WO2010030342A2 - Polymères d'héparosane et leurs procédés de fabrication et d'utilisation destinés à l'amélioration de composés thérapeutiques - Google Patents

Polymères d'héparosane et leurs procédés de fabrication et d'utilisation destinés à l'amélioration de composés thérapeutiques Download PDF

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WO2010030342A2
WO2010030342A2 PCT/US2009/005050 US2009005050W WO2010030342A2 WO 2010030342 A2 WO2010030342 A2 WO 2010030342A2 US 2009005050 W US2009005050 W US 2009005050W WO 2010030342 A2 WO2010030342 A2 WO 2010030342A2
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heparosan
polymer
drug
conjugate
therapeutic
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PCT/US2009/005050
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WO2010030342A3 (fr
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Paul L. Deangelis
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The Board Of Regents Of The University Of Oklahoma
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Priority to CA2773755A priority Critical patent/CA2773755C/fr
Priority to EP09813350.7A priority patent/EP2341941A4/fr
Publication of WO2010030342A2 publication Critical patent/WO2010030342A2/fr
Publication of WO2010030342A3 publication Critical patent/WO2010030342A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

Definitions

  • the presently claimed and disclosed invention(s) relates, in general, to the field of therapeutics and, more particularly but without limiting, to novel compositions and methods for making heparosan biomaterials that are suitable for conjugation to therapeutics for the purpose of enhancing drug action and/or delivery as well as bioreactive agents for biotechnical applications.
  • the presently claimed and disclosed invention(s) relates generally to the field of therapeutics and, more particularly, to the development of enhanced therapeutics through the use of modifying and/or coupling agents and, in particular but without limitation, natural polysaccharides and oligosaccharides such as heparosan.
  • modifying and/or coupling agents and, in particular but without limitation, natural polysaccharides and oligosaccharides such as heparosan.
  • PEG poly[ethylene glycol]
  • FDA Food & Drug Administration
  • PEGylation The process of adding PEG to a drug, i.e., "PEGylation,” has been very successful, as shown in Table 1.
  • the hydrophilic chains of PEG polymers increase the solubility of the cargo in water, protect the cargo when in the human body and prolong the therapeutic action of the cargo. Due to its artificial nature, its chemical synthesis, and its potential harmful effects when ingested in large quantities over long periods of time, the use of PEG has significant drawbacks and alternatives have been sought.
  • the presently disclosed and claimed invention is directed to such alternative modifying and/or coupling agents, which overcome the defects and disadvantages of the prior art.
  • the patent or application file contains at least one drawing executed in color.
  • FIG. 1 graphically depicts the structures of heparosan and polyethylene glycol.
  • FIG. 2A is a graphical representation of the pharmacokinetics (pK) of radioactive heparosan conjugate in plasma in a rat model.
  • Rats were injected intravenously with 125 l-heparosan polymer (100 kDa mass) at 'Time 0', and at various times, blood was drawn, and the radioactivity in the plasma was measured.
  • the data indicate that 100 kDa heparosan, the active molecule of HEPylation, has a long lifetime (half-life of approximately 2 days) in the mammalian bloodstream.
  • Fig. 2B is a graphical representation of the pharmacokinetics of radioactive heparosan conjugate in plasma in a rat model. Rats were injected intravenously with 125 I- heparosan polymer (60 kDa monodisperse polymer) at 'Time 0', and at various times, blood was drawn, and the radioactivity in the plasma was measured. The data indicate that 60 kDa heparosan, the active molecule of HEPylation, has a long lifetime (half-life of approximately 15 hours) in the mammalian bloodstream. In addition, upon comparison of the data in Figures 2A and 2B, the size of the polymer determines the plasma half-life, thus allowing tuning of drug-conjugate pharmacokinetics.
  • I- heparosan polymer 60 kDa monodisperse polymer
  • Fig. 3 is a graphical representation of the fate of a radioactive heparosan conjugate in a rat model. Rats were injected intravenously with 100 kDa 125 l-heparosan polymer at 'Time 0', and at various times, the radioactivity in blood (Plasma or red and white blood cells, 'R&W BC), organs (liver, kidney, spleen, heart, bladder, brain), and excreted waste (urine, feces) was measured.
  • Fig. 4 is a pictorial representation showing heparosan is very stable in the mammalian bloodstream.
  • the 100 kDa 125 l-heparosan conjugate was injected intravenously into rats, and at various times, blood was withdrawn, and the plasma was isolated.
  • the samples were deproteinized and analyzed by agarose gel (1.5%) electrophoresis and autoradiography.
  • the molecular weight of starting probe (lane H; arrow) and the polymer in plasma samples are equivalent even after approximately 1-2 days time. Over time, the polymer is removed from circulation within the mammal and then metabolized/excreted.
  • Fig. 5 is graphical representation showing synthesis of monodisperse heparosan polymers.
  • Alternative embodiments include (a) activating heparosan produced by fermentation of bacteria and then coupling to cargo or (b) coupling the activated short acceptor to a cargo, then elongating via polymer grafting to a useful, desired size heparosan chain with heparosan synthase PmHSl.
  • FIG. 7 are pictorial representations of SDS-PAGE gels illustrating the production of HEPylated BSA molecules (left panel) and degradation thereof with heparosan lyase (right panel).
  • FIG. 8 are pictorial representations of an SDS-PAGE gel (left panel) and gel filtration chromatography profile (right panel) illustrating production of a series of higher molecular weight products corresponding to a series of HEPylated BSA molecules.
  • Fig. 9 is a pictorial representation of an SDS-PAGE gel illustrating the production of HEPylated IgG molecules (see arrow area).
  • Fig. 10 is a pictorial representation of a PAGE gel visualized by virtue of ultraviolet-induced fluorescence, demonstrating production of HEPylated fluorescein molecules (see arrow). Unreacted FITC (fluorescein isothiocyanate) is bracketed.
  • Fig. 11 is a graphical representation of thin layer chromatography (TLC) of short heparosan acceptor coupled to a radioactive cargo and its subsequent elongated product.
  • TLC thin layer chromatography
  • Hep4 (middle lane) and its subsequent elongation by polymer grafting with PmHSl synthase into a heparosan vehicle (left lane) suitable for prolonging residence time in the mammalian blood stream.
  • BH 125 I Bolton-Hunter reagent
  • Hep4 heparosan tetrasaccharide
  • Poly
  • Fig. 12 is a graphic depiction (right panel) of the production of HEPylated cargo by polymer grafting, and a pictorial representation (left panel) of an SDS-PAGE gel that demonstrates the use of said method to produce HEPylated BSA molecules (see bracket area).
  • Fig. 13 is a pictorial representation of an SDS-PAGE gel demonstrating the production of HEPylated BSA molecules (see bracket area) utilizing naturally occurring heparosan obtained from in vivo microbial fermentation as the source of the vehicle.
  • Fig. 14 is a graphical representation of an agarose gel analysis of heparosan coupled to radioactive cargo.
  • This gel was stained with a sugar detection reagent (Stains-all) as well as exposed to X-ray film (Autorad; 2 exposure times - short or long) to illustrate the defined synthesis of a radioactive cargo coupled to approximately 220 kDa heparosan (same polymer as in the TLC of Fig. 11).
  • the narrow size distribution is demonstrated by loading of both a low (Ix) and a high (1Ox) concentration of HEPylated probe as well as overexposure of the X-ray film.
  • Fig. 15 is a graphical representation of the total distribution of the measured radioactivity from Fig. 16 for i.m. dosing.
  • Fig. 16 is a graphical representation illustrating the curve-fit for pK analysis of the elimination of a radioactive heparosan compound from the plasma for i.m. dosing.
  • Fig. 17 is a pictorial representation of an agarose gel demonstrating the stability of the heparosan vehicle utilized in Figs. 15-16 in the extracellular compartments of the mammalian body.
  • Fig. 18 is a graphical representation of TLC of Urine Metabolites.
  • Fig. 19 is a graphical representation of heparosan metabolite excretion into feces.
  • Excretion into feces and urine accounts for the metabolized heparosan vehicle indicating that heparosan does not accumulate in a mammalian patient (as shown in Fig. 3).
  • the size of the radioactive polymers in the feces was less than 3 kDa (or less than 15 sugar units) as measured by ultrafiltration, thereby indicating that heparosan is degraded and then excreted over time.
  • heparosan a natural polysaccharide related to heparin.
  • Heparosan can be synthesized in a step-wise, reproducible, and defined manner so as to provide all of the advantages of PEG without its potential side effects.
  • Heparosan is soluble in water, biocompatible, and bio-inert within the human body.
  • heparosan heparosan
  • PEGylation a process termed herein as "HEPylation”
  • heparosan has a higher water solubility than PEG
  • d) as a naturally occurring polysaccharide, heparosan's degradation products are biocompatible
  • heparosan is not immunogenic.
  • PEG polymers having a molecular weight of 6,000 Da or 6 kDa have a shorter half-life in blood serum than PEG polymers having a molecular weight of 170,000 Da or 170 kDa (PEG-170).
  • the half-life of PEG molecules in blood serum is directly dependent upon the size of the polymer.
  • ADA adenosine Enzon (March 1990) replacement, reduced immune combined deaminase) reverses symptoms response immunodeficiency of ADA deficiency disease
  • Granulocyte Pegfilgrastim (NEULASTA/ Stimulation of Longer half-life, Prophylaxis against colony- Amgen (January 2002) neutrophil self-regulating severe neutropenia stimulating production clearance and its complications factor during myelosuppressive chemotherapy
  • PEG Heparosan HEPylation a. Extend Cargo Half-life in Bloodstream? Yes Yes (e.g., avoid renal clearance if larger molecular weight) b. Protect Cargo from Degradation? Yes Yes (e.g., by proteases) c. Shield Cargo from Immune Response? Yes Yes (e.g., prevent antibody generation) d. Trap Cargo in Cancerous Regions? Yes Yes (e.g., due to altered tumor vasculature) e. Enhance Solubility of Cargo? Yes Yes, increased solubility potential (e.g., especially hydrophobic chemotherapy than PEG due to its more agents)? h ⁇ drophilic nature f. Variety of Cargo Coupling Chemistries?
  • Yes Yes (e.g., amine, sulfhydryl reactive)
  • Exhance Cargo Stability? Yes Yes e.g., prevent protein unfolding events
  • h. Reduce Dosage and Maintain Constant Yes Yes Blood Concentrations? (e.g., avoid peaks and troughs; predictable dosing plateau in desired range)
  • Suitability for a Range of Cargo (e.g., platform technology) proteins, peptides? Yes Yes Yes small MW drugs? Yes Yes liposomes? Yes Yes Yes Yes Yes Yes
  • GAGs Glycosaminoglycans
  • GAGs are long linear polysaccharides comprising disaccharide repeats that contain an amino sugar. GAGs are well known to be essential in vertebrates.
  • the GAG structures possess a significant number of negative groups and hydroxyl groups and are, therefore, highly hydrophilic.
  • the GAGs are structural, adhesion, and/or signaling elements in humans.
  • a few microbes also produce extracellular polysaccharide coatings called capsules that are composed of GAG chains and that serve as virulence factors. The capsule assists in the microbe's evasion of host defenses such as phagocytosis and complement.
  • host defenses such as phagocytosis and complement.
  • the microbial polysaccharide is identical or very similar to the host GAG, the antibody response to the microbe is either very limited or non-existent.
  • heparosan also called N-acetylheparosan or unsulfated, unepimerized heparin; [4-GlcUA-beta-l,4-GlcNAc-alpha-l-] n ; shown in Fig. 1
  • the bacterial-derived enzymes used to produce heparosan for use in one embodiment of the presently claimed and disclosed invention(s) synthesize heparosan as their final product.
  • PmHSl is a robust enzyme that efficiently makes polymers up to ⁇ 1 MDa (1,000 kDa or ⁇ 5,000 monosaccharide units) in vitro.
  • Escherichia coli K5 at least two enzymes, KfiA, the alpha-GlcNAc transferase, and KfiC, the beta-GlcUA-transferase, (and perhaps KfiB, a protein of unknown function) work in concert to form the disaccharide repeat of heparosan.
  • the E. coli enzyme complex is not as efficient as the PmHSl enzyme as it is more difficult to produce the long polymer chains with the E. coli enzyme complex.
  • any method which produces heparosan may be used.
  • heparosan it is not the method of producing heparosan that is determinative - rather, it is the conjugation of heparosan from any source or method of production (e.g., fermented heparosan produced by native or recombinant microbes, as well as chemoenzymatic syntheses or organic chemical syntheses) to a target molecule (i.e., the cargo) for increased solubility in water, bioavailability and dwell time within the patient that is presently disclosed and claimed.
  • any source or method of production e.g., fermented heparosan produced by native or recombinant microbes, as well as chemoenzymatic syntheses or organic chemical syntheses
  • a target molecule i.e., the cargo
  • a key advantage to using heparosan is that it has increased biostability in the extracellular matrix when compared to other GAGs such as hyaluronic acid and chondroitin. As with most compounds synthesized in the body, new molecules are typically made, and after serving their purpose, are broken down into smaller constituents for recycling. [0040] Heparin and heparan sulfate, for example, are degraded by a single enzyme known as heparanase.
  • heparosan or any of its fragments (generated by reactive oxygen species, etc.) is internalized into the lysosome, then the molecules will be degraded by resident beta-glucosidase and beta-hexosaminidase enzymes (which remove one sugar at a time from the non-reducing termini of the GAG chain), similar to the degradation of heparin or hyaluronic acid. Therefore, the heparosan polymer is biodegradable and will not permanently reside in the body and thereby cause a lysosomal storage problem.
  • heparin/heparan sulfate The normal roles of heparin/heparan sulfate in vertebrates include binding coagulation factors (inhibiting blood clotting) and growth factors (signaling cells to proliferate or differentiate).
  • the key structures of heparin/heparan sulfate that are recognized by these factors include a variety of O-sulfation patterns and the presence of iduronic acid [IdoUA]; in general, polymers without these modifications do not stimulate clotting or cell growth.
  • Heparosan-based materials which do not have such O-sulfation patterns, therefore, do not provoke unwanted clotting or cellular growth/modulation.
  • HEPylated drug conjugates do not initiate clotting and/or cell growth processes and remain solely bio-reactive as per the drug or cargo constituent — the heparosan is thus termed or deemed to be biologically inert.
  • coli K5 utilize heparosan coatings to ward off host defenses by acting as molecular camouflage. Indeed, scientists had to resort to using capsule-specific phages or selective GAG-degrading enzymes to type these heparosan-coated microbes since a conventional antibody or serum could not be generated - the heparosan is thus termed or deemed non- immunogenic or non-antigenic.
  • any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the presently claimed and disclosed invention(s), and vice versa.
  • compositions of the presently claimed and disclosed invention(s) can be used to achieve methods of the presently claimed and disclosed invention(s).
  • compositions and/or methods disclosed and claimed herein refers to all permutations and combinations of the listed items preceding the term.
  • A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
  • expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
  • All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure.
  • Heparosan is a sugar polymer of the formula -[GlcNAc-alpha4-GlcUA-beta4] n - where n is from 2 to about 5,000.
  • oligosaccharide generally denotes n being from about 1 to about 11 while the term “polysaccharide” denotes n being equal to or greater than 12.
  • conjuggate refers to a complex created between two or more compounds by covalent or weak bonds.
  • carrier refers to the carrier of the cargo (e.g., the heparosan polymer) in the conjugate.
  • active agent(s), active ingredient(s),
  • pharmaceutically active ingredient(s), “therapeutic,” “medicant,” “medicine,” “biologically active compound” and “bioactive agent(s)” are defined as drugs and/or pharmaceutically active ingredients.
  • the presently claimed and disclosed invention(s) may be used to encapsulate, attach, bind or otherwise be used to affect the storage, stability, longevity and/or release of any of the following drugs as the pharmaceutically active agent in a composition.
  • bioactive agents such as, acetaminophen, aspirin, salicylic acid, methyl salicylate, choline salicylate, glycol salicylate, 1-menthol, camphor, mefenamic acid, fluphenamic acid, indomethacin, diclofenac, alclofenac, ibuprofen, ketoprofen, naproxene, pranoprofen, fenoprofen, sulindac, fenbufen, clidanac, flurbiprofen, indoprofen, protizidic acid, fentiazac, tolmetin, tiaprofenic acid,
  • (B) Drugs having an action on the central nervous system for example sedatives, hypnotics, antianxiety agents, analgesics and anesthetics, such as, chloral, buprenorphine, naloxone, haloperidol, fluphenazine, pentobarbital, phenobarbital, secobarbital, amobarbital, cydobarbital, codeine, lidocaine, tetracaine, dyclonine, dibucaine, cocaine, procaine, mepivacaine, bupivacaine, etidocaine, prilocaine, benzocaine, fentanyl, nicotine, and the like.
  • Local anesthetics such as, benzocaine, procaine, dibucaine, lidocaine, and the like.
  • Antihistaminics or antiallergic agents such as, diphenhydramine, dimenhydrinate, perphenazine, triprolidine, pyrilamine, chlorcyclizine, promethazine, carbinoxamine, tripelennamine, brompheniramine, hydroxyzine, cyclizine, meclizine, clorprenaline, terfenadine, chlorpheniramine, and the like.
  • Anti-allergenics such as, antazoline, methap ⁇ rilene, chlorpheniramine, pyrilamine, pheniramine, and the like. Decongestants such as, phenylephrine, ephedrine, naphazoline, tetrahydrozoline, and the like.
  • Antipyretics such as, aspirin, salicylamide, non-steroidal anti-inflammatory agents, and the like.
  • Antimigrane agents such as, dihydroergotamine, pizotyline, and the like.
  • Acetonide anti-inflammatory agents such as hydrocortisone, cortisone, dexamethasone, fluocinolone, triamcinolone, medrysone, prednisolone, flurandrenolide, prednisone, halcinonide, methylprednisolone, fludrocortisone, corticosterone, paramethasone, betamethasone, ibuprophen, naproxen, fenoprofen, fenbufen, flurbiprofen, indoprofen, ketoprofen, suprofen, indomethacin, piroxicam, aspirin, salicylic acid, diflunisal, methyl salicylate, phenylbuta
  • Steroids such as, androgenic steriods, such as, testosterone, methyltestosterone, fluoxymesterone, estrogens such as, conjugated estrogens, esterified estrogens, estropipate, 17- ⁇ estradiol, 17- ⁇ estradiol valerate, equilin, mestranol, estrone, estriol, 17 ⁇ ethinyl estradiol, diethylstilbestrol, progestational agents, such as, progesterone, 19-norprogesterone, norethindrone, norethindrone acetate, melengestrol, chlormadinone, ethisterone, medroxyprogesterone acetate, hydroxyprogesterone caproate, ethynodiol diacetate, norethynodrel, 17- ⁇ hydroxyprogesterone, dydrogesterone, dimethisterone, ethinylest
  • Respiratory agents such as, theophylline and ⁇ 2 -adrenergic agonists, such as, albuterol, terbutaline, metaproterenol, ritodrine, carbuterol, fenoterol, quinterenol, rimiterol, solmefamol, soterenol, tetroquinol, and the like.
  • Sympathomimetics such as, dopamine, norepinephrine, phenylpropanolamine, phenylephrine, pseudoephedrine, amphetamine, propylhexedrine, arecoline, and the like.
  • Antimicrobial agents including antibacterial agents, antifungal agents, antimycotic agents and antiviral agents; tetracyclines such as, oxytetracycline, penicillins, such as, ampicillin, cephalosporins such as, cefalotin, aminoglycosides, such as, kanamycin, macrolides such as, erythromycin, chloramphenicol, iodides, nitrochanoin, nystatin, amphotericin, fradiomycin, sulfonamides, purrolnitrin, clotrimazole, miconazole chloramphenicol, sulfacetamide, sulfamethazine, sulfadiazine, sulfamerazine, sulfamethizole and sulfisoxazole; antivirals, including idoxuridine; clarithromycin; and other anti-infectives including nitrofurazone, and the like.
  • Antihypertensive agents such as, clonidine, ⁇ -methyldopa, reserpine, syrosingopine, rescinnamine, cinnarizine, hydrazine, prazosin, and the like.
  • Antihypertensive diuretics such as, chlorothiazide, hydrochlorothrazide, bendoflumethazide, trichlormethiazide, furosemide, tripamide, methylclothiazide, penfluzide, hydrothiazide, spironolactone, metolazone, and the like.
  • Cardiotonics such as, digitalis, ubidecarenone, dopamine, and the like.
  • Coronary vasodilators such as, organic nitrates such as, nitroglycerine, isosorbitol dinitrate, erythritol tetranitrate, and pentaerythritol tetranitrate, dipyridamole, dilazep, trapidil, trimetazidine, and the like.
  • Vasoconstrictors such as, dihydroergotamine, dihydroergotoxine, and the like, ⁇ -blockers or antiarrhythmic agents such as, timolol pindolol, propranolol, and the like.
  • Humoral agents such as, the prostaglandins, natural and synthetic, for example PGEl, PGE2 ⁇ , and PGF2 ⁇ , and the PGEl analog misoprostol.
  • Antispasmodics such as, atropine, methantheline, papaverine, cinnamedrine, methscopolamine, and the like.
  • (I) Calcium antagonists and other circulatory organ agents such as, aptopril, diltiazem, nifedipine, nicardipine, verapamil, bencyclane, ifenprodil tartarate, molsidomine, clonidine, prazosin, and the like.
  • Anti-convulsants such as, nitrazepam, meprobamate, phenytoin, and the like.
  • Agents for dizziness such as, isoprenaline, betahistine, scopolamine, and the like.
  • Tranquilizers such as, reserprine, chlorpromazine, and antianxiety benzodiazepines such as, alprazolam, chlordiazepoxide, clorazeptate, halazepam, oxazepam, prazepam, clonazepam, flurazepam, triazolam, lorazepam, diazepam, and the like.
  • Antipsychotics such as, phenothiazines including thiopropazate, chlorpromazine, triflupromazine, mesoridazine, piperracetazine, thioridazine, acetophenazine, fluphenazine, perphenazine, trifluoperazine, and other major tranqulizers such as, chlorprathixene, thiothixene, haloperidol, bromperidol, loxapine, and molindone, as well as those agents used at lower doses in the treatment of nausea, vomiting, and the like.
  • phenothiazines including thiopropazate, chlorpromazine, triflupromazine, mesoridazine, piperracetazine, thioridazine, acetophenazine, fluphenazine, perphenazine, trifluoperazine, and other major tranqulizers such as, chlorprathixene, thio
  • (K) Drugs for Parkinson's disease, spasticity, and acute muscle spasms such as levodopa, carbidopa, amantadine, apomorphine, bromocriptine, selegiline (deprenyl), trihexyphenidyl hydrochloride, benztropine mesylate, procyclidine hydrochloride, baclofen, diazepam, dantrolene, and the like.
  • Respiratory agents such as, codeine, ephedrine, isoproterenol, dextromethorphan, orciprenaline, ipratropium bromide, cromglycic acid, and the like.
  • Non-steroidal hormones or antihormones such as, corticotropin, oxytocin, vasopressin, salivary hormone, thyroid hormone, adrenal hormone, kallikrein, insulin, oxendolone, and the like.
  • Vitamins such as, vitamins A, B, C, D, E and K and derivatives thereof, calciferols, mecobalamin, and the like for dermatologically use.
  • Enzymes such as, lysozyme, urokinaze, and the like.
  • Herbal medicaments or crude extracts such as, Aloe vera, and the like.
  • Antitumor agents such as, 5-fluorouracil and derivatives thereof, krestin, picibanil, ancitabine, cytarabine, and the like.
  • Anti-estrogen or anti-hormone agents such as, tamoxifen or human chorionic gonadotropin, and the like.
  • Miotics such as pilocarpine, and the like.
  • (N) Cholinergic agonists such as, choline, acetylcholine, methacholine, carbachol, bethanechol, pilocarpine, muscarine, arecoline, and the like.
  • Antimuscarinic or muscarinic cholinergic blocking agents such as, atropine, scopolamine, homatropine, methscopolamine, homatropine methylbromide, methantheline, cyclopentolate, tropicamide, propantheline, anisotropine, dicyclomine, eucatropine, and the like.
  • (O) Mydriatics such as, atropine, cyclopentolate, homatropine, scopolamine, tropicamide, eucatropine, hydroxyamphetamine, and the like.
  • Psychic energizers such as 3-
  • Antidepressant drugs such as, isocarboxazid, phenelzine, tranylcypromine, imipramine, amitriptyline, trimipramine, doxepin, desipramine, nortriptyline, protriptyline, amoxapine, maprotiline, trazodone, and the like.
  • Anti-diabetics such as, insulin, and anticancer drugs such as, tamoxifen, methotrexate, and the like.
  • Anorectic drugs such as, dextroamphetamine, methamphetamine, phenylpropanolamine, fenfluramine, diethylpropion, mazindol, phentermine, and the like.
  • (S) Anti-malarials such as, the 4-aminoquinolines, alphaaminoquinolines, chloroquine, pyrimethamine, and the like.
  • Protein therapeutics such as enzymes, cytokines, growth factors, hormones, receptors, antibodies, immune complexes, and the like. Also included are protein derivatives that enhance or block the activity of any of the naturally-occurring or isolated molecules listed herein or interacting components in the biochemical or cellular pathways.
  • Anti-ulcerative agents such as, misoprostol, omeprazole, enprostil, and the like. Antiulcer agents such as, allantoin, aldioxa, alcloxa, N-methylscopolamine methylsuflate, and the like. Antidiabetics such as insulin, and the like.
  • Anti-cancer agents such as, cis-platin, actinomycin D, doxorubicin, vincristine, vinblastine, etoposide, amsacrine, mitoxantrone, tenipaside, taxol, colchicine, cyclosporin A, phenothiazines or thioxantheres.
  • antigens for use with vaccines, one or more antigens, such as, natural, heat-killer, inactivated, synthetic, peptides and even T cell epitopes (e.g., GADE, DAGE, MAGE, etc.) and the like.
  • antigens such as, natural, heat-killer, inactivated, synthetic, peptides and even T cell epitopes (e.g., GADE, DAGE, MAGE, etc.) and the like.
  • Example therapeutic or active agents also include water soluble or poorly soluble drugs of molecular weights from 40 to 1,100 including the following: Hydrocodone, Lexapro, Vicodin, Effexor, Paxil, Wellbutrin, Bextra, Neurontin, Lipitor, Percocet, Oxycodone, Valium, Naproxen, Tramadol, Ambien, Oxycontin, Celebrex, Prednisone, Celexa, Ultracet, Protonix, Soma, Atenolol, Lisinopril, Lortab, Darvocet, Cipro, Levaquin, Ativan, Nexium, Cyclobenzaprine, Ultram, Alprazolam, Trazodone, Norvasc, Biaxin, Codeine, Clonazepam, Toprol, Zithromax, Diovan, Skelaxin, Klonopin, Lorazepam, Depakote, Diazepam, Albuterol, Topamax, Seroquel
  • the above drugs may be used either in the free form or, if capable of forming salts, in the form of a salt with a suitable acid or base. If the drugs have a carboxyl group, their esters may be employed.
  • the "suitable acid” may be an organic acid, for example, methanesulfonic acid, lactic acid, tartaric acid, fumaric acid, maleic acid, acetic acid, or an inorganic acid, for example, hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid.
  • the base may be an organic base, for example, ammonia, triethylamine, or an inorganic base, for example, sodium hydroxide or potassium hydroxide.
  • the esters may be alkyl esters, aryl esters, aralkyl esters, and the like.
  • the heparosan conjugate may be administered parenterally, intraperitoneal ⁇ , intraspinally, intravenously, intramuscularly, intravaginally, subcutaneously, intranasally, rectally, or intracerebrally.
  • Dispersions of the heparosan conjugate may be prepared in glycerol, liquid poly[ethylene glycols], and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, such preparations of the heparosan conjugate may also contain a preservative to prevent the growth of microorganisms.
  • compositions suitable for injection use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the heparosan conjugate may be used in conjunction with a solvent or dispersion medium containing, for example, water, ethanol, poly-ol (for example, glycerol, propylene glycol, and liquid poly[ethylene glycol], and the like), suitable mixtures thereof, vegetable oils, and combinations thereof.
  • a solvent or dispersion medium containing, for example, water, ethanol, poly-ol (for example, glycerol, propylene glycol, and liquid poly[ethylene glycol], and the like), suitable mixtures thereof, vegetable oils, and combinations thereof.
  • the proper fluidity of the heparosan conjugate may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and/or by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.
  • Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.
  • Sterile injectable solutions may be prepared by incorporating the heparosan conjugate in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the heparosan conjugate into a sterile carrier that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the methods of preparation may include vacuum drying, spray drying, spray freezing and freeze-drying that yields a powder of the active ingredient (i.e., the heparosan conjugate) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the heparosan conjugate may be orally administered, for example, with an inert diluent or an assimilable edible carrier.
  • the heparosan conjugate and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet.
  • the heparosan conjugate may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • the percentage of the heparosan conjugate in the compositions and preparations may, of course, be varied as will be known to the skilled artisan.
  • the amount of the heparosan conjugate in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of heparosan conjugate calculated to produce the desired therapeutic effect.
  • the specification for the dosage unit forms of the presently claimed and disclosed invention(s) are dictated by and directly dependent on (a) the unique characteristics of the heparosan conjugate and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such a therapeutic compound for the treatment of a selected condition in a subject.
  • Aqueous compositions of the present invention comprise an effective amount of the nanoparticle, nanofibril or nanoshell or chemical composition of the presently claimed and disclosed invention(s) dissolved and/or dispersed in a pharmaceutically acceptable carrier and/or aqueous medium.
  • the biological material should be extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where appropriate.
  • the active compounds may generally be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, intralesional, and/or even intraperitoneal routes.
  • compositions that contains an effective amount of the nanoshell composition as an active component and/or ingredient will be known to those of skill in the art in light of the present disclosure.
  • such compositions may be prepared as injectables, either as liquid solutions and/or suspensions; solid forms suitable for using to prepare solutions and/or suspensions upon the addition of a liquid prior to injection may also be prepared; and/or the preparations may also be emulsified.
  • the heparosan vehicle can be used to enhance a secondary vehicle (e.g., liposomes, nanoparticles, etc.) that acts as a carrier or adjuvant for a drug.
  • GAG synthesis and heparosan synthesis in particular is rather versatile with respect to chemical functionality as well as size control.
  • U.S. Publication No. US 2008/0109236 Al discloses a methodology for polymer grafting utilizing heparin/heparosan synthases from Pasteurella in order to provide heparosan polymers having a targeted size and that are substantially monodisperse at the desired size ranges.
  • appropriate reactive moieties may be added to the heparosan polymer at the reducing or non-reducing termini or throughout the sugar chain. Having one reactive group/chain is preferable when conjugating the
  • heparosan polymers suitable for HEPylation with a cargo molecule can be produced.
  • Table 4 lists different HEPylation polymer chemistries which are available and/or suitable for modifying the heparosan polymer to make it more acceptable or suitable for conjugating with specific cargo molecules. It is not the nature or manner of the complexation or conjugation between heparosan and the drug (by any covalent chemical or weak bond) that is controlling; rather, it is the particular use to which the heparosan will be put.
  • PmHSl (SEQ ID NOS: 1 and 2, the amino acid and nucleotide sequences, respectively) was expressed as a carboxyl terminal fusion to maltose binding protein (MBP) using the pMAL-c2X vector (New England BioLabs).
  • MBP maltose binding protein
  • E. coli XJa Zymo Research
  • E. coli XJa Zymo Research
  • Cultures were grown in Superior Broth (AthenaES) at 30°C with ampicillin (100 ⁇ g/ml), and L-arabinose (3.25 mM).
  • IPTG isopropyl ⁇ -D-1-thiogalactopyranoside
  • a heparosan polysaccharide (having a molecular weight of approximately
  • P. multocida Type D cells were grown in a proprietary synthetic media at 37°C in shake flasks for approximately 24 hrs. Spent culture medium (the liquid part of culture after microbial cells were removed) was harvested (by centrifugation at 10,000 x g, 20 min) and deproteinized (solvent extraction with chloroform).
  • the very large anionic heparosan polymer (“fermentation heparosan,” having a molecular weight of approximately 200-300 kDa) was isolated via ultrafiltration (30 kDa molecular weight cut-off; Amicon) and ion exchange chromatography (NaCl gradient on Q-Sepharose; Pharmacia). Heparosan from E. coli K5 cultures can also be used, but the polymers are initially lower molecular weight than P. multocida Type D.
  • oligosaccharides were verified by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-ToF MS). Alternatively, acid hydrolysis or enzymatic cleavage yields oligosaccharides that can also be employed for use.
  • MALDI-ToF MS matrix assisted laser desorption ionization time of flight mass spectrometry
  • the dry sugar was dissolved in anhydrous methanol (0.71 mg/ml w/v or 0.93 itiM final) under sonication.
  • the Amino-heparosan polymer may be further reacted with various activated bifunctional N-hydroxysuccinimide esters to thereby add desirable groups including maleimides, a sulfhydryl selective reagent, etc.
  • the amino-heparosan polymer was reacted with approximately 10-fold molar excess of (Pierce) in 10% dimethylsulfoxide, 0.1 M potassium phosphate, 0.15 M NaCl, pH 7.4, for 2 hrs at room temperature.
  • the target compound in the reaction mixture was purified by gel filtration chromatography on Sephadex G-25 resin (PD-IO, Pharmacia) as detailed above.
  • the amino-HEP4 products may also be reductively aminated by treatment with adipic acid dihydrazide (30 eq) and sodium cyanoborohydride (100 eq) at 50-60 0 C in 1 M sodium phosphate buffer, pH 5.5. After desalting, the obtained hydrazide amino-HEP4 may be further purified by strong anion exchange chromatography using Sepharose Q (Pharmacia) with an ammonium bicarbonate gradient elution. The hydrazide amino-HEP4 primer may also be extended by the PmHSl enzyme in order to produce polymers having varying sizes as described below.
  • fermentation heparosan (or its derivatives) may be used.
  • heparosan from any source or produced by any methodology is intended to be within the presently claimed and disclosed invention.
  • it is not the nature or manner of the complexation between heparosan and the drug (by any chemical or weak bond) that is controlling; rather it is the particular use to which the heparosan will be put.
  • the yield and molecular weight size distribution of the heparosan is checked by (a) carbazole assays for uronic acid; and (b) agarose gel electrophoresis (IX TAE buffer, 0.8-1.5% agarose) followed by Stains-All detection.
  • the carbazole assay is a spectrophotometric chemical assay that measures the amount of uronic acid in the sample via production of a pink color; every other sugar in the heparosan chain is a glucuronic acid (GIcUA).
  • the heparosan polymer size is determined by comparison to monodisperse HA size standards (HA Lo-Ladder, Hyalose, LLC) run on gels.
  • the detection limit of the carbazole and the gel assays is approximately 5-15 micrograms of polymer. Any endotoxin is removed by passage through an immobilized polymyxin column (Pierce); the material is then tested with a Limulus amoebacyte-based assay (www.Cambrex.com) to assure that the heparosan contains ⁇ 0.05 endotoxin units/mg solid (based on USP guidelines).
  • Mw/Mn weight average molecular mass
  • Mn number average molecular weight
  • the polydispersity value for an ideal monodisperse polymer equals 1.
  • the parallel reaction without an acceptor (lane 0) resulted in a large product that was significantly polydisperse, i.e., it contains heparosan polymers of varying size and length.
  • the polymerization by synthases in the presence of an acceptor is a synchronized process.
  • Reactions without acceptor exhibit a lag period interspersed with numerous, out of step initiation events that yield a short heparosan oligosaccharide.
  • the heparosan polymer is elongated rapidly.
  • Other new chains that arise later during the lag period are also elongated rapidly, but the size of these younger chains never catches up to the older chains in a reaction with a finite amount of UDP-sugars.
  • all heparosan chains are elongated in parallel in a nonprocessive fashion resulting in a more homogenous final polymer population.
  • the synthase adds all available UDP-sugar precursors to the nonreducing termini of acceptors as in the equation: n UDP-GIcUA + n UDP-GIcNAc + z [GICUA-GICNAC] X ⁇ 2n UDP + z [GlcUA-GlcNAc] x+(n/z)
  • the size distribution of the heparosan polymers produced was determined by high performance size exclusion chromatography-multi angle laser light scattering (SEC- MALLS). Polymers (2.5 to 12 ⁇ g mass; 50 ⁇ l injection) were separated on PL aquagel-OH 30 (8 ⁇ m), -OH 40, -OH 50, -OH 60 (15 ⁇ m ) columns (7.5 x 300 mm, Polymer Laboratories) in tandem or alone as required by the size range of the polymers to be analyzed. The columns were eluted with 50 mM sodium phosphate, 150 mM NaCI, pH 7 at 0.5 ml/min.
  • MALLS analysis of the eluant was performed by a DAWN DSP Laser Photometer in series with an OPTILAB DSP lnterferometric Refractometer (632.8 nm; Wyatt Technology).
  • the ASTRA software package was used to determine the absolute average molecular mass using a dn/dc coefficient of 0.153 determined for HA, a polymer with the exact same sugar composition as heparosan, by Wyatt Technology.
  • the Mw and polydispersity values from at least two SEC-MALLS runs were averaged in order to obtain a final approximation of the Mw and polydispersity of the heparosan molecule.
  • doxorubicin and taxol are useful chemotherapy agents for treating several cancers. Taxol is only slightly soluble in water (i.e., approximately 0.4 micrograms per mL), and such solubility issues can be improved through conjugation with the hydrophilic heparosan polymer.
  • the carbonyl groups found on the taxol or doxorubicin molecule allows the drug to couple monovalently to the heparosan polymer, thereby providing a heparosan drug conjugate.
  • the drug molecule is also attachable to multiple positions on the heparosan polymer.
  • a dihydrazide may also be added to the drug-heparosan conjugate in order to create a time-release formulation.
  • the heparosan-doxorubicin or taxol- heparosan conjugate is water-soluble and nontoxic; as heparosan is slowly degraded in blood pH, the linkage releases free active doxorubicin or taxol in a specific and controlled manner.
  • protein targets include enzymes, cytokines, interferon, antibodies, receptors and growth factors as well as modified derivatives (e.g., with either chemical or molecular genetic changes).
  • Bovine serum albumin, BSA is a useful surrogate for testing and modeling a protein therapeutic conjugated with heparosan.
  • the BSA protein does not have intrinsic glycosylation and facilitates analysis of the addition of one or more heparosan chains to the BSA-heparosan conjugate.
  • the use of heparosan as vehicle for drug conjugation is also applicable to recombinant proteins with a bioengineered extra cysteine or an exposed sulfhydryl group, such as antibodies, resulting in an improved strategy to couple such cargo.
  • the cysteine's sulfhydryl group is coupled to the monovalent heparosan-maleimide to provide the heparosan conjugate.
  • heparosan- thiopyridyl may be used if protein release is desirable due to a reversible disulfide linkage between the sugar polymer (i.e., the heparosan polymer) and the cargo (i.e., recombinant proteins, etc.).
  • the aldehyde of heparosan chains may be coupled to amines of proteins via reductive amination with sodium cyanoborohydride; a useful process for the conjugation of growth factors and interferon.
  • the molecule is challenged with (a) proteases and (b) antibodies (e.g., anti-BSA antibody).
  • proteases e.g., a) proteases and (b) antibodies (e.g., anti-BSA antibody).
  • samples of protein or protein-heparosan are treated with dilution series of trypsin, an aggressive serine protease, for 0-60 min, then run on the SDS-PAGE gel. Relative resistance to digestion occurs for protein-heparosan.
  • protein or protein-heparosan are incubated with anti-protein IgG beads (e.g., anti-BSA IgG beads from Sigma) for 1 hour in saline, then the supernatant analyzed by PAGE.
  • anti-protein IgG beads e.g., anti-BSA IgG beads from Sigma
  • soluble anti-protein reagent can be incubated with test samples and run on native gels (similar to standard gels except that sample buffer lacks reducing agent and will not be boiled).
  • a higher molecular weight complex forms when the antibody builds to the protein-heparosan conjugate causing a "super-shift".
  • the protein-heparosan conjugate is resistant to anti-protein if the heparosan blocks its epitope. The overall goal is to assess, and to optimize, as needed the reaction parameters to produce the heparosan-conjugate.
  • heparosan vehicle and therapeutic cargo involving various chemistries which include, but are not limited to, the examples listed previously in Table 4.
  • two proteins, BSA and IgG antibody, and two small molecules, fluorescein and Bolton-Hunter reagent were used as cargo for coupling various monodisperse or polydisperse heparosan polymers produced either via fermentation in vivo or chemoenzymatic synthesis in vivo.
  • Figs. 7-8 illustrate the production of HEPylated BSA molecules via chemoenzymatic synthesis.
  • Fig. 7-8 illustrate the production of HEPylated BSA molecules via chemoenzymatic synthesis.
  • radioactive bovine serum albumin [BSA] 125 I-BoItOn- Hunter labeled; migration marked with arrow) protein was reacted via reductive amination with sodium cyanoborohydride with two different reactive 20 kDa heparosan polysaccharides, 'H' or 'N' (unmodified BSA starting material is lane O').
  • Each reactive heparosan was made by extending a short oligosaccharide acceptor into a longer 20 kDa polymer with PmHSl enzyme and UDP-sugars.
  • the acceptors were derived from heparosan polysaccharide ( ⁇ 200-300 kDa) by two different methods: for H, a heparosan tetrasaccharide formed by HCI cleavage with general structure [GICUA-GICNAC] 2 was used while for N, a heparosan tetrasaccharide formed by base treatment followed by nitrous acid cleavage with general structure [GlcUA-GlcNAc]-GlcUA-anhydromannitol was used.
  • bovine serum albumin [BSA] protein was reacted with Traut's reagent (T; iminothiolane) to convert some of its amino groups (lysines and amino termini) into free sulfhydryl groups forming T-BSA; in this case, ⁇ l-3 residues on average were predicted to be modified based on the reaction stoichiometry employed and the general completeness of the reaction.
  • T-BSA material was incubated with a reactive 75 kDa maleimide heparosan.
  • the reactive heparosan was made by (i) converting a heparosan tetrasaccharide ([GlcUA-GlcNAc]-GlcUA-anhydromannitol) into an amino derivative using reductive amination with sodium cyanoborohydride in the presence of ammonia, (ii) extending this sugar into a longer polymer with PmHSl enzyme and UDP-sugars, and (ii) reacting the long amino-polymer with a N-hydroxysuccinimide ester of a maleimide- containing compound.
  • T-BSA chemically modified T-BSA
  • ⁇ 1 to 3 T reagents/BSA molecule are formed in a rather uncontrolled chemical reaction
  • a natural protein or a genetically engineered molecule e.g., with an extra free cysteine residue
  • any sulfhydryl moiety could be used on the cargo (protein or other small molecule or secondary vehicles) as well as any alternative sulfhydryl-reactive reagent on the heparosan polymer including p ⁇ ridylthiols or haloacids.
  • the control wells did not have any BSA competitor, thus representing a 'maximal binding * signal.
  • BSA alone competed for binding with the radioactive probe to immobilized antibody; the signal was substantially reduced by 25 ng of BSA competitor and greatly reduced with 500 nanograms of BSA.
  • T-BSA without heparosan competed in a fashion similar to normal BSA.
  • more HEPylated BSA was needed to partially inhibit the radioactive signal, indicating that the antibody did not recognize or bind to the HEPylated molecules as well as BSA or T-BSA. Therefore, HEPylation will help shield cargo from the full brunt of immunological defenses in the mammalian body.
  • T-BSA 500 ng 140 190
  • Fig. 9 illustrates the production of HEPylated IgG molecules.
  • a preparation of radioactive immunoglobulins [IgG] ( 125 l-Bolton-Hunter labeled; migration marked with bracket) was oxidized with sodium periodate to create new aldehydes on the IgG sugar moieties on the Fc region. This oxidized glycoprotein was reacted via reductive amination with reactive hydrazide heparosan polysaccharide using sodium cyanoborohydride.
  • heparosan tetrasaccharide acceptor derived from nitrous acid as described earlier
  • adipic dihydrazide a compound with 2 terminal hydrazide functional groups; one end couples with sugar and the other end remains free for reaction with cargo
  • Fig. 10 illustrates the production of HEPylated IgG molecules.
  • a preparation of reactive h ⁇ drazide 75 kDa heparosan (similar reagent as in Fig. 9) was reacted with fluorescein isothiocyanate (FITC).
  • FITC fluorescein isothiocyanate
  • heparosan without the reactive hydrazide group was also treated with the same FITC reagent (lane O').
  • FITC reagent lane O'
  • the PAGE gel visualized by virtue of ultraviolet-induced fluorescence, a higher molecular weight fluorescent product was observed in the 'Hy' lane, corresponding to HEPylated fluorescein molecules (see arrow) (unreacted FITC starting material is bracketed).
  • This data demonstrates yet another example of coupling heparosan vehicle to a small molecule that is a proxy for a therapeutic cargo.
  • the hydrazide linkage is meta-stable at physiological pH thus the HEPylated cargo will break down over time, facilitating time- release delivery of free small molecule.
  • certain toxic therapeutics such as cancer chemotherapy drugs, this is a useful dosing feature.
  • the cargo can first be coupled to the reactive acceptor, and then the heparosan chain added by polymer grafting with PmHSl (e.g., elongate the acceptor while coupled to cargo) due to the mild reaction conditions as shown in the example of Fig. 11.
  • PmHSl polymer grafting with PmHSl
  • FIG. 12 radioactive bovine serum albumin ( 125 l-Bolton-Hunter labeled BSA) protein was reacted via reductive amination with sodium cyanoborohydride with two different reactive oligosaccharide acceptors derived from heparosan (same acceptors as in Fig. 7).
  • H a heparosan tetrasaccharide formed by HCl cleavage with general structure [GICUA-GICNAC] 2 was used while for N, a heparosan tetrasaccharide formed by base treatment followed by nitrous acid cleavage with general structure [GlcUA-GlcNAcJ-GlcUA- anhydromannitol was used. Then the short oligosaccharide acceptor covalently attached onto the BSA was extended via polymer grafting into a longer heparosan polymer with PmHSl enzyme and UDP-sugars.
  • heparosan produced by bacteria in vivo can be purified and (a) coupled via its reducing end aldehyde or (b) activated to couple to cargo (this latter approach with fermentation-derived heparosan results in functional, but more heterogeneous final products with higher polydispersity).
  • this latter approach with fermentation-derived heparosan results in functional, but more heterogeneous final products with higher polydispersity.
  • heparosan As seen by the SDS-PAGE gel visualized by autoradiography, higher molecular weight product was observed in the reaction lanes (pH 5, 7.2, or 9) corresponding to HEPylated BSA molecules (see bracketed area). Therefore, in addition to chemoenzymatic synthetically derived heparosan vehicles, naturally occurring heparosan from microbes may also be used as the source of vehicle.
  • Such polymers include, but are not limited to, recombinant microbial hosts (e.g., Escherichia, Bacillus) with heparosan synthases such as PmHSl or other native microbes that produce heparosan such as Escherichia coli K5.
  • Typical elongation reactions contain: 50 mM Tris, pH 7.2, 1 mM MnCl 2 , 1 to 50 mM UDP-sugars, 0.1 mg/ml PmHSl enzyme and a primer; the stoichiometric ratio of primer to UDP-sugars controls the heparosan chain size.
  • 125 !-Heparosan A heparosan oligosaccharide primer with a 125 l-Bolton-Hunter reagent (a proxy for the therapeutic cargo) was elongated to any desired length with the PmHSl enzyme. A series of polymers having distinct sizes, but equal radiochemical specific activity (approximately 70 Ci/mmol) were generated; an example of one such radioactive conjugate is depicted in Fig. 14.
  • the radioactive tetrasaccharide primer was made from heparosan polysaccharide by partial de-acetylation in base, nitrous acid cleavage, reductive amination with ammonia, then coupling to NHS ester of 125 l-Bolton-Hunter reagent (Perkin Elmer NEN).
  • NHS ester of 125 l-Bolton-Hunter reagent (Perkin Elmer NEN).
  • the strong gamma-rays were readily detectable in samples of blood or tissues without additional processing.
  • a series of 12S l-heparosan probes were created to monitor the activity and functionality of the heparosan conjugate.
  • the rat was used as the model to track the fate of the heparosan conjugate after injection, but other mammals such as man are expected to behave similarly.
  • heparosan conjugate in the mammalian body is shown in the example of Fig. 3.
  • the samples were placed directly in test tubes and measured with a solid-state gamma scintillation counter for 1 minute.
  • the counting interval was extended to 5 or 10 minutes per sample (with a comparable blank value subtracted). The amount of radioactivity in the various samples overtime was used to calculate half-life in various compartments.
  • Rats were anesthetized by isoflurane inhalation (5-2% to effect) before being administered 0.2 ml of the radiolabeled test compound by i.v. infusion into the right jugular vein. Following compound infusion, the i.v.
  • Rats were then placed into holding cages until being reanesthetized just before a terminal blood draw and organ collection. [00122] The following groups of rats were dosed:
  • each of plasma and blood cells were transferred into plastic culture tubes for subsequent determination of radioactivity.
  • liver (3 tubes), spleen (1 tube), kidneys (1 tube/kidney), bladder (empty - 1 tube), heart (1 tube), lungs (1 tube/lung), brain (perfusion groups only - 2 tubes) and any urine (1 tube) or fresh fecal pellets (1 tube) were collected and prepared for radioactive counting.
  • the samples were placed into a gamma counter were the total radioactivity was converted into counts per minute (CPM). Similar studies were done for i.m. and i.p. injection.
  • Transcardial Perfusion At the post-dosing time-points listed above, following blood withdrawal under isoflurane anesthesia, rats were euthanized via cardiac perfusion with 200 ml of ice-cold sterile saline. For the perfusion, anesthetized rats were placed on a wire mesh cage top over a sink. Their fore limbs were then taped to the cage top so that the chest cavity was exposed. Using forceps to stabilize the skin, an initial cut with scissors was used to expose the musculature of the chest and upper abdomen. The caudal tip of the sternum was then grasped, and the diaphragm was rapidly punctured with the scissors, followed by cutting through the sternum to expose the heart.
  • the perfusion system consisted of a peristaltic pump (Masterflex, Cole-Parmer, Vernon Hills, IL) used at a setting of 'T with tubing connected to a 16 gauge needle that was inserted into the left ventricle of the heart which was gently held in place with forceps to deliver ice-cold sterile saline. The right atrium was then cut to allow the blood and saline to exit the circulatory system.
  • the quality of the perfusion was dependent on the amount of time the heart remained beating following insertion of the perfusion needle and could be monitored by the loss of color from the tail, hind limbs and liver.
  • pK values The blood plasma half-life (T 1 Z 2 or KlO) of the test compound was determined with WinNon ⁇ n software (version 5.2.1) using a Gauss-Newton modeling algorithm (#7), as shown below. Additional derived pK values including the area under the curve, Cmax, and body clearance were calculated by the same software package.
  • Calculation of total activity Total activity was determined at each time point based on the averaged activity for each listed sample based on the following calculations:
  • Plasma ratio ml of plasma/ml of total blood withdrawn
  • Platelet ratio ml of blood cells/ml of total blood withdrawn
  • Total blood cell CPM blood cell CPM/100 ⁇ l x 1000 ⁇ l/1 ml x est. total blood cell volume
  • pK analysis Probe 1 Fig. 2A shows the curve-fit for the elimination of the radioactivity from the plasma for Probe 1. From the software analysis of the data, the Ti/ 2 (half-life) or the elimination constant was 49.8 hours. The area under the curve (AUC) was 93.3 (hr)(pmol/ml) with a Cmax of 1.30 pmol/ml and a clearance of 0.014 ml/hr.
  • pK analysis Probe 2 Fig. 2B shows the curve-fit for the elimination of the radioactivity from the plasma for Probe 2. From the software analysis of the data, the Ti/ 2 for the elimination constant was 15.4 hours.
  • the area under the curve was 33.0 (hr)(pmol/ml) with a Cmax of 1.48 pmol/ml and a clearance of 0.040 ml/hr.
  • the heparosan molecular weight is stable in the mammalian bloodstream as shown in Fig. 4.
  • Fig. 15 depicts an analysis of the blood plasma half-life and the blood absorption half-life of a radioactive heparosan compound following injection thereof into rats.
  • Male Sprague-Dawley rats received two 0.1 ml injections of the radioactive heparosan compound (i.m.) in both hind limb calves.
  • This test compound 100 kDa was radiolabeled with 125 I (70 Ci/mmol).
  • the activity of the radiolabel was set at 4.0 ⁇ Ci per 0.2 ml, however testing demonstrated that 10% of the probe was retained within the syringes, thus the effective dose was 3.6 ⁇ Ci.
  • Fig. 15 shows the total distribution of the measured radioactivity for i.m. dosing. As shown, the majority of the activity remains in the plasma with relatively constant activity in the red cells and organs.
  • the amount of radioactivity in the different organs dissected from the rats was also measured.
  • the activity in the liver was less than 3.0% of total activity at all time points.
  • Total activity in the spleen was always less than 0.3%. This data indicates that i.m. dosing does not accumulate in these organs.
  • Activity in the kidneys from remained about 2% of the total activity.
  • Activity in the bladder was minimal ( ⁇ 0.2%) at all time points.
  • the probe did not accumulate in either the heart or the lungs as activity in both organs was 1% or less throughout the study. Based on the perfusion tests where residual blood was washed out of organs, this small amount of radioactivity was due to trapped blood (for i.m. and i.p. studies, no perfusion was performed).
  • radioactive heparosan was administered to animals via an abdominal injection.
  • Male Sprague-Dawley rats received a single 0.2 ml injection of the 100 kDa heparosan (i.p.) into the peritoneum.
  • 0.5 ml of whole blood was collected from the tail vein, while at 48, 72, 96 or 120 hr post-injection the rats were euthanized with blood and organs collected to determine distribution of the probe in a similar to the i.m. study.
  • the T 1 / 2 was at least 2 days when heparosan was injected i.p.
  • the activity in the liver was less than 3% all time-points. Similar to the i.m. dosing, total activity in the spleen was always less than 0.3%.
  • the activity in the kidneys was 2% or less at all time-points.
  • Activity in the bladder was minimal ( ⁇ 0.1%).
  • the probe did not accumulate in either the heart or the lungs. This data indicates that i.p. dosing does not accumulate in these organs.
  • both the urine and the fecal pellets were radioactive, demonstrating that i.p. dosing excretion began within the first hour post-dosing and continued throughout the testing period.
  • TLC thin layer chromatography
  • Feces After injection into rats, heparosan metabolites were excreted in feces over time as shown in Fig. 19. Water extracts of feces from various time points after injection were subjected to ultrafiltration with various molecular weight cut-off membranes (3, 10, or 50 kDa; Amicon Microcon) and gamma counting of the retained and eluted fractions. The size of the radioactive metabolites were inferred by the ability to penetrate the pores of the membrane; the vast majority of the radioactivity passed through the 3 kDa membranes; thus, the heparosan fragments that passed through the intestines was smaller than 3 kDa.
  • the aldehyde of heparosan polymers (e.g., either the natural reducing end or via periodate oxidation) is coupled directly to therapeutic proteins via its N-terminal amino groups using reductive amination with Na cyanoborohydride.
  • the reaction is done in 0.1 M Na acetate, pH 5, at 4° to 37°C; the buffer pH used de-selects the lysine groups with higher pKa (need an unprotonated amine for nucleophilic attack) in favor of the amino terminus.
  • Lower temperatures e.g. 4° to 10 0 C is used to preserve protein folding.
  • reactions at pH 7-9 are used to add multple heparosan chains to the lysines as well as the N- terminus of the protein cargo.
  • This methodology is therapeutically and commercially successful for proteins like interferons and GCSF (Neulasta).
  • chemistries are useful for successful conjugate synthesis; the basic requirements are (a) there is an appropriate reactive or activated group on the heparosan polymer vehicle (either short acceptor or longer polymers) that will react or interact with a group on the cargo (i.e., therapeutic agent or drug), or if desired, a secondary vehicle (e.g., liposome or nanoparticle), and (b) suitable mild reaction conditions that preserve the integrity and functionality of both the vehicle and the cargo.
  • the heparosan polymer vehicle either short acceptor or longer polymers
  • a secondary vehicle e.g., liposome or nanoparticle

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Abstract

La présente invention concerne des compositions, des procédés, et des systèmes destinés au développement et à l'utilisation de l'héparosane, un polymère naturel apparenté à l'héparine, comme nouvel agent ou support de modification thérapeutique qui peut moduler les comportements pharmacocinétiques de la charge de médicaments et le comportement d'un patient mammifère.
PCT/US2009/005050 2008-09-09 2009-09-09 Polymères d'héparosane et leurs procédés de fabrication et d'utilisation destinés à l'amélioration de composés thérapeutiques WO2010030342A2 (fr)

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CA2773755A CA2773755C (fr) 2008-09-09 2009-09-09 Polymeres d'heparosane et leurs procedes de fabrication et d'utilisation destines a l'amelioration de composes therapeutiques
EP09813350.7A EP2341941A4 (fr) 2008-09-09 2009-09-09 Polymères d'héparosane et leurs procédés de fabrication et d'utilisation destinés à l'amélioration de composés thérapeutiques

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WO2014043496A2 (fr) 2012-09-17 2014-03-20 Technip France Amortissement de vibrations induites par un tourbillon de plateforme de haute mer à entretoises comportant des plaques verticales
WO2014060401A1 (fr) * 2012-10-15 2014-04-24 Novo Nordisk Health Care Ag Polypeptides de facteur vii de coagulation
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US8980608B2 (en) 2012-03-30 2015-03-17 The Board Of Regents Of The University Of Oklahoma High molecular weight heparosan polymers and methods of production and use thereof
WO2015055692A1 (fr) * 2013-10-15 2015-04-23 Novo Nordisk Health Care Ag Polypeptides du facteur vii de la coagulation
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US20150225710A1 (en) * 2014-02-12 2015-08-13 Novo Nordisk A/S Coagulation Factor IX Conjugates
US9603945B2 (en) 2008-03-19 2017-03-28 The Board Of Regents Of The University Of Oklahoma Heparosan polymers and methods of making and using same for the enhancement of therapeutics
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US8580290B2 (en) 2001-05-08 2013-11-12 The Board Of Regents Of The University Of Oklahoma Heparosan-based biomaterials and coatings and methods of production and use thereof
US9629914B2 (en) 2001-05-08 2017-04-25 The Board Of Regents Of The University Of Oklahoma Heparosan-based biomaterials and coatings and methods of production and use thereof
US9925209B2 (en) 2008-03-19 2018-03-27 The Board Of Regents Of The University Of Oklahoma Heparosan-polypeptide and heparosan-polynucleotide drug conjugates and methods of making and using same
US9603945B2 (en) 2008-03-19 2017-03-28 The Board Of Regents Of The University Of Oklahoma Heparosan polymers and methods of making and using same for the enhancement of therapeutics
US9687559B2 (en) 2008-03-19 2017-06-27 The Board Of Regents Of The University Of Oklahoma Heparosan polymers and methods of making and using same for the enhancement of therapeutics
US8980608B2 (en) 2012-03-30 2015-03-17 The Board Of Regents Of The University Of Oklahoma High molecular weight heparosan polymers and methods of production and use thereof
US10392642B2 (en) 2012-03-30 2019-08-27 The Board Of Regents Of The University Of Oklahoma High molecular weight heparosan polymers and methods of production and use thereof
US9885072B2 (en) 2012-03-30 2018-02-06 The Board Of Regents Of The University Of Oklahoma High molecular weight heparosan polymers and methods of production of use thereof
WO2014043496A2 (fr) 2012-09-17 2014-03-20 Technip France Amortissement de vibrations induites par un tourbillon de plateforme de haute mer à entretoises comportant des plaques verticales
CN104661685A (zh) * 2012-10-15 2015-05-27 诺和诺德保健Ag（股份有限公司） 因子vii缀合物
JP2015533152A (ja) * 2012-10-15 2015-11-19 ノヴォ・ノルディスク・ヘルス・ケア・アーゲー 第ｖｉｉ因子コンジュゲート
US10179905B2 (en) 2012-10-15 2019-01-15 Novo Nordisk Health Care Ag Factor VII conjugates
WO2014060397A1 (fr) * 2012-10-15 2014-04-24 Novo Nordisk Health Care Ag Conjugués de facteur vii
WO2014060401A1 (fr) * 2012-10-15 2014-04-24 Novo Nordisk Health Care Ag Polypeptides de facteur vii de coagulation
WO2015055692A1 (fr) * 2013-10-15 2015-04-23 Novo Nordisk Health Care Ag Polypeptides du facteur vii de la coagulation
US9370583B2 (en) 2013-10-15 2016-06-21 Novo Nordisk Healthcare Ag Coagulation factor VII polypeptides
US9371370B2 (en) 2013-10-15 2016-06-21 Novo Nordisk Healthcare Ag Coagulation factor VII polypeptides
CN105979972A (zh) * 2014-02-12 2016-09-28 诺和诺德股份有限公司 因子viii缀合物
US20150225710A1 (en) * 2014-02-12 2015-08-13 Novo Nordisk A/S Coagulation Factor IX Conjugates
US20150224203A1 (en) * 2014-02-12 2015-08-13 Novo Nordisk A/S Factor VIII Conjugates

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EP2341941A2 (fr) 2011-07-13
CA2773755C (fr) 2017-04-25
WO2010030342A3 (fr) 2010-06-17
EP2341941A4 (fr) 2014-12-10

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