Huaizhong Pan

Targeting angiogenesis-dependent calcified neoplasms using combined polymer therapeutics.

Background There is an immense clinical need for novel therapeutics for the treatment of angiogenesis-dependent calcified neoplasms such as osteosarcomas and bone metastases. We developed a new therapeutic strategy to target bone metastases and calcified neoplasms using combined polymer-bound angiogenesis inhibitors. Using an advanced “living polymerization” technique, the reversible addition-fragmentation chain transfer (RAFT), we conjugated the aminobisphosphonate alendronate (ALN), and the potent anti-angiogenic agent TNP-470 with N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer through a Glycine-Glycine-Proline-Norleucine linker, cleaved by cathepsin K, a cysteine protease overexpressed at resorption sites in bone tissues. In this approach, dual targeting is achieved. Passive accumulation is possible due to the increase in molecular weight following polymer conjugation of the drugs, thus extravasating from the tumor leaky vessels and not from normal healthy vessels. Active targeting to the calcified tissues is achieved by ALNs affinity to bone mineral. Methods and Finding The anti-angiogenic and antitumor potency of HPMA copolymer-ALN-TNP-470 conjugate was evaluated both in vitro and in vivo. We show that free and conjugated ALN-TNP-470 have synergistic anti-angiogenic and antitumor activity by inhibiting proliferation, migration and capillary-like tube formation of endothelial and human osteosarcoma cells in vitro. Evaluation of anti-angiogenic, antitumor activity and body distribution of HPMA copolymer-ALN-TNP-470 conjugate was performed on severe combined immunodeficiency (SCID) male mice inoculated with mCherry-labeled MG-63-Ras human osteosarcoma and by modified Miles permeability assay. Our targeted bi-specific conjugate reduced VEGF-induced vascular hyperpermeability by 92% and remarkably inhibited osteosarcoma growth in mice by 96%. Conclusions This is the first report to describe a new concept of a narrowly-dispersed combined polymer therapeutic designed to target both tumor and endothelial compartments of bone metastases and calcified neoplasms at a single administration. This new approach of co-delivery of two synergistic drugs may have clinical utility as a potential therapy for angiogenesis-dependent cancers such as osteosarcoma and bone metastases.

Biodistribution and pharmacokinetic studies of bone-targeting N-(2-hydroxypropyl)methacrylamide copolymer-alendronate conjugates

The biodistribution and pharmacokinetics of bone-targeting N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-alendronate conjugates were evaluated following intravenous administration of radioiodinated conjugates to young healthy BALB/c mice. The synthesis of a polymerizable and cathepsin K cleavable alendronate derivative, N-methacryloylglycylglycylprolylnorleucylalendronate, enabled the preparation of HPMA copolymer-alendronate conjugates with varying composition. Using the RAFT (reversible addition-fragmentation chain transfer) polymerization technique, four conjugates with different molecular weight and alendronate content and two control HPMA copolymers (without alendronate) with different molecular weight were prepared. The results of biodistribution studies in mice demonstrated a strong binding capacity of alendronate-targeted HPMA copolymer conjugates to bone. Conjugates with low (1.5 mol%) alendronate content exhibited a similar bone deposition capacity as conjugates containing 8.5 mol % of alendronate. The molecular weight was an important factor in the biodistribution of the HPMA copolymer conjugates. More conjugate structures need to be evaluated, but the data suggest that medium molecular weights (50-100 kDa) might be effective drug carriers for bone delivery.

Backbone Degradable Multiblock N-(2-Hydroxypropyl)methacrylamide Copolymer Conjugates via Reversible Addition Fragmentation Chain Transfer Polymerization and Thiol-ene Coupling Reaction

Telechelic water-soluble HPMA copolymers and HPMA copolymer-doxorubicin (DOX) conjugates have been synthesized by RAFT polymerization mediated by a new bifunctional chain transfer agent (CTA) that contains an enzymatically degradable oligopeptide sequence. Postpolymerization aminolysis followed by chain extension with a bis-maleimide resulted in linear high molecular weight multiblock HPMA copolymer conjugates. These polymers are enzymatically degradable; in addition to releasing the drug (DOX), the degradation of the polymer backbone resulted in products with molecular weights similar to the starting material and below the renal threshold. The new multiblock HPMA copolymers hold potential as new carriers of anticancer drugs.

Biorecognition and subcellular trafficking of HPMA copolymer-anti-PSMA antibody conjugates by prostate cancer cells.

A new generation of antibodies against the prostate specific membrane antigen (PSMA) has been proven to bind specifically to PSMA molecules on the surface of living prostate cancer cells. To explore the potential of anti-PSMA antibodies as targeting moieties for macromolecular therapeutics for prostate cancer, fluorescently labeled HPMA (N-(2-hydroxypropyl)methacrylamide) copolymer-anti-PSMA antibody conjugates (P-anti-PSMA) were synthesized and the mechanisms of their endocytosis and subcellular trafficking in C4-2 prostate cancer cells were studied. Radioimmunoassays showed the dissociation constants of P-anti-PSMA for C4-2 prostate cancer cells in the low nanomolar range, close to values for free anti-PSMA. It indicated that conjugation of anti-PSMA to HPMA copolymers did not compromise their binding affinity. The rate of endocytosis of P-anti-PSMA was much faster than that of control HPMA copolymer conjugates containing nonspecific IgG. Selective pathway inhibitors of clathrin-mediated endocytosis and of macropinocytosis inhibited the internalization of P-anti-PSMA. Inhibition of clathrin-mediated endocytosis was further evidenced by down-regulation of clathrin heavy chain expression by siRNA. Using a dominant-negative mutant of dynamin (Dyn K44A) to abolish the clathrin-, caveolae-independent endocytic pathway, we found that some of P-anti-PSMA adopted this pathway to be endocytosed into C4-2 cells. Thus multiple receptor-mediated endocytic pathways, including clathrin-mediated endocytosis, macropinocytosis, and clathrin-, caveolae-independent endocytosis, were involved in the internalization of P-anti-PSMA. The extent of the participation of each pathway in P-anti-PSMA endocytosis was estimated. Membrane vesicles containing P-anti-PSMA rapidly colocalized with membrane vesicles overexpressing Rab7, a late endosome localized protein, demonstrating that a part of P-anti-PSMA was transported to late endosomes.

Enhanced anti-tumor activity and safety profile of targeted nano-scaled HPMA copolymer-alendronate-TNP-470 conjugate in the treatment of bone malignances

Bone neoplasms, such as osteosarcoma, exhibit a propensity for systemic metastases resulting in adverse clinical outcome. Traditional treatment consisting of aggressive chemotherapy combined with surgical resection, has been the mainstay of these malignances. Therefore, bone-targeted non-toxic therapies are required. We previously conjugated the aminobisphosphonate alendronate (ALN), and the potent anti-angiogenic agent TNP-470 with N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer. HPMA copolymer-ALN-TNP-470 conjugate exhibited improved anti-angiogenic and anti-tumor activity compared with the combination of free ALN and TNP-470 when evaluated in a xenogeneic model of human osteosarcoma. The immune system has major effect on toxicology studies and on tumor progression. Therefore, in this manuscript we examined the safety and efficacy profiles of the conjugate using murine osteosarcoma syngeneic model. Toxicity and efficacy evaluation revealed superior anti-tumor activity and decreased organ-related toxicities of the conjugate compared with the combination of free ALN plus TNP-470. Finally, comparative anti-angiogenic activity and specificity studies, using surrogate biomarkers of circulating endothelial cells (CEC), highlighted the advantage of the conjugate over the free agents. The therapeutic platform described here may have clinical translational relevance for the treatment of bone-related angiogenesis-dependent malignances.

Endocytic uptake of a large array of HPMA copolymers: Elucidation into the dependence on the physicochemical characteristics

Endocytic uptake and subcellular trafficking of a large array of HPMA (N-(2-hydroxypropyl)methacrylamide) based copolymers possessing positively or negatively charged residues, or hydrophobic groups were evaluated by flow cytometry and living cell confocal microscopy in cultured prostate cancer cells. The degrees of cellular uptake of various copolymer fractions with narrow polydispersities were quantified. The copolymer charge was the predominant physicochemical feature in terms of cellular uptake. Fast and efficient uptake occurred in positively charged copolymers due to non-specific adsorptive endocytosis, whereas slow uptake of negatively charged copolymers was observed. The uptake of copolymers was also molecular weight dependent. The copolymers were internalized into the cells through multiple endocytic pathways: positively charged copolymers robustly engaged clathrin-mediated endocytosis, macropinocytosis and dynamin-dependent endocytosis, while weakly negatively charged copolymers weakly employed these pathways; strongly negatively charged copolymers only mobilized macropinocytosis. HPMA copolymer possessing 4 mol% of moderately hydrophobic functional groups did not show preferential uptake. All copolymers ultimately localized in late endosomes/lysosomes via early endosomes; with varying kinetics among the copolymers. This study indicates that cell entry and subsequent intracellular trafficking of polymeric drug carriers are strongly dependent on the physicochemical characteristics of the nanocarrier, such as charge and molecular weight.

Water-soluble HPMA copolymer - Prostaglandin E1 conjugates containing a cathepsin K sensitive spacer

A novel bone targeting, N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer based, prostaglandin E1 (PGE1) delivery system was designed, synthesized and characterized. PGE1 was bound to the polymer backbone via a spacer, composed of a cathepsin K sensitive tetrapeptide (Gly-Gly-Pro-Nle) and a self-eliminating 4-aminobenzyl alcohol structure. The HPMA copolymer conjugates were prepared by photo–initiated free radical copolymerization of HPMA, PGE1-containing macromonomer, and optionally a comonomer containing a reactive p-nitrophenyl ester group. The latter group was used as attachment points for the d-aspartic acid octapeptide targeting moieties. Incubation of the PGE1-containing macromonomer and HPMA copolymer-PGE1 conjugates with cathepsin K resulted in release of unmodified PGE1. The rate of release depended on the composition of the conjugate. The higher the PGE1 content in the conjugate, the slower the PGE1 release. This appeared to be the result of association of hydrophobic side-chains in aqueous media, which rendered the formation of the enzyme substrate complex more difficult. The data seems to indicate that HPMA copolymer-PGE1 conjugates have a potential in the treatment of osteoporosis and other bone diseases.

Synthesis of Long-Circulating, Backbone Degradable HPMA Copolymer–Doxorubicin Conjugates and Evaluation of Molecular-Weight-Dependent Antitumor Efficacy

Backbone degradable, linear, multiblock N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (DOX) conjugates are synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization followed by chain extension via thiol-ene click reaction. The examination of molecular-weight-dependent antitumor activity toward human ovarian A2780/AD carcinoma in nude mice reveals enhanced activity of multiblock, second-generation, higher molecular weight conjugates when compared with traditional HPMA copolymer-DOX conjugates. The examination of body weight changes during treatment indicates the absence of non-specific adverse effects.

Synthesis and Characterization of Enzymatically Degradable PEG‐Based Peptide‐Containing Hydrogels

Biodegradable hydrogels were synthesized by the click reaction of 4-arm azido-terminated PEG differing in molecular weight (2,100 and 8,800) and two alkyne-terminated peptides: [alkyne]-GFLGK-[alkyne] and ([alkyne]-GFLG)(2)K. The physical properties of in situ formed hydrogels were examined. The hydrogels were highly elastic as determined by rheological and microrheological studies. Swelling degree and enzymatic degradation by papain were dependent on the molecular weight of the PEG, but not the peptide. For PEG8800-based hydrogels, time-course analysis of degradation showed that the molecular weight of the soluble fraction quickly reached the PEG precursor value. These findings may guide future design of hydrogels with controllable mechanical properties and enzymatic degradability.

Efficiency of high molecular weight backbone degradable HPMA copolymer–Prostaglandin E1 conjugate in promotion of bone formation in ovariectomized rats

Multiblock, high molecular weight, linear, backbone degradable HPMA copolymer-prostaglandin E1 (PGE1) conjugate has been synthesized by RAFT polymerization mediated by a new bifunctional chain transfer agent (CTA), which contains an enzymatically degradable oligopeptide sequence flanked by two dithiobenzoate groups, followed by postpolymerization aminolysis and thiol-ene chain extension. The multiblock conjugate contains Asp8 as the bone targeting moiety and enzymatically degradable bonds in the polymer backbone; in vivo degradation produces cleavage products that are below the renal threshold. Using an ovariectomized (OVX) rat model, the accumulation in bone and efficacy to promote bone formation was evaluated; low molecular weight conjugates served as control. The results indicated a higher accumulation in bone, greater enhancement of bone density, and higher plasma osteocalcin levels for the backbone degradable conjugate.