Patent Application
20140364368
Homeostatic regulation pervades diverse processes which play critical roles in human health, including hormone release, ionic balance, and cell-mediated immunity. In particular, the body employs negative feedback loops to keep these processes within physiologic limits while preventing runaway amplification cascades or positive feedback cycles. A key example of a homeostatically-regulated process with significant medical relevance is blood coagulation, the protease-driven positive-feedback cascade by which clots are formed to stop blood loss from a damaged vessel. Dysregulation of this process, whether pathological or drug-induced, leads to two adverse outcomes: too little coagulation may lead to life-threatening hemorrhage and hypovolemic shock, while overactive coagulation may lead to thrombosis (clotting within the blood vessel), the potentially fatal medical condition underlying pulmonary embolism, stroke, and organ infarction.
An essential step in the coagulation, or blood clotting, cascade is the proteolytic cleavage of fibrinogen to release fibrinopeptides A and B. These peptides, in turn, lead to the generation of fibrin which can undergo polymerisation to form a hemostatic plug, or ‘blood clot’. Although the blood coagulation cascade may be modulated at numerous different sites, a rate limiting step in this process is the cleavage of fibrinogen, which is catalyzed by the trypsin-like serine protease thrombin. Anti-coagulants are pharmacological agents inhibit thrombin activity, thereby preventing the action of thrombin in the blood coagulation cascade. The most common side effect of anti-coagulants is the occurrence of hemorrhagic complications, which can on occasion prove fatal.
The need for tight control of coagulation explains the narrow therapeutic windows of anti-coagulants, even when they are administered with strict dose titration and monitoring. One example of such anti-coagulant is unfractionated heparin (UFH), a mainstay in the hospital setting, which is particularly difficult to dose properly because of its unpredictable pharmacokinetics. Clotting time measurements and dose re-adjustment may be required up to every 3-4 hours to maintain UFH levels within the therapeutic range (Hirsh J. et al., Chest (2001), 119:64S-94S).
For the past century, pharmaceutical development of anti-coagulants has focused on targeting novel molecular entities within the coagulation cascade, yet regardless of the target, these drugs globally inhibit clot formation and inherently increase the risk of bleeding (Mannucci P. et al., Annals of Medicine (2011), 43:116-123; Melnikova I., Nature Reviews Drug Discovery (2009), 8:353-4). Several examples of engineering approaches to systemic anti-coagulant delivery have been demonstrated in the literature to date, although none directly addresses the serious bleeding side-effects. Methods utilizing nanoparticle delivery have focused on improving the drug efficacy through multivalent interactions or by targeting anti-coagulant particles to locations prone to thrombus formation, but still lack active mechanisms to limit bleeding (Peters et al., PNAS (2009), 106:9815-9819; Shiang et al., Angew. Chemie (2011), 50:7660-7665). Polymer microsphere formulations have been tested as oral delivery vehicles for heparin, which has low gastrointestinal absorption, but do not alter the bleeding profile of the drug itself (Jiao et al., J. Pharm. Sci. (2002), 91:760-768); Jiao et. al., Circulation (2002), 105:230-235). Other strategies have explored the use of biodegradable polymers for controlled release of anticoagulants (Vasudev et al., Biomaterials (1997), 18:375-81; Gutowska et al., J. Biomed. Mater. Res. (1995), 29:811-21; Baldwin et al., J. Biomed. Mater. Res. (2012), 100A:2106-18). The aforementioned approaches all rely on the classic paradigm of passive delivery mechanisms that are wholly dissociated from the disease state and therefore not designed to mitigate the current bleeding risks of anticoagulation. Further, such open-loop systems deliver their cargo without any form of feedback regulation and cannot autonomously titrate the release of drugs in response to the dynamic circulatory conditions within the body.
An alternative strategy to wholesale, unrestricted anticoagulation employed engineering of an active mechanism for releasing an anti-coagulant at the time and site of a thrombotic event in order to maximize therapeutic efficacy while offsetting bleeding risk. Similar strategies have been applied to the engineering of bioresponsive thrombolytics, another arm of antithrombotic therapy designed to dissolve existing clots where therapeutic activation is initiated by a proteolytic or biophysical triggers associated with thrombosis. Previously, a recombinant thrombin-activated variant of human plasminogen was introduced that demonstrated selective generation of plasmin, a component of the anti-clot cascade, localized to newly formed clots without affecting established clots or bleeding time (Dawson et al., J. of Biol. Chem. (1994), 269:15989-92; corner et al., J. of Thrombosis and Haemostasis (2005), 3:146-153). This technology has since entered testing in clinical trials (Curtis et al., J. of Thrombosis and Haemostasis (2005), 3:1180-1186; Gibson et al., J. of Thrombosis and Thrombolysis (2006), 22:13-21). Recently, shear-activated microparticles were designed that released tissue plasminogen activator (tPA) only in thrombosed vessels upon exposure to local shear stresses one to two orders of magnitude higher than those present in normal vasculature (Korin et al., Science (2012), 337:738-742). This technology required lower doses of the drug and produced fewer side-effects than treatment with free tPA in mouse models of embolism.
Although the thrombin-activated plasminogen and the shear-activated microparticles represent bioresponsive thrombolytics that may be used to dissolve already existing clots, there still exists a need in the art for more efficacious bioresponsive anticoagulants that can be used prophylactically to prevent the formation of future clots. There also exists a need in the art for anticoagulants that would have predictable dosing profiles and reduced side effects.
The present invention provides stimulus responsive nanocomplexes comprising a therapeutic agent and a masking moiety. The masking moiety prevents the therapeutic agent from exerting its biological activity and also comprises a sensor responsive to a stimulus. When the sensor is modified in the presence of the stimulus, the masking moiety is no longer able to prevent the therapeutic agent from exerting its biological activity. The present invention also provides methods for treating subjects in need thereof, using these nanocomplexes, e.g., treating subjects suffering from or prone to hypercoagulable states.
Accordingly, in one aspect, the present invention provides a stimulus responsive nanocomplex. The nanocomplex includes a therapeutic agent; and a masking moiety comprising a sensor responsive to a stimulus, wherein the masking moiety prevents the therapeutic agent from exerting its biological activity, and wherein the sensor is modified in the presence of the stimulus, thereby allowing the therapeutic agent to exert its biological activity. In some embodiments, the masking moiety comprises a proteinaceous compound. In some embodiments, the proteinaceous compound comprises a peptide.
In certain embodiments, the sensor comprises a protease sensitive sequence. In one embodiment, the protease sensitive sequence is a thrombin cleavage sequence.
In some embodiments, the stimulus is an agent capable of cleaving the protease sensitive sequence. In some embodiments, the agent capable of cleaving the protease sensitive sequence is a clot-forming agent. In a further embodiment, the clot-forming agent is a protease. In one specific embodiment, the protease is thrombin.
In some embodiments, the therapeutic agent is a blood homeostasis agent. In certain embodiments, the blood homeostasis agent is an anti-coagulant. In specific embodiments, the anti-coagulant is heparin or bivalirudin.
In some embodiments, the nanocomplex is self-assembling. In other embodiments, the nanocomplex is self-titrating.
In certain embodiments, the therapeutic agent and the masking moiety interact directly with each other to form the nanocomplex.
In some embodiments, the therapeutic agent is a charged therapeutic agent, and the masking moiety is a charged moiety. In certain embodiments, the charged therapeutic agent is negatively charged and the charged masking moiety is positively charged. In other embodiments, the charged therapeutic agent is positively charged, and the charged masking moiety is negatively charged.
In some embodiments, the masking moiety is a peptide. In certain embodiments, the therapeutic agent is a blood homeostasis agent. In some aspects, the blood homeostasis agent is an anti-coagulant. In one specific embodiment, the anti-coagulant is heparin.
In certain aspects, the sensor comprises a protease sensitive sequence. In a specific embodiment, the protease sensitive sequence is a thrombin cleavage sequence.
In some embodiments, the therapeutic agent and the masking moiety interact indirectly with each other. In some embodiments, the stimulus responsive nanocomplex further comprises a nanoparticle. In a specific embodiment, the nanoparticle is an iron oxide nanoparticle.
In some embodiments, the therapeutic agent and the masking moiety both interact with the nanoparticle. In one embodiment, the masking moiety is a peptide.
In certain embodiments, the therapeutic agent is a blood homeostasis agent. In some embodiments, the blood homeostasis agent is an anti-coagulant. In a specific embodiment, the anti-coagulant is bivalirudin.
In some embodiments, the sensor comprises a protease sensitive sequence. In one embodiment, the protease sensitive sequence is a thrombin cleavage sequence.
In certain embodiments, the masking moiety in the stimulus responsive nanocomplex comprises a peptide. In some embodiments, the peptide is further conjugated to a polymeric agent. In one specific embodiment, the polymeric agent is polyethylene glycol (PEG).
In another aspect, the present invention provides a stimulus responsive nanocomplex, including a charged therapeutic agent; and a charged peptide comprising a sensor responsive to a stimulus, wherein the charge of the peptide is opposite to the charge of the charged therapeutic agent thereby allowing the formation of the nanocomplex and wherein in the presence of the stimulus the sensor is modified thereby releasing the charged therapeutic agent from the nanocomplex. In some embodiments, the charged therapeutic agent is negatively charged and the charged peptide is positively charged. In other embodiments, the charged therapeutic agent is positively charged and the charged peptide is negatively charged.
In certain embodiments, the charged therapeutic agent is a blood homeostasis agent. In some embodiments, the blood homeostasis agent is an anti-coagulant. In one specific embodiment, the anti-coagulant is heparin.
In some embodiments, the sensor is a protease sensitive sequence. In a specific embodiment, the protease sensitive sequence is a thrombin cleavage sequence.
In certain aspects, the stimulus is an agent capable of cleaving the protease sensitive sequence. In some embodiments, the agent is a clot-forming agent. In certain embodiments, the clot-forming agent is a protease. In one specific embodiment, the protease is thrombin.
In some embodiments, the charged peptide masks the biological activity of the charged therapeutic agent.
In yet another aspect, the present invention provides a nanocomplex for the treatment or prevention of thrombosis, the nanocomplex including a negatively charged anti-thrombotic agent; and a positively charged peptide, wherein the peptide comprises a protease sensitive sequence.
In one embodiment, the anti-thrombotic agent is heparin. In some embodiments, the peptide is in excess of the anti-thrombotic agent within the nanocomplex.
In some embodiments, the nanocomplex is formed by the self-assembly of the anti-thrombotic agent and the peptide. In further embodiments, the peptide masks the negative charge of the anti-thrombotic agent. In one embodiment, the protease sensitive sequence is a thrombin cleavage sequence.
In a further aspect, embodiment, the present invention provides a nanocomplex for the treatment or prevention of thrombosis, the nanocomplex including heparin; and a positively charged peptide comprising a thrombin cleavable sequence.
In some embodiments, the peptide is in excess of the heparin within the nanocomplex. In some embodiments, the nanocomplex is self-assembling. In certain embodiments, the peptide masks the negative charge of the heparin.
In another aspect, the present invention provides a nanocomplex for the treatment or prevention of thrombosis, the nanocomplex including bivalirudin; a peptide-polyethylene glycol conjugate comprising a thrombin cleavable sequence; and a nanoparticle, wherein both the bivalirudin and the peptide-polyethylene conjugate are attached to the nanoparticle.
In some embodiments, the present invention also provides a method of treating a subject in need thereof, the method comprising administering to the subject an effective amount of the nanocomplex of the invention, thereby treating the subject.
In some embodiments, the subject is suffering from a hypercoagulable state. In certain embodiments, the hypercoagulable state is hypertension or cardiovascular disease. In some aspects, the cardiovascular disease is coronary occlusion, arteriosclerotic heart disease (ASHD) or coronary thrombosis.
In one embodiment, the subject is a human.
The present invention is further illustrated by the following detailed description and drawings.
The present invention provides stimulus responsive nanocomplexes comprising a therapeutic agent and a masking moiety. In the absence of the stimulus, the masking moiety interacts directly or indirectly with the therapeutic agent and prevents the therapeutic agent from exerting its biological activity. When the stimulus is present, the masking moiety no longer masks the therapeutic agent, thereby allowing the therapeutic agent to exert its biological activity.
In some embodiments, the therapeutic agent may be a blood homeostasis agent, e.g., an anti-coagulant. In such exemplary embodiments, the benefits provided by the nanocomplexes of the present invention include selective activation of the exogenous anti-coagulant only in response to inappropriate thrombotic events, while smaller-scale clotting in response to everyday compromises of the endothelium, such as bruising or cuts, is not affected. In certain embodiments, the nanocomplexes comprise heparin, an anionic anti-coagulant, that forms charge-based complexes with a cationic thrombin-sensitive peptide (
Furthermore, because the underlying chemistry used to produce the nanocomplexes is very well established, the manufacture of the nanoparticles may be cheaper and simpler, as compared to the other agents known in the art, such as thrombin-activated plasminogen and the shear-activated microparticles.
In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.
The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.
As used herein, the term “stimulus responsive nanocomplex” is a complex that comprises a therapeutic agent which becomes available to exert its biological activity in response to a stimulus. In some embodiments, the stimulus responsive nanocomplex comprises a therapeutic agent and a masking moiety. The masking moiety comprises a sensor responsive to a stimulus. In the absence of the stimulus, the masking moiety prevents the therapeutic agent from exerting its biological activity. In the presence of the stimulus, the sensor is modified, thereby allowing the therapeutic agent to exert its biological activity. The therapeutic agent and the masking moiety may interact directly to form the nanocomplex or they may interact indirectly, e.g., through a nanoparticle, to form the nanocomplex. Thus, in some embodiments, the stimulus responsive nanocomplex further comprises a nanoparticle, e.g., an iron oxide nanoparticle. In one embodiment, the stimulus responsive nanocomplex is thrombin-activatable plasminogen or a thrombin-activatable plasminogen analog.
As used herein, the term “therapeutic agent” includes any biologically active agent that may be used in the nanocomplexes of the present invention. In some embodiments, the therapeutic agent may be a small molecule, a peptide, an oligosaccharide, an oligonucleotide, or a protein, e.g., an antibody or a fragment thereof. The therapeutic agent may carry an overall negative charge, a positive charge or may be neutral at physiological conditions. In some embodiments, the therapeutic agent is a blood homeostasis agent, e.g., an anti-coagulant, useful in treating a hypercoagulable state. Specific examples of such agents include, but are not limited to, acenocoumarol (Sinthrome®), apixaban, aspirin, bivalirudin (Angiox®), clopidogrel (Plavix®), fondaparinux sodium (Arixtra®), low molecular weight heparins (e.g., semuloparin, bemiparin, dalteparin, and enoxaparin), heparin, heparin sodium, dabigatran etexilate mesylate (Pradaxa®), danaparoid sodium (Orgaran), epoprostenol sodium (Flolan®), tinzaparin sodium (Innohep®), warfarin (Marevan®), menadiol sodium phosphate, rivaroxaban (BAY 59-7939, Xarelto®), prasugrel, ticagrelor, ticlopidine, argatroban, lepidurin, anagrelide, apixaban, cilostazol, and dipyridamole. In one specific embodiment, the therapeutic agent is heparin. In another specific embodiment, the therapeutic agent is bivalirudin.
As used herein, the term “masking moiety” includes any moiety that, when present as a part of a nanocomplex of the invention, prevents the therapeutic agent from exerting its biological activity, e.g., an anti-coagulant activity. In some embodiments, the masking moiety may interact directly with the therapeutic agent via any type of interaction known in the art. For example, the masking moiety may interact with the therapeutic agent via electrostatic interactions, hydrogen bonding interactions, covalent interactions, Van der Waals interactions or hydrophobic interactions. In one embodiment, both the masking moiety and the therapeutic agent are charged, and interact with each other directly via electrostatic interactions.
In other embodiments, the masking moiety interacts indirectly with the therapeutic agent. In one such embodiment, both the masking moiety and the therapeutic agent are conjugated to a nanoparticle.
The masking moiety may be any entity, such as a small molecule, a peptide, a polypeptide, an oligosaccharide, an oligonucleotide, a peptide nucleic acid (PNA), or a protein, e.g., an antibody or a fragment thereof. In some embodiments, the masking moiety is a peptide, such as a charged peptide, e.g., a positively or a negatively charged peptide. In one specific embodiment, the peptide is positively charged.
In one embodiment, the masking moiety is not a higher ordered structure. In a further embodiment, the masking moiety is not an encapsulating particle or a protein cage, i.e., a structure with an interior cavity which is either naturally accessible to a solvent or can be made to be so by altering solvent concentration, pH or equilibria ratios. In a specific embodiment, the masking moiety is not a virion protein cage. In yet another embodiment, the masking moiety is not, and the nanocomplex does not comprise, a transmembrane polypeptide that naturally comprises a pore (i.e., a channel).
The masking moiety comprises a sensor responsive to a stimulus. The “sensor responsive to a stimulus”, as used herein, is any molecular entity that is modified in response to a stimulus, thereby allowing the therapeutic agent to exert its biological activity. For example, the sensor may undergo a conformation change, a cleavage, a binding, or a degradation in response to a stimulus. In one embodiment, the sensor comprises a protease sensitive sequence, e.g., a thrombin cleavage sequence, and is cleaved in response to exposure to the stimulus, e.g., thrombin.
“Biological activity”, as used herein, is well known in the art and includes any activity by a therapeutic agent, as described herein, that elicits a response from living tissue or an organism. In some embodiments, the biological activity includes any activity exerted by a therapeutic agent comprised in the nanocomplexes as described herein. In a specific embodiment, the biological activity is an anti-coagulant activity that prevents, reduces or inhibits blood clotting.
A “stimulus”, as used herein, includes any set of conditions that produce a change in the sensor. For example, a stimulus may be a specific pH, a specific temperature or a change in temperature, or an agent capable of interacting with the sensor and present in a location at which the activity of the therapeutic agent is needed and/or desired. In some embodiments, the stimulus is an agent capable of cleaving a protease sensitive sequence. In further embodiments, the stimulus is a clot forming agent, e.g., a protease, such as thrombin.
A “hypercoagulable state”, or “thrombophilia”, as used herein, refers to any blood clotting disorder that is characterized by excessive coagulation or any other condition associated with excessive coagulation. In one embodiment, the conditions associated with excessive coagulation include, but are not limited to, any condition characterized by an increased risk of myocardial infarction, pulmonary embolism or a stroke, such as hypertension or cardiovascular disease, e.g., coronary occlusion, arteriosclerotic heart disease (ASHD) or coronary thrombosis.
A hypercoagulable state may be a genetic (inherited) or an acquired condition. Examples of genetic hypercoagulable states include, but are not limited to, Factor V Leiden; conditions caused by prothrombin gene mutation; deficiencies of natural proteins that prevent clotting (such as antithrombin, protein C and protein S); conditions characterized by elevated levels of homocysteine, elevated levels of fibrinogen or by dysfunctional fibrinogen (dysfibrinogenemia); conditions characterized by elevated levels of factor VIII and other factors including factor IX and XI; conditions characterized by abnormal fibrinolytic system, including hypoplasminogenemia, dysplasminogenemia, and elevation in levels of plasminogen activator inhibitor (PAI-1). Acquired hypercoagulable conditions may include, but are not limited to, cancer and associated conditions caused by some medications used to treat cancer, such as tamoxifen, bevacizumab, thalidomide and lenalidomide; recent trauma or surgery; central venous catheter placement; obesity; pregnancy; conditions caused by supplemental estrogen use, including oral contraceptive pills (birth control pills); conditions characterized by hormone replacement therapy; conditions characterized by prolonged bed rest or immobility; heart attack, congestive heart failure, stroke and other illnesses that lead to decreased activity; heparin-induced thrombocytopenia (decreased platelets in the blood due to heparin or low molecular weight heparin preparations); conditions caused by lengthy airplane travel, also known as “economy class syndrome”; antiphospholipid antibody syndrome; previous history of deep vein thrombosis or pulmonary embolism; myeloproliferative disorders such as polycythemia vera or essential thrombocytosis; paroxysmal nocturnal hemoglobinuria; inflammatory bowel syndrome; HIV/AIDS; and nephrotic syndrome characterized by excessive protein in the urine).
The present invention provides stimulus responsive nanocomplexes comprising a therapeutic agent and a masking moiety that prevents the therapeutic agent from exerting its biological activity. The masking moiety comprises a sensor responsive to a stimulus. The sensor is modified in the presence of the stimulus, such that the masking moiety no longer prevents the therapeutic agent from exerting its biological activity.
The therapeutic agent may be any biologically active agent that may be used in the nanocomplexes of the present invention. In some embodiments, the therapeutic agent is a blood homeostasis agent, e.g., an anti-coagulant, such as heparin or bivalirudin. In some embodiments, the therapeutic agent may be a charged therapeutic agent, e.g., a positively charged therapeutic agent or a negatively charged therapeutic agent, or a neutral therapeutic agent. In a specific embodiment, the therapeutic agent is a negatively charged therapeutic agent, e.g., heparin. In another specific embodiment, the therapeutic agent is a therapeutic agent with no charge, e.g., bivalirudin.
The masking moiety may be any moiety that, when present in a nanocomplex of the invention, prevents the therapeutic agent from exerting its biological activity. The masking moiety may be any entity, e.g., a small molecule, a peptide, a polypeptide, an oligosaccharide, an oligonucleotide, a peptide nucleic acid (PNA), or a protein, e.g., an antibody or a fragment thereof. In one embodiment, the masking moiety is a peptide. In a further embodiment, the masking moiety is a charged peptide, e.g., a positively or a negatively charged peptide. The positively charged peptide may comprise positively charged amino acids, e.g., arginine or lysine. In a specific embodiment, a positively charged peptide may comprise stretches of alternating arginines and lysines.
In some embodiments, the masking moiety and the therapeutic agent are present in the nanocomplex at a ratio of between about 1:1 to about 10:1 masking moiety:therapeutic agent, e.g., about 1:1, about 1.5:1, about 2:1, about 2.5 to 1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, about 5.5:1, about 6:1, about 6.5:1, about 7:1, about 7:5:1, about 8:1, about 8.5:1, about 9:1, about 9.5:1, or about 10:1. In a specific embodiment, the masking moiety, e.g., a peptide, such as a positively charged peptide, and the therapeutic agent, e.g., a negatively charged therapeutic agent, such as heparin, are present at a ratio of 5:1.
In some embodiments, the masking moiety, e.g., a peptide, may be further conjugated to an additional agent, e.g., a polymeric agent. Conjugation of the masking moiety, e.g., a peptide, to the additional agent, e.g., a polymeric agent may be desirable to prevent the instability and aggregation of nanocomplexes in physiological solutions and at high concentrations. The instability and aggregation of nanocomplexes can negatively impact in vivo performance by decreasing circulation time and increasing risk of lung entrapment.
The polymeric agent may comprise any number of hydrophilic non-fouling polymers. Examples of such polymers include, but are not limited to, polyethylene glycols (PEGs), polyoxazolines, poly(amino acids), N-(2-hydroxylpropyl)methacrylamide (HPMA), polybetaines, polyglycerols, polysaccharides (e.g., hyaluronic acid, dextran and chitosan), and polypeptides.
In some embodiments, the polymeric agent is a member of a family of polyethylene glycols (PEGs). Polyethylene glycols are a family of polymers produced from the condensation of ethylene glycol, and have the general formula H(OCH2CH2)nOH where n, the number of ethylene glycol groups, is greater than or equal to 4. Generally, the designation of a polyethylene glycol (PEG) includes a number that corresponds to its average molecular weight. For example, polyethylene glycol 1500 refers to a mixture of polyethylene glycols having an average value of n between 29 and 36 and a molecular weight range of 1300 to 1600 grams/mole. In a specific embodiment, the PEG has an average molecular weight of 5000 grams/mole. PEGs may further be covalently linked to additional functional groups, e.g., groups that may allow the PEGs to be linked to other moieties, e.g., a therapeutic agent. In a specific embodiment, the PEG is a poly(ethylene glycol)-succinimidyl valerate.
In some embodiments, the additional agent, e.g., a polymeric agent such as PEG, and the masking moiety, e.g., a peptide are present in the nanocomplex at a ratio of between about 1:1 to about 25:1 additional agent:masking moiety, e.g., about 1:1, about 2:1, about 3:1, about 4 to 1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1 or about 25:1. In a specific embodiment, the additional agent, e.g., a polymeric agent such as PEG, and the masking moiety, e.g., a peptide, are present in the nanocomplex at a ratio of 25:1. In another specific embodiment, the additional agent, e.g., a polymeric agent such as PEG, and the masking moiety, e.g., a peptide, are present in the nanocomplex at a ratio of 5:1.
The masking moiety may interact directly or indirectly with the therapeutic agent. Direct interactions of the masking moiety with the therapeutic agent may involve electrostatic interactions, hydrogen bonding interactions, covalent interactions, Van der Waals interactions or hydrophobic interactions. In some embodiments, where both the masking moiety and the therapeutic agent are charged, e.g., the masking moiety is negatively charged and the therapeutic agent is positively charged, or the masking moiety is positively charged and the therapeutic agent is negatively charged, the interactions between the masking moiety and the therapeutic agent are electrostatic interactions. In a specific embodiment, the masking moiety is a positively charged peptide, e.g., a peptide having the sequence comprising stretches of alternating positively-charged amino acids, arginine and lysine, such as rkrkLVPRGrkrkLVPRGrkrkLVPRGrkrk (with lower-case letters denoting d-amino acids), that interacts with the negatively charged therapeutic agent, e.g., heparin.
The masking moiety and the therapeutic agent may also interact indirectly. In a specific embodiment, the masking moiety and the therapeutic agent may both be conjugated to a third agent, e.g., a nanoparticle. Examples of nanoparticles that can serve as third agents include, but are not limited to, gold nanoparticles, silica nanoparticles, dextran, albumin, PEG, dendrimers and PLGA particles. The masking moiety, e.g., a peptide, and the therapeutic agent, e.g. bivalirudin, may be conjugated to the third agent, e.g., a nanoparticle, through a number of different chemistries that may comprise, but are not limited to, NHS-amine coupling, maleimide-sulfhydryl coupling and click chemistry.
In one embodiment, the third agent is an iron oxide nanoparticle. Accordingly, in one example, the nanocomplex of the invention comprises the masking moiety, e.g., a peptide, and the therapeutic agent, e.g., bivalirudin, that are both conjugated to a nanoparticle, e.g., an iron oxide nanoparticle.
The “sensor responsive to a stimulus”, as comprised in the masking moiety of the nanocomplexes of the invention, is any molecular entity that is modified in response to a stimulus. For example, the sensor may undergo a conformation change, a cleavage, a binding, or a degradation in response to a stimulus. In one embodiment, the sensor comprises a protease sensitive sequence, e.g., a thrombin cleavage sequence, and is cleaved in response to exposure to the stimulus, e.g., thrombin. For example, the sensor may comprise the thrombin cleavage sequence: P4-P3-P2-P1-P1′-P′ comprising arginine (R) in position P1. Examples of such thrombin cleavage sequences may include P4-P3-P2-P1-P1′-P2′ comprising arginine (R) in position Pi and glycine (G) in position P2 and position P1; or P4-P3-P2-P1-P1′-P2′ comprising hydrophobic residues in position P4 and position P3, proline (P) in position P2, arginine (R) in position P1, and non-acidic amino-acids in position P1′ and position P2′. In a specific embodiment, the sensor comprises the peptide sequence: leucine-valine-proline-arginine-glycine (LVPRG), a well-known thrombin substrate.
In some embodiments, the nanocomplexes of the invention are self-assembling. The self-assembling nanocomplexes are spontaneously formed after their components, e.g., the masking moiety and the therapeutic agent, are mixed together. Formation of the self-assembling nanocomplexes does not require additional manipulations, e.g., chemical reaction or conjugation steps. For example, the nanocomplex comprising a charged masking moiety, e.g., a positively charged peptide, and a charged therapeutic agent, e.g., heparin, is self-assembling.
In some embodiments, the nanocomplexes of the invention are also self-titrating. A self-titrating nanocomplex responds to changes in the strength of a stimulus, thereby modulating the level of the resulting biological activity by the therapeutic agent. For example, in nanocomplexes comprising a peptide as a masking moiety and heparin as the therapeutic agent, the amount of heparin released from the nanocomplex in response to the stimulus, e.g., thrombin, is proportional to the amount of thrombin activity present. Such nanocomplex deploys more anti-coagulant under thrombotic conditions, yet, is more tolerant of healthy coagulation processes (see
Although the nanocomplexes of the invention may be used without additional carriers, nanocomplexes of the invention may also be formulated as pharmaceutical compositions further comprising a pharmaceutically acceptable carrier or diluent. As used herein, a “pharmaceutical composition” can be a formulation containing the nanocomplexes, in a form suitable for administration to a subject. Suitable pharmaceutically acceptable carriers may contain inert ingredients which do not inhibit the biological activity of the therapeutic agents contained in the nanocomplex. The pharmaceutically acceptable carriers that may be used in the pharmaceutical composition are any carriers that are biocompatible, i.e., non-toxic, non-inflammatory, non-immunogenic and devoid of other undesired reactions upon the administration to a subject. Standard pharmaceutical formulation techniques can be employed, such as those described in Remington: the Science and Practice of Pharmacy, 19th edition, Mack Publishing Co., Easton, Pa. (1995).
The pharmaceutical composition can be in bulk or in unit dosage form. The unit dosage form can be in any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial. The quantity of a nanocomplex in a unit dose is the effective amount of the nanocomplex that can vary according to the chosen administration route. A variety of routes are contemplated, including topical, oral, transmucosal or parenteral, including transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal and intranasal. For oral administration, the nanocomplex may be combined with a suitable solid or liquid carrier or diluent to form capsules, tablets, pills, powders, syrups, solutions, suspensions, or the like.
The tablets, pills, capsules, and the like can contain from about 1 to about 99 weight percent of the active ingredient and a binder such as gum tragacanth, acacias, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch or alginic acid; a lubricant such as magnesium stearate; and/or a sweetening agent such as sucrose, lactose or saccharin. When a dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil.
For parental administration, a nanocomplex may be combined with sterile aqueous or organic media to form injectable solutions or suspensions. For example, solutions in sesame or peanut oil, aqueous propylene glycol and the like can be used, as well as aqueous solutions of water-soluble pharmaceutically-acceptable salts of the compounds. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. In one embodiment, the pharmaceutical composition is not a hydrogel composition. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
In addition to the formulations described above, a formulation can optionally include, or be co-administered with one or more additional drugs, e.g., anti-coagulants, antihypertensives, anti-inflammatories, antibiotics, antifungals, antivirals, immunomodulators, antiprotozoals, steroids, decongestants, bronchodilators, antihistamines, anticancer agents, and the like. The formulation may also contain preserving agents, solubilizing agents, chemical buffers, surfactants, emulsifiers, colorants, odorants and sweeteners.
The present invention also provides methods for treating or preventing a disease or a condition, e.g., a hypercoagulable state, in a subject. The invention also provides methods for treating a subject in need of anti-thrombotic therapy or prophylaxis. The methods include administering to the subject a therapeutically effective amount or prophylactically effective amount of a nanocomplex of the invention.
As used herein, a “subject” includes either a human or a non-human animal, preferably a vertebrate, and more preferably a mammal. A subject may include a transgenic organism. Most preferably, the subject is a human, such as a human suffering from or predisposed to developing thrombosis or a hypercoagulable state.
A “hypercoagulable state”, or “thrombophilia”, as used herein, refers to any blood clotting disorder that is characterized by excessive coagulation or any other condition associated with excessive coagulation. In one embodiment, the conditions associated with excessive coagulation include, but are not limited to, any condition characterized by an increased risk of myocardial infarction, pulmonary embolism or a stroke, such as hypertension or cardiovascular disease, e.g., coronary occlusion, arteriosclerotic heart disease (ASHD) or coronary thrombosis.
The nanocomplexes of the invention may be administered to a subject using any mode of administration known in the art, including, but not limited to subcutaneous, intravenous, intramuscular, intraocular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combinations thereof. In one embodiment, the nanocomplexes are administered parenterally, e.g., intravenously. In a further embodiment, the nanocomplexes are administered intravenously by a bolus dose, via continuous infusion, e.g., via an intravenous drip.
The nanocomplexes of the invention may also be administered using a dosing schedule that comprises an initial dose and one or more subsequent maintenance doses. For example, the schedule may include an initial intravenous bolus dose, followed by one or more maintenance doses administered intravenously by continuous infusion, e.g., via an intravenous drip. The exact dosing schedule for a nanocomplex of the invention will depend on the dosing schedule recommended for the specific therapeutic agent comprised in the nanocomplexes.
Other modes of administration include epidural, intracerebral, intracerebroventricular, nasal administration, intraarterial, intracardiac, intraosseous infusion, intrathecal, and intravitreal, and pulmonary. The mode of administration may be appropriately determined by a one of skill in the art, e.g., a physician, in the course of the treatment.
In some embodiments, the nanocomplex is administered to a subject in an amount effective to prevent, reduce or inhibit clot formation in a subject. Such amount includes the amount of the nanocomplex that releases the dose of the therapeutic agent that is effective to treat or prevent a hypercoagulable state, e.g., reduce a risk of a myocardial infarction. Such amount also includes the amount of the nanocomplex that maintains the desired concentration of the therapeutic agent in the blood of a subject that is effective to treat or prevent a hypercoagulable state, e.g., reduce a risk of a myocardial infarction.
An “effective amount”, as used herein, includes the amount of a nanocomplex that, when administered to a subject for treating a condition, e.g., a hypercoagulable state, provides the amount of therapeutic agent that is sufficient to effect treatment of a condition, e.g., a hypercoagulable state, (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease).
The “effective amount,” as used herein, includes the amount of a nanocomplex that, when administered to a subject who is at risk of developing or may be predisposed to a disease or condition, e.g., a hypercoagulable state, such as a cardiovascular disease, provides the amount of the therapeutic agent that is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. The “effective amount,” as used herein, also includes the amount of a nanocomplex that, when administered to a subject who is at risk of developing or may be predisposed to a disease or condition, e.g., a hypercoagulable state, such as a cardiovascular disease, is able to maintain the concentration of the therapeutic agent in the blood of the subject that is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease.
The “effective amount” may vary depending on the exact nature of the nanocomplex, how the nanocomplex is administered, the rate and the efficiency of release of the therapeutic agent from the nanocomplex, the amount of the therapeutic agent present in the nanocomplex, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.
In one embodiment, the contemplated dose range for the nanocomplex comprising heparin is the range that provides the dose of heparin from about 1 U/kg to about 500 U/kg, e.g., about 1 U/kg, about 5 U/kg, about 10 U/kg/ about 15 U/kg, about 20 U/kg, about 25 U/kg, about 30 U/kg, about 35 U/kg, about 40 U/kg, about 45 U/kg, about 50 U/kg, about 55 U/kg, about 60 U/kg, about 65 U/kg, about 70 U/kg, about 75 U/kg, about 80 U/kg, about 85 U/kg, about 90 U/kg, about 95 U/kg, about 100 U/kg, about 110 U/kg, about 120 U/kg, about 130 U/kg, about 140 U/kg, about 150 U/kg, about 160 U/kg, about 170 U/kg, about 180 U/kg, about 190 U/kg, about 200 U/kg, about 210 U/kg, about 220 U/kg, about 230 U/kg, about 240 U/kg, about 250 U/kg, about 250 U/kg, about 260 U/kg, about 270 U/kg, about 280 U/kg, about 290 U/kg, about 300 U/kg, about 310 U/kg, about 320 U/kg, about 330 U/kg, about 340 U/kg, about 350 U/kg, about 360 U/kg, about 370 U/kg, about 380 U/kg, about 390 U/kg, about 400 U/kg, about 410 U/kg, about 420 U/kg, about 430 U/kg, about 440 U/kg, about 450 U/kg, about 460 U/kg, about 470 U/kg, about 480 U/kg, about 490 U/kg, or about 500 U/kg.
In another embodiment, the recommended dose range of the nanocomplex comprising heparin is the range that provides the dose of heparin from about 1 U/kg to about 160 U/kg, e.g., about 1 U/kg, about 5 U/kg, about 10 U/kg, about 15 U/kg, about 20 U/kg, about 25 U/kg, about 30 U/kg, about 35 U/kg, about 40 U/kg, about 45 U/kg, about 50 U/kg, about 55 U/kg, about 60 U/kg, about 65 U/kg, about 70 U/kg, about 75 U/kg, about 80 U/kg, about 85 U/kg, about 90 U/kg, about 95 U/kg, about 100 U/kg, about 110 U/kg, about 120 U/kg, about 130 U/kg, about 140 U/kg, about 150 U/kg, about 160 U/kg. These doses of the nanocomplex may be administered intravenously, e.g., as bolus doses.
The initial administration of the bolus dose of the nanocomplex comprising heparin may be followed by one or more maintenance doses required to maintain therapeutic levels of heparin in the blood of the subject. Maintenance of the therapeutic levels may be accomplished by administering the nanocomplexes of the invention via intravenous infusion, e.g., an intravenous drip. Maintenance doses of the nanocomplexes may range from about 0.1 to about 30 U/kg/hour, e.g., about 0.1 U/kg/hour, about 1 U/kg/hour, about 3 U/kg/hour, about 4 U/kg/hour, about 5 U/kg/hour, about 6 U/kg/hour, about 7 U/kg/hour, about 8 U/kg/hour, about 9 U/kg/hour, about 10 U/kg/hour, about 11 U/kg/hour, about 12 U/kg/hour, about 13 U/kg/hour, about 14 U/kg/hour, about 15 U/kg/hour, about 16 U/kg/hour, about 17 U/kg/hour, about 18 U/kg/hour, about 19 U/kg/hour, about 20 U/kg/hour, about 21 U/kg/hour, about 22 U/kg/hour, about 23 U/kg/hour, about 24 U/kg/hour, about 25 U/kg/hour, about 26 U/kg/hour, about 27 U/kg/hour, about 28 U/kg/hour, about 29 U/kg/hour or about 30 U/kg/hour.
The recommended dose range of the nanocomplex comprising bivalirudin is the range that provides the dose of bivalirudin of from about 0.01 U/kg to about 10 mg/kg, e.g., about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, or about 10 mg/kg. These doses of the nanocomplex may be administered intravenously, e.g., as bolus doses.
The initial administration of the bolus dose of the nanocomplex comprising bivalirudin may be followed by one or more maintenance doses required to maintain therapeutic levels of bivalirudin in the blood of the subject. The maintenance doses of the nanocomplexes comprising bivalirudin may range from about 0.01 mg/kg/hour to about 10 mg/kg/hour, e.g., about 0.01 U/kg/hour, about 0.02 U/kg/hour, about 0.03 U/kg/hour, about 0.04 U/kg/hour, about 0.05 U/kg/hour, about 0.06 U/kg/hour, about 0.07 U/kg/hour, about 0.08 U/kg/hour, about 0.09 U/kg/hour, about 0.1 U/kg/hour, about 0.2 U/kg/hour, about 0.3 U/kg/hour, about 0.4 U/kg/hour, about 0.5 U/kg/hour, about 0.6 U/kg/hour, about 0.7 U/kg/hour, about 0.8 U/kg/hour, about 0.9 U/kg/hour, about 1 U/kg/hour, about 2 U/kg/hour, about 3 U/kg/hour, about 4 U/kg/hour, about 5 U/kg/hour, about 6 U/kg/hour, about 7 U/kg/hour, about 8 U/kg/hour, about 9 U/kg/hour, or about 10 U/kg/hour. The maintenance doses of the nanocomplexes comprising bivalirudin may be administered for a length of time ranging from about 4 hours to about 20 hours after the initial administration of the bolus dose.
It may be necessary to use doses of nanocomplex outside the ranges disclosed herein in some cases, as will be apparent to those of ordinary skill in the art. Furthermore, it is noted that the clinician or treating physician will know how and when to interrupt, adjust, or terminate therapy in conjunction with individual patient response.
In some embodiments, the nanocomplex is administered in combination with other therapeutic agents or other therapeutic regimens. For example, other agents or other therapeutic regimens suitable for treating a hypercoagulable state, e.g., a cardiovascular disease, may include angiotensin converting enzyme inhibitors (ACE inhibitors), angiotension II receptor blockers, antiarrhythmics, antiplatelet drugs, anti-hypertensives, e.g., beta-blockers, calcium channel blocker drugs, anti-coagulants, digoxin, diuretics or nitrates. In one embodiments, the nanocomplex of the invention is administered in combination with aspirin.
The present invention is next described by means of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified form. The invention is not limited to any particular preferred embodiments described herein. Many modifications and variations of the invention may be apparent to those skilled in the art and can be made without departing from its spirit and scope. The contents of all references, patents and published patent applications cited throughout this application, including the figures, are incorporated herein by reference.
Heparin is a strongly anionic material which may be neutralized in the clinical setting via sequestration by cationic, arginine-rich protamine peptides that bind heparin to form non-reactive nanocomplexes (Rossmann P. et al., Virchows Archiv B Cell Pathol., 1982, 40:81-98). Synthetic peptide antidotes of heparin require a minimum amount of positive charge in order to completely neutralize the functional activity of heparin (DeLucia A. et al., J. of Vascular Surgery, 1993, 18:49-58). Accordingly, a long peptide with multiple cationic regions separated by protease-cleavable sequences was designed. It was expected that this peptide would veil heparin function while intact, but will break down into fragments that would be too small to inhibit heparin activity in response to thrombin-induced cleavage. Sequence of the peptide contained LVPRG, a well-known thrombin substrate separated by stretches of alternating positively-charged amino acids, arginine and lysine, and was as follows: rkrkLVPRGrkrkLVPRGrkrkLVPRGrkrk (lower-case letters denote d-amino acids). Peptides were synthesized by standard FMOC solid-phase peptide synthesis at Koch Institute Biopolymers Core Facility or at Tufts University Core Facility, lyophilized, and resuspended at the concentration of 5 mg/mL in ddH2O.
A formulation of the peptide-heparin that is free from the instability and aggregation characteristics of charged nanocomplexes in physiological solutions and at high concentrations was then developed. The instability and aggregation can negatively impact in vivo performance by decreasing circulation time and increasing risk of lung entrapment, as evidenced by bare protamine-heparin nanocomplexes that rapidly deposit in locations such as the glomerular basement membrane and do not remain in circulation (Rossmann et al., Virchows Archive B Cell Pathol (1982), 40:81-98). To enhance stability of nanocomplexes in ionic solutions, to prevent non-specific protein interactions, to improve biodistribution and to increase circulation time through steric stabilization, the FDA-approved polymer, poly(ethylene glycol) (PEG), a biocompatible polymer widely used in drug delivery applications, was conjugated to the peptides via amine-ester chemistry. Specifically, 5 kDa PEG was covalently attached to the lysines of the peptides by incubating 5 mg/mL stock peptide solution with amine-reactive 5,000 Da poly(ethylene glycol)-succinimidyl valerate (PEG-SVA, Laysan Bio Inc.) for 1 hour at room temperature.
The PEG-conjugated peptides as described in Example 1 were combined with heparin (sodium salt from porcine mucosa, Sigma) at a fixed concentration of 20 U/mL (−0.1 mg/mL) unless reported otherwise. To determine the optimal ratio of PEG:peptide:heparin, nanocomplexes with peptide:heparin ratios between 1:1 to 10:1 and PEG:peptide ratios between 1:1 to 25:1 were formed, incubated in PBS buffer or 10% serum for 1 hour, and their size and zeta potential was measured by dynamic light scattering. For measurements in ionic solutions, 10×PBS stock was added to pre-formed nanocomplex solutions for a final concentration of 1×PBS. For measurements in serum, bovine serum (Gibco) was added to a concentration of 10% (v/v). Mean hydrodynamic diameter was determined via dynamic light scattering of a 50 μL sample at 20 U/mL heparin (ZetaSizer Nano Series, Malvern) and is shown in
Increasing the peptide:heparin ratios from 5:1 to 10:1 increased the size of nanocomplexes in PBS and 10% serum by an order of magnitude (from ˜100 nm to 1000 nm), possibly due to complex aggregation (See
Since cationic peptides may be cytotoxic (Ellerby et al., Nat Med (1999), 5: 1032-1038; Hancock, R. E. W., Lancet (1997), 349(9049):418-422; Wyman et al., Biochemistry (1997), 36(10):3008-3017), the cytotoxicity of nanoparticles at 25:5:1 PEG:peptide:heparin ratio (25:5:1 LVPR.RK4 particles) was determined. For the cytotoxicity assays, Human umbilical vein endothelial cells (HUVEC, Passage 9) were cultured in EGM-2 media (Lonza) on a 96-well plate. When the cells reached 70% confluency, nanocomplexes, free peptide, or free heparin were added as 9×stocks in PBS, diluted in EGM-2. After 24 hours elapsed, cell viability was quantified by an MTS Assay (CellTiter AQueous One, Promega) based on OD490 after 1 hour incubation. No cell toxicity was observed up to 10 U/mL, which corresponds to a ˜1000 U/kg heparin dose in the bloodstream (See
The purpose of this experiment was to show that the nanocomplexes could function to self-regulate the release of heparin in response to clotting activity. The LVPR.RK4 peptides were designed with three thrombin-cleavable stretches to enable release of heparin in response to thrombin activity.
First, to characterize thrombin-triggered disassembly of the nanocomplexes, nanocomplexes formulated as described above using fluorescently labeled heparin (FITC heparin, Polysciences) were incubated for 30 minutes at 37° C. with or without thrombin (500 nM) at 37° C. and were assayed using analytical FPLC. Specifically, analytical samples of nanocomplexes, nanocomplexes incubated with thrombin and of free heparin were applied to a Superdex 200 column pre-equilibrated with PBS. Absorbance of the column effluent was monitored at a wavelength of 488 nm by a UV flow-through detector.
As is shown in
To test for full sequestration of heparin, the Azure II assay was used. Azure II is a metachromatic dye that exhibits a shift in absorbance upon electrostatic interactions. Samples containing intact nanocomplexes, nanocomplexes incubated with thrombin or free heparin were mixed with 0.1 mg/ml Azure II solution at a 1:10 volumetric ratio, and the absorbance was read at 530 nm with SpectraMAX Plus spectrophotometer (Molecular Devices).
As shown in
The sequestration of heparin charge by uncleaved peptide and release of heparin charge upon peptide cleavage indicates that the heparin function will be unveiled in response to thrombin activity. The Factor Xa (FXa) assay (anti-FXa assay) was used to determine the functional state of the heparin in samples containing intact and thrombin-cleaved nanocomplexes. This assay uses a chromogenic substrate to measure the FXa enzyme activity of the sample and to determine the amount of heparin in the sample, which is inversely proportional to anti-coagulant levels. The anti-FXa assay (Sekisui Diagnostics) was performed according to the manufacturer's instructions. The release of heparin was determined by incubating nanocomplexes with various concentrations of human thrombin (Haemotologic Technologies) at 37° C. for the various amounts of time. The activity of the released heparin was then determined using the anti-FXa assay. In the anti-FXa assay, a test sample or heparin standard is added to a fixed amount of antithrombin III (ATIII). A fixed amount of factor Xa is then added to the sample, resulting in the formation of inactive ATIII-Xa complexes. Residual Xa is then measured using either a clotting-based assay or chromogenic assay. The residual Xa activity is inversely proportional to the heparin concentration in the sample and can be quantitated from a calibration curve.
As is shown in
Anti-FXa assay was also used to determining the kinetics of heparin release when the nanocomplexes are incubated with varying concentrations of thrombin over time. In whole blood or plasma, thrombin concentrations as low as ˜1 nM are required to initiate the subsequent burst in thrombin generation (up to 100-500 nM) that is needed to produce a stable fibrin clot (Brummel et al., Blood (2002), 100:148-152; Orfeo et al., PLOS One (2011), 6(11):e27852). Over-anticoagulation may delay or dampen this thrombin generation cycle and disrupt fibrin formation, resulting in prolonged bleeding, whereas insufficient anticoagulation may fail to inhibit the unchecked coagulation activity that leads to thrombosis. Therefore, the purpose of this experiment was to probe the response of nanocomplexes to dynamic thrombin levels.
As is shown in
The effect of the nanocomplexes on a physiologic coagulation pathway was tested using a standard activated partial thromboplastin time (aPTT) clotting assay, which is also used to monitor heparin levels of patients. This assay measures the amount of time needed for a plasma sample to form a clot in vitro when the contact pathway of coagulation is triggered by addition of an exogenous aPTT reagent. For the assay, varying concentrations of nanocomplexes or free heparin were added to 50 μl of APTT reagent (Thermo Scientific) at 37° C. for 3 minutes. Subsequently, 50 μl of 25 mM CaCl2 (Sigma) pre-incubated at 37° C. were added to samples and clotting was monitored via absorbance at 605 nm with a SpectraMAX Plus spectrophotometer (Molecular Dynamics).
As can be seen in
Current clinical guidelines for aPTT monitoring of heparin dosing recommend dose ranges that yield an aPTT approximately ˜1.5-2.3× of the control bleeding time (Eikelboom, J. W., Hirsh, J., Thromb Haemost (2006), 96(5):547-52), affording a relatively narrow therapeutic range. Moreover, even these guidelines are non-standardized, necessitating that each laboratory establish their own reference and therapeutic ranges, which further complicates the management of drug administration. In human plasma samples spiked with free heparin, the clotting time increased dramatically in a dose-dependent manner from ˜29 s in the absence of heparin (0 U/mL) up to ˜44s (˜1.5 fold over control) with exposure to only 0.8 U/mL, and as long as ˜136 s (˜5 fold over control) for 2.0 U/mL (
In the bloodstream, the nanocomplexes must face a complex milieu of proteins and plasma components without systemic release of their cargo until exposed to sites of thrombus formation. To demonstrate that the anti-coagulant property of the nanocomplexes remains suppressed in vivo under healthy conditions, two circulation time experiments were performed. In the first experiment, mice were injected with nanocomplexes containing fluorescently labeled heparin or free heparin (100 U/kg, n=3), and their blood was sampled over time by measuring the amount of fluorescence in the plasma. Specifically, mice (n=3 per condition) were injected via tail vein with nanocomplexes or free heparin formulated with 1×PBS. Blood samples collected thorough retro-orbital blood draw and centrifuged at 2,900×g for 5 minutes to isolate plasma. The plasma was then analyzed by fluorimetry using the Spectramax Gemini EM Fluorescence Microplate Reader (Molecular Devices) at excitation/emission wavelengths of 485/538 nm.
The results, as presented in
In the second experiment, mice were again injected with nanocomplexes or free heparin and, and their plasma was collected and evaluated for functional heparin activity. Specifically, healthy female Swiss Webster mice (3-4 months, n=3 per condition) were injected via tail vein with LVPR.RK4 nanocomplexes or free heparin at 100 U/kg, formulated in isotonic 5% dextrose solution in water (D5W). Blood samples were collected in tubes containing 3.2% sodium citrate (Sigma) for a final volume ratio of 9:1 (blood:citrate) through retro-orbital blood draws and centrifuged at 2,900×g for 5 minutes to isolate the plasma. Heparin activity was then measured using the anti-FXa assay.
The results, as presented in
The purpose of the next experiment was to test if designing the nanocomplexes to release more heparin in response pathological levels of thrombin activity would temper its impact on bleeding time during hemostatic events. Moderate to potentially fatal bleeding is the primary side effect of nearly all clinical anti-coagulants, including heparin, and hemostasis is largely driven by platelet activation and plug formation, a process requiring significantly lower thrombin concentrations than needed for fibrin clot formation (Dawson et al., J. of Biol. Chem. (1994), 269:15989-92).
To assess the effect of nanocomplex administration on bleeding tail transection on mice was performed five minutes after they were treated intravenously with nanocomplexes. Specifically, mice (n=5-7 per condition) were anesthetized with isofluorane gas and administered nanocomplexes (200 U/kg, n=5), heparin (200 U/kg, n=7), or PBS control (n=5). After 5 minutes, 2 mm of distal mouse tail was removed by scalpel. Bleeding time was determined by lightly dabbing the tail with kimwipe tissues (Kimtech) until bleeding fully ceased for at least 1 minute. All experimental protocols involving animals were approved by the MIT Committee on Animal Care.
As is shown in
To evaluate the ability of nanocomplexes to prevent thrombosis in vivo, a thromboplastin-induced model of pulmonary thromboembolism was utilized. As is demonstrated in
To ascertain the initial dosing ranges for the nanocomplexes, mice were dosed with thromboplastin and escalating doses of free heparin (40 U/kg, 100 U/kg and 200 U/kg heparin). Deposition of VT750-fibrin in the lungs of the mice was quantified using ex vivo infrared imaging. The results, presented in
Mice (n=5 per condition) were anesthetized with isofluorane gas and co-administered nanocomplexes (200 U/kg), free heparin (200 U/kg), or PBS control and 1 nmol of VT750-fibrinogen via tail vein injection. After 5 minutes, mice were injected with thromboplastin (2 uL/g). After 30 minutes, mice were euthanized with CO2 and the lungs were harvested and imaged on the LI-COR Odyssey Infrared Imaging System. Fibrin deposition was then quantified using ImageJ software. For histologic analysis, paraffin-embedded sections of lungs were prepared (Koch Institute Histology Core). Lungs were first fixed by incubating in 4% paraformaldehyde overnight. Hematoxylin and eosin immunochemical staining of lung sections was used to visualize clots in the lungs.
The results of the in vivo experiments are shown in
As is shown in
To further analyze the anticoagulant mechanism of the nanocomplexes, we performed real-time thrombin generation assays in human plasma. For this assay, varying concentrations of nanocomplexes or free heparin were added to 20 μL of control normal human plasma (Thermo Scientific). Real time thrombin generation was measured using the Technothrombin TGA kit (Technoclone) using the RD reagent according to manufacturer instructions. The fluorescence was monitored using a TECAN Infinite M200 Pro and thrombin generation was calculated using the corresponding Excel evaluation software provided by Technoclone.
As can be seen in
This application claims the benefit of U.S. Provisional Application No. 61/831,916, filed on Jun. 6, 2013. The entire contents of the aforementioned priority application are hereby incorporated herein by reference.
This invention was made with government support under grant number 1-R01-CA124427, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61831916 | Jun 2013 | US |
