Delivery of Therapeutic Proteins to the Brain

Protein therapeutics have tremendous potentials to treat disorders in central nervous system (CNS). However, delivery of therapeutic dose of proteins to the brain is challenging due to the blood-brain barrier (BBB). In Chapter I, I summarize the current strategies applied to enhance the brain delivery of therapeutic proteins, including strategies to open the BBB, penetrate the BBB, or bypass the BBB. The advantages and challenges for each technology are described. In Chapter II, we explore strategies to improve the brain delivery of leptin for treatment of obesity. Leptin is an adipocyte-secreted hormone that is delivered via a saturable transporter system across the BBB to the brain where it acts on receptors in hypothalamus to control appetite and thermogenesis. Peripheral resistance to leptin due to its impaired brain delivery prevents therapeutic use of leptin in overweight and moderately obese patients. To address this problem, we modify the N-terminal amine of leptin with Pluronic P85 (LepNP85) and administer this conjugate intranasally using the nose-to-brain route to bypass the BBB. We compare this conjugate with the native leptin, the N-terminal leptin conjugate with poly(ethylene glycol) (LepNPEG5K), and two conjugates of leptin with Pluronic P85 attached randomly to the lysine amino groups of the hormone. Our work suggests that selective modification at the N-terminal preserves leptin activity and modification with P85 enhances the nose-to-brain transport of leptin. In conclusion, LepNP85 with optimized conjugation chemistry is a promising candidate for treatment of obesity. In Chapter III, we demonstrate for the first time that naïve macrophage exosomes interact with intercellular adhesion molecule 1 (ICAM-1) and C-type lectin receptors on brain endothelial cells that form BBB. Upregulation of ICAM-1, a common process in inflammation, promotes macrophage exosomes uptake in the BBB cells. We further demonstrate in vivo that naïve macrophage exosomes, after intravenous (IV) administration, cross the BBB and deliver a cargo protein to the brain parenchyma. The delivery is enhanced in the presence of brain inflammation. Taken together, macrophage exosomes are promising nanocarriers for brain delivery of therapeutic proteins.Doctor of Philosoph

Pluronic modified leptin with increased systemic circulation, brain uptake and efficacy for treatment of obesity

Modification of hydrophilic proteins with amphiphilic block copolymers capable of crossing cell membranes is a new strategy to improve protein delivery to the brain. Leptin, a candidate for the treatment of epidemic obesity, has failed in part because of impairment in its transport across the blood–brain barrier (BBB) that develops with obesity. We posit that modification of leptin with poly(ethylene oxide)-b-poly(propylene oxide)-b-poly (ethylene oxide), Pluronic P85 (P85) might permit this protein to penetrate the BBB independently of its transporter, thereby overcoming peripheral leptin resistance. Here we report that peripherally administered leptin–P85 conjugates exhibit biological activity by reducing food intake in mouse models of obesity (ob/ob, and diet-induced obese mouse). We further generated two new leptin–P85 conjugates: one, Lep(ss)–P85(L), containing one P85 chain and another, Lep(ss)–P85(H), containing multiple P85 chains. We report data on their purification, analytical characterization, peripheral and brain pharmacokinetics (PK). Lep(ss)–P85(L) crosses the BBB using the leptin transporter, and exhibits improved peripheral PK along with increased accumulation in the brain compared to unmodified leptin. Lep(ss)–P85(H) also has improved peripheral PK but in a striking difference to the first conjugate penetrates the BBB independently of the leptin transporter via a non-saturable mechanism. The results demonstrate that leptin analogs can be developed through chemical modification of the native leptin with P85 to overcome leptin resistance at the level of the BBB, thus improving the potential for the treatment of obesity

Macrophage exosomes as natural nanocarriers for protein delivery to inflamed brain

Recent work has stimulated interest in the use of exosomes as nanocarriers for delivery of small drugs, RNAs, and proteins to the central nervous system (CNS). To overcome the blood-brain barrier (BBB), exosomes were modified with brain homing peptides that target brain endothelium but likely to increase immune response. Here for the first time we demonstrate that there is no need for such modification to penetrate the BBB in mammals. The naïve macrophage (Mϕ) exosomes can utilize, 1) on the one hand, the integrin lymphocyte function-associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1), and, 2) on the other hand, the carbohydrate-binding C-type lectin receptors, to interact with brain microvessel endothelial cells comprising the BBB. Notably, upregulation of ICAM-1, a common process in inflammation, promotes Mϕ exosomes uptake in the BBB cells. We further demonstrate in vivo that naïve Mϕ exosomes, after intravenous (IV) administration, cross the BBB and deliver a cargo protein, the brain derived neurotrophic factor (BDNF), to the brain. This delivery is enhanced in the presence of brain inflammation, a condition often present in CNS diseases. Taken together, the findings are of interest to basic science and possible use of Mϕ-derived exosomes as nanocarriers for brain delivery of therapeutic proteins to treat CNS diseases

Pharmacokinetics and Pharmacodynamics Modeling and Simulation Systems to Support the Development and Regulation of Liposomal Drugs

Liposomal formulations have been developed to improve the therapeutic index of encapsulated drugs by altering the balance of on- and off-targeted distribution. The improved therapeutic efficacy of liposomal drugs is primarily attributed to enhanced distribution at the sites of action. The targeted distribution of liposomal drugs depends not only on the physicochemical properties of the liposomes, but also on multiple components of the biological system. Pharmacokinetic–pharmacodynamic (PK–PD) modeling has recently emerged as a useful tool with which to assess the impact of formulation- and system-specific factors on the targeted disposition and therapeutic efficacy of liposomal drugs. The use of PK–PD modeling to facilitate the development and regulatory reviews of generic versions of liposomal drugs recently drew the attention of the U.S. Food and Drug Administration. The present review summarizes the physiological factors that affect the targeted delivery of liposomal drugs, challenges that influence the development and regulation of liposomal drugs, and the application of PK–PD modeling and simulation systems to address these challenges

Luteinizing Hormone Releasing Hormone-Targeted Cisplatin-Loaded Magnetite Nanoclusters for Simultaneous MR Imaging and Chemotherapy of Ovarian Cancer

Given the superior soft tissue contrasts obtained by MRI and the long residence times of magnetic nanoparticles (MNPs) in soft tissues, MNP-based theranostic systems are being developed for simultaneous imaging and treatment. However, development of such theranostic nanoformulations presents significant challenges of balancing the therapeutic and diagnostic functionalities in order to achieve optimum effect from both. Here we developed a simple theranostic nanoformulation based on magnetic nanoclusters (MNCs) stabilized by a bisphosphonate-modified poly­(glutamic acid)-<i>b</i>-(ethylene glycol) block copolymer and complexed with cisplatin. The MNCs were decorated with luteinizing hormone releasing hormone (LHRH) to target LHRH receptors (LHRHr) overexpressed in ovarian cancer cells. The targeted MNCs significantly improved the uptake of the drug in cancer cells and decreased its IC₅₀ compared to the nontargeted formulations. Also, the enhanced LHRHr-mediated uptake of the targeted MNCs resulted in enhancement in the T₂-weighted negative contrast in cellular phantom gels. Taken together, the LHRH-conjugated MNCs show good potential as ovarian cancer theranostics

Pluronic modified leptin with increased systemic circulation, brain uptake and efficacy for treatment of obesity

Modification of hydrophilic proteins with amphiphilic block copolymers capable of crossing cell membranes is a new strategy to improve protein delivery to the brain. Leptin, a candidate for the treatment of epidemic obesity, has failed in part because of impairment in its transport across the blood–brain barrier (BBB) that develops with obesity. We posit that modification of leptin with poly(ethylene oxide)-b-poly(propylene oxide)-b-poly (ethylene oxide), Pluronic P85 (P85) might permit this protein to penetrate the BBB independently of its transporter, thereby overcoming peripheral leptin resistance. Here we report that peripherally administered leptin–P85 conjugates exhibit biological activity by reducing food intake in mouse models of obesity (ob/ob, and diet-induced obese mouse). We further generated two new leptin–P85 conjugates: one, Lep(ss)–P85(L), containing one P85 chain and another, Lep(ss)–P85(H), containing multiple P85 chains. We report data on their purification, analytical characterization, peripheral and brain pharmacokinetics (PK). Lep(ss)–P85(L) crosses the BBB using the leptin transporter, and exhibits improved peripheral PK along with increased accumulation in the brain compared to unmodified leptin. Lep(ss)–P85(H) also has improved peripheral PK but in a striking difference to the first conjugate penetrates the BBB independently of the leptin transporter via a non-saturable mechanism. The results demonstrate that leptin analogs can be developed through chemical modification of the native leptin with P85 to overcome leptin resistance at the level of the BBB, thus improving the potential for the treatment of obesity