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WO1997018840A2 - Liberation d'agents therapeutiques au moyen de composes auto-assembles pour former des microstructures a rapport axial eleve_ - Google Patents

Liberation d'agents therapeutiques au moyen de composes auto-assembles pour former des microstructures a rapport axial eleve_ Download PDF

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Publication number
WO1997018840A2
WO1997018840A2 PCT/US1996/018850 US9618850W WO9718840A2 WO 1997018840 A2 WO1997018840 A2 WO 1997018840A2 US 9618850 W US9618850 W US 9618850W WO 9718840 A2 WO9718840 A2 WO 9718840A2
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WIPO (PCT)
Prior art keywords
therapeutic
compounds
har
microstructures
harfm
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PCT/US1996/018850
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English (en)
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WO1997018840A3 (fr
Inventor
Paul Yager
Michael H. Gelb
Paul A. Carlson
Anatoly N. Lukyanov
Alex S. Goldstein
Kyujin C. Lee
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University Of Washington
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Priority to AU12738/97A priority Critical patent/AU1273897A/en
Publication of WO1997018840A2 publication Critical patent/WO1997018840A2/fr
Publication of WO1997018840A3 publication Critical patent/WO1997018840A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1274—Non-vesicle bilayer structures, e.g. liquid crystals, tubules, cubic phases or cochleates; Sponge phases
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6919—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a ribbon or a tubule cochleate

Definitions

  • the present invention concerns compounds, compositions and methods useful for delivering therapeutics .
  • the delivery approaches include: (1) external delivery systems, such as external mechanical pumps and osmotic patches; (2) internal osmotic pumps; and (3) implantable or ingestible polymeric structures that can include erodible hydrogels.
  • external delivery systems such as external mechanical pumps and osmotic patches
  • internal osmotic pumps such as internal osmotic pumps
  • implantable or ingestible polymeric structures that can include erodible hydrogels.
  • continuous release can be set by the pump design or by controlling the motor.
  • Continuous drug delivery using continuous infusion with an i.v . line (the only viable method for some chemotherapeutic drugs) is costly and restricts the patient's movement.
  • Implanted catheters and pumps are an expensive solution, the considerable risk of which is only balanced by the importance of continuous delivery of the drug in question .
  • Using implantable macroscopic devices for drug delivery restricts the site of delivery to one that can accommodate the object.
  • the NORPLANT ® contraceptive system effective though it is, requires a large insertion site and must be surgically recovered after use.
  • Liposomes are one example of a self-assembled microstructure, and encapsulating drugs in liposomes has proven useful in some circumstances. Ostro, Liposomes : From
  • liposomes can be used to deliver drugs to skin.
  • Phosphatidylglycerols have been modified with a wide range of peptide and non-peptide drugs (in particular AZT) with the assumption that they would self assemble into liposomes, and would be trapped by macrophages in the reticuloendothehal system after injection into the bloodstream.
  • Lipid tubules are a recently discovered self-organizing system in which lipids crystallize into tightly packed bilayers that spontaneously form hollow cylinders less than 1 ⁇ m in diameter.
  • the basic subunit of the tubule is a helical ribbon of lipid bilayer and, in some cases, open helical structures of the same diameter can be seen .
  • polymerizable diacetylenic phosphatidylcholines such as 1 ,2-di-(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (referred to as DC 8,9 PC) were discovered by Yager and Schoen to form novel hollow tubular
  • Diacetylenic lipid tubules are straight, rigid, about 0.75 ⁇ m in diameter, and can be made to range in length from a few ⁇ m to nearly 1 mm, depending on the conditions used to form the microstructure . Further, the walls of the tubules may be as thin as a single bilayer.
  • the lumen (the open space in a tubular organ or device) is generally open, allowing free access by diffusion from the ends of the microstructures .
  • Kunitake et al demonstrated that a positively charged chiral amphiphile based on glutamate forms structures similar to those formed by DC 8,9 PC. Kunitake et al .'s Helical Superstructures are Formed from Chiral Ammonium Bilayer Membranes, 1709-1712 (Chem Lett., 1984). Helices and tubules of much smaller diameters ( ⁇ 300 ⁇ ) were found by Yamada et al . to form from related synthetic two-chain amphiphiles with oligopeptides (such as 12-14-mers of glutamic and aspartic acid) as hydrophilic headgroups.
  • oligopeptides such as 12-14-mers of glutamic and aspartic acid
  • Lipid tubules have been "decorated” with inorganic materials, including metals [See, for instance,
  • the drug delivery approach described herein is distinctly different, and potentially much more widely applicable, than any of the prior known methods for continuously delivering therapeutics.
  • the invention provides therapeutic materials which are themselves capable of forming high axial ratio microstructures, particularly tubules, cochleate cylinders, helical ribbons and twisted ribbons.
  • HARFM comprises high axial ratio forming molecules, i.e., lipid molecules that are capable of self-assembling into such microstructures
  • Th is a therapeutic covalently or otherwise coupled to the HARFM.
  • the therapeutic can be any agent now known or hereafter developed that does not interfere with the formation of high axial ratio (HAR) microstructures.
  • the Th may be selected from the group consisting of peptides, nucleic acids, antigens and conventional pharmaceuticals.
  • R 1 and R 2 are alkyl, alkenyl (i.e., compounds that include at least one double bond), alkynyl (i.e., compounds that include at least one triple bond) or heteroalkyl, heteroalkenyl or heteroalkynyl chains having from about 10 to about 25 carbon atoms.
  • Heteroalkyl, heteroalkenyl and heteroalkynyl compounds are compounds that include heteroatoms, such as, without limitation, nitrogen, oxygen and sulfur.
  • X is a hydrophilic group.
  • R 1 and R 2 preferably include at least one site of unsaturation, and generally are coupled to the carbon atom by functional groups that include heteroatoms, particularly but not necessarily, esters and amides.
  • R 1 and R 2 also can be attached to a chiral carbon.
  • X is a polypeptide, such as polyglutamate or polyaspartate.
  • spacers can be used to couple therapeutics to HARFMs .
  • suitable spacers are polypeptides that include enzyme cleavage sites, such as protease cleavage sites recognized by trypsin, trypsin-like enzymes and elastase.
  • R 1 and R 2 are hydrophobic alkyl, alkenyl or alkynyl chains having from about 10 to about 25 carbon atoms, Y is selected from the group consisting of
  • R 1 and R 2 may both include at least one site of unsaturation.
  • the present invention also provides compositions useful for delivering therapeutic agents.
  • the compositions comprise plural constituent molecules self assembled into HAR microstructures. Each constituent molecule satisfies the formula
  • the therapeutic may be coupled to the HARFM using a spacer (S), i.e. HARFM-S-Th .
  • S spacer
  • compositions may self assemble so that only a portion of the plural constituent molecules have therapeutics coupled to HARFMs .
  • the plural constituent molecules self-assembled into HAR microstructures can have plural different therapeutics. The result is a microstructure having plural different therapeutics associated therewith .
  • the present invention also provides a method for delivering therapeutic agents, particularly in a steady, continuous manner.
  • the method comprises administering to a person or animal effective amounts of compounds or compositions made in accordance with the present invention comprising plural constituent molecules self-assembled into HAR microstructures.
  • the method can comprise administering effective amounts of compounds satisfying the formulas discussed above, including the use of spacers .
  • the compounds or compositions may be administered by any number of methods including, but not limited to, topically, orally, such as in the case of vaccines, intramuscularly, intranasally, subcutaneously, intraperitoneally, intralesionally or intravenously.
  • the compositions may further comprise conventional materials known in the pharmaceutical field, including materials selected from the group consisting of aqueous buffers, stabilizers, diluents and adjuvants.
  • An object of the invention is to develop a device-free method by which drugs can be released into the body, particularly in a continuous manner (0-order kinetics) through association with HARFMs.
  • Another object of this invention is to form compounds and compositions comprising drugs or prodrugs associated with HARFMs that continuously release drugs either through dissolution of the molecules from the ends of the microstructures or through enzymatic cleavage.
  • Still another object of the present invention concerns using a homogeneous population of HARFMs to dissolve (or be enzymatically degraded) in such a manner that the rate of release of the constituent molecules (or parts thereof) is constant until the microstructures are consumed.
  • Still another object of the present invention is to ligate an appropriate hydrophobic anchoring moiety to water-soluble molecules and clinically significant therapeutics, such as conventional pharmaceuticals and bioactive polypeptides, and to allow such compounds to self-associate into HAR microstructures.
  • Still another object of the present invention is to provide compounds and compositions comprising therapeutics coupled to HARFMs by spacers.
  • a particularly suitable class of spacers are peptides or polypeptides (polypeptides are defined herein to mean an amino acid chain having at least two amino acids linked by amide bonds). Such spacers also can include enzyme recognition sites .
  • Still another object of the present invention is to provide materials and methods useful for oral delivery of materials to the gut, such as delivery of therapeutics and vaccines to the small intestine, wherein such materials are generally impervious to the low pH and proteolytic activity of the stomach.
  • FIG. 1 is a schematic drawing illustrating non-liposomal microstructures of bilayer-forming amphiphiles.
  • FIG. 2 is a schematic representation illustrating the dissolution of a therapeutic drug from the ends of a cylindrical microstructure.
  • FIG. 3 is a schematic representation illustrating therapeutic release from a cylindrical microstructure under the influence of an enzyme-catalyzed hydrolysis reaction .
  • FIG. 4 is a schematic representation illustrating the use of spacers for coupling therapeutics to cylindrical microstructures.
  • FIG. 5 is a schematic representation of a monolayer array of lipids at the edge of a tubule representing the enzymatic cleavage of a spacer.
  • FIG. 6 is a graph that compares the kinetics of dissolution of spheres, infinitely long solid cylinders (no diffusion from the ends) and flat slabs (for modeling dissolution from the ends of tubules).
  • FIG . 7 is a graph illustrating the calculated degradation of a flat sheet having a 10:1 axial ratio that is degrading from its edges at a rate proportional to the length of its edges as a model of the degradation of cochleate cylinders.
  • FIG. 8 is a graph of time versus % hydrolysis for suspensions of DC 8,9 PC tubules and DPPC liposomes by 160 nM cobra venom PLA 2 .
  • FIG. 9 is a graph of time versus concentration of micellar DC 8,9 PC illustrating the time course for the solubihzation of a 0.5 mM suspension of DC 8.9 PC lipid tubules in the presence of 50 mM OG.
  • FIG. 10 is a graph of time versus the mole fraction of DC 8,9 PC remaining in tubule microstructures as a function of time .
  • FIG . 11 is a graph of time versus concentration of DC 8,9 PC within tubules (mM) for various concentrations of solubihzng detergent.
  • FIG. 12 is a graph of time versus concentration of DC 8,9 PC within tubules (mM) illustrating the temperature dependence on the concentration of DC 8,9 PC withm tubule microstructures.
  • FIG . 13 is an Ahhrenius plot of the solubihztion rate versus inverse temperature.
  • FIG . 14 is a graph showing the total concentration of 10, 12-tricosadiynolic acid (DC 8,9 PC) over time following the action of P L A 2 on a suspension of DC 8,9 PC.
  • HAR microstructure refers to microstructures wherein the ratio of the major axes is from about 2 to 5,000, and more typically from about 2 to 1 ,000.
  • HFA-cerebroside cochleate cylinder having a diameter of about 0.1 ⁇ m
  • there are about 20 lipid bilayer "wraps" in the structure This means that at the end of the cochleate cylinder there is about 3 ⁇ m of linear bilayer edge exposed.
  • FIG. 1 provides a schematic representation of tubules and cochleate microstructures.
  • the therapeutic compounds may be coupled to materials capable of forming HAR microstructures, one example being covalently bonding therapeutic compounds to lipids capable of self assembling into HAR microstructures .
  • Noncovalent attachment also may be used to associate the therapeutics with the lipids.
  • the lipid components generally are, or are similar to, ceramides, phosphatidylcholines, amino acids and fatty acids, the structural components are generally intended to be completely metabolized into nontoxic products .
  • HARFMs solve many continuous drug delivery problems, and are useful for the continuous release of drugs.
  • One reason for this is that the geometry of drug particles affects the kinetics of drug release.
  • the environment in which the compounds undergo hydrolysis or enzymatic cleavage also can effect the kinetics of the reaction. This is discussed in more detail below.
  • HAR microstructures could prevent them from being used in all applications.
  • a suspension of HAR microstructures should be usable in all circumstances for which macroscopic polymeric drug delivery systems are currently
  • This method of controlled release avoids pumps or incorporation of drug into a macroscopic rigid matrix of a particular shape.
  • the small diameter of HAR microstructures allows them to be placed into cavities in the body using a needle or catheter, whereas their great length will immobilize them after injection.
  • a wide range of HARM-based antitumor drugs could be injected into tumors (intralesionally) using small needles, perhaps avoiding the need for major surgery in some cases.
  • the present compounds and compositions comprise HARFMs having therapeutics associated therewith HARFMs might themselves be useful therapeutics.
  • therapeutics will be attached, such as by covalent bonding, to HARFMs to produce composite compounds according to the formulas HARFM-Th or HARFM-S-Th, wherein "HARFM” stands for high axial ratio forming molecules, "Th” is a therapeutic, and "S” is a spacer.
  • HARFM-Th and HARFM-S-Th compounds form suitable microstructures when subjected to microstructure-forming regimens.
  • HARFMs, therapeutics and spacers each will be discussed separately below.
  • HARFMs there are a number of HARFMs currently known, and these HARFMs likely can be used for the synthesis of composite (i .e., HARFM-Th ; HARFM-S-Th) compounds. Other compounds also are capable of forming HAR microstructures .
  • the HARFMs currently deemed preferable for use in producing composite compounds for the delivery of therapeutics will be glutamate-based amphiphiles (Formula 1), polyglutamate-based amphiphiles (Formula 2), phosphatidylcholine with tricosadiynoyl fatty acyl chains, referred to as DC 8 ,9 PC (Formula 3), NFA-Gal-cer (Formula 4), and derivatives of these compounds .
  • NFA-Gal-cer can have an hydroxyl group ⁇ to the amide bond (this compound is referred to as HFA-Gal-Cer) .
  • the compounds represented by Formulas 1-4 can be synthesized according to published procedures or are commercially available. More specifically, DC 8,9 PC is commercially available from Avanti Polar Lipids, Birmingham AL, and NFA-Gal cer and HFA-Gal-cer are commercially available from Sigma Chemical Company.
  • Glutamate-based amphiphiles (represented by Formula 1) can be synthesized using procedures published by Kunitake. See, for instance, Kunitake et al .'s Helical Superstructures are Formed from Chiral Ammonium Bilayers, 1709-1712 (Chem Lett., 1984).
  • the compounds shown in Formulas 1-4 also can be modified to form additional compounds useful for forming HAR microstructures.
  • the alkyl chains in each of the compounds shown in Formulas 1-4 can be changed to have different numbers of carbon atoms, as long as these modifications do not prevent such compounds from forming HAR microstructures under the appropriate conditions.
  • the alkyl chain lengths for the compound of Formula 2 have been varied to be C-12, C-14 or C-16 (see compounds of Table 1 below) and these compounds appear to form HARFMs in accordance with the present invention.
  • ceramide type compounds also have been synthesized and are HARFMs .
  • the ceramides comprise sphingosine acylated with fatty acids . Good results have been obtained using nervonic acid, or fatty acids similar thereto, coupled to sphingosine, or derivatives thereof, to provide N-nervonoyl ceramides .
  • Nervonic acid was chosen for several reasons. First, it is present naturally in the body, and therefore should not be toxic. Second, it includes a site of unsaturation, i.e., a double bond, which currently is believed to favor formation of HAR microstructures relative to compounds which do not include sites of unsaturation .
  • Various HARFMs also can be made by selectively coupling compounds to the 1 ° hydroxyl group provided by sphingosine .
  • the compounds synthesized to date are shown below, and the synthesis of these compounds is further discussed in Example 5 .
  • the compounds shown below also can be acylated .
  • a therapeutic compound also must be selected .
  • the therapeutic compound (Th) is coupled, such as by covalent bonding, to individual HARFMs to form composite compounds.
  • the therapeutic compounds can be conventional pharmaceuticals, peptides (such as oligopeptides, enzymes, etc.), nucleic acids (such as DNA and RNA), cells, antigens, etc.
  • peptides such as oligopeptides, enzymes, etc.
  • nucleic acids such as DNA and RNA
  • cells antigens, etc.
  • antigens etc.
  • the following is a partial list of therapeutics that can be attached to HARFMs to form composite compounds .
  • candidate peptides for attachment to HARFM include insulin, vasopressin, growth hormone, and any other natural or synthetic peptide ligand now known or hereafter discovered or synthesized for endogenous receptors.
  • Peptides also can be used to form vaccines, such as orally administered vaccines.
  • Vaccine generally refers to systems that deliver an antigen, generally a protein, in a controlled manner to elicit an immune response.
  • steroids Another example of a class of compounds commonly used as therapeutics are the steroids.
  • candidate steroids for attachment to HARFMs include estrogen, progesterone and testosterone.
  • Synthetic and/or semi-synthetic derivatives eg estrone or methyl-testosterone
  • Combinations of these steroids also may be used, such as are used in birth control formulations, and methylprednisolone, which is used as an anti inflammatory corticosteroid.
  • Another class of candidate agents for attachment to CFMs are the conventional organic pharmaceuticals. Examples of such compounds, without limitation, include
  • antihypertensives e.g., calcium channel blockers such as nifedipine and verapamil.
  • vasodilators such as nitroglycerin.
  • diuretics such as lasix and hydrochlorothiazide.
  • stimulants such as methylphenidate.
  • antidepressants such as doxepin or serotonin specific re-uptake inhibitors including Prozac.
  • antipsychotics such as lithium and haloperidol .
  • antiemetics such as chlorpromazine or scopolamine .
  • analgesics such as acetaminophen and acetylsalicylic acid.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • indomethacin or naproxen non-steroidal anti-inflammatory drugs
  • histamine antagonists such as cimetidine, ranitidine and diphenhydramine .
  • narcotics such as morphine and demerol.
  • the therapeutic compounds selected for coupling to the HARFMs can be directly coupled to the CFM.
  • the therapeutic can be coupled to the HARFM using a spacer (spacers also are referred to as tethers and linkers), i.e., HARFM-S-Th.
  • Spacers appear to uncouple the steric interactions of the agents from the packing of the HAR-forming lipids.
  • the spacer might also provide a cleavage site recognized by an enzyme that is either dispensed in combination with the HARFMs-Ths compounds, or is endogenous to the environment in which the HARFMs-Ths are administered. See FIG. 4, which provides a schematic representation illustrating the use of spacers for coupling therapeutics to HAR microstructures.
  • Polypeptides are an example of a class of spacers useful for the present invention.
  • Such polypeptides generally will include a sequence known to be susceptible to attack by a protease, such as, without limitation, trypsin and trypsin-like enzymes (trypsin cleaves on the carboxyl side of lysine and arginine residues) and elastase (which recognizes Ala-Ala-Ala sequences) at the site of use.
  • a protease such as, without limitation, trypsin and trypsin-like enzymes (trypsin cleaves on the carboxyl side of lysine and arginine residues) and elastase (which recognizes Ala-Ala-Ala sequences) at the site of use.
  • trypsin and trypsin-like enzymes trypsin cleaves on the carboxyl side of lysine and arginine residues
  • elastase which recognizes Ala-Ala-Ala sequences
  • Polypeptides are not the only compounds potentially useful as spacers for the purpose of separating the steric interaction between the HARFM and therapeutics .
  • the spacer might include a functional group of limited stability against cleavage at the site of use.
  • the spacer might simply comprise alkyl, alkenyl or alkynyl carbon chains having a functionality that is readily cleaved in the environment in which the composite compounds are administered.
  • Such compounds might be esters, as long as the ester functionality is sufficiently labile in the environment in which the composite compounds are administered to release Th upon hydrolysis.
  • the spacers might comprise carbohydrates or polyoxyalkylenes, particularly polyoxymethylene and polyoxyethylene.
  • HARFMs to form the composite HARFM-Th or HARFM-S-Th.
  • Specific guidance as to the means for attaching Th to a particular HARFM depends upon several factors, including the nature of the HARFM, the Th, and on the environment in which the composite compounds will be administered.
  • the head group of the HARFMs include nucleophilic groups, such as amine and hydroxyl groups. These nucleophilic groups can be reacted with electrophilic species to couple the agents to the HARFMs. 1. Peptides
  • Peptides such as insulin and enkephalins
  • Peptides of any desired sequence can be synthesized using standard synthetic techniques, such as solid-phase synthesis using Applied Biosystems Peptide Synthesizers or other available devices.
  • the peptide is prepared with its N-terminus and all of its reactive side chains in protected form .
  • the peptide includes a free C-terminal carboxyl group. This is accomplished using a special peptide synthesis resin called super acid-sensitive resin, known as SASRIN, which is available from Bachem, Inc.
  • SASRIN super acid-sensitive resin
  • the fully protected peptide is cleaved from the resin with mild acid, such as 1 % trifluoroacetic acid in methylene chloride. This leaves the side chain and N-terminus protecting groups intact.
  • Peptide synthesis is accomplished with the ⁇ -amino groups of the amino acids protected, such as with a fluorenylmethyloxycarbonyl (FMOC) protecting group, and bearing standard side-chain protecting groups that are removed with strong acid (i.e ., trityl, t-butyl, etc .) .
  • FMOC fluorenylmethyloxycarbonyl
  • the FMOC group can be left on and removed along with the side chain protecting groups after the peptide is coupled to the lipid.
  • the FMOC protecting group can be removed while the peptide is still bound to the resin. This allows modifications of the N-terminus, such as by modifying the N-terminus with probes. Probes containing an N-hydroxylsuccinimide ester or an isothiocyanate can be used for attachment to the peptide N-terminus .
  • the polypeptide is cleaved from the SASRIN resin, it is then coupled to the ⁇ -amino group of dialkylated glutamine compounds or glutamic acid lipids using either dicyclohexylcarbodiimide or diethyl phosphorylcyanate in a solvent such as DMF or methylene chloride.
  • the coupling is monitored by observing the loss of the lipid NH 2 group using the Kaiser test . Kaiser et al .'s Color Test for Detection of Free Terminal Amino Groups in the Solid-Phase Synthesis of Peptides, 34:595-598 (Anal. Biochem ., 1970).
  • the crude material is treated with neat trifluoroacetic acid containing the appropriate scavengers (thioamsole, 1 ,2-dithioethane, etc. , depending on the structure of the side-chain protecting groups) .
  • the crude lipidic-peptides are purified by HPLC on a reverse-phase column.
  • Nucleic acids useful in the practice of the present invention comprise isolated nucleic acids .
  • An "isolated" nucleic acid has been substantially separated or purified away from other nucleic acid sequences in the cell of the organism from which it naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA .
  • the term thus encompasses nucleic acids purified by standard nucleic acid purification means. It also embraces nucleic acids prepared by recombinant expression in a host cell and chemically synthesized nucleic acids. Also included are nucleic acids that are substantially similar to such nucleic acids. Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Carruthers, Tetra. Letts .
  • nucleic acids may be synthesized, for example, on commercial automated oligonucleotide synthesizers .
  • Desired nucleic acid compounds can be attached to the HARFMs by a variety of methods .
  • nucleic acids can be coupled to HARFMs using the 5'-hydroxyl group. This hydroxyl group can be used to link nucleic acids to the HAR-forming lipids via an ester functionality. Because a number of the HAR-forming lipids used for the present invention have amines at the head group (See, for instance, the compounds of Table 1), an additional group containing a free carboxyl group must be used to couple the nucleic acids to the HAR-forming lipids.
  • peptide spacers comprising amino acids having a side-chain carboxyl group could be used to couple nucleic acids to the HARFMs .
  • Aspartic and glutamic acid are examples of amino acids having a carboxyl functionality that could be included in the peptide spacer to link nucleic acids to the HARFM-forming molecules .
  • Conventional pharmaceuticals also can be attached to the HARFMs .
  • the method for attachment depends on the particular HARFM and therapeutic selected . However, solely by way of example, the following provides a discussion concerning the attachment of particular classes of conventional therapeutics to HARFMs.
  • Steroids generally have a hydroxyl group in the A ring (the first 6-membered ring) .
  • This hydroxyl group can be used to link steroids to the HAR-forming lipids via an ester functionality as discussed above for nucleic acids.
  • an additional group containing a free carboxyl group must be used to couple the steroid to the HAR-forming lipids .
  • Amino acids having a carboxyl group in a side chain could be included in peptide spacers to link steroids to the HAR-forming molecules.
  • Acetylsalicylic acid (aspirin) is an additional example of a conventional therapeutic that could be delivered using HAR-forming lipids.
  • Acetyl-salicylic acid includes a carboxyl group that could be used to form an amide with an amine or an ester with a hydroxyl group.
  • a number of the HAR-forming lipids have amines at the head group of the lipid. The amine could be used to form an amide with the carboxyl group of acetylsalicylic acid.
  • HAR-forming lipids that include hydroxyl groups could be directly attached to acetylsalicylic acid via an ester
  • HAR-forming lipids that have amines at the head group generally will be coupled to compounds such as acetylsalicylic acid using spacers.
  • polypeptide spacers could be used for this purpose wherein at least one of the amino acids in the polypeptide includes a side chain having an hydroxyl group, such as serine.
  • the side-chain hydroxyl group could be coupled to the carboxyl group of acetylsalicylic acid via an ester functionality.
  • HARFMs are then subjected to HAR-forming regimens .
  • the conditions required to form the desired microstructures may differ from compound to compound, although all the surfactants synthesized form aggregates in water because of their hydrophobic tails .
  • the following procedures have proved most useful for inducing the HAR microstructures in the compounds tested to date.
  • microstructures precipitate directly from the mixture (Georger et al .);
  • microstructures formed in accordance with the general procedures outlined above, and as described in more detail in the examples, can be confirmed using a light microscope for lipid microstructures having dimensions larger than about 1 ⁇ m.
  • a Zeiss ICM-405 inverted microscope has been equipped for epifluorescence illumination, brightfield, and phase contrast imaging, attachments include a 63 ⁇ 1.40 NA Planapochromat, a 35mm camera, and a Peltier effect microscope stage for sample temperature control (-20 to + 100°C, +/-0.1 °C) .
  • a Dage 66 SIT video camera (with S-VHS VCR and monitor) allows video imaging through the microscope in all imaging modes. Image processing and printing from live or stored video is possible using a Data Translation QuickCapture frame grabber board in a Macintosh II . This system allows imaging of HAR microstructures at video rates.
  • TEM transmission electron microscopy
  • Imaging can be either direct or with a phosphotungstic acid negative stain.
  • Freeze fracture replicas can be made using a Balzers 360 belonging to the Imaging Center. Additional techniques can be used to characterize the compounds formed, including circular dichroism (CD) and Raman spectroscopy .
  • HARFMs formed as described above were subjected to tests to determine the thermal stability of the compounds at physiological temperatures and physiological pH. Examples 14-15 provide more detail concerning how thermal and physiological-fluid tests were conducted.
  • HAR therapeutics formed in accordance with the present invention were stable at physiological pH and physiological temperatures, particularly those materials having T Ms greater than physiolgoical temperature.
  • the HARFM comprises an HAR-forming surfactant with a therapeutic covalently attached to its headgroup.
  • the surfactant would be a lipidated drug if it were active in its intact form.
  • the compounds of the present invention function as lipidated prodrugs.
  • the constant rate of cylinder dissolution appears to be controlled largely by the solubility of the lipidated drug in the surrounding medium. The greater the ratio of head-group area to hydrocarbon chain surface area, the more rapid will be the dissolution and delivery.
  • the drug moiety is attached to the HAR-forming surfactant via a cleavable spacer (sometimes referred to as a tether) as discussed in more detail below
  • spacers might be a polypeptide with a sequence known to be susceptible to attack by a protease at the site of use, or a functional group of limited stability against cleavage when exposed to the solution at the site of use.
  • the drugs are packed tightly enough at the surface of the HARFM microstructure to prevent access by a protease .
  • HARFMs particularly tubules and cochleate structures, generally are crystalline materials and tend to dissolve only from the surfaces and ends thereof, or perhaps from regions of imperfection in the HAR microstructure.
  • the end-dominated dissolution model and lysis was evaluated both theoretically and empirically .
  • Theoretical dissolution rates of three structures - a solid sphere, an infinitely long solid cylinder, and a slab were used to model the kinetics for dissolution at HAR microstructures, particularly tubule and cochleate ends .
  • the dissolution rate is proportional to the exposed surface area.
  • the three are drastically different when considering one particle or a homogeneous population of particles .
  • heterogeneity in particle size softens the distinction between the models.
  • FIG. 6 shows that the relative release rate depends upon the morphology of the system.
  • the rate of appearance of dissolved surfactant or surfactant breakdown products from tubules appears to remain substantially constant until the number of tubules (and ends) declines.
  • the rate of drug release to the tissue is limited by the rate of release from the ends of tubules, so that drug release rate generally is constant (0-order), as opposed to the more conventional first-order kinetics found with a wide range of other geometries .
  • Cochleate cylinders consist of one or more bilayers that have wrapped in a helical manner to form the cochleate microstructure .
  • Cochleate cylinders therefore have two types of "free edges", those at the microstructure ends, and one or two bilayer edges along the length of the microstructure.
  • an appropriate model for the dissolution or enzymatic degradation of cochleate cylinders is the unrolled flat sheet that comprises the microstructure .
  • very long and very short cochleate cylinders both can degrade with kinetics similar to those of the lipid tubules.
  • the sheet that wraps to form the cochleate cylinder has an axial ratio of about 10: 1 , there is only an 18% decrease in the hydrolysis or degradation rate before the microstructure is completely hydrolyzed or degraded (if the ratio is greater than 10: 1 , then the decrease in the hydrolysis rate or degradation rate is concomitantly decreased).
  • the rate of drug release generally will only be constant to the extent that the HAR microstructure population is homogeneous in length. While is it possible to form HAR microstructures with unimodal length distributions using particular crystallization methods [See, for instance, the crystallization protocol discussed in Yager et al .'s Helical and Tubular
  • PLA 2 is known to hydrolyze the well-studied dipalmitoyl phosphatidylcholine (DPPC) below its Tm. An experiment was designed to determine whether PLA 2 can work on DC 8,9 PC below its T M in tubule form.
  • DPPC dipalmitoyl phosphatidylcholine
  • Small unilamellar vesicles were prepared from 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) .
  • the T M of DPPC at 41 .3°C is similar to that of DC 8,9 PC, and is only slightly depressed in SUVs. Because they have identical head groups, comparison of hydrolysis of DPPC vesicles and DC 8,9 PC tubules allows isolation of those effects unique to a tubular microstructure.
  • microstructures observed by TEM reflect a more complex process. Shortly after addition of enzyme, helical ribbons emerging from what appear to be fractured tubules are visible. Even though a few intact tubules are still present at the 50% hydrolysis point, the types of microstructures present include small filaments, helical ribbons, and elongated sheets . Tubules appear to remain intact until certain fraction of reaction products is reached within a local region of the tubule bilayer. The point when product accumulation can no longer support the specific asymmetric curvature required to form a one micrometer diameter tubule, the product regions fracture and unwrap to form smaller helices, filaments and flat sheets .
  • Fluorescent PLA 2 also has been used to track reaction progress .
  • 5-carboxyfluorescein-tagged PLA 2 was used.
  • enzyme appears to distribute uniformly over the tubule surface.
  • the product microstructures show strong fluorescence, which implies enhanced PLA 2 binding to product-risk microstructures .
  • enzyme reaction with tubules is not limited to reaction at the end of tubules, the reaction progress nevertheless is still more constant and slower than that of SUVs . This makes the tubules attractive drug delivery agents.
  • Bile salts occuring naturally in humans act similarly to detergents .
  • the kinetics of HAR microstructure dissolution in detergents is a good model for the oral administration of therapeutics and vaccines for delivery to the gastrointestinal tract.
  • the kinetics of dissolution in detergent solutions has been investigated.
  • the commercially-available tubule-forming phospholipid 1 ,2-bis(10, 12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC 8,9 PC) and the non-ionic detergent octyl ⁇ -D-glucoside (OG) were used as the model system.
  • DC 8,9 PC Upon precipitation from ethanol, DC 8,9 PC forms multi-lamellar tubules with an average diameter of 0.75 ⁇ m, a length distribution ranging from 30-50 ⁇ m, and a melting temperature (T m ) of 43.8° C.
  • the tubule morphology is composed of helically-wrapped lipid bilayers that close to form straight, hollow, ngid tubes. Tubules can appear, however, in the presence of minority structures such as open helical ribbons . If given time to anneal, the lipids form closed and uniform tubules.
  • the tight crystalline packing of the tubule wall will hinder release of monomeric lipid from the microstructure and insertion of detergent into the tubule except at regions of defects in the crystalline packing such as must occur at tubule ends or at "helical" defects.
  • FIG . 9 shows the concentration of DC 8,9 PC solubilized into OG detergent micelles as a function of time.
  • an aqueous suspension of DC 8,9 PC tubules was added to an aqueous suspension of OG detergent micelles to create a final solution having a 0.5 mM concentration of DC 8,9 PC and a 50 mM concentration of OG.
  • the reaction vessel was at room temperature (approx. 21 ° C) .
  • Tubule microstructures were much larger than detergent micelles so a 0.2 ⁇ m filter was used to separate the two phases prior to analysis.
  • DC 8,9 PC absorbs strongly from 190 to 254 nm because of the diacetylene groups present in the hydrocarbon tails.
  • micellar DC 8,9 PC The amount of micellar DC 8,9 PC was determined with a UV-Vis spectrometer by calculating the second derivative of the optical density with respect to wavelength at 250 nm, which was a mathematical step that reduced errors introduced from scattering artifacts. The concentration could be determined by comparing this value to those obtained from a calibration curve. Throughout the course of solubilization, aliquots of the suspension were removed, passed through a 0.2 ⁇ m filter, and assayed for the amount of solubilized DC 8,9 PC.
  • FIG. 10 shows the mole fraction of DC 8,9 PC remaining within a tubule as a function of time and depicts the nature of the solubilization process.
  • the logarithm of the DC 8,9 PC tubule concentration depends linearly with time, which suggests that tubule disintegration is a first order process. Furthermore, changes in solution turbidity, as determined by measuring the optical density at 400 nm, correlates well (e.g. linearly) with the amount of DC 8,9 PC within the tubule.
  • Multilamellar PC tubules, L interact with detergent, OG, to form mixed micelles, M .
  • the effective concentration of detergent that is available for solubilization does not change throughout the course of the reaction (e.g. detergent is not consumed by the reaction, mixed micelles can contain many phospholipids, etc.)
  • the kinetics can be described as a first order process.
  • FIG. 1 1 shows the time course of solubilization as a function of OG concentration.
  • An estimate for the value for the second order rate constant, k 2 can be determined from the slope of the line created when the measured first- order rate constant k 1 , is plotted against detergent concentration.
  • FIG. 12 shows the concentration of DC 8,9 PC within a tubule as a function of time as determined from measuring the O.D. at 400 nm.
  • a solution containing a 100-fold molar excess of OG (40 mM) was added to a stirred quartz cuvette and placed in a temperature-controlled UV-Vis spectrometer. Once the detergent suspension had reached thermal equilibrium, an aqueous suspension of DC 8,9 PC tubules (0.4 mM) was quickly added.
  • the temperature dependence of the rate of tubule solubilization was determined by measuring the decrease in turbidity (e.g. changes in O.D.) with time.
  • HAR microstructures will be used for the continuous administration of therapeutics to animals and patients
  • HAR microstructure based continuous release can be used for administering therapeutics, for example and without limitation, topically, orally, intramuscularly, intranasally, subcutaneously, lntraperitoneally, intralesionally, intravenously, or any other administration means now known or hereafter developed that allow for the compounds to remain in HAR lipid microstructures .
  • the safety and comfort of the patient also must be considered. Larger diameter HAR microstructues (about 1 ⁇ m diameter) are unlikely to be safe for injection into the circulatory system because of possible clogging of capillaries . All other internal and external sites of drug delivery are possible, however. Most of the tubule mass is in the wall.
  • Multi-bilayer tubules or cochleate cylinders thus may be better suited for circumstances where high drug loading is necessary. Smaller and more flexible tubules and cochleate cylinders have less wasted space and may also be small enough to pass through the capillary beds.
  • HAR-microstructure-based therapeutic delivery systems can provide controlled release in topical or subcutaneous applications.
  • the relatively long length of some of the microstructures can immobilize them without a rigid polymeric matrix
  • HAR microstructures also can be used in mucosal and oral delivery .
  • the tight packing of the lipid molecules in the HAR microstructure could afford protection of certain drugs such as peptides from the premature enzymatic hydrolysis that now plagues peptide delivery systems as has been shown for calcium induced cochleate cylinders .
  • proteolytic and lipolytic enzymes While there are often ample concentrations of proteolytic and lipolytic enzymes present in the interstitial fluid in vivo, these enzymes are often inhibited to prevent uncontrolled cell damage .
  • HAR microstructures could be co-suspended with hydrolytic enzymes.
  • HAR microstructures placed in subcutaneous sites could act as long-acting vaccines that deliver antigen long enough to create a strong immune response.
  • the compound tested was Pro 3 -glutamic acid didodecylamide, and was administered to the animals in an aqueous solution comprising 120 nM NaCl at a pH of 7.2.
  • the control for the study was 120 mM NaCl at a pH of 7.2.
  • mice 13 mice were used, divided into three groups ( 1 group of three animals, and 2 groups of 5 animals). The rear flanks of the animals were shaved prior to receiving injections .
  • the group 1 animals received a single 250 microliter injection of the control, and were euthanised at 15 days.
  • the group two and three animals received a single 250 microliter control injection at a first site, and a 250 microliter injection, 100 micrograms of the test material, at a second site .
  • the group two animals were euthanised at eight days, and the group three animals were euthanised at 15 days.
  • the health of the animals was monitored daily.
  • Histology analysis was performed on tissue collected from the injection sites and preserved in 10% neutral buffered formalin solution. Cross sections from skin injection sites and surrounding tissue were procesed by normal paraffin embedding and staining by Hematoxyhn and Eosin .
  • This example describes the synthesis of glutamine-based amphiphiles using hexadecylamine to form dihexadecyl glutamides .
  • the synthesis described can gnerally be used for the synthesis of a variety of compounds wherein the length of the side chains is varied.
  • the first step in the synthesis of dihexadecyl glutamides involved forming an activated ester from glutamic acid with the amino group protected with a CBZ protecting group. This allows the activated ester to be coupled with hexadecylamine.
  • N-hydroxysuccimmide was used to activate the ester .
  • the di-N-hydroxysuccinimide-Z-glutamic acid ester was then ready for coupling with an amine.
  • 1.0 (0.10 mmoles) gram of the di-N-hydroxysuccinimide-Z-glutamic acid was dissolved in 25 milliliters of chloroform ester.
  • 1.1 equivalents of hexyadecylamine available commercially
  • the resulting solution was washed with saturated sodium bicarbonate, brine, and dried over sodium sulfate. Diethyl ether was then added to precipitate a solid.
  • the solid was resuspended in ethyl acetate, and the semi-solid product was filtered, washed (3X) with ether, and dried .
  • the product was purified using a silica gel column, the eluting solution comprising chloroform-5 % methanol . This provided compounds comprising the hexadecylamine side chains coupled to the glutamic acid core, but with the CBZ protecting group still intact .
  • the resulting product was then resuspended in ethyl ether and filtered, and then dried in vacuo to produce the bromide salt .
  • the free amine was produced by first dissolving the product in chloroform, and then adding saturated sodium bicarbonate, followed by filtration and drying in vacuo.
  • This example desenbes coupling a tetrapeptide to a glutamine-based lipid, which can be produced as discussed above in Example 1.
  • a tetrapeptide (boc-gly-lys-e-CBZ-sar-pro) was purchased from Anapec of St. Jose, CA. The tetrapeptide was purified using a silica gel column and a chloroform methanol acetic acid (9:1 :0.2) elutant system. The product was collected and then extracted with methylene chloride.
  • 60 milligrams of the protected tetrapeptide-lipid were then dissolved in 800 ⁇ l of methylene chloride. The solution was then cooled to about 0°C. 2 milliliters of HCl/dioxane (4 molar) were added to the solution. The solution was kept at 0°C for about 2 hours. The solution was then concentrated in vacuo. A fraction of the product was purified using an analytical C-4 HPLC column, using acetonitrile/water/0.6% TFA . The product produced was the salt of the amine.
  • the free amine was liberated by dissolving 45 milligrams of the tetrapeptide-lipid in
  • proline derivatives In a manner similar to that described in Example 2, additional peptides and single amino acids derivatives, such as proline derivatives, also have been made .
  • proline with an FMOC protecting group has been coupled to the C-12 glutamine lipid shown in Table 1 using EDC, the water-soluble derivative of DCC).
  • This example concerns the synthesis of cylinder forming molecules having peptide spacers coupled thereto, wherein the spacer incudes an enzyme cleavage site .
  • N ⁇ -Glycyl-N ⁇ -(2,2,5,7,8-pentamethylchromane -6-sulfonyl)-arginyl-alanyl-glycyl-glycyl-alanyl-alanyl-prolyl-prolyl-2-chlorotrityl resin was purchased as a custom order from the University of Washington immunology biopolymer facility.
  • N ⁇ -Glycyl-N ⁇ -(2,2,5,7,8-pentamethylchromane-6-sulfonyl)-arginyl-alanyl-glycyl-glycyl-alanyl-alanyl-prolyl-prolyl-2-chlorotrityl resin (200mg) was washed with CH 2 CL 2 and reacted with acetic anhydride (41 5ul, 0.44mmol) and dnsopropyleihylamine (95.8ul, 0.55mmol) in CH 2 CL 2 for 2 hrs in a manual solid phase peptide synthesis vessel on a rocker for 2 hrs. The peptide resin was washed with CH 2 CL 2 three times and dried in vacuo to produce
  • N ⁇ -Acetyl-glycyl-N ⁇ -(2,2,5,7,8-pentamethylchromane-6-sulfonyl)-arginyl-alanyl-glycyl-glycyl-alanyl-alanyl-prolyl-prolyl-2-chlorotrityl resin (80mg) was treated with 1 :1 :8 acetic acid:trifluoroethanol CH 2 CL 2 (2ml) at room temperature for 30 min. After filtration of the cleaved peptide, the resin was retreated with the same cleavage mixture for 30 min. The combined filtrates were evaporated, dissolved in H 2 O, and dried in Speed- Vac.
  • This example describes the synthesis of radiolabelled materials, particularly ⁇ , ⁇ - Dihexadecyl [5- 3 H]prolyl-prolyl-prolyl-glutamide hydrochloride .
  • proline 11.50mg
  • dioxane 4.0mg, 99.9umol
  • NaOH 4.0mg, 99.9umol
  • reaction mixture was diluted with H 2 O, washed with hexane, CH 2 CL 2 added at 0°C, acidified to pH 1 to 2 with 1 N HCL, extracted with CH 2 CL 2 , washed with H 2 O, dried under Na 2 SO 4 , filtered, evaporated, and dried in vacuo to give a 65 % yield of
  • N ⁇ -tert-butoxycarbonyl-[5- 3 H]proline TLC CHCL3: MeOH (9:1) : Rf 0.25.
  • ⁇ , ⁇ -dihexadecyl prolyl-prolyl-glutamide hydrochloride 48.8mg, 59.14umol
  • N ⁇ -tere-butoxycarbonyl-[5- 3 H]proline (14.0mg, 65.06umol) was added followed by hydroxy benzotriazole (8.8mg, 65.06umol).
  • reaction mixture was diluted with CHCL 3 and washed with sat'd NH 4 CL, H 2 O, sat'd NaHCO 3 , H 2 O, brine, dried over Na 2 SO, filtered, evaporated, and further purified by silica gel flash chromatography with CHCL 3 :MeOH (97.5 :2.5): to give a 93 % yiedl of ⁇ , ⁇ -Dihexadecyl N ⁇ -tert-butoxycarbonyl-[5- 3 H]prolyl-prolyl-prolyl-glutamide.
  • Fluorophores and tritiated aceryl groups can be coupled to the terminal amino group of polypeptides bound to molecules capable of forming cylindrical lipid microstructures. This allows the detection of therapeutics, such as polypeptides, once they have been released from the cyhndrical microstructure .
  • a suitable fluorophore is O-aminobenzoic acid.
  • O-aminobenzoic acid first was protected with a BOC protecting group using known chemistry to produce BOC-aminobenzoic acid. This protected fluorophore was then coupled to the tetrapeptide derivative as produced in Example 2 using EDC. Likewise, a tritiated acetyl derivative can be made by reacting the terminal amino group of the tetrapeptide with tritiated acetic anhydride.
  • This example describes the synthesis of ceramide derivatives made from sphingosine.
  • Phase contrast optical micrographs were taken using a Zeiss ICM 405 (Carl Zeiss, Inc. , Thornwood, NY) with 40x (NA 0.75) or 63x (NA 1.4, oil) phase contrast lenses. Sonication was performed using a bath sonicator (Laboratory Supplies &. Co. , Inc ., Hicksville, NY, output 80 KC) .
  • Nervonic acid 0.558 g, 1.52 mmol
  • N-hydroxy succinimide 0.175 g, 1.52 mmol in 60 mL anhyd EtOAc was stirred overnight with dicyclohexylcarbodumide (0.314 g, 1.52 mmol) .
  • the white precipitate was removed and the supernatant evaporated in vacuo.
  • N-nervonoyl ceramide N-nervonoyl ceramide
  • N-hydroxy succinimide ester of nervonic acid (0.092 g, 198.4 ⁇ mol) and sphingosine (0.062 g, 207.0 ⁇ mol) were dissolved in 10 mL anhyd THF and stirred overnight under Ar .
  • N-nervonoyl ceramide (0.018 g, 27.8 ⁇ mol), triphenylmethyl chloride (0.015 g, 55.5 ⁇ mol) and N,N-dimethyl-4-aminopyridine (0.007 g, 55.5 ⁇ mol) in 20 mL anhyd toluene was refluxed for 16 h under Ar.
  • N-nervonoyl-1-O-triphenylmethyl ceramide (0.108 g, 0.12 mmol), imidazole (0.066 g, 0.97 mmol), and t-butylchlorodiphenylsilane (0.79 mL, 3.03 mmol) was stirred 19.5 h in 25 mL anhyd DMF under Ar. Added 25 mL of H 2 O and extraced with 3 ⁇ 15 mL Et 2 O Washed the ether layer with 10 mL H 2 O and 10 mL satd NaCl (aq) . Flash chromatography (15:1-2: 1 Hexane.
  • N-nervonoyl-1-O-triphenylmethyl-3-O-[diphenyl-r-butylsilyl] ceramide (0.093 g, 82.4 ⁇ mol) was stirred for 4 h with p-toluenesulfonic acid monohydrate (0.010 g, 49.4 ⁇ mol) in 20 mL 1 : 1 MeOH CH 2 Cl 2 . Added Et 2 O (40 mL) and washed the solution with 10 mL 5 % NaHCO 3 (aq) and 10 mL H 2 O.
  • N-nervonoyl-3-O-[diphenyl-t-butylsilyl] ceramide (0.021 g, 23.7 ⁇ mol), N-acetyl -glycine (0.006 g, 47.4 ⁇ mol), and N,N-dimethyl-4-aminopyridine (0.06 g, 47.4 ⁇ mol) in 21 mL 2:5 CH 3 CN :CH 2 Cl 2 (anhyd) was stirred for 2 h under Ar whereupon dicyclohexylcarbodumide (0.010 g, 47.4 ⁇ mol) was added and the reaction stirred for 24 h under Argon. The solvent was removed in vacuo.
  • N-nervonoyl-3-O-[diphenyl-t-butylsilyl] ceramide (0.034 g, 38 .4 ⁇ mol), N-acetyl-L-proline (0.010 g, 63.6 ⁇ mol), and N,N-dimethyl-4-aminopyridine (0.011 g, 90 .0 ⁇ mol) in 15 mL
  • N-nervonoyl-3-O-[diphenyl-t-butylsilyl] ceramide (0.041 g, 46.2 ⁇ mol), N-t-butylcarbamate-L-proline (0.011 g, 50 9 ⁇ mol) and N,N-dimethyl-4-aminopyridine (0.006 g, 50.9 ⁇ mol) was stirred for 4 h under Ar in 7 mL anhyd Ch 3 CN and 17 mL anhyd CH 2 Cl 2 .
  • N-nervonoyl-1-O-(N-acetyl-glycine)-3-O-[diphenyl-t-butyl-silyl] ceramide (0.009 g, 9 1 ⁇ mol) in 10 mL anyyd THF and 0,01 mL 1.0 M n-butylammonium fluoride (in THF) was stirred for 1 h under Argon The solvent was removed by rotary evaporation and the residue purified by flash chromatography (2 1-0.1 Hexane:EtOAc) to provide 1-O-(N-acetyl-glycine)-nervonoyl-ceramide as a white solid (0.002 g, 29%): R f (EtOAc) 0.25; 1 H NMR (499 MHz) 6.1 1
  • N-nervonoyl-1-O-(N-acetyl-L-proline)-3-O-[diphenyl-:-butyl-silyl] ceramide (0.021 g, 20.5 ⁇ mol) in 12 mL anhyd THF and 0.01 mL 1.0 M n-butylammonium fluoride (in THF) was stirred for 2 h under Argon.
  • This example describes a general HAR microstructure forming regimen .
  • Amphiphile 0.1 mg was dissolved in anhyd DMF so that the concentration was 1.0 mM . Water was added in ⁇ 10 ⁇ L increments until the solution became cloudy . The test tube was then covered and allowed to sit at 20° C for 2-24 h undisturbed . For larger amounts of amphiphile, water was added with vortex mixing ( ⁇ 3 sec) between additions.
  • This example describes a general HAR microstructure forming regimen.
  • Amphiphile 0.1 mg was dissolved in pyridine so that the concentration was 1.0 mM . Water was added in ⁇ 10 ⁇ L increments until the solution became cloudy . The test tube was allowed to sit at 20° C so that the solvent could evaporate over time.
  • This example describes a general HAR microstructure forming regimen.
  • This example describes a general HAR microstructure forming regimen.
  • Amphiphile was placed in ethylene glycol water (either 19 : 1 or 1:1 v/v) for a final concentration of 1 mg/mL.
  • This example describes a particular HAR microstructure forming regimen
  • Samples of 0 2 milligrams of NH 2 -Gly-Lys-Sar-Pro-Glu(NH-C 12 H 25 ) 2 or (Pro) 3 -Ghi(NH-C 12 H 15 ) 2 were dissolved in 40 ⁇ l of MeOH were added to 400 ⁇ l of HEPES buffered saline at pH 7.4 while vortexing and incubated for 2 hours at room temperature
  • HEPES buffered saline pH 7.4
  • 150 ⁇ l of a 1 mg/ml MeOH solution of the peptide lipid was mixed with 350 ⁇ l of HEPES buffered saline (HBS), and incubated overnight Before microscopy the obtained peptide-2 particles were transferred to HBS using centrifugal- driven filtration To do this, particles were centrifuge
  • This example describes how to make HAR microstructures from Ac-NH-Lys-Ala-Sar-Pro-Glu(NH-C 12 H 25 ) 2 and NH 2 Gly-Lys-Sar-Pro-Glu(NH-C 12 H 25 ) 2 by heating and cooling in HBS/EtOH mixtures.
  • 0.2 milligrams of Ac-NH-Lys-Ala-Sar-Pro-Glu(NH-C 12 H 25 ) 2 and NH 2 Gly-Lys-Sar-Pro-Glu(NH-C 12 H 25 ) 2 were dissolved in 50 ⁇ l of EtOH.
  • This example describes forming HAR microstructures by heating and cooling in
  • HBS/MeOH mixtures 0.1 milligram samples of (Pro) 3 -Glu-(NH-C 14 H 29 ) 2 or (Pro) 3 -Glu-(NH -C 14 H 29 ) 2 dissolved in 20 ⁇ l of MeOH each were added to 200 ⁇ l of HBS at pH 7.4 while vortexing . Concentrations of MeOH in the samples were adjusted to be between 20 and 50 percent, by volume . Sealed samples were then heated to 65°C, and slowly (within about 4 hours) cooled to room temperature. The obtained particles were separated from MeOH/HBS mixtures by centrifugation at 3000 X g for 15 minutes at room temperature . The obtained pellets were reconstituted in 1 milliliter of HBS. After overnight incubation the particles were filtered on centrifugal-driven filtration units and reconstituted in 150 ⁇ l of HBS each. The slow cooling technique resulted in close to 100% conversion of the peptide lipids to particles having high axial ratios.
  • This example describes a stability study to determine the stability of the cylinders at physiological temperatures
  • Tubules of (Pro) 3 -Glu(NH-C 12 H 2 s) 2 were formed by dilution of MeOH solutions as described above in Example 12.
  • Tubules of (Pro) 3 -Glu(NH-C 16 H 33 ) 2 were formed by heating and cooling in HBS/MeOH mixtures as described above in Example 12.
  • tubules were then incubated in HBS for 1 hour at 38°C .
  • the results indicate that the stability of the tubules correlates with the T M , i. e., if the T M is greater than the temperature of the environment, then the tubules are stable.
  • the T M of (Pro) 3 Glu(NH-C 16 H 33 ) : is about 59°C, and the incubation of these tubules did not convert the tubules to different microstructures.
  • the T M of tubules of (Pro) 3 Glu(NH-C 12 H 25 ) 2 is about 29 9°C, and incubation of such tubules at physiological temperature converted the tubules into semi-clear micellar solutions.
  • This example describes a stability study of tubules at physiological pH .
  • Such tubules were then incubated for 45 hours at 40°C in the presence of fetal calf serum (FCS) or sonicated dioleyoyl-phosphatidylcholine (DOPC) liposomes in HBS at pH 7.4.
  • FCS fetal calf serum
  • DOPC dioleyoyl-phosphatidylcholine
  • This example describes the cleavage of a peptide coupled to ditetradecyl glutamide, namely ⁇ , ⁇ -Ditetradecyl N ⁇ -acetyl-glycyl-arginyl-alanyl-glycyl-gylcyl-alanyl-alanyl-prolyl-prolyl-prolyl-glutamide trifluoroacetate (substrate) . 5.46 nmoles of the
  • Tubules were precipitated by drop-wise addition of water to a 5 mM solution of the lipid in ethanol until the volume fraction of water reached 70%. The tubules were washed 7 times in distilled/deionized water by repeated centrifugation to remove traces of ethanol. The final pellet of tubules was resuspended in 150 ⁇ M NaCl, 50 mM Tris-HCl (pH 8.0) in the presence of 10 mM CaCl 2 .
  • the tubules were then incubated at 30°C in Tris-HCl buffer at pH 8.0 at a lipid concentration of 0.5 mM in the presence of 10 mM Ca + + .
  • Tris-HCl buffer pH 8.0 at a lipid concentration of 0.5 mM in the presence of 10 mM Ca + + .
  • 4 units (2.24 ⁇ g/ml) of Naja naja venom PLA 2 (Sigma Chemicals) were added to the tubules .
  • 100 ⁇ l aliquots were removed and quenched with 25 mM EDTA, which scavenges Ca ++ and stops the PLA 2 reaction.
  • FIG. 14 shows that the hydrolysis rate is substantially constant over the time period tested. The constant rate of hydrolysis continues until nearly all of the substrate is consumed.
  • a tritiated polypeptide derivative is prepared by reacting tritiated acetic anhydride with terminal amino group of a polypeptide attached to a cylinder-forming lipid, which was synthesized as stated above.
  • the tritiated derivative is then mjected subcutaneously into multiple rabbits.
  • the feces and urine of the test animals is then monitored for the presence of tritiated derivatives.
  • test animals are sacrificed for determining the total presence of tritiated derivatives in tissue samples from the test animals.
  • the continuous delivery of therapeutics using the cylindrical lipid microstructures as delivery vehicles is demonstrated.

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Abstract

L'invention concerne des agents thérapeutiques comprenant plusieurs composés thérapeutiques auto-assemblés pour former des microstructures à rapport axial élevé. Lesdits composés thérapeutiques correspondant à la formule HARFM-Th, HARFM étant un matériau à rapport axial élevé et Th une substance thérapeutique associée à celui-ci. L'agent thérapeutique peut également correspondre à la formule HARFM-S-Th, S étant un espaceur. La libération de la substance thérapeutique par l'agent suit généralement une cinétique d'ordre 0 ou une pseudo-cinétique de premier ordre. L'invention porte également sur un procédé d'administration d'une dose efficace d'une substance thérapeutique auto-assemblée pour former une microstructure HAR, à un animal ou à une personne.
PCT/US1996/018850 1995-11-22 1996-11-21 Liberation d'agents therapeutiques au moyen de composes auto-assembles pour former des microstructures a rapport axial eleve_ WO1997018840A2 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1146855A4 (fr) * 1998-12-22 2004-06-16 Univ Washington Apport de substance therapeutique au moyen de composes auto-assembles pour former des microstructures a rapport axial eleve
WO2009074569A1 (fr) * 2007-12-11 2009-06-18 Bracco International Bv Composés de ciblage et thérapeutiques avec un espaceur comprenant de la polyproline et microvésicules remplies de gaz comprenant lesdits composés
CN106083637A (zh) * 2016-06-06 2016-11-09 大连民族大学 味精类脂、味精脂质体的合成方法及其应用

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US5004566A (en) * 1987-02-06 1991-04-02 Geo-Centers, Inc. Process for fabrication of lipid microstructures from dry organic solvent
US5840707A (en) * 1993-10-04 1998-11-24 Albany Medical College Stabilizing and delivery means of biological molecules

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1146855A4 (fr) * 1998-12-22 2004-06-16 Univ Washington Apport de substance therapeutique au moyen de composes auto-assembles pour former des microstructures a rapport axial eleve
WO2009074569A1 (fr) * 2007-12-11 2009-06-18 Bracco International Bv Composés de ciblage et thérapeutiques avec un espaceur comprenant de la polyproline et microvésicules remplies de gaz comprenant lesdits composés
CN106083637A (zh) * 2016-06-06 2016-11-09 大连民族大学 味精类脂、味精脂质体的合成方法及其应用

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WO1997018840A3 (fr) 1997-10-09

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