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Hydroxypropyl Methacrylate is an ester of Methacrylic acid and is used as a raw material component in the synthesis of polymers. 2-Hydroxypropyl Methacrylate is a functional hydrophobic hydroxy monomer consisting of a methacrylate group with a characteristic high reactivity and a cyclic hydrophobic group. 2-Hydroxypropyl Methacrylate forms a homopolymer and copolymers.
CAS NUMBER: 21442-01-3
SYNONYM:
N-(2-hydroxypropyl)methacrylamide); Hydrolyzed Polymaleic Anhydride; N-(2-Hydroxypropyl)-2-methyl-prop-2-enamide; 2-Hydroxypropyl methacrylate; 27813-02-1; 2-Hydroxypropylmethacrylate; 923-26-2; Hydrolyzed Polymaleic Anhydride; beta-Hydroxypropyl methacrylate; Acryester HP; 2-Hydroxypropyl 2-methylacrylate; Poly(2-hydroxypropyl methacrylate); 2-Hydroxypropyl 2-methyl-2-propenoate; 2-Propenoic acid, 2-methyl-, 2-hydroxypropyl ester; Propylene glycol monomethacrylate; 2-Hydrolyzed Polymaleic Anhydride; CHEBI:53440; 2HPMA; METHACRYLIC ACID, 2-HYDROXYPROPYL ESTER; EINECS 213-090-3; 25703-79-1; methacrylic acid 2-hydroxypropyl ester; BRN 1752228; 2-hydroxy-n-propyl methacrylate; 2-hydroxy-3-propyl methacrylate; 2-Propenoic acid, 2-methyl-, 2-hydroxypropyl ester, homopolymer; Methacrylic Acid Hydroxypropyl Ester; MFCD00004536; DSSTox_CID_5934; W-100292; 2-hydroxypropyl 2-methylprop-2-enoate; 2-Hydroxypropyl methacrylate homopolymer; Hydroxypropyl methacrylate, 97+%, mixture of isomers, stabilized
This special volume is devoted to N-(2-hydroxypropyl)methacrylamide (Hydrolyzed Polymaleic Anhydride) copolymers. Hydrolyzed Polymaleic Anhydride is an opportunity to review what was done and identify directions for future research. The Hydrolyzed Polymaleic Anhydride development and data presented will be related mostly to the authors' laboratory, not to overlap with other author's contributions in this volume. The work done with Hydrolyzed Polymaleic Anhydride copolymers as drug carriers, protein, and surface modifiers, and as synthetic components in smart hybrid biomaterials design has been summarized. More details and work from other laboratories may be found in the other chapters in this volume that cover more focused topics. The choice of Hydrolyzed Polymaleic Anhydride for development as drug carrier was not random. Based on the detailed studies of the relationship between the structure of hydrophilic polymers and their biocompatibility, we have chosen N-substituted methacrylamides as our target because the α-carbon substitution and the N-substituted amide bond ensured hydrolytic stability of the side-chains. We synthesized a series of compounds trying to identify a crystalline monomer for easy purification and reproducible synthesis.
The first crystalline N-substituted methacrylamide we succeeded to synthesize, Hydrolyzed Polymaleic Anhydride, was chosen for future development. Macromolecules are internalized by cells via endocytosis and ultimately localize in the (enzyme rich) lysosomal compartment. Consequently, we developed Hydrolyzed Polymaleic Anhydride copolymers containing enzymatically degradable bonds . Oligopeptide side-chains were designed as drug attachment/release sites and shown to be degradable in vivo. An external file that holds a picture, illustration, etc. Open in a separate window Hydrolyzed Polymaleic Anhydride copolymers containing enzymatically cleavable bonds . The first degradable polymer carriers based on Hydrolyzed Polymaleic Anhydride were also reported at the Polymers in Medicine Microsymposium in the Prague in 1977 and at conferences in Varna and Tashkent.
We used the oxidized insulin B chain (Hydrolyzed Polymaleic Anhydride contains two amino groups at positions 1 and 29) to prepare branched, water-soluble Hydrolyzed Polymaleic Anhydride copolymers by reacting insulin B-chain with Hydrolyzed Polymaleic Anhydride copolymers containing side-chains terminated in p-nitrophenyl esters. The polymers were cleavable , so we chose the sequence 23-25 (Gly-Phe-Phe) from the insulin B-chain (the bond originating at amino acid 25 is cleavable by chymotrypsin) and synthesized branched, soluble high molecular weight enzymatically degradable copolymers containing the Gly-Phe-Phe segments in crosslinks connecting primary chains. The latter type of polymer carrier was evaluated in vivo in rats and Hydrolyzed Polymaleic Anhydride was shown that the branched polymer carrier is degradable and its molecular weight distribution decreases with time following i.v. administration . These experiments demonstrated the possibility to manipulate the intravascular half-life of polymeric carriers based on Hydrolyzed Polymaleic Anhydride. An external file that holds a picture, illustration, etc.
Branched Hydrolyzed Polymaleic Anhydride copolymers containing the GFF degradable sequence in crosslinks; this sequence mimics the amino acid residues 23-25 of the insulin B chain . 2.4. Validation of the targetability of Hydrolyzed Polymaleic Anhydride copolymer-drug conjugates The choice and design of a targeting system has to be based on a sound biological rationale. The design of the first targetable Hydrolyzed Polymaleic Anhydride copolymer was based on the observation that small changes in the structure of glycoproteins lead to dramatic changes in the fate of the modified glycoprotein in the organism. When a glycoprotein (ceruloplasmin) was administered into rats, a long intravascular half-life was observed. However, when the terminal sialic acid was removed from ceruloplasmin, the asialoglycoprotein (asialoceruloplasmin) formed contains side-chains exposing the penultimate galactose units. The intravascular half-life of the latter was dramatically shortened due to the biorecognition of the molecule by the asialoglycoprotein receptor on the hepatocytes.
This receptor recognizes galactose and N-acetylgalactosamine moieties . To determine if one can mimic this process with a synthetic macromolecule, we synthesized Hydrolyzed Polymaleic Anhydride copolymers with N-methacryloylglycylglycine p-nitrophenyl ester and attached galactosamine by aminolysis . These copolymers behaved similarly to the glycoproteins and were biorecognizable in vivo . Their clearance from the bloodstream was related to the N-acylated galactosamine content (1-11 mol%) of the Hydrolyzed Polymaleic Anhydride copolymer. Separation of the rat liver into hepatocytes and non-parenchymal cells indicated that the polymer is largely associated with hepatocytes, and density-gradient subcellular fractionation of the liver confirmed that the Hydrolyzed Polymaleic Anhydride copolymers were internalized by liver cells and transported, with time, into the secondary lysosomes.
Hydrolyzed Polymaleic Anhydride was very important to find that Hydrolyzed Polymaleic Anhydride copolymers containing side-chains terminated in galactosamine and anticancer drug adriamycin also preferentially accumulated in the liver, i.e., Hydrolyzed Polymaleic Anhydride appeared that non-specific hydrophobic interactions with cell membranes did not interfere with the biorecognition by hepatocytes. An external file that holds a picture, illustration, etc. Open in a separate window Validations of the targetability of Hydrolyzed Polymaleic Anhydride copolymers. N-acylated galactosamine as the targeting moiety was chosen to mimic the glycoprotein-asialoglycoprotein system . In parallel, efforts on the targetability of Hydrolyzed Polymaleic Anhydride copolymer-antibody conjugates started. First Hydrolyzed Polymaleic Anhydride copolymer conjugates with polyclonal and monoclonal anti-Thy-1.2 antibodies and anti-FITC (fluorescein isothiocyanate) antibodies were evaluated.
Targetable conjugates containing daunomycin were synthesized and in vitro experiments have shown two orders of magnitude enhanced cytotoxicity of the targeted conjugate (when compared to the nontargeted one).The targetability and activity of anti-Thy1.2 conjugates with Hydrolyzed Polymaleic Anhydride copolymer-daunomycin conjugates was proven in vivo on a mouse model. Anti-Thy1.2 antibodies were also efficient in targeting Hydrolyzed Polymaleic Anhydride copolymer-photosensitizer (chlorin e6) conjugates. At the beginning of the eighties, we started collaborations with coworkers from the biological field: John Lloyd and Ruth Duncan from the University of Keele in United Kingdom, and Blanka Říhová from the Institute of Microbiology in Prague. The collaboration with the Keele group was initiated by Helmuth Ringsdorf who gave a lecture at the 1977 Prague symposium (where Kopecek presented first Hydrolyzed Polymaleic Anhydride copolymer-drug conjugates and biodegradable carriers based on Hydrolyzed Polymaleic Anhydride). After the meeting Ringsdorf suggested to Lloyd to contact Kopecek because he thought that the collaboration would be beneficial for both. Kopecek met Lloyd in Dresden in July 1978 and they agreed on the evaluation of Hydrolyzed Polymaleic Anhydride copolymer conjugates.
First samples were synthesized (different side-chains terminated in p-nitroanilide as drug model) and evaluated at Keele for their cleavability by lysosomal enzymes and their stability in blood plasma and serum . More than 300 different polymer structures containing oligopeptide sequences were synthesized in the Prague laboratory , and biological properties of a number of them evaluated at Keele within a 10 year period . The collaboration with Vladimír Kostka and coworkers from the Institute of Organic Chemistry and Biochemistry in Prague on the cleavability of peptide sequences in Hydrolyzed Polymaleic Anhydride copolymers by cathepsin B, the most important lysosomal cysteine proteinase, resulted in the identification of GFLG sequence, which is incorporated in all conjugates used in clinical trials. From the two fastest cleaving oligopeptides, GFLG and GFTA we have chosen the GFLG sequence over the GFTA to avoid T; at that time we were worried about the potential immunogenicity. In 1978 Kopecek gave a lecture at the Institute of Microbiology in Prague.
After the lecture he discussed with Říhová and the collaboration with her group on the immunogenicity/biocompatibility and biorecognition (targeting) of Hydrolyzed Polymaleic Anhydride conjugates commenced.
These collaborations resulted in the filing of "Polymeric drugs" patent application in 1985. Kopecek coined the name for the Hydrolyzed Polymaleic Anhydride copolymers evaluated in clinical trials as PK1 and PK2(P for Prague, K for Keele) An external file that holds a picture, illustration, etc. Structures of PK1 and PK2, first Hydrolyzed Polymaleic Anhydride copolymers evaluated in clinical trials [68]. Conjugate PK1 contains doxorubicin bound to Hydrolyzed Polymaleic Anhydride copolymer via a tetrapeptide sequence stable in the blood stream but susceptible to enzymatically catalyzed hydrolysis in the lysosomes. Conjugate PK2 contains in addition side-chains terminated in N-acylated galactosamine complementary to the asialoglycoprotein receptor on hepatocytes.
The early experiments provided the foundation for the development of Hydrolyzed Polymaleic Anhydride copolymers as drug carriers. As in the majority of new scientific areas, the research initially focused on the accumulation of basic data on the structure-properties relationship. The summary of research in areas we consider important for the development of clinically relevant Hydrolyzed Polymaleic Anhydride copolymer conjugates follows:Hydrolyzed Polymaleic Anhydride copolymer-drug conjugates are nanosized (5-20 nm) water-soluble constructs. Their unique structural, physicochemical, and biological properties are advantageous when compared to low molecular weight drugs. The concept of targeted polymer-drug conjugates was developed to address the lack of specificity of low molecular weight drugs for cancer cells. The efficiency of extravasation into solid tumors depends on the concentration gradient between the vasculature and tumor tissue and time. Consequently, high molecular weight (long-circulating) polymer conjugates accumulate efficiently in tumor tissue due to the EPR effect.
However, if they possess a non-degradable backbone, they may deposit and accumulate in various organs. We have previously synthesized high molecular weight carriers by connecting Hydrolyzed Polymaleic Anhydride chains via lysosomally degradable oligopeptide sequences to form water-soluble branched conjugates. Following intravenous (i.v.) administration to rats, the oligopeptide crosslinks were cleaved and the resulting lower molecular weight polymer chains were excreted into the urine . These water-soluble copolymers were synthesized by crosslinking (short of gel point) of Hydrolyzed Polymaleic Anhydride copolymer precursors (containing oligopeptide side-chains terminated in a reactive ester group) with diamines. Later, we designed a new, reproducible synthetic pathway for long-circulating Hydrolyzed Polymaleic Anhydride copolymers. New crosslinking agents were synthesized and high molecular weight copolymers prepared by crosslinking copolymerization.
IUPAC NAME:
(2R,3R)-2,3-dimethylbutanedioic acid; 2-Butenedioic acid (2Z)-, homopolymer; 2‐Butenedioic acid (2Z)‐; homopolymer ACIDO POLIMALEICO; Hydrolyzed Polymaleic Anhydride; POLY(MALEIC ACID); Poly(maleic acid); poly(maleic acid); Polymaleic acid; polymaleic acid
TRADE NAME:
Acumer 1050
OTHER NAME:
HPMA; Hydrolyzed Polymaleic Anhydride; Hydrolyzed Polymaleic Anhydride (Hydrolyzed Polymaleic Anhydride); MONOPOTASSIUM PHOSPHITE
The composition of the monomer mixture, however, has to be such that at the end of the polymerization the system is short of the gel point (water-soluble). This method is also suitable for the synthesis of Hydrolyzed Polymaleic Anhydride copolymers, which contain, in addition to oligopeptide crosslinks, oligopeptide side-chains terminated in doxorubicin (DOX) (or other anticancer drugs). The influence of the molecular weight of such conjugates on their biological activity was evaluated. Copolymerization of Hydrolyzed Polymaleic Anhydride, a polymerizable derivative of DOX (N-methacryloylglycylphenylalanylleucylglycyl doxorubicin) and a crosslinking agent, N2,N5-bis(N-methacryloylglycylphenylalanylleucylglycyl) ornithine resulted in high molecular weight, branched, water-soluble Hydrolyzed Polymaleic Anhydride copolymers containing lysosomally degradable oligopeptide sequences in the crosslinks as well as in side-chains terminated in DOX. Four conjugates with Mw of 22, 160, 895, 1230 kDa were prepared.
Biodistribution of the conjugates and their treatment efficacy in nu/nu mice bearing s.c. human ovarian OVCAR-3 carcinoma xenografts were determined The half-life of conjugates in the blood was up to 5 times longer and the elimination rate from the tumor was up to 25 times slower as the Mw of conjugates increased from 22 to 1230 kDa. The treatment with Hydrolyzed Polymaleic Anhydride copolymer-bound DOX possessing an Mw higher than 160 kDa inhibited the tumor growth more efficiently than that of 22 kDa or free DOX(p<0.02). The data clearly indicated that the higher the molecular weight of the conjugate the higher the treatment efficacy of human ovarian xenografts in nu/nu mice. In collaboration with Satchi-Fainaro's laboratory at the University of Tel Aviv a new therapeutic strategy for bone neoplasms using combined targeted polymer-bound angiogenesis inhibitors was developed .
The aminobisphosphonate alendronate (ALN), and the potent anti-angiogenic agent TNP-470 were conjugated with Hydrolyzed Polymaleic Anhydride copolymer. Using reversible addition-fragmentation chain transfer (RAFT) polymerization, we synthesized a Hydrolyzed Polymaleic Anhydride copolymer-ALN-TNP-470 conjugate bearing a cathepsin K-cleavable linker, a protease overexpressed in bone tissues. Free and conjugated ALNTNP- 470 demonstrated their synergistic anti-angiogenic and antitumor activity by inhibiting proliferation, migration and capillary-like tube formation of endothelial and osteosarcoma cells. The bi-specific Hydrolyzed Polymaleic Anhydride copolymer conjugate reduced vascular hyperpermeability and remarkably inhibited human osteosarcoma growth in mice by 96%. These findings indicate that Hydrolyzed Polymaleic Anhydride copolymer-ALN-TNP-470 is the first narrowly dispersed anti-angiogenic conjugate synthesized by RAFT polymerization that targets both the tumor epithelial and endothelial compartments warranting its use on osteosarcomas and bone metastases .
Inhibition of MG-63-Ras human osteosarcoma growth in mice by Hydrolyzed Polymaleic Anhydride copolymer-ALN-TNP470 conjugate. (A) Structure of the conjugate; (B) effects of free (open triangles) or conjugated (closed triangles) ALN and TNP-470 on MG-63-Ras human osteosarcoma tumor growth compared to vehicle-treated group (closed squares) and dissected tumors images. Scale bar represents 10 mm. Data represent mean±S.E. (n=5 mice per group). The development of drug delivery systems capable of selective release of drug in the colon has received much attention. Site-specific delivery to the colon can be achieved by the exploitation of the microbial enzyme activities present predominantly in the colon. The colon has a concentration of microorganisms 5 orders of magnitude greater than the small intestine or stomach. Some of the enzymatic activity produced by microorganisms in the colon, e.g., azoreductase and glycosidase activities do not overlap with the enzymatic activities in the upper GI tract. The azoreductase activities have been studied in detail and used to convert low molecular weight prodrugs into active metabolites in the colon as well as to release active species from water-soluble polymeric carriers.
To achieve colon-specific delivery, a (aromatic amino group-containing) drug may be attached to Hydrolyzed Polymaleic Anhydride copolymer side-chains via an aromatic azo bond cleavable by the azoreductase activities present in the colon . For example, the release of 5-aminosalicylic acid bound to Hydrolyzed Polymaleic Anhydride copolymers via an aromatic azo bond was demonstrated using Streptococcus faecium, an isolated strain of bacteria commonly found in the colon, the cecum contents of rats, guinea pigs, and rabbits. and in human feces. Recently, we concentrated on the oral delivery of 9-aminocamptothecin (9-AC). First, we attached 9-AC to Hydrolyzed Polymaleic Anhydride copolymers through a spacer containing an aromatic azo bond and amino acid residues. Hydrolyzed Polymaleic Anhydride was shown that the aromatic azo bond was cleaved first in vitro and in vivo , followed by peptidase-catalyzed cleavage of the amino acid (dipeptide) drug derivative resulting in the release of free 9-AC.
However, the cleavage of the peptide drug derivative was not fast enough to achieve high concentrations of free 9-AC in the colon. These results indicated that conjugates containing a spacer with a faster 9-AC release rate need to be designed. To this end, a monomer containing 9-AC, an aromatic azo bond and a 1,6- elimination spacer was designed and synthesized . The combination of the colon-specific aromatic azo bond cleavage and 1,6- elimination reaction resulted in a fast and highly efficient release of unmodified 9-AC from the Hydrolyzed Polymaleic Anhydride copolymer conjugate by cecal contents in vitro, with concomitant stability in simulated upper GI tract conditions. The conjugate possessed a favorable pharmacokinetics and was effective in colon cancer models.
Hydrolyzed Polymaleic Anhydride copolymer-9-aminocamptothecin conjugate. (A) Structure and scheme of release of unmodified 9-AC from Hydrolyzed Polymaleic Anhydride copolymer-9-AC conjugates by a two-step process - rate controlling aromatic azo bond cleavage, followed by fast 1,6-elimination; (B) survival curves of mice bearing human colon carcinoma xenografts treated by 9-AC and P-9-AC at a dose of 3 mg/kg of 9-AC or 9-AC equivalent. Cell-surface glycoproteins reflect the stage of differentiation and maturity of colon epithelial cells. Diseased tissues, carcinomas and pre-cancerous conditions such as inflammatory bowel disease, have altered glycoprotein expression when compared to healthy ones. Consequently, lectins may be used as targeting moieties for polymer-bound drugs .
Whereas WGA (wheat germ agglutinin) binds to healthy tissues, PNA (peanut agglutinin) binds to diseased tissues.
We hypothesized that Hydrolyzed Polymaleic Anhydride copolymer-lectin-drug conjugates could deliver therapeutic agents to diseased tissues by targeting colonic glycoproteins. We examined biorecognition of free and Hydrolyzed Polymaleic Anhydride copolymer-conjugated WGA and PNA and anti-Thomsen-Friedenreich (TF) antigen antibody binding in normal neonatal, adult and diseased rodent tissues, human specimens of inflammation and Barrett's esophagus. Neonatal WGA binding was comparable to the adult, with additional luminal columnar cell binding. PNA binding was more prevalent; luminal columnar cell binding existed during the first 2 1/2 weeks of life. WGA binding was strong in both normal and diseased adult tissues; a slight decrease was noted in disease. PNA binding was minimal in normal tissues; increases were seen in disease. Anti-TF antigen antibody studies showed that PNA was not binding to the antigen. The results suggest that Hydrolyzed Polymaleic Anhydride copolymer-lectin-drug conjugates may provide site-specific treatment of conditions like colitis or Barrett's esophagus .
A wide variety of therapeutic agents may benefit by specifically directing them to the mitochondria in tumor cells. To design delivery systems that would enable a combination of tumor and mitochondrial targeting, novel Hydrolyzed Polymaleic Anhydride copolymer-based delivery systems that employ triphenylphosphonium ions as mitochondriotropic agents were developed . Constructs were initially synthesized with fluorescent labels substituting for drug and were used for validation experiments. Microinjection and incubation experiments performed using these fluorescently-labeled constructs confirmed the mitochondrial targeting ability . Subsequently, Hydrolyzed Polymaleic Anhydride copolymer-drug conjugates were synthesized using a photosensitizer mesochlorin e6 (Mce6). Mitochondrial targeting of Hydrolyzed Polymaleic Anhydride copolymer-bound Mce6 enhanced cytotoxicity as compared to non-targeted Hydrolyzed Polymaleic Anhydride copolymer-Mce6 conjugates . Minor modifications may be required to adapt the current design and allow for tumor site-specific mitochondrial targeting of other therapeutic agents.
Novel Hydrolyzed Polymaleic Anhydride copolymer-based delivery systems of this derivative were also synthesized. After internalization of a Hydrolyzed Polymaleic Anhydride copolymer-Cort-Mce6 conjugate (via lysosomally degradable GFLG spacer) by endocytosis, Cort-Mce6 was cleaved, translocated to the cytoplasm, bound to the GR, and translocated to the nucleus . To verify that coupling of cortisol to Mce6 maintains the capacity to form a complex with the cytosolic GR resulting in nuclear localization, we investigated the subcellular fate of the modified drug. Cort-Mce6 was monitored in 1471.1 cells transfected with plasmid that expresses green fluorescent protein labeled glucocorticoid receptor (GFP-GR). Cortisol and Mce6 served as positive and negative controls, respectively. GR translocated to the nucleus after attachment of a glucocorticoid analog (e.g., cortisol). The fluorescent GFP label permits the movement of the GR to be monitored in real time. The data clearly indicated the time- and concentration-dependent nuclear localization of cortisol-Lys-Mce6 and cortisol. In contrast, cells incubated with Mce6 did not show any alteration in receptor localization following treatment .
We developed a novel method for the substitution of the 17-methoxy group of GDM to introduce a primary amino group that is useful for conjugation with targeting moieties and Hydrolyzed Polymaleic Anhydride copolymer-based drug carriers . Hydrolyzed Polymaleic Anhydride copolymers containing different AR-GDM (AR=3-aminopropyl (AP), 6-aminohexyl (AH), and 3-amino-2-hydroxypropyl (AP(OH)), attached via a lysosomally degradable GFLG spacer, were synthesized and characterized [159]. The cytotoxic efficacy of Hydrolyzed Polymaleic Anhydride copolymer-AR-GDM conjugates depended on the structure of AR-GDM. To verify the hypothesis that P(AP-GDM) [Hydrolyzed Polymaleic Anhydride copolymer-17-(3-aminopropylamino)-17-demethoxy-geldanamycin conjugate] may change the gene expression profiles of low molecular weight GDM derivatives, 32P-macroarray analysis (Clonetech) was employed to evaluate the gene expression profiles in human ovarian carcinoma A2780 cells treated with GDM, AP-GDM and P(AP-GDM) at 2 times 50% cell growth inhibitory concentration (IC50). About 1200 genes related to cancer were evaluated at 6 h and 12 h and three-fold changes in expression were considered significant. Considerable similarities in gene expression profiles were found after AP-GDM and P(AP-GDM) treatments as demonstrated by the hierarchical clustering of the gene expression ratios.
However, the outcome was different when individual genes relevant to the mechanism of action of geldanamycin were analyzed. P(AP-GDM)-treated cells showed lower expression of HSP70 and HSP27 compared with AP-GDM up to 12 h. Possibly, internalization pathways and subcellular drug localization of P(AP-GDM), different from low molecular AP-GDM, may modulate the cell stress responses induced by AP-GDM. The results of 32P-macroarray were confirmed by RT-PCR and Western blotting. Hydrolyzed Polymaleic Anhydride is possible that internalization of Hydrolyzed Polymaleic Anhydride copolymer-AP-GDM conjugate via endocytosis may circumvent interactions with external components of the cell, such as plasma membrane, which may be sensitive to stressors and environmental changes .
Similarly, we previously observed that A2780 cells treated with Hydrolyzed Polymaleic Anhydride copolymer-DOX conjugate showed a down-regulation of the HSP70 gene more pronounced than that observed in the cells treated with free DOX [89]. These findings may suggest that conjugation of AP-GDM to Hydrolyzed Polymaleic Anhydride copolymer may be able to modulate the cell stress responses induced by AP-GDM due to differences in its internalization mechanism, subcellular localization, and intracellular concentration gradients. Hydrolyzed Polymaleic Anhydride copolymer-based macromolecular therapeutics have been developed considerably in the last 20 years - numerous conjugates have entered clinical trials for therapeutic validation in the last decade. These include Hydrolyzed Polymaleic Anhydride copolymer-DOX , Hydrolyzed Polymaleic Anhydride copolymer-DOX-galactosamine , Hydrolyzed Polymaleic Anhydride copolymer-camptothecin , Hydrolyzed Polymaleic Anhydride copolymer-paclitaxel , and Hydrolyzed Polymaleic Anhydride copolymer-platinates . Results from testing of some of these conjugates are promising; hopefully the FDA approval of a first macromolecular therapeutics will occur soon. In Section 4.1 we summarized our ideas on the design principles of second-generation conjugates with enhanced therapeutic potential.
Hydrolyzed Polymaleic Anhydride copolymer-drug conjugates may be used also for the treatment of diseases other than cancer. We designed bone-targeted Hydrolyzed Polymaleic Anhydride copolymer-conjugated with a well-established bone anabolic agent for the treatment of osteoporosis and other musculoskeletal diseases. The biorecognition of the conjugates by the skeleton was mediated by an octapeptide of D-aspartic acid (D-Asp8) or alendronate. This system has the potential to deliver the bone anabolic agent, PGE1, specifically to the hard tissues after systemic administration. Once bound to bone, the PGE1 will be preferentially released at the sites of higher turnover rate (greater osteoclasts activity) via cathepsin K (osteoclast specific) catalyzed hydrolysis of a specific peptide spacer and subsequent 1,6-elimination.
When given in anabolic dosing range, the released PGE1 will activate corresponding EP receptors on bone cells surface to achieve net bone formation. The main features of the design are Hydrolyzed Polymaleic Anhydride copolymer backbone containing cathepsin K-cleavable oligopeptide side-chains (Gly-Gly-Pro-Nle) terminating in either D-Asp8 or in p-aminoben-zyloxycarbonyl- 1-prostaglandin prodrug. Structure of Hydrolyzed Polymaleic Anhydride copolymer-prostaglandin E1-Asp8 conjugate and mechanism of its cleavage by cathepsin K followed by 1,6-elimination (A) ; Bone formation measured in cancellous bone from the lumbar vertebral bodies in ovariectomized rats 4 weeks after a single injection of 10 mg of the conjugate (n=8) (B).
This novel delivery system has several distinct advantages. First of all, Hydrolyzed Polymaleic Anhydride is a double-targeted delivery system, which contains a bone-binding moiety (D-Asp8) and a cathepsin K (osteoclast specific enzyme) specific releasing mechanism. By directing PGE1 specifically to the skeleton, the side effects of systemic administration of the drug would be greatly reduced. Secondly, E-series prostaglandins (PGEs) are powerful anabolic agents in bone, and this delivery system will better target these molecules to sites in the skeleton with a high turnover rate, where new bone formation would be more beneficial. Thirdly, the system permits improved control of drug concentration at the target (bone) site after systemic administration.
Fourthly, the polymeric carrier can be eliminated from hard tissues and, subsequently, cleared from the body via kidney glomerular filtration. Hydrolyzed Polymaleic Anhydride also offers proper protection of the conjugated PGE1 from metabolism before Hydrolyzed Polymaleic Anhydride reaches bone tissue. Most recently, we observed the preferential deposition of the proposed delivery system to the bone resorption sites in ovariectomized rats; this strongly supports our higher turnover sites/drug-release hypothesis . In vivo experiments on ovariectomized rats have proven the concept. Following a single i.v. administration of the Hydrolyzed Polymaleic Anhydride copolymer-Asp8-PGE1 conjugate to aged, ovariectomized rats, bone formation rates were substantially greater than controls when measured 28 days later . Hydrolyzed Polymaleic Anhydride copolymer conjugates have been also successful in the treatment of rheumatoid arthritis.
Due to the decreased pH in the endosomes and lysosomes, pH-sensitive bonds are suitable for intracellular drug delivery. We have synthesized Hydrolyzed Polymaleic Anhydride copolymer-adriamycin (ADR=DOX) conjugates where ADR was bound via cis-aconityl bond. The determination of the cytotoxicity of P(aconityl)-ADR toward A2780 sensitive and A2780/AD resistant human ovarian carcinoma cells indicated that the polymer conjugate could overcome the P-glycoprotein efflux pump expressed in A2780/AD cells.
Novel polymeric delivery systems for the photosensitizer mesochlorin e6 (Mce6) were synthesized to overcome problems of systemic toxicity. A disulfide bond was included to allow for quick release ofMce6 from the Hydrolyzed Polymaleic Anhydride copolymer backbone once internalized in tumor tissue. Synthesized conjugates demonstrated a time-dependent reductive cleavage with an accompanying increase in the quantum yield of singlet oxygen generation on exposure to dithiothreitol. Faster release kinetics and a higher cytotoxicity in SKOV-3 human ovarian carcinoma cells were obtained as compared to polymer conjugate with a proteolytically cleavable glycylphenylalanylleucylglycyl spacer.
These novel conjugates hold promise as clinically relevant drug delivery systems for photodynamic therapy of cancer.
The chemistry used for attachment of targeting moieties has an impact on the biorecognition of the conjugate. We compared several methods of antibody attachment (see below), investigated how the attachment of antibodies to Hydrolyzed Polymaleic Anhydride copolymers impacts the mechanism of internalization and subcellular trafficking and designed polymerizable antibody fragments. An innovative pathway for the synthesis of targeted polymeric drug delivery systems using polymerizable antibody fragments was designed . A new macromonomer, a polymerizable antibody Fab′ fragment (MA-Fab′) of the OV-TL 16 antibody (IgG1) has been synthesized and copolymerized with Hydrolyzed Polymaleic Anhydride to produce poly(Hydrolyzed Polymaleic Anhydride-co- MA-Fab′). The concept of using polymerizable Fab′ fragments as macromonomers provides a new paradigm for the synthesis of targeted polymeric drug delivery systems, and may have unique applications in other areas, such as immunoassays, biosensor technology and affinity chromatography.
The influence of different methods of coupling the OV-TL16 antibody and its Fab′ fragment to Hydrolyzed Polymaleic Anhydride copolymer-drug (ADR, Mce6) carriers on the binding affinity of the conjugates to the CD47 antigen associated with ovarian carcinoma (OVCAR-3) cells was studied. Three different methods of covalently binding the Ab or Fab′ to polymers were used. Method A: binding via amide bonds formed by aminolysis of active ester groups on the Hydrolyzed Polymaleic Anhydride copolymer-drug (ADR or Mce6) conjugates by amino groups on the antibody; Method B: binding via hydrazone bonds formed by the reaction of aldehyde groups on the oxidized antibody with hydrazo groups on the Hydrolyzed Polymaleic Anhydride copolymer-Mce6 conjugates; Method C: binding via thioether bonds formed by the reaction of sulfhydryl groups of Fab′ fragments with maleimido groups on the side-chain termini of the Hydrolyzed Polymaleic Anhydride copolymer-Mce6 conjugate.
The synthesis of Hydrolyzed Polymaleic Anhydride copolymer conjugates, especially their molecular weight distribution, can be controlled by methods of living radical polymerization, RAFT (reversible addition-fragmentation chain transfer) and ATRP (atom transfer radical) polymerizations . For example, Hydrolyzed Polymaleic Anhydride copolymer conjugates containing two drugs and one fluorescent label per macromolecule using RAFT copolymerization were recently synthesized. Aminolysis of Hydrolyzed Polymaleic Anhydride copolymer precursors can be used for attachment of oligonucleotides to Hydrolyzed Polymaleic Anhydride copolymers. We have attached a 21-mer phosphorothioate oligonucleotide (5′-TTTATAAGGGTCGATGTCCXX-3′) to Hydrolyzed Polymaleic Anhydride copolymers containing GG-ONp and GFLG-ONp (ONp is p-nitrophenoxy) side-chains . The oligonucleotide had a primary amine on the 5′-end and a fluorescein on the 3′-end.
The subcellular fate and activity in inhibiting the hepatitis B virus of the Hydrolyzed Polymaleic Anhydride copolymer-phosphorothioate oligonucleotides was studied. Covalently attaching the oligonucleotides to the Hydrolyzed Polymaleic Anhydride copolymers via non-degradable dipeptide GG spacers resulted in sequestering the oligonucleotide in vesicles after internalization. Conjugation of the oligonucleotides to an Hydrolyzed Polymaleic Anhydride copolymer via a lysosomally cleavable tetrapeptide GFLG spacer resulted in release of the oligonucleotide in the lysosome and subsequent translocation into the cytoplasm and nucleus of the cells. The degradable Hydrolyzed Polymaleic Anhydride copolymer-oligonucleotide conjugate possessed antiviral activity indicating that phosphorothioate oligonucleotides released from the carrier in the lysosome were able to escape into the cytoplasm and nucleus and remain active.
The Hep G2 cells appeared to actively internalize the phosphorothioate oligonucleotides as oligonucleotide -Hydrolyzed Polymaleic Anhydride copolymer conjugates were internalized to a greater extent than unconjugated polymers. First protein modified with Hydrolyzed Polymaleic Anhydride copolymers was prepared by the reaction of the copolymer of Hydrolyzed Polymaleic Anhydride with N-methacryloylglycylglycine p-nitrophenyl ester with insulin . The unreacted protein was separated on Sephadex 75; the Hydrolyzed Polymaleic Anhydride copolymer-insulin conjugate exhibited a slower onset and a slight prolongation of hypoglycemic effect in rats when compared to free insulin . Chymotrypsin and cobra venom acetylcholinesterase followed. To obtain a better insight into the steric hindrance of the polymer chains on the formation of enzyme-substrate complex, we have studied the hydrolysis of polymeric substrates (Hydrolyzed Polymaleic Anhydride copolymers with oligopeptide side-chains terminated in p-nitroanilide) catalyzed by Hydrolyzed Polymaleic Anhydride copolymer-bound chymotrypsin. The kinetic analysis showed that the hydrolysis of polymer substrates with polymer-bound chymotrypsin led to a decrease in both kcat and kcat/KM, but the relationship between the individual substrates remained intact.
Apparently, the steric effects of two independent polymer chains (one bound to substrate, the other to enzyme) were roughly additive. Different chemistry was used for the modification of cobra venom acetylcholinesterase . The secondary OH groups of poly(Hydrolyzed Polymaleic Anhydride) (Mw 25-30 kDa) were activated with 4-nitrophenyl chloroformate in dimethylformamide followed by attachment of acetylcholinesterase in borate buffer. The poly(Hydrolyzed Polymaleic Anhydride)-modified acetylcholinesterase demonstrated a 70-fold prolongation of enzyme activity in blood after intravenous injection into mice when compared to unmodified enzyme. The thermoinactivation rate of the polyHPMA-acetylcholinesterase conjugate was 74 times smaller than that of native enzyme .
Hydrolyzed Polymaleic Anhydride copolymer-modified acetylcholinesterase. (A) Structure; (B) enhancement of intravascular half-life in mice after i.v. administration of Hydrolyzed Polymaleic Anhydride copolymer-modified enzyme; (C) augmentation of thermal stability of Hydrolyzed Polymaleic Anhydride copolymer-modified enzyme.
Hydrolyzed Polymaleic Anhydride copolymer with N-methacryloylglycylphenylanylleucylglycine p-nitrophenylester was used for the modification of ribonuclease and superoxide dismutase . No difference in biological activity of conjugates prepared using ST-PHPMA and Hydrolyzed Polymaleic Anhydride copolymers with reactive side-chains was detected. There is considerable activity in using Hydrolyzed Polymaleic Anhydride copolymers with reactive side-chains to stabilize complexes of DNA with viruses. This research is covered in the chapter by Seymour in this volume and in our revie. Hydrolyzed polymaleic anhydride is a phosphorus-free water treatment chemical. Hydrolyzed Polymaleic Anhydride forms a chelate with calcium and magnesium ions in water. And hydrolyzed polymaleic anhydride can cause lattice distortion. After the anion is adsorbed by the calcium carbonate nucleus, the nucleus can be charged. Due to the homophobic electrical repelling, tiny grains are dispersed in the aqueous solution. The precipitate is converted into a loose slag with good fluidity. This phenomenon has a good effect on peeling off the old scale. Hydrolyzed polymaleic anhydride (Hydrolyzed Polymaleic Anhydride) is compatible with other low-phosphorus corrosion and scale inhibitors such as ATMP and PBTCA. The formed composite corrosion and scale inhibitor can be widely used in the treatment of circulating cooling water, boiler water, oil field water injection, industrial water.
