HPMA

HPMA

 

CAS No.: 21442-01-3

Synonyms: 

N-(2-hydroxypropyl)methacrylamide); HPMA; N-(2-Hydroxypropyl)-2-methyl-prop-2-enamide; 2-Hydroxypropyl methacrylate; 27813-02-1; 2-Hydroxypropylmethacrylate; 923-26-2; HPMA; 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-HPMA; 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; ACMC-20ah6g; Epitope ID:131322; 2-Propenoic acid, 2-methyl-, monoester with 1,2-propanediol, homopolymer; DSSTox_RID_77971; DSSTox_RID_78619; DSSTox_RID_78792; DSSTox_GSID_25934; DSSTox_GSID_27936; DSSTox_GSID_29629; SCHEMBL19017; 9086-85-5; KSC205K5R; CHEMBL1873783; DTXSID1029629; Methacrylic acid 2-hydroxypropyl; .beta.-hydroxypropyl methacrylate; CTK1A5558; 1,2-Propanediol, 1-methacrylate; 2-Hydroxypropyl 2-methylacrylate #; KS-00000VL9; Tox21_200694; Tox21_201232; Tox21_202531; ANW-73190; AKOS015899917; FCH1116000; LS-1083; ACM27813021; NCGC00090806-01; NCGC00090806-02; NCGC00090806-03; NCGC00258248-01; NCGC00258784-01; NCGC00260080-01; AK105956; AS-59279; CAS-923-26-2; CC-11007; LS-89931; AX8019918; CAS-25703-79-1; CAS-27813-02-1; FT-0694519; M0512; NS00014926; 128399-EP2277565A2; 128399-EP2277566A2; 128399-EP2277567A1; 128399-EP2277568A2; 128399-EP2277569A2; 128399-EP2277570A2; 128399-EP2292280A1; 128399-EP2295401A2; 140484-EP2277565A2; 140484-EP2277566A2; 140484-EP2277567A1; 140484-EP2277568A2; 140484-EP2277569A2; 140484-EP2277570A2; 140484-EP2292280A1; 2-Propenoic acid,2-methyl-,2-hydroxypropyl ester; C-33889; Hydroxypropyl methacrylate(7.4cp(30 degrees c)); Q27124054; N-(2-Hydroxypropyl)methacrylamide; 21442-01-3; N-(2-hydroxypropyl)-2-methylprop-2-enamide; 2-Propenamide,N-(2-hydroxypropyl)-2-methyl-; Poly(n-(2-hydroxypropyl)methacrylamide); 2-Propenamide, N-(2-hydroxypropyl)-2-methyl-; N-(2-HYDROXYPROPYL) METHACRYLAMIDE; Duxon; UNII-R3F262Z4E0; R3F262Z4E0; N-(2-Hydroxypropyl)methacrylamide polymer; SCHEMBL16097; CTK4E6787; KS-00002BES; DTXSID30944024; FCH920532; MFCD00080531; AKOS006344692; HY-W077028; SS-4855; VZ32912; 40704-75-4; OR311087; 2-Hydroxypropyl methacrylamide, 99% (GC); DB-045587; CS-0115777; FT-0602791; N-(2-hydroxypropyl)-2-methyl-2-propenamide; X3687; 2-methyl-N-(2-oxidanylpropyl)prop-2-enamide; N-(2-Hydroxypropyl)-2-methyl-prop-2-enamide; A815380; C-02233; N-(2-Hydroxypropyl)-2-methylprop-2-enimidic acid; Q27287739; 2-Propenamide, N-(2-hydroxypropyl)-2-methyl-, homopolymer; N-(2-Hydroxypropyl)-2-methyl-prop-2-enamide, AldrichCPR; rocryl410; Photomer 2317; 2-Hydroxypropyl meth; HYDROXYPROPYL METHACRYLATE; Hydroxy propyl ethacrylate; 2-HYDROXYPROYL METHACRYLATE; 2-HYDROXYPROPYL METHACRYLATE; Methacrylsurehydroxypropylester; Hydroxypropyl Methacrylate HPMA; Hydroxypropyl Methacrylate; S-(2-HYDROXYPROPYL)MERCAPTURIC ACID; N-ACETYL-S-(2-HYDROXYPROPYL)CYSTEINE, DICYCLOHEXYLAMMONIUM SALT; S-(2-Hydroxypropyl)mercapturic Acid, HPMA;N-Acetyl-3-[(2-hydroxypropyl)thio]alanine DicyclohexylaMMoniuM Salt; N-Acetyl-S-(2-hydroxypropyl)-L-Cysteine Dicyclohexylammonium Salt; doxorubicin-HPMA; doxorubicin-HPMA copolymer conjugate; HPMA-doxorubicin; 1-methacryloylamino-2-hydroxypropane; N-(2-HYDROXYPROPYL)METHACRYLAMIDE; N-(2-Hydroxypropyl)-2-methyl-prop-2-enamide; N-(2-Hydroxypropyl)-2-methyl-prop-2-enamide AldrichCPR; 2-Hydroxypropyl methacrylamide 99% (GC); 2-Hydroxypropyl methacrylamide; 2-Propenamide,N-(2-hydroxypropyl)-2-methyl-; N-(2-hydroxypropyl)methacrylamide); HPMA; N-(2-Hydroxypropyl)-2-methyl-prop-2-enamide; 2-Hydroxypropyl methacrylate; 27813-02-1; 2-Hydroxypropylmethacrylate; 923-26-2; HPMA; 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-HPMA; 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; ACMC-20ah6g; Epitope ID:131322; 2-Propenoic acid, 2-methyl-, monoester with 1,2-propanediol, homopolymer; DSSTox_RID_77971; DSSTox_RID_78619; DSSTox_RID_78792; DSSTox_GSID_25934; DSSTox_GSID_27936; DSSTox_GSID_29629; SCHEMBL19017; 9086-85-5; KSC205K5R; CHEMBL1873783; DTXSID1029629; Methacrylic acid 2-hydroxypropyl; .beta.-hydroxypropyl methacrylate; CTK1A5558; 1,2-Propanediol, 1-methacrylate; 2-Hydroxypropyl 2-methylacrylate #; KS-00000VL9; Tox21_200694; Tox21_201232; Tox21_202531; ANW-73190; AKOS015899917; FCH1116000; LS-1083; ACM27813021; NCGC00090806-01; NCGC00090806-02; NCGC00090806-03; NCGC00258248-01; NCGC00258784-01; NCGC00260080-01; AK105956; AS-59279; CAS-923-26-2; CC-11007; LS-89931; AX8019918; CAS-25703-79-1; CAS-27813-02-1; FT-0694519; M0512; NS00014926; 128399-EP2277565A2; 128399-EP2277566A2; 128399-EP2277567A1; 128399-EP2277568A2; 128399-EP2277569A2; 128399-EP2277570A2; 128399-EP2292280A1; 128399-EP2295401A2; 140484-EP2277565A2; 140484-EP2277566A2; 140484-EP2277567A1; 140484-EP2277568A2; 140484-EP2277569A2; 140484-EP2277570A2; 140484-EP2292280A1; 2-Propenoic acid,2-methyl-,2-hydroxypropyl ester; C-33889; Hydroxypropyl methacrylate(7.4cp(30 degrees c)); Q27124054; N-(2-Hydroxypropyl)methacrylamide; 21442-01-3; N-(2-hydroxypropyl)-2-methylprop-2-enamide; 2-Propenamide,N-(2-hydroxypropyl)-2-methyl-; Poly(n-(2-hydroxypropyl)methacrylamide); 2-Propenamide, N-(2-hydroxypropyl)-2-methyl-; N-(2-HYDROXYPROPYL) METHACRYLAMIDE; Duxon; UNII-R3F262Z4E0; R3F262Z4E0; N-(2-Hydroxypropyl)methacrylamide polymer; SCHEMBL16097; CTK4E6787; KS-00002BES; DTXSID30944024; FCH920532; MFCD00080531; AKOS006344692; HY-W077028; SS-4855; VZ32912; 40704-75-4; OR311087; 2-Hydroxypropyl methacrylamide, 99% (GC); DB-045587; CS-0115777; FT-0602791; N-(2-hydroxypropyl)-2-methyl-2-propenamide; X3687; 2-methyl-N-(2-oxidanylpropyl)prop-2-enamide; N-(2-Hydroxypropyl)-2-methyl-prop-2-enamide; A815380; C-02233; N-(2-Hydroxypropyl)-2-methylprop-2-enimidic acid; Q27287739; 2-Propenamide, N-(2-hydroxypropyl)-2-methyl-, homopolymer; N-(2-Hydroxypropyl)-2-methyl-prop-2-enamide, AldrichCPR; rocryl410; Photomer 2317; 2-Hydroxypropyl meth; HYDROXYPROPYL METHACRYLATE; Hydroxy propyl ethacrylate; 2-HYDROXYPROYL METHACRYLATE; 2-HYDROXYPROPYL METHACRYLATE; Methacrylsurehydroxypropylester; Hydroxypropyl Methacrylate HPMA; Hydroxypropyl Methacrylate; S-(2-HYDROXYPROPYL)MERCAPTURIC ACID; N-ACETYL-S-(2-HYDROXYPROPYL)CYSTEINE, DICYCLOHEXYLAMMONIUM SALT; S-(2-Hydroxypropyl)mercapturic Acid, HPMA;N-Acetyl-3-[(2-hydroxypropyl)thio]alanine DicyclohexylaMMoniuM Salt; N-Acetyl-S-(2-hydroxypropyl)-L-Cysteine Dicyclohexylammonium Salt; doxorubicin-HPMA; doxorubicin-HPMA copolymer conjugate; HPMA-doxorubicin; 1-methacryloylamino-2-hydroxypropane; N-(2-HYDROXYPROPYL)METHACRYLAMIDE; N-(2-Hydroxypropyl)-2-methyl-prop-2-enamide; N-(2-Hydroxypropyl)-2-methyl-prop-2-enamide AldrichCPR; 2-Hydroxypropyl methacrylamide 99% (GC); 2-Hydroxypropyl methacrylamide; 2-Propenamide,N-(2-hydroxypropyl)-2-methyl- 

HPMA

This special volume is devoted to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. It is an opportunity to review what was done and identify directions for future research. The HPMA 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 HPMA 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 HPMA 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 [11-21], 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, HPMA, was chosen for future development [22,23].

2.2. First HPMA copolymer drug and/or protein conjugates

Macromolecules are internalized by cells via endocytosis and ultimately localize in the (enzyme rich) lysosomal compartment. Consequently, we developed HPMA copolymers containing enzymatically degradable bonds (Fig. 3) [34]. Oligopeptide side-chains were designed as drug attachment/release sites [35] and shown to be degradable in vivo [36]. An external file that holds a picture, illustration, etc. Object name is nihms159442f3.jpg Open in a separate window Fig. 3 HPMA copolymers containing enzymatically cleavable bonds [30,34,37-45,47-49,55].

The first degradable polymer carriers based on HPMA were also reported at the Polymers in Medicine Microsymposium in the Prague in 1977 [52] and at conferences in Varna [53] and Tashkent [54]. We used the oxidized insulin B chain (it contains two amino groups at positions 1 and 29) to prepare branched, water-soluble HPMA copolymers by reacting insulin B-chain with HPMA copolymers containing side-chains terminated in p-nitrophenyl esters. The polymers were cleavable (Fig. 4), 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 [38]. The latter type of polymer carrier was evaluated in vivo in rats and it was shown that the branched polymer carrier is degradable and its molecular weight distribution decreases with time following i.v. administration [36]. These experiments demonstrated the possibility to manipulate the intravascular half-life of polymeric carriers based on HPMA.

An external file that holds a picture, illustration, etc. Object name is nihms159442f4.jpg Fig. 4 Branched HPMA copolymers containing the GFF degradable sequence in crosslinks; this sequence mimics the amino acid residues 23-25 of the insulin B chain [38,52]. 2.4. Validation of the targetability of HPMA 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 HPMA copolymer was based on the observation [56] 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 [56]. To determine if one can mimic this process with a synthetic macromolecule, we synthesized HPMA copolymers with N-methacryloylglycylglycine p-nitrophenyl ester and attached galactosamine by aminolysis [57]. These copolymers behaved similarly to the glycoproteins and were biorecognizable in vivo (Fig. 5). Their clearance from the bloodstream was related to the N-acylated galactosamine content (1-11 mol%) of the HPMA copolymer [57-59]. 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 HPMA copolymers were internalized by liver cells and transported, with time, into the secondary lysosomes [59,60]. It was very important to find that HPMA copolymers containing side-chains terminated in galactosamine and anticancer drug adriamycin also preferentially accumulated in the liver, i.e., it appeared that non-specific hydrophobic interactions with cell membranes did not interfere with the biorecognition by hepatocytes [61]. An external file that holds a picture, illustration, etc. Object name is nihms159442f5.jpg Open in a separate window Fig. 5 Validations of the targetability of HPMA copolymers. N-acylated galactosamine as the targeting moiety was chosen to mimic the glycoprotein-asialoglycoprotein system [57-59]. In parallel, efforts on the targetability of HPMA copolymer-antibody conjugates started. First HPMA 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) [62]. The targetability and activity of anti-Thy1.2 conjugates with HPMA copolymer-daunomycin conjugates was proven in vivo on a mouse model [63]. Anti-Thy1.2 antibodies were also efficient in targeting HPMA copolymer-photosensitizer (chlorin e6) conjugates [64].

2.5. Early interdisciplinary collaborations

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 HPMA copolymer-drug conjugates and biodegradable carriers based on HPMA). 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 HPMA 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 [42,65] and their stability in blood plasma and serum [46]. More than 300 different polymer structures containing oligopeptide sequences were synthesized in the Prague laboratory [24,25,35,47], and biological properties of a number of them evaluated at Keele within a 10 year period [66,67]. The collaboration with Vladimír Kostka and coworkers from the Institute of Organic Chemistry and Biochemistry in Prague on the cleavability of peptide sequences in HPMA copolymers by cathepsin B [44, Fig. 4], 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 (see Fig. 3, example 5), 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 [69-72] and biorecognition (targeting) [62-64] of HPMA conjugates commenced. These collaborations resulted in the filing of “Polymeric drugs” patent application in 1985 [68]. Kopecek coined the name for the HPMA copolymers evaluated in clinical trials as PK1 and PK2(P for Prague, K for Keele) (Fig. 6). An external file that holds a picture, illustration, etc Object name is nihms159442f6.jpg

Structures of PK1 and PK2, first HPMA copolymers evaluated in clinical trials [68]. Conjugate PK1 contains doxorubicin bound to HPMA 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.

3. HPMA copolymer-drug conjugates

The early experiments provided the foundation for the development of HPMA 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 HPMA copolymer conjugates follows:

HPMA 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 [85] due to the EPR effect [79,100]. However, if they possess a non-degradable backbone, they may deposit and accumulate in various organs [18]. We have previously synthesized high molecular weight carriers by connecting HPMA chains via lysosomally degradable oligopeptide sequences [34] to form water-soluble branched conjugates [36,38-41,101-103]. 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 [36]. These water-soluble copolymers were synthesized by crosslinking (short of gel point) of HPMA 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 HPMA copolymers [85,104]. New crosslinking agents were synthesized and high molecular weight copolymers prepared by crosslinking copolymerization. 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 [104] is also suitable for the synthesis of HPMA 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 [85]. Copolymerization of HPMA, 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 HPMA 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 (Fig. 7). 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 HPMA 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 [85].

An external file that holds a picture, illustration, etc. Object name is nihms159442f7.jpg Open in a separate window Fig. 7 Long-circulating HPMA copolymer-DOX (P-DOX) conjugates of different molecular weight (Mw). (A) Chemical structure of HPMA copolymer-doxorubicin conjugate containing glycylphenylalanylleucylglycine side-chains and N2,N5-bis(N-methacryloylglycylphenylalanylleucylglycyl)ornithine crosslinker [104]; (B) concentration of DOX in OVCAR-3 carcinoma xenografts in nu/nu mice after i.v. bolus of free DOX or P-DOX of different Mw; (C) growth inhibition of s.c. human ovarian OVCAR-3 carcinoma xenografts in nu/nu mice by long-circulating P-DOX conjugates. The mice received i.v. injection of 2.2 mg/kg DOX equivalent dose as P-DOX of different Mw [85].

We hypothesized that HPMA copolymer-bound DOX [P(GFLG)- DOX] (P is the HPMA copolymer backbone) would behave differently than free DOX during long term incubation with cancer cells. To verify the hypothesis, we have studied the effect of free DOX and P(GFLG)- DOX on the induction of multidrug resistance and changes in metabolism in human ovarian carcinoma A2780 cells during repeated cyclic (chronic) exposure [111]. Such experiments are of therapeutic relevance. The development of multidrug resistance during adaptation of sensitive human ovarian carcinoma A2780 cells to free DOX and P(GFLG)-DOX was analyzed. Adaptation of sensitive A2780 cells to repeated action of free DOX augmented cellular resistance to DOX and finally led to the over-expression of the MDR1 gene. On the other hand, P(GFLG)-DOX induced neither the multidrug resistance with or without MDR1 gene expression, nor the adaptation of the sensitive A2780 cells to free DOX [111].

An external file that holds a picture, illustration, etc. Object name is nihms159442f8.jpg Fig. 8 Effect of free DOX (squares) and HPMA copolymer-bound DOX (triangles) on the growth of sensitive A2780 and multidrug resistant A2780/AD human ovarian carcinoma xenografts in female nu/nu mice. Mice were treated i.p. 6 times over 3 weeks (1st and 4th day of each week) with the maximum tolerated dose of free DOX (5 mg/kg) and P(GFLG)- DOX (25 mg/kg). Circles – control tumor. Means±SE are shown [89].

Finally, we have demonstrated the advantages of targeted combination chemotherapy and photodynamic therapy using OV-TL16- targeted HPMA copolymer-DOX and HPMA copolymer-mesochlorin e6 conjugates. OV-TL16 antibodies are complementary to the OA-3 antigen (CD47) present on the majority of ovarian cancers. The immunoconjugates (Fig. 9) preferentially accumulated in human ovarian carcinoma OVCAR-3 xenografts in nude mice with a concomitant increase in therapeutic efficacy when compared with non-targeted conjugates [83]. The targeted conjugates suppressed tumor growth for the entire length of the experiment (>60 days; unpublished data).

An external file that holds a picture, illustration, etc. Object name is nihms159442f9.jpg Open in a separate window Fig. 9 Efficacy of combination chemotherapy and photodynamic therapy of OVCAR-3 xenografts in nude mice with non-targeted and OV-TL16 antibody-targeted HPMA copolymer conjugates. Therapeutic efficacy of combination therapy of HPMA copolymer-bound Mce6 (P(GFLG)-Mce6) and DOX (P(GFLG)-DOX) targeted with OV-TL 16 antibodies toward OVCAR-3 xenografts was compared to non-treated xenografts and non-targeted combination chemotherapy and photodynamic therapy. Equivalent doses of targeted combination therapy enhanced the tumor-suppressive effect as compared to non-targeted combination therapy. Dose administered: 2.2 mg/kg DOX equivalent and 1.5 mg/kg Mce6 equivalent. Irradiation for photodynamic therapy: 650 nm, 200 mW/cm2 18 h after administration [83, unpublished].

The combination index (CI) analysis was used to quantify the synergism, antagonism, and additive effects of binary combinations of free and HPMA copolymer-bound anticancer drugs, 2,5-bis(5-hydroxymethyl- 2-thienyl)furan (SOS), DOX, and mesochlorin e6 mono-ethylenediamine (Mce6) in anticancer effect toward human renal carcinoma A498 cells. The combination of SOS+DOX proved to be synergistic over all cell growth inhibition levels. All other combinations exhibited synergism in a wide range of drug effect levels [117]. Similarly, the targeted (using Fab′ of OV-TL16 antibody) and nontargeted targeted HPMA copolymer-drug conjugates, P(GFLG)-Mce6 and P(GFLG)-SOS, were evaluated against human ovarian carcinoma OVCAR-3 cells. The observations that most combinations produced synergistic effects will be important for clinical translation [118].

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 [119]. The aminobisphosphonate alendronate (ALN), and the potent anti-angiogenic agent TNP-470 were conjugated with HPMA copolymer. Using reversible addition-fragmentation chain transfer (RAFT) polymerization, we synthesized a HPMA 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 HPMA copolymer conjugate reduced vascular hyperpermeability and remarkably inhibited human osteosarcoma growth in mice by 96%. These findings indicate that HPMA 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 (Fig. 10) [119].

Inhibition of MG-63-Ras human osteosarcoma growth in mice by HPMA 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). Adapted from [119].

3.4. Novel targeting strategies

As discussed in 3.1, HPMA copolymer-drug conjugates accumulate passively in solid tumors as a result of the (molecular weight dependent) enhanced permeation and retention (EPR) effect [85]. Active targeting of HPMA copolymer-drug conjugates can be achieved with the incorporation of cancer cell-specific ligands, such as carbohydrates, lectins, antibodies, antibody fragments, and peptides, resulting in enhanced uptake of conjugates by cancer cells through receptor-mediated endocytosis with concomitant improvement of therapeutic efficacy [120,121].

Among different cancer targeting molecules, peptides are of particular interest. Enhanced peptide targeting efficiency can be achieved through multivalent interactions [122] between targets and HPMA copolymer-peptide conjugates containing multiple copies of peptides within a single polymer chain (Fig. 11) [123].

Multivalency effect in the biorecognition of HPMA copolymer-peptide-DOX conjugates. Inhibition of Raji B cell growth by exposure to HPMA copolymer-DOX (P (GFLG)-DOX) conjugate containing varying amount of targeting peptide, EDPGFFN-VEIPEF, per macromolecule. (A) Structure of conjugate; (B) inhibition of Raji B cell growth by P(GFLG)-DOX (no targeting peptide), P(GFLG)-DOX containing 1.9 mol% targeting peptide, and P(GFLG)-DOX containing 3.9 mol% targeting peptide. Adapted from [123].

Combinatorial approaches, such as phage display or synthetic peptide libraries, are suitable for the identification of targeting peptides. Overexpression of the CD21 receptor was found on lymphoblastoid cell lines such as Raji cells; consequently, we have used these techniques to identify targeting moieties for lymphomas [124,125]. With phage display, five distinctive peptides (RMWPSSTVNLSAGRR, PNLDFSPTCSFRFGC, GRVPSMFGGHFFFSR, RLAYWCFSGLFLLVC, and PVAAVSFVPYLVKTY) were identified as ligands of CD21 receptor. The dissociation constants of selected peptides were determined to be in the micromolar range [124]. Using a synthetic chemical combinatorial technique, one-bead one-compound (OBOC) method, we identified four heptapeptides (YILIHRN, PTLDPLP, LVLLTRE, and IVFLLVQ) as ligands for the CD21 receptor [125]. The dissociation constants were found to be similar to peptides selected by phage display. Importantly, the peptides retained their biorecognizability towards CD21 receptor after they were conjugated to HPMA copolymers and demonstrated a multivalency effect [125]. Several peptide-targeted HPMA copolymer- drug conjugates displayed anticancer activity [123,126,127]. The combinatorial chemistry approach (OBOC), when combined with a high-stringency screening method, is able to identify peptides with a picomolar affinity [128,129].

3.4.1. Oral, colon-specific delivery of drugs

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 [130]. To achieve colon-specific delivery, a (aromatic amino group-containing) drug may be attached to HPMA copolymer side-chains via an aromatic azo bond cleavable by the azoreductase activities present in the colon [51,131-138]. For example, the release of 5-aminosalicylic acid bound to HPMA copolymers via an aromatic azo bond was demonstrated using Streptococcus faecium, an isolated strain of bacteria commonly found in the colon [131], the cecum contents of rats, guinea pigs, and rabbits [133], and in human feces [133].

Recently, we concentrated on the oral delivery of 9-aminocamptothecin (9-AC). First, we attached 9-AC to HPMA copolymers through a spacer containing an aromatic azo bond and amino acid residues [134,135]. It was shown that the aromatic azo bond was cleaved first in vitro [134] and in vivo [135], 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 [51]. 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 HPMA copolymer conjugate by cecal contents in vitro, with concomitant stability in simulated upper GI tract conditions. The conjugate possessed a favorable pharmacokinetics [136,137] and was effective in colon cancer models (Fig. 12) [138].

HPMA copolymer-9-aminocamptothecin conjugate. (A) Structure and scheme of release of unmodified 9-AC from HPMA copolymer-9-AC conjugates by a two-step process – rate controlling aromatic azo bond cleavage, followed by fast 1,6-elimination [51]; (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 [138].

3.4.1.1. Targeting in the gastrointestinal tract

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 [139-141]. Whereas WGA (wheat germ agglutinin) binds to healthy tissues, PNA (peanut agglutinin) binds to diseased tissues. We hypothesized that HPMA copolymer-lectin-drug conjugates could deliver therapeutic agents to diseased tissues by targeting colonic glycoproteins. We examined biorecognition of free and HPMA 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 HPMA copolymer-lectin-drug conjugates may provide site-specific treatment of conditions like colitis or Barrett’s esophagus [141].

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 HPMA copolymer-based delivery systems that employ triphenylphosphonium ions as mitochondriotropic agents [147] were developed [142]. 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 [148]. Subsequently, HPMA copolymer-drug conjugates were synthesized using a photosensitizer mesochlorin e6 (Mce6). Mitochondrial targeting of HPMA copolymer-bound Mce6 enhanced cytotoxicity as compared to non-targeted HPMA copolymer-Mce6 conjugates [142]. Minor modifications may be required to adapt the current design and allow for tumor site-specific mitochondrial targeting of other therapeutic agents.

Novel HPMA copolymer-based delivery systems of this derivative were also synthesized [143]. After internalization of a HPMA 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 [143]. 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 (Fig. 13) 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 [143].

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 HPMA copolymer-based drug carriers [158]. HPMA 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 HPMA copolymer-AR-GDM conjugates depended on the structure of AR-GDM [160].

To verify the hypothesis that P(AP-GDM) [HPMA 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 [91]. 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 [91]. It is possible that internalization of HPMA 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 (Fig. 15). Similarly, we previously observed that A2780 cells treated with HPMA 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 HPMA 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 [91].

3.7. Cancer: clinical trials

HPMA 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 HPMA copolymer-DOX [163-165], HPMA copolymer-DOX-galactosamine [166], HPMA copolymer-camptothecin [167], HPMA copolymer-paclitaxel [168], and HPMA copolymer-platinates [169]. 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.

3.8. HPMA copolymer conjugates in the treatment of non-cancerous diseases

HPMA copolymer-drug conjugates may be used also for the treatment of diseases other than cancer. We designed bone-targeted HPMA copolymer-conjugated with a well-established bone anabolic agent (prostaglandin E1; PGE1) for the treatment of osteoporosis and other musculoskeletal diseases [50,170-175]. The biorecognition of the conjugates by the skeleton was mediated by an octapeptide of D-aspartic acid (D-Asp8) or alendronate [170,172].

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 [50,176]. 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 HPMA 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 E1, a PGE1 prodrug (Fig. 17A).

Structure of HPMA copolymer-prostaglandin E1-Asp8 conjugate and mechanism of its cleavage by cathepsin K followed by 1,6-elimination (A) [50]; 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) [174].

This novel delivery system has several distinct advantages. First of all, it 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 [171,173]. Fourthly, the polymeric carrier can be eliminated from hard tissues and, subsequently, cleared from the body via kidney glomerular filtration. It also offers proper protection of the conjugated PGE1 from metabolism before it 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 [172]. In vivo experiments on ovariectomized rats have proven the concept. Following a single i.v. administration of the HPMA copolymer-Asp8-PGE1 conjugate to aged, ovariectomized rats, bone formation rates were substantially greater than controls when measured 28 days later (Fig. 17B) [173]. HPMA copolymer conjugates have been also successful in the treatment of rheumatoid arthritis [175,177].

3.9. Methods of synthesis of HPMA copolymer conjugates

The topic of conjugate synthesis will be extensively covered throughout this volume. We have demonstrated several approaches through this chapter. Consequently, we shall just mention some of the important points, which were not covered.

3.9.1. Hydrolytically cleavable bond between drug and HPMA copolymer

Due to the decreased pH in the endosomes and lysosomes, pH-sensitive bonds are suitable for intracellular drug delivery. We have synthesized HPMA copolymer-adriamycin (ADR=DOX) conjugates where ADR was bound via cis-aconityl bond [177] (Fig. 18). 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 [178].

3.9.2. Disulfide-linked HPMA copolymer-mesochlorin e6 conjugates

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 HPMA copolymer backbone once internalized in tumor tissue (Fig. 19). 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 [179].

3.9.3. Attachment of targeting moieties

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 HPMA copolymers impacts the mechanism of internalization and subcellular trafficking [180] and designed polymerizable antibody fragments [84].

3.9.4. Polymerizable antibody fragments

An innovative pathway for the synthesis of targeted polymeric drug delivery systems using polymerizable antibody fragments was designed [84]. A new macromonomer, a polymerizable antibody Fab′ fragment (MA-Fab′) of the OV-TL 16 antibody (IgG1) has been synthesized and copolymerized with HPMA to produce poly(HPMA-co- MA-Fab′) (Fig. 20). 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 [84].

3.9.5. Impact of chemistry of attachment of antibodies to HPMA copolymers on the binding affinity of the conjugates

The influence of different methods of coupling the OV-TL16 antibody and its Fab′ fragment to HPMA 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 [181]. Method A: binding via amide bonds formed by aminolysis of active ester groups on the HPMA 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 HPMA 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 HPMA copolymer-Mce6 conjugate. Differences in Ka were observed as shown in Fig. 21.

The synthesis of HPMA copolymer conjugates, especially their molecular weight distribution, can be controlled by methods of living radical polymerization, RAFT (reversible addition-fragmentation chain transfer) [182] and ATRP (atom transfer radical) polymerizations [183]. For example, HPMA copolymer conjugates containing two drugs and one fluorescent label per macromolecule using RAFT copolymerization were recently synthesized [119; Fig. 10].

3.9.7. Attachment of oligonucleotides

Aminolysis of HPMA copolymer precursors can be used for attachment of oligonucleotides to HPMA copolymers. We have attached a 21-mer phosphorothioate oligonucleotide (5′-TTTATAAGGGTCGATGTCCXX-3′) to HPMA copolymers containing GG-ONp and GFLG-ONp (ONp is p-nitrophenoxy) side-chains [184]. 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 HPMA copolymer-phosphorothioate oligonucleotides was studied. Covalently attaching the oligonucleotides to the HPMA copolymers via non-degradable dipeptide GG spacers resulted in sequestering the oligonucleotide in vesicles after internalization. Conjugation of the oligonucleotides to an HPMA 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 HPMA 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 -HPMA copolymer conjugates were internalized to a greater extent than unconjugated polymers [184].

3.9.8. Attachment of cell penetrating peptides (CPP)

These peptides, such as the Tat peptide (48GRKKRRQRRR57) which originates from HIV-1 Tat protein, a potent activator of HIV-1 transcription, have been attached using various chemical designs. One approach is to add several amino acid residues, e.g. to produce 48GRKKRRQRRR57YK(FITC)C. The latter may be attached to HPMA copolymer side-chains terminated in maleimide via thioether bonds [185]. We have recently reviewed the biological implications of CPP attachment [144], so we shall not discuss it here.

 

4.1. Modification with semitelechelic poly(HPMA)

Poly(HPMA) [189] exhibits similar properties as poly(ethylene glycol) [190] when used for modification of enzymes or vesicular carriers. The first report on semitelechelic poly(HPMA) (ST-PHPMA) was published in 1995 [191]. Modification of nanospheres, based on a copolymer of methyl methacrylate, maleic anhydride, and methacrylic acid, with ST-PHPMA resulted in decreased protein adsorption in vitro and increased intravascular half-life, as well as decreased accumulation in the liver, after intravenous administration into rats. The higher the molecular weight of ST-PHPMA, the more pronounced the changes in these properties (Fig. 22). These data seem to indicate the influence of the hydrodynamic thickness of the coating layer on the process of opsonization and capture by Kupffer cells of the liver and macrophages of the spleen [191].

First synthesis and application of semitelechelic poly(HPMA) to modify surface of nanospheres. (A) Structure; (B) adsorption isotherms of IgG onto surface-modified P (MMA-MA-MAA) nanospheres in 1/15 M saline at 25 °C for 3 h. Each point represents the mean±SD (n=3); (C) body distribution profiles for unmodified and modified [14C]-P(MMA-MA-MAA) nanospheres in rats 24 h after i.v. administration and the correlation with Mn of ST-PHPMA. Each point represents the mean±SD (n=5) [191].

Similarly, carboxyl and amino group modification of chymotrypsin with ST-PHPMA-CONHNH2 and ST-PHPMA-COOSu (N-hydroxysuccinimide ester) produced conjugates [189] with comparable properties to PEG-modified chymotrypsin [187]. Ulbrich et al. used ST-PHPMA to modify ribonuclease, chymotrypsin [192], and superoxide dismutase [193]. The proteolytic stability of modified protein increased and their immunogenicity decreased [192,193].

4.2. Modification with HPMA copolymers containing multiple reactive side-chains

First protein modified with HPMA copolymers was prepared by the reaction of the copolymer of HPMA with N-methacryloylglycylglycine p-nitrophenyl ester with insulin [28]. The unreacted protein was separated on Sephadex 75; the HPMA copolymer-insulin conjugate exhibited a slower onset and a slight prolongation of hypoglycemic effect in rats when compared to free insulin [28]. Chymotrypsin [29,30] and cobra venom acetylcholinesterase [194] 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 (HPMA copolymers with oligopeptide side-chains terminated in p-nitroanilide) catalyzed by HPMA 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 [30].

Different chemistry was used for the modification of cobra venom acetylcholinesterase [194]. The secondary OH groups of poly(HPMA) (Mw 25-30 kDa) were activated with 4-nitrophenyl chloroformate in dimethylformamide followed by attachment of acetylcholinesterase in borate buffer. The poly(HPMA)-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 (Fig. 23) [194].

HPMA copolymer-modified acetylcholinesterase. (A) Structure; (B) enhancement of intravascular half-life in mice after i.v. administration of HPMA copolymer-modified enzyme; (C) augmentation of thermal stability of HPMA copolymer-modified enzyme [194].

HPMA copolymer with N-methacryloylglycylphenylanylleucylglycine p-nitrophenylester was used for the modification of ribonuclease [195] and superoxide dismutase [193]. No difference in biological activity of conjugates prepared using ST-PHPMA and HPMA copolymers with reactive side-chains was detected [193].

There is considerable activity in using HPMA 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 review [196].

5.1. HPMA-based hydrogels

First HPMA-based hydrogels were synthesized by crosslinking copolymerization of HPMA and methylene-bis-acrylamide or ethylene- bis-methacrylamide in the early 70s [197]. The kinetic course of copolymerization, interaction parameter polymer-water, the modulus of elasticity, and concentration of elastically effective chains were characterized.

Degradable hydrogels containing oligopeptide crosslinks susceptible to chymotrypsin-catalyzed hydrolysis were synthesized by crosslinking HPMA copolymers containing reactive side-chains (terminated in p-nitrophenoxy groups) with oligopeptide (GGY, GFY, GLF, AGVY, and AGFY)-containing diamines [49]. The degradability of hydrogels was dependent on the length and detailed structure of the oligopeptide sequence and on the network density, and thus the equilibrium degree of swelling (Fig. 24). The higher the degree of swelling, the faster the rate of degradation. The degree of swelling also has an impact on surface vs. bulk degradation of the hydrogel. If the enzyme cannot diffuse into the hydrogel interior, only surface degradation takes place. This was demonstrated by a comparison of the degradation of HPMA copolymer crosslinked with AGVY-containing sequences catalyzed by chymotrypsin and HPMA copolymer-bound chymotrypsin. Only surface degradation of the hydrogel was observed in the latter case due to the larger size of polymer-modified enzyme [49]. HPMA-based hydrogels containing the sequence GFYAA in the crosslinks were cleavable with cathepsin B, a lysosomal thiol proteinase [44]. In further experiments, HPMA-based hydrogels with degradable crosslinks were shown to release FITC-dextran and daunomycin during incubation with a mixture of lysosomal enzymes (Tritosomes) or chymotrypsin [198].

Crosslinking copolymerization of HPMA with N,O-dimethacryloyl-hydroxylamine produced hydrolytically degradable hydrogels [199]. These hydrogels were used as a depo for anticancer drug (DOX); results of combination therapy using DOX release from hydrogels followed by antibody-targeted therapy has been effective in the treatment of terminal stages of Bcl1 leukemia in mice [200].

Hennink et al. synthesized ABA triblock copolymers, where block A is thermosensitive poly(HPMA lactate) and block B is PEG. Heating of the copolymer results in the formation of a viscoelastic material, which may be stabilized (crosslinked) by photopolymerization [202]. These materials are suitable for protein delivery [203].

Self-assembly of HPMA graft copolymers, CCK-P and CCE-P, into hybrid hydrogels mediated by antiparallel heterodimer coiled-coil formation. Two distinct pentaheptad peptides (CCE and CCK) were designed to create a dimerization motif and serve as physical crosslinkers (P is the HPMA copolymer backbone). Aqueous solutions of CCE-P or CCK-P did not form gels. In contrast, gel-like materials were formed from equimolar mixtures of CCE-P/CCK-P at low concentrations [211].

HPMA

Bu özel cilt, N- (2-hidroksipropil) metakrilamid (HPMA) kopolimerlerine ayrlmtr. Neyin yapldn gözden geçirmek ve gelecekteki aratrmalar için yön belirlemek için bir frsattr. HPMA geliimi ve sunulan veriler, bu ciltteki dier yazarlarn katklaryla örtümeyecek ekilde, çounlukla yazarlarn laboratuvaryla ilgili olacaktr. laç tayclar, protein ve yüzey deitiriciler olarak ve akll hibrit biyomateryal tasarmnda sentetik bileenler olarak HPMA kopolimerleriyle yaplan çalmalar özetlenmitir. Dier laboratuvarlardan daha fazla ayrnt ve çalma, bu cildin daha odaklanm konular kapsayan dier bölümlerinde bulunabilir.

laç taycs olarak gelitirme için HPMA seçimi rastgele deildi. Hidrofilik polimerlerin yaps ile biyouyumluluklar [11-21] arasndaki ilikinin ayrntl çalmalarna dayanarak, hedefimiz olarak N-ikameli metakrilamidleri seçtik çünkü α-karbon ikamesi ve N-ikameli amid ba hidrolitik stabiliteyi salad. yan zincirler. Kolay saflatrma ve yeniden üretilebilir sentez için bir kristal monomer tanmlamaya çalan bir dizi bileik sentezledik. Sentezlemeyi baardmz ilk kristalin N-ikameli metakrilamid, HPMA, gelecekteki gelitirme için seçildi [22,23].

2.2. lk HPMA kopolimer ilac ve / veya protein konjugatlar

Makromoleküller, hücreler tarafndan endositoz yoluyla içselletirilir ve nihayetinde (enzim açsndan zengin) lizozomal bölmede lokalize olur. Sonuç olarak, enzimatik olarak parçalanabilir balar içeren HPMA kopolimerleri gelitirdik (ekil 3) [34]. Oligopeptid yan zincirleri, ilaç balanma / salm bölgeleri olarak tasarland [35] ve in vivo bozunabilir olduu gösterildi [36]. Bir resim, illüstrasyon vb. çeren harici bir dosya. Nesne ad nihms159442f3.jpg Ayr bir pencerede aç ekil 3 Enzimatik olarak parçalanabilen balar içeren HPMA kopolimerleri [30,34,37-45,47-49,55].

HPMA’ya dayal ilk bozunabilir polimer tayclar, 1977’de Prag’daki Polymers in Medicine Microsymposium’da [52] ve Varna [53] ve Takent’teki [54] konferanslarda da bildirildi. nsülin B-zincirini p-nitrofenil esterlerde sonlandrlm yan zincirler içeren HPMA kopolimerleri ile reaksiyona sokarak dall, suda çözünür HPMA kopolimerlerini hazrlamak için oksitlenmi insülin B zincirini (pozisyon 1 ve 29’da iki amino grubu içerir) kullandk. Polimerler bölünebilirdi (ekil 4), bu yüzden insülin B-zincirinden 23-25 ​​(Gly-Phe-Phe) dizisini seçtik (amino asit 25’ten kaynaklanan ba kimotripsin tarafndan bölünebilir) ve sentezlenmi dall, çözünür yüksek birincil zincirleri balayan çapraz balarda Gly-Phe-Phe segmentlerini içeren moleküler arlk enzimatik olarak bozunabilir kopolimerler [38]. kinci tip polimer tayc, sçanlarda in vivo deerlendirildi ve dall polimer taycnn bozunabildii ve moleküler arlk dalmnn, i.v. yönetim [36]. Bu deneyler, HPMA’ya dayal polimerik tayclarn intravasküler yarlanma ömrünü deitirme olasln gösterdi.

Bir resim, illüstrasyon, vb. çeren harici bir dosya. Nesne ad nihms159442f4.jpg’dir. ekil 4 Çapraz balantlarda GFF bozunur sekans içeren dallanm HPMA kopolimerleri; bu dizi, insülin B zincirinin 23-25 ​​amino asit kalntlarn taklit eder [38,52]. 2.4. HPMA kopolimer-ilaç konjugatlarnn hedeflenebilirliinin dorulanmas Bir hedefleme sisteminin seçimi ve tasarm, salam bir biyolojik manta dayanmaldr. lk hedeflenebilir HPMA kopolimerinin tasarm, glikoproteinlerin yapsndaki küçük deiikliklerin organizmada modifiye edilmi glikoproteinin kaderinde dramatik deiikliklere yol açt gözlemine [56] dayanyordu. Sçanlara bir glikoprotein (seruloplazmin) uygulandnda, uzun bir intravasküler yar ömür gözlenmitir. Bununla birlikte, terminal sialik asit seruloplazminden çkarldnda oluan asialoglikoprotein (asialoseruloplazmin), sondan bir önceki galaktoz birimlerini aça çkaran yan zincirler içerir. kincisinin intravasküler yar ömrü, molekülün hepatositler üzerindeki asialoglikoprotein reseptörü tarafndan biyolojik olarak tannmasna bal olarak önemli ölçüde ksalmtr. Bu reseptör galaktoz ve N-asetilgalaktozamin parçalarn tanr [56]. Bu süreci sentetik bir makromolekül ile taklit edip edemeyeceimizi belirlemek için, HPMA kopolimerlerini N-metakrililglisilglisin p-nitrofenil ester ile sentezledik ve aminoliz yoluyla galaktozamini ekledik [57]. Bu kopolimerler, glikoproteinlere benzer ekilde davrandlar ve in vivo biyo-tannabilirdi (ekil 5). Bunlarn kan dolamndan temizlenmesi, HPMA kopolimerinin N-asillenmi galaktozamin içerii (% 1-11 mol) ile ilikiliydi [57-59]. Sçan karacierinin hepatositlere ve parankimal olmayan hücrelere ayrlmas, polimerin büyük ölçüde hepatositler ile ilikili olduunu gösterdi ve karacierin younluk-gradyan hücre alt fraksiyonasyonu, HPMA kopolimerlerinin karacier hücreleri tarafndan içselletirildiini ve zamanla ikincil hücreye tandn dorulad. lizozomlar [59,60]. Galaktozamin ve antikanser ilaç adriamisin ile sonlandrlan yan zincirler içeren HPMA kopolimerlerinin ayrca tercihli olarak karacierde biriktiini bulmak çok önemliydi, yani hücre zarlaryla spesifik olmayan hidrofobik etkileimlerin hepatositler tarafndan biyo-tanmaya müdahale etmedii görüldü [61 ]. Bir resim, illüstrasyon vb. çeren harici bir dosya. Nesne ad nihms159442f5.jpg’dir Ayr bir pencerede açn ekil 5 HPMA kopolimerlerinin hedeflenebilirliinin dorulanmas. Hedefleme parças olarak N-asillenmi galaktozamin, glikoprotein-asialoglikoprotein sistemini taklit etmek için seçildi [57-59]. Buna paralel olarak, HPMA kopolimer-antikor konjugatlarnn hedeflenebilirlii için çabalar balad. Poliklonal ve monoklonal anti-Thy-1.2 antikorlar ve anti-FITC (fluorescein isothiocyanate) antikorlar içeren ilk HPMA kopolimer konjugatlar deerlendirildi. Daunomisin içeren hedeflenebilir konjugatlar sentezlendi ve in vitro deneyler, hedeflenen konjugatn sitotoksisitesini arttran iki büyüklük derecesini gösterdi (hedeflenmeyenle karlatrldnda) [62]. Anti-Thy1.2 konjugatlarnn HPMA kopolimer-daunomisin konjugatlaryla hedeflenebilirlii ve aktivitesi, bir fare modelinde in vivo kantlanmtr [63]. Anti-Thy1.2 antikorlar, HPMA kopolimer-a duyarllatrc (klorin e6) konjugatlarnn hedeflenmesinde de etkili olmutur [64].

2.5. Erken disiplinler aras ibirlikleri

Seksenlerin banda, biyolojik alandaki i arkadalarmzla ibirliine baladk: Birleik Krallk’taki Keele Üniversitesi’nden John Lloyd ve Ruth Duncan ve Prag’daki Mikrobiyoloji Enstitüsü’nden Blanka Říhová. Keele grubu ile ibirlii, 1977 Prag sempozyumunda bir konferans veren Helmuth Ringsdorf tarafndan balatld (Kopecek burada HPMA’ya dayal ilk HPMA kopolimer-ilaç konjugatlarn ve biyolojik olarak parçalanabilir tayclar sundu). Toplantdan sonra Ringsdorf, Lloyd’a Kopecek ile iletiime geçmesini önerdi çünkü ibirliinin her ikisi için de faydal olacan düündü. Kopecek, Temmuz 1978’de Dresden’de Lloyd ile tant ve HPMA kopolimer konjugatlarnn deerlendirilmesi üzerinde anlatlar. lk örnekler sentezlendi (ilaç modeli olarak p-nitroanilide sonlandrlan farkl yan zincirler) ve lizozomal enzimler [42,65] tarafndan bölünebilirlikleri ve kan plazmas ve serumdaki stabiliteleri [46] açsndan Keele’de deerlendirildi. Prag laboratuvarnda oligopeptid sekanslar içeren 300’den fazla farkl polimer yaps sentezlendi [24,25,35,47] ve bunlarn bir ksmnn biyolojik özellikleri 10 yllk bir süre içinde Keele’de deerlendirildi [66,67]. Vladimír Kostka ve Prag’daki Organik Kimya ve Biyokimya Enstitüsü’nden meslektalar ile HPMA kopolimerlerindeki peptit dizilerinin en önemli lizozomal sistein proteinaz olan katepsin B [44, ekil 4] tarafndan bölünebilirlii konusunda ibirlii, GFLG’nin tanmlanmasyla sonuçland. klinik deneylerde kullanlan tüm konjugatlara dahil edilen dizi. En hzl bölünen iki oligopeptitten, GFLG ve GFTA’dan (baknz ekil 3, örnek 5), T’yi önlemek için GFTA yerine GFLG dizisini seçtik; o srada potansiyel immünojenisite konusunda endieliydik. 1978’de Kopecek, Prag’da Mikrobiyoloji Enstitüsü’nde bir konferans verdi. Konferanstan sonra Říhová ile tartt ve grubuyla HPMA konjugatlarnn immünojenitesi / biyouyumluluu [69-72] ve biyo-tanma (hedefleme) [62-64] üzerine ibirlii balad. Bu ibirlikleri 1985 ylnda “Polimerik ilaçlar” patent bavurusunun yaplmasyla sonuçland [68]. Kopecek, klinik çalmalarda deerlendirilen HPMA kopolimerlerinin adn PK1 ve PK2 (Prag için P, Keele için K) olarak almtr (ekil 6). Resim, illüstrasyon vb. çeren harici bir dosya Nesne ad nihms159442f6.jpg’dir.

PK1 ve PK2’nin yaplar, ilk HPMA kopolimerleri klinik çalmalarda deerlendirildi [68]. Konjugat PK1, kan aknda stabil olan ancak lizozomlarda enzimatik olarak katalize edilmi hidrolize duyarl bir tetrapeptid dizisi araclyla HPMA kopolimerine balanan doksorubisin içerir. Konjugat PK2 ek olarak, hepatositler üzerindeki asialoglikoprotein reseptörüne tamamlayc olan N-asillenmi galaktozamin ile sonlandrlm yan zincirler içerir.

3. HPMA kopolimer-ilaç konjugatlar

lk deneyler, ilaç tayclar olarak HPMA kopolimerlerinin gelitirilmesi için temel oluturdu. Yeni bilimsel alanlarn çounda olduu gibi, aratrma balangçta yap-özellikler ilikisi üzerine temel verilerin toplanmasna odakland. Klinik olarak ilgili HPMA kopolimer konjugatlarnn gelitirilmesi için önemli olduunu düündüümüz alanlardaki aratrmalarn özeti aadaki gibidir:

HPMA kopolimer-ilaç konjugatlar, nano boyutlu (5-20 nm) suda çözünür yaplardr. Benzersiz yapsal, fizikokimyasal ve biyolojik özellikleri, düük moleküler arlkl ilaçlarla karlatrldnda avantajldr. Hedeflenen polimer-ilaç konjugatlar kavram, kanser hücreleri için düük moleküler arlkl ilaçlarn özgüllük eksikliini gidermek için gelitirilmitir.

Kat tümörlere ekstravazasyonun etkinlii, vaskülatür ve tümör dokusu arasndaki konsantrasyon gradyanna ve zamana baldr. Sonuç olarak, yüksek moleküler arlkl (uzun sirkülasyonlu) polimer konjugatlar EPR etkisine [79,100] bal olarak tümör dokusunda [85] verimli bir ekilde birikir. Ancak bozunmayan omurgaya sahiplerse çeitli organlarda birikebilir ve birikebilirler [18]. Suda çözünür dall konjugatlar [36,38-41,101-103] oluturmak için HPMA zincirlerini lizozomal olarak bozunabilir oligopeptid dizileri [34] araclyla balayarak daha önce yüksek moleküler arlkl tayclar sentezledik. Sçanlara intravenöz (i.v.) uygulamay takiben, oligopeptit çapraz balar yarld ve ortaya çkan düük moleküler arlkl polimer zincirleri idrarla atld [36]. Bu suda çözünür kopolimerler, HPMA kopolimer öncülerinin (reaktif bir ester grubunda sonlandrlm oligopeptit yan zincirleri içeren) diaminlerle çapraz balanmasyla (jel noktasndan ksa) sentezlendi.

Daha sonra, uzun süre dolaan HPMA kopolimerleri için yeni, yeniden üretilebilir bir sentetik yol tasarladk [85,104].

Yeni çapraz balama maddeleri sentezlendi ve yüksek moleküler arlkl kopolimerler çapraz balanarak kopolimerizasyon ile hazrland. Bununla birlikte, monomer karmnn bileimi, polimerizasyonun sonunda sistemin jel noktasndan (suda çözünür) ksa olaca ekilde olmaldr. Bu yöntem [104], oligopeptit çapraz balarna ek olarak doksorubisin (DOX) (veya dier antikanser ilaçlar) ile sonlandrlan oligopeptit yan zincirlerini içeren HPMA kopolimerlerinin sentezi için de uygundur.

Bu tür konjugatlarn moleküler arlnn biyolojik aktiviteleri üzerindeki etkisi deerlendirildi [85]. DOX’un polimerize edilebilir bir türevi olan HPMA’nn kopolimerizasyonu (N-metakriloilglisilfenilalanilleukilglisil doksorubisin) ve bir çapraz balama maddesi, N2, N5-bis (N-metakriloilglisilfenilalanilalanilösililglisil) ornitin içeren kopolimerizasyonu, yüksek moleküler moleküler-moleküler-moleküler-moleküler-moleküler-moleküler-moleküler-moleküler-moleküler-moleküler-moleküler arlkl MA ile sonuçland. çapraz balarn yan sra DOX’ta sonlandrlan yan zincirlerde. 22, 160, 895, 1230 kDa’lk Mw’ye sahip dört konjugat hazrland. Konjugatlarn biyolojik dalm ve s.c. içeren nu / nu farelerde tedavi etkinlii. insan yumurtalk OVCAR-3 karsinom ksenograftlar belirlendi (ekil 7). Kandaki konjugatlarn yarlanma ömrü 5 kata kadar daha uzundu ve tümörden eliminasyon oran, konjugatlarn Mw’si 22’den 1230 kDa’ya çktkça 25 kata kadar daha yavat. 160 kDa’dan daha yüksek bir Mw’ye sahip HPMA kopolimer bal DOX ile tedavi, tümör büyümesini 22 kDa veya serbest DOX’unkinden (p <0.02) daha verimli bir ekilde inhibe etti. Veriler, konjugatn moleküler arl ne kadar yüksekse, nu / nu farelerde insan yumurtalk ksenograftlarnn tedavi etkinliinin de o kadar yüksek olduunu açkça göstermitir [85].

Bir resim, illüstrasyon, vb. çeren harici bir dosya. Nesne ad nihms159442f7.jpg Ayr bir pencerede aç ekil 7 Farkl moleküler arlktaki (Mw) uzun dolaan HPMA kopolimer-DOX (P-DOX) konjugatlar. (A) Glisilfenilalanilösilglisin yan zincirleri ve N2, N5-bis (N-metakriloilglisilfenilalanilleukilglisil) ornitin çapraz balayc [104] içeren HPMA kopolimer-doksorubisin konjugatnn kimyasal yaps; (B) i.v.’den sonra nu / nu farelerinde OVCAR-3 karsinom ksenograftlarnda DOX konsantrasyonu. serbest DOX veya farkl Mw P-DOX bolusu; (C) s.c.’nin büyüme inhibisyonu nu / nu farelerindeki insan yumurtalk OVCAR-3 karsinom ksenograftlar, uzun dolaan P-DOX konjugatlar tarafndan. Fareler i.v. 2.2 mg / kg DOX edeer dozunun farkl Mw P-DOX olarak enjeksiyonu [85].

HPMA kopolimer bal DOX [P (GFLG) – DOX] (P, HPMA kopolimer omurgasdr) kanser hücreleri ile uzun süreli inkübasyon srasnda serbest DOX’ten farkl davranacan varsaydk. Hipotezi dorulamak için, serbest DOX ve P (GFLG) – DOX’un çoklu ilaç direncinin indüksiyonu üzerindeki etkisini ve tekrarlanan döngüsel (kronik) maruziyet srasnda insan yumurtalk karsinomu A2780 hücrelerinde metabolizmadaki deiiklikleri inceledik [111]. Bu tür deneyler, terapötik açdan önemlidir. Hassas insan yumurtalk karsinomu A2780 hücrelerinin serbest DOX ve P (GFLG) -DOX’a adaptasyonu srasnda çoklu ilaç direncinin geliimi analiz edildi. Duyarl A2780 hücrelerinin, serbest DOX artrlm hücresel direncin DOX’a tekrarlanan etkisine adaptasyonu ve sonunda MDR1 geninin ar ekspresyonuna yol açt. Öte yandan, P (GFLG) -DOX, MDR1 gen ekspresyonu olan veya olmayan çoklu ilaç direncini veya duyarl A2780 hücrelerinin serbest DOX’a adaptasyonunu indüklememitir [111].

Bir resim, illüstrasyon, vb. çeren harici bir dosya. Nesne ad nihms159442f8.jpg ekil 8 Serbest DOX (kareler) ve HPMA kopolimer bal DOX (üçgenler) ‘in hassas A2780 ve çoklu ilaca dirençli A2780 / AD insan büyümesi üzerindeki etkisi dii nu / nu farelerinde yumurtalk karsinomu ksenograftlar. Fareler i.p. Maksimum tolere edilen serbest DOX (5 mg / kg) ve P (GFLG) – DOX (25 mg / kg) dozu ile 3 haftada 6 kez (her haftann 1. ve 4. günü). Daireler – tümörü kontrol eder. Ortalama ± SE gösterilir [89].

Son olarak, OV-TL16 hedefli HPMA kopolimer-DOX ve HPMA kopolimer-mezoklorin e6 konjugatlarn kullanarak hedefli kombinasyon kemoterapi ve fotodinamik tedavinin avantajlarn gösterdik. OV-TL16 antikorlar, yumurtalk kanserlerinin çounda bulunan OA-3 antijenine (CD47) tamamlaycdr. mmünokonjugatlar (ekil 9), hedeflenmemi konjugatlarla karlatrldnda terapötik etkinlikte elik eden bir artla birlikte çplak farelerde tercihen insan yumurtalk karsinomu OVCAR-3 ksenograftlarnda birikmitir [83]. Hedeflenen konjugatlar, deneyin tüm uzunluu boyunca tümör büyümesini basklad (> 60 gün; yaynlanmam veriler).

Bir resim, illüstrasyon vb. çeren harici bir dosya. Nesne ad nihms159442f9.jpg Ayr bir pencerede aç ekil 9 Hedeflenmemi ve OV-TL16 antikorlu çplak farelerde OVCAR-3 ksenograftlarnn kombinasyon kemoterapisi ve fotodinamik tedavisinin etkinlii hedeflenen HPMA kopolimer konjugatlar. OVCAR-3 ksenograftlarna yönelik OV-TL 16 antikorlar ile hedeflenen HPMA kopolimere bal Mce6 (P (GFLG) -Mce6) ve DOX (P (GFLG) -DOX) kombinasyon tedavisinin terapötik etkinlii, tedavi edilmemi ksenograftlar ve Hedeflenen kombinasyon kemoterapi ve fotodinamik terapi. Hedefli kombinasyon terapisinin edeer dozlar, hedeflenmemi kombinasyon terapisine kyasla tümör basklayc etkiyi arttrd. Uygulanan doz: 2,2 mg / kg DOX edeeri ve 1,5 mg / kg Mce6 edeeri. Fotodinamik terapi için nlama: 650 nm, 200 mW / cm2 uygulamadan 18 saat sonra [83, yaynlanmam].

Kombinasyon indeksi (CI) analizi, serbest ve HPMA kopolimer bal antikanser ilaçlarn ikili kombinasyonlarnn, 2,5-bis (5-hidroksimetil-2-tienil) furan’n (SOS) sinerjizmi, antagonizmas ve ilave etkilerini ölçmek için kullanld. nsan böbrek karsinomu A498 hücrelerine kar antikanser etkisinde, DOX ve mezoklorin e6 mono-etilendiamin (Mce6). SOS + DOX kombinasyonunun, tüm hücre büyümesi inhibisyon seviyelerinde sinerjik olduu kantland. Dier tüm kombinasyonlar, geni bir ilaç etki seviyeleri aralnda sinerji sergilemitir [117]. Benzer ekilde, hedeflenen (OV-TL16 antikorunun Fab’si kullanlarak) ve hedeflenmemi hedeflenen HPMA kopolimer-ilaç konjugatlar, P (GFLG) -Mce6 ve P (GFLG) -SOS, insan yumurtalk karsinomu OVCAR-3 hücrelerine kar deerlendirildi. Çou kombinasyonun sinerjik etkiler ürettii gözlemleri, klinik çeviri için önemli olacaktr [118].

Satchi-Fainaro’nun Tel Aviv Üniversitesi’ndeki laboratuvar ile ibirlii içinde, kombine hedeflenmi polimere bal anjiyogenez inhibitörleri kullanan kemik neoplazmalar için yeni bir terapötik strateji gelitirildi [119]. Aminobisfosfonat alendronat (ALN) ve güçlü anti-anjiyojenik ajan TNP-470, HPMA kopolimeri ile konjuge edildi. Tersine çevrilebilir ekleme-parçalanma zincir transferi (RAFT) polimerizasyonu kullanarak, kemik dokularnda ar ifade edilen bir proteaz olan bir katepsin K-parçalanabilir balayc tayan bir HPMA kopolimeri-ALN-TNP-470 konjugat sentezledik. Serbest ve konjuge ALNTNP-470, endotelyal ve osteosarkom hücrelerinin proliferasyonunu, göçünü ve klcal benzeri tüp oluumunu inhibe ederek sinerjik anti-anjiyojenik ve antitümör aktivitelerini gösterdi. Bi-spesifik HPMA kopolimer konjugat, vasküler ar geçirgenlii azaltt ve farelerde insan osteosarkom büyümesini% 96 orannda önemli ölçüde inhibe etti. Bu bulgular, HPMA kopolimeri-ALN-TNP-470’in, hem tümör epitel hem de endotel kompartmanlarn hedefleyen RAFT polimerizasyonu ile sentezlenen ve osteosarkomlar ve kemik metastazlarnda kullanmn garantileyen ilk dar dalm anti-anjiyojenik konjugat olduunu göstermektedir (ekil 10) [119] .

Farelerde MG-63-Ras insan osteosarkom büyümesinin HPMA kopolimer-ALN-TNP470 konjugat tarafndan inhibisyonu. (A) Konjugatn yaps; (B) serbest (açk üçgenler) veya konjuge (kapal üçgenler) ALN ve TNP-470’in araçla tedavi edilen grup (kapal kareler) ve kesilmi tümör görüntüleri ile karlatrldnda MG-63-Ras insan osteosarkom tümör büyümesi üzerindeki etkileri. Ölçek çubuu 10 mm’yi temsil eder. Veriler ortalama ± S.E’yi temsil eder. (n = grup bana 5 fare). [119] ‘dan uyarlanmtr.

3.4. Yeni hedefleme stratejileri

3.1’de tartld gibi, HPMA kopolimer-ilaç konjugatlar (moleküler arla bal) arttrlm geçirgenlik ve tutma (EPR) etkisinin bir sonucu olarak kat tümörlerde pasif olarak birikir [85]. HPMA kopolimer-ilaç konjugatlarnn aktif hedeflenmesi, karbonhidratlar, lektinler, antikorlar, antikor fragmanlar ve peptitler gibi kanser hücresine özgü ligandlarn dahil edilmesiyle elde edilebilir, bu da, reseptör aracl endositoz yoluyla kanser hücreleri tarafndan konjugatlarn almnn artmasyla sonuçlanr. terapötik etkinlikte e zamanl iyileme [120,121].

Farkl kanseri hedefleyen moleküller arasnda peptitler özellikle ilgi çekicidir. Gelitirilmi peptit hedefleme verimlilii, hedefler ve tek bir polimer zinciri içinde çoklu peptit kopyalar içeren HPMA kopolimer-peptit konjugatlar arasndaki multivalent etkileimler [122] yoluyla elde edilebilir (ekil 11) [123].

HPMA kopolimer-peptid-DOX konjugatlarnn biyo-tanmasnda çoklu deerlilik etkisi. Makromolekül bana deien miktarda hedefleme peptidi, EDPGFFN-VEIPEF içeren HPMA kopolimer-DOX (P (GFLG) -DOX) konjugatna maruz braklarak Raji B hücre büyümesinin inhibisyonu. (A) konjugatn yaps; (B) Raji B hücre büyümesinin P (GFLG) -DOX (hedefleyici peptit yok),% 1.9 mol hedefleme peptidi içeren P (GFLG) -DOX ve% 3.9 mol hedefleme peptidi içeren P (GFLG) -DOX tarafndan inhibisyonu. [123] ‘den uyarlanmtr.

Faj gösterimi veya sentetik peptit kitaplklar gibi kombinatoryal yaklamlar, hedefleme peptitlerinin tanmlanmas için uygundur. CD21 reseptörünün ar ekspresyonu, Raji hücreleri gibi lenfoblastoid hücre çizgilerinde bulundu; sonuç olarak, bu teknikleri lenfomalar için hedefleme parçalarn belirlemek için kullandk [124,125]. Faj gösterimi ile, be farkl peptid (RMWPSSTVNLSAGRR, PNLDFSPTCSFRFGC, GRVPSMFGGHFFFSR, RLAYWCFSGLFLLVC ve PVAAVSFVPYLVKTY) CD21 reseptörünün ligandlar olarak tanmland. Seçilen peptitlerin ayrma sabitlerinin mikromolar aralkta olduu belirlendi [124]. Sentetik bir kimyasal kombinatoryal teknik, tek boncuklu tek bileik (OBOC) yöntemi kullanarak, CD21 reseptörü için ligand olarak dört heptapeptid (YILIHRN, PTLDPLP, LVLLTRE ve IVFLLVQ) belirledik [125]. Ayrlma sabitlerinin, faj gösterimi ile seçilen peptitlere benzer olduu bulundu. Önemlisi, peptidler, HPMA kopolimerlerine konjuge edildikten ve çok deerlikli bir etki gösterdikten sonra CD21 reseptörüne kar biyo-tannabilirliklerini korumutur [125]. Birkaç peptit hedefli HPMA kopolimer-ilaç konjugat antikanser aktivite gösterdi [123,126,127]. Kombinatoryal kimya yaklam (OBOC), yüksek kesinlikte bir tarama yöntemi ile birletirildiinde pikomolar afiniteye sahip peptitleri belirleyebilir [128,129].

3.4.1. laçlarn oral, kolona özgü teslimi

Kolonda seçici ilaç salm yapabilen ilaç verme sistemlerinin gelitirilmesi büyük ilgi görmütür. Kolona bölgeye özgü iletim, arlkl olarak kolonda bulunan mikrobiyal enzim aktivitelerinin kullanlmasyla salanabilir. Kolon, ince barsak veya mideden 5 kat daha büyük bir mikroorganizma konsantrasyonuna sahiptir. Kolondaki mikroorganizmalar tarafndan üretilen enzimatik aktivitenin bir ksm, örnein, azoredüktaz ve glikosidaz aktiviteleri, üst GI kanalndaki enzimatik aktivitelerle örtümez. Azoredüktaz aktiviteleri ayrntl olarak çallm ve düük moleküler arlkl ön ilaçlar kolondaki aktif metabolitlere dönütürmek ve ayrca suda çözünür polimerik tayclardan aktif türleri salmak için kullanlmtr [130]. Kolona özgü datm elde etmek için, bir (aromatik amino grubu içeren) ilaç, kolonda bulunan azoredüktaz aktiviteleri ile bölünebilen bir aromatik azo ba araclyla HPMA kopolimer yan zincirlerine balanabilir [51,131-138]. Örnein, aromatik bir azo ba yoluyla HPMA kopolimerlerine balanan 5-aminosalisilik asidin salnm, kolon [131], sçanlarn, kobaylarn ve tavanlarn çekum içerii olan izole bir bakteri türü olan Streptococcus faecium kullanlarak gösterilmitir. [133] ve insan dksnda [133].

Son zamanlarda, 9-aminokamptotesin (9-AC) oral yolla verilmesine odaklandk. lk olarak, aromatik azo ba ve amino asit kalntlar içeren bir ayrc araclyla 9-AC’yi HPMA kopolimerlerine baladk [134,135]. Aromatik azo bann ilk olarak in vitro [134] ve in vivo [135] ayrld, ardndan amino asit (dipeptid) ilaç türevinin peptidazla katalize edilen bölünmesinin serbest 9-AC salmyla sonuçland gösterilmitir. Bununla birlikte, peptid ilaç türevinin bölünmesi, kolonda yüksek konsantrasyonlarda serbest 9-AC elde etmek için yeterince hzl deildi. Bu sonuçlar, daha hzl bir 9-AC salm oranna sahip bir aralayc içeren konjugatlarn tasarlanmas gerektiini gösterdi. Bu amaçla, 9-AC, aromatik azo ba ve 1,6-eliminasyon aralayc içeren bir monomer tasarland ve sentezlendi [51]. Kolona özgü aromatik azo ba klivaj ve 1,6-eliminasyon reaksiyonunun kombinasyonu, in vitro olarak çekal içerii ile HPMA kopolimer konjugatndan modifiye edilmemi 9-AC’nin hzl ve yüksek verimli bir ekilde salnmasna neden olurken, simüle edilmi üst GI kanalnda e zamanl stabilite ile sonuçland. koullar. Konjugat, olumlu bir farmakokinetie [136,137] sahipti ve kolon kanseri modellerinde etkiliydi (ekil 12) [138].

HPMA kopolimer-9-aminokamptotesin konjugat. (A) HPMA kopolimer-9-AC konjugatlarndan modifiye edilmemi 9-AC’nin iki aamal bir ilemle salnmasnn yaps ve emas – hz kontrollü aromatik azo ba bölünmesi, ardndan hzl 1,6-eliminasyonu [51]; (B) 3 mg / kg 9-AC veya 9-AC edeeri dozunda 9-AC ve P-9-AC ile tedavi edilen insan kolon karsinomu ksenograftlar tayan farelerin hayatta kalma erileri [138].

3.4.1.1. Gastrointestinal sistemde hedefleme

Hücre yüzeyi glikoproteinleri, kolon epitel hücrelerinin farkllama ve olgunlama aamasn yanstr. Hastalkl dokular, karsinomlar ve iltihapl barsak hastal gibi kanser öncesi durumlar, salkl olanlarla karlatrldnda glikoprotein ifadesini deitirmitir. Sonuç olarak, lektinler, polimere bal ilaçlar için hedefleme parçalar olarak kullanlabilir [139-141]. WGA (buday tohumu aglutinini) salkl dokulara balanrken, PNA (fstk aglutinin) hastalkl dokulara balanr. HPMA kopolimer-lektin-ilaç konjugatlarnn kolonik glikoproteinleri hedefleyerek hastalkl dokulara terapötik ajanlar verebileceini varsaydk. Normal neonatal, yetikin ve hastalkl kemirgen dokularnda, insan inflamasyon örneklerinde ve Barrett’s özofagusunda serbest ve HPMA kopolimer konjuge WGA ve PNA ve anti-Thomsen-Friedenreich (TF) antijen antikor balanmasnn biyo-tanmasn inceledik. Yenidoan WGA balanmas, ilave lüminal kolumnar hücre balanmas ile yetikin ile karlatrlabilirdi. PNA balanmas daha yaygnd; lüminal kolumnar hücre balanmas, yaamn ilk 2 1/2 haftasnda mevcuttu. WGA balanmas hem normal hem de hastalkl yetikin dokularda güçlüydü; hastalkta hafif bir azalma kaydedildi. Normal dokularda PNA balanmas minimaldi; hastalkta artlar görüldü. Anti-TF antijen antikor çalmalar, PNA’nn antijene balanmadn gösterdi. Sonuçlar, HPMA kopolimer-lektin-ilaç konjugatlarnn kolit veya Barrett’s özofagusu gibi durumlarn bölgeye özgü tedavisini salayabileceini göstermektedir [141].

Çok çeitli terapötik maddeler, onlar tümör hücrelerindeki mitokondriye spesifik olarak yönlendirerek fayda salayabilir. Tümör ve mitokondriyal hedeflemenin bir kombinasyonunu mümkün klacak uygulama sistemlerini tasarlamak için, mitokondriotropik ajanlar olarak trifenilfosfonyum iyonlarn kullanan [147] yeni HPMA kopolimer bazl iletim sistemleri gelitirilmitir [142]. Yaplar balangçta ilacn yerine geçen floresan etiketlerle sentezlendi ve dorulama deneyleri için kullanld. Bu floresan etiketli yaplar kullanlarak gerçekletirilen mikroenjeksiyon ve inkübasyon deneyleri, mitokondriyal hedefleme yeteneini dorulad [148]. Daha sonra, HPMA kopolimer-ilaç konjugatlar, bir a duyarllatrc mezoklorin e6 (Mce6) kullanlarak sentezlendi. HPMA kopolimerine bal Mce6’nn mitokondriyal hedeflemesi, hedeflenmemi HPMA kopolimer-Mce6 konjugatlarna kyasla sitotoksisiteyi artrd [142]. Mevcut tasarm uyarlamak ve dier terapötik ajanlarn tümör bölgesine özgü mitokondriyal hedeflemesine izin vermek için küçük modifikasyonlar gerekebilir.

Bu türevin yeni HPMA kopolimer bazl datm sistemleri de sentezlendi [143]. Bir HPMA kopolimer-Cort-Mce6 konjugatnn (lizozomal olarak bozunabilir GFLG ayrc yoluyla) endositoz yoluyla içselletirilmesinden sonra, Cort-Mce6 bölündü, sitoplazmaya yer deitirdi, GR’ye baland ve çekirdee yer deitirdi [143]. Kortizolün Mce6’ya balanmasnn, nükleer lokalizasyonla sonuçlanan sitozolik GR ile bir kompleks oluturma kapasitesini koruduunu dorulamak için, modifiye edilmi ilacn hücre alt kaderini aratrdk. Cort-Mce6, yeil floresan protein etiketli glukokortikoid reseptörü (GFP-GR) ifade eden plazmid ile transfekte edilmi 1471.1 hücrede izlendi. Kortizol ve Mce6 srasyla pozitif ve negatif kontroller olarak görev yapt. GR, bir glukokortikoid analounun (örnein, kortizol) eklenmesinden sonra çekirdee yer deitirmitir. Floresan GFP etiketi, GR’nin hareketinin gerçek zamanl olarak izlenmesine izin verir. Veriler (ekil 13), kortizol-Lys-Mce6 ve kortizolün zamana ve konsantrasyona bal çekirdek lokalizasyonunu açkça gösterdi. Bunun aksine, Mce6 ile inkübe edilen hücreler, tedaviyi takiben reseptör lokalizasyonunda herhangi bir deiiklik göstermedi [143].

Hedefleme parçalar ve HPMA kopolimer bazl ilaç tayclar [158] ile konjugasyon için yararl olan bir birincil amino grubunu tantmak üzere GDM’nin 17-metoksi grubunun ikamesi için yeni bir yöntem gelitirdik. Farkl AR-GDM (AR = 3-aminopropil (AP), 6-aminoheksil (AH) ve 3-amino-2-hidroksipropil (AP (OH)) içeren, lizozomal olarak parçalanabilir bir GFLG ayrc araclyla balanan HPMA kopolimerleri sentezlendi ve karakterize edilmi [159] HPMA kopolimer-AR-GDM konjugatlarnn sitotoksik etkinlii AR-GDM’nin yapsna balyd [160].

P (AP-GDM) [HPMA kopolimer-17- (3-aminopropilamino) -17-demetoksi-geldanamisin eleniinin] düük moleküler arlkl GDM türevlerinin gen ekspresyon profillerini deitirebilecei hipotezini dorulamak için, 32P-makro-dizi analizi (Clonetech) GDM, AP-GDM ve P (AP-GDM) ile 2 kat% 50 hücre büyümesi inhibe edici konsantrasyonda (IC50) tedavi edilen insan yumurtalk karsinomu A2780 hücrelerinde gen ekspresyon profillerini deerlendirmek için kullanld. Kanserle ilgili yaklak 1200 gen, 6. ve 12. saatte deerlendirildi ve ekspresyondaki üç kat deiiklik önemli kabul edildi. Gen ekspresyon oranlarnn hiyerarik kümelenmesinin gösterdii gibi, AP-GDM ve P (AP-GDM) tedavilerinden sonra gen ekspresyon profillerinde önemli benzerlikler bulundu [91].

Bununla birlikte, geldanamisinin etki mekanizmas ile ilgili tek tek genler analiz edildiinde sonuç farklyd. P (AP-GDM) ile tedavi edilen hücreler, 12 saate kadar AP-GDM ile karlatrldnda HSP70 ve HSP27’nin daha düük ekspresyonunu gösterdi. Muhtemelen, düük moleküler AP-GDM’den farkl olarak P’nin (AP-GDM) içselletirme yollar ve hücre alt ilaç lokalizasyonu, AP-GDM tarafndan indüklenen hücre stresi yantlarn modüle edebilir. 32P-makro-dizisinin sonuçlar RT-PCR ve Western blot ile dorulanmtr [91]. HPMA kopolimer-AP-GDM konjugatnn endositoz yoluyla içselletirilmesinin, stresörlere ve çevresel deiikliklere duyarl olabilen plazma membran gibi hücrenin d bileenleri ile etkileimleri engellemesi mümkündür (ekil 15). Benzer ekilde, daha önce, HPMA kopolimer-DOX konjugat ile muamele edilen A2780 hücrelerinin, HSP70 geninde, serbest DOX ile tedavi edilen hücrelerde gözlenenden daha belirgin bir aa regülasyon gösterdiini gözlemledik [89]. Bu bulgular, AP-GDM’nin HPMA kopolimerine konjugasyonunun, AP-GDM tarafndan indüklenen hücre stres yantlarn, içselletirme mekanizmasndaki, hücre alt lokalizasyonundaki ve hücre içi konsantrasyon gradyanlarndaki farkllklar nedeniyle modüle edebileceini düündürebilir [91].

3.7. Kanser: klinik aratrmalar

HPMA kopolimer bazl makromoleküler terapötikler, son 20 ylda önemli ölçüde gelitirilmitir – çok sayda konjugat, son on ylda terapötik dorulama için klinik denemelere girmitir. Bunlar, HPMA kopolimer-DOX [163-165], HPMA kopolimer-DOX-galaktozamin [166], HPMA kopolimer-kamptotesin [167], HPMA kopolimer-paklitaksel [168] ve HPMA kopolimer-platinatlar [169] içerir. Bu konjugatlarn bazlarnn test edilmesinden elde edilen sonuçlar umut vericidir; umarm ilk makromoleküler terapötiklerin FDA onay yaknda gerçekleecektir. Bölüm 4.1’de, iyiletirilmi terapötik potansiyele sahip ikinci nesil konjugatlarn tasarm ilkeleri hakkndaki fikirlerimizi özetledik.

3.8. Kanserli olmayan hastalklarn tedavisinde HPMA kopolimer konjugatlar

HPMA kopolimer-ilaç konjugatlar, kanser dndaki hastalklarn tedavisi için de kullanlabilir. Osteoporoz ve dier kas-iskelet sistemi hastalklarnn tedavisi için iyi bilinen bir kemik anabolik ajan (prostaglandin E1; PGE1) ile konjuge kemik hedefli HPMA kopolimeri tasarladk [50,170-175]. Konjugatlarn iskelet tarafndan biyo-tanmasna D-aspartik asit (D-Asp8) veya alendronatn bir oktapeptidi araclk etti [170,172].

Bu sistem, sistemik uygulamadan sonra kemik anabolik ajan PGE1’i spesifik olarak sert dokulara verme potansiyeline sahiptir. Kemie balandktan sonra, PGE1 tercihen spesifik bir peptit ayrcnn katepsin K (osteoklast spesifik) katalize edilmi hidrolizi ve ardndan 1,6-eliminasyonu yoluyla daha yüksek devir hzna (daha fazla osteoklast aktivitesi) sahip bölgelerde salnacaktr [50,176]. Anabolik doz aralnda verildiinde, salnan PGE1, net kemik oluumunu salamak için kemik hücrelerinin yüzeyindeki karlk gelen EP reseptörlerini aktive edecektir. Tasarmn ana özellikleri, D-Asp8’de veya bir PGE1 ön ilac olan p-aminoben-ziloksikarbonil-1-prostaglandin E1’de sonlanan katepsin K-parçalanabilir oligopeptit yan zincirleri (Gly-Gly-Pro-Nle) içeren HPMA kopolimer omurgasdr. (ekil 17A).

HPMA kopolimer-prostaglandin E1-Asp8 konjugatnn yaps ve bunun katepsin K ile bölünmesinin mekanizmas ve ardndan 1,6-eliminasyonu (A) [50]; Tek bir 10 mg konjugat (n = 8) (B) enjeksiyonundan 4 hafta sonra yumurtalklar alnm sçanlarda lomber vertebral cisimlerden alnan süngerimsi kemikte kemik oluumu ölçülmütür [174].

Bu yeni uygulama sisteminin birkaç belirgin avantaj vardr. Her eyden önce, bir kemik balama parças (D-Asp8) ve bir katepsin K (osteoklast spesifik enzim) spesifik salm mekanizmas içeren çift hedefli bir salm sistemidir. PGE1’in spesifik olarak iskelete yönlendirilmesiyle, ilacn sistemik uygulanmasnn yan etkileri büyük ölçüde azaltlacaktr. kinci olarak, E-serisi prostaglandinler (PGE’ler) kemikte güçlü anabolik ajanlardr ve bu iletim sistemi, bu molekülleri iskeletteki yeni kemik oluumunun daha faydal olaca yüksek devir hzna sahip bölgelere daha iyi hedefleyecektir. Üçüncüsü, sistem, sistemik uygulamadan sonra hedef (kemik) bölgede ilaç konsantrasyonunun gelimi kontrolüne izin verir [171,173]. Dördüncü olarak, polimerik tayc sert dokulardan elimine edilebilir ve ardndan böbrek glomerüler filtrasyon yoluyla vücuttan atlabilir. Ayrca, konjuge PGE1’in kemik dokusuna ulamadan önce metabolizmadan uygun ekilde korunmasn salar. Son zamanlarda, önerilen verme sisteminin yumurtalklar alnm sçanlarda kemik erimesi bölgelerine tercihli birikimini gözlemledik; bu, daha yüksek cirolu sitelerimizi / ilaç salm hipotezimizi kuvvetle desteklemektedir [172].

Ovariektomi uygulanm fareler üzerinde yaplan in vivo deneyler, konsepti kantlamtr. Tek bir i.v. HPMA kopolimer-Asp8-PGE1 konjugatnn yal, yumurtalklar alnm sçanlara uygulanmasnda, kemik oluum oranlar 28 gün sonra ölçüldüünde kontrollerden önemli ölçüde daha yüksekti (ekil 17B) [173]. HPMA kopolimer konjugatlar, romatoid artrit tedavisinde de baarl olmutur [175,177].

3.9. HPMA kopolimer konjugatlarnn sentez yöntemleri

Konjugat sentezi konusu bu cilt boyunca kapsaml bir ekilde ilenecektir. Bu bölüm boyunca birkaç yaklam gösterdik. Sonuç olarak, ele alnmayan baz önemli noktalara deineceiz.

3.9.1. laç ve HPMA kopolimeri arasnda hidrolitik olarak bölünebilen ba

Endozomlarda ve lizozomlarda azalm pH nedeniyle, pH’a duyarl balar hücre içi ilaç iletimi için uygundur. ADR’nin cis-akonitil ba [177] yoluyla baland HPMA kopolimer-adriamisin (ADR = DOX) konjugatlarn sentezledik (ekil 18). P (aconityl) -ADR’nin A2780’e duyarl ve A2780 / AD’ye dirençli insan yumurtalk karsinomu hücrelerine kar sitotoksisitesinin belirlenmesi, polimer konjugatnn A2780 / AD hücrelerinde eksprese edilen P-glikoprotein da akm pompasnn üstesinden gelebileceini gösterdi [178].

3.9.2. Disülfür bal HPMA kopolimer-mezoklorin e6 konjugatlar

Sistemik toksisite sorunlarnn üstesinden gelmek için a duyarllatrc mezoklorin e6 (Mce6) için yeni polimerik datm sistemleri sentezlendi. Tümör dokusunda içselletirildikten sonra Mce6’nn HPMA kopolimer omurgasndan hzl salnmasna izin vermek için bir disülfür ba dahil edildi (ekil 19). Sentezlenen konjugatlar, ditiyotreitole maruz kalndnda singlet oksijen üretiminin kuantum veriminde elik eden bir artla birlikte zamana bal indirgeyici bir bölünme gösterdi. Daha hzl salm kinetii ve SKOV-3 insan yumurtalk karsinom hücrelerinde daha yüksek bir sitotoksisite, proteolitik olarak bölünebilen bir glisilfenilalanilleusilglisil aralayc ile polimer konjugatna kyasla elde edildi. Bu yeni konjugatlar, kanserin fotodinamik tedavisi için klinik olarak ilgili ilaç datm sistemleri olarak umut vaat etmektedir [179].

3.9.3. Hedefleme parçalarnn eki

Hedefleme parçalarnn balanmas için kullanlan kimya, konjugatn biyo-tannmas üzerinde bir etkiye sahiptir. Birkaç antikor balanma yöntemini karlatrdk (aaya baknz), antikorlarn HPMA kopolimerlerine eklenmesinin içselletirme ve hücre alt trafii [180] mekanizmasn nasl etkilediini aratrdk ve polimerize edilebilir antikor parçalar tasarladk [84].

3.9.4. Polimerize edilebilir antikor fragmanlar

Polimerize edilebilir antikor fragmanlar kullanlarak hedeflenen polimerik ilaç verme sistemlerinin sentezi için yenilikçi bir yol tasarland [84]. OV-TL 16 antikorunun (IgG1) polimerize edilebilir bir antikor Fab ′ fragman (MA-Fab ′) olan yeni bir makromonomer, poli (HPMA-co-MA-Fab ′) üretmek için HPMA ile sentezlenmi ve kopolimerize edilmitir (ekil 20 ). Polimerize edilebilir Fab ′ fragmanlarn makromonomerler olarak kullanma kavram, hedeflenen polimerik ilaç verme sistemlerinin sentezi için yeni bir paradigma salar ve immünolojik testler, biyosensör teknolojisi ve afinite kromatografisi gibi dier alanlarda benzersiz uygulamalara sahip olabilir [84].

3.9.5. Antikorlarn HPMA kopolimerlerine balanmasnn kimyasnn konjugatlarn balanma afinitesi üzerindeki etkisi

OV-TL16 antikorunun ve onun Fab ′ fragmannn HPMA kopolimer-ilaç (ADR, Mce6) tayclarna balanmasna yönelik farkl yöntemlerin, konjugatlarn yumurtalk karsinomu (OVCAR-3) hücreleriyle ilikili CD47 antijenine balanma afinitesi üzerindeki etkisi, okudu. Ab veya Fab ′’nn polimerlere kovalent olarak balanmas için üç farkl yöntem kullanld [181]. Yöntem A: antikor üzerindeki amino gruplar tarafndan HPMA kopolimer-ilaç (ADR veya Mce6) konjugatlar üzerinde aktif ester gruplarnn aminolizi ile oluturulan amid balar yoluyla balanma; Yöntem B: oksitlenmi antikor üzerindeki aldehit gruplarnn HPMA kopolimeri-Mce6 konjugatlar üzerindeki hidrazo gruplar ile reaksiyonu sonucu oluan hidrazon balar yoluyla balanma; Yöntem C: Fabp fragmanlarnn sülfhidril gruplarnn HPMA kopolimeri-Mce6 konjugatnn yan zincir uçlar üzerindeki maleimido gruplar ile reaksiyonu sonucu oluan tiyoeter balar yoluyla balanma. ekil 21’de gösterildii gibi Ka’daki farkllklar gözlendi.

HPMA kopolimer konjugatlarnn sentezi, özellikle moleküler arlk dalm, canl radikal polimerizasyon yöntemleri, RAFT (tersine çevrilebilir ekleme-fragmantasyon zincir transferi) [182] ve ATRP (atom transfer radikali) polimerizasyonlar [183] ​​ile kontrol edilebilir. Örnein, RAFT kopolimerizasyonu kullanlarak makromolekül bana iki ilaç ve bir floresan etiket içeren HPMA kopolimer konjugatlar yakn zamanda sentezlenmitir [119; ekil 10].

3.9.7. Oligonükleotidlerin balanmas

HPMA kopolimer öncülerinin aminolizi, oligonükleotitlerin HPMA kopolimerlerine balanmas için kullanlabilir. GG-ONp ve GFLG-ONp (ONp p-nitrofenoksidir) yan zincirleri içeren HPMA kopolimerlerine 21-mer fosforotioat oligonükleotidi (5′-TTTATAAGGGTCGATGTCCXX-3 ′) ekledik [184]. Oligonükleotid, 5′-ucunda bir birincil amine ve 3′-ucunda bir flüoreseine sahipti. HPMA kopolimer-fosforotioat oligonükleotidlerinin hepatit B virüsünün inhibe edilmesindeki hücre alt kaderi ve aktivitesi incelenmitir. Oligonükleotitlerin, bozunmayan dipeptit GG aralayclar araclyla HPMA kopolimerlerine kovalent olarak balanmas, oligonükleotidin, içselletirmeden sonra veziküllerde tutulmasyla sonuçland. Oligonükleotitlerin, lizozomal olarak bölünebilen bir tetrapeptit GFLG aralaycs araclyla bir HPMA kopolimerine konjugasyonu, lizozomdaki oligonükleotidin salnmasna ve ardndan hücrelerin sitoplazmas ve çekirdeine translokasyon ile sonuçland. Parçalanabilir HPMA kopolimer-oligonükleotid konjugat, lizozomdaki taycdan salnan fosforotioat oligonükleotidlerin sitoplazma ve çekirdee kaçabildiini ve aktif kalabildiini gösteren antiviral aktiviteye sahipti. Hep G2 hücrelerinin, oligonükleotid -HPMA kopolimer konjugatlar konjuge olmayan polimerlerden daha büyük ölçüde içselletirildii için fosforotioat oligonükleotidleri aktif olarak içselletirdii görülmütür [184].

3.9.8. Hücreye nüfuz eden peptidlerin (CPP) balanmas

HIV-1 transkripsiyonunun güçlü bir aktivatörü olan HIV-1 Tat proteininden kaynaklanan Tat peptidi (48GRKKRRQRRR57) gibi bu peptitler, çeitli kimyasal tasarmlar kullanlarak eklenmitir. Bir yaklam, birkaç amino asit kalnts, ör. 48GRKKRRQRRR57YK (FITC) C üretmek için. Sonuncusu, maleimidde tiyoeter balar yoluyla sonlandrlan HPMA kopolimer yan zincirlerine balanabilir [185]. Yakn zamanda CPP balanmasnn biyolojik etkilerini gözden geçirdik [144], bu yüzden burada tartmayacaz.

 

4.1. Semitelechelic poly (HPMA) ile modifikasyon

Poli (HPMA) [189], enzimlerin veya veziküler tayclarn modifikasyonu için kullanldnda poli (etilen glikol) [190] ile benzer özellikler gösterir. Yar selekelik poli (HPMA) (ST-PHPMA) hakkndaki ilk rapor 1995 ylnda yaynland [191]. ST-PHPMA ile metil metakrilat, maleik anhidrit ve metakrilik asidin bir kopolimerine dayanan nanosferlerin modifikasyonu, in vitro olarak protein adsorpsiyonunun azalmasna ve intravasküler yarlanma ömrünün artmasna ve ayrca intravenöz uygulamadan sonra karacierde birikimin azalmasna neden oldu. sçanlar. ST-PHPMA’nn moleküler arl ne kadar yüksekse, bu özelliklerdeki deiiklikler o kadar belirgindir (ekil 22). Bu veriler, kaplama tabakasnn hidrodinamik kalnlnn, karacierin Kupffer hücreleri ve dalan makrofajlar tarafndan opsonizasyon ve yakalama süreci üzerindeki etkisini gösteriyor gibi görünmektedir [191].

Nanosfer yüzeyini deitirmek için ilk yar-ekelik poli (HPMA) sentezi ve uygulamas. (Yap; (B) IgG’nin yüzey deitirilmi P (MMA-MA-MAA) nanosferleri üzerine 25 ° C’de 3 saat boyunca 1/15 M salin içinde adsorpsiyon izotermleri. Her nokta ortalama ± SD’yi (n = 3) temsil eder; (C) i.v.’den 24 saat sonra farelerde modifiye edilmemi ve modifiye edilmi [14C] -P (MMA-MA-MAA) nanosferleri için vücut dalm profilleri. ST-PHPMA’nn Mn ile yönetimi ve korelasyonu. Her nokta ortalama ± SD’yi (n = 5) temsil eder [191].

Benzer ekilde, kimotripsin’in ST-PHPMA-CONHNH2 ve ST-PHPMA-COOSu (N-hidroksisüksinimid ester) ile karboksil ve amino grubu modifikasyonu, PEG ile modifiye edilmi kimotripsin [187] ile karlatrlabilir özelliklere sahip konjugatlar [189] üretti. Ulbrich vd. ribonükleaz, kimotripsin [192] ve süperoksit dismutaz [193] modifiye etmek için ST-PHPMA kulland. Modifiye edilmi proteinin proteolitik stabilitesi artm ve immünojenisitesi azalmtr [192,193].

4.2. Birden fazla reaktif yan zincir içeren HPMA kopolimerleriyle modifikasyon

HPMA kopolimerleri ile modifiye edilen ilk protein, HPMA kopolimerinin N-metakrililglisilglisin p-nitrofenil ester ile insülin ile reaksiyona sokulmasyla hazrlanmtr [28]. Reaksiyona girmemi protein, Sephadex 75 üzerinde ayrld; HPMA kopolimer-insülin konjugat, serbest insüline kyasla sçanlarda daha yava bir balangç ​​ve hafif bir hipoglisemik etki uzamas gösterdi [28]. Bunu kimotripsin [29,30] ve kobra zehiri asetilkolinesteraz [194] izledi.

Polimer zincirlerinin enzim-substrat kompleksi oluumu üzerindeki sterik engeline daha iyi bir fikir edinmek için, HPMA kopolimerine bal olarak katalize edilen polimerik substratlarn (p-nitroanilide sonlandrlm oligopeptid yan zincirlerine sahip HPMA kopolimerleri) hidrolizini inceledik. kimotripsin. Kinetik analiz, polimer substratlarn polimere bal kimotripsin ile hidrolizinin hem kcat hem de kcat / KM’de bir azalmaya yol açtn, ancak tek tek substratlar arasndaki ilikinin bozulmadan kaldn gösterdi. Görünüe göre, iki bamsz polimer zincirinin (biri substrata, dieri enzime bal) sterik etkileri kabaca katk maddesi idi [30].

Kobra zehiri asetilkolinesterazn modifikasyonu için farkl kimya kullanlmtr [194]. Poli (HPMA) ‘nn (Mw 25-30 kDa) ikincil OH gruplar, dimetilformamid içinde 4-nitrofenil kloroformat ile aktive edildi, ardndan borat tamponuna asetilkolinesteraz eklendi. Poli (HPMA) ile modifiye edilmi asetilkolinesteraz, modifiye edilmemi enzim ile karlatrldnda farelere intravenöz enjeksiyondan sonra kandaki enzim aktivitesinin 70 kat uzadn gösterdi. PoliHPMA-asetilkolinesteraz konjugatnn termoinaktivasyon hz, doal enziminkinden 74 kat daha küçüktü (ekil 23) [194].

HPMA kopolimeri ile modifiye edilmi asetilkolinesteraz. (Yap; (B) i.v.’den sonra farelerde intravasküler yar ömür art. HPMA kopolimeri ile modifiye edilmi enzimin uygulanmas; (C) HPMA kopolimeri ile modifiye edilmi enzimin termal stabilitesinin artrlmas [194].

Ribonükleaz [195] ve süperoksit dismutazn [193] modifikasyonu için N-metakrililglisilfenilfenilglisin p-nitrofenilester içeren HPMA kopolimeri kullanlmtr. ST-PHPMA ve reaktif yan zincirlere sahip HPMA kopolimerleri kullanlarak hazrlanan konjugatlarn biyolojik aktivitesinde hiçbir fark tespit edilmemitir [193].

DNA komplekslerini virüslerle stabilize etmek için reaktif yan zincirlere sahip HPMA kopolimerlerinin kullanmnda önemli bir aktivite vardr. Bu aratrma, bu ciltte ve incelememizde [196] Seymour’un bölümünde yer almaktadr.

5.1. HPMA bazl hidrojeller

lk HPMA bazl hidrojeller, 70’lerin banda HPMA ve metilen-bis-akrilamid veya etilen-bis-metakrilamidin çapraz balanmas ile sentezlendi [197]. Kopolimerizasyonun kinetik seyri, polimer-su etkileim parametresi, esneklik modülü ve elastik olarak etkili zincirlerin konsantrasyonu karakterize edildi.

Kimotripsin katalizli hidrolizine duyarl oligopeptit çapraz balar içeren bozunabilir hidrojeller, reaktif yan zincirler (p-nitrofenoksi gruplarnda sonlandrlr) içeren HPMA kopolimerlerinin oligopeptitle (GGY, GFY, GLF, AGVY ve diaminler [49]) çapraz balanmasyla sentezlendi. . Hidrojellerin bozunabilirlii, oligopeptit dizisinin uzunluuna ve ayrntl yapsna ve a younluuna ve dolaysyla denge ime derecesine balyd (ekil 24). ime derecesi ne kadar yüksekse, bozulma oran o kadar hzldr. ime derecesi ayn zamanda hidrojelin toplu bozunmasna kar yüzey üzerinde bir etkiye sahiptir. Enzim hidrojelin iç ksmna dalamazsa, sadece yüzey bozulmas gerçekleir. Bu, kimotripsin ve HPMA kopolimer bal kimotripsin ile katalize edilen AGVY içeren dizilerle çapraz balanan HPMA kopolimerinin degradasyonunun karlatrlmasyla gösterilmitir. Polimerle modifiye edilmi enzimin daha büyük boyutuna bal olarak, hidrojelin sadece yüzeyde degradasyonu gözlenmitir [49]. Çapraz balarda GFYAA dizisini içeren HPMA bazl hidrojeller, bir lizozomal tiyol proteinaz olan katepsin B ile bölünebiliyordu [44]. Dier deneylerde, bozunabilir çapraz balara sahip HPMA bazl hidrojellerin, lizozomal enzimler (Tritozomlar) veya kimotripsin [198] karm ile inkübasyon srasnda FITC-dekstran ve daunomisin sald gösterilmitir.

HPMA’nn N, O-dimetakriloil-hidroksilamin ile çapraz balanma kopolimerizasyonu hidrolitik olarak parçalanabilir hidrojeller üretti [199]. Bu hidrojeller, antikanser ilaç (DOX) için bir depo olarak kullanld; Hidrojellerden DOX salmn kullanan kombinasyon terapisinin sonuçlar ve ardndan antikor hedefli tedavi, farelerde Bcl1 lösemisinin terminal aamalarnn tedavisinde etkili olmutur [200].

Hennink vd. sentezlenmi ABA triblok kopolimerleri, burada blok A sya duyarl poli (HPMA laktat) ve blok B PEG’dir. Kopolimerin stlmas, fotopolimerizasyon ile stabilize edilebilen (çapraz balanabilen) viskoelastik bir materyalin oluumuyla sonuçlanr [202]. Bu malzemeler protein verilmesi için uygundur [203].

HPMA graft kopolimerleri, CCK-P ve CCE-P’nin antiparalel heterodimer kvrml-sarmal oluumunun araclk ettii hibrit hidrojellere kendi kendine montaj. ki farkl pentaheptad peptidi (CCE ve CCK), bir dimerizasyon motifi oluturmak ve fiziksel çapraz balayclar olarak hizmet etmek için tasarlanmtr (P, HPMA kopolimer omurgasdr). Sulu CCE-P veya CCK-P çözeltileri jel oluturmad. Buna karlk, düük konsantrasyonlarda CCE-P / CCK-P’nin emolar karmlarndan jel benzeri malzemeler oluturulmutur [211].