The Development of a Macromolecular Analgesic for Arthritic Pain.

The addictive potential of clinically used opioids as a result of their direct action on the dopaminergic reward system in the brain has limited their application. In an attempt to reduce negative side effects as well as to improve the overall effectiveness of these analgesics, we have designed, synthesized, and evaluated an N-(2-hydroxypropyl)methacrylamide (HPMA)-based macromolecular prodrug of hydromorphone (HMP), a commonly used opioid. To this end, P-HMP was synthesized via RAFT polymerization and a subsequent polymer analogous reaction. Its interaction with inflammatory cells in arthritic joints was evaluated in vitro using a RAW 264.7 cell culture, and subsequent confocal microscopy analysis confirmed that P-HMP could be internalized by the cells via endocytosis. In vivo imaging studies indicated that the prodrug can passively target the arthritic joint after systemic administration in a rodent model of monoarticular adjuvant-induced arthritis (MAA). The inflammatory pain-alleviating properties of the prodrug were assessed in MAA rats using the incapacitance test and were observed to be similar to dose-equivalent HMP. Analgesia through mechanisms at the spinal cord level was further measured using the tail flick test, and it was determined that the prodrug significantly reduced spinal cord analgesia versus free HMP, further validating the peripheral restriction of the macromolecular prodrug. Immunohistochemical analysis of cellular uptake of the P-HMP within the MAA knee joint proved the internalization of the prodrug by phagocytic synoviocytes, colocalized with HMP's target receptor as well as with pain-modulating ion channels. Therefore, it can be concluded that the novel inflammation-targeting polymeric prodrug of HMP (P-HMP) has the potential to be developed as an effective and safe analgesic agent for musculoskeletal pain.

The Evaluation of Therapeutic Efficacy and Safety Profile of Simvastatin Prodrug Micelles in a Closed Fracture Mouse Model.

PURPOSE: To evaluate the therapeutic efficiency of a micellar prodrug formulation of simvastatin (SIM/SIM-mPEG) and explore its safety in a closed femoral fracture mouse model. METHODS: The amphiphilic macromolecular prodrug of simvastatin (SIM-mPEG) was synthesized and formulated together with free simvastatin into micelles. It was also labeled with a near infrared dye for in vivo imaging purpose. A closed femoral fracture mouse model was established using a three-points bending device. The mice with established closed femoral fractures were treated with SIM/SIM-mPEG micelles, using free simvastatin and saline as controls. The therapeutic efficacy of the micelles was evaluated using a high-resolution micro-CT. Serum biochemistry and histology analyses were performed to explore the potential toxicity of the micelle formulation. RESULTS: Near Infrared Fluorescence (NIRF) imaging confirmed the passive targeting of SIM/SIM-mPEG micelles to the bone lesion of the mice with closed femoral fractures. The micelle was found to promote fracture healing with an excellent safety profile. In addition, the accelerated healing of the femoral fracture also helped to prevent disuse-associated ipsilateral tibia bone loss. CONCLUSION: SIM/SIM-mPEG micelles were found to be an effective and safe treatment for closed femoral fracture repair in mice. The evidence obtained in this study suggests that it may have the potential to be translated into a novel therapy for clinical management of skeletal fractures and non-union.

Macromolecular glucocorticoid prodrug improves the treatment of dextran sulfate sodium-induced mice ulcerative colitis.

A macromolecular prodrug (P-Dex) of dexamethasone (Dex) was developed to improve the treatment of inflammatory bowel disease (IBD). Colonic inflammation was induced by feeding mice with dextran sulfate sodium. Mice were treated with daily i.p. injection of free Dex or single i.v. injection of P-Dex, PBS or free polymer. Both P-Dex and free Dex could lower disease activity index and histology scores when compared to the controls. A single injection of P-Dex with 1/4 equivalent Dex dose had a better therapeutic effect than daily free Dex treatment. Mechanism study found that P-Dex could target the inflamed colon, and be retained by epithelial cells and local inflammatory infiltrates, suggesting that the improved efficacy of P-Dex may be attributed to its inflammation targeting, subcellular processing and activation. Collectively, these data support our hypothesis that the development of macromolecular prodrug of glucocorticoid may have the potential to improve the clinical management of IBD.

Safe and effective subcutaneous adipolysis in minipigs by a collagenase derivative.

Adipocytes attached to the extracellular matrix (ECM) mainly consist of collagen in adipose tissues, while the degradation of ECM by collagenase induces the apoptosis of adipocytes, leading to a decrease in local subcutaneous adipose. To achieve this goal, we are developing a mutant collagenase H (ColH) to remove local subcutaneous fat such as submental fat (SMF). Three vectors were constructed for expressing rColH(FM, mutant for fat melting, with 6xHis tag), rColH(WT, wild-type, with 6xHis tag), and rColH(E451D, E451D mutant, without 6xHis tag) in Escherichia coli. rColH(FM) & rColH(WT) were purified by Ni Sepharose on a laboratory scale, while rColH(E451D) was purified by five chromatography purification steps on a large scale. Then, the stability of rColH(FM) and rColH(WT) was tested by SDS-PAGE to investigate the influence of the E451D mutation on stability. Afterwards, the enzyme kinetics of ColH (mutant or wild-type, with or without His tag) were investigated and compared. Finally, the adipolysis of rColH(E451D) at various doses was tested in vitro and in vivo. The ultrasound results in minipigs suggested that effective adipolysis was induced by rColH(E451D) compared with the negative control, and the histological results suggest dose-dependent fibrosis, necrosis, inflammation and cholesterol cleft formation. These findings indicate the possibility of rColH(E451D) becoming a new injectable drug to safely remove subcutaneous adipose.

Simvastatin prodrug micelles target fracture and improve healing.

Simvastatin (SIM), a widely used anti-lipidemic drug, has been identified as a bone anabolic agent. Its poor water solubility and the lack of distribution to the skeleton, however, have limited its application in the treatment of bone metabolic diseases. In this study, an amphiphilic macromolecular prodrug of SIM was designed and synthesized to overcome these limitations. The polyethylene glycol (PEG)-based prodrug can spontaneously self-assemble to form micelles. The use of SIM trimer as the prodrug's hydrophobic segment allows easy encapsulation of additional free SIM. The in vitro studies showed that SIM/SIM-mPEG micelles were internalized by MC3T3 cells via lysosomal trafficking and consistently induced expression of both BMP2 and DKK1 mRNA, suggesting that the prodrug micelle retains the biological functions of SIM. After systemic administration, optical imaging suggests that the micelles would passively target to bone fracture sites associated with hematoma and inflammation. Furthermore, flow cytometry study revealed that SIM/SIM-mPEG micelles had preferred cellular uptake by inflammatory and resident cells within the fracture callus tissue. The treatment study using a mouse osteotomy model validated the micelles' therapeutic efficacy in promoting bone fracture healing as demonstrated by micro-CT and histological analyses. Collectively, these data suggest that the macromolecular prodrug-based micelle formulation of SIM may have great potential for clinical management of impaired fracture healing.

Macromolecular prodrug of dexamethasone prevents particle-induced peri-implant osteolysis with reduced systemic side effects.

Aseptic implant loosening related to implant wear particle-induced inflammation is the most common cause of failure after joint replacement. Modulation of the inflammatory reaction to the wear products represents a rational approach for preventing aseptic implant failure. Long-term treatment using anti-inflammatory agents, however, can be associated with significant systemic side effects due to the drugs' lack of tissue specificity. To address this issue, N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-dexamethasone conjugate (P-Dex) was developed and evaluated for prevention of wear particle-induced osteolysis and the loss of fixation in a murine prosthesis failure model. Daily administration of free dexamethasone (Dex) was able to prevent wear particle-induced osteolysis, as assessed by micro-CT and histological analysis. Remarkably, monthly P-Dex administration (dose equivalent to free Dex treatment) was equally effective as free dexamethasone, but was not associated with systemic bone loss (a major adverse side effect of glucocorticoids). The reduced systemic toxicity of P-Dex is related to preferential targeting of the sites of wear particle-induced inflammation and its subcellular sequestration and retention by local inflammatory cell populations, resulting in sustained therapeutic action. These results demonstrate the feasibility of utilizing a macromolecular prodrug with reduced systemic toxicity to prevent wear particle-induced osteolysis.

A dexamethasone prodrug reduces the renal macrophage response and provides enhanced resolution of established murine lupus nephritis.

We evaluated the ability of a macromolecular prodrug of dexamethasone (P-Dex) to treat lupus nephritis in (NZB × NZW)F1 mice. We also explored the mechanism underlying the anti-inflammatory effects of this prodrug. P-Dex eliminated albuminuria in most (NZB × NZW)F1 mice. Furthermore, P-Dex reduced the incidence of severe nephritis and extended lifespan in these mice. P-Dex treatment also prevented the development of lupus-associated hypertension and vasculitis. Although P-Dex did not reduce serum levels of anti-dsDNA antibodies or glomerular immune complexes, P-Dex reduced macrophage recruitment to the kidney and attenuated tubulointerstitial injury. In contrast to what was observed with free dexamethasone, P-Dex did not induce any deterioration of bone quality. However, P-Dex did lead to reduced peripheral white blood cell counts and adrenal gland atrophy. These results suggest that P-Dex is more effective and less toxic than free dexamethasone for the treatment of lupus nephritis in (NZB × NZW)F1 mice. Furthermore, the data suggest that P-Dex may treat nephritis by attenuating the renal inflammatory response to immune complexes, leading to decreased immune cell infiltration and diminished renal inflammation and injury.