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WO1998000161A1 - Fibrin-based systems for the controlled release of medicinals - Google Patents

Fibrin-based systems for the controlled release of medicinals Download PDF

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Publication number
WO1998000161A1
WO1998000161A1 PCT/US1997/008909 US9708909W WO9800161A1 WO 1998000161 A1 WO1998000161 A1 WO 1998000161A1 US 9708909 W US9708909 W US 9708909W WO 9800161 A1 WO9800161 A1 WO 9800161A1
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WIPO (PCT)
Prior art keywords
medicinal
protein
gel
inhibitor
fibrin
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PCT/US1997/008909
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French (fr)
Inventor
Garfield P. Royer
Original Assignee
Buford Biomedical, Inc.
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Publication date
Application filed by Buford Biomedical, Inc. filed Critical Buford Biomedical, Inc.
Priority to AU31431/97A priority Critical patent/AU3143197A/en
Publication of WO1998000161A1 publication Critical patent/WO1998000161A1/en

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    • 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
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/2063—Proteins, e.g. gelatin
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1658—Proteins, e.g. albumin, gelatin

Definitions

  • This invention relates generally to a fibrin based bio-erodible delivery system which enables timed release of medicinals including proteins and small molecules. Fibrinogen is converted to fibrin in the presence of the medicinal which is entrapped when the fibrin gels.
  • Proteins either currently being administered by physicians or under investigation include growth factors, interferons, colony stimulating factors, and interleukins. In nature, these molecules may act locally as paracrine agents; i.e., they interact with and activate nearby cells. Further, they can be pleiotropic, i.e., they can activate or stimulate more than one kind of cell.
  • BGH bovine growth hormone
  • Bio-erodible polymers have been used for encapsulation of numerous classes of drugs (U.S. Patent No. 4,349,530; Royer, G.P., et al, J. Parenteral Science & Technol.. 37, 34 (1983); Lee, T.K., et al, Science. 213, 233 (1981); WO91/06287 (1991); WO93/25221 (1993), all of which are hereby incorporated by reference).
  • Synthetic polymers and copolymers of lactic acid and glycolic acid have been extensively investigated (U.S. Patent 5,122,367; Langer, Science, 249, 1927 (1990); U . S . Patent 4,983,393 (1991)).
  • U.S. Patent 4,983,393 discloses a composition for use as an intra-vaginal insert comprising agarose, agar, saline solution glycosaminoglycans, collagen, fibrin and an enzyme.
  • U.S. Patent 3,089,815 discloses an injectable pharmaceutical preparation composed of fibrinogen and thrombin. No provisions are made for protection of the medicinals from the action of thrombin.
  • PCT Application WO/92/09301 discloses fibrin glue that is supplemented with at least one growth factor.
  • U.S. Patents, 4,377,572 and 4,627,879 describe the use of fibrin in externally applied tissue sealant and topical wound treatments.
  • bio-erodible carriers are hydrophobic in nature and produce acid in conjunction with their hydrolysis. Examples include polymers of alpha hydroxy acids and various dicarboxylic acids. Although useful for delivery of small molecules and some polypeptides of low molecular weight, PLGA (co-polymer of lactic acid and glycol ic acid), for example, is not generally useful for proteins (Langer, R., Science 249, 1527 (1990).
  • fibrinogen is converted to fibrin which aggregates to form a strong gel.
  • factor XIII is present the gel is further stabilized by a specific enzyme-catalyzed transpeptidation reaction which covalently links fibrin segments. Formation of a fibrin -natri- ⁇ from fibrinogen
  • thrombin catalyzes the conversion of fibrinogen to fibrin, it also activates Factor XIII to become Factor XIII.
  • the primary function of thrombin is to convert fibrinogen to fibrin Similar to most proteases, thrombin will catalyze the rapid hydrolysis of small molecules and proteins, other than its natural substrate, examples include
  • Pre-formed fibrin has been used as an excipient for formation of implants combination with various inorganic chemicals (Brown, R G , et al , US Patent 4,393,0 1) These implants have been used for delivering steroids in farm animals Fibrin is isolated, dried at high temperature and powdered to form the starting material A drug, of low molecular weight, and other components are mixed prior to pelletization Similar pre-formed fibrin preparations of bov ine origin hav e been used (again in combination with other materials) for formation of human prosthetic devices (Capperauld, R., et al., Surgery. Gyn. and Ob. 144, 3 (1977)).
  • Plasmin results from the activation of the precursor protein plasminogen. This process is inhibited by low molecular weight compounds such as amino caproic acid and trans-4-(aminomethyl) cyclohexanecarboxylic acid (AMCHA). The latter compound is more potent than amino caproic acid and is the active ingredient in an FDA approved hemostatic having the trade name "Cyklokapron.”
  • AMCHA trans-4-(aminomethyl) cyclohexanecarboxylic acid
  • the subject invention relates to a medicinal delivery system based on the enzyme-catalyzed conversion of fibrinogen to fibrin, which forms a gel thus entrapping the medicinal.
  • a thrombin inhibitor is added along with the medicinal protein to protect the medicinal protein from fragmentation by the action of thrombin.
  • the erosion rate of the matrix is altered by incorporation of fibrinolysis inhibitors, adjusting the concentration of matrix components, and including Factor XIII at various levels.
  • the invention also includes methods for obtaining sustained release of a medicinal and treating pain comprising administering the delivery systems of the invention to a mammal.
  • the invention includes methods of synthesizing a drug delivery system comprising the steps of mixing fibrinogen, thrombin, medicinal, and other optional components such as inhibitors of fibrinolysis, Factor XIII, albumin and collagen, and shaping the mixture.
  • kits comprising the components needed to make the delivery system.
  • FIGURE 1 shows an idealized release profile of the subject invention.
  • FIGURE 2 shows a release profile of azo-albumin from a disc of fibrin matrix (13mm x 3mm).
  • the attributes of a delivery system for medicinal proteins include.
  • the release profile is adjustable by varying preparative conditions, components, and component concentrations.
  • the present invention relates to a fibrin based delivery system for the controlled release of medicinals including proteins and small molecules.
  • Thrombin catalyzed polymerization of fibrinogen can be employed to entrap medicinal proteins to produce bio-erodible dosage forms in a variety of shapes. Absence of chemical cross-linking agents prevents potentially harmful modification of the medicinal protein or non-protein drug
  • Fibrinogen is available commercially or it can be isolated from plasma, for example, by alcohol fractionation. Cryoprecipitates containing fibrinogen are alternatively used.
  • thrombin is also commercially available Alternatively, some fractions of fibrinogen contain sufficient thrombin to catalyze the gelation reaction in the presence of added CaCl 2 .
  • Thrombin inhibitors such as antithrombin III, hirudin and hirudin fragments (see 1995 Sigma Catalog) are commercially available. These inhibitors are useful when the medicinal protein can be cleaved (and rendered inactive) by thrombin Calcium is available in the form of CaCl 2 solution, CaCl 2 finely ground, poorly soluble calcium compounds such as encapsulated calcium CaCO 3 , and calcium propionate. Calcium ion can be withheld until after the dosage form is shaped. For example, after dispersion of the mixture (Ca-free) into droplets, finely ground Ca salts or solutions of Ca 2+ can be added to bring about gelation.
  • Biocompatible additives such as albumin, collagen, globulin, polyvinylpyrrolidone, polyethyleneglycol, polylysine, etc. are optionally added to alter the release profile of the delivery system.
  • Fibrinolysis catalyzed by the enzyme plasmin, is slowed by Factor XIII- induced stabilization. Therefore, including Factor XIII will reduce the erosion rate and extend the lifetime of the dosage form.
  • the erosion rate is reduced and the lifetime of the dosage form is extended by incorporation of fibrinolysis inhibitors such as alpha-2-antiplasmin, aprotinin, and AMCHA.
  • fibrinogen 10-90 mg/ml
  • medicinal protein .01-15 mg/ml
  • thrombin inhibitor 0.01 -10 unit/ml
  • thrombin 0.01-10 unit/ml
  • calcium .1-1M
  • the medicinal protein is mixed and allowed to react at room temperature.
  • the medicinal protein and the optional thrombin inhibitor are added and the mixture dispersed with stirring into a water-immiscible medium such as mineral oil or vegetable oil.
  • the temperature is optionally raised, for example, to 37°C. The temperature should not be high enough to denature the protein.
  • Factor XIII typically, .1-1 ⁇ M is used.
  • an amine-acceptor is used with the Factor XIII such as a lysine containing peptide (e.g., polylysine).
  • a fibrinolysis inhibitor is optionally added.
  • the delivery system can be formed as beads, granules, microspheres (100-200 microns), threads, cylinders, disks, films or cell-sized microspheres (less than 100 microns) using techniques presented herein and known to those skilled in the art.
  • Microbeads can be formed by dispersing a mixture of components in a water- immiscible medium with stirring. Subsequently, bead stabilization is accelerated by increasing the temperature to 37°C. Other geometric forms are produced using a similar two-step process. Adjustments of component concentrations and order of addition also permit direct injection of a reaction mixture which solidifies at 37°C in the body.
  • Implantable discs can be made, for example, by mixing all components at room temperature or below, pouring the mixture into a cylindrical mold and then curing at 37°C. The extruded fibrin cylinder is then sliced into discs.
  • Sub-batches of beads can be prepared with relatively low levels of Factor XIII or an inhibitor of fibrinolysis. These sub-batches can be used to constitute blends of beads. To illustrate, when these blends of beads contain proportionately more of Factor XIII, the release is relatively slow. Blends of beads weighted toward low levels of Factor XIII release drug relatively quickly in proportion to the faster erosion rate.
  • proportions of thrombin and fibrinogen see e.g., Example 3
  • direct injection of a liquid is possible.
  • fibrinogen, thrombin, calcium chloride, along with optional additives such as Factor XIII, a fibrinolysis inhibitor, albumin, etc. are mixed and allowed to react. Prior to gelation, the medicinal protein and the optional thrombin inhibitor are added. The mixture is injected, s.c, after which the formulation becomes a solid at body temperature.
  • Fibrinolysis inhibitors are optionally added to extend the residence time of the formulation.
  • Inhibitors can be included in microbeads which are included within the formulation or which are delivered separately. These "guardian" beads release inhibitor at a rate which controls erosion of the fibrin matrix.
  • Simultaneous use of free and bead-entrapped fibrinolysis inhibitor such as AMCHA can be used advantageously to control fibrinolysis and therefore the residence time.
  • this approach can be employed to preserve the fibrin depot for a period sufficiently long to permit release of most of the drug AMCHA can be entrapped in PLGA (50:50) microspheres which are small enough to be injected using commonly used procedures and instruments.
  • Fibrin dosage forms of the subject invention exhibit residence times in vivo of up to three weeks or more.
  • the term “medicinal” includes proteins as well as small molecule agents.
  • the term “protein” includes naturally occurring proteins, recombinant proteins, protein derivatives and polypeptides.
  • Medicinal proteins useful in the subject invention include colony stimulating factors (CSF) including G-CSF, GM-CSF, and M-CSF; erythropoietin (EPO); interleukins, IL-2, IL-4, IL-6, etc; interferons; growth factors (GF) including epidermal-GF, nerve-GF; tumor necrosis factor (TNF); hormones/bioactive peptides; ACTH; angiotensin, atrial natriuretic peptides, bradykynin, dynorphins/endorphins/ ⁇ -lipotropin fragments, enkephalin, gastrointestinal peptides including gastrin and glucacon; growth hormone and growth hormone releasing factors; luteinizing hormone and releasing hormone; melanocyte stimulating hormone; neuroten
  • the matrices of the invention are very suitable for the continuous release of therapeutically active proteins and peptides (see table below) over extended periods of time, i.e., 1-6 days, 1, 2, 3 or 4 weeks or 1-2 months.
  • extended periods of time i.e., 1-6 days, 1, 2, 3 or 4 weeks or 1-2 months.
  • the extent of the systemic (parenteral) treatment indications in humans is reflected by the examples in the following table.
  • G-CSF Hematopoietic growth factor bone marrow failure.
  • G-CSF, GM-CSF, M-CSF, IL-3 Also im uno adjuvant, adjunct to myelosuppressive chemotherapy
  • adenosine deaminase heritable immunodeficiencies nucleoside phosphorylase
  • enzymes such as ceredase heritable lysosomal diseases such as Gaucher disease enzymes such as arylsulfatases heritable mucopolysaccharidoses such as Hurlers
  • pituitary hormones such as pituitary failure syndromes (hypothalamic growth hormone, gonadotrophins, pituitary disorders) vasopressin, et al
  • cytokines such as TNF, or immunoadjuvant in vaccine administration GM-CSF, interleukins such as hepatitis, herpes viruses, parasitic as IL-l, IL-2, IL-12 diseases, and cancer
  • interferons alpha, beta and hairy cell leukemia, viral infections gamma
  • blood clotting factors such as bleeding disorders such as hemophilia factor VIII
  • vaccines such as hepatitis prevention of bacterial viral and vaccines, herpes vaccines, parasitic diseases, cancers such as antimicrobial vaccines, cancer melanoma vaccines
  • the medicinal protein is protected as described in commonly owned U.S. Serial No. 08/571,613 hereby incorporated by reference in its entirety.
  • Medicinal proteins can be protected from proteolysis or other reactions by chemical modification of sidechains adjacent to susceptible bonds or which constitute reactive functional groups.
  • the medicinal protein can be physically protected, that is, it can be employed as a crystal or solid matrix such as gelatin micro-beads. Such small particles can be dispersed into the fibrin liquid prior to gelation and then trapped within the depot. Any matrix which does not react with medicinal can be employed as long as it is safe, bio- erodible, and does not interfere with the release.
  • microbeads of the subject invention containing medicinal are encapsulated in the delivery system described in commonly owned U.S. Ser. No. 08/571,613, for example microbeads of the subject invention in a delivery system comprising albumen and/or collagen crosslinked with oxidized dextran.
  • the present delivery system is also applicable to formulations with non-protein medicinals, including but not limited to alkaloids, steroids, terpenoids, amino acid derivatives, nucleoside/nucleotide derivatives, polynucleotides, carbohydrates, polysaccharides, lipids, lipopolysaccharides, purines, pyrimidines and derivatives of same.
  • non-protein medicinals including but not limited to alkaloids, steroids, terpenoids, amino acid derivatives, nucleoside/nucleotide derivatives, polynucleotides, carbohydrates, polysaccharides, lipids, lipopolysaccharides, purines, pyrimidines and derivatives of same.
  • Advantageous small molecule drugs include: analgesics, anesthetics (e.g., lidocaine or pramocaine), antialcohol preparations, anti-infectives, antiseptics (e.g., compounds of monovalent silver, silver benzoate, PVP-I 2 ), anticoagulants, anticancer drugs, antidepressants, antidiabetic agents, antihypertensive drugs, antiinflammatory agents (e.g., hydrocortisone), antinauseants, anorexics, antiulcer drugs, cardiovascular drugs, contraceptives, decongestants, diuretics, hormones/antihormones, immunosuppressives, narcotic detoxification agents, uricosuric agents, and wound healing promoters such as deoxyribonucleosides (see table below).
  • analgesics e.g., lidocaine or pramocaine
  • antialcohol preparations e.g., anti-infectives, antiseptics (
  • the delivery systems of the invention are also suitable for the prolonged, controlled release of non-proteinaceous compounds with therapeutic activity.
  • a matrix can be constructed that will enable the steady release of a local anesthetic such as lidocaine over several weeks such that an adequate concentration can be maintained for 1-6 days, 1, 2, 3 or 4 weeks or 1-2 months, in the immediate vicinity of nerve roots damaged by a pathologic vertebral fracture due to cancer thus relieving severe pain without causing irreparable loss of function.
  • fibrin discs containing local anesthetic are fibrin discs containing local anesthetic.
  • the discs e.g., 0.8cm in dia. x .3cm
  • anesthetic e.g., lidocaine or marcaine
  • one disc is implanted laterally and one disc medially. This approach obviates systemic use of narcotic pain killers.
  • antibiotics such as penicillins, bacterial infections, e.g., bone and joint beta-lactams, amino-glycosides, infections, soft tissue infections in areas macrolides, tetracyclines, etc. not sufficiently vascularized, Streptococcal sore throat or E. Coli urinary tract infection
  • Antifungals such as nystatin thrush and other localized fungal infections
  • immunosuppressants such as organ/tissue heterotransplantation cyclosporin
  • anticoagulants such as heparin, pulmonary embolus, vascular diseases coumadin diuretics such as furosemide cardiac failure
  • vermifuge such as mebendazole various helminth parasitism
  • antimalarials such as treatment and prophylaxis of malarial chloroquine parasitism
  • anesthetics such as lidocaine, cancer pain, arthritis pain, post surgical pain marcaine or pramocaine
  • Formulations made possible by this invention enable the local therapeutic application of medicinals.
  • infected wounds or chronically infected ulcers such as those seen in patients with diabetes or vascular insufficiency to the lower extremities benefit from preformed films based on this invention that release debriding enzymes in combination with antibiotics and growth factors such as TGF- ⁇ or PDGF, to promote healing.
  • the healing of clean surgical incisions is enhanced by the local application of beads or implants that release growth factors and optionally anti-infectives or a local anesthetic to relieve patient pain
  • Particularly advantageous compounds for use in the subject invention are those in crystalline form.
  • Preformed films for transdermal delivery or for topical application as bandages can also be used.
  • the film may be used to deliver non-proteinaceous drugs such as anti-infectives and wound healing promoters.
  • FIG. 1 An idealized release profile is shown in Figure 1.
  • the concentration of medicinal in the vicinity of the bead reflects the rate of internal degradation of the three different classes of gel matrices.
  • the profile shown depicts the system with identical medicinal concentration in all classes. For example, to have higher levels of medicinal released at a later period, more medicinal would be incorporated in the Class III beads as shown in this example. Release profiles can be obtained from zero order release to those involving late-stage bursts. It is also possible to administer more than one medicinal in the same treatment regimen. The drugs can be released simultaneously or sequentially.
  • rate DA(d[m]/dx) (1)
  • D is the diffusion coefficient
  • A is the surface area d[m]/dx is the medicinal gradient Stokes law is also applicable
  • r is the radius of the medicinal
  • v is the viscosity of the medium.
  • the diffusion coefficient (D) is inversely related to the viscosity of the medium (v), the size of medicinal (r), and also the erosion rate of the device.
  • the matrix density and degree of cross-linking influence the viscosity. As the fibrin matrix is broken down, v is reduced which increases D which in turn increases the rate of diffusion. In the extreme case involving a dense matrix and a large medicinal molecule, the rate will be very low or zero until the breakdown occurs.
  • the volume of a sphere is given by (4)
  • V 4/3 ⁇ r 3 (4)
  • a bead of a given volume will have less surface area than multiple beads with the same aggregate volume.
  • a blend of beads with a range of radii will tend to give a release profile resembling zero order.
  • Medicinal matrix of the invention is administered to a human or other mammal as beads, disks, threads and implants of various other shapes using techniques known to those skilled in the art Beads would be normally administered via needle subcutaneously, intramuscularly, intraperitoneally, or intravenously for cell-sized microbeads Tablets and capsules are used for oral delivery
  • a liquid formulation can be directly injected subcutaneously, intramuscularly and intraperitoneally, which solidifies soon after injection
  • the liquid formulations are made such that the mixture remains a liquid until after injection See Example 3
  • Films can be cast using the preparations described herein, for example the preparation of Example 4
  • One approach is to use a multi-channel pipette and apply the preparation to a glass plate
  • the texture of the resulting material varies depending on the humidity and temperature
  • the preparation is applied directly to the wound or to a bandage which is in turn placed on the wound
  • Medicinals for topical preparations include, for example, anesthetics (lidocaine, pramocaine), antiseptics (PVP-I 2 , compounds of monovalent silver, polymyxin B, neomycin, gramicidin) and living cells.
  • the preparations of the invention provide beneficial osmotic draw as well as protection, pain relief and infection control
  • agents such as growth factors or cultured cells
  • Medicinal matrix can be administered concomitant with surgical procedures. Examples of this include 1) an antibiotic matrix following abdominal surgery, 2) matrix containing cytotoxic chemotherapeutic drug following tumor removal, and 3) matrices containing adjuvants/antigens following tumor removal.
  • implants can be placed under the skin adjacent to the joint capsule, or elsewhere following orthopedic surgery.
  • Solution A 400 ⁇ l was made containing fibrinogen (25 mg/ml) in buffer-- Hepes, 30 mM, pH 7.2, 0.15 M in NaCI.
  • Solution B 100 ⁇ l was made containing the enzyme thrombin (about 5 units) and CaCl 2 (1.6 M) in the same buffer.
  • Solution C (lOO ⁇ l) was made containing protein (azo-albumin)(2mg/ml), and antithrombin III/heparin in sufficient quantity to neutralize the thrombin. Solutions were equilibrated to temperature (20°C).
  • Microspheres were made using known methods with or without low concentrations of surfactants.
  • the mixture was injected into the stirred water-immiscible medium at room temperature The temperature was then raised to 37°C and the stirring was continued for 3 hr.
  • the microspheres were collected by centrifugation and then washed quickly with ether on a filter.
  • thrombin concentration is adjusted to allow direct injection.
  • Solutions A, B and C are mixed as previously described and the mixture is injected s.c. into a mammal.
  • Example 4
  • Example 1 The procedure of Example 1 is repeated using other geometric configurations including films containing antibiotics and wound healing promoters.
  • the reaction mixture is poured onto a flat glass plate with borders to provide boundaries of the desired dimensions.
  • the glass plate is warmed to 37°C and allowed to stand for 3-8 hours in a humidity controlled chamber.
  • Solution A 400 ⁇ l was made containing fibrinogen (25mg/ml) in buffer— Hepes, 30 M, pH 7.2, 0 15 M in NaCI. Finely ground lidocaine (50 mg) was then suspended therein.
  • Solution B (lOO ⁇ l) was made containing thrombin (5 units) and CaCl 2 (1.6 M) in the same buffer. Solutions A and B were equilibrated to 20°C and mixed When using a matrix protectant, such as AMCHA, it is included in Solution A by direct dissolution or by suspension if in the form of PLGA microspheres AMCHA should be at least 10 ⁇ g/ml in the immediate vicinity of the drug depot
  • Example 7 The procedure of Example 1 was repeated but with 10-40% by weight of a crystalline or amorphous solid medicinal protein suspended in the reaction mixture.
  • Example 7 The procedure of Example 1 was repeated but with 10-40% by weight of a crystalline or amorphous solid medicinal protein suspended in the reaction mixture.
  • Fibrin stock solution (1 ml/3%) was mixed with 50 mg of gentamicin sulfate.
  • Thrombin solution (25 ⁇ l/2.5 units) was added and the mixture was dispensed in 200 ⁇ l aliquots to screw-cap vials. After incubation for 1 hr at 37° the vials were allowed to stand at room temperature overnight. The samples were stored at 0-4°.
  • the buffer (1 ml/PBS with 0.01% azide) was pipetted into four vials, two of which served as controls. The buffer was changed at 24-hr intervals. Gentamicin concentration was determined using a standard curve prepared with known amounts using the TNBS test (Habeeb, A.F.S. A. 1966 Anal. Biochem. 14, 328). A release profile is shown below:

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Abstract

A fibrin-based bio-erodible matrix for the controlled release of medicinals including protein therapeutics is disclosed. A method for controlled drug release is also disclosed.

Description

FIBRIN BASED SYSTEMS FOR THE CONTROLLED RELEASE OF MEDICINALS
FIELD OF THE INVENTION
This invention relates generally to a fibrin based bio-erodible delivery system which enables timed release of medicinals including proteins and small molecules. Fibrinogen is converted to fibrin in the presence of the medicinal which is entrapped when the fibrin gels.
BACKGROUND OF THE INVENTION
The rapid progress in recombinant DNA technology has provided researchers and clinicians with a variety of newly discovered proteins in amounts sufficient to enable laboratory and clinical study (Cytokines. A. Meager, Prentis Hall, 1991). Proteins either currently being administered by physicians or under investigation include growth factors, interferons, colony stimulating factors, and interleukins. In nature, these molecules may act locally as paracrine agents; i.e., they interact with and activate nearby cells. Further, they can be pleiotropic, i.e., they can activate or stimulate more than one kind of cell.
Delivery of these highly potent molecules for treatment of disease remains a challenge. Serious toxicity, low maximum tolerated doses (MTD), and limited therapeutic windows have been observed when given parenterally. As noted above, since some of these molecules are paracrine agents, localized delivery is another issue (Golumbek, P.T., et al, Cancer Research. 53, 5341 (1993)). As an example, systemic use of certain colony stimulating factors may result in autoimmunity and tissue damage from intense inflammatory reactions. Temporary relief of illness may be followed by permanent damage to the immune system. Many novel proteins now being investigated for clinical use have very short half-lives. Clearance from the circulation can occur in a few hours or even a few minutes. Hence, prolonged release of effective doses below toxic levels would be advantageous.
Recombinant hormones such as bovine growth hormone (BGH) are widely used in dairy cattle. BGH is currently administered biweekly by injection. Controlled release of protein components in veterinary vaccines is desirable. Reduction of the frequency of injection and improvement in performance of the bio-active protein would be advantageous.
Bio-erodible polymers have been used for encapsulation of numerous classes of drugs (U.S. Patent No. 4,349,530; Royer, G.P., et al, J. Parenteral Science & Technol.. 37, 34 (1983); Lee, T.K., et al, Science. 213, 233 (1981); WO91/06287 (1991); WO93/25221 (1993), all of which are hereby incorporated by reference). Synthetic polymers and copolymers of lactic acid and glycolic acid have been extensively investigated (U.S. Patent 5,122,367; Langer, Science, 249, 1927 (1990); U.S. Patent 4,983,393 (1991)). Autologous albumin and gelatin are also exemplified in the literature (U.S. Patent No. 4,349,530; Royer, G.P., et al, J, Parental Science & Technol.. 37, 34 (1983); Lee, T.K., et al, Science. 213, 233 (1981)). Cross-linking with glutaraldehyde is known to stabilize albumin and gelatin matrices. Glutaraldehyde, however, is non-specific in its reaction with proteins. In solution glutaraldehyde forms a polymer which contains Michael addition sites. Functional groups such as amines and thiols react at these sites to form stable addition products. As a result, the protein drug can be inactivated or covalently bound to the matrix components. The latter reaction lowers the effective amount of deliverable drug or creates an antigenic molecule. A negative consequence of the latter chemical reaction is the development of autoimmunity. U.S. Patent 4,983,393 discloses a composition for use as an intra-vaginal insert comprising agarose, agar, saline solution glycosaminoglycans, collagen, fibrin and an enzyme.
U.S. Patent 3,089,815 discloses an injectable pharmaceutical preparation composed of fibrinogen and thrombin. No provisions are made for protection of the medicinals from the action of thrombin.
PCT Application WO/92/09301 discloses fibrin glue that is supplemented with at least one growth factor. U.S. Patents, 4,377,572 and 4,627,879 describe the use of fibrin in externally applied tissue sealant and topical wound treatments.
Many bio-erodible carriers are hydrophobic in nature and produce acid in conjunction with their hydrolysis. Examples include polymers of alpha hydroxy acids and various dicarboxylic acids. Although useful for delivery of small molecules and some polypeptides of low molecular weight, PLGA (co-polymer of lactic acid and glycol ic acid), for example, is not generally useful for proteins (Langer, R., Science 249, 1527 (1990).
Matrices stabilized by covalent cross-links (Lee, T. et al, Science 213 233 (1981); Royer, US Patent 4,349,530; Golumbeck, P.K., Cancer Research 53, 5841 (1993), suffer since the medicinal protein can be inactivated or coupled to the matrix as a result of reaction with the cross-linking reagent. Moreover, harmful immunogens can be generated by these reactions.
In the final steps of blood clotting, fibrinogen is converted to fibrin which aggregates to form a strong gel. When factor XIII is present the gel is further stabilized by a specific enzyme-catalyzed transpeptidation reaction which covalently links fibrin segments. Formation of a fibrin -natri-ε from fibrinogen
Figure imgf000006_0001
lysis
The enzyme thrombin cataly zes the conversion of fibrinogen to fibrin, it also activates Factor XIII to become Factor XIII. The primary function of thrombin is to convert fibrinogen to fibrin Similar to most proteases, thrombin will catalyze the rapid hydrolysis of small molecules and proteins, other than its natural substrate, examples include
Thrombin
Susceptible Bonds Proteins
Arg-A≤p Secreπn
Arg-Val Cholecystokin
Arg-Cys, Arg-Asp, Arg-Gly human chorionic gonadotrophin
Arg-Gly Insulin B-Chain
Pre-formed fibrin has been used as an excipient for formation of implants combination with various inorganic chemicals (Brown, R G , et al , US Patent 4,393,0 1) These implants have been used for delivering steroids in farm animals Fibrin is isolated, dried at high temperature and powdered to form the starting material A drug, of low molecular weight, and other components are mixed prior to pelletization Similar pre-formed fibrin preparations of bov ine origin hav e been used (again in combination with other materials) for formation of human prosthetic devices (Capperauld, R., et al., Surgery. Gyn. and Ob. 144, 3 (1977)).
Plasmin results from the activation of the precursor protein plasminogen. This process is inhibited by low molecular weight compounds such as amino caproic acid and trans-4-(aminomethyl) cyclohexanecarboxylic acid (AMCHA). The latter compound is more potent than amino caproic acid and is the active ingredient in an FDA approved hemostatic having the trade name "Cyklokapron."
Failures of conventional delivery systems for proteins are typically attributable to lack of design for controlled release, denaturation/inactivation of the medicinal protein in the matrix, adverse immunological reactions, or chemical modification of the medicinal during formulation.
OBJECTS OF THE INVENTION
It is an object of the invention to provide a bio-erodible delivery system which enables timed release of medicinals.
It is an object of the invention to provide a delivery system for proteins which does not alter the biological activity of the proteins.
It is a further object of the invention to provide a delivery system where the release profile is easily altered. SUMMARY OF THE INVENTION
The subject invention relates to a medicinal delivery system based on the enzyme-catalyzed conversion of fibrinogen to fibrin, which forms a gel thus entrapping the medicinal. For certain protein medicinals, after adequate fibrinogen conversion but prior to gelation, a thrombin inhibitor is added along with the medicinal protein to protect the medicinal protein from fragmentation by the action of thrombin. The erosion rate of the matrix is altered by incorporation of fibrinolysis inhibitors, adjusting the concentration of matrix components, and including Factor XIII at various levels.
The invention also includes methods for obtaining sustained release of a medicinal and treating pain comprising administering the delivery systems of the invention to a mammal.
The invention includes methods of synthesizing a drug delivery system comprising the steps of mixing fibrinogen, thrombin, medicinal, and other optional components such as inhibitors of fibrinolysis, Factor XIII, albumin and collagen, and shaping the mixture.
Additionally, the invention includes kits comprising the components needed to make the delivery system.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 shows an idealized release profile of the subject invention.
FIGURE 2 shows a release profile of azo-albumin from a disc of fibrin matrix (13mm x 3mm). DETAILED DESCRIPTION OF THE INVENTION
The attributes of a delivery system for medicinal proteins include.
-mild, non-denaturing preparative conditions -hospitable environment—hydrophilic, neutral -bio-erodible
-non-immunogenic, non-pyrogenic -stable, sterilizable
-programmable, i.e., the release profile is adjustable by varying preparative conditions, components, and component concentrations.
The present invention relates to a fibrin based delivery system for the controlled release of medicinals including proteins and small molecules. Thrombin catalyzed polymerization of fibrinogen can be employed to entrap medicinal proteins to produce bio-erodible dosage forms in a variety of shapes. Absence of chemical cross-linking agents prevents potentially harmful modification of the medicinal protein or non-protein drug
Fibrinogen is available commercially or it can be isolated from plasma, for example, by alcohol fractionation. Cryoprecipitates containing fibrinogen are alternatively used.
The enzyme thrombin is also commercially available Alternatively, some fractions of fibrinogen contain sufficient thrombin to catalyze the gelation reaction in the presence of added CaCl2.
Thrombin inhibitors such as antithrombin III, hirudin and hirudin fragments (see 1995 Sigma Catalog) are commercially available. These inhibitors are useful when the medicinal protein can be cleaved (and rendered inactive) by thrombin Calcium is available in the form of CaCl2 solution, CaCl2 finely ground, poorly soluble calcium compounds such as encapsulated calcium CaCO3, and calcium propionate. Calcium ion can be withheld until after the dosage form is shaped. For example, after dispersion of the mixture (Ca-free) into droplets, finely ground Ca salts or solutions of Ca2+ can be added to bring about gelation.
Biocompatible additives such as albumin, collagen, globulin, polyvinylpyrrolidone, polyethyleneglycol, polylysine, etc. are optionally added to alter the release profile of the delivery system.
Fibrinolysis, catalyzed by the enzyme plasmin, is slowed by Factor XIII- induced stabilization. Therefore, including Factor XIII will reduce the erosion rate and extend the lifetime of the dosage form.
The erosion rate is reduced and the lifetime of the dosage form is extended by incorporation of fibrinolysis inhibitors such as alpha-2-antiplasmin, aprotinin, and AMCHA.
Production of the Delivery System
It is advantageous to use a two step process consisting of a formation step followed by a gelation step. Examples of ratios of components are as follows: fibrinogen ( 10-90 mg/ml) medicinal protein (.01-15 mg/ml)/thrombin inhibitor (0.01 -10 unit/ml) thrombin (.01-10 unit/ml) calcium (.1-1M)
All of the above components except the medicinal protein are mixed and allowed to react at room temperature. Prior to gelation, the medicinal protein and the optional thrombin inhibitor are added and the mixture dispersed with stirring into a water-immiscible medium such as mineral oil or vegetable oil. After the microspheres have formed, the temperature is optionally raised, for example, to 37°C. The temperature should not be high enough to denature the protein.
Further stabilization occurs in the presence of Factor XIII (typically, .1-1 μM is used). Optionally, an amine-acceptor is used with the Factor XIII such as a lysine containing peptide (e.g., polylysine). A fibrinolysis inhibitor is optionally added.
The delivery system can be formed as beads, granules, microspheres (100-200 microns), threads, cylinders, disks, films or cell-sized microspheres (less than 100 microns) using techniques presented herein and known to those skilled in the art. Microbeads can be formed by dispersing a mixture of components in a water- immiscible medium with stirring. Subsequently, bead stabilization is accelerated by increasing the temperature to 37°C. Other geometric forms are produced using a similar two-step process. Adjustments of component concentrations and order of addition also permit direct injection of a reaction mixture which solidifies at 37°C in the body.
Implantable discs can be made, for example, by mixing all components at room temperature or below, pouring the mixture into a cylindrical mold and then curing at 37°C. The extruded fibrin cylinder is then sliced into discs.
Sub-batches of beads (or other shapes) can be prepared with relatively low levels of Factor XIII or an inhibitor of fibrinolysis. These sub-batches can be used to constitute blends of beads. To illustrate, when these blends of beads contain proportionately more of Factor XIII, the release is relatively slow. Blends of beads weighted toward low levels of Factor XIII release drug relatively quickly in proportion to the faster erosion rate. By adjusting proportions of thrombin and fibrinogen, see e.g., Example 3, direct injection of a liquid is possible. In this embodiment, fibrinogen, thrombin, calcium chloride, along with optional additives such as Factor XIII, a fibrinolysis inhibitor, albumin, etc., are mixed and allowed to react. Prior to gelation, the medicinal protein and the optional thrombin inhibitor are added. The mixture is injected, s.c, after which the formulation becomes a solid at body temperature.
Fibrinolysis inhibitors are optionally added to extend the residence time of the formulation. Inhibitors can be included in microbeads which are included within the formulation or which are delivered separately. These "guardian" beads release inhibitor at a rate which controls erosion of the fibrin matrix. Simultaneous use of free and bead-entrapped fibrinolysis inhibitor such as AMCHA can be used advantageously to control fibrinolysis and therefore the residence time. For delivery of small molecules, this approach can be employed to preserve the fibrin depot for a period sufficiently long to permit release of most of the drug AMCHA can be entrapped in PLGA (50:50) microspheres which are small enough to be injected using commonly used procedures and instruments.
Fibrin dosage forms of the subject invention exhibit residence times in vivo of up to three weeks or more.
Medicinal Proteins
As used herein, the term "medicinal" includes proteins as well as small molecule agents. The term "protein" includes naturally occurring proteins, recombinant proteins, protein derivatives and polypeptides. Medicinal proteins useful in the subject invention include colony stimulating factors (CSF) including G-CSF, GM-CSF, and M-CSF; erythropoietin (EPO); interleukins, IL-2, IL-4, IL-6, etc; interferons; growth factors (GF) including epidermal-GF, nerve-GF; tumor necrosis factor (TNF); hormones/bioactive peptides; ACTH; angiotensin, atrial natriuretic peptides, bradykynin, dynorphins/endorphins/β-lipotropin fragments, enkephalin, gastrointestinal peptides including gastrin and glucacon; growth hormone and growth hormone releasing factors; luteinizing hormone and releasing hormone; melanocyte stimulating hormone; neurotensin; opiode peptides; oxytocin, vasopressin and vasotocin, somatostatin; substance P; clotting factors such as Factor VIII, enzymes used for "replacement therapy," e.g., glucocerebrosidase, hexoseaminidase A; and antigens used in preventative (such as tetanus toxoid and diphtheria toxoid), and therapeutic vaccines.
The matrices of the invention are very suitable for the continuous release of therapeutically active proteins and peptides (see table below) over extended periods of time, i.e., 1-6 days, 1, 2, 3 or 4 weeks or 1-2 months. The extent of the systemic (parenteral) treatment indications in humans is reflected by the examples in the following table.
Human Parenteral Treatment Indications: Proteins and Peptides
Medicinal Clinical Indication
Hematopoietic growth factor bone marrow failure. G-CSF, GM-CSF, M-CSF, IL-3 Also im uno adjuvant, adjunct to myelosuppressive chemotherapy
erythropoietin bone marrow failure; chronic anemia, kidney disease
adenosine deaminase, heritable immunodeficiencies nucleoside phosphorylase
enzymes such as HGRPT, inborn errors of metabolism such as hypoxanthine-guanine Lesch-Nyhan syndrome phosphoriboxyl transferase
enzymes such as ceredase heritable lysosomal diseases such as Gaucher disease enzymes such as arylsulfatases heritable mucopolysaccharidoses such as Hurlers
pituitary hormones, such as pituitary failure syndromes (hypothalamic growth hormone, gonadotrophins, pituitary disorders) vasopressin, et al
pituitary hormone analogs, disorders such as prostate such as analogs of LH-RH hyperplasia and cancer, breast cancer, See e.g., Sigma Catalog #s endometriosis L0637, L800L, L8886
insulin diabetes
cytokines such as TNF, or immunoadjuvant in vaccine administration GM-CSF, interleukins such such as hepatitis, herpes viruses, parasitic as IL-l, IL-2, IL-12 diseases, and cancer
interferons (alpha, beta and hairy cell leukemia, viral infections gamma) blood clotting factors such as bleeding disorders such as hemophilia factor VIII
various immunoglobulins, therapy or prophylaxis in specific disease hyper immune sera exposure or immunodeficiency state
vaccines such as hepatitis prevention of bacterial viral and vaccines, herpes vaccines, parasitic diseases, cancers such as antimicrobial vaccines, cancer melanoma vaccines
In another embodiment of the invention, the medicinal protein is protected as described in commonly owned U.S. Serial No. 08/571,613 hereby incorporated by reference in its entirety. Medicinal proteins can be protected from proteolysis or other reactions by chemical modification of sidechains adjacent to susceptible bonds or which constitute reactive functional groups. In addition to chemical protection, the medicinal protein can be physically protected, that is, it can be employed as a crystal or solid matrix such as gelatin micro-beads. Such small particles can be dispersed into the fibrin liquid prior to gelation and then trapped within the depot. Any matrix which does not react with medicinal can be employed as long as it is safe, bio- erodible, and does not interfere with the release.
In another embodiment of the invention, microbeads of the subject invention containing medicinal are encapsulated in the delivery system described in commonly owned U.S. Ser. No. 08/571,613, for example microbeads of the subject invention in a delivery system comprising albumen and/or collagen crosslinked with oxidized dextran.
There are many analogous parenteral and local therapies that are enhanced by this invention in veterinary medicine, especially when biologically active proteins and polypeptides are used, e.g., bovine and porcine growth hormone, tetanus toxoid, C. perfringens antigens (B,C,D), feline leukemia antigens and leptospirosis antigens.
Non-Protein Delivery
The present delivery system is also applicable to formulations with non-protein medicinals, including but not limited to alkaloids, steroids, terpenoids, amino acid derivatives, nucleoside/nucleotide derivatives, polynucleotides, carbohydrates, polysaccharides, lipids, lipopolysaccharides, purines, pyrimidines and derivatives of same.
Advantageous small molecule drugs include: analgesics, anesthetics (e.g., lidocaine or pramocaine), antialcohol preparations, anti-infectives, antiseptics (e.g., compounds of monovalent silver, silver benzoate, PVP-I2), anticoagulants, anticancer drugs, antidepressants, antidiabetic agents, antihypertensive drugs, antiinflammatory agents (e.g., hydrocortisone), antinauseants, anorexics, antiulcer drugs, cardiovascular drugs, contraceptives, decongestants, diuretics, hormones/antihormones, immunosuppressives, narcotic detoxification agents, uricosuric agents, and wound healing promoters such as deoxyribonucleosides (see table below). The delivery systems of the invention are also suitable for the prolonged, controlled release of non-proteinaceous compounds with therapeutic activity. For example, a matrix can be constructed that will enable the steady release of a local anesthetic such as lidocaine over several weeks such that an adequate concentration can be maintained for 1-6 days, 1, 2, 3 or 4 weeks or 1-2 months, in the immediate vicinity of nerve roots damaged by a pathologic vertebral fracture due to cancer thus relieving severe pain without causing irreparable loss of function.
Especially useful for post-surgical implantation are fibrin discs containing local anesthetic. The discs (e.g., 0.8cm in dia. x .3cm) release anesthetic (e.g., lidocaine or marcaine) over a period of at least three days. For example, following arthroscopic surgery of the knee, one disc is implanted laterally and one disc medially. This approach obviates systemic use of narcotic pain killers.
The extent and wide variety of uses of the delivery systems of the invention in conjunction non-proteinaceous molecules can be appreciated from the examples given in the following table.
Human Parenteral Treatment Indications: Non-proteinaceous Molecules
Medicinal Clinical Indication
antibiotics such as penicillins, bacterial infections, e.g., bone and joint beta-lactams, amino-glycosides, infections, soft tissue infections in areas macrolides, tetracyclines, etc. not sufficiently vascularized, Streptococcal sore throat or E. Coli urinary tract infection
Antifungals such as nystatin thrush and other localized fungal infections
antiinflammatories such as inflammation due to arthritis, etc. hydrocortisone
hormones such as testosterone, contraception, various testicular, ovarian, estrogens, adrenal steroids adrenal failure states immunosuppressants such as organ/tissue heterotransplantation cyclosporin
anticoagulants such as heparin, pulmonary embolus, vascular diseases coumadin diuretics such as furosemide cardiac failure
vermifuge such as mebendazole various helminth parasitism
antimalarials such as treatment and prophylaxis of malarial chloroquine parasitism
anesthetics such as lidocaine, cancer pain, arthritis pain, post surgical pain marcaine or pramocaine
Analogous uses of small molecules in veterinary medicine are also included in this invention.
* * *
Formulations made possible by this invention enable the local therapeutic application of medicinals. For example, infected wounds or chronically infected ulcers such as those seen in patients with diabetes or vascular insufficiency to the lower extremities benefit from preformed films based on this invention that release debriding enzymes in combination with antibiotics and growth factors such as TGF-β or PDGF, to promote healing. The healing of clean surgical incisions is enhanced by the local application of beads or implants that release growth factors and optionally anti-infectives or a local anesthetic to relieve patient pain
Particularly advantageous compounds for use in the subject invention are those in crystalline form. Preformed films for transdermal delivery or for topical application as bandages can also be used. In this case the film may be used to deliver non-proteinaceous drugs such as anti-infectives and wound healing promoters.
Release of Medicinals and Modes of Administration
An idealized release profile is shown in Figure 1. Here the concentration of medicinal in the vicinity of the bead reflects the rate of internal degradation of the three different classes of gel matrices. The profile shown depicts the system with identical medicinal concentration in all classes. For example, to have higher levels of medicinal released at a later period, more medicinal would be incorporated in the Class III beads as shown in this example. Release profiles can be obtained from zero order release to those involving late-stage bursts. It is also possible to administer more than one medicinal in the same treatment regimen. The drugs can be released simultaneously or sequentially.
While not wishing to be bound by a particular theory, the rate of diffusion of a medicinal from a matrix to the therapeutic compartment is given by equation (1) rate = DA(d[m]/dx) (1)
D is the diffusion coefficient A is the surface area d[m]/dx is the medicinal gradient Stokes law is also applicable (2)
D = 1/rv (2)
r is the radius of the medicinal v is the viscosity of the medium. The diffusion coefficient (D) is inversely related to the viscosity of the medium (v), the size of medicinal (r), and also the erosion rate of the device. The matrix density and degree of cross-linking influence the viscosity. As the fibrin matrix is broken down, v is reduced which increases D which in turn increases the rate of diffusion. In the extreme case involving a dense matrix and a large medicinal molecule, the rate will be very low or zero until the breakdown occurs.
Another means of control of the release profile is the geometry of the delivery device. The surface area of sphere for example is given by (3)
A = 4πr2 (3)
The volume of a sphere is given by (4)
V = 4/3 πr3 (4)
Combining (3) and (4) gives
A V = l/r (5)
As shown in equation (5), a bead of a given volume will have less surface area than multiple beads with the same aggregate volume. A blend of beads with a range of radii will tend to give a release profile resembling zero order.
Computer modeling of this system with adjustable parameters can be used to generate a set of hypothetical release profiles for a given therapeutic protein. Solid Dosage Forms
Medicinal matrix of the invention is administered to a human or other mammal as beads, disks, threads and implants of various other shapes using techniques known to those skilled in the art Beads would be normally administered via needle subcutaneously, intramuscularly, intraperitoneally, or intravenously for cell-sized microbeads Tablets and capsules are used for oral delivery
Injectabte kiqtiid
In addition to implants and injectable beads, a liquid formulation can be directly injected subcutaneously, intramuscularly and intraperitoneally, which solidifies soon after injection The liquid formulations are made such that the mixture remains a liquid until after injection See Example 3
Preformed Films
Films can be cast using the preparations described herein, for example the preparation of Example 4 One approach is to use a multi-channel pipette and apply the preparation to a glass plate The texture of the resulting material varies depending on the humidity and temperature Alternatively, the preparation is applied directly to the wound or to a bandage which is in turn placed on the wound Medicinals for topical preparations include, for example, anesthetics (lidocaine, pramocaine), antiseptics (PVP-I2, compounds of monovalent silver, polymyxin B, neomycin, gramicidin) and living cells. For abrasions and lacerations, the preparations of the invention provide beneficial osmotic draw as well as protection, pain relief and infection control For burn treatment other agents such as growth factors or cultured cells are optionally included Medicinal matrix can be administered concomitant with surgical procedures. Examples of this include 1) an antibiotic matrix following abdominal surgery, 2) matrix containing cytotoxic chemotherapeutic drug following tumor removal, and 3) matrices containing adjuvants/antigens following tumor removal. Also, implants can be placed under the skin adjacent to the joint capsule, or elsewhere following orthopedic surgery.
The following Examples are illustrative, but not limiting of the compositions and methods of the present invention. Other suitable modifications and adaptations of a variety of conditions and parameters normally encountered in clinical therapy which are obvious to those skilled in the art are within the spirit and scope of this invention.
EXAMPLES Example 1
Production of Implantable Discs or Cylinders
Solution A (400 μl) was made containing fibrinogen (25 mg/ml) in buffer-- Hepes, 30 mM, pH 7.2, 0.15 M in NaCI. Solution B (100 μl) was made containing the enzyme thrombin (about 5 units) and CaCl2 (1.6 M) in the same buffer. Solution C (lOOμl) was made containing protein (azo-albumin)(2mg/ml), and antithrombin III/heparin in sufficient quantity to neutralize the thrombin. Solutions were equilibrated to temperature (20°C).
Solutions A and B were mixed and allowed to react approximately 2 minutes (or until the viscosity had noticeably increased prior to gelation). Solution C was then added. After mixing, the resulting combination was injected into the mold. Cylindrical molds were made by cutting off the needle ends of disposable syringes. The modified syringe was clamped in a vertical position and filled with the mixture of solutions A and B which had cured for three hours at 37°C Step-wise extrusion and slicing allowed production of implantable discs. Small syringes of narrow bore can be used in a similar fashion to produce cylinders.
Controlled release occurs over a three week period. See Figure 2
Example 2
Microsphere Preparation
Microspheres were made using known methods with or without low concentrations of surfactants. About 500ml of the water-immiscible medium, such as vegetable oil, was charged into a round-bottom flask with baffled sides. Agitation was provided by an overhead mechanical stirrer Solutions A, B, and C were mixed as in Example 1 using the same proportions with the total final volume of 2 ml. The mixture was injected into the stirred water-immiscible medium at room temperature The temperature was then raised to 37°C and the stirring was continued for 3 hr. The microspheres were collected by centrifugation and then washed quickly with ether on a filter.
Example 3
Direct Injection
The thrombin concentration is adjusted to allow direct injection. Solutions A, B and C are mixed as previously described and the mixture is injected s.c. into a mammal. Example 4
Films
The procedure of Example 1 is repeated using other geometric configurations including films containing antibiotics and wound healing promoters. The reaction mixture is poured onto a flat glass plate with borders to provide boundaries of the desired dimensions. The glass plate is warmed to 37°C and allowed to stand for 3-8 hours in a humidity controlled chamber.
Example 5
Fibrin Based Formulation of Lidocaine
Solution A (400μl) was made containing fibrinogen (25mg/ml) in buffer— Hepes, 30 M, pH 7.2, 0 15 M in NaCI. Finely ground lidocaine (50 mg) was then suspended therein. Solution B (lOOμl) was made containing thrombin (5 units) and CaCl2 (1.6 M) in the same buffer. Solutions A and B were equilibrated to 20°C and mixed When using a matrix protectant, such as AMCHA, it is included in Solution A by direct dissolution or by suspension if in the form of PLGA microspheres AMCHA should be at least 10 μg/ml in the immediate vicinity of the drug depot
Example 6
Beads Containing Dispersed Solid Medicinal
The procedure of Example 1 was repeated but with 10-40% by weight of a crystalline or amorphous solid medicinal protein suspended in the reaction mixture. Example 7
Release Profile of an Antibiotic
A. Matrix Preparation
Fibrin stock solution (1 ml/3%) was mixed with 50 mg of gentamicin sulfate. Thrombin solution (25 μl/2.5 units) was added and the mixture was dispensed in 200 μl aliquots to screw-cap vials. After incubation for 1 hr at 37° the vials were allowed to stand at room temperature overnight. The samples were stored at 0-4°.
B. Release of Gentamicin
The buffer (1 ml/PBS with 0.01% azide) was pipetted into four vials, two of which served as controls. The buffer was changed at 24-hr intervals. Gentamicin concentration was determined using a standard curve prepared with known amounts using the TNBS test (Habeeb, A.F.S. A. 1966 Anal. Biochem. 14, 328). A release profile is shown below:
Time(Days) % Released
1 32
2 13
3 5
4 6
5 6
It will be readily apparent to those skilled in the art that numerous modifications and additions may be made to both the present invention, the disclosed device, and the related system without departing from the invention disclosed.

Claims

WHAT IS CLAIMED IS
1. A gel medicinal delivery system comprising (i) fibrin, and
(ii) a protein medicinal not cleavable by thrombin.
2. A gel medicinal delivery system comprising (i) fibrin,
(ii) a protein medicinal, and (iii) a thrombin inhibitor.
. A gel medicinal delivery system comprising (i) fibrin,
(ii) a medicinal, and
(iii) an inhibitor of fibrinolysis.
4. A gel medicinal delivery system comprising (i) fibrin,
(ii) a medicinal, and
(iii) Factor XIII optionally with an amine acceptor.
5. A system as in Claim 4 wherein said amine acceptor is a lysine containing peptide.
6. A system as in Claim 1 further comprising a compound selected from the group consisting of albumin, collagen, polyvinyl-pyrrolidone, polyethylene glycol and polylysine.
7. A system as in Claim 1 encapsulated in a gel matrix comprising at least one matrix protein selected from the group consisting of gelatin and albumin, and a polymeric stabilizer and/or an external cross linker
8. A system as in Claim 1 further comprising Factor XIII.
9. A system as in Claim 3 wherein said inhibitor of fibrinolysis is selected from the group consisting of alpha-2-antiplasmin, aprotinin, pancreatic trypsin inhibitor and AMCHA.
10. A system as in Claim 1 wherein said system is in the form of a bead.
1 1 A system as in Claim 1 wherein said system is in the form of a disc.
12. A system as in Claim 1 wherein said protein medicinal is a protected protein.
13. A system as in Claim 1 wherein said protein medicinal is encapsulated in non-covalently linked microbeads.
14. A system as in Claim 2 wherein said protein medicinal is encapsulated in non-covalently linked microbeads.
15. A system as in claim 13 wherein said microbeads are gelatin microbeads.
16. A system as in Claim 1 wherein said protein medicinal is in crystalline form.
17. A system as in Claim 3 further comprising a thrombin inhibitor
18. A system as in Claim 2 wherein said thrombin inhibitor is antithrombin III/heparin.
19. A system as in Claim 1 wherein component (i) comprises 0-80% by weight fibrin and 0-80% by weight medicinal.
20. A sustained release delivery system comprising:
(a) a first gel matrix comprising:
(i) fibrin,
(ii) an inhibitor of fibrinolysis, and (iii) a medicinal protein, and
(b) a second gel matrix comprising:
(i) fibrin,
(ii) an inhibitor of fibrinolysis at a different level than is said first gel matrix, and, (iii) said medicinal protein.
21. A blend of gel matrices for sustained release of a medicinal comprising at least two gel matrices according to Claim 3 wherein said gel matrices have different levels of inhibitor of fibrinolysis.
22. A blend of gel matrices for sustained release of a medicinal comprising at least two gel matrices according to Claim 4 wherein said at least two said gel matrices have different levels of Factor XIII.
23. A blend of gel matrices for sustained release of a medicinal comprising at least two gel matrices according to Claim 1 wherein said at least two said gel matrices have different levels of gel density.
24. A method for obtaining sustained release of a medicinal protein comprising administering the system of Claim 1 to a mammal.
25. A method for obtaining sustained release of a medicinal protein comprising administering the system of Claim 3 to a mammal.
26. A method for obtaining sustained release of a medicinal protein comprising administering the system of Claim 20 to a mammal.
27. A method of treating pain in a mammal in need of such treatment comprising administering the system of Claim 3 to said mammal wherein said medicinal is an anesthetic.
28. A method of treating pain in a mammal in need of such treatment comprising administering the system of Claim 4 to said mammal wherein said medicinal is an anesthetic.
29. A method as in Claim 24 wherein said administration is subcutaneously.
30. A method as in Claim 25 wherein said administration is subcutaneously.
31. A method as in Claim 25 wherein said inhibitor of fibrinolysis is administered separately.
32. A method as in Claim 25 wherein said inhibitor of fibrinolysis is in the form of microbeads.
33. A method of synthesizing a drug delivery system comprising the steps of: a) mixing fibrin, medicinal protein, calcium ion to form a gel matrix, b) shaping said gel matrix, c) curing said gel matrix.
34. A method as in Claim 33 wherein said mixing step includes mixing thrombin.
35. A kit comprising:
(i) a vial containing fibrinogen,
(ii) a vial containing thrombin and calcium ion, and
(iii) a vial containing medicinal protein and a thrombin inhibitor.
36. A kit comprising:
(i) a vial containing fibrinogen,
(ii) a vial containing thrombin and calcium ion, and
(iii) a vial containing medicinal protein not cleaved by thrombin.
37. A kit comprising:
(i) a vial containing fibrinogen,
(ii) a vial containing thrombin and calcium ion, and
(iii) a vial containing medicinal protein and an inhibitor of fibrinolysis.
38. A kit as in Claim 35 further comprising a vial containing a Factor XIII.
PCT/US1997/008909 1996-05-24 1997-05-27 Fibrin-based systems for the controlled release of medicinals WO1998000161A1 (en)

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