of the structural heterogeneity of heparin and LMWHs, correlating their activity with a particular structure or structural motif has been a challenging task. Herein, we demonstrate a practical analytical method that enables the measurement of a structural correlate to in vivo anticoagulant function. With this understanding we have developed LMWHs with increased anticoagulant activity and decreased polydispersity. In addition to the pronounced anti-Xa and anti-IIa activity of these LMWHs, we also demonstrate that they possess desirable in vivo pharmacokinetic properties, the ability to cause the release of tissue factor pathway inhibitor (TFPI) from the endothelium, complete bioavailability through s.c. delivery, and the ability to inhibit both venous and arterial thromboses. Importantly, from a clinical safety point of view, unlike LMWHs presently used in the clinic, we show that these LMWHs are rapidly and completely neutralized by protamine. Together, the findings presented herein demonstrate a facile approach for the creation of designer LMWHs with optimal activity profiles.