Targeted oral peptide delivery using multi-unit particulates: Drug and permeation enhancer layering approach.

Oral delivery of peptides is a challenge due to their instability and their limited transport and absorption characteristics within the gastrointestinal tract. In this work, we used layering techniques in a fluidized bed dryer to create a configuration in which the active peptide, permeation enhancers, and polymers are coated to control the release of the peptide. Formulations were developed to disintegrate at pH values of 5.5 and 7.0. In addition, sustained-release or mucoadhesive polymers were coated to trigger release at a desired site in the gastrointestinal tract. Dissolution studies with a USP Type I (basket) apparatus confirmed the duration of release. Pharmacokinetic studies were performed in beagle dogs to evaluate bioavailability. A high-disintegration pH was found to be advantageous in enhancing bioavailability.

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 ß-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 ß-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. ß-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.

Freeze-thaw stress of Alhydrogel ® alone is sufficient to reduce the immunogenicity of a recombinant hepatitis B vaccine containing native antigen.

Preventing losses in vaccine potency due to accidental freezing has recently become a topic of interest for improving vaccines. All vaccines with aluminum-containing adjuvants are susceptible to such potency losses. Recent studies have described excipients that protect the antigen from freeze-induced inactivation, prevent adjuvant agglomeration and retain potency. Although these strategies have demonstrated success, they do not provide a mechanistic understanding of freeze-thaw (FT) induced potency losses. In the current study, we investigated how adjuvant frozen in the absence of antigen affects vaccine immunogenicity and whether preventing damage to the freeze-sensitive recombinant hepatitis B surface antigen (rHBsAg) was sufficient for maintaining vaccine potency. The final vaccine formulation or Alhydrogel(®) alone was subjected to three FT-cycles. The vaccines were characterized for antigen adsorption, rHBsAg tertiary structure, particle size and charge, adjuvant elemental content and in-vivo potency. Particle agglomeration of either vaccine particles or adjuvant was observed following FT-stress. In vivo studies demonstrated no statistical differences in IgG responses between vaccines with FT-stressed adjuvant and no adjuvant. Adsorption of rHBsAg was achieved; regardless of adjuvant treatment, suggesting that the similar responses were not due to soluble antigen in the frozen adjuvant-containing formulations. All vaccines with adjuvant, including the non-frozen controls, yielded similar, blue-shifted fluorescence emission spectra. Immune response differences could not be traced to differences in the tertiary structure of the antigen in the formulations. Zeta potential measurements and elemental content analyses suggest that FT-stress resulted in a significant chemical alteration of the adjuvant surface. This data provides evidence that protecting a freeze-labile antigen from subzero exposure is insufficient to maintain vaccine potency. Future studies should focus on adjuvant protection. To our knowledge, this is the first study to systematically investigate how FT-stress to adjuvant alone affects immunogenicity. It provides definitive evidence that this damage is sufficient to reduce vaccine potency.

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

PURPOSE: To evaluate the effects of pirfenidone nanoparticles on corneal re-epithelialization and scarring, major clinical challenges after alkali burn. METHODS: Effect of pirfenidone on collagen I and α-smooth muscle actin (α-SMA) synthesis by TGFß 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. RESULTS: Pirfenidone prevented (P<0.05) increase in TGF ß 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. CONCLUSION: 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.

Local delivery of biodegradable pirfenidone nanoparticles ameliorates bleomycin-induced pulmonary fibrosis in mice.

Our purpose was to assess sustained delivery and enhanced efficacy of pirfenidone-loaded nanoparticles after intratracheal instillation.Poly(lactide-co-glycolide) nanoparticles containing pirfenidone (NPs) were prepared and characterized. Biodistribution of NPs and solution was assessed using LC-MS after intratracheal administration in C57Bl/6 mice at 3 and 24 h and 1 week post-administration. Efficacy was tested in C57Bl/6 mice in a bleomycin-induced pulmonary fibrosis model. Mice received 10 µg pirfenidone intratracheally in solution or NPs, once a week, for 3 weeks after bleomycin administration. Drug effects were monitored on day 28. Lung hydroxyproline content, total number of cells, and numbers of macrophages, lymphocytes, and neutrophils in bronchoalveolar lavage (BAL) were assessed. Numbers of macrophages, lymphocytes, and neutrophils were assessed in the lung as well.NPs sustained significantly higher levels of pirfenidone in the lungs and BAL at 24 h and 1 week, compared to the solution group. Pirfenidone solution and NPs significantly reduced hydroxyproline levels by 57 and 81%, respectively, compared to bleomycin alone. At the end of 4 weeks, BAL cellularity was reduced by 25.4% and 56% with solution and NP treatment, respectively. The numbers of lymphocytes and neutrophils in the BAL were also reduced by 58.9 and 82.4% for solution and 74.5% and 89.7% for NPs, respectively. The number of inflammatory macrophages in the lung was reduced by 62.8% and the number of neutrophils was reduced by 59.1% in the NP group and by 37.7% and 44.5%, respectively, in the solution group, compared to bleomycin alone.In conclusion, nanoparticles sustain lung pirfenidone delivery and enhance its anti-fibrotic efficacy.

Functionalized STAT1 siRNA nanoparticles regress rheumatoid arthritis in a mouse model.

AIM: To develop and characterize an RGD peptide functionalized poly(lactide-co-glycolytic) acid (PLGA) nanosystem to deliver a STAT1 siRNA to joint tissues in a mouse model of rheumatoid arthritis. METHODS: RGD-PLGA polymer was synthesized and used in preparing functionalized nanoparticles loaded with either tracking material or siRNA. The properties of the nanoparticles and stability of siRNA after encapsulation was assessed. Nanoparticle distribution was determined both noninvasively and based on analysis of dissected organs from arthritic and healthy mice. Arthritic mice were treated with weekly doses of STAT1 siRNA-loaded nanoparticles or controls. Clinical disease was assessed. Paws of arthritic mice were sectioned for histology or processed for RNA. STAT1, Mrc-1, and IL-10 mRNA abundance was determined by quantitative PCR. RESULTS: Nanoparticles protected the siRNA from serum degradation. The presence of RGD peptide on the nanoparticles increased paw tissue uptake in arthritic mice. Furthermore, RGD functionalization increased lung delivery of nanoparticles in arthritic mice but not in control mice. Disease regressed in the STAT1 siRNA-treated animals and progressed in all control groups. STAT1 mRNA levels were decreased in paws of treated animals, while Mrc-1 and IL-10 mRNA levels were increased. CONCLUSION: RGD functionalized PLGA nanoparticles encapsulating STAT1-targeted siRNAs are efficacious in the treatment of established arthritis, possibly through a selective inhibition of macrophage and dendritic cell activation.

Delivery of SAR 1118 to the retina via ophthalmic drops and its effectiveness in a rat streptozotocin (STZ) model of diabetic retinopathy (DR).

PURPOSE: To determine the pharmacokinetics of SAR 1118, a small-molecule antagonist of leukocyte function-associated antigen (LFA)-1, after administration of ophthalmic drops in normal rats, and to determine its pharmacologic activity by assessing the inhibition of retinal leukostasis and vascular leakiness in a streptozotocin (STZ)-induced diabetic retinopathy model. METHODS: The ocular pharmacokinetics of SAR 1118 were studied in rats after a single topical dose of (14)C-SAR 1118 (1 mg/eye; 40 µCi; 15.5 µL). SAR 1118 concentration time profiles in plasma and ocular tissues were quantified by liquid scintillation counting (LSC). The pharmacologic activity of SAR 1118 eye drops administered thrice daily for 2 months at 1% (0.3 mg/eye/d) and 5% (1.5 mg/eye/d) was assessed in an STZ-induced diabetic rat model by determining retinal leukostasis and blood-retinal barrier breakdown. Diabetic rats treated with periocularly administered celecoxib microparticles served as the positive control, and vehicle-treated rats served as the negative control. RESULTS: A single dose of 6.5% (14)C-radiolabeled SAR 1118 ophthalmic drops delivered retinal drug levels greater than 1 µM in less than 30 minutes and sustained levels greater than 100 nM for 8 hours. SAR 1118 eye drops significantly reduced leukostasis and blood-retinal barrier breakdown in a dose-dependent manner. CONCLUSIONS: SAR 1118 ophthalmic drops administered thrice daily deliver therapeutic levels of SAR 1118 in the retina and can alleviate the retinal complications associated with diabetes.

Nanomicellar formulations for sustained drug delivery: strategies and underlying principles.

Micellar delivery systems smaller than 100 nm can be readily prepared. While micelles allow a great depth of tissue penetration for targeted drug delivery, they usually disintegrate rapidly in the body. Thus, sustained drug delivery from micellar nanocarriers is a challenge. This article summarizes various key strategies and underlying principles for sustained drug delivery using micellar nanocarriers. Comparisons are made with other competing delivery systems such as polymeric microparticles and nanoparticles. Amphiphilic molecules self-assemble in appropriate liquid media to form nanoscale micelles. Strategies for sustained release nanomicellar carriers include use of prodrugs, drug polymer conjugates, novel polymers with low critical micellar concentration or of a reverse thermoresponsive nature, reverse micelles, multi-layer micelles with layer by layer assembly, polymeric films capable of forming micelles in vivo and micelle coats on a solid support. These new micellar systems are promising for sustained drug delivery.

Targeted drug and gene delivery systems for lung cancer therapy.

PURPOSE: To evaluate the efficacy of a novel docetaxel derivative of deslorelin, a luteinizing hormone-releasing hormone (LHRH) agonist, and its combination in vivo with RGD peptide conjugated nanoparticles encapsulating an antiangiogenic, anti-vascular endothelial growth factor (VEGF) intraceptor (Flt23k; RGD-Flt23k-NP) in H1299 lung cancer cells and/or xenografts in athymic nude BALB/c mice. EXPERIMENTAL DESIGN: The in vitro and in vivo efficacy of the deslorelin-docetaxel conjugate was evaluated in H1299 cells and xenografts in athymic nude mice. Coadministration of deslorelin-docetaxel conjugate and RGD-Flt23k-NP was tested in vivo in mice. Tumor inhibition, apoptosis, and VEGF inhibition were estimated in each of the treatment groups. RESULTS: The conjugate enhanced in vitro docetaxel efficacy by 13-fold in H1299 cells compared with docetaxel at 24 hours, and this effect was inhibited following reduction of LHRH receptor expression by an antisense oligonucleotide. Combination of the conjugate with the RGD-Flt23k-NP in vivo resulted in an 82- and 15-fold tumor growth inhibition on day 39 following repeated weekly i.v. injections and a single intratumoral (i.t.) injection, respectively. These effects were significantly greater than individual targeted therapies or docetaxel alone. Similarly, apoptotic indices for the combination therapy were 14% and 10% in the i.v. and i.t. groups, respectively, and higher than the individual therapies. Combination therapy groups exhibited greater VEGF inhibition in both the i.v. and i.t. groups. CONCLUSIONS: Docetaxel efficacy was enhanced by LHRH receptor-targeted deslorelin conjugate and further improved by combination with targeted antiangiogenic nanoparticle gene therapy. Combination of novel targeted therapeutic approaches described here provides an attractive alternative to the current treatment options for lung cancer therapy.