Injectable Biodegradable Silica Depot for Controlled Subcutaneous Delivery of Antisense Oligonucleotides with beyond Monthly Administration.

Single-stranded antisense oligonucleotides (ASOs) are typically administered subcutaneously once per week or monthly. Less frequent dosing would have strong potential to improve patient convenience and increase adherence and thereby for some diseases result in more optimal therapeutic outcomes. Several technologies are available to provide sustained drug release via subcutaneous (SC) administration. ASOs have a high aqueous solubility and require relatively high doses, which limits the options available substantially. In the present work, we show that an innovative biodegradable, nonporous silica-based matrix provides zero-order release in vivo (rats) for at least 4 weeks for compositions with ASO loads of up to about 100 mg/mL (0.5 mL injection) without any sign of initial burst. This implies that administration beyond once monthly can be feasible. For higher drug loads, substantial burst release was observed during the first week. The concentrations of unconjugated ASO levels in the liver were found to be comparable to corresponding bolus doses. Additionally, infusion using a minipump shows a higher liver exposure than SC bolus administration at the same dose level and, in addition, clear mRNA knockdown and circulating protein reduction comparable to SC bolus dosing, hence suggesting productive liver uptake for a slow-release administration.

Topical cis-urocanic acid prevents ocular surface irritation in both IgE -independent and -mediated rat model.

PURPOSE: Our purpose was to investigate the effect of locally administered cis-urocanic (cis-UCA) in two experimental models of allergic conjunctivitis. METHODS: The compound 48/80 (C48/80)-induced ocular irritation model (IgE-independent) and the ovalbumin (OA)-induced ocular allergy model (IgE-mediated) were used to test and compare the effect of cis-UCA on dexamethasone, ketotifen and olopatadine. In the C48/80 model, clinical severity scoring from photographs, immunohistochemical analysis of nuclear Ki-67 antigen to quantify actively proliferating epithelial cells and of caspase-3 enzyme to identify apoptotic activity in the conjunctival tissue were used. In the OA model, an Evans Blue stain concentration of conjunctival tissue was used to evaluate vascular leakage due to allergic reaction. RESULTS: The cis-UCA was well tolerated and effective in both the IgE-independent and -mediated rat models. Treatment with C48/80 caused conjunctival hyperaemia, which was significantly inhibited by ketotifen at the 6 h time point (p = 0.014) and by dexamethasone and cis-UCA 0.5% at 12 (p = 0.004) and 24 (p = 0.004) hour time points. In a comparison between the active drug treatments, only ketotifen showed a significant difference (p = 0.023) to cis-UCA treatment at the 1 h time point, otherwise there were no statistically significant differences between the active drugs. Ketotifen, dexamethasone and cis-UCA 0.5% significantly inhibited the C48/80-induced nuclear accumulation of Ki-67, without differences between the active treatment groups. In the OA model, cis-UCA 0.5% did not inhibit the vascular leakage of conjunctiva, whereas cis-UCA 2.5% of was at least equally effective compared to olopatadine, abolishing the allergic vascular leakage response almost completely. CONCLUSIONS: The present findings in the two AC models suggest that cis-UCA might have anti-allergic potency both in immediate and delayed-type allergic reactions in the eye.

Intravesical treatment with cis-urocanic acid improves bladder function in rat model of acute bladder inflammation.

AIMS: The aim was to study the effect of intravesically instilled cis-urocanic acid (cis-UCA) on bladder function in an experimental rat model of acute bladder inflammation. Hyaluronic acid (HA) was used as a comparator compound. METHODS: Bladder irritation was induced in female rats by intravesical hydrochloric acid (HCl) infusion. Vehicle, 0.5% HA, or 2% cis-UCA solutions were infused intravesically twice a day for three consequent days. On the fourth day, urodynamical measurements were performed, the animals were sacrificed, and the bladders were removed for histopathological assessment. RESULTS: HCl treatment caused significant impairment of bladder function indicated by decreased micturition intervals and voided urine volumes and induced severe voiding dysfunction observed as occurrence of overflow incontinence. These functional changes were accompanied by increased bladder weight, hemorrhage, and infiltration of inflammatory cells into the urothelium. Intravesical cis-UCA treatment recovered bladder function by significantly prolonging the micturition interval, increasing the voided volume, and reducing the occurrence of overflow incontinence. All these changes were comparable to the effects of HA. CONCLUSIONS: Intravesical administration of cis-UCA was able to partially recover bladder function impaired by chemical irritation. Cis-UCA may offer a novel intravesical treatment option in some inflammatory conditions of the bladder. Neurourol. Urodynam. 35:786-791, 2016. © 2015 Wiley Periodicals, Inc.

Three randomised phase I/IIa trials of 5% cis-urocanic acid emulsion cream in healthy adult subjects and in patients with atopic dermatitis.

New treatment modalities are needed in atopic dermatitis. We evaluated the pharmacokinetics, safety, tolerability, and efficacy of topical cis-urocanic acid (cis-UCA) cream in randomised vehicle-controlled double-blinded clinical trials. The subjects received 5% cis-UCA emulsion cream and control vehicle on volar forearms after right-left randomisation. Study 1: 16 healthy subjects received one dose on the skin and, a week later, on DMSO-irritated skin. Study 2: 16 healthy subjects received 2 daily doses for 10 days. Study 3: 13 patients with mild to moderate disease were treated on selected skin lesions twice daily for 28 days. Study treatments were well tolerated. cis-UCA remained close to endogenous levels in plasma and urine. cis-UCA reduced transepidermal water loss (TEWL) both in healthy subjects and in the patients. Eczema area severity index and physician's global assessment improved from baseline with both treatments. cis-UCA cream improved skin barrier function and suppressed inflammation in the human skin.

Antitumor effects of cis-urocanic acid on experimental urothelial cell carcinoma of the bladder.

PURPOSE: We determined the effect of protodynamic therapy against bladder cancer cells in vitro and in vivo. We investigated cis-urocanic acid in rat bladder cancer cell cultures and in an orthotopic rat urothelial carcinoma model to assess its safety and antiproliferative activity. MATERIALS AND METHODS: The rat bladder cancer cell line AY-27 was exposed to cis-urocanic acid (BioCis Pharma, Turku, Finland) at pH 6.5 or 7.4 for 2 hours. Cell viability was measured by colorimetric assay at 24 and 48 hours. For in vivo experiments AY-27 cells were instilled into the acid treated bladder of 17 rats. After 4, 7 and 10 days 14 rats were treated intravesically with cis-urocanic acid 6% (weight per volume) or vehicle. Rats were sacrificed on day 12 and the bladders were dissected. Immunohistochemical staining was done to assess apoptosis (caspase-3) and cell proliferation (Ki-67) in vivo. RESULTS: Cis-urocanic acid caused dose dependent, pH dependent inhibition of AY-27 cell proliferation, showing the protodynamic action at concentrations of 0.5% and 1%. At higher cis-urocanic acid doses complete cell death was observed. All tumors detected in animals treated with vehicle were muscle invasive (stage T2 or greater) but only 43% of tumors were muscle invasive in the cis-urocanic acid treated group (p=0.049). There was no difference in the percent of apoptotic or proliferating tumor cells between treatment groups. No signs of toxicity were observed. CONCLUSIONS: Cis-urocanic acid showed direct antiproliferative activity against rat bladder cancer cells in vitro and antitumor effects in vivo. It may have therapeutic potential as an intravesical agent for nonmuscle invasive bladder cancer.

Injectable Biodegradable Silica Depot: Two Months of Sustained Release of the Blood Glucose Lowering Peptide, Pramlintide.

Diabetes mellitus is a major healthcare challenge. Pramlintide, a peptide analogue of the hormone amylin, is currently used as an adjunct with insulin for patients who fail to achieve glycemic control with only insulin therapy. However, hypoglycemia is the dominant risk factor associated with such approaches and careful dosing of both drugs is needed. To mitigate this risk factor and compliance issues related to multiple dosing of different drugs, sustained delivery of Pramlintide from silica depot administered subcutaneously (SC) was investigated in a rat model. The pramlintide-silica microparticle hydrogel depot was formulated by spray drying of silica sol-gels. In vitro dissolution tests revealed an initial burst of pramlintide followed by controlled release due to the dissolution of the silica matrix. At higher dosing, pramlintide released from subcutaneously administered silica depot in rats showed a steady concentration of 500 pM in serum for 60 days. Released pramlintide retained its pharmacological activity in vivo, as evidenced by loss of weight. The biodegradable silica matrix offers a sustained release of pramlintide for at least two months in the rat model and shows potential for clinical applications.

Cis-urocanic acid inhibits SAPK/JNK signaling pathway in UV-B exposed human corneal epithelial cells in vitro.

PURPOSE: The cornea is sensitive to ultraviolet B (UV-B) radiation-induced oxidative stress and inflammation. Its clinical manifestations are photokeratitis and climatic droplet keratopathy. Urocanic acid (UCA) is a major endogenous UV-absorbing chromophore in the epidermis and it is also an efficacious immunosuppressant. We have previously shown that cis-UCA can suppress UV-B-induced interleukin-6 and -8 secretion and cytotoxicity in human corneal epithelium (HCE) cells. In the current study, we further wanted to investigate the effects of cis-UCA on UV-B-induced inflammatory and apoptotic responses in HCE-2 cells, focusing on the nuclear factor kappa B (NF-κB) and AP-1 (subunits c-Fos and c-Jun) signaling pathways. METHODS: After exposing HCE-2 cells to UV-B and cis-UCA, DNA binding of c-Fos, c-Jun and NF-κB was measured with ELISA. In addition, the endogenous levels of phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase (phospho-SAPK/JNK) and phospho-c-Jun were determined. The proliferative capacity of HCE-2 cells was also quantified, and the cytotoxicity of the cis-UCA and UV-B treatments was monitored by measuring the release of lactate dehydrogenase enzyme in the culture medium. RESULTS: UV-B irradiation induced the binding of transcription factors c-Jun, c-Fos, and NF-κB to DNA. Cis-UCA inhibited the binding of c-Jun and c-Fos but not that of NF-κB. Moreover, UV-B increased the levels of phospho-c-Jun and phospho-JNK, and the expression of both was attenuated by cis-UCA. Cis-UCA also alleviated the UV-B-induced apoptosis and proliferative decline in human corneal cells. CONCLUSIONS: The results from this study suggest that cis-UCA suppresses JNK signaling pathway, which provides potential for treating UV-B-induced inflammatory defects in human corneal cells.

Sustained In-Vivo Release of Triptorelin Acetate from a Biodegradable Silica Depot: Comparison to Pamorelin® LA.

Triptorelin acetate was encapsulated into silica microparticles by spray-drying a mixture of colloidal silica sol and triptorelin acetate solution. The resulting microparticles were then combined with another silica sol containing silica nanoparticles, which together formed an injectable silica-triptorelin acetate depot. The particle size and surface morphology of the silica-triptorelin acetate microparticles were characterized together with the in vitro release of triptorelin, injectability and rheology of the final injectable silica-triptorelin acetate depot. In vivo pharmacokinetics and pharmacodynamics of the silica-triptorelin acetate depot and Pamorelin® were evaluated and compared in Sprague-Dawley male rats after subcutaneous administration. Serum samples up to 91 days were collected and the plasma concentrations of triptorelin and testosterone were analyzed with ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). In vivo pharmacokinetics showed that injections of the silica-triptorelin acetate depot gave 5-fold lower Cmax values than the corresponding Pamorelin® injections. The depot also showed a comparable sustained triptorelin release and equivalent pharmacodynamic effect as the Pamorelin® injections. Detectable triptorelin plasma concentrations were seen with the depot after the 91-day study period and testosterone plasma concentrations remained below the human castration limit for the same period.

Molecular targets for the protodynamic action of cis-urocanic acid in human bladder carcinoma cells.

BACKGROUND: cis-urocanic acid (cis-UCA) is an endogenous amino acid metabolite capable of transporting protons from the mildly acidic extracellular medium into the cell cytosol. The resulting intracellular acidification suppresses many cellular activities. The current study was aimed at characterizing the molecular mechanisms underlying cis-UCA-mediated cytotoxicity in cultured cancer cells. METHODS: 5367 bladder carcinoma cells were left untreated or treated with cis-UCA. Cell death was assessed by measuring caspase-3 activity, mitochondrial membrane polarization, formation and release of cytoplasmic histone-associated DNA fragments, and cellular permeabilization. Cell viability and metabolic activity were monitored by colorimetric assays. Nuclear labelling was used to quantify the effects of cis-UCA on cell cycle. The activity of the ERK and JNK signalling pathways was studied by immunoblotting with specific antibodies. Phosphatase activity in cis-UCA-treated cells was determined by assay kits measuring absorbance resulting from the dephosphorylation of an artificial substrate. All statistical analyses were performed using the two-way Student's t-test (p < 0.05). RESULTS: Here we report that treatment of the 5637 human bladder carcinoma cells with 2% cis-UCA induces both apoptotic and necrotic cell death. In addition, metabolic activity of the 5637 cells is rapidly impaired, and the cells arrest in cell cycle in response to cis-UCA. Importantly, we show that cis-UCA promotes the ERK and JNK signalling pathways by efficiently inhibiting the activity of serine/threonine and tyrosine phosphatases. CONCLUSIONS: Our studies elucidate how cis-UCA modulates several cellular processes, thereby inhibiting the proliferation and survival of bladder carcinoma cells. These anti-cancer effects make cis-UCA a potential candidate for the treatment of non-muscle invasive bladder carcinoma.

Protodynamic therapy for bladder cancer: in vitro results of a novel treatment concept.

OBJECTIVE: To present a novel treatment approach for urinary bladder cancer, protodynamic therapy, which comprises inhibition of cancer cell proliferation by intracellular acidification; cis-urocanic acid (cis-UCA) was investigated as a protodynamic drug in bladder cancer cell cultures and compared with conventional chemotherapeutic agents. MATERIALS AND METHODS: The moderately differentiated cell line 5637 and the poorly differentiated T24 cell line were exposed to cis-UCA for 0.25-2 h, and to epirubicin, doxorubicin, cisplatin and paclitaxel for 2 h, to simulate drug exposure on intravesical instillation. The combination of cis-UCA and chemotherapeutic agents was also studied. Cell viability was measured with a colorimetric assay. RESULTS: cis-UCA inhibited proliferation and suppressed the survival of cells at an extracellular pH pK(a2), as suggested by the protodynamic theory. cis-UCA caused dose-dependent, irreversible termination of cell proliferation. The number of viable surviving BC cells decreased by >85% with 2%cis-UCA (P < 0.001). Viable cells disappeared completely with 4% and 6%cis-UCA after a 2-h treatment, and by 90% with 6%cis-UCA within a 15-min exposure. These effects were associated with distinct morphological changes. The other drugs tested had a clearly lower effect on cell survival. Interestingly, when combined, cis-UCA markedly enhanced the cytotoxic effect of epirubicin. CONCLUSION: cis-UCA is a potent antiproliferative agent in bladder cancer cell cultures. As our previous non-clinical studies showed that cis-UCA is locally and systemically well tolerated, protodynamic therapy with cis-UCA is a promising intravesical treatment for bladder cancer.