Pirfenidone nanoparticles improve corneal wound healing and prevent scarring following alkali burn.

To evaluate the effects of pirfenidone nanoparticles on corneal re-epithelialization and scarring, major clinical challenges after alkali burn.Effect of pirfenidone on collagen I and α-smooth muscle actin (α-SMA) synthesis by TGFβ induced primary corneal fibroblast cells was evaluated by immunoblotting and immunocytochemistry. Pirfenidone loaded poly (lactide-co-glycolide) (PLGA) nanoparticles were prepared, characterized and their cellular entry was examined in primary corneal fibroblast cells by fluorescence microscopy. Alkali burn was induced in one eye of Sprague Dawley rats followed by daily topical treatment with free pirfenidone, pirfenidone nanoparticles or vehicle. Corneal re-epithelialization was assessed daily by flourescein dye test; absence of stained area indicated complete re-epithelialization and the time for complete re-epithelialization was determined. Corneal haze was assessed daily for 7 days under slit lamp microscope and graded using a standard method. After 7 days, collagen I deposition in the superficial layer of cornea was examined by immunohistochemistry.Pirfenidone prevented (P<0.05) increase in TGF β induced collagen I and α-SMA synthesis by corneal fibroblasts in a dose dependent manner. Pirfenidone could be loaded successfully within PLGA nanoparticles, which entered the corneal fibroblasts within 5 minutes. Pirfenidone nanoparticles but not free pirfenidone significantly (P<0.05) reduced collagen I level, corneal haze and the time for corneal re-epithelialization following alkali burn.Pirfenidone decreases collagen synthesis and prevents myofibroblast formation. Pirfenidone nanoparticles improve corneal wound healing and prevent fibrosis. Pirfenidone nanoparticles are of potential value in treating corneal chemical burns and other corneal fibrotic diseases

The chemomodulatory effects of glufosfamide on docetaxel cytotoxicity in prostate cancer cells

Background. Glufosfamide (GLU) is a glucose conjugate of ifosfamide in which isophosphoramide mustard is glycosidically linked to the β-D-glucose molecule. Based on GLU structure, it is considered a targeted chemotherapy with fewer side effects. The main objective of the current study is to assess the cytotoxic potential of GLU for the first time in prostate cancer (PC) cells representing different stages of the tumor. Furthermore, this study examined the potential synergistic activity of GLU in combination with docetaxel (DOC). Methods. Two different cell lines were used, LNCaP and PC-3. Concentration-response curves were assessed. The tested groups per cell line were, control, GLU, DOC and combination. Treatment duration was 72 h. Cytotoxicity was assessed using sulforhodamine B (SRB) assay and half maximal inhibitory concentration (IC50) was calculated. Synergy analyses were performed using Calcusyn®software. Subsequent mechanistic studies included β-glucosidase activity assay, glucose uptake and apoptosis studies, namely annexin V-FITC assay and the protein expression of mitochondrial pathway signals including Bcl-2, Bax, Caspase 9 and 3 were assessed. Data are presented as mean ± SD; comparisons were carried out using one way analysis of variance (ANOVA) followed by Tukey-Kramer’s test for post hoc analysis. Results. GLU induced cytotoxicity in both cell lines in a concentration-dependent manner. The IC50 in PC-3 cells was significantly lower by 19% when compared to that of LNCaP cells. The IC50 of combining both drugs showed comparable effect to DOC in PC-3 but was tremendously lowered by 49% compared to the same group in LNCaP cell line. β-glucosidase activity was higher in LNCaP by about 67% compared to that determined in PC-3 cells while the glucose uptake in PC-3 cells was almost 2 folds that found in LNCaP cells. These results were directly correlated to the efficacy of GLU in each cell line. Treatment of PC cells with GLU as single agent or in combination with DOC induced significantly higher apoptosis as evidenced by Annexin V-staining. Apoptosis was significantly increased in combination group by 4.9 folds and by 2.1 Folds when compared to control in LNCaP cells and PC-3 cells; respectively. Similarly, the expression of Bcl-2 was significantly decreased while Bax, caspase 9 and 3 were significantly increased in the combined treatment groups compared to the control. Conclusion. GLU has a synergistic effect in combination with DOC as it increases the cell kill which can be attributed at least partially to apoptosis in both the tested cell lines and it is suggested as a new combination regimen to be considered in the treatment of the prostate cancer. Further experiments and clinical investigations are needed for assessment of that regimen

Pirfenidone prevents α-SMA expression in corneal fibroblasts: Western blot analysis shows reduction in α-SMA expression by pirfenidone from the TGF β induced corneal fibroblast in a dose dependent manner; Lane1- Untreated, Lane2- TGF β, Lane3- TGF β and 2.5 µg/ml free pirfenidone, Lane4- TGF β and 5 µg/ml free pirfenidone, Lane5- TGF β and 10 µg/ml free pirfenidone.

<p><i>Data is expressed as Mean ± SEM, N = 3, * p<0.05 vs Untreated, # p<0.05 vs TGF β.</i></p

Characteristics of nanoparticles.

<p><i>Data expressed as Mean ± SD</i>.</p

Pirfenidone suppresses Collagen I expression in corneal fibroblasts: (A) Western blot analysis shows reduction of Collagen I expression by pirfenidone in TGF β induced corneal fibroblasts in a dose dependent manner; Lane1- Untreated, Lane2- TGF β, Lane3- TGF β and 2.5 µg/ml free pirfenidone, Lane 4- TGF β and 5 µg/ml free pirfenidone, Lane5- TGF β and 10 µg/ml free pirfenidone.

<p><i>Data is expressed as Mean ± SEM, N = 3, * p<0.05 vs Untreated, # p<0.05 vs TGF β.</i> (B) Western blot also shows that suppression of collagen I was similar in free pirfenidone and pirfenidone NP treated corneal fibroblasts; Lane1- Untreated, Lane2- TGF β, Lane3- TGF β and 5 µg/ml free pirfenidone, Lane4- TGF β and 5 µg/ml pirfenidone NP, Lane5- TGF β and 10 µg/ml free pirfenidone, Lane6- TGF β and 10 µg/ml pirfenidone NP. <i>Data is expressed as Mean ± SEM, N = 3, * p<0.05 vs Untreated, # p<0.05 vs TGF β.</i> (C) Imunocytochemistry also shows suppression of collagen I expression by pirfenidone from TGF β induced corneal fibroblasts in a dose dependent manner. The suppression was similar in free pirfenidone and pirfenidone NP treated cells.</p

Surface morphology of NPs: (A) AFM and (B) TEM pictures show smooth surface of the NPs.

<p>Surface morphology of NPs: (A) AFM and (B) TEM pictures show smooth surface of the NPs.</p

Collagen 1 expression was reduced in the pirfenidone NP treated cornea: Immunohistochemistry shows increased expression of collagen I in the superficial layer of corneas from control and free pirfenidone treated rats, and a reduced expression in the corneas from pirfenidone NP treated rats.

<p>Corresponding DAPI stained sections are also presented.</p

Effect of free pirfenidone and Pirfenidone NPs on corneal healing after alkali exposure: (A) Representative pictures of control, free pirfenidone and pirfenidone NP treated corneas after 1 hr, 4 th day and 7 th day of alkali treatment.

<p>(B) Corneal re-epithelialization time: Corneal re-epithelialization time was significantly (P<0.05) reduced in pirfenidone NP treated eyes compared to the control eyes, but the corneal re-epithelialization time in free pirfenidone treated eyes was not significantly different from the control eyes. <i>Data is expressed as Mean ± SEM, N = 6.</i> (C) Corneal haze: A significant (P<0.05) reduction in corneal haze score was evidenced in pirfenidone NP treated eyes compared to the control eyes. <i>Data is expressed as Mean ± SEM, N = 6.</i></p

Delivery of SAR 1118 to the Retina via Ophthalmic Drops and its Effectiveness in a Rat Streptozotocin (STZ) Model of Diabetic Retinopathy (DR)

The delivery and efficacy of SAR 1118 were examined in a rat model of diabetic retinopathy