Poly-beta-amino-ester licofelone conjugates development for osteoarthritis treatment

Disease-modifying osteoarthritis drugs (DMOADs) are a new therapeutic class for osteoarthritis (OA) prevention or inhibition of the disease development. Unfortunately, none of the DMOADs have been clinically approved due to their poor therapeutic performances in clinical trials. The joint environment has played a role in this process by limiting the amount of drug effectively delivered as well as the time that the drug stays within the joint space. The current study aimed to improve the delivery of the DMOADs into cartilage tissue by increasing uptake and retention time of the DMOADs within the tissue. Licofelone was used a model DMOAD due to its significant therapeutic effect against OA progression as shown in the recent phase III clinical trial. For this purpose licofelone was covalently conjugated to the two different A16 and A87 poly-beta-amino-ester (PBAEs) polymers taking advantage of their hydrolysable, cytocompatible, and cationic nature. We have shown cartilage uptake of the licofelone–PBAE conjugates increased 18 times and retention in tissues was prolonged by 37 times compared to the equivalent dose of the free licofelone. Additionally, these licofelone conjugates showed no detrimental effect on the chondrocyte viability. In conclusion, the cationic A87 and A16 PBAE polymers increased the amount of licofelone within the cartilage, which could potentially enhance the therapeutic effect and pharmacokinetic performance of this drug and other DMOADs clinically.


Materials
Cartilage media materials that were purchased from Thermo Fisher Scientific are Dulbecco's Modified Eagle Medium (DMEM, 11885-084), insulin-transferrin-selenium, and minimum non-essential amino acids (MEMNEAA) by Gibco, while penicillin/streptomycin, amphotericin B, and proline were purchased from Sigma Aldrich.

Buffers preparation
Phosphate buffered saline (PBS, pH 7.4) was prepared by dissolving a 100 g tablet of PBS in 100 ml of deionized water (dH 2 O).The sodium acetate buffer (pH 5) was obtained by mixing (volume to volume) 30% of (5.8 ml acetic acid in 994.2 ml of dH 2 O) with 70% of (13.6 g sodium acetate trihydrate in 1000 ml of dH2O).

Cartilage digestion solution
The digestion buffer was prepared by mixing 2 mM of DTT and 0.3 mg/ml of papain in a pH 6.8 buffer, which contains 20 mM sodium phosphate buffer and 1 mM of EDTA.(1)The 1 ml of digestion solution contains 900 μl digestion buffer and 100 μl DMSO.

Preparation of cartilage complete medium
The complete medium contains 500 ml DMEM, 5 ml insulin-transferrin-selenium, 5 ml penicillin/streptomycin, 5 ml MEMNEAA, 0.5 ml amphotericin B, 0.5 ml ascorbic acid, and 0.5 ml proline.The 250 µg of amphotericin B, 20 mg of ascorbic acid, and 4 moles of proline were separately dissolved in 1 ml of sterile PBS pH 7.4.(2) The XTT assay sample preparation 0.135 % DMSO was used to solubilize 1 and 2.7 μg/ml licofelone, whereas the A87licofelone and A87 polymer were soluble in complete medium.Therefore, the cytotoxicity of 0.135 % DMSO was investigated.At 24 and 48 hours, 0.135 % DMSOtreated cartilage showed no statistical difference compared to the untreated cartilage (control) (Figure S. 6), so no toxicity associated with the use of 0.135 % DMSO was observed.A healthy cartilage uptake of 0.95 mg/ml of NBQX salt in PBS pH 7.4 or NBQX hydrate in DMSO was observed to determine the effect of DMSO on the drug uptake.
The reason for this is that DMSO must be used in licofelone studies since the licofelone is not soluble in aqueous buffer.There was no effect observed between the uptake of NBQX dissolved in PBS and DMSO.Both NBQX were purchased from Sigma Aldrich.

Figure S. 4 :
Figure S.4: The calibration curve of polyethylene glycol standards Bars represent (Mean ± SD of n=2)

Figure S. 6 :
Figure S. 6: The calibration curve of licofelone Bars represent (Mean ± SD of n=2)

Figure S. 8 :
Figure S. 8: The effect of 0.135% DMSO on cell viability.Bars represent (Mean ± SD of n=3)

Figure S. 11 :
Figure S. 11: The hydrolysis of A16 and A87 at pH 7.4 and pH 5; The A graphs represent the hydrolysis study of A16.The blue (■) and bars represent the hydrolysis at pH 7.4, and the black (■) and bars represent the hydrolysis at pH 5 (mean ± SD of n=3).The B graphs represent the hydrolysis study of A87.The blue (▲) and bars represent the hydrolysis at pH 7.4, and the black (▲) and bars represent the hydrolysis at pH 5 (mean ± SD of n=3).The A.1 and B.1 graphs compare the Mw at pH 7.4 and pH 5 with the respect to the time points of A16 and A87, respectively.The A.2 and B.2 graphs compare the average of Mw at 1 hour to other time points at pH 7.4.The A.3 and B.3 graphs compare the average of Mw at

Figure
Figure S. 12: The RP-HPLC chromatograms of licofelone A) RP-HPLC chromatogram shows components involve in the uptake and retention time study, B) RP-HPLC chromatogram of licofelone alone (Rt=4.1 min), and C) RP-HPLC chromatogram shows the separation of licofelone retention time from other components (Rt=4.1 min).