EP1073419A2 - Composition a liberation prolongee contenant un polymre amorphe - Google Patents

Composition a liberation prolongee contenant un polymre amorphe

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Publication number
EP1073419A2
EP1073419A2 EP99912785A EP99912785A EP1073419A2 EP 1073419 A2 EP1073419 A2 EP 1073419A2 EP 99912785 A EP99912785 A EP 99912785A EP 99912785 A EP99912785 A EP 99912785A EP 1073419 A2 EP1073419 A2 EP 1073419A2
Authority
EP
European Patent Office
Prior art keywords
composition
particles
pharmaceutical substance
substance
antisolvent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99912785A
Other languages
German (de)
English (en)
Other versions
EP1073419A4 (fr
Inventor
Theodore W. Randolph
Mark C. Manning
Richard F. Falk
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Technology Corp
Original Assignee
University of Technology Corp
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Filing date
Publication date
Application filed by University of Technology Corp filed Critical University of Technology Corp
Publication of EP1073419A2 publication Critical patent/EP1073419A2/fr
Publication of EP1073419A4 publication Critical patent/EP1073419A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • 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
    • A61K47/54—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 compound
    • A61K47/541—Organic ions forming an ion pair complex with the pharmacologically or therapeutically active agent
    • 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
    • A61K47/54—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 compound
    • A61K47/554—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 compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • 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
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • 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
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/145—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic compounds
    • 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
    • A61K9/1682—Processes
    • A61K9/1694—Processes resulting in granules or microspheres of the matrix type containing more than 5% of excipient

Definitions

  • the present invention concerns a highly amorphous sustained-release composition for sustained release of a pharmaceutical substance; an antisolvent precipitation method for making the composition: products made using the composition: and uses of the composition.
  • compositions may be introduced into a human or animal host for therapeutic or curative purposes in a number of ways.
  • the pharmaceutical substance is administered in the form of solid particles.
  • a micropump may be used in some applicauons for prolonged treatment by slowly injecting a suspension of small particles in a liquid.
  • small particles having both a pharmaceutical substance and a biodegradable polymer may be placed within tissue for sustained release of the pharmaceutical substance, with the biodegradable polymer acting to control the release of the pharmaceutical substance.
  • small particles may be inhaled to lodge in tissue of the lungs, permitting the pharmaceutical substance to then enter the circulatory system or to be released for local treatment.
  • nebulizauon a liquid having the pharmaceutical substance in solution is sprayed at a high velocity and inhaled.
  • nebulizauon may involve spraying a powder as fine particles propelled by a carrier gas, with the particles being inhaled.
  • Panicles administered by both these nebulizauon methods may have a wide distribution of droplet or panicle sizes. resulting in a very low utiiizauon of the pharmaceutical substance. Panicles, or droplets, which are too large tend to lodge in the throat and mouth during inhalation and are not.
  • lyophilization involves rapid freezing of the pharmaceutical substance with water, followed by rapid dehydration of the frozen material to produce dry particles of the pharmaceutical substance. This technique has been used with proteins and other polypeptides, but the low temperatures involved may reduce the biological activity of some polypeptide molecules. Also, the particles produced by lyophilization tend to be large and clumping and are often not suitable for pharmaceutical delivery methods which require smaller particles. It is possible to grind the lyophilized particles to produce smaller particles, but such grinding may damage some pharmaceutical substances, especially proteins.
  • gas antisolvent precipitation One method which has been proposed for making small particles of a pharmaceutical substance is called gas antisolvent precipitation.
  • a pharmaceutical substance is dissolved in an organic solvent which is then sprayed into an antisolvent fluid, such as carbon dioxide, under supercritical conditions.
  • the antisolvent fluid rapidly invades spray droplets, causing precipitation of very small pharmaceutical particles.
  • the gas antisolvent precipitation technique requires that the pharmaceutical substance be soluble in the organic solvent.
  • hydrophobic pharmaceutical substances this generally presents no problem because those substances can readily be dissolved in relatively mild, non-polar organic solvents.
  • Hydrophilic pharmaceutical substances are substantially insoluble in such relatively mild organic solvents.
  • insulin a hydrophilic protein
  • DMSO dimethylsulfoxide
  • DMF N,N-dimethylformamide
  • One problem with such a process is that highly polar solvents such as DMSO and DMF tend to unfold protein molecules from their native tertiary structure, or conformation. These protein molecules would, therefore, also be precipitated in an unfolded state for incorporation into the solid particles. Such unfolding could seriously reduce the biological activity of a protein or other polypeptide, especially if stored as a solid particle in the unfolded state for any appreciable time.
  • cationic surfactants can be used to conjugate nucleic acids to enzymes and to purify nucleic acids. See U.S. Patents Nos. 4,873,187 and 5,010,183. In particular, the latter patent teaches that the cationic surfactants and nucleic acids form hydrophobic complexes that can be dissolved or dispersed in polar solvents for purification of the nucleic acids.
  • the morphology of the particles may detrimentally effect performance.
  • M. Mayajim et al. Effect of Polymer Crystalliniry on Paperverine Release from Poly(l-lactic acid) Matrix, describe a problem encountered with amorphous poly(l-lactic acid) in sustained-release compositions.
  • the amorphous polymer had an undesirable tendency to crystallize during drug release, thereby altering the drug release characteristics of the composition.
  • a method for placing a pharmaceutical substance into solution in an organic solvent in the form of a hydrophobic ion pair complex with an amphiphilic material.
  • the resulting solution may then be subjected to gas antisolvent precipitation using a near critical or supercritical fluid to produce a precipitate of particles comprising the pharmaceutical substance.
  • Particles may be produced with a relatively narrow size distribution in a variety of sizes, thereby permitting flexibility in preparing particles for effective utilization in a variety of pharmaceutical applications.
  • the present invention permits pharmaceutical substances which are ordinarily substantially not soluble in an organic solvent to be solubilized. which facilitates further processing to prepare pharmaceutical powders.
  • the method is particularly preferred for use with proteins and other polypeptide molecules. Those molecules may be dissolved in a relatively mild, relatively non-polar organic soivenu thereby decreasing the potential for the reduction in biological activity which oui ⁇ rea ⁇ irom use of a strong, highly polar organic solvent in which the hydrophilic molecules are directly soluble.
  • a biodegradable polymer may be co- dissolved in the organic solvent along with the pharmaceutical substance and the amphiphilic material.
  • the particles produced comprise an intimate mixture of the biodegradable polymer with the pharmaceutical substance and the amphiphilic material. Problems of compositional variation or concentration of the pharmaceutical substance near the surface of the particle are, therefore, reduced relative to processes which require processing of a pharmaceutical substance in a suspension.
  • a highly amorphous sustained-release composition may be made including a biocompatible polymer, typically also biodegradable, and a pharmaceutical substance in the form of a hydrophobic ion pair complex with an amphiphilic material.
  • the polymer is highly amorphous, preferably with no greater than about 25% crystallinity and more preferably with an even lower crystallinity.
  • the composition typically includes a very high loading of the pharmaceutical substance and the amphiphilic material, which together in the hydrophobic ion pair complex typically comprise greater than about 15 weight percent of the composition, and preferably even more
  • the composition exhibits desirable release cnaracte ⁇ stics for sustained release of the Dnarmaceutical substance and does not appear to have a problem with crystaihzauon of the polymer during drug release, as has been reported by others.
  • the amorphous cnaracter of the polymer should be less likely to provoke an immune response that could cause inflammation.
  • the composition typically includes low or insignificant levels of residual solvent from the manufacture process.
  • the composition may be incorporated into a variety of product forms for administration to a mammalian patient. Preferred methods for use of the composition include inhalation for pulmonary delivery, subcutaneous placement, intraperitoneal placement and intraocular placement.
  • the antisolvent precipitation process is controlled to provide the desired characteristics in the composition.
  • a high ratio of volumetric flow of antisolvent fluid to volumetric flow of liquid feed (in which the pharmaceuucal substance, amphiphilic mate ⁇ al and polymer are codissolved) is preferred, with a ratio of from about 20 to about 30 being particularly preferred. Lower flow ratios tend to increase crystallinity of the polymer m the compos on.
  • the preferred method of operation of the process is to contact the liquid feed and antisolvent fluid at subcritical conditions, but preferably at a reduced temperature of greater than about 0. .
  • a pha ⁇ naceutical substance havmg panicles comprising a pharmaceutical substance and an amphiphilic material in a hydrophobic ion pa:r complex.
  • the solid panicles are hollow and have a substantially elongated, fiber-like shape. These elongated particles are advantageous in that they should have a longer retention time, compared to substantially spheroidal particles, in the stomach of a human or animal host following tngesuon. Therefore, the particles may be advantageously used for sustained release applications for delivery of a pharmaceutical substance in the stomach region.
  • the admmistranon mav be by inhalation of the solid particles, by injection of a suspension of the solid particles in a liquid medium or by ingestion of the solid particles.
  • the invention also provides cationic surfactants having the formula:
  • P is a biocompatible hydrophobic moiety
  • C is a biocompatible cationic moiety
  • L is a biodegradable linkage linking P and C.
  • These cationic surfactants are substantially less toxic than currently existing cationic surfactants and can be used for a ⁇ ministration of pharmaceutical substances to animals and in other situations where cell survival is important. In particular, they can be used as the amphiphilic material in the methods and compositions described above. In addition, these cationic surfactants can be used to deliver nucleic acids into cells, making them useful in genetic engineering techniques, including gene therapy.
  • Fig. 1 shows the log of the apparent partition coefficient for the dipeptide Gly-Phe- NH : .
  • Fig. 2 shows the log of the apparent partition coefficient for 8-Arg-vasopressin (AVP).
  • Fig. 3 shows the log of the apparent partition coefficient for insulin.
  • Fig. 4 shows the CD spectra of a 6:1 SDS-insulin complex in 1-octanol.
  • Fig. 5 shows the CD spectra of insulin extracted from 1 -octanol using an aqueous solution of 0.10 M HC1.
  • Fig. 6 shows the effect of temperature on the denaturation of insulin dissolved in
  • Fig. 7 shows the logarithm of the apparent partition coefficient of bovine pancreatic trypsin inhibitor (BPTI) from pH 4 water into 1 -octanol.
  • BPTI bovine pancreatic trypsin inhibitor
  • Fig. 8 shows the UV-visible absorption spectrum of human serum albumin (HSA) in NMP (50: 1 SDS to HSA ratio).
  • Fig. 9 shows the melting point of the SDS:insulin HIP complex as a function of the molar ratio of SDS to insulin.
  • Fig. 10 shows a CD scan for a 9:1 SDS:insulin molar ratio at 222 nm as a function of temperature.
  • Fig. 11 shows an absorbance scan for a 9: 1 SDS:insulin molar ratio at 222 run as a function of temperature.
  • Fig. 12 shows a process flow diagram for one embodiment of an antisolvent precipitation method for producing pharmaceutical powders.
  • Fig. 13 shows a process flow diagram for batch processing for gas antisolvent precipitation relating to Examples 19-29.
  • Fig. 14 is an SEM photomicrograph of a particle of the present invention comprising imipramine.
  • Fig. 15 is another SEM photomicrograph of a particle of the present invention comprising imipramine.
  • Fig. 16 is a SEM photomicrograph of a particle of the present invention comprising ribonuclease and poly(ethyleneglycol).
  • Fig. 17 is a SEM photomicrograph of particles of the present invention comprising a-chymotrypsin.
  • Fig. 18 is a SEM photomicrograph of particles of the present invention comprising pentamidine.
  • Fig. 19 shows a process flow diagram for continuous processing for gas antisolvent precipitation relating to Examples 30-32.
  • FIGS 20A-G illustrate schemes for the synthesis of arginine esters.
  • CBZ is phenylmethoxycarbonyl and tBOC is t-butyloxycarbonyl.
  • FIGS 21A-F illustrate schemes for the synthesis of cholesterol esters and carbamates.
  • THF is tetrahydrofuran.
  • Me is methyl.
  • Mel is methyliodide.
  • MEK is methyl ethyl ketone.
  • Figure 22 A is a graph of surface tension versus concentration for arginine octyl ester.
  • Figure 22B is a graph of surface tension versus concentration for arginine dodecyl ester.
  • Figure 23A is a graph of OD 90 versus concentration comparing cytotoxicity of arginine dodecyl ester and tetradecyltrimethylammonium bromide (CTAB) in CCRF-CEM cells.
  • Figure 2'B is a graph of OD 490 versus concentration comparing cytotoxicity of arginine dodecyl ester and tetradecyltrimethylammonium bromide (CTAB) in COS-7 cells.
  • CTAB tetradecyltrimethylammonium bromide
  • Figure 24A is a graph showing the time dependence of DNA transfection using arginine dodecyl ester.
  • Figure 24B is a graph of luciferase intensity versus concentration showing the effect of arginine dodecyl ester concentration on DNA transfection.
  • Figure 25 A is a graph of OD 490 versus concentrauon showing lack of cytotoxicity of CC-cholesterol in COS-7 cells.
  • Figure 25B is a graph of OD 490 versus concentration showing lack of cytotoxicity of CC-cholesterol in J ⁇ G-3 cells.
  • Figure 26 shows the steroid backbone.
  • Figure 27 illustrates a scheme for the synthesis of a ketal starting with 4-cholesten-3- one.
  • X represents a cationic moiety.
  • the present invention permits a pharmaceutical substance to be solubi zed in an organic solvent by associating the pha ⁇ naceutical substance with an amphiphilic mate ⁇ al.
  • the pharmaceutical substance is substantially not directly soluble in the organic solvent, but becomes soluble in association with the amphiphilic mate ⁇ al. It should be appreciated that by substantially not soluble it is not meant that the pharmaceutical substance is utterly insoluble in an organic solvent. Rather, it is meant that the direct solubility of the pharmaceutical substance in the organic solvent is limited and that it would be desirable to dissolve an amount of the pha ⁇ naceutical substance over and above that amount which is directly soluble.
  • the solubility of the pha ⁇ naceutical substance in the organic solvent may be increased by an order of magnitude or more, and is often increased by more than two orders of magnitude relative to direct dissolution of the pharmaceutical substance into the organic solvent, in the absence of the amphiphilic mate ⁇ al
  • the pharmaceutical substance and the amphiphilic material are m a true, homogeneous soluuon in the organic solvent
  • a true, homogeneous solution it is meant that the pharmaceutical substance, the amphiphilic mate ⁇ al and the organic solvent form a smgle liquid phase
  • the present invention is, therefore, distinguishable from the preparation of emulsions, micellar systems and
  • the pharmaceutical substance and the amphiphilic mate ⁇ al are associated in the form of a complex between the amphiphilic material and the pharmaceuucal substance, with the complex being substantially not soluble aqueous liquids at a physiological pH.
  • the amphiphilic mate ⁇ al and the pha ⁇ naceutical substance have oppositely charged ionic portions which associate to form an ion pair complex.
  • Such an ion pair complex is referred to as a hydrophobic ion pair (HIP) complex.
  • the pharmaceutical substance may comprise a canonic portion which associates with an anionic portion of the amphiphilic material or an anionic portion which associates with a cauonic portion of the amphiphilic material.
  • the pharmaceutical substance may be any substance which may be administered to a human or animal host for medical purpose, which is normally a cura ⁇ ve, therapeutic, preventive, or diagnostic purpose.
  • the pharmaceutical substance is preferably directly soluble to some meaningful degree in an aqueous liquid at a physiological pH.
  • a physiological pH is a pH of from about 1 to about 8.
  • the pharmaceutical substance exhibits a charged character when dissolved in an aqueous liquid at a physiological pH.
  • a pharmaceuucal substance includes various salt forms of a substance as well as ionic forms and dissociation products, such as may be found in an aqueous soluuon.
  • the pharmaceutical substance may comp ⁇ se a protein or other polypeptide. a nucleic acid, an analgesic or another mate ⁇ al.
  • a nucleic acid a nucleic acid, an analgesic or another mate ⁇ al.
  • representaUve types of pharmaceutical substances which may be used with the present lnvenuon. with a few specific examples listed for each type of pha ⁇ naceutical substance: cholinergic agonists
  • antichoiinesterase agents neostigmine, physostigmine
  • antimusca ⁇ nic drugs atropme, scopalamine
  • antiadrenergics tolazohne, phentolamine, propranolol, atenolol
  • ganglioruc stimulating agents tonicotine, tnmethaphan
  • neuromuscular blocking agents gallamine, succinylcho ne
  • local anesthetics procaine, lidocaine, cocaine
  • benzodiazepines triazolam
  • antipsychotics chlorpromazine, t ⁇ fiupromaz ⁇ ne
  • antidepressants fluoxetme.
  • imipramine amit ⁇ ptyline, phenelzine
  • antiparkinson's drugs L-dopa, dopamine
  • opioids and anti-opoids morphine, naloxone, naltrexone, methadone
  • CNS stimulants theophyllme, strychnine
  • autocoids and anti-autocoids histamine. betazole, chlo ⁇ henira ⁇ une, cimeudine
  • anti-inflammato ⁇ es tolmetin, piroxicam
  • anu-hypertensives clonidine. hydralazine, minoxidil
  • diuretics metalozone, bumetamide
  • polypeptides lysopressm.
  • vasopress oxytccin, insulin, calcitonin. gene-related peptide, LHRH agonists, ACTH, growth hormone
  • antifungals clotrimazole. miconazole
  • antimalarials chloroquine, primaquine
  • antiprotozoals pentamidine, melarsoprol
  • antihelminthics piperazine, oxamniquine
  • antimicrobials streptomycin, erythromycin, cefaclor, ceftriaxone, oxytetracycline.
  • rifampicin, isoniazid, dapsone aminoglycosides (gentamycin, neomycin, streptomycin): antineoplastics (mechlorethamine. melphalan, doxorubicin, cisplatin); anticoagulants (heparin); nucleic acids (genes, antisense RNAs, ribozymes, plasmids).
  • the pharmaceutical substance may be a sympathomimetic drug such as catecholamines (epinephrine. norepinephrine); noncatecholamines (amphetamine, phenylephrine); and ⁇ -adrenergics (terbutaline, albuterol).
  • macromolecules such as polymers, nucleic acids, proteins or polypeptides.
  • One advantage of the present invention is that the pharmaceutical substance, when in solution with the amphiphilic material in the organic solvent, retains a substantially native conformation. This is particularly important for materials, such as proteins and ribozymes, which are highly susceptible to loss of activity due to loss of native conformational structure.
  • the amphiphilic material may be any material with a hydrophobic portion and a hydrophilic portion. These materials are typically surfactants.
  • the hydrophilic portion is ionic under the conditions of use.
  • the hydrophobic portion may be any hydrophobic group, such as an alkyl, aryl or alkylaryl group.
  • the amphiphilic material associates with the pharmaceutical substance to form a hydrophobic ion pair which is soluble in the organic solvent when the pha ⁇ naceutical substance itself is substantially not soluble in the organic solvent
  • amphiphilic material includes different salt forms of a material as well as ionic forms and dissociation products of a material, such as may be present in a solution.
  • Preferred amphiphilic aiatsr ⁇ -Is are those posing little or substantially no toxicological problem for a human or animal host.
  • anionic amphiphilic materials include sulfates, sulfonates, phosphates (including phospholipids), carboxylates, and sulfosuccinates.
  • Some specific anionic amphiphilic materials useful with the present invention include: sodium dodecyl sulfate
  • SDS bis-(2-ethylhexyl) sodium sulfosuccinate
  • AOT cholesterol sulfate and sodium laurate.
  • Particularly preferred anionic amphiphilic materials are SDS and AOT.
  • Preferred cationic amphiphilic materials are the cationic surfactants of the invention (see below).
  • Specific cationic amphiphilic materials include the arginine and cholesterol esters, carbamates, carbonates and ketals (see below).
  • the solution of the pharmaceutical substance and the amphiphilic material in the organic solvent may be prepared in any suitable manner.
  • small amounts of the amphiphilic material may be added to an aqueous solution, in which the pharmaceutical substance is initially dissolved, until a precipitate forms of an HIP complex of the pharmaceutical substance and the amphiphilic material.
  • the precipitate may then be recovered and dissolved in an organic solvent to provide the desired solution.
  • the aqueous liquid and the organic solvent may then be contacted to effect a partitioning of the pharmaceutical substance into the organic solvent to form an HIP complex with the amphiphilic material.
  • aqueous liquid may then be contacted with an organic solvent to partition into the organic solvent at least some of the pharmaceutical substance and the amphiphilic material in the form of an HIP complex.
  • the organic solvent may be any organic liquid in which the pharmaceutical substance and the amphiphilic material, together, are soluble, such as in the form of an HIP complex.
  • organic solvents monohydric alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 1-hexanol, 1- octanol, trifluoroethanol); polyhydric alcohols (propylene glycol, PEG 400, 1,3-propanediol); ethers (tetrahydrofuran (THF), diethyl ether, diglyme); alkanes (decalin, isooctane, mineral oil); aromatics (benzene, toluene, chlorobenzene, pyridine); amides (n-methyl pyrrolidone (NMP), N,N-dimethylformamide (DMF)); esters (ethyl acetate, methyl acetate); chlorocarbons (CH-C1 2 , CHC1 3 , CC1
  • monohydric alcohols methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 1-he
  • the present invention involves the use of amphiphilic materials as ion pairing agents to modulate the solubility and partitioning behavior of pharmaceutical substances such as polypeptides, proteins, nucleic acids, and drugs.
  • Complexes are formed by stoichiometric interaction of an amphiphilic material, such as a detergent or other surfactant (e.g., alkyl sulfate. such as sodium dodecyl sulfate (SDS), or arginine ester), with the ionic functional groups of a polypeptide, protein, nucleic acid, or organic molecule that are accessible for ion pairing.
  • a detergent or other surfactant e.g., alkyl sulfate. such as sodium dodecyl sulfate (SDS), or arginine ester
  • the basic group may be an amine (as found in the lysine amino acid residue or the N-terminal amino group of a polypeptide) or a guanidinium group (as in arginine).
  • the acidic group may be a carboxyl group or phosphate group.
  • An ion pair is subsequently formed, refe ⁇ ed to as a hydrophobic ion pair (HIP) complex.
  • the HIP complex formed vviii have reduced aqueous solubility, but enhanced solubility in organic solvents. It has been discovered that an HIP complex may be dissolved in an organic solvent to form a true homogeneous solution.
  • the method of the invention for forming a true homogeneous solution is fundamentally different from any other method for placing proteins into organic solvents, such as those which use suspensions, micelles, microemulsions, or chemical modifications of the protein.
  • This discovery holds important implications in the area of drug delivery and release, including delivery to the body by inhalation and dispersion in a hydrophobic biodegradable matrix. While the decreased aqueous solubility of the HIP complex has been observed previously, the use of an HIP complex precipitate for improved drug delivery is novel.
  • HIP complex out of aqueous solution may be controlled for the production of uniform HIP complex particles of a desired size. These particles may then be formed into a suspension.
  • This invention also includes a method of obtaining HIP complex particles of specific sizes by controlling the conditions of HIP complex precipitation.
  • the size of KIP complexes is controlled by controlling the rates of the mixing of a protein solution and the addition of an anionic or cationic detergent to the protein solution.
  • the HIP complex can produce very fine suspensions which have limited solubility in water, and the technology can be used to produce particles of varying specific size.
  • the particle size of the HIP complex which is formed in water will depend on the degree of agitation of the protein solution and the rate of counterion addition. The smallest particles are produced with high shear being applied to the aqueous protein solution and slow addition of detergent.
  • This approach is also important in pulmonary drug delivery, where the particle size is critical to delivery to certain sites within the lung.
  • a high speed homogenizer can be used to stir the protein solution and a surfactant is added dropwise to the agitated solution. Particles in the 2-10 micron range can be obtained using this procedure. Particles of this size are required to get a sufficient amount of protein delivered to the lung to have a beneficial effect.
  • the particles once formed can be separated by centrifugation or filtration. Larger particles will be formed with slow agitation speeds and more rapid addition of surfactant.
  • DNase an enzyme currently being used by cystic fibrosis patients to dissolve viscous fluid build-up in the lung.
  • Other examples include protein and peptide enzyme inhibitors currently being tested for the treatment of emphysema.
  • antituberculosis drugs e.g.. streptomycin, isoniazid, pyrazinamide, ethambutol.
  • transgenes used to transfect lung cells for gene therapy.
  • the invention includes a ethcd of controlling the release of a protein from a suspension by controlling the size of the HIP complex panicle.
  • T e release rate of protein into an aqueous solution from an HIP complex will be much slower than that of the protein itself. This rate will be a function of the particle size of the complex and the solubility of the complex in water or biological fluid.
  • the solubility is a function of the amphiphilic material used and the strength of its association with the protein. Therefore, extended (controlled) release of the protein from the suspension can be achieved. This property permits proteins to be formulated as a suspension for depot injection.
  • This invention also includes the discovery that imcomplexed protein released from the HIP complex can be extracted back into aqueous medium with retention of its native structure.
  • the native uncomplexed protein can be reclaimed by dissolution in an aqueous solution which contains an excess of chloride or other counterion, indicating that the complexation is an entirely reversible process. It has been discovered that the protein of the HIP complex subsequently extracted back into an aqueous medium retains its native structure. This makes HIP methodology useful in the delivery of proteins for use as therapeutic agents.
  • HIP complexes display greatly enhanced thermal stability relative to the native protein, both with respect to chemical degradation and denaturation. This suggests that the HIP complex is useful for long term storage of the protein. Further, this aspect of the invention permits high temperature (steam) sterilization of proteins without the loss of biological activity, which until now, could not be accomplished.
  • poiymer delivery systems for proteins are usually sterilized by radiation as proteins are destroyed by heat.
  • the present invention discloses a method by which proteins may be processed by heating at sterilizing temperatures.
  • the enhanced thermal stability of the present invention may be important for the formulation of proteins in maintaining an active enzyme in an organic solvent and for long term storage of sensitive proteins.
  • Included in this invention is a method of uniformly distributing a drug throughout a hydrophobic polymer comprising adding a sufficient amount of a detergent to an organic molecule to form a precipitate, isolating the precipitate, and co-dissolving the precipitate and a hydrophobic polymer in an organic solvent to form a homogeneous distribution of the organic molecule within the polymer.
  • the water-soluble drug is leached out of the polymer by biological fluids (rather than its controlled release as the polymer is slowly degraded).
  • the invention provides a new method for distributing a drug uniformly through a hydrophobic polymer.
  • HIP complex formation permits both proteins and hydrophobic polymers to possess similar solubility parameters, thus facilitating incorporation of the protein into the polymer matrix.
  • the inventors have discovered that HIP complexes may be uniformly distributed in biodegradable polymers as they possess a solubility in solvents that will also dissolve the polymer. Where the HIP complex does not dissolve in the solvent used it will suspend easily as a result of its hydrophobic surface.
  • the invention wherein the drugs being delivered are included in the polymer matrix in an HIP complex represents three advantages over the biodegradable polymer systems: (1) the hydrophobic polymers can be better mixed with the drug in its lipophilic ion-pair state; (2) the drug forms hydrophobic particles within the polymer, and avoids the problem of the formation of a concentration of polar particles at the interface of the polymer leading to the "burst" effect; (3) the hydrophobic particles dispersed within the biodegradable polymer are not leached out by biological fluids which result in a predictable release rate.
  • the inventors have discovered the use of the HIP complex to control (retard or extend) the release of a drug at a predictable rate, resulting in part from a more uniform formulation.
  • One embodiment of this invention includes a method for achieving a true homogeneous solution of biologically active proteins and polypeptides in a organic solvent. None of the methods by which enzymatic activity is achieved in a nonaqueous environment employs a true protein solution.
  • the inventors have discovered that the HIP complex can be redissolved in an organic solvent such that a true homogeneous solution is formed. This discovery has important ramifications for controlling the enzymatic activity of proteins in the body. Through the formation of HIP complexes, enzymes and other proteins can be solubilized in a variety of organic solvents, including ethanol, propylene glycol and glycols in general. N-methyl pyrrolidone (NMP) and others.
  • NMP N-methyl pyrrolidone
  • HIP complex dissolved in organic solvent can be extracted back into aqueous medium with retention of the native protein structure.
  • This discovery has potential use in the purification of proteins.
  • a protein having a pH different from others in a mixture may be extracted or preferentially precipitated from the mixture by HIP complex formation.
  • the invention further includes a method of obtaining a stabilized protein comprising precipitating a protein in the HIP complex.
  • a stabilized protein comprising precipitating a protein in the HIP complex.
  • the HIP complex may, in many cases, provide a simple alternative to obtaining a stabilized protein.
  • a protein in the solid HIP complex has enhanced stability and resistance to degradation through storage, shipping, and handling. Chemical stability is conferred because the amount of water present is relatively low, as in lyophilized powders.
  • a diluent containing a significant chloride concentration e.g. phosphate buffered saline (PBS) or normal saline.
  • the protein can also be stored as a stable entity by dissolving or suspending the HIP complex in an organic solvent or solvent mixture. To form an aqueous solution of the protein, the solution or suspension can be shaken with water containing chloride. In cases where the organic solvent is immiscible with water, the protein will partition into the water.
  • An additional embodiment of this invention is a method of incorporating proteins and other drugs into lipid vesicies, liposomes. or deterger.: .-icslles. Shaking of an oil-water mixture with an HIP complex of a protein leads to emulsification, indicating that a HIP complex can more easily be introduced into emulsion delivery systems than the drug alone.
  • Systems for such use can be designed using either the insoluble material in suspension formulations or in oil formulation, such as oil in water emulsions.
  • Other examples include nasal and pulmonary aerosols, ophthalmic suspensions, transdermal patches, lozenges, chewing gum, buccal and sublingual systems, and suppositories.
  • Another aspect of this invention is the reduction of the bitter taste of drugs inco ⁇ orated into HIP complexes, since only compounds in solution are tasted. Therefore, this invention includes a method for improving the taste of orally administered drugs by formation of insoluble HIP complexes with such drugs.
  • the taste of a substance is detected by receptors in the tongue.
  • a major approach to modifying the taste of a drug is to alter its solubility in saliva. If the solubility is sufficiently low the taste will not be noted.
  • the low solubility of the HIP complex in biological fluids, including saliva can be used to mask the flavor of a drug, optionally, the HIP complexes may be incorporated into a polymer to further mask the taste of the drug.
  • Another way to mask taste is to partition the drug into an oil. such as olive oil. This can then be given as an oil in water emulsion with flavoring agents added to the outer water phase. HIP complex formation would provide the drug with the necessary high oil to water partition coefficient.
  • hydrophobic ion-pairing refers to the interaction between an amphiphilic material and a pharmaceutical substance.
  • Preferred amphiphilic materials include detergents which interact with proteins, other polypeptides and nucleic acids.
  • HIP complex derivatives are substances modified by formation of a hydrophobic ion-pair. The detergent interacts with an oppositely charged compound, such as a polypeptide or nucleic acid. This interaction has been termed HIP because it appears to be primarily electrostatic in nature.
  • anionic detergents encompasses any hydrophobic material that is a salt of an acid which can be employed to modify solubility properties in the described way. including sulfates, sulfonates, phosphates, and carboxylates. Sulfates are the salts of the stronger acids in this series and, therefore, the most efficient at forming ion pairs. Provided that the alkyl chains or aryl rings are of 8-18 carbons in length, they are potential candidates for HIP methodology.
  • cationic surfactants encompasses any material having a hydrophobic moiety and a cationic moiety which can be employed to modify solubility properties in the described way.
  • cationic surfactants Prefe ⁇ ed are the biocompatible cationic surfactants of the invention (see below).
  • the solution having the HIP complex dissolved in the organic solvent is itself a valuable product
  • the solution may also be used in the preparation of additional pharmaceutical products.
  • the solution may be used to prepare a powder of solid particles comprising the pharmaceutical substance and the amphiphilic material.
  • the solution is subjected to antisolvent precipitation processing to prepare a powder of solid particles.
  • Powders may be prepared having particles of an ultrafine size and a relatively narrow size distribution.
  • hollow elongated, fiber-like particles of a small size may be prepared. These particles have unique properties which may be desirable for various pharmaceutical applications.
  • a liquid feed solution 102 is provided having a pharmaceutical substance and an amphiphilic material dissolved together in an organic solvent, which is used as a carrier liquid for processing of the pharmaceutical substance.
  • the liquid feed solution 102 is subjected to antisolvent precipitation 104 in which the liquid feed solution 102 is contacted with an antisolvent fluid 106.
  • the antisolvent fluid 106 invades the organic solvent of the liquid feed solution 102, resulting in precipitation of solid particles comprising the pharmaceutical substance and the amphiphilic material.
  • the resulting mixture 108, having the precipitated particles is subjected to separation 110 in which solid particles 112 are separated from the exiting fluid
  • the antisolvent fluid is a fluid in which the pharmaceutical substance and the amphiphilic material, in association, are substantially not soluble. It should be understood that it is possible that the antisolvent fluid may be capable of dissolving some amount of the pharmaceutical substance and the amphiphilic material without departing from the scope of the present invention.
  • the antisolvent fluid is substantially incapable of dissolving a significant portion of the pha ⁇ naceutical substance and the amphiphilic material from the liquid feed solution such that at least a significant portion of pharmaceutical substance and the amphiphilic material are, in effect, not soluble in the antisolvent fluid.
  • the antisolvent fluid is at least partially miscible with the organic solvent such that the antisolvent fluid is capable of penetrating into the organic solvent sufficiently to cause the desired precipitation of the pharmaceutical substance and the amphiphilic material.
  • the antisolvent precipitation 104 is conducted under thermodynamic conditions which are near critical or supercritical relative to the antisolvent fluid.
  • the antisolvent precipitation is such that the antisolvent fluid is at a reduced pressure of greater than about 0.5, with the reduced pressure being the ratio of the total pressure during the antisolvent precipitation 104 to the critical pressure of the antisolvent fluid 106. More preferably, the contacting occurs at a reduced pressure of from about 0.8 to about 2.0 relative to the antisolvent fluid and even more preferably at a reduced pressure of from about 0.8 to about 1.2.
  • the antisolvent precipitation 104 is at a reduced temperature of greater than about 0.75, with the reduced temperature being the ratio of the temperature (in K) during the antisolvent precipitation 104 to the critical temperature (in K) of the antisolvent fluid 106. More preferably, the contacting occurs at a reduced temperature of greater than about 0.85, even more preferably greater than about 0.9 and most preferably greater than about 0.95. Typically, the reduced temperature is smaller than about 1.2.
  • the antisolvent fluid may comprise any suitable fluid for near critical or supercritical processing.
  • These fluids include carbon dioxide, ammonia, nitrous oxide, methane, ethane, ethylene, propane, butane, pentane, benzene, methanol, ethanol, isopropanol, isobutanol, fluorocarbons (including chiorotrifluoromethane. monofiuoromethane. hexafluoraethane and 1,1-difluoroethylene), toluene, pyridine, cyclohexane. m-cresol, decalin, cyclohexanol, o- xylene, tetralin, aniiin.
  • acetylene chlorotrifluorosilane, xenon, sulfur hexafluoride, propane, and others.
  • Carbon dioxide, ethane, propane, butane and ammonia are prefe ⁇ ed antisolvent fluids.
  • the antisolvent fluid should preferably have a critical temperature of from about 0 C C to about 50 °C. Included in this category of antisolvent fluids are carbon dioxide, nitrous oxide, ethane, ethylene, chlorotrifluoromethane. monofluoromethane, acetylene. 1.1-difluoroethylene, hexafluoroethane. chlorotrifluorosilane. and xenon.
  • a particularly preferred antisolvent fluid is carbon dioxide because it is readily available, non-toxic, and has a critical temperature of 31 °C and a critical pressure of 72.9 atm, which permits processing under relatively mild conditions.
  • the contacting of the liquid feed solution 102 with the antisolvent fluid 106 during the antisolvent precipitation 104 may be accomplished using any suitable contacting technique and contacting apparatus.
  • the liquid feed solution 102 is sprayed as small droplets into the antisolvent fluid 106.
  • a sonicated spray nozzle which is vibrated ultrasonically. has been found to work well because it is capable of producing very small droplets of a relatively uniform size and is, therefore, conducive to preparation of ultrafine powders having particles of a na ⁇ ow size distribution.
  • the contacting may be performed in a batch operation or continuously. Also, continuous operation could involve contacting by concurrent flow or countercu ⁇ ent flow.
  • the separation 110 may be accomplished using any suitable separation technique and apparatus. For example, the separation may involve simple density separation, filtration or use of a centrifuge.
  • the antisolvent precipitation process of the present invention may be used to produce ultrafine particles of a narrow size distribution and which are often of spheroidal shape. These it ⁇ - .-. ⁇ r.! 1c- .- ⁇ s _; . ' ⁇ z zz -.;_;::: l ⁇ micrcss cr may be 1 micron or smaller.
  • the size of the particles produced will depend upon the particular pharmaceutical substance and the processing conditions used.
  • panicle size becomes larger as the viscosity and surface tension of the organic solvent increases.
  • the use of ethanol as an organic solvent would generally produce smaller particles than the use of isopropanol as an organic solvent.
  • particles generally tend to become larger in the vicinity of the critical temperature as the process temperature approaches the critical temperature from above. If the process temperature is too high, however, then particle sizes generally tend to become larger again.
  • using carbon dioxide the smallest particles seem to be produced around a temperature of about 35 °C, with larger particles generally being produced at substantially higher and lower temperatures.
  • the pressure is preferably within the range of from about 70 bars to about 90 bars.
  • the method of the present invention may be used to produce particles of a na ⁇ ow size distribution.
  • particles produced in the gas antisolvent precipitation method of the present invention are such that greater than about 90 weight percent of the particles are within about 50 percent larger or smaller than a weight average particle size.
  • hollow fiber-like particles may be made according to the present invention, the length of which may vary depending upon processing conditions.
  • These fiber-like particles have a tubular quality in that they comprise an elongated body, of a substantially rounded cross-section, which has a hollow interior, which typically is open at least one end of the elongated body, and is preferably open at both ends of the elongated body.
  • these fiber-like particles tend to form when the pharmaceutical substance is subjected to gas antisolvent precipitation at a very high concentration in the organic solvent such that the molecules of the pharmaceutical substance tend to be entangled when dissolved in the organic solvent.
  • Macromolecules are particularly susceptible to such entanglement in solution and are, therefore, prefe ⁇ ed for making these fiber-like particles.
  • Such macromolecules include polymers and polypeptides, including proteins. The concentrations required for any particular pharmaceutical substance will depend upon the specific pharmaceutical substance being processed, but concentrations of 5 to 10 weight percent or higher, relative to the organic solvent, may be required for many polypeptide macromolecules.
  • the fiber-like particles typically have a diameter of smaller than about 100 microns, preferably smaller than about 50 microns. In some cases, the diameter may be as small as 10 microns or less. Length may vary from about 0.3 mm or less to as long as 1 cm or more, and is preferably longer than about 0.5 mm and more preferably longer than about 1 mm.
  • hollow, fiber-like particles offer a number of advantages for use in the pharmaceutical industry, one advantage is that these fiber-like particles have a shape that will not upon ingestion, pass as easily as a spheroidal particle through the stomach. The fiberlike particles should, therefore, tend to have a longer retention time in the stomach region and would, accordingly, be available in a stomach region for a longer period of time for the desired pharmaceutical treatment.
  • Another advantage of the fiber-like particles is that, because they are hollow, it is possible to place smaller particles of another pharmaceutical substance inside the hollow interiors. For example, small particles of mo ⁇ hine or pentamidine could be loaded into the hollow interiors of a protein-based fiber-like particle.
  • a biodegradable polymer may also be inco ⁇ orated into the solid particles of the present invention, as noted previously, for controlled release of the pharmaceutical substance.
  • a biodegradable polymer may be inco ⁇ orated in the antisolvent precipitation method of the present invention by co-dissolving the biodegradable polymer in the organic solvent along with the pharmaceutical substance and the amphiphilic material. The panicles produced during antisolvent precipitation will then contain the biodegradable polymer as well as the amphiphilic material"and the pharmaceutical substance.
  • the biodegradable polymer may be used in any convenient amount relative to the pharmaceutical substance.
  • the weight ratio of the biodegradable polymer to the pha ⁇ naceutical substance could vary from about 0.1 to 1 to about 100,000 to 1 depending upon the application. Most controlled release applications, however, will involve a ratio of from about 10 to 1 to about 100 to 1.
  • Inco ⁇ oration of the biodegradable polymer into the solid particles may be used to delay release of the pharmaceutical substance and to permit sustained release of the pharmaceutical substance over some extended period of time. It has been found that the release profile from a particle of the present invention in an aqueous buffer solution for the pharmaceutical substance is relatively constant and that a sudden initial release, or "burst effect.” is avoided. This indicates that the pharmaceutical substance is not concentrating near the surface of the particle and that the panicle comprises an intimate and homogeneous mixture of the pharmaceutical substance, the amphiphilic material and the biodegradable polymer. Any biodegradable polymer may be used which may be co-dissolved in the organic solvent along with the pharmaceutical substance and the amphiphilic material.
  • biodegradable polymers include those having at least some repeating units representative of polymerizing at least one of the following: an alphahydroxycarboxylic acid, a cyclic diester of an alphahydroxycarboxylic acid, a dioxanone, a lactone, a cyclic carbonate, a cyclic oxalate, an epoxide, a glycol. and anhydrides.
  • Preferred is a biodegradable polymer comprising at least some repeating units representative of polymerizing at least one of lactic acid, glycolic acid, lactide, glycolide. ethylene oxide and ethylene glycol.
  • the biodegradable polymers may be a homopolymer or a copolymer of two or more different monomers. Prefe ⁇ ed homopolymers include poly(lactic acid), polylactide, poly(glycolic acid), pc glycolide and poiy(ethyie ⁇ e glycol).
  • solid particles having the pharmaceutical substance and the amphiphilic material according to the present invention are introduced into a human or animal host.
  • the solid particles are inhaled for pulmonary delivery.
  • pulmonary delivery it is prefe ⁇ ed that greater than about 90 weight percent of all of the solid particles in an aciministered pharmaceutical formulation are of a size smaller than about 10 microns and more preferably at least about 90 weight percent of said particles are smaller than about 6 microns, and even more preferably at least about 90 weight percent of all of said solid particles are from about 1 micron to about 6 microns.
  • Particularly prefe ⁇ ed for pulmonary delivery applications are particles of from about 2 microns to about 5 microns in size. These particles may also comprise a biodegradable polymer for delayed and/or sustained release of the pharmaceutical substance.
  • the ultrafine size and na ⁇ ow size distribution of the solid particles of the present invention permit a much higher utilization of the pharmaceutical substance for pulmonary delivery than the low utilization experienced with present methods for pulmonary delivery of pharmaceutical substances.
  • current aerosol and nebulizauon techniques may use only 10 percent of a pharmaceutical substance which is administered, with the particles of the present invention, 80 percent or more of a pharmaceutical substance which is administered may be utilized.
  • the solid particles of the present invention may also be placed in a suspension and the suspension injected into the host
  • the particles will often comprise a biodegradable polymer for sustained release of the pha ⁇ naceutical substance.
  • the particles should be less than about 100 microns in size, most preferably less than about 50 microns in size, although smaller or larger particles may be used in some applications.
  • substantially all particles should be of a size smaller than about 1 micron so that the particles will not be susceptible to creating a blockage within the circulatory system.
  • Tne particles may comprise a biodegradable polymer, if desired.
  • greater than about 90 weight percent of the particles are preferably smaller than about 1 micron to reduce problems associated with settling of the solid particles. More preferably, substantially all panicles are smaller than about 1 micron.
  • the fiber-like particles shouid be useful in a number of pharmaceutical applications to deliver a pharmaceutical substance to a location where it is needed. For example, due to their hollow, fibrous shape, these panicles should tend to absorb water due to capillary action.
  • the fiber-like particles may, therefore accelerate biodegradation of a biodegradable polymer relative to a particle which is not hollow.
  • the fiber-like particles could be woven or spun, alone or with other fibrous materials, to inco ⁇ orate a pharmaceutical substance into a medical product using the woven or spun materials.
  • the fiber- like particles could be made to include a growth factor. Some of the fiber-like particles then may be used in making wound coverings, from which the growth factor could be delivered to the wound.
  • the fiber-like particles could be used as a support for the growth of cells.
  • the fiber-like particles could be inco ⁇ orated into grafts, such as arterial grans, by spinning with other fibers such as DacronTM or another material.
  • the fiber-like particles could include a pharmaceutical substance to enhance healing in the vicinity of the graft or the acceptance of the graft.
  • the fiber-like particles could be used in the manufacture of patches for delivery of a pharmaceutical substance, including patches for sublingual or buccal delivery of a pharmaceutical substance.
  • Particles of the present invention may also be used to enhance properties of immune system boosters to e ⁇ cit an immune system response.
  • a suspension of the ion-paired particles of the present invention could be used.
  • the particles of the present invention could be used in cements, to deliver a growth factor to help heal broken bones or teeth.
  • the invention further provides novel cationic surfactants having the formula:
  • P is a biocompatible hydrophobic moiety
  • C is a biocompatible cationic moiety
  • L is a biodegradable linkage linking P and C.
  • Biocompatible is used herein to mean that the hydrophobic or cationic moiety is naturally-occurring in, or is well-tolerated by, cells (including prokaryotic and eukaryotic cells) or an organism (including animals (e.g., humans) and plants).
  • a "biodegradable linkage” is one which is degraded by normal conditions or processes found in a cell or organism.
  • the biodegradable linkage of a cationic surfactant of the invention is degraded into two biocompatible components in a cell or organism to which the cationic surfactant is delivered.
  • the cationic surfactants of the invention are much less toxic than currently existing cationic surfactants.
  • P is preferably a saturated or u ⁇ saturated.
  • linear, branched or cyclic hydrocarbon e.g., cyciic alkyl, aryl, or combinations thereof
  • P which is an alkyl containing 10-20 carbon atoms.
  • P which comprises the steroid backbone, the steroid backbone preferably being substituted with C-L- at C3 and or containing at least one double bond, P most preferably being the cholesterol nucleus.
  • steroid backbone is meant the fused tetracyclic structure common to all steroids (shown Figure 26).
  • cholesterol nucleus is meant cholesterol without the hydroxyl group at C3 and being substituted at C3 with C-L-.
  • P may be substituted or unsubstituted.
  • the substituent may be any moiety that has at least some degree of hydrophobicity and is of low toxicity to cells or in vivo. Suitable substituents include alkyl, cyclic alkyl, aryl, alkyl esters, alkyl amines, alkyl ethers, etc. L is preferably an ester, carbonate, carbamate or ketal linkage.
  • C must be positively charged at pH 7.4 or less.
  • C preferably comprises a guanidinium group or one or more primary, secondary, tertiary or quaternary amines.
  • C may be an arginine, lysine, choline. ethanolamine, or ethylene diamine residue.
  • C is most preferably an arginine residue.
  • Particularly prefe ⁇ ed cationic surfactants are arginine esters having the following formula:
  • R which may be substituted or unsubstituted, is a saturated or unsaturated, linear, branched or cyclic hydrocarbon (e.g.. alkyl. cyclic alkyl. aryl, or combinations thereof) containing at least 8 carbon atoms. More preferably R, contains 8-40 carbon atoms, most preferably 10-30 carbon atoms. Presently preferred is a P which is an alkyl containing 10-20 carbon atoms or is the cholesterol nucleus. Suitable substituents are those listed above for P.
  • R may comprise one or more neutral amino acids.
  • R is H, one or more neutral or basic amino acids, including additional arginines. or a linear, branched or cyclic hydrocarbon (e.g...
  • alkyl cyclic alkyl, aryl, or combinations thereof containing at least 1, preferably 1-15. most preferably 2-10, carbon atoms and also, optionally, containing at least one amine group within the hydrocarbon, attached to the hydrocarbon (including at either end), or both.
  • Preferred amine groups are quaternary amines and guanidinium groups.
  • R, and R2 are preferably chosen so that they are not immunogenic.
  • R, or R 2 is a peptide. it will preferably comprise fewer than 6 amino acids. Methods of making peptides are. of course, well known (also see below). Suitable peptides can also be purchased commercially.
  • R may also be linked to the arginine residue through other biodegradable linkages.
  • prefe ⁇ ed linkages include ketal, carbonate and carbamate linkages.
  • the arginine esters of the invention may be synthesized by known methods of synthesizing arginine esters. See, e.g., Guglielmi et al..2. Physiol. Chem., 352, 1617-1630 (1971) and U.S. Patents Nos. 5,364,884 and 4,308,280, the complete disclosures of which are inco ⁇ orated herein by reference. These prior syntheses have been limited to short-chain alkyl and benzyl esters (six carbons or less), but the methods can be employed for synthesis of the arginine esters of the invention.
  • the arginine esters may be prepared by the reaction of R 2 -arginine with an alcohol, RIOH, in the presence of dry gaseous hydrogen chloride or using thionyl chloride (see Figures 20A-E). It has been found necessary to modify these syntheses by using sulfuric acid to catalyze the ester formation when more hydrophobic
  • Arginine esters of the invention can also be synthesized using the conditions described in Mitsunobu, Synthesis 1981, 1-28, with R : -arginine first being protected as in peptide synthetic methods and then deblocked after the formation of the ester (see Figures 20F-G).
  • Other possible methods include the use of protected arginine derivatives and dicyclohexylcarbodiimide as the coupling agent and the use of Lewis acids, such as BF 3 etherate.
  • cationic cholesterol surfactants having the following formula:
  • CHOL is the cholesterol nucleus.
  • L is an ester, carbamate, carbonate or ketal linkage.
  • R 3 is a linear, branched or cyclic hydrocarbon (e.g., alkyl, cyclic alkyl, aryl, or combinations thereof) containing at least 1 , preferably 1-15, most preferably 2- 10, carbon atoms and also containing at least one amine group within the hydrocarbon, attached to the hydrocarbon (including at either end), or both.
  • Prefe ⁇ ed amine groups are quaternary amines and guanidinium groups. Most preferred is an arginine residue (-CH(NH : )-CH -CH 2 -CH ; -NH- CCNH ⁇ NRT).
  • R may be substituted with neutral or other basic groups, including alkyls, aryls, amides, ester groups, and ether groups containing no more than 10 carbon atoms.
  • the cationic surfactants of the invention can be used for the same pu ⁇ oses as prior art cationic surfactants. However, due to their much lower toxicity compared to the prior art cationic surfactants, the cationic surfactants of the invention are especially useful in pharmaceutical preparations and in other situations where cell survival is important. In particular, they can be used as the amphiphilic material in the methods and compositions described above.
  • the cationic surfactants of the invention can be used to deliver negatively charged compounds, such as acidic proteins and nucleic acids, into cells. This is accomplished by simply contacting the cells with a cationic surfactant of the invention and a compound desired to be delivered into the cell.
  • the cells may be any type of eukaryotic or prokaryotic cell, but is preferably a mammalian cell, including human cells. The contacting may take place in vitro or in vivo.
  • the cationic surfactants are particularly suitable for trarisforming cells.
  • the cells may be transformed with any type of nucleic acid, including recombinant DNA molecules coding for a desired protein or polypeptide, recombinant DNA molecules coding for a desired antisense RNA or ribozyme, cloning vectors, expression vectors, viral vectors, plasmids, a transgene for producing transgenic animals or for gene therapy, antisense RNA, and ribozymes.
  • the cells may be any type of cell, but are preferably microorganisms (e.g., bacteria and yeast and other fungi) and animal (including human) cells (e.g., cell lines, pluripotent stem cells and fertilized embryos). The contacting may take place in vitro or in vivo.
  • the cell is contacted with a nucleic acid and a surfactant according to the invention.
  • the nucleic acid and surfactant are combined and incubated together before contacting them with the cell.
  • the time of incubation is that time sufficient to allow the nucleic acid and surfactant to complex. This time can be determined empirically. A time of about 45 minutes has been found to be sufficient for incubation of arginine dodecyl ester and a plasmid (see Example 39).
  • the cell is contacted with the nucleic acid and surfactant for a time sufficient to allow the nucleic acid to be delivered into at least some of the cells. This time can also be determined empirically.
  • a time of about 30 hours has been found to be sufficient when using the combination of arginine dodecyl ester and plasmid (see Example 39).
  • Other conditions for contacting the cell with the nucleic acid and surfactant are known in the art or may be determined empirically.
  • the cationic surfactants of the invention may be used alone to transform cells. Preferably, however, they are used in combination with helper lipids for transforming cells.
  • the lipids may be any of those lipids known in the art to be useful in transforming cells, including dioleoyl phosphatidyl ethanolamine (DOPE) and cholesterol.
  • DOPE dioleoyl phosphatidyl ethanolamine
  • the lipid should preferably promote fusion of the nucleic acid/surfactant/lipid complex with the membrane of the cell so that the nucleic acid may be transported into the interior of the cell.
  • the cell is contacted with a nucleic acid, a surfactant according to the invention and a lipid.
  • the nucleic acid, surfactant and lipid are combined and incubated together before contacting them with the cell.
  • the three may be combined simultaneously or sequentially (in any possible order of the three).
  • the time of incubation is that time sufficient to allow the nucleic acid, surfactant and lipid to complex. This time can be determined empirically.
  • the cell is contacted with the nucleic acid/surfactant/lipid for a time sufficient to allow the nucleic acid to be delivered into at least some of the cells. This time can also be determined empirically. Other conditions for contacting the cell with the nucleic acid, surfactant and lipid are known in the an or may be determined empirically.
  • the cationic surfactants of the invention may also be used, with or without helper lipids, in combination with other methods of transformation, such as electroporation. This may be particularly advantageous in transformation of plant cells.
  • the cells may be cultured to produce a desired protein, polypeptide or RNA.
  • the cells may be injected into an animal for gene therapy.
  • the cells may be allowed to grow and differentiate into a transgenic animal or plant.
  • the cationic surfactant or the lipid are preferably selected or modified so that they are targeted to selected cells to be transformed.
  • the nucleic acid surfactant combination could be inco ⁇ orated into liposomes composed of the lipids.
  • the liposomes could be targeted to particular cells by having an antibody specific for a molecule on the surface of the cells attached to the exterior of the liposomes.
  • the invention also provides a kit for delivering nucleic acids or other negatively charged compounds into cells.
  • This kit comprises a container of a cationic surfactant of the invention.
  • the kit may further comprise a container containing a nucleic acid, such as a cloning vector, expression vector or gene.
  • the kit may further comprise other reagents and materials normally used for transforming cells, such as restriction enzymes, lipids, polymerase chain reaction reagents, and buffers.
  • the antisolvent precipitation method of the present invention may be operated to produce compositions having particularly desirable characteristics for sustained release of a pharmaceutical substance.
  • the composition is characterized as including a pharmaceutical material, in the form of an HIP complex with an amphiphilic material, and a biocompatible polymer, with the biocompatible polymer being highly amo ⁇ hous.
  • the highly amo ⁇ hous character of the polymer is particularly desirable for reducing immune system responses and, therefore, reducing the likelihood of causing significant inflammation during use by a human or other mammalian patient.
  • the highly amo ⁇ hous sustained-release composition also provides a desirably stable sustained release profile for release of the pharmaceutical substance, and typically with little or negligible burst effect.
  • the highly amo ⁇ hous sustained-release composition is particularly well suited for delivery of a pharmaceutical substance for sustained release by pulmonary delivery, subcutaneous placement lntrape ⁇ toneal placement and intraocular placement.
  • the antisolvent process for making particles of the i-thh amo ⁇ hous sustamed-reiease composition, the particles and composition so made, product forms lnco ⁇ oraung the composition, and uses of the composition for administration to a mammalian patient for sustamed-reiease pu ⁇ oses are all within the scope of the present invention.
  • the sustained-release composition includes the biocompatible polymer in a highly amo ⁇ hous form.
  • highly amo ⁇ hous it is meant that the polymer is typically no more than about 25% crystalline. Most often, however, it will be desirable to keep the crystalline content of the biocompatible poiymer as low as possible.
  • the sustamed-reiease composition may be prepared typically with the biocompatible polymer being no more than about 20% crystalline, preferably no more than about 15% crystalline, more preferably no more than about 10% crystalline, and even more preferably no more than about 5% crystalline.
  • Particularly prefe ⁇ ed is for the biocompatible polymer to be substantially entirely amo ⁇ hous.
  • the crystalline content of the biocompatible polymer is as calculated based or. x-ray defraction results, a technique well known in the art.
  • Careful control of the antisolvent precipitation process is important for making the highly amo ⁇ hous sustained-release composition of the present mvention with the desirable features noted above.
  • the volumet ⁇ c ratio of the antisolvent fluid flow rate to the liquid feed flow rate should typically be larger than about 5, and more typically be m a range of from about 5 to about 100. Prefe ⁇ ed amo ⁇ hous characte ⁇ stics.
  • volumet ⁇ c ratio of antisolvent fluid flow rate to liquid feed flow rate of larger than about 15, more preferably larger than about 20, and even more preferably larger than about 25.
  • a particularly prefe ⁇ ed range for the volumet ⁇ c ratio of antisolvent fluid flow rate to liquid feed flow rate for preparing the highly amo ⁇ hous sustained-release composi ⁇ on is from about 10 to about 50, more parucularly from about 15 to about 40, even more particularly from about 15 to about 30, and most particularly from about 20 to about
  • the manner m which the antisolvent fluid and the liquid feed are contacted is also important. Although contacting may be in counterflow, it has been found that concurrent flow, or co- flow, of the liquid feed and the antisolvent fluid generally produces a superior product.
  • control of the relative concentrations of the biocompatible polymer, the pharmaceutical substance and the amphiphilic material is an important consideration when making the highly amo ⁇ hous sustained release composition.
  • the relative amounts in the liquid feed of the HIP complex, comprised of the pharmaceutical substance and the amphiphilic material, and the biocompatible polymer will depend upon the specific application.
  • the HIP complex may comprise about 0.5 weight percent or more of the total weight of the HIP complex and the biocompatible polymer, although amounts of greater than I weight percent are more common and greater than 5 weight percent are even more common.
  • the HIP complex should typically comprise at least about 15 weight percent preferably at least about 20 weight percent, more preferably at least about 25 weight percent, and even more preferably at least about 30 weight percent.
  • the HIP complex content will be no larger than about 70 weight percent, preferably no larger than about 60 weight percent and more preferably no larger than about 50 weight percent.
  • the highly amo ⁇ hous sustained-release composition will typically include a very high loading of the HIP complex material.
  • the highly amo ⁇ hous sustained-release composition will comprise the pharmaceutical substance and the amphiphilic material, in the form of a HIP complex, in an amount of at least about 15 weight percent preferably at least about 20 weight percent, more preferably at least about 25 weight percent and even more preferably at least about 30 weight percent.
  • the structural integrity of the composition may be compromised. Therefore, the HIP content will typically comprise no greater than about 70 weight percent of the composition, preferably no greater than about 60 weight percent of the composition and even more preferably no greater than about 50 weight percent of the composition.
  • the biocompatible polymer typically makes up the balance of the composition.
  • the antisolvent precipitation process of manufacture permits manufacture of the composition with the biocompatible polymer in a highly amo ⁇ hous state and with a heavy loading of the HIP complex material. Furthermore, 4
  • the HIP complex material is. nevertheless, typically substantially homogeneously dispersed throughout a matrix of the polymer, such that the HIP complex and the biocompatible polymer are in an intimate mixture on a microscopic level.
  • This intimate and homogeneous mixture is very important to achieving a stable release profile for release of the pharmaceutical substance from the sustained-release composition, as is the high level of loading of HIP complex material in the composition, as is discussed further below.
  • the mass content of the amphiphilic material in the HIP complex material is typically as large as or larger than the mass content of the pharmaceutical substance.
  • the mass ratio of the amphiphilic material to the pharmaceutical substance in the composition will typically be larger than about 1, and is often in a range of about 1 to about 5 with a range of from about 2 to about 6 being prefe ⁇ ed.
  • the pharmaceutical substance typically comprises greater than about 5 weight percent of the composition, preferably greater than about 10 weight percent of the composition, more preferably greater than about 15 weight percent of the composition, and even more preferably greater than about 20 weight percent of the composition.
  • the antisolvent fluid for making the highly amo ⁇ hous sustained-release composition will typically be carbon dioxide. It is prefe ⁇ ed, however, that contacting of the antisolvent fluid and the liquid feed occur at subcritical conditions.
  • the temperature is subcriticaJ with the reduced temperature, relative to the antisolvent fluid, preferably being in a range having a lower limit of about 0.75, more preferably about 0.85, even more preferably about 0.90, and most preferably about 0.95; and having an upper limit of about 0.95 and more preferably about 0.99 and even more preferably about 0.995.
  • the reduced pressure, relative to the antisolvent fluid is typically from about 0.5 to about 2.
  • the temperature during the antisolvent precipitation step is preferably in a range of from about 20°C to about 30°C, and more preferably from about 20°C to about 30°C.
  • the pha ⁇ naceutical substance may be any ionic pha ⁇ naceutical material and the amphiphilic material may be any compatible surfactant. Any of the pharmaceutical substances or amphiphilic materials described previously may be used. Prefened pharmaceutical substances include antibiotics, chemotherapeutic agents, and biologic agents. Prefe ⁇ ed surfactants are AOT and SDS, as well as the cationic materials discussed previously.
  • the solvent may be any suitable solvent but often includes at least one of methylene chloride or trichloromethane.
  • the biocompatible polymer may be any polymer that may be processed in the antisolvent precipitation process to form the composition such that the polymer is in a highly amo ⁇ hous state, as discussed previously. Most often, the biocompatible polymer is a biodegradable polymer, as discussed previously. When biodegradable, the polymer is most often hydrolytically degradable.
  • biocompatible polymer as it is commercially available, is frequently available only in a highly crystalline state. The polymer is, however, converted to a highly amo ⁇ hous state during the antisolvent precipitation process.
  • Prefe ⁇ ed polymers for the biocompatible polymer are poly(lactic acid) homopolymers, including poly(l-lactic acid) and poly(d-lactic acid), poly(glycolic acid) homopolymer, polyanhydrides, such as poly(sebacic acid), poly(carboxyphenoxyhexane), polybutyrates and cellulosic polymers such as polyhydroxypropyl ethylcellulose. Any suitable molecular weight polymer may be used that is soluble in the solvent used for the liquid feed. Typical molecular weights are from about
  • the biocompatible polymer could be a mixture of two or more different polymers or a copolymer of two or more different monomers.
  • the polymers are typically not prepared from the acids, but from the cyclic diesters, lactide or glycolide, as the case may be.
  • poly(lactic acid) and poly(glyco!ic acid) polymers are inclusive of polymers prepared directly by condensation polymerization of the acids or by ring-opening polymerization of the cyclic diesters.
  • the polymers made from ring-opening polymerization of lactide and glycolide are often refe ⁇ d to as polylactide and polyglycolide.
  • the highly amo ⁇ hous sustained- release composition will be in paniculate form.
  • the paniculate product may include particles of a variety of sizes and shapes. In that regard, any of the paniculate products having particle characteristics as previously described may be made in the form of the highly amo ⁇ hous sustained-release composition.
  • the paniculate product preferably includes ultrafine particles of a spheroidal shape and with greater than about 90 weight percent of the particles being smaller than about 10 microns, more preferably smaller than about 6 microns, and even more preferably of a size of from about 1 micron to about 6 microns.
  • the highly amo ⁇ hous sustained-release composition may be made in the form of the fiber-like particles, or in the form of extremely fine particles of a size smaller than about 1 micron.
  • the highly amo ⁇ hous. sustained-release composition may be inco ⁇ orated into a variety of product forms for use.
  • One product form is a macrostructure formed by agglomeration of particles of the paniculate product prepared by the antisolvent precipitation method.
  • the agglomeration is typically accomplished by compression.
  • cylinders, pellets, beads (e.g., spheroidal, ellipsoidal or other shape), discs and other macrostructures could be prepared from smaller particles.
  • Prefe ⁇ ed uses for such macrostructures are for subcutaneous and intraperitoneal surgical implantation.
  • the macrostructure will typically have a mass in a range with a lower limit of about 0.01 gram, about 0.05 gram, about 0.1 gram, about 0.5 gram or about 1 gram: and an upper limit of about 100 grams, about 50 grams, about 10 grams or about 1 gram. Any mass range having any one of the stated lower limits and any one of the stated upper limits is within the scope of the present invention, so long as the upper limit is larger than the lower limit. For example, for many applications, the macrostructure mass will be in a range of from about 0.05 to about 0.5 gram. To the extent that more of the pharmaceutical substance is desired than contained in a single macrostructure, then multiple macrostructures may be implanted together.
  • the macrostructure will typically have a mass in a range of from about 1 to about 10 grams, although a larger mass may be desirable at times. Also, depending upon the specific application, the macrostructure will typically occupy a volume within a range having a lower limit of about 0.01 cubic centimeter. 0.05 cubic centimeter, 0.1 cubic centimeter, 0.5 cubic centimeter or 1 cubic centimeter; and an upper limit of about 100 cubic centimeters, about 50 cubic centimeters, about 10 cubic centimeters or about 1 cubic centimeter. Any volume range having any one of the stated lower limits and any one of the stated upper limits is within the scope of the present invention, so long as the upper limit is larger than the lower limit.
  • Another product form is a suspension of particles of the highly amo ⁇ hous sustained- release composition in a liquid vehicle.
  • Prefe ⁇ ed uses for such suspensions are for placement by injection subcutaneously. intraperitoneally and intraocular ly .
  • Another preferred use for the liquid vehicle is for oral administration, for example, when uptake by gastrointestinal tissue is desired.
  • the particles of the highly amo ⁇ hous sustained-release composition should typically be smaller than about 50 microns, more preferably smaller than about 20 microns and most preferably smaller than about 10 microns. Particles of a size of from about 0.5 micron to about 10 microns are preferred for most injection applications.
  • the liquid vehicle for the suspension may be an aqueous liquid or an organic liquid.
  • the particles of the highly amo ⁇ hous sustained-release composition and the liquid vehicle are preferably mixed immediately before administration to a patient. Otherwise, significant release of the pharmaceutical substance into the aqueous liquid vehicle could occur prior to aciministration.
  • the particles and the liquid vehicle could be provided in a kit including the particles in one container and the aqueous liquid in a second container for easy mixing prior to use. For most applications, however, it will be desirable to use an organic liquid vehicle that is premixed with the particles and stored for later use, without significant release of the pharmaceutical substance during storage. Examples of prefe ⁇ ed organic liquid vehicles are ethanol and propylene glycol.
  • a powder of the highly amo ⁇ hous sustained-release composition could be packaged in a nebulizer or other aerosol-producing device for pulmonary delivery applications. Particle sizes for pulmonary delivery are preferably as discussed previously.
  • the product forms of macrostructures, liquid suspensions and for aerosol generation are prefe ⁇ ed even for compositions in which the biocompatible polymer may not have the desired highly amo ⁇ hous character.
  • a major advantage of the highly amo ⁇ hous sustained-release composition of the present invention is that because of the highly amo ⁇ hous character of the biocompatible polymer, the composition is less likely to cause an immune response when administered to a patient. More highly crystalline materials are more likely to cause an immune response and, therefore, accompanying inflammation.
  • the highly amo ⁇ hous sustained-release composition of the present invention exhibits very favorable release characteristics for sustained release of the pharmaceutical substance. This desirable result is believed to be related to the highly amo ⁇ hous character of the composition and to the heavy loading of the composition with the HIP complex material.
  • the highly amo ⁇ hous sustained-release composition of the present invention when immersed in a phosphate buffer solution at a temperature of about 37°C, typically exhibits a release profile for release of the pharmaceutical substance that plots as a single substantially straight line when plotted as cumulative pharmaceutical substance released versus the square root of time.
  • the highly amo ⁇ hous sustained-release composition therefore, exhibits only a single diffusion- controlled stage of release, and does not exhibit the occurrence of multiple diffusion- controlled stages of release, as has been reported for other amo ⁇ hous compositions due to crystallization of polymer during use. Even though the release profile of the composition of the present invention exhibits substantially only a single stage of diffusion-controlled release, it should be recognized that anomalies in the release profile may be expected to occur at the very beginning of release. Also, during later stages of release, degradation of the polymer, in the case of a biodegradable polymer, will affect the release profile.
  • the highly amo ⁇ hous sustained-release composition of the present invention typically greater than about 70 percent preferably greater than about 80 percent and more preferably greater than about 90 percent of the pharmaceutical substance is released during the aforementioned single diffusion-controlled stage of release when immersed in the phosphate buffer solution at the noted temperature.
  • the phosphate buffer solution for comparison pu ⁇ oses, should typically be an aqueous solution including about 0.9 gram per liter of sodium chloride, about
  • Yet another significant advantage of the highly amo ⁇ hous sustained-release composition to the present invention is that it typically exhibits little, if any, significant burst effect during the initial stages or release of the pharmaceutical substance.
  • the highly amo ⁇ hous sustained-release composition when immersed in the phosphate buffer solution, described previously, at a temperature of about 37°C, typically no more than about 15% of the pharmaceutical substance is released during the first 24 hours following immersion, preferably no more than about 10% and even more preferably no more than about 5%.
  • the highly amo ⁇ hous sustained- release composition will typically exhibit this low burst effect as manufactured by the antisolvent precipitation process.
  • compositions may be subjected to a post-manufacture wash with a buffer solution to remove pharmaceutical substance that may be adhering to the outside surface of particles.
  • a wash may be accomplished, for example, by sonication for a short duration in a bath of buffer solution. Other methods for washing the particles are also possible.
  • the noted advantages of the highly amo ⁇ hous sustained-release composition concerning pharmaceutical release characteristics are believed to be attributable, at least in part, to the highly amo ⁇ hous character of the composition and to the heavy loading of the composition with the HIP complex.
  • the amo ⁇ hous character of the composi ⁇ on is desirable because sustained-release composition made with highly crystalline polymers often have pharmaceutical release rates that are too slow.
  • the amo ⁇ hous character of the sustained-reiease composition of the present invention appears to be retained for at least a significant time during release.
  • the initial formation of the highly amo ⁇ hous biocompatible polymer in the composition and the apparent maintenance of a highly amo ⁇ hous character during use are believed to be due. at least in pan. to heavy loading with the HIP complex material.
  • both the HIP complex material and the biocompatible polymer both typically have a high hydrophobicity, they typically form a homogeneous and intimate mixture which may retard crystallization of the polymer during use.
  • Still a further significant advantage of the highly amo ⁇ hous sustained-release composition of the present invention is that it typically includes only very small quantities of residual organic solvent.
  • residual organic solvent levels, as manufactured are typically less than about 50 parts per million by weight, preferably less than about 25 parts per million by weight and even more preferably less than about 10 pans per million by weight. Frequently, residual organic solvent levels are less than about 3 pans per million by weight.
  • the antisumble precipitation process is preferably conducted such that after the particles have been precipitated in a reactor vessel, they are retained in the reactor vessel and flushed with a volume of substantially pure antisolvent fluid for a sufficient time to reduce the residual solvent content to the desired level.
  • a post-precipitation flush with a volume of substantially pure antisolvent fluid should preferably include at lease about one reactor volume of antisolvent fluid, with a flush of at least about two times the reactor volume being more prefe ⁇ ed.
  • One particularly prefe ⁇ ed embodiment for the highly amo ⁇ hous sustained-release composition of the present invention includes poly(l-lactic acid) as the biocompatible polymer.
  • the prefe ⁇ ed solvent is either methylene chloride or trichloromethane, although ethyl acetate may also be used if the poly
  • (1-lactic acid) has been suitably end-capped or otherwise modified for enhanced solubility.
  • the pharmaceutical substance may be any of the pha ⁇ naceutical substances previously listed.
  • the pharmaceutical substance in the highly amo ⁇ hous sustained-release composition is isoniazid, which is preferably in the form of an ion pair with AOT.
  • This composition is particularly prefe ⁇ ed for treating tuberculosis.
  • the prefe ⁇ ed biocompatible polymer is poly(l-lactic acid).
  • the prefe ⁇ ed method for administering the highly amo ⁇ hous sustained-release composition with isoniazid is via subcutaneous placement typically by implantation of a macrostructure, as previously described, comprised of agglomerated panicles made by the antisolvent precipitation process.
  • the antisolvent precipitation process may be used to make particles including a highly amo ⁇ hous biocompatible polymer, even in the absence of a HIP complex material.
  • a pharmaceutical substance that is not in an HIP complex form could be suspended in or, for a few pharmaceuticals, co-dissolved in a solvent containing the polymer.
  • particles made by the antisolvent precipitation process could be of substantially pure biocompatible polymer.
  • the prefe ⁇ ed processing conditions with respect to flow rates, conditions for contacting the solvent and antisolvent pressure, temperature, antisolvent fluids, solvents and biocompatible polymers are as previously described for the highly amo ⁇ hous sustained-release composition including an HIP material.
  • compositions without the HIP complex material are no; preferred because the presence of the HIP complex material, as previously discussed, significantly enhances the properties of the composition for use in sustained-release applications.
  • the biocompatible polymer in the particles typically preferably is not more than about 25% crystalline, preferably not more than about 20 weight percent more preferably not more than about 15% crystalline, even more preferably not more than about 10% crystalline, and most preferably not more than about 5% crystalline.
  • Example 1 The methods used for measuring apparent partitioning coefficients are described in Example 1.
  • the measurement of the behavior of the Gly-Phe-NH 2 :SDS complex is described in Example 2.
  • the behavior of the 8-Argvasopressin:SDS complex, leuprolide:SDS complex. neurotensin:SDS complex, and bradykinin:SDS complex are described in Example 3.
  • the behavior of the insulin:SDS complex is described in Example 4.
  • Example 7 describes the CD s'-c.tr m :f t i ir.sulin:SDS complex.
  • Example 8 describes the thermal stability of the insulin:SDS complex.
  • Example 9 describes the behavior of other large proteins with SDS, specifically, human growth hormone.
  • the behavior of bovine pancreatic trypsin inhibitor with SDS is described in Example 10
  • Example 11 describes the behavior of human serum albumin with SDS.
  • the melting point of the SDS:insulin HIP complex was studied (Example 12).
  • Example 13 describes a method for forming a fine HIP complex suspension suitable for pulmonary delivery.
  • Example 14 describes a method for achieving uniform distribution of a protein throughout a hydrophobic polymer suitable for use as an injectable implant.
  • Example 15 describes the use of the HIP complex for improved storage of proteins.
  • the use of protein precipitation in the HIP complex for protein purification is described in Example 16.
  • a method of administering a protein dissolved as an HIP complex in organic solvent is described in Example 17.
  • Example 18 describes the preparation of a drug with reduced bitter taste.
  • Examples 19-29 demonstrate batch preparation of particles using gas antisolvent precipitate.
  • Examples 30-32 demonstrate continuous preparation of particles using gas antisolvent precipitation.
  • Examples 33-40 describe the preparation, characterization and use of cationic surfactants of the invention.
  • the relative solubilities in two phases is given in terms of an apparent partition coefficient.
  • the apparent partition coefficient is defined as the ratio of the equilibrium concentration in an organic phase to that in an aqueous phase.
  • the actual value of the apparent partition coefficient, P is dependent on the two solvent systems employed. In all cases herein described, the organic phase is 1 -octanol and the aqueous phase is water alone or with a minimal amount of HC1 added.
  • Results are described as logarithms of the apparent partition coefficient.
  • a log P value of 0 means that the compound is equally soluble in water and the organic phase.
  • a positive log P value means the peptide is more soluble in the organic phase than in water and a negative log P values indicate a greater aqueous solubility than in the organic solvent All of the log P values reported herein have been co ⁇ ected for slight changes in solubility with pH.
  • the polypeptide In order for HIP to occur, the polypeptide must contain at least one basic group (either a lysine or arginine side chain or a free N-terminal amino group).
  • Gly-Phe-NH contains a single basic group, and at pH 7 forms a 1:1 complex with SDS. The complex precipitates from aqueous solution, but readily partitions into 1 -octanol. as shown in Fig. 1.
  • Fig. 1 For Gly-Phe itself, which exists in a zwitterionic form at neutral pH. a complex with SDS is formed with difficulty, and little enhancement of the partition coefficient is observed.
  • AVP is a nonapeptide hormone which controls water and salt elimination in the body. It contains two basic groups, the N-terminal amino group and the guanidinium side chain of Arg 8 , and no acidic groups. Stoichiometric addition of SDS produces a precipitate from an aqueous solution (pH 7) which readily partitions into a 1-octanol (Fig.2). At a mole ratio of 2:1 (SDS:peptide). the solubility in 1-octanol actually exceeds the solubility in water by more than tenfold (i.e., log P > 1). Overall, the apparent partition coefficient for AVP was increased by nearly four orders of magnitude.
  • SDS a nonapeptide hormone which controls water and salt elimination in the body. It contains two basic groups, the N-terminal amino group and the guanidinium side chain of Arg 8 , and no acidic groups. Stoichiometric addition of SDS produces a precipitate from an aqueous solution (pH 7) which readily partitions
  • Polypeptides which contain both acidic and basic groups can also form hydrophobic ion pairs.
  • Insulin contains six basic groups (one Arg, one Lys, two His, and two F-terminal amino groups) and four acidic groups. By lowering the pH to 2.5, all of the acidic groups
  • Example 5 Dissolution of Insulin-SDS complex as a Function of the Organic Solvent Dissolution of insulin-SDS complexes in other solvents was investigated as well (Table i). Precipitates of SDS-insulin complexes were isolated and added to various organic solvents. Some degree of polarity appears to be necessary to obtain measurable solubility in the organic phase, as partitioning into chlorocarbons (CH 2 C1 2 1-chlorooctane, and CC1 4 ) and alkanes (mineral oil, hexane) could not be detected using UV-visible abso ⁇ tion spectroscopy. Besides alcohols. SDS-insulin complexes are soluble in N-methylpy ⁇ olidone (NMP), trimethylphosphate (TMP). polyethylene glycol, ethanol, and t-butanol.
  • NMP N-methylpy ⁇ olidone
  • TMP trimethylphosphate
  • Leuprolide acetate is a luteinizing hormone releasing hormone (LHRH) agonist used in the treatment of endometriosis. It contains 9 amino acid residues and two basic functionalities (a histidine and an arginine group). Both termini are blocked.
  • Stoichiometric amounts of SDS were added to an aqueous solution of leuprolide (0 and 0.5 mg ml, pH 6.0), resulting in formation of a precipitate.
  • the apparent partition coefficient of the SDS-leuproiid complex (Fig. 2) exhibited a log P into 1 -octanol greater than 1.0.
  • Example 7 CD Spectrometrv of the SDS-Insulin Complex. Two important considerations for proteins dissolved in non-aqueous solvents are whether native structures are retained and whether the material can be extracted back into an aqueous phase.
  • the secondary composition of a 6:1 SDS-insulin complex dissolved in neat 1-octanol at 5°C is shown in Fig. 3. The insulin concentration was 61 ug ml.
  • CD spectra were recorded on an Aviv 62DS spectrophotometer equipped with a thermoelectric temperature unit. All temperatures were measured ⁇ 0.2° C. Samples were placed in strain-free quartz cells (pathlength of 1 mm) and spectra obtained taking data every 0.25 run using a three second averaging time, and having a spectral bandwidth of 1 nm.
  • 222 nm band to the 208 nm band is similar to that observed for insulin at high concentrations (Pocker and Biswas (1980) supra). This represent the first example of native-like structure in a protein dissolved in a neat organic solvent.
  • Fig.4 shows the far ultraviolet CD spectrum of insulin extracted from 1-octanol into an aqueous solution of 0.10 M HCl.
  • the pathlength was 1 mm, the sample concentration 53 ug/ml. and the sample temperature 5°C.
  • insulin can be extracted back into the aqueous phase, presumably due to replacement of the SDS counterion with chloride.
  • Lower HCl concentrations did not affect extraction of insulin from 1 -octanol.
  • Examination of the CD spectrum of the redissolved material (Fig. 4) indicates an overall structure similar to that of native insulin.
  • Example 9 Behav : or of Lary? P-oteins Com iled v ; th SDS.
  • hGH redissolves, presumably via micellar solubilization.
  • the hGH precipitate was not found to be soluble in 1-octanol, as determined by spectrophotometric assay, however, it was easily suspended in water and various oils, such as olive oil.
  • Bovine pancreatic trypsin inhibitor is a small basic protein (MW 5900) with a well defined and stable structure (Wlodawer ⁇ laL (1984) J. Mol. Biol. 180:301-329, and (1987) J. Mol. Biol. 122:145-156).
  • the melting point (MP of SDSinsuiin ion pairs in 1-octanol was studied at SDS:insulin ratio ranging from 1 : 1 to 1 :24.
  • Insulin at 1 mg ml in 0.005 N HCl was prepared containing SDS at 1, 2, 3, 4. 5, 6, 7, 8, 9, 12, 15, 18, 21 and 24 moles of SDS per mole of insulin. Equal volumes of octanol were added to each SDS:insulin solution to partition the insulin into the octanol phase. The concentration of the SDSinsuiin complex extracted into the octanol was estimated by its absorbance at 278 nm and the solution diluted to 200 ug/ml. The melting point of the various insulin in octanol solutions was then determined with an AVIV 62DS circular dichroism spectrometer.
  • Fig. 9 shows the graph of melting point as a function of SDSinsuiin molar ratios, with an apparent maximum at 6:1 molar ratio and a melting point of about 116°C.
  • the molar ratio of 6:1 is also the stoichiometric ratio and show the highest thermal stability for insulin in octanol.
  • Fig. 10 shows a typical CD scan at 222 nm as a function of temperature. A melting point of 106° C was determined by the maxima of the first derivative of the pictured data.
  • Fig. 11 shows a typical absorbance scan at 222 nm as a function of temperature and effectively mimics the CD scan, showing a melting point of 106°C.
  • Example 13 Formation of a Fine Suspension HIP Complex for Pulmonary Delivery.
  • a protein solution is stirred vigorously using a homogenizer.
  • SDS is added dropwise to the agitated solution.
  • Particles in the 2-10 micron range are obtained. These particles are separated from the mixture by centrifugation or filtration.
  • the particles are then suspended in a mixture of Freon* 11 and 12, such than when placed in a meter dose inhaler, a therapeutic amount of protein is delivered on each actuation.
  • Example 14 Uniform Distribution of Protein Throughout a Hydrophobic Polvmer for Use in an Iniectable Implant.
  • the biodegradable polymer consisting of a 50:50 mixture of poly-lactic acid and poly- glycolic acid is dissolved in a volatile organic solvent such as N-methyl-py ⁇ oiidone (NMP).
  • NMP N-methyl-py ⁇ oiidone
  • An appropriate amount of an HIP-protein complex such as insulin-SDS (0.5%-5.0% by weight relative to the polymer) is dissolved in the same solvent.
  • the two solutions are mixed and sti ⁇ ed for one hour.
  • the solvent is removed by evaporation. This is done in a mold to form an implant or by a spray drying procedure to form small uniform particles for injection.
  • the resulting solid material can also be ground to a powder and formulated as an injectable suspension.
  • the protein is released from these systems as the polymer biodegrades and the HIP complex hydrolyses.
  • the HIP complex is formed by dissolving the protein or polypeptide in water at minimal ionic strength.
  • the pH is adjusted to as low a pH value as is practical to ensure stability and activity.
  • a stock solution of SDS is added so that the number of equivalents of
  • SDS matches the number of basic groups.
  • the pH is adjusted to 2.5, and 6 molar equivalents of SDS are used per mole of insulin.
  • the resulting complex precipitates from solution, is collected, and dried at room temperature.
  • the solid HIP complex may be stored at higher humidities and temperatures than the native proteins without noticeable loss of activity.
  • Dissolution in a non-reactive organic solvent such as 1-octanol produces a true solution of a protein.
  • the HIP complex of insulin stored in 1-octanol is much more stable than insulin in water, as shown by its enhanced thermal stability.
  • Example 16 Use of HIP Complex Formation for Protein Purification.
  • the hydrophobicities of HIP complexes of proteins will differ according to the fraction of the protein ' s surface covered by the alkyl sulfate molecules.
  • the HIP protein complexes are separated using a variety of methods, including hydrophobic interaction columns.
  • proteins may be purified by selective precipitation out of solution.
  • a protein is separated from additives such as human serum albumin (HSA), which may be present in amounts 20-50 times greater than the protein. Since HSA does not precipitate out of solution at pH 5.0 with SDS, a basic protein may be selectively precipitated and purified from HSA under those conditions.
  • HSA human serum albumin
  • Example 17 Use of HIP Complex Dissolved in an Organic Solution for Administration of a Protein to a Patient.
  • HIP complexes administered to a patient may be accomplished in a number of ways.
  • a biodegradable polymer/HIP complex system may be dissolved in an organic solvent for example N-methyl py ⁇ olidone, and injected subcutaneously to form an implant, processed to form microspheres which can be injected subcutaneously or intramuscularly, processed to form an implant which is placed surgically under the skin, or given orally as pan of an oral delivery system for peptides and proteins.
  • the solid HIP complex may also be prepared as a suspension or a non-aqueous solution, which may be injected or placed on the skin where the complex may partition into the skin.
  • the HIP complex may also be nebulized and administered to a patient via inhalation, for pulmonary drug delivery.
  • the HIP complex may also be formulated to be given orally, such that it is protected from degradation in the stomach via an enterically coated capsule, and released in either the upper or lower intestinal tract
  • the HIP complex may be loaded alone or in conjunction with oils, bile salts, or other enhancers to increase abso ⁇ tion.
  • the HIP complex may also be suspended or dissolved in oil and introduced to the patient as a rectal or vaginal suppository.
  • Example 18 Preparation of a Drug With Reduced Bitter Taste.
  • the low solubility of the HIP complex results in diminished taste of bitter tasting drugs taken orally.
  • the HIP complex may also be dissolved in oil so as to further reduce bitter taste.
  • Examples 19-29 demonstrate batch manufacture of particles having a pharmaceutical substance and an amphiphilic material using supercritical carbon dioxide as a gas antisolvent.
  • Fig. 13 shows a process flow diagram for the batch processing of Examples 19-29.
  • the test solution 136 for each example has a pha ⁇ naceutical substance and an amphiphilic material dissolved together as a hydrophobic ion pair complex in an organic solvent. Some examples have a biodegradable polymer also dissolved in the organic solvent. Solid particles which precipitate are allowed to settle, with all valves closed, onto a scanning electron microscope (SEM) stub in the antisolvent chamber 124. The antisolvent chamber 124 is then slowly depressurized through the valve 130 and the SEM stub is removed for analysis. Any remaining solid particles from the antisolvent chamber 124 are collected on the filter 144.
  • SEM scanning electron microscope
  • Figs. 14 and 15 are SEM photomicrographs of imipramine particles of Example 22, showing the elongated fiber-like shape of the particles. In Fig. 15 it may be seen that the fiber-like particle has a hollow interior in which small particles of another pharmaceutical substance could be loaded for some pharmaceutical applications.
  • Fig 16. is a SEM photomicrograph of a particle of ribonuclease and poly (ethylene glycol) of Example 27, showing an opening in the end of the particle into a hollow interior space.
  • Fig. 17 is a SEM photomicrograph of particles of ⁇ -chymotrypsin of Example 19, showing ultrafine spheroidal particles of a size smaller than about 10 microns, with many of a size of around 1 micron.
  • Fig. 18 is a SEM photomicrograph of pentamidine particles of Example 29 of a size smaller than about 1 micron.
  • Examples 30-32 show continuous manufacture of solid particles comprising a pha ⁇ naceutical substance and an amphiphilic material.
  • Fig. 19 shows a process flow diagram for the continuous manufacture test for Examples 30-32.
  • the antisolvent chamber 124 is first pressurized with an automatic syringe, pump 126 with a back pressure regulator 146 adjusted maintain the desired antisolvent pressure in the antisolvent chamber 124 at a given antisolvent flow rate through the system. This initial pressurization is performed with the valve 148, the valve 134 and the valve 130 closed and with the valve 150 and the valve 132 open.
  • One of two methods for metering the solution 136 into the antisolvent chamber 124 is used for each example.
  • One method is to load the pump 152 with pure solvent and to spray the pure solvent into the antisolvent chamber 124 until a steady state is achieved.
  • the solution 11.6 is then loaded into the injection port 138 and spiked into the solvent delivery line 154 to the antisolvent chamber 124.
  • the second method is to load the pump 152 with the solution and, bypassing the injection port, to deliver the solution to the antisolvent chamber 124.
  • Both delivery techniques are operated at a flow rate of 1 milliliter per minute with a carbon dioxide flow rate of 20 milliliters per minute.
  • the solution enters the antisolvent chamber 124 through the sonicated orifice 142.
  • carbon dioxide is vented from the top of the antisolvent chamber to allow particles to settle and not be entrained in the exiting carbon dioxide. Any particles that are washed out of the antisolvent chamber 124 are collected on the filter 144.
  • valves 150 and 130 are closed and valves 134 and 148 are opened and carbon dioxide is metered into the antisolvent chamber 124 from bottom to top to flush any residual solvent from the antisolvent chamber 124.
  • the system is then slowly depressurized and particles which have precipitated are collected from either the antisolvent chamber 124 or the filter 144.
  • Table 4 shows the makeup of the solution for each of Examples 30-32 and results of the examples, including a description of particles which are produced.
  • This example describes the synthesis of arginine octyl ester. This ester was synthesized by the in situ generation of the acid chloride of arginine, followed by direct esterification with the appropriate alcohol (see Figure 20A).
  • the powder was found to be insoluble in a variety of organics, including alcohols, hydrocarbons, aromatics, DMF and pyridine.
  • the powder was also insoluble in water, and would only dissolve in 0.1 N or stronger HCl.
  • TLC Assay A M (Sigma) showed distinct differences in mobility for substrate and product (the product traveled with the solvent front).
  • product and substrate were dissolved in 0.1 N HCl at 1 mg ml, and the product and substrate solutions were then spatted onto a Selecto silica gel TLC plate which was placed in a vapor-saturated vessel containing 60% isopropanol. 15% methyl ethyl ketone, and 25% 1 N HCl.
  • the chromatograms were developed with ninhydrin.
  • the molecular structure of the product was verified by NMR and fast atom bombardment (FAB) mass spectrometry to be arginine octyl ester dihydrochloride.
  • the melting point was 155°C.
  • the yield was approximately 100%.
  • Example 35 Synthesis Of Arginine Dodecvl Ester. This ester was synthesized using approximately the same procedure as described in
  • Example 33 for the octyl ester 1-Dodecanol (Aldrich) was used in place of the 1-octanol.
  • the substrate did not disappear as in the octyl synthesis. As the mixture was heated to approximately 80°C, the substrate began to clump together. Additional rounds of thionyl chloride addition did not change the appearance of the clumped substrate. TLC of the supernatant showed some product. Five volumes of diethyl ether caused some opaque precipitate to form, but it did not coagulate as in the octyl synthesis. Attempts using Whatman filter paper to filter out the precipitate by both gravity and Buchner filtration were unsuccessful, so the precipitate was collected by centrifugation. The resulting pellet had a gummy appearance like the octyl product.
  • Example 37 Synthesis Of A Cholesterol Carbonate N,N-dimethyl ethanolamine (Aldrich; 0.24 ml.2.44 mmol) was added dropwise over the course of 30 minutes at room temperature to a sti ⁇ ed solution of cholesterol chloroformate (Aldrich; 1.0 g. 2.2 mmol) in dichloromethane (Fisher; 30 ml). The resulting white suspension was stined at room temperature for 10 minutes, at which time TLC (20: 1 hexanes:ethyl acetate) showed the reaction to be complete. Saturated sodium bicarbonate solution ( 10 ml) was added to the suspension, at which point a clear solution resulted.
  • CC-CHOL has the following formula:
  • Stock solutions of the arginine esters were made by first dissolving the powder in 0.1 N HCl to give a 10 mM solution and then raising the pH to a value between 5 and 6. The pH should not be raised above 8.
  • arginine octyl ester is a relatively poor detergent with a critical micelle concentration (cmc) of about 6 mM (2.2 mg/ml) (see Figure 22A).
  • cmc critical micelle concentration
  • the dodecyl ester is a much better surfactant with a cmc of approximately 0.3 mM (0.10 mg/ml)
  • C. Cvtotoxicitv The cytotoxicity of arginine dodecyl ester was investigated in cell culture with two types of cells (see Cory et al., Cancer Commun., 3, 207-212 (1991)): CCRF-CEM cells, a human T-cell leukemia cell line that grows in suspension (obtained from the American Type Culture Collection, ATCC); and a green monkey kidney cell line (COS-7) that grows in monolayers (also obtained from ATCC). For comparison, the cells were also exposed to tetradecyltrimethylammonium bromide (DTAB) (Sigma).
  • DTAB tetradecyltrimethylammonium bromide
  • Cells were plated into 96-well plates (Coming) in a total of 200 ⁇ L Dulbecco's modified minimal essential medium for COS-7 cells, RPMI 1640 for CEM cells, supplemented with penicillin G (50 U/ml). streptomycin sulfate (50 ⁇ g/ml), and 10% fetal calf serum, at 10,000 cells/well for COS-7 amd 50,000 cells/well for CEM cells. The plates were incubated at 37°C for 24 hours after plating. The cells were then exposed to various concentrations of the detergents. Each detergent concentration was used in 8 replicate wells. After 2-6 hours, media/detergent solutions were aspirated, and the wells were washed twice with PBS.
  • CEM suspension cells centrifugation of the suspension at 1000 x g for 5 min between each wash was required. After washing, 200 ⁇ L of fresh medium were added, and the cells were incubated for 72 hours. After 72 hours, cell proliferation was determined using the Promega CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay. To do so, cells were exposed to MTS substrate (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium for 3 hours. Cellular respiration was assessed by monitoring the appearance of a soluble formazan reduction product by spectrophotometry at 490 nm.
  • Absorbance was read using a Molecular Devices spectrophotometric plate reader. Absorbance was directly proportional to the number of living cells in each well. Survival was plotted versus detergent concentration, with the untreated control group representing 100% survival. Detergent concentrations producing half-maximal growth inhibition (IC 50 values) were extrapolated from the resulting curves.
  • the plasmid used was pRSV4001uc. It was obtained from Dr. David Gordon. Div. Endocrinology, University' of Colorado School of Medicine, Denver, CO. It was propagated in Escherichia coli strain DH5a (ATCC), isolated by a standard alkaline-SDS lysis procedure, and purified twice by isopycnic centrifugation on CsCl gradients (Sambrook et al., Molecular
  • Intensity of luminescence should be proportional to the amount of expressed luciferase and, therefore, the efficiency of transfection "Background” is the reading from just the substrate mixture on the luminometer before addition of cell lysate. Average background is approximately 50 units. Any reading over 100 units is considered significant
  • CC-CHOL was tested for cytotoxicity as desc ⁇ bed in Example 38 using COS-7 and JEG-3 cells.
  • JEG-3 cells are a human cho ⁇ ocarcinoma cell lme available from ATCC.
  • the culture medium was Eagle's minimum essenual medium containing 10% serum.
  • va ⁇ ous embodiments of the present invention have been described in detail, it should be understood that any feature of any embodiment may be combined with any other feature of any other embodiment.
  • Any compatible combmauon of pharmaceutical substance, amphiphilic mate ⁇ al, polymer and/or solvent may be used
  • any feature of any processing method may be used with any solvent.
  • the hollow, fiber-like particles may be prepared for any suitable combmauon of pharmaceuucal substance and amphiphilic mate ⁇ al
  • the fiber-like panicles may be made of a biocompatible polymer, alone or in combmation with other mate ⁇ als. or a pharmaceutical substance, alone or in combination with other materials, which are directly soluble in the organic solvent Such features are expressly included within the scope of the present invention.

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Abstract

La présente invention concerne une composition permettant la libération prolongée d'une substance pharmaceutique. Cette composition contient un polymère biocompatible hautement amorphe dans un complexe d'ions hydrophobe associé à un matériau amphiphile. L'invention concerne également un procédé utilisant un antisolvant comprimé pour fabriquer la composition, différentes formes de produit incorporant la composition, et diverses utilisations convenant à la composition.
EP99912785A 1998-03-18 1999-03-18 Composition a liberation prolongee contenant un polymre amorphe Withdrawn EP1073419A4 (fr)

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US78390P 1998-03-18
PCT/US1999/006198 WO1999047543A2 (fr) 1998-03-18 1999-03-18 Composition a liberation prolongee contenant un polymre amorphe

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FR2815540B1 (fr) † 2000-10-19 2005-06-10 Separex Sa Procede de fabrication de tres fines particules constituees d'un principe insere dans une molecule hote
NZ530315A (en) * 2001-07-10 2007-01-26 Corixa Corp Compositions and methods for delivery of proteins and adjuvants encapsulated in microspheres
GB0216780D0 (en) * 2002-07-19 2002-08-28 Bradford Particle Design Ltd Methods of particle formation
EP1452177A1 (fr) * 2003-02-27 2004-09-01 Boehringer Ingelheim International GmbH Compositions pharmaceutiques contenant du laurylsulfate de sodium comme agent qui masque l'amertume
JP2007516259A (ja) * 2003-12-09 2007-06-21 メッドクリスタルフォームズ、エルエルシー 活性剤との混合相共結晶の調製方法
US8399007B2 (en) 2006-12-05 2013-03-19 Landec Corporation Method for formulating a controlled-release pharmaceutical formulation
WO2008070118A1 (fr) 2006-12-05 2008-06-12 Landec Corporation Administration de médicaments
US8114883B2 (en) 2007-12-04 2012-02-14 Landec Corporation Polymer formulations for delivery of bioactive materials
WO2009100222A1 (fr) 2008-02-08 2009-08-13 Qps Llc Compositions non polymères pour l'administration contrôlée de médicament
US20160175313A1 (en) * 2013-08-06 2016-06-23 Dongkook Pharmaceutical Co., Ltd., Entecavir microspheres and pharmaceutical composition for parenteral administration containing same
AU2015316252A1 (en) * 2014-09-10 2017-03-09 Double Bond Pharmaceuticals Ab Targeted delivery of hydrophilic drugs
WO2017153052A1 (fr) * 2016-03-07 2017-09-14 Double Bond Pharmaceuticals Ab Administration ciblée de médicaments hydrophiles dans les poumons

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WO1996029998A1 (fr) * 1995-03-28 1996-10-03 Fidia Advanced Biopolymers S.R.L. Nanospheres contenant un polysaccharidique biocompatible
WO1997038698A1 (fr) * 1996-04-18 1997-10-23 University Technology Corporation Procedes pour le traitement de troubles de l'oreille interne et moyenne

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US4582820A (en) * 1982-12-23 1986-04-15 Research Corporation Orally administered biologically active peptides and proteins
ATE37983T1 (de) * 1982-04-22 1988-11-15 Ici Plc Mittel mit verzoegerter freigabe.
CH683149A5 (fr) * 1991-07-22 1994-01-31 Debio Rech Pharma Sa Procédé pour la préparation de microsphères en matériau polymère biodégradable.
WO1994008599A1 (fr) * 1992-10-14 1994-04-28 The Regents Of The University Of Colorado Appariement d'ions de medicaments pour ameliorer l'efficacite et l'administration

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Publication number Priority date Publication date Assignee Title
WO1996029998A1 (fr) * 1995-03-28 1996-10-03 Fidia Advanced Biopolymers S.R.L. Nanospheres contenant un polysaccharidique biocompatible
WO1997038698A1 (fr) * 1996-04-18 1997-10-23 University Technology Corporation Procedes pour le traitement de troubles de l'oreille interne et moyenne

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JP2002506876A (ja) 2002-03-05
AU3108299A (en) 1999-10-11
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