WO2000064483A2 - Releasable linkage and compositions containing same - Google Patents
Releasable linkage and compositions containing same Download PDFInfo
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- WO2000064483A2 WO2000064483A2 PCT/US2000/010830 US0010830W WO0064483A2 WO 2000064483 A2 WO2000064483 A2 WO 2000064483A2 US 0010830 W US0010830 W US 0010830W WO 0064483 A2 WO0064483 A2 WO 0064483A2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—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
- A61K47/6911—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 liposome
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—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
- A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—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
- A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—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
- A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
- A61K47/544—Phospholipids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
Definitions
- the present invention relates to a compound comprised of a hydrophilic polymer, such as polyethyleneglycol, cleavably linked to an amine-containing ligand, which in preferred embodiments can be an amine-containing lipid, drug or protein.
- a hydrophilic polymer such as polyethyleneglycol
- the compounds are cleavable under mild thiolytic conditions to regenerate the amine- containing ligand in its original form.
- Hydrophilic polymers such as polyethylene glycol (PEG) have been used for modification of various substrates, such as polypeptides, drugs and liposomes, in order to reduce immunogenicity of the substrate and/or to improve its blood circulation lifetime.
- PEG polyethylene glycol
- parenterally administered proteins can be immunogenic and may have a short pharmacological half-life. Proteins can also be relatively water insoluble. Consequently, it can be difficult to achieve therapeutically useful blood levels of the proteins in patients. Conjugation of PEG to proteins has been described as an approach to overcoming these difficulties. Davis et al. in U.S. Pat. No.
- PEG-protein conjugates described to date suffer from several disadvantages. For example, modification of the protein with PEG often inactivates the protein so that the resulting conjugate has poor biological activity. Typically in the prior art to date, it is desired to have the PEG stably linked to the protein so that the beneficial properties provided by PEG remain. Another problem with some protein PEG conjugates is that upon decomposition of the conjugate undesirable products may be formed. PEG has also been described for use in improving the blood circulation lifetime of liposomes (U.S. Patent No. 5,103,556). Here, the PEG is covalently attached to the polar head group of a lipid in order to mask or shield the liposomes from being recognized and removed by the reticuloendothelial system. Liposomes having releasable PEG chains have also been described, where the PEG chain is released from the liposome upon exposure to a suitable stimulus, such as a change in pH
- the invention includes a compound having the general structure:
- R 1 is a hydrophilic polymer comprising a linkage for attachment to the dithiobenzyl moiety
- R 2 is selected from the group consisting of H, alkyl and aryl
- R 4 comprises an amine-containing ligand
- R 5 is selected from the group consisting of H, alkyl and aryl; and where orientation of CH 2 -R 3 is selected from the ortho position and the para position.
- R 5 is H and R 2 is selected from the group consisting of CH 3' C 2 H 5 and C 3 H 8 . In another embodiment, R 2 and R 5 are alky Is.
- the amine-containing ligand R 4 is selected from the group consisting of a polypeptide, an amine-containing drug and an amine-containing lipid.
- the lipid includes either a single hydrocarbon tail or a double hydrocarbon tail.
- the lipid is a phospholipid having a double hydrocarbon tail.
- the hydrophilic polymer R 1 can be, in yet another embodiment, selected from the group consisting of polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, poly e thy loxazoline , polyhydroxypropy loxazoline , polyhydroxypropy 1-methacry lamide , poly me thacry lamide, poly dimethyl-acry lamide, poly hydroxypropylmethacry late, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, polyaspartamide, copolymers thereof, and polyethyleneoxide- polypropylene oxide.
- the hydrophilic polymer R 1 is polyethyleneglycol. In another embodiment, when R 1 is polyethylene glycol, R 5 is H and R 2 is CH 3 or C 2 H 5
- the amine-containing ligand R 4 is a polypeptide.
- the polypeptide can be, in another embodiment, a recombinant polypeptide.
- Exemplary and preferred polypeptides include cytokines, such as interferons, interleukins, and growth factors, and enzymes.
- the invention includes a composition comprising a conjugate obtainable by reaction with a compound having the general structural formula:
- R 1 is a hydrophilic polymer comprising a linkage for attachment to the dithiobenzyl moiety
- R 2 is selected from the group consisting of H, alkyl and aryl
- R 4 comprises a leaving group
- R 5 is selected from the group consisting of H, alkyl and aryl
- orientation of CH 2 -R 3 is selected from the ortho position and the para position.
- the composition also includes a pharmaceutically-acceptable carrier, such as saline, buffer or the like.
- R 2 is selected from the group consisting of CH 3 , C 2 H 5 and C 3 H 8 .
- the leaving group in another embodiment, is derived from a compound selected from the group consisting of chloride, p ⁇ r -nitrophenol, ⁇ rt/z ⁇ -nitrophenol, N- hydroxy-tetrahydrophthalimide, N-hydroxysuccinimide, N-hydroxy-glutarimide, N- hydroxynorbornene-2,3-dicarboxyimide, 1-hydroxybenzotriazole, 3-hydroxypyridine, 4- hydroxypyridine, 2-hydroxypyridine, l-hydroxy-6-trifluoromethylbenzotriazole, immidazole, triazole, N-methyl-imidazole, pentafluorophenol, trifluorophenol and trichlorophenol.
- the claimed compound is reacted with an amine-containing ligand that displaces R 4 to form a conjugate that includes the amine-containing ligand.
- the amine-containing ligand can be a phospholipid.
- the hydrophilic polymer R 1 is polyethyleneglycol
- R 5 is H
- R 2 is CH 3 or C 2 H 5 .
- the composition containing the conjugate comprises a liposome.
- the liposome can further comprise an entrapped therapeutic agent.
- the amine-containing ligand comprises a polypeptide.
- the invention includes a liposome composition comprising liposomes which include a surface coating of hydrophilic polymer chains wherein at least a portion of the hydrophilic polymer chains have the general structure:
- R 1 is a hydrophilic polymer comprising a linkage for attachment to the dithiobenzyl moiety
- R 2 is selected from the group consisting of H, alkyl and aryl
- R 4 comprises an amine-containing ligand
- R 5 is selected from the group consisting of H, alkyl and aryl
- orientation of CH 2 -R 3 is selected from the ortho position and the para position.
- the liposomes have a longer blood circulation lifetime than liposomes having hydrophilic polymer chains joined to the liposome via an aliphatic disulfide linkage.
- the liposome further comprises an entrapped therapeutic agent.
- the invention includes a method for improving the blood circulation lifetime of liposomes having a surface coating of releasable hydrophilic polymer chains.
- the method includes preparing liposomes that have between about 1 % to about 20% of a compound having the general structure:
- R 1 , R 2 , R 3 , and R 5 are as described above and R 4 comprises an amine- containing lipid.
- R 5 is H and R 2 is selected from the group consisting of CH 3 , C 2 H 5 and C 3 H 8 .
- the amine-containing lipid comprises a phospholipid and R 1 is polyethyleneglycol.
- the liposomes can further comprise an entrapped therapeutic agent.
- Fig. 1A shows an embodiment of the invention where the dithiobenzyl (DTB) links a methoxy-polyethyelene glycol (mPEG) moiety and the amine-containing ligand;
- Fig. IB shows the products after thiolytic cleavage of the compound in Fig. 1A;
- Fig. 2 illustrates a synthetic reaction scheme for synthesis of the mPEG-DTB- amine-lipid, where the amine-ligand is the lipid distearoylphosphatidylethanolamine (DSPE);
- DTB dithiobenzyl
- mPEG methoxy-polyethyelene glycol
- Fig. 3 illustrates the thiolytic cleavage mechanism of a /j ⁇ ra-dithiobenzyl urethane (DTB)-linked mPEG-DSPE conjugate;
- Figs. 4A-4B show a synthetic reaction scheme for preparation of an mPEG-DTB- DSPE compound in accord with the invention where the DTB linkage is sterically hindered by an alkyl group;
- Fig. 5 shows another synthetic reaction scheme for preparation of an mPEG- DTB-ligand compound in accord with the invention
- Fig. 6 A is a synthetic reaction scheme for synthesis of an mPEG-DTB-lipid which upon thiolytic cleavage yields a cationic lipid;
- Fig. 6B shows the products after thiolytic cleavage of the compound in Fig. 6A;
- Fig. 7A shows the rate of cleavage of ⁇ rt/. ⁇ -1- ⁇ PEG-DTB-DSPE and para-mPEG- DTB-DSPE conjugates in solution to form micelles in buffer alone (ort/zo-conjugate (*); /? ⁇ r ⁇ -conjugate (+)) and in the presence of 150 ⁇ M cysteine ( ⁇ rtb ⁇ -conjugate (open circles); /? ⁇ r ⁇ -conjugate (open squares);
- Fig. 7B shows the rate of cleavage of micellar mPEG-DTB-DSPE conjugates as described in Fig. 7A and of ⁇ rtb ⁇ -mPEG-DTB-DSPE (solid circles) and para-mPEG- DTB-DSPE (solid squares) conjugates formulated in liposomes and incubated in the presence of 150 ⁇ M cysteine;
- Figs. 8A-8B show percentage of content release of entrapped fluorophore from liposomes comprised of DOPE:ortbo-mPEG-DTB-DSPE (Fig. 8A) or of OOPE ⁇ ara- mPEG-DTB-DSPE (Fig. 8B) incubated in the presence of cysteine at the indicated concentrations;
- Fig. 9 A shows normalized percent release of entrapped fluorophore as a function of time for liposomes comprised of DOPE and ⁇ ra-mPEG-DTB-DSPE.
- the percent release of entrapped fluorophore is normalized with respect to percent release of fluorophore from liposomes incubated in the absence of cysteine.
- the release rate from liposomes incubated in the presence of cysteine at concentrations of 15 ⁇ M (solid squares), 75 ⁇ M (open triangles), 150 ⁇ M (X symbols), 300 ⁇ M (open circles), 1500 ⁇ M (solid circles), 3000 ⁇ M (+ symbols), and 15000 ⁇ M (open diamonds) is shown;
- Fig. 9B shows normalized percent release of entrapped fluorophore as a function of time for liposomes comprised of DOPE and /7-zr ⁇ -mPEG-MeDTB-DSPE.
- the percent release of entrapped fluorophore is normalized with respect to percent release of fluorophore from liposomes incubated in the absence of cysteine.
- the release rate for liposomes incubated in the presence of cysteine at concentrations of 15 ⁇ M (solid squares), 75 ⁇ M (open triangles), 150 ⁇ M (X symbols), 300 ⁇ M (open circles), 1500 ⁇ M (solid circles), 3000 ⁇ M (+ symbols), and 15000 ⁇ M (open diamonds) is shown; Fig.
- FIG. 9C shows normalized percent release of entrapped fluorophore as a function of time for liposomes comprised of DOPE and mPEG-meDTB-distearoyl-glycerol compound of Fig. 6A.
- the percent release of entrapped fluorophore is normalized with respect to percent release of fluorophore from liposomes incubated in the absence of cysteine.
- Fig. 10 is a plot showing the amount of liposomes, in counts per minute/mL of liposomes containing entrapped In 111 , in blood samples taken from mice at various times after injection of liposomes comprised of PHPC: cholesterol :mPEG-DTB-DSPE
- One group of animals received a 200 ⁇ L injection of 200 mM cysteine at time zero (solid squares).
- the control group was injection with saline at time zero (open circles);
- Fig. 11A shows a synthetic reaction scheme for synthesis of an mPEG-DTB- protein compound in accord with another embodiment of the invention
- Fig. 11B shows the decomposition products after thiolytic cleavage of the compound in Fig. 11 A;
- Fig. 12 is a rendering of a photograph of an sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profile of lysozyme reacted for 15 minutes (Lane 1) or for 1 hour (Lane 2) with mPEG-MeDTB- nitrophyenylchloroformate to form a mPEG-MeDTB-lysozyme conjugate, native lysozyme (Lane 3), lysozyme reacted for 1 hour with mPEG-nitrophenylchloroformate (Lane 4), molecular weight markers (Lane 5), and the samples of Lanes 1-4 treated with 2% ⁇ -mercaptoethanol for 10 minutes at 70°C (Lanes 6-9);
- Fig. 13 shows the decomposition products after thiolytic cleavage of the an mPEG-DTB- -nitroanilide conjugate
- Fig. 14A shows the absorbence as a function of wavelength, in nm, of mPEG- MeDTB-para-nitroanilide (closed diamonds) and after in vitro incubation with 5 mM cysteine for 2 minutes (closed squares), 5 minutes (x symbols), 10 minutes (open squares), 20 minutes (triangles), 40 minutes (open diamonds) and 80 minutes (closed circles); and
- Fig. 14B shows the amount of ⁇ r -nitroanilide, in mole/L, released in vitro as a function of time, in minutes, from mPEG-MeDTB-p ⁇ ra-nitroanilide conjugate incubated in the presence of 5 mM cysteine (closed circles), 1 mM cysteine (closed squares) and 0.15 mM cysteine (closed diamonds).
- Polypeptide refers to a polymer of amino acids and does not refer to a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, and enzyme are included within the definition of polypeptide. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like.
- Ammonia-containing intends any compound having a moiety derived from ammonia by replacing one or two of the hydrogen atoms by alkyl or aryl groups to yield general structures RNH 2 (primary amines) and R 2 NH (secondary amines), where
- R is any hydrocarbyl group.
- Hydrophilic polymer refers to a polymer having moieties soluble in water, which lend to the polymer some degree of water solubility at room temperature.
- exemplary hydrophilic polymers include polyvinylpyrrolidone, poly vinylme thy lether, poly methy loxazoline, poly ethy loxazoline, polyhydroxypropy loxazoline , polyhydroxypropy 1-methacry lamide , polymethacrylamide , poly dime thy 1-acry lamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, polyaspartamide, copolymers of the above-recited polymers, and polyethyleneoxide-polypropylene oxide copolymers. Properties and reactions with many of these polymers are described in U.S. Patent Nos. 5,395,619 and 5,631 ,018.
- Polymer comprising a reactive functional group or “polymer comprising a linkage for attachment” refers to a polymer that has been modified, typically but not necessarily, at a terminal end moiety for reaction with another compound to form a covalent linkage. Reaction schemes to functionalize a polymer to have such a reactive functional group of moiety are readily determined by those of skill in the art and/or have been described, for example in U.S. Patent No. 5,613,018 or by Zalipsky et al. , in for example, Eur. Polymer. J. , 19(12): 1177-1183 (1983); Bioconj. Chem. , 4(4):296-299 (1993).
- Alkyl as used herein intends a group derived from an alkane by removal of a hydrogen atom from any carbon atom: "C n H 2n+ 1 " .
- the groups RCH 2 -, R 2 CH- (R not equal to H), and R 3 C- (R not equal to H) are primary, secondary and tertiary alkyl groups respectively.
- Aryl refers to a substituted or unsubstituted monovalent aromatic radical having a single ring (e.g. , benzene) or two condensed rings (e.g. , naphthyl). This term includes heteroaryl groups, which are aromatic ring groups having one or more nitrogen, oxygen, or sulfur atoms in the ring, such as furyl, pyrrole, pyridyl, and indole.
- substituted is meant that one or more ring hydrogens in the aryl group is replaced with a halide such as fluorine, chlorine, or bromine; with a lower alkyl group containing one or two carbon atoms; nitro, amino, memylamino, dimethylamino, methoxy, halomethoxy, halomethyl, or haloethyl.
- a halide such as fluorine, chlorine, or bromine
- R' and R" are linear or branched alkyl chains that may be further substituted.
- PEG poly (ethylene glycol); mPEG, methoxy-PEG; DTB, dithiobenzyl; MeDTB, methyl-dithiobenzyl, EtDTB, ethyl- dithiobenzyl; DSPE, distearoyl phosphatidylethanolamine; DOPE, dioleoyl phosphatidylethanolamine; PHPC, partially hydrogenated phosphatidylcholine; MALDI- TOFMS, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry.
- the invention comprises a compound of the form:
- R 1 comprises a hydrophilic polymer including functional group suitable for covalently attaching the polymer to the dithiobenzyl moiety.
- R 2 and R 5 are independently selected to be H, an alkyl or an aryl, and, as will be seen, can be varied to tailor the rate of disulfide cleavage. For example, to achieve a faster rate of cleavage, R 2 and R 5 are hydrogen. A slower rate of cleavage is achieved by sterically hindering the disulfide by selecting an alkyl or aryl for one or both of R 2 and R 5 .
- R 3 comprises a linking moiety joined to R 4 , which comprises an amine-containing ligand.
- the amine-containing ligand R 4 can be a primary or a secondary amine and can be selected from any number of substrates, including, but not limited to lipids, drugs, polypeptides, viruses, surfaces of biomaterials and aminoglycosides.
- R 4 is a primary or secondary amine-containing lipid, drug or polypeptide.
- the orientation of the group CH 2 -R 3 can be either ortho or para. Fig.
- R 1 is the hydrophilic polymer methoxy-polyetheylene glycol
- mPEG CH 3 O(CH 2 CH 2 O) n
- n is from about 10 to about 2300, which corresponds to molecular weights of about 440 Daltons to about 100,000 Daltons.
- the molecular weight of the polymer depends to some extent on the selection of R 3 .
- R 3 is an amine-containing lipid for use in a liposome
- a preferred range of PEG molecular weight is from about 750 to about 10,000 Daltons, more preferably from about 2,000 to about 5,000 Daltons.
- the mPEG in this embodiment includes a urethane linking moiety.
- R 3 is an amine-containing polypeptide
- a preferred range of PEG molecular weight is from about 2,000 to about 40,000 Daltons, more preferably from about 2,000 to about 20,000 Daltons.
- R 1 can be selected from a variety of hydrophilic polymers, and exemplar polymers are recited above. It will also be appreciated that for some ligands, such as polypeptides, the molecular weight of the polymer may depend on the number of polymer chains attached to the ligand, where a larger molecular weight polymer is often selected when the number of attached polymer chains is small.
- R 2 and R 5 in this exemplary compound are H, however either or both R 2 and R 5 can also be a straight chain or branched alkyl or an aryl group.
- R 5 is H and R 2 is an alkyl, and several examples are given below.
- Fig. IB shows the mechanism of thiolytic cleavage of the mPEG-DTB-(NH 2 - ligand) compound of Fig. 1A.
- the ortho- or p ⁇ ra-dithiobenzyl carbamate moiety is cleavable under mild thiolytic conditions, such as in the presence of cysteine or other naturally-occurring reducing agents.
- the amine-containing ligand is regenerated in its natural, unmodified form.
- Studies in support of the invention, described below, show that natural, physiologic conditions in vivo are sufficient to initiate and achieve cleavage of the DTB linkage. It will be appreciated that a reducing agent can also be administered to artificially induce thiolytic conditions sufficient for cleavage and decomposition of the compound.
- the amine-containing ligand comprises an amine-containing polypeptide, drug or lipid. Examples of these embodiments will now be described.
- the amine-containing ligand is an amine-containing lipid.
- Lipids as referred to herein intend water-insoluble molecules having at least one acyl chain containing at least about eight carbon atoms, more preferably an acyl chain containing between about 8-24 carbon atoms.
- a preferred lipid is a lipid having an amine-containing polar head group and an acyl chain.
- Exemplary lipids are phospholipids having a single acyl chain, such as stearoylamine, or two acyl chains.
- Preferred phospholipids with an amine-containing head group include phosphatidylethanolamine and phosphatidylserine.
- the lipid tail(s) can have between about 12 to about 24 carbon atoms and can be fully saturated or unsaturated.
- One preferred lipid is distearoylphosphatidylethanolamine (DSPE), however those of skill in the art will appreciate the wide variety of lipids that fall within this description. It will also be appreciated that the lipid can naturally include an amine group or can be derivatized to include an amine group. Other lipid moieties that do not have an acyl tail, such as cholesterolamine, are also suitable.
- mPEG derivatives (MW 2000 and 5000 Daltons) having a methoxycarbonyldithioalkyl end group were prepared by reacting 2- (methoxycarbonyldithio)ethaneamine with mPEG-chloroformate, which was readily prepared by phosgenation of dried mPEG-OH solution (Zalipsky, S. , et al., Biotechnol. Appl. Biochem. 15:100-114 (1992).).
- the former compound was obtained through 2- aminoethanethiol hydrochloride reaction with an equivalent amount of mefhoxycarbonylsulfenyl chloride, according to published procedures (Brois, S.J., et al., J. Amer. Chem. Soc. 92:7629-7631 (1970); Koneko, T., et al., Bioconjugate Chem. 2:133-141 (1991)).
- Both the para and ortho isomers of mercaptobenzyl alcohol (Grice, R., et al, J. Chem. Soc. 1947-1954 (1963)) coupled cleanly with the resulting PEG-linked acyldisulfide, yielding mPEG bearing a dithio benzyl alcohol end group.
- Fig. 3 shows the mechanism of thiolytic cleavage of the mPEG-DTB-DSPE conjugate. Upon cleavage, the phosphatidylethanolamine lipid is regenerated in its natural, unmodified form.
- Figs. 4A-4B show a reaction scheme for synthesis of mPEG-DTB-DSPE conjugates having an alkyl group adjacent the disulfide linkage, e.g. , a more hindered disulfide linkage.
- mPEG-OH in dichloromethane was reacted with p-nitrophenylchloroformate in the presence of triethylamine (TEA) to form mPEG-nitrophenyl carbonate.
- TAA triethylamine
- mPEG-methyl-dithiobenzyl- nitrophenyl chloroformate was reacted with DSPE to form the desired compound.
- the nitrophenyl chloroformate moiety in the mPEG-methyl-dithiobenzyl-nitrophenyl chloroformate compound acts as a leaving group to yield the desired product upon reaction with a selected lipid.
- the invention contemplates, in another aspect, a composition that comprises a compound produced by reaction with a compound such as mPEG-methyl-dithiobenzyl-R 3 , where R 3 represents a leaving group joined through a linking moiety to the benzene ring.
- the leaving group is displaced upon reaction with an amine-containing ligand, such as DSPE, a polypeptide or an amine-containing drug.
- the leaving group is selected according to the reactivity of the amine in the ligand, and is preferably derived from various acidic alcohols that have a hydroxy- or oxy-containing leaving group.
- Example 2B describes preparation of an mPEG-EtDTB-lipid conjugate where the disulfide linkage is hindered by an ethyl moiety.
- Fig. 5 shows another synthetic reaction scheme for preparation of an mPEG- DTB-ligand compound in accord with the invention.
- the details of the reaction procedure are given in Examples 3A-3B. Briefly, cold l-amino-2-propanol was reacted with sulfuric acid to form 2-amino-l-methylethyl hydrogen sulfate. This product was reacted with carbon disulfide and sodium hydroxide in aqueous ethanol to yield 5- methylthiazolidine-2-thione. An aqueous solution of hydrochloric acid was added to the 5-methylthiazolidine-2-thione and heated. After refluxing for one week, the product, 1- mercapto(methyl)ethyl ammonium chloride, was crystallized and recovered. This product was reacted with methoxy carbonylsulfenyl chloride to yield 2- (methoxycarbonyldithio)ethaneamine. Reaction of the 2-
- Fig. 6A shows a reaction scheme for preparation of another mPEG-DTB-lipid compound in accord with the invention.
- the reaction details are provided in Example 4.
- the lipid 1,2-distearoyl-sn-glycerol is activated for reaction with mPEG-DTB-nitropheynl, prepared as described in Fig. 4A or Fig. 5.
- the resulting mPEG-DTB-lipid differs from the compounds described above in the absence of a phosphate head group.
- the mPEG- DTB-lipid of Fig. 6A is neutral prior to cleavage.
- Fig. 6B upon thiolytic reduction of the disulfide bond, the compound decomposes to yield a cationic lipid.
- the positively-charged lipid provides for electrostatic interaction in vivo and commensurate advantages in in vivo targeting.
- R 5 of the claimed compound is H.
- R 5 is an alkyl or an aryl moiety.
- the acetylthio group (-SCOCH 3 ) is hydrolyzed to a thiol group (-SH), which is then reacted with methyl (chlorosulfenyl)formate (ClSCOOCH 3 ), generating a methoxycarbonyl diothio group (- SSCOOCH 3 );
- this intermediate is then reacted with p-mercapto benzyl alcohol to give the N-PEG-substituted ⁇ -(dithiobenzyl alcohol) amide (having the structure PEG-NH- CO-CH 2 CR'R"-SS-/?-phenyl-CH 2 OH).
- the benzyl alcohol moiety is then reacted with nitrophenyl chloroformate to give the
- the ortho- and para- compounds represented by the open circles and the open squares, respectively, in the presence of 150 ⁇ M cysteine cleave as shown in Fig. 7A.
- the ort/r ⁇ -compound exhibited a slightly faster rate of decomposition than its para counterpart (T 1/2 «12 minutes vs. «18 minutes).
- Liposome Compositions Comprising an mPEG-DTB-lipid Compound a. In vitro Characterization
- the mPEG-DTB-lipid compound is formulated into liposomes.
- Liposomes are closed lipid vesicles used for a variety of therapeutic purposes, and in particular, for carrying therapeutic agents to a target region or cell by systemic administration of liposomes.
- liposomes having a surface coating of hydrophilic polymer chains, such as polyethylene glycol (PEG) are desirable as drug carries, since these liposomes offer an extended blood circulation lifetime over liposomes lacking the polymer coating.
- the polymer chains in the polymer coating shield the liposomes and form a "stiff brush" of water solvated polymer chains about the liposomes.
- the polymer acts as a barrier to blood proteins, preventing binding of the protein and recognition of the liposomes for uptake and removal by macrophages and other cells of the reticuloendothelial system.
- liposomes having a surface coating of polymer chains are prepared by including in the lipid mixture between about 1 to about 20 mole percent of the lipid derivatized with the polymer.
- the actual amount of polymer derivatized lipid can be higher or lower depending on the molecular weight of the polymer.
- liposomes are prepared by adding between about 1 to about 20 mole percent of the polymer-DTB-lipid conjugate to other liposome lipid bilayer components.
- liposomes containing the polymer- DTB-lipid conjugate of the invention have a blood circulation lifetime the is longer than liposomes containing a polymer-lipid conjugate where the polymer and lipid are joined by a cleavable aliphatic disulfide bond.
- liposomes comprised of the vesicle-forming lipid partially hydrogenated phosphatidyl choline along with cholesterol and the ⁇ rt/z ⁇ -mPEG-DTB-DSPE or the ⁇ ra-mPEG-DTB-DSPE compound were prepared as described in Example 6. Cysteine-mediated cleavage of the mPEG-DTB- DSPE compounds was monitored in the presence and absence of 150 ⁇ M cysteine in an aqueous buffer. The results are shown in Fig. 7B, which includes the data of Fig. 7 A for comparison. In Fig.
- the ortho- and /? ⁇ ra-compounds in micellar form in the absence of cysteine show no cleavage, which indicates stability of the conjugate in the absence of thiols.
- the open circles and the open squares correspond to the ortho- and/? ⁇ ra-compounds, respectively, in micellar form in the presence of cysteine, as discussed above with respect to Fig. 7A.
- the solid circles and the solid squares correspond to the ortho- and ⁇ r ⁇ -compounds, respectively, in liposomal form in the presence of cysteine.
- Fig. 7B shows that both the ortho and para compound were slightly more resistant to thiolytic cleavage when incorporated into liposomes.
- Examination of the thiolysis reaction products by TLC sica gel G, chloroform / methanol / water 90: 18:2) (Dittmer, J.C. , et al , J. Lipid Res. 5:126-127 (1964)) showed DSPE as the sole lipid component and another spot corresponding to a thiol-bearing, lip id-free mPEG derivative.
- liposomes were prepared from the lipid dioleoyl phosphatidylethanolamine (DOPE) and either the ortho-mPEG- DTB-DSPE or the ⁇ ra-mPEG-DTB-DSPE compound were prepared.
- DOPE is a hexagonal phase lipid which alone does not form lipid vesicles.
- liposomes will form when DOPE is combined with a few mole percent of the mPEG-DTB-DSPE compound. Cleavage of the mPEG-DTB-DSPE compound triggers decomposition of the liposomes and release of liposomally-entrapped contents.
- the content release characteristics of such liposomes provides for a convenient quantitative evaluation of cleavable PEG-bearing liposomes.
- Liposomes comprised of DOPE and the ortho- or r ⁇ ra-mPEG-DTB-DSPE compound were prepared as described in Example 7A with entrapped fluorophores, p- xylene-bis-pyridinium bromide and trisodium 8-hydroxypyrenetrisulfonate. Release of the fluorophores from liposomes incubated in the presence of cysteine at various concentrations was monitored as described in Example 7B.
- Fig. 8A results for liposomes comprising the ⁇ rt/r ⁇ -compound are shown in Fig. 8A, where percentage of content release of entrapped fluorophore from liposomes incubated in the presence of cysteine at concentrations of 15 ⁇ M (solid diamonds), 150 ⁇ M (solid inverted triangles), 300 ⁇ M (solid triangles) and 1.5 mM (solid circles) are shown.
- Fig. 8B is a similar plot for liposomes comprising fhe ⁇ r ⁇ -compound, where the liposomes are incubated in cysteine at concentrations of 15 ⁇ M (solid diamonds), 300 ⁇ M (solid triangles), 1 ⁇ M (solid squares) and 1.5 mM (solid circles).
- Figs. 8A results for liposomes comprising the ⁇ rt/r ⁇ -compound are shown in Fig. 8A, where percentage of content release of entrapped fluorophore from liposomes incubated in the presence
- liposomes comprised of DOPE and three different mPEG-DTB-lipid compounds were prepared.
- the liposomes were prepared as described in Example 7 and included and entrapped fluorophore.
- the three mPEG-DTB-lipid compounds were mPEG-DTB-DSPE as shown in Fig. 1A; mPEG- MeDTB-DSPE as shown in Fig.
- Fig. 6A The liposomes were comprised of 97 mole percent DOPE and 3 mole percent of one of the mPEG-DTB-lipid compounds. Cysteine-mediated rate of cleavage of the compounds was determined by monitoring the release of entrapped fluorophore as a function of time in the presence of various cysteine concentrations. The results are shown in Figs. 9A-9C where the percent release of entrapped fluorophore is normalized for the release rate from liposomes incubated in buffer alone. Fig.
- FIG. 9A shows the percent release of entrapped fluorophore as a function of time for liposomes comprised of DOPE and /? r ⁇ -mPEG-DTB-DSPE (compound of Fig. 1A).
- the release rate from liposomes containing the conjugate and incubated in the presence of cysteine at concentrations of 15 ⁇ M (solid squares), 75 ⁇ M (open triangles), 150 ⁇ M (X symbols), 300 ⁇ M (open circles), 1500 ⁇ M (solid circles), 3000 ⁇ M (+ symbols), and 15000 ⁇ M (open diamonds) is shown.
- FIG. 9B shows the percent release of entrapped fluorophore as a function of time for liposomes comprised of DOPE and para mPEG-MeDTB-DSPE (compound of Fig. 4B).
- the release rate of the fluorophore from liposomes incubated in the presence of cysteine at concentrations of 15 ⁇ M (solid squares), 75 ⁇ M (open triangles), 150 ⁇ M (X symbols), 300 ⁇ M (open circles), 1500 ⁇ M (solid circles), 3000 ⁇ M (+ symbols), and 15000 ⁇ M (open diamonds) is shown.
- Fig. 9C is a similar plot for liposomes formed with DOPE and mPEG-MeDTB- distearoyl glycerol (compound of Fig. 6A).
- the release rate of dye from liposomes incubated in the presence of cysteine at concentrations of 15 ⁇ M (solid squares), 75 ⁇ M (open triangles), 150 ⁇ M (X symbols), 300 ⁇ M (open circles), 1500 ⁇ M (solid circles), 3000 ⁇ M (+ symbols), and 15000 ⁇ M (open diamonds) is shown.
- Figs. 9A-9C show that the rate of mPEG-MeDTB-lipid cleavage is cysteine- concentration dependent, with a slow rate of cleavage, as evidenced by release of entrapped fluorophore, at cysteine concentrations of 15-75 ⁇ M.
- the mPEG-MeDTB-DSPE compound Fig. 9B
- Fig. 9A cleaves approximately 10 times more slowly than the mPEG-DTB-DSPE compound (Fig. 9A).
- the rate of cleavage can be tailored according to the R moiety (see Fig. 2) in the DTB linkage.
- cleavage of the polymer-DTB-lipid compound of the invention results in regeneration of the original lipid in unmodified form. This is desirable since unnatural, modified lipids can have undesirable in vivo effects. At the same time, the compound is stable when stored in the absence of reducing agents.
- the blood circulation lifetime of liposomes containing the mPEG-DTB-lipid were compared to liposomes containing a polymer-lipid conjugate where the polymer and lipid are joined by a cleavable aliphatic disulfide bond.
- Aliphatic disulfide linkages are readily cleaved in vivo and the blood circulation lifetime of liposomes having polymer chains grafted to their surface by an aliphatic disulfide typically do not have the extended blood circulation lifetime observed for liposomes having stably linked polymer chains.
- the dithiolbenzyl linkage of the invention and in particular the more hindered DTB linkages, are more stable in vivo and achieve a longer blood circulation lifetime than liposomes with polymer chains attached via an aliphatic disulfide linkage.
- the invention includes a compound as described with respect to Fig. 1A, where the aimne-containing ligand is a polypeptide.
- a synthetic reaction scheme showing preparation of a polymer-DTB-polypeptide is shown in Fig. 11A, with mPEG as the exemplary polymer.
- a mPEG-DTB-leaving group compound is prepared according to one the synthetic routes described above in Figs. 2, 4A and 5.
- the leaving group can be nitrophenyl carbonate or any one of the others described above.
- the mPEG-DTB-nitrophenyl carbonate compound is coupled to an amine moiety in a polypeptide by a urethane linkage.
- the R group adjacent the disulfide in the compound can be H, CH 3 , C 2 H 5 or the like and is selected according to the desired rate of disulfide cleavage.
- Fig. 1 IB shows the decomposition products upon cysteine-mediated cleavage of the compound. As seen the native protein with no modification to the protein amine group is regenerated upon cleavage.
- polymer chains such as PEG
- Attachment of polymer chains, such as PEG, to a polypeptide often diminishes the enzymatic or other biological activity, e.g. , receptor binding, of the polypeptide.
- polymer modification of a polypeptide increases the blood circulation lifetime of the polypeptide.
- the polymer-modified polypeptide is administered to a subject.
- physiologic reducing conditions such as blood cysteine and other in vivo thiols
- the biological activity of the polypeptide is gradually restored. In this way, the polypeptide initially has a sufficient blood circulation lifetime for biodistribution, and over time regains its full biological activity as the polymer chains are cleaved.
- lysozyme was used as a model polypeptide and an mPEG-MeDTB-lysozyme conjugate was prepared by a synthetic route similar to those described above. Lysozyme was incubated with mPEG-MeDTB- nitrophenylcarbonate in 0.1 M borate, at pH 9 at a 2: 1 ratio of nitrophenylcarbonate to amino group of lysozyme. After reactions times of 15 minutes and 3 hours, samples were characterized by SDS-PAGE. A comparative compound was prepared by reacting lysozyme under the same conditions for 60 minutes with a conjugate of mPEG-nitropheny] carbonate, which will form a stable mPEG-lysozyme conjugate.
- Fig. 12 shows a rendering of the SDS-PAGE gel.
- Lane 1 corresponds to the compound formed after 15 minutes reaction of lysozyme with mPEG-MeDTB- nitrophyenylcarbonate and
- Lane 2 represents the compound formed after a 1 hour reaction time of the same compounds.
- Lane 3 represents native lysozyme and Lane 4 corresponds to lysozyme reacted for 1 hour with mPEG-nitrophenylcarbonate.
- the molecular weight markers in Lane 5 are as follows, from the top down:
- Lanes 6-9 of the SDS-PAGE profile correspond to the samples in Lanes 1-4 after treatment with 2% ⁇ -mercaptoethanol for 10 minutes at 70°C.
- the stable mPEG-lysozome compound was not affected upon incubation with a reducing agent, as evidenced by the agreement in the profile in Lane 9 and Lane 4.
- any of the hydrophilic polymers described above are contemplated for use.
- the molecular weight of the polymer is selected depending on the polypeptide, the number of reactive amines on the polypeptide and the desired size of the polymer-modified compound.
- Polypeptides contemplated for use are unlimited and can be naturally-occurring or recombinantly produced polypeptides. Small, human recombinant polypeptides are preferred, and polypeptides in the range of 10-30 KDa are preferred.
- Exemplary polypeptides include cytokines, such as tumor necrosis factor (TNF), inter leukins and interferons, erythropoietin (EPO), granulocyte colony stimulating factor (GCSF), enzymes, and the like.
- Viral polypeptides are also contemplated, where the surface of a virus is modified to include one or more polymer chain linked via a DTB reversible linkage. Modification of a virus containing a gene for cell transfection would extend the circulation time of the virus and reduce its immunogenicity, thereby improving delivery of an exogeneous gene.
- a compound of the form polymer-DTB- amine-containing drug is contemplated.
- the compound is of the structure described above, and in particular with respect to Fig. 1A where the -imine-containing ligand in the figure is the amine-containing drug. Modification of therapeutic drugs with PEG is effective to improve the blood circulation lifetime of the drug and to reduce any immunogenicity.
- a polymer-DTB-amine-containing drug is prepared according to any of the reaction schemes described above, with modifications as necessary to provide for the particular drug.
- a wide variety of therapeutic drugs have a reactive amine moiety, such as mitomycin C, bleomycin, doxorubicin and ciprofloxacin, and the invention contemplates any of these drugs with no limitation. It will be appreciated that the invention is also useful for drugs containing an alcohol or carboxyl moiety.
- the polymer-DTB moiety can be linked to the drug via urethane, ester, ether, thioether or thioester linkages.
- the polymer-DTB-drug compound after administration in vivo thiolytically decomposes to regenerate the amine-containing drug in its native, active form, therapeutic activity of the compound after modification and prior to administration is not necessary.
- modification of the drug with the DTB-polymer causes a reduction or loss of therapeutic activity, after administration and cleavage of the DTB- polymer from the drug, activity of the drug is regained.
- the drug nitroanilide was reacted with mPEG-MeDTB-nitrophenylcarbonate to form an mPEG-MeDTB- ⁇ ra-nitroanilide compound, as shown in Fig. 13.
- Decomposition of the compound upon exposure to a reducing agent yields the products shown in the figure, with the drug p ⁇ ra-nitroanilide regenerated in an unmodified state.
- Fig. 14A Seen in the figure are samples measured at the following time points: time zero (closed diamonds), 2 minutes (closed squares), 5 minutes (x symbols), 10 minutes (open squares), 20 minutes (triangles), 40 minutes (open diamonds) and 80 minutes (closed circles).
- time zero closed diamonds
- 2 minutes closed squares
- 5 minutes x symbols
- 10 minutes open squares
- 20 minutes triangles
- 40 minutes open diamonds
- 80 minutes closed circles
- the compounds of the invention comprise an ami-ne-containing ligand reversibly joined to a hydrophilic polymer via an ortho or para-disulfide of a benzyl urethane linkage.
- This linkage when subjected to mild thiolytic conditions is cleaved to regenerate the original amine-containing ligand in its unmodified form.
- the rate of cleavage can be controlled by steric hinderance of the disulfide in the linkage and/or by controlling the thiolytic conditions in vivo.
- the compounds prior to cleavage of the dithiobenzyl linkage are provided with an increased blood circulation lifetime, improved stability and reduced immunogenicity.
- the ortho conjugate differed only in benzyl and aromatic signals at 5.11 (s, CH 2 , 2H), and 7.31 (d, 1H), 7.39 (m, 2H) 7.75(d, 1H) ppm.
- 5 MALDI-TOFMS produced a distribution of ions spaced at equal 44 Da intervals, corresponding to the ethylene oxide repeating units.
- the average molecular weights of the compounds was 3127 and 3139 Da o para and ortho isomers respectively (theoretical molecular weight -3100 Da).
- Acetic acid (445.57 ⁇ l, 7.42 mmol, 3.75 eq) was added to the reaction mixture to neutralize excess of sodium hydride. The solution became thick and whitish. Solvent was evaporated and the solid was recrystallized with ethyl acetate (30 ml) and isopropanol (70ml). The product mixture did not dissolve completely, while precipitate filtered off. Then the product mixture was recrystallyzed with isopropanol/tert-butyl alcohol (100ml/20ml). Yield: 8.87 g (84%).
- MALDI-TOFMS produced a bell shaped distribution of ions spaced at equal 44 Da intervals, corresponding to the ethylene oxide repeating units.
- the average molecular mass of the conjugate and mPEG-thiol (mostly cleaved disulfide) is 6376 and 5368 Da (theoretical molecular mass " 6053, and 5305 Daltons).
- N-hydroxy-s-norbornene-2,3-dicarboxylic acid imide (HONB) (48 mg, 0.269 mmol) was added to DSPE (55 mg, 0.073 mmol) in CHC1 3 (3 ml) at 50°C (oil bath temperature). After 3-4 minutes it became a clear solution. Then mPEG-EtDTB- nitrophenylchloroformate (334 mg, 0.134 mmol) was added, followed by triethylamine (TEA, 45 ⁇ l, 0.329 mmol).
- TEA triethylamine
- the reaction was a heterogeneous mixture, with the starting material as oil which was denser than water. After one week the oil starting material was gone, although still visible on TLC.
- the reaction was removed from heat and allowed to cool to room temperature, and then was refrigerated to crystallize starting material. The crystallized starting material was filtered. Filtrate was evaporated and it was dried over P 2 O 5 and NaOH to remove all water and HCI.
- the crude product was washed with two portions of diethyl ether (50 ml each) to remove all starting material. It was again dried over P 2 O 5 . Yield: 2.83 g (45 %).
- the reaction was refluxed (85°C) for 40 minutes, after which time it turned bright yellow and a thick precipitate was formed. Ethanol was evaporated and then the aqueous solution was warmed and then filtered through a Buchner funnel to remove all water-soluble impurities. The remaining crystals were dissolved in warm ethanol and then warm water was added until the solution was 80% water. The mixture was allowed to cool and then refrigerated, yielding needle-like crystals.
- Example 5 In vitro Cleavage of mPEG-DTB-DSPE Compound Ortbo-mPEG-DTB-DSPE and para- mPEG-DTB-DSPE (prepared as described in
- Example 1 were added to a buffered aqueous solution (pH 7.2) in the presence and absence of 150 ⁇ M cysteine. Disappearance of the conjugates was monitored by HPLC
- lipids partially hydrogenated phosphatidylcholine (PHPC), cholesterol and ortho- or ⁇ ra-mPEG-DTB-DSPE prepared as described in Example 1 , mPEG
- a suitable organic solvent typically cholorfo ⁇ n/methanol in a 1: 1 or 1:3 ratio.
- the solvent was removed by rotary evaporation to form a dried lipid film.
- the film was hydrated with aqueous buffer to from liposomes that were sized via extrusion to an average diameter of 120 nm.
- the liposomes were incubated in phosphate buffered saline, pH 7.2, containing 5 mM EDTA at 37°C in the presence of 150 ⁇ M cysteine. Disappearance of the conjugates was monitored by HPLC (Phenomenex C 8 Prodigy, 4.6 x 50 mm column, detection at 277 nm, mobile phase methanol / water 95:5 with 0.1 % trifluoroacetic acid at 1 mL/min). Results are shown in Fig. 7B where the liposomes comprising the ortho- conjugate are represented by the solid circles and liposomes comprising the para- conjugate by the solid squares.
- Example 7 In vitro Cleavage of o- and p-mPEG-DTB-DSPE Compound in Liposomes
- the solvent was removed by rotary evaporation to form a dried lipid film.
- the lipid film was hydrated with an aqueous solution containing 30 mM each of the fluorophores 7-xylene-bis-pyridinium bromide and trisodium 8-hydroxypyrenetrisulfonate. was hydrated with aqueous buffer to from liposomes that were sized via extrusion to an average diameter of 100 nm.
- PHPC partially hydrogenated phosphatidylcholine
- mice were divided into two study groups.
- the liposome composition described above was injected into all test animals.
- One group of the test animals also received a 200 ⁇ L injection of 200 mM cysteine at 1 , 3 and 5 hours post liposome injection.
- the other test group received an injection of saline at the same time points.
- Liposome content in the blood was determined by monitoring blood samples for In 111 . The results are shown in Fig. 10.
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Abstract
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IL14604700A IL146047A0 (en) | 1999-04-23 | 2000-04-21 | Releasable linkage and compositions containing same |
EP00923572A EP1173221A2 (en) | 1999-04-23 | 2000-04-21 | Releasable linkage and compositions containing same |
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Cited By (7)
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WO2002076428A1 (en) * | 2001-03-26 | 2002-10-03 | Alza Corporation | Liposome composition for improved intracellular delivery of a therapeutic agent |
EP1279406A1 (en) * | 2000-04-03 | 2003-01-29 | Santen Pharmaceutical Co., Ltd. | Transporters and drug delivery system by using the same |
WO2005053749A2 (en) * | 2003-11-26 | 2005-06-16 | Alza Corporation | Thiol-cleavable linkage between polymer and ligand |
US7276248B2 (en) | 1999-04-23 | 2007-10-02 | Alza Corporation | Conjugate having a cleavable linkage for use in a liposome |
US7303760B2 (en) | 1999-04-23 | 2007-12-04 | Alza Corporation | Method for treating multi-drug resistant tumors |
US7932294B2 (en) | 2004-08-26 | 2011-04-26 | Apparao Satyam | Prodrugs containing novel bio-cleavable linkers |
WO2018180914A1 (en) | 2017-03-30 | 2018-10-04 | 日油株式会社 | Hydrophilic polymer derivative having self-immolative acetal linker and composite using same |
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KR20070019941A (en) * | 2003-09-03 | 2007-02-16 | 교와 핫꼬 고교 가부시끼가이샤 | Compound modified with glycerol derivative |
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Also Published As
Publication number | Publication date |
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AU770390B2 (en) | 2004-02-19 |
EP1173221A2 (en) | 2002-01-23 |
NO20015169D0 (en) | 2001-10-23 |
WO2000064483A3 (en) | 2001-08-02 |
NO20015169L (en) | 2001-12-19 |
AU4367200A (en) | 2000-11-10 |
IL146047A0 (en) | 2002-07-25 |
JP2002542386A (en) | 2002-12-10 |
CA2368793A1 (en) | 2000-11-02 |
NZ514990A (en) | 2004-01-30 |
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