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WO2005115338A2 - Inhibiteurs de glyoxylase i macromoleculaires actives par le glutathion (gsh) - Google Patents

Inhibiteurs de glyoxylase i macromoleculaires actives par le glutathion (gsh) Download PDF

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Publication number
WO2005115338A2
WO2005115338A2 PCT/US2005/013361 US2005013361W WO2005115338A2 WO 2005115338 A2 WO2005115338 A2 WO 2005115338A2 US 2005013361 W US2005013361 W US 2005013361W WO 2005115338 A2 WO2005115338 A2 WO 2005115338A2
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hydroxycarbamoyl
prodrug
glutathione
sulfoxide
ethyl
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PCT/US2005/013361
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WO2005115338A3 (fr
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Donald J. Creighton
Zhe-Bin Zheng
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University Of Maryland, Baltimore County
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Priority to US11/587,160 priority Critical patent/US20070287672A1/en
Publication of WO2005115338A2 publication Critical patent/WO2005115338A2/fr
Publication of WO2005115338A3 publication Critical patent/WO2005115338A3/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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/167—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction with an outer layer or coating comprising drug; with chemically bound drugs or non-active substances on their surface

Definitions

  • This invention relates to macromolecular prodrugs having antitumor activity when activated by GSH, and methods involving administering these to patients. More particularly, this invention relates to, inter alia , the synthesis and use of polyacrylamide carriers to target anticancer prodrugs to tumors, and to release active antitumor agents selectively in tumor cells.
  • This detoxification pathway is to remove cytotoxic methylglyoxal from cells as D-lactate via the sequential action of the isomerase glyoxalase I (Glxl) and the thioester hydrolase glyoxalase II (GlxII) , as shown in Scheme 1 below (Creighton et al, "Glutathione-Dependent Aldehyde Oxidation Reactions", In Molecular Structure and Energetics : Principles of Enzyme Activity, Liebman et al, Eds.; VCH Publishers, 9: 353-386 (1988)).
  • Methylglyoxal is a highly reactive alpha-ketoaldehyde that arises as a normal by-product of carbohydrate metabolism (Richard et al, Biochemistry, 30:4581-4585 (1991)) and is capable of covalently modifying proteins and nucleic acids critical to cell viability (Reiffen et al, J. Cancer Res . Clin . Oncol . , 107:206-219 (1984); Ayoub et al, Leuk. Res . , 17:397-401 (1993); Baskaran administrat et al, Biochem . Int . , 212:166-174 (1990); Ray et al, Int . J.
  • Inhibitors of Glxl have been investigated as anticancer agents because of their potential to induce elevated concentrations of methylglyoxal in tumor cells (Creighton et al (2000) , supra) , and because of the observation that rapidly dividing tumor cells are exceptionally sensitive to the cytotoxic effects of exogenous methylglyoxal (Ray et al, supra ; White et al, supra ; and Papoulis et al, supra) .
  • methylglyoxal is able to covalently modify nucleotide bases in DNA (Papoulis et al, supra)
  • methylglyoxyl is probably genotoxic as well.
  • inhibitors of Glxl that are hydrolytically destroyed by the thioester hydrolase GlxII, which then offer a selective strategy for specifically inhibiting tumor cells, as normal cells contain much higher concentrations of GlxII than tumor cells.
  • Table 1 shows a comparison of the activities of Glxl and GlxII in normal versus cancer cells (Creighton et al (200-0) , supra) .
  • Table 1 Reported Glyoxalase Activities in Normal Cells versus Cancer Cells Tissue Glyoxalase Activity r (mU/mg protein)
  • Glxl GlxIIC Glxl/GlxII
  • normal cells may be able to detoxify thioester inhibitors of hGlxI more rapidly than the corresponding tumor cells, resulting in higher steady-state concentrations of the inhibitors in tumor cells.
  • the diethylester prodrug form of S- (N-p-chlorophenyl-N- hydroxycarbamoyl) glutathione, CHG(Et) 2 which is both an inhibitor of hGlxI and a substrate for hGlxII (Murthy et al, J. Med. Chem .
  • an antitumor strategy targeting hGlxI provides benefits over more established chemotherapies that attack rapidly dividing tumor cells at various stages of mitosis, or that arrest tumor cells at some stage in the cell cycle.
  • many of the small molecule antitumor drugs currently in use target rapidly dividing tumor cells by either directly or indirectly inhibiting DNA and/or protein synthesis.
  • a known class of transition state analogue inhibitors of human Glxl are S- (N-aryl/alkyl-N- hydroxycarbamoyl) glutathiones . These thioester derivatives of GSH mimic the stereoelectronic features of the tightly bound transition state species that flank the ene-diolate intermediate that forms along the reaction coordinate of the enzyme .
  • transition state analogue inhibitors are also slow substrates for bovine liver GlxII, which suggests that these compounds may selectively inhibit tumor cells over normal cells (Murthy et al, supra) .
  • the effectiveness of the transition state analogue • inhibitors can be measured by their specificity for the Glxl active site and by the time they occupy the active site, thereby blocking access of the enzyme's natural substrate (GSH-methylglyoxal thiohemiacetal) .
  • an inhibitor with a low competitive inhibition constant associates with the active site of an enzyme with higher affinity and greater specificity, and therefore, occupies the active site for a longer period of time than inhibitors with higher i values .
  • prodrug strategies have been investigated to enhance the cell permeability of these compounds.
  • the transition state analogue inhibitors are lethal to different human and murine tumor cell lines in culture when administered as diethyl ester prodrugs (US 5,616,563) (Kavarana et al, supra) . After diffusion into cells, the prodrugs undergo esterase-catalyzed de- esterification inside the cell to give the di-acid form of the transition state analogue.
  • a second strategy for delivering the transition state analogue inhibitors to tumors is as the membrane-permeable sulfoxide prodrug S- (N-4- chlorophenyl-N-hydroxycarbamoyl) ethyl sulfoxide, shown as (2) in scheme 2 below, which undergoes an acyl-interchange reaction with GSH to give CHG, shown as (1) in scheme 2 below (Hamilton et al, J. Med. Chem . 42:1823-1827(1999)).
  • High molecular weight copolymer-prodrugs have been used to better direct cancer chemotherapeutic agents to tumor tissue via the so-called “enhanced permeability and retention” (EPR) effect (Brocchini and Duncan, “Pendent drugs, release from polymers,” In Encyclopedia of Controlled Drug Delivery, Mathiowitz Ed., pp 786-816, John Wiley and Sons, New York (1999); Duncan, Nat . Rev. Drug Discoc , 2:347- 360 (2003) ; Thanou and Duncan, Curr. Opin . Investig.
  • EPR enhanced permeability and retention
  • a typical strategy for designing copolymer prodrugs is to covalently attach the drug to the polymer via a peptide linkage, which will undergo catalyzed hydrolysis when the copolymer-prodrug- filled endosomes fuse with the peptidase-fil-led lysosomes . The free drug is then available to diffuse out of the lysosomes into the cell cytosol .
  • prodrug conjugates of poly- hydroxypropylmethacrylamide (HPMA) have been designed to deliver the anticancer drugs doxorubicin (Seymour et al, Br. J. Cancer, 63_: 859-866 (1991)), daunomycin (Duncan et al, Br. J.
  • GSH-activated prodrugs are intriguing for cancer therapy because GSH concentrations are often elevated by as much as two-fold in tumor tissues (Cook et al, ancer Res . , 51:4287-4294 (1991); Bl ⁇ ir et al, Cancer Res . , 57:152-155 (1997); Kosower and Kosower, Int . Rev. Cyt .
  • GSH concentration differences could give rise to preferential activation of a strategically designed GSH-activated macromolecular prodrug in tumor cells .
  • the adventitious activation of a GSH- activated macromolecular prodrug in circulating human plasma should be minimal, as GSH concentrations are typically 1-2 ⁇ M (Anderson, "Enzymatic and Chemical Methods for Determination of Glutathione," In Gluta thi one : Chemi cal , Bi ochemi cal , and Medical Aspects , Vol IIIA, Dolphin et al, Eds., John Wiley and Sons, Toronto (1989)) .
  • lysosomes contain transporter systems for importing cysteine and cysteinyl dipeptides like cysteinyl-glycine into lysosomes (Foster and Lloyd, Biochim . Biophys . Acta 947:465-491 (1988) ) .
  • cysteine-dependent transporter For example, a highly specific cysteine-dependent transporter has been found in human fibroblast lysosomes with a K m of 0.5 mM, and evidence has been presented for the efflux of cystine and cysteine from these lysosomes (Pisoni et al, J Cell Biol . 110:327-335 (1990)).
  • An important role of cysteine is to activate intralysosomal thiol-dependent proteases that function to breakdown proteins taken up by endocytosis .
  • GSH also stimulates intralysosomal protein breakdown was attributed to a GSH transporter in the lysosomal membrane (Mego, Biochem J. 218:775-783 (1984)).
  • An object of the present invention is to provide macromolecular prodrugs of GSH-based antitumor agents that will accumulate preferentially in tumor tissue, and will be activated preferentially in tumor cells .
  • this object has been met by covalently linking sulfoxide prodrugs to macromolecular carriers, which upon administration to a cancer patient, will help target the prodrug to tumor tissue via the "enhanced permeability and retention effect.”
  • These sulfoxide prodrugs which will be taken up largely via endocytosis by tumor cells, contain a sulfoxide group adjacent to an acyl group which undergoes an acyl interchange reaction with glutathione present in the enodosomal or lysosomal compartment.
  • Another object of the present invention is to provide an effective antitumor pharmaceutical composition with less adverse side-effects than current chemotherapies .
  • this object has been met by macromolecular GSH- activated prodrugs in combination with a pharmaceutically acceptable diluent.
  • a further object of the present invention is to provide a method of treating a subject having a neoplastic condition. According to one embodiment of the invention, this object has been met by a method comprising the step of administering to a subject having a neoplastic condition a pharmaceutically effective amount of a macromolecular GSH-activated prodrug. As described above, these prodrugs accumulate preferentially in tumor tissue.
  • a still further object of the present invention is to provide a method of inhibiting the proliferation of a tumor cell.
  • this object has been met by a method comprising the step of contacting a tumor cell with an amount of a macromolecular GSH-activated prodrug that effectively inhibits proliferation of said tumor cell .
  • the macromolecular prodrug is activated by endosomal or lysosomal GSH, after endocytotic uptake, allowing the active Glxl inhibitor to diffuse into the cytoplasm and exhibit antitumor activity.
  • Another object of the invention is to provide a method of forming an active Glxl inhibitor from a macromolecular prodrug in the presence of glutathione .
  • this object has been met by providing a sulfoxide prodrug covalently linked to a macromolecular carrier, such that, in the presence of glutathione, an active Glxl inhibitor will be formed and released from said carrier.
  • An additional object of the invention is to provide methods of synthesizing macromolecular copolymer prodrugs .
  • this object has been met by a method comprising the following steps .
  • this object has been met by reacting 2- hydroxymethyl-2-endocyclic enone and methacryloyl chloride to form 2-methacyloyloxymethyl-2-endocyclic enone, and co-polymerizing the 2- methacyloyloxymethyl-2-endocyclic enone with an acrylamide to form the copolymer prodrug.
  • FIG. 1 Mechanisms by which a Glxl inhibitor and an alkylating exocyclic enone could be generated from a copolymer-prodrug conjugate in the presence of GSH.
  • Figure 2 Synthesis of HPMA polyacrylamide conjugates.
  • Figure 3 Spectral data of an HPMA-copolymer containing both the sulfoxide and 2- methylenecyclohexenone functions: (top) IR(ATR, AMTIR) ; (bottom) X H NMR 300 MHz (methanol-d 4 /TMS) spectra. Spectral lines unique to the three functional groups in the copolymers are indicated in the spectra.
  • Figure 4 Mol % compositions and spectrophotometrically determined second-order rate constants (k 2 ) for the reaction of GSH with the sulfoxide and cyclohexenone functions of different copolymer prodrugs .
  • Kinetic constants were calculated from the first-order rate of loss of the functional group under pseudo-first-order conditions (>20-fold excess of GSH) , and are the average of triplicate determinations ⁇ standard deviation.
  • Figure 5 Elution profile from a reverse-phase HPLC column of a reaction mixture obtained after incubating P8 with 10 mM GSH in potassium phosphate buffer (0.1 M) , pH 6.5, for 15 min. Abscissa: percent total absorbancy (200-400 nm) .
  • Figure 7 Rate profiles for the reaction of copolymer P6 ( Figure 7A) (0.05 mM in cyclohexenyl equivalents) with GSH (1 mM) , and copolymer P3 ( Figure 7B) (0.025 mM in 8-sulfoxide equivalents) with GSH (0.5 mM) .
  • the rate constants are the best- fit values to the expression for a first-order exponential decay (solid line through the data) .
  • Figure 8. In vitro inhibition of B16 melanotic melanoma by different HPMA copolymer prodrug conjugates versus the unpolymerized prodrugs.
  • Figure 9 Growth inhibition of B16 melanoma in the presence of PI ( Figure 9A) or P4 ( Figure 9B) .
  • Data for copolymer drug conjugates are indicated by squares; data for polymer controls with no appended drug are indicated by triangles .
  • Drug concentration is calculated, on the basis of equivalents of drug/L.
  • the present invention provides prodrugs useful as anti-tumor agents that comprise a macromolecular carrier and at least one precursor of a Glxl inhibitor covalently linked to the macromolecular carrier.
  • the precursor contains a sulfoxide adjacent to an acyl group which, in the presence of glutathione, allows the formation and release of an active Glxl inhibitor from the macromolecular carrier as a result of an acyl interchange reaction with the thiol of a glutathione at the acyl group of the precursor.
  • the prodrug Due to the macromolecular carrier, the prodrug accumulates preferentially in tumor tissue via the enhanced permeability and retention effect, which is due in part to the fact that higher molecular weight species tend to remain in circulating plasma longer than low molecular weight species, and the fact that blood vessels in rapidly growing tumors are more permeable to high molecular weight species than are blood vessels in normal, established tissues. Further, after endocytosis of the macromolecular prodrug by a tumor cell, endosomal or lysosomal glutathione will initiate release and activation of the active drug, which may then diffuse into the cytoplasm of the cell to target the Glxl enzyme .
  • the efficacy of the invention described herein for the first time demonstrates that the concentration of GSH inside lysosomes and/or endosomes is sufficient to form active Glxl inhibitor from the corresponding sulfoxide prodrug (Scheme 2) , as well as cytotoxic exocyclic enone from the corresponding endocyclic enone (Scheme 3).
  • the macromolecular carrier employed with the invention has an average molecular mass of greater than 2.5 kDa.
  • the macromolecular carrier has an average molecular mass within the range of 5 kDa to 70 kDa, more preferably in the range of 10 kDa to 50 kDa, and most preferably in the range of 15 kDa to 32 kDa.
  • larger molecular weight species will accumulate in tumor tissue via the EPR effect (Brocchini and Duncan, surpra (1999) ) .
  • Macromolecular prodrugs with a molecular weight of 20 kDa or greater may penetrate selectively through the leaky blood vessels in tumor tissue. Further, macromolecular prodrugs with a molecular weight of about 10 kDa or greater may avoid loss through the renal tubules .
  • Macromolecular carriers of the invention include carriers that are , linear or branched polymers .
  • the macromolecular carrier is polymerized from polymeric units having an amide.
  • the prodrug may be formed by radical polymerization of the polymeric amide unit and the methacrylamide derivative (s) of sulfoxide prodrugs and/or endocyclic enones.
  • Preferred carriers in this regard are polyacrylamide and polymethacrylamide.
  • HPMA N- (2-hydroxypropyl)methacrylamide
  • HPMA exhibits little i munogenicity (Rihova et al . , Makromol . Chem . 9:13-24 (1985).
  • HPMA is commercially available from Polysciences, Inc (Warrington, PA) .
  • various derivatives ⁇ of acrylamide may be used in accordance with the invention which are known in the art and which are commercially available .
  • macromolecular carriers that may be employed with the invention, and which are well-known in the art, include: polyethylene glycol (PEG) ; DIVEMA (Brocchini and Duncan, surpra (1999)); polysaccharides such as dextran, chitosan, carboxymethylchitin, carboxymethylpullulan, and alginate; polyaminoacids; polyesters; block copolymers; and alternating polymers such as PEG lysine.
  • PEG polyethylene glycol
  • DIVEMA Brocchini and Duncan, surpra (1999)
  • polysaccharides such as dextran, chitosan, carboxymethylchitin, carboxymethylpullulan, and alginate
  • polyaminoacids such as polyaminoacids
  • polyesters such as block copolymers
  • block copolymers such as PEG lysine.
  • the precursors that may be covalently linked to macromolecular carriers in accordance with the invention are those that contain a sulfoxide adjacent to an acyl group, such that an acyl interchange reaction with the thiol of a glutathione will simultaneously form and release an active Glxl inhibitor from the carrier .
  • Preferred precursors contain an N- hydroxycarbamoyl moiety, which binds tightly to the human Glxl enzyme active site.
  • Particularly preferred are precursors defined by the formula S- (N-aryl/alkyl-N- hydroxycarbamoyl) alkyl sulfoxide. The synthesis of these ethyl sulfoxide prodrugs is known in the art (Hamilton et al, J. Med.
  • the alkyl sulfoxide group may be a C ⁇ -C 2u alkyl sulfoxide, preferably a C ⁇ -C 10 alkyl sulfoxide.
  • ethyl, propyl, butyl, pentyl, or hexyl sulfoxides are particularly preferred, with ethyl sulfoxide precursors being the most preferred.
  • the precursors may be S- (N-aryl-N- hydroxycarbamoyl) alkyl sulfoxides, where aryl is a carbocyclic or heterocyclic group, which may be substituted or unsubstituted, with at least one ring having a conjugated ⁇ -electron system, and containing up to two conjugated or fused ring systems.
  • Heterocyclic aryls may contain C, N, 0, or S atoms. Carbocyclic aryls are preferred, with substituted or unsubstituted phenyl being particularly preferred.
  • the precursors may be S- (N-alkyl-N- hydroxycarbamoyl) alkyl sulfoxides, where N-alkyl is a C ⁇ -C 2 o alkyl, and preferably a C ⁇ -C ⁇ 0 alkyl. Of these, ethyl, propyl, butyl, pentyl and hexyl are particularly preferred.
  • Particularly preferred precursors include: S- (N-p-chlorophenyl-N- ydroxycarbamoyl) ethyl sulfoxide, S- (N-4-chlorophenyl-N- hydroxycarbamoyl) propyl sulfoxide, S-(N-4- chlorophenyl-N-hydroxycarbamoyl) butyl sulfoxide, S- (N-p-bromophenyl-N-hydroxycarbamoyl) ethyl sulfoxide, S- (N-p-iodophenyl-N-hydroxycarbamoyl) ethyl sulfoxide, S- (N-phenyl-N-hydroxycarbamoyl) ethyl sulfoxide, S- (N-hydroxy-N-methylcarbamoyl) ethyl sulfoxide, S- (N-methyl-N-hydroxycarbamoy
  • the precursors of the invention give rise,, to active Glxl inhibitors via an acyl interchange reaction with glutathione.
  • Preferred precursors form an active Glxl inhibitor of the formula S- (N- aryl/alkyl-N-hydroxycarbamoyl) glutathione, which are transition state analogues of the Glxl enzyme.
  • aryl is a carbocyclic or heterocyclic group, which may be substituted or unsubstituted, with at least one ring having a conjugated ⁇ -electron system, and containing up to two conjugated or fused ring systems.
  • Heterocyclic aryls may contain C, N, O, or S atoms.
  • N-alkyl is a C ⁇ -C 2 o alkyl, and preferably a C ⁇ -C ⁇ 0 alkyl. Of these, ethyl, propyl, butyl, pentyl and hexyl are particularly preferred.
  • Particularly preferred active Glxl inhibitors of the formula S- (N-aryl-N- hydroxycarbamoyl) glutathione include: S- (N-p- chlorophenyl-N-hydroxycarbamoyl) glutathione, S- (N-p- bromophenyl-N-hydroxycarbamoyl) glutathione, S- (N-p- iodophenyl-N-hydroxycarbamoyl) glutathione, S- (N- phenyl-N-hydroxycarbamoyl) glutathione .
  • S- (N-aryl-N- hydroxycarbamoyl) glutathione include: S- (N-p- chlorophenyl-N-hydroxycarbamoyl) glutathione, S- (N-p- bromophenyl-N-hydroxycarbamoyl) glutathione, S- (N-p- iodophenyl-N-
  • S- (N-hydroxycarbamoyl) glutathione derivatives including: S- (N-p-chlorophenyl-N- hydroxycarbamoyl) glutathione, S- (N-p-bromophenyl-N- hydroxycarbamoyl) glutathione, S- (N-p-iodophenyl-N- hydroxycarbamoyl) glutathione, S- (N-phenyl-N-hydroxycarbamoyl) glutathione.
  • Glxl inhibitors that may be used in accordance with the invention include irreversible inactivators , which acylate the active site of the human glyoxylase I enzyme. These compounds, including the synthesis thereof, have been described in WO 2005/007079, the disclosure of which is hereby incorporated by reference .
  • Irreversible inactivators include those of the formula S- (CH 2 C (O) OROC (O) CH 2 X) glutathione, where R is selected from the group consisting of alkylene, (CH 2 CH 2 0) ⁇ -2o, (CH 2 CH 2 N) 1-20/ and arylene .
  • X represents a halogen.
  • Preferred irreversible inactivators are compounds of the formula CH 2 C (O) O (CH 2 ) n OC (O) CH 2 X) glutathione, wherein n is 2 through 6 and wherein X represents a halogen.
  • Particularly preferred irreversible inactivators are S- (bromoacetoxy butyl acetoxy) glutathione and S- (bromoacetoxy propyl acetoxy) glutathione .
  • Computational docking of these compounds into the X-ray crystal structure of hGlxI indicates that the S-substituents are ideally positioned to alkylate the sulfhydryl group of Cys60 in the active site, which is located about 12 to 13 Angstroms from the sulfur atom of the bound inactivators .
  • irreversible inactivators are also accommodated by the hGlxI active site, especially where the S- substituent is able to assume a "bowed" conformation in the active site, allowing the haloacetyl function to be positioned near Cys ⁇ O.
  • Preferred irreversible inactivators have bromoacetoxy, chloroacetoxy, acryloyl and crotonyl groups. Particularly preferred are irreversible inactivators having a bromoacetoxy group.
  • these irreversible inactivators may be employed as sulfoxide prodrugs, analogous to the transition state analogues described supra , such that the inactivator is formed and released from a macromolecular carrier through an acyl interchange reaction with GSH.
  • Glxl inhibitor which may be formed by an acyl interchange reaction between a sulfoxide precursor and glutathione may be employed according to the invention
  • hydrophobicity of the resulting S- substituent of the active drug correlates with a higher affinity for the human Glxl active site (Kalsi 2000, supra).
  • the S- (N-aryl-N- hydroxycarbamoyl) glutathione derivatives bind especially tightly to the active site of Glxl, as these compounds mimic the stereoelectronic features of the tightly bound transition state formed along the reaction coordinate of the enzyme during normal catalysis .
  • Glxl inhibitor that are catalytically hydrolyzed by human GlxII, a thioester hydrolase that is abundant in normal tissues, but deficient in tumor tissues. This is an additional basis for tumor targeting, since such compounds are believed to accumulate specifically in tumor cells .
  • S- (N-p-chlorophenyl-N- hydroxycarbamoyl) glutathione, S- (N-p-bromophenyl-N- hydroxycarbamoyl) glutathione , S- (N-phenyl-N- hydroxycarbamoyl) glutathione, and S- (N-methyl-N- hydroxycarba oyl) glutathione are substrates for bovine liver GlxII.
  • the prodrugs of the invention may comprise two or more precursors which may be the same or different.
  • the prodrugs may also comprise one or more alkylating agents, such as an endocyclic enone that gives rise to the exocyclic enone through a Michael addition reaction with glutathione. These compounds will also be released from the carrier as a result of the Michael addition reaction. As with the Glxl inhibitors, these compounds may be activated preferentially in tumor cells having elevated concentrations of glutathione.
  • the macromolecular prodrug is in the form of an HPMA copolymer with one or more sulfoxide precursors and one or more endocyclic enones .
  • Preferred endocyclic enones that may act as precursors to alkylating agents are 2-substituted cyclohexenone, 2-substituted cycloheptenone, 2- substituted eyelopentenone, 2-substituted benzoquinone, 2-substituted napthoquinone , and 2- substituted anthroquinone .
  • Other alkylating agents include the COMC derivatives COMC-5, COMC-6, COMC-7, and COMC-8 (Hamilton et al . , J. Am . Chem . Soc. 25:15049 (2003) ) .
  • Preferred embodiments of the invention include prodrugs comprising copolymers of HPMA along with one or more of the following precursors of transition state analogues: S- (N-p-chlorophenyl-N- hydroxycarbamoyl) ethyl sulfoxide, S-(N-4- chlorophenyl-N-hydroxycarbamoyl)propyl sulfoxide, S- (N- -chloropheny1-N-hyrdoxycarbamoyl) utyl sulfoxide, S- (N-p-bromophenyl-N- hydroxycarba oyl) ethyl sulfoxide, S- (N-p-iodophenyl- N-hydroxycarbamoyl) ethyl sulfoxide, S- (N-phenyl-N- hydroxycarbamoyl) ethyl sulfoxide, S- (N-hydroxy-
  • the mol% of the sulfoxide precursor and/or endocyclic enone in the macromolecular prodrug of the invention is at least 1.5, more preferably at least 3 , and still more pre erably at least 8. Most preferably, the sulfoxide precursor and/or endocyclic enone in the macromolecular prodrug is about 10 mol%.
  • the present invention further provides pharmaceutical compositions comprising •• a macromolecular prodrug of the invention together with a pharmaceutically acceptable diluent, such as physiological saline.
  • the pharmaceutically acceptable diluent includes DMSO as needed to solubilize the prodrug.
  • the present invention further provides methods of treating a subject having a neoplastic condition comprising administering to a subject in need of such treatment a pharmaceutically effective amount of a macromolecular GSH-activated prodrug.
  • a pharmaceutically effective amount is a dose of from 0.01 g of macromolecular prodrug containing from 8 to 10 mol% inhibitor and/or alkylating agent to about 1.0 g of macromolecular prodrug.
  • a preferred dose is from 0.1 g to about 1.0 g.
  • compositions of the invention may be administered to treat a neoplastic condition.
  • the compositions of the present invention can be used to treat any cancerous condition.
  • Preferred conditions are members selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, kidney cancer, liver cancer, brain , cancer, and haemopoetic tissue cancer. More preferred cancers are prostate, colon and lung tumors, which overexpress Glxl,.
  • the tumor to be treated is particularly susceptible to the "enhanced permeability and retention effect," such that the macromolecular prodrug accumulates in the tumor tissue selectively.
  • the compositions of the present invention may be administered in any favorable fashion, intravenous, subcutaneous, and intramuscular administration are preferred. Most preferably, the compositions of the invention are administered intravenously.
  • the composition of the present invention may be administered by continuous i.v. infusion or bolus i.v. infusion to a subject having a neoplastic condition.
  • the present invention also provides methods of inhibiting the proliferation of a tumor cell comprising contacting a tumor cell with an amount of a composition of the invention effective to inhibit proliferation of said tumor cell.
  • the present invention includes inhibiting the proliferation of a tumor cell in vitro as well as in vivo.
  • An effective amount of the macromolecular prodrug of the invention for inhibiting proliferation of a tumor cell in vivo is that which provides a concentration of drug that results in a 50% decrease in tumor volume over the course of treatment .
  • a pharmaceutically effective amount may be from 0.01 g of macromolecular prodrug containing from 8 to 10 mol% inhibitor and/or alkylating agent to about 1.0 g of macromolecular prodrug.
  • a preferred dose is from 0.1 g to about 1.0 g.
  • the particular amount administered varies depending on the age, weight, sex of the subject, the mode of administration, and the particular neoplastic condition being treated.
  • An effective amount of a macromolecular prodrug of the invention effective to inhibit proliferation of a tumor cell in vi tro is that which provides a concentration of drug in a range of about 50 nM to about 1 mM, and preferably 300 nM or less.
  • the invention provides methods of forming active Glxl inhibitor and/or alkylating exocyclic enone from a macromolecular prodrug.
  • active Glxl inhibitor and/or alkylating exocyclic enone is formed by contacting the prodrug with glutathion .
  • the prodrug may be contacted with glutathione in vivo or in vitro.
  • the macromolecular prodrug of the invention is contacted in vitro with a 20-fold excess of glutathione
  • the present invention also provides methods of synthesizing copolymer prodrugs.
  • the method comprises the following steps . Performing an acyl interchange reaction between S- (N- aryl/alkyl/hydroxy-N-hydroxycarbamoyl) alkyl sulfoxide and a thioamine to form S- (N-aryl/alkyl-N- hydroxycarbamoyl) thioalkylamine.
  • S- (N-aryl/alkyl-N- hydroxycarbamoyl) thioalkylamine is then reacted with methacryloyl chloride and pyridine to form S- (N- aryl/alkyl-N- hydroxycarbamoylthioalkyl)methacrylamide, which is then polymerized with an acrylamide to form a copolymer prodrug.
  • the thioamine is cystea ine.
  • S- (N- aryl/alkyl-N- hydroxycarba oylthioalkyl) ethacrylamide is copolymerized with HPMA in the presence of azobisisobutylnitrile in an organic solvent such as acetone to form the copolymer prodrug.
  • the invention also provides methods of synthesizing copolymer prodrugs which comprise endocyclic enones capable of forming the alkylating exocyclic enone upon reaction with GSH.
  • copolymer prodrugs may be produced by reacting 2-hydroxymethyl-2-endocyclic enone and methacryloyl so as to form 2-methacryloyloxymethyl- 2-endocyclic enone, and then co- olymerizing the 2- methacryloyloxymethyl-2-endocyclic enone with an acrylamide to form a copolymer prodrug.
  • 2- methacryloyloxymethyl-2-endocyclic enone is formed in a reaction with 4-methylmorpholine .
  • methacryloyloxymethyl-2- endocyclic enone is copolymerized with HPMA in the presence of azobisisobutylnitrile in an organic solvent such as acetone .
  • aryl/alkyl-N- hydroxycarbamoylthioalkyl) ethacrylamide is copolymerized with HPMA and methacryloyloxymethyl-2- endocyclic enone in the presence of azobisisobutylnitrile in an organic solvent such as acetone.
  • NMR spectra were taken on a GE QE-300 NMR spectrometer.
  • IR spectra measured with a ThermoNicolet Avatar 370 FTIR spectrometer using a Pike MIRacle ATR accessory (AMTIR crystal) .
  • Mass spectral data were obtained at the Center for Biomedical and Bio-organic Mass Spectrometry, Washington University.
  • UV spectra were recorded using a Beckman DU 640 spectrophotometer.
  • HPLC was carried out using a Waters High-Performance Liquid Chromatography System composed of a 600 Controller, Delta 600 Pumps and 996 Photodiode Array Detector.
  • Analytical HPLC was performed using a Waters Nova-Pak C ⁇ 8 , 4 ⁇ m, 3.9 x 150 mm column or Symmetry C 18 , 5 ⁇ m, 4.6 x 150 mm column.
  • Preparative HPLC was performed using a SymmetryPrep C 18 , 7 ⁇ m, 19 x 150 mm column.
  • the molecular weights of the HPMA copolymers were estimated by gel permeation chromatograph (GPC) using a High Performance Gel Permeation Column (Tricorn Superose 12 10/300 GL) from Amersham Biosciences. Materials . Dextran molecular weight standards were purchased from Sigma Chem. Co. (1,000, 5,000, and 12,000 Da) and Polysciences, Inc. (40,000 Da).
  • Lyophilized human serum and GSH were purchased from Sigma Chem. Co.
  • HPMA was purchased from Polysciences, Inc.
  • Azobisisobutylnitrile (AIBN) methacryloyl chloride, 4-methylmorpholine and 3- chloroperoxybenzoic acid (80-85% pure) were purchased from Aldrich Chem. Co. All other reagents were of the highest purity commercially available.
  • Example 1 Synthesis of Compounds and HPMA copolymers S- (N-4-Chlorophenyl-N- hydroxycarbamoyl) thioethylamine (shown as (9) in Figure 2) was synthesized from the corresponding ethyl sulfoxide prodrug using the following procedure .
  • S- (N-4-Chlorophenyl-N- hydroxycarbamoylthioethyl) methacrylamide (shown as (8) in Figure 2) was synthesized from S- (N-4- Chlorophenyl-N-hydroxycarbamoyl) thioethylamine using the following procedure.
  • Methacryloyl chloride (318 ⁇ L, 3.29 mmol) was added slowly over 30 minutes to a stirring solution of S- (N-4-Chlorophenyl-N- hydroxycarbamoyl) thioethylamine (410 mg, 1.66 mmol) in 26 mL anhydrous DMF and 13 mL pyridine at 0°C, and the reaction mixture stirred at room temperature for an additional 20 minutes. The solvent was removed in vacuo and the residue fractionated by reverse-phase HPLC, using 50% acetonitrile in water, containing 0.1% trifluoroacetic acid, as a running solvent.
  • 2-Methacryloyloxymethyl-2-eyelohexenone (shown as (7) in Figure 2) was synthesized in the following manner .
  • Methacryloyl chloride (0.79 ml, 8 mmol) was added dropwise over about 30 minutes to a stirring solution of 2-hydroxymethyl-2-cyclohexenone (shown as (10) in Figure 2) (504 mg, 4 mmol) and 4- methylmorpholine (1 mL, 9 mmol) in 10 mL CH 2 C1 2 at 0°C.
  • the reaction mixture was allowed to stir for an additional 20 minutes.
  • Copolymer Pi The methacryloyl derivative shown as (8) in Figure 2 (41 mg, 0.13 mmol), HPMA (110 mg, 0.77 mmol) and AIBN (6 mg) were dissolved in 0.75 ml acetone under argon in a closed vial, and incubated at 50-55°C for 24 hours. The white precipitate was recovered by filtration and was dried under vacuum for 30 min. The crude product was dissolved in 0.25 mL of methanol and then precipitated by the slow addition of excess acetone: diethyl ether (3:1). The precipitation procedure was repeated and the precipitate dried under vacuum over night : Yield 63 mg.
  • IR (ATR, AMTIR) br 3349, 2972, 2930, 2886, S1636, S1527, 1487 (S-O) , 1203 cm " x . P3, compound (8) of Figure 2 (42 mg, 0.13 mmol), HPMA (282 mg, 2 mmol) and AIBN (18 mg) dissolved in 1.6 mL acetone: Yield 32 mg.
  • IR(ATR, AMTIR) br 3353, 2972, 2934, 2905, S1638, S1527, 1487 (S-O), 1202 cm “1 .
  • X H NMR 300 MHz, methanol-d 4 /TMS
  • the spectra were all very similar to one another, with significant line broadening due to the high molecular weights of the copolymers .
  • the chemical shift assignments were based on comparisons with the NMR spectra of poly HPMA and compound (8) of Figure 2; relative integrated intensities varied as a function of the mole % of the 8-sulfoxide function: ⁇ 1.01 (s, CH 3 C(C) 3 ), 1.16 (d, CH 3 CO-), 1.6-2.0 (m, - (CH 2 ) -HPMA) , 2.9-3.3 (m, -NCH a H b CO- ; N- CH 2 CH 2 -S ), 3.87 (m, -HCO-) , 7.40 (d, arom.
  • P6, compound (7) of Figure 2 (15 mg, 0.076 mmol), HPMA (87 mg, 0.61 mmol) and AIBN (5 mg) dissolved in 0.5 mL acetone. Yield 52 mg.
  • the mol % of the cyclohexenone function was estimated from the integrated intensities of the cyclohexenone ring protons C(5)H 2 , C(6)H 2 ( ⁇ 9.85) versus that of H-C-0 ( ⁇ 10.02): P4, -27; P5, - 20; P6, - 12. Copolymer P7.
  • the white precipitate was recovered by filtration and brought to dryness to give 70 mg crude product.
  • the crude product was dissolved in 2 ml methanol at 0°C and m-chloroperoxybenzoic acid (9.1 mg, 0.042 mmol) in 0.1 mL of diethyl ether was added dropwise.
  • the reaction mixture was allowed to stir for an additional lh, the solvent was removed in vacuo and the residue was dissolved in 0.4 mL methanol.
  • the product was precipitated by the dropwise addition of acetone : diethyl ether (3:1). The precipitation procedure was repeated, and the final copolymer product was dried under vacuum overnight: Yield 52 mg.
  • Copolymer P8 was prepared by the same general procedure used to prepare P7 starting with a reaction mixture composed of compound (7) (16 mg,
  • the gel permeation column was eluted with 50 mM sodium phosphate buffer (pH 7.0) containing 0.25 M NaCl at a flow rate of one ml/min.
  • Molecular weights were interpolated from standard curves (log M n and log M w versus retention time) obtained using polydextran molecular weight standards . The results are shown in Figure .
  • the mol% sulfoxide and cyclohexenone functions in the copolymers were determined as follows.
  • Ethanolic solutions of copolymer were prepared and 20 ⁇ L aliquots were mixed with 80 ⁇ L of 10 mM GSH in potassium phosphate buffer (0.1 M, pH 7.5) and incubated at room temperature for 10 minutes to convert the 8-sulfoxide and/or cyclohexenyl functions to compounds (1) and the GSH adduct (5) of Figure 1, respectively.
  • a 10 ⁇ L aliquot of this solution was then fractionated by reverse-phase HPLC (Nova-Pak C 18 , 4 ⁇ m, 3.9 x 150 mm column) .
  • the running solvent was 25% acetonitrile in water, containing 0.1% trifluoroacetic acid; for 5, the running solvent was 13% acetonitrile in water, containing 0.1% trifluoroacetic acid.
  • the areas under the peaks corresponding to compounds (1) and (5) were converted to mole quantities by comparison with standard curves of peak areas versus moles of authentic (1) and (5) injected onto the same column.
  • the amounts of compounds (1) and (5) were then used to calculate mole fractions of 8-sulfoxide and cyclohexenyl groups in the original copolymer. The results are shown in Figure 4.
  • the sulfoxide and cyclohexenone functions in the mixed function copolymers P7 and P8 independently react with free GSH, as the rate constants for the individual reactive groups are similar in magnitude to those for the copolymers containing only one of the reactive groups ( Figure 4) .
  • EXAMPLE 3 Stability of Copolymers in Human Serum To 0.45 L human serum at 37°C, was added P8 to an initial concentration of approximately 1 mM in 8- sulfoxide and cyclohexenyl groups. As a function of time, 30 ⁇ L aliquots of the incubation mixture were transferred to 20 ⁇ L of potassium phosphate buffer (0.1 M, pH 7.5) containing 1.6 mM GSH to convert the 8-sulfoxide and cyclohexenyl functions to compounds (1) and (5) , respectively. After incubation at room temperature for 5 minutes, the samples were deproteinized by the addition of 100 ⁇ L ethanol.
  • potassium phosphate buffer 0.1 M, pH 7.5
  • the protein precipitate was sedimented by centrifugation at 13,000g, and the supernatant was fractionated by reverse-phase HPLC, as described in Example 2 above .
  • the transition state analogue, compound (1) , and the GSH adduct, compound (5) were quantified, as described above in Example 2.
  • the rate constants were calculated from the first-order rate of loss of 8-sulfoxide or cyclohexenyl groups as a function of time. In this manner, the likely chemical stabilities of the 8-sulfoxide and cyclohexenyl groups in the copolymers under physiological conditions were estimated by determining the time-dependent loss of these groups from P8 during incubation with human serum at 37°C.
  • the stability of the copolymer prodrugs in circulating human plasma is an important aspect of drug efficacy in humans.
  • the approximate half-lives for the reaction of the sulfoxide- and cyclohexenone-containing copolymers with free GSH ( ⁇ 2 ⁇ M) (Anderson, (1989) supra) that applies in circulating human plasma at pH 7.5 are estimated to be about 175 and 17400 minutes, respectively, using the rate constants in Figure 4.
  • Murine B16 melanotic melanoma was obtained from the DCT Tumor Repository (NCI-Cancer Research and Development Center, Frederick, MD) and was maintained in RPMI 1640 medium containing L- glutamate (Gibco BRL, Gaithersburg, MD) , supplemented with 10% heat-inactivated fetal calf serum and genta ycin (10 ⁇ g/mL) , under 37 °C humidified air containing 5% C0 2 . Under these conditions B16 cells have a doubling time of about 26 hours.
  • the simple ethyl, propyl and butyl sulfoxide prodrugs (2, 2a, and 2b) have IC 50 values that are roughly five to ten-fold lower than that of HPMA-8- sulfoxide (Pi) , while the IC 50 value of compound (3) is over 7000-fold lower than that of HPMA-7 (P4) .
  • the IC 50 values for PI, P4 and the copolymer containing both prodrugs (P7) differ by no more than a factor of three.
  • the antitumor activities of the HPMA-copolymer prodrugs listed in Figure 8 are most likely due to the copolymers themselves and not to toxic contaminants in the copolymer preparations .
  • Antitumor activity is unlikely to arise from unreacted sulfoxide-8 and/or compound (7) , as neither NMR nor HPLC analysis of the copolymer preparations indicate that these species are present.
  • compound (1) and/or (4) might form in the growth medium during the efficacy studies, due to reaction of the copolymers with contaminating GSH (perhaps arising from cell lysis) .
  • this is an unlikely source of antitumor activity, because highly charged S-substituted GSH derivatives like compounds (1) and (4) of Figure 1 do not readily diffuse across cell membranes near neutral pH (Kavarana et al, J. Med. Chem . 42 : 221-228 (1999)).
  • the in vi tro antitumor activity of sulfoxide copolymer PI implies the presence of intralysosomal GSH to react with PI to give the cytotoxic transition state analogue inhibitor (1) in Figure 1. This follows from reports that yeast Glxl is highly specific for GSH and will not use cysteine or cysteinylglycine as cofactors (Kermack and Matheson, Biochem . J. 65:48-58 (1957)).
  • the cysteine and/or cysteinylglycine homologs of compound (1) might form inside the lysosomes, diffuse across the lysosomal membrane into the cytosol and then form (1) via acyl-interchange with cytosolic GSH.
  • This kind of cofactor specificity is not required to explain the cytotoxicity of the cyclohexenone-containing copolymer P7, . as intralysosomal GSH, cysteine or cysteinylglycine could all react with P7 to give cytotoxic exocyclic enones .
  • Both the sulfoxide- and cyclohexenone- containing copolymer prodrugs are significantly less potent than the low molecular weight prodrugs designed to enter cells by diffusion across the cell membrane.
  • HPMA-8 sulfoxide (Pi) is about 6.5-fold less potent than sulfoxides 2, 2a, and 2b
  • HPMA- 7 (P4) is about 7 x 10 3 -fold less potent that the simple 2-substituted cyclohexenone, compound 3. This is not a new phenomenon.
  • the HPMA copolymer of 6- (3-aminopropyl) -ellipticine (APE) is >75-fold less potent than APE with B16F10 melanoma in vi tro, although the copolymer is significantly more potent in tumor-bearing mice (Searle et al, Bioconj ugate Chem . 12:711-718 (2001)). This has been attributed to the slow rate of endocytotic uptake of the copolymer and the slow rate of peptide hydrolysis of the linker, which can have half-lives on the order of many hours.
  • endocytotic uptake of the copolymers described herein might also be a slow process; the rate constants for formation of compounds (1) and (4) of Figure 1 at pH 5.3 (lysosomes) will be approximately 50-fold smaller that the rate constants that apply in the cytosol with a pH of 7 (cytosol) .
  • efficacy might also be limited by the rate of expulsion of the prodrugs from the lysosomes into the cytoplasm of the cell.
  • the copolymer prodrugs containing mixed functional groups for example, copolymers P7 .and P8) provide an elegant manner of administering combination chemotherapy.

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Abstract

La présente invention concerne des promédicaments macromoléculaires présentant une activité antitumorale lorsqu'ils sont activés par le GHS ainsi que des méthodes de synthèse et d'administration de ces derniers à des patients. De manière plus spécifique, cette invention se rapporte, entre autres, à la synthèse et à l'utilisation de vecteurs polyacrylamide pour cibler les promédicaments anticancéreux sur les tumeurs et pour libérer sélectivement les agents antitumoraux, dans les cellules tumorales. Ces agents antitumoraux actifs ciblent le site actif de la glyoxalase I détoxifiant le méthylglyoxal pour ainsi provoquer la régression de la/des tumeurs.
PCT/US2005/013361 2004-04-20 2005-04-20 Inhibiteurs de glyoxylase i macromoleculaires actives par le glutathion (gsh) WO2005115338A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106146612A (zh) * 2015-04-17 2016-11-23 成都大学 一类乙二醛酶i不可逆抑制剂及其制备方法和用途
CN113101278A (zh) * 2021-04-14 2021-07-13 中山大学附属第七医院(深圳) 具有gsh和酯酶肿瘤微环境双响应的靶向纳米粒及其制备方法和应用

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US5616563A (en) * 1992-12-07 1997-04-01 University Of Maryland Baltimore Campus Glutathione N-hydroxycarbamoyl thioesters and method of inhibiting neoplastic growth
US5969174A (en) * 1998-01-07 1999-10-19 University Of Maryland At Baltimore County Competitive inhibitors of glyoxalase I and method of generating such competitive inhibitors inside tumor cells

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106146612A (zh) * 2015-04-17 2016-11-23 成都大学 一类乙二醛酶i不可逆抑制剂及其制备方法和用途
CN106146612B (zh) * 2015-04-17 2019-09-27 成都大学 一类乙二醛酶i不可逆抑制剂及其制备方法和用途
CN113101278A (zh) * 2021-04-14 2021-07-13 中山大学附属第七医院(深圳) 具有gsh和酯酶肿瘤微环境双响应的靶向纳米粒及其制备方法和应用
CN113101278B (zh) * 2021-04-14 2023-05-26 中山大学附属第七医院(深圳) 具有gsh和酯酶肿瘤微环境双响应的靶向纳米粒及其制备方法和应用

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