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.

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.

Silica microparticles for sustained zero-order release of an anti-CD40L antibody.

Silica microparticle hydrogel depot (HG) formulation was prepared using spray drying of silica-based sol-gels for the sustained delivery of MR1 antibody which binds to CD40 ligand (CD40L). The formulation was tested in vitro for antibody release, surface morphology, particle size, rheology, and injectability. In vivo pharmacokinetic evaluation was performed for the microparticle formulation and free MR1 antibody in BALB/c female mice. Serum samples up to day 62 were assessed using an enzyme-linked immunosorbent assay. In vitro release indicated that the MR1 antibody was uniformly encapsulated in silica microparticles, and less than 5% burst release of the antibody was observed. In vivo pharmacokinetics showed a zero-order release up to 62 days from the MR1 silica microparticle HG-controlled release composition.

The effects of a HTR2B stop codon and testosterone on energy metabolism and beta cell function among antisocial Finnish males.

Herein, we examined insulin resistance (IR), insulin sensitivity (IS), beta cell activity, and glucose metabolism in subjects with antisocial personality disorder (ASPD), and whether the serotonin 2B (5-HT2B) receptor and testosterone have a role in energy metabolism. A cohort of subjects belonging to a founder population that included 98 ASPD males, aged 25-30, was divided into groups based on the presence of a heterozygous 5-HT2B receptor loss-of-function gene mutation (HTR2B Q20*; n = 9) or not (n = 89). Serum glucose and insulin levels were measured in a 5 h oral glucose tolerance test (75 g) and indices describing IR, IS, and beta cell activity were calculated. Body mass index (BMI) was also determined. Concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid were measured in cerebrospinal fluid, and testosterone levels from serum. An IR-like state comprising high IR, low IS, and high beta cell activity indices was observed among ASPD subjects without the HTR2B Q20* allele. By contrast, being an ASPD HTR2B Q20* carrier appeared to be preventive of these pathophysiologies. The HTR2B Q20* allele and testosterone predicted lower BMI independently, but an interaction between HTR2B Q20* and testosterone lead to increased insulin sensitivity among HTR2B Q20* carriers with low testosterone levels. The HTR2B Q20* allele also predicted reduced beta cell activity and enhanced glucose metabolism. Reduced 5-HT2B receptor function at low or normal testosterone levels may be protective of obesity. Results were observed among Finnish males having an antisocial personality disorder, which limits the generality.

Effects of capsid-modified oncolytic adenoviruses and their combinations with gemcitabine or silica gel on pancreatic cancer.

Conventional cancer treatments often have little impact on the course of advanced pancreatic cancer. Although cancer gene therapy with adenoviruses is a promising developmental approach, the primary receptor is poorly expressed in pancreatic cancers which might compromise efficacy and thus targeting to other receptors could be beneficial. Extended stealth delivery, combination with standard chemotherapy or circumvention of host antiadenoviral immune response might improve efficacy further. In this work, capsid-modified adenoviruses were studied for transduction of cell lines and clinical normal and tumor tissue samples. The respective oncolytic viruses were tested for oncolytic activity in vitro and in vivo. Survival was studied in a peritoneally disseminated pancreas cancer model, with or without concurrent gemcitabine while silica implants were utilized for extended intraperitoneal virus delivery. Immunocompetent mice and Syrian hamsters were used to study the effect of silica mediated delivery on antiviral immune responses and subsequent in vivo gene delivery. Capsid modifications selectively enhanced gene transfer to malignant pancreatic cancer cell lines and clinical samples. The respective oncolytic viruses resulted in increased cell killing in vitro, which translated into a survival benefit in mice. Early proinfammatory cytokine responses and formation of antiviral neutralizing antibodies was partially avoided with silica implants. The implant also shielded the virus from pre-existing neutralizing antibodies, while increasing the pancreas/liver gene delivery ratio six-fold. In conclusion, capsid modified adenoviruses would be useful for testing in pancreatic cancer trials. Silica implants might increase the safety and efficacy of the approach.

Intraepidermal nerve fibre density in cancer patients receiving adjuvant chemotherapy.

BACKGROUND: Chemotherapy-induced neuropathy is a common adverse event in patients receiving vinca alcaloids, platinum derivatives and taxanes. However, the underlying pathogenetic mechanisms have not been completely elucidated. We set up a prospective pilot study on skin biopsies in newly diagnosed cancer patients receiving neurotoxic chemotherapeutic agents as adjuvant treatment in order to study the occurrence of small-fibre pathology and its relationship to clinical symptoms. PATIENTS AND METHODS: Skin biopsies from distal leg were performed in 12 patients before, during and after chemotherapy. Using light microscopy, the intraepidermal nerve fibre (IENF) density was determined from the skin biopsies by counting morphometrically the immunopositive nerves per epidermal area. RESULTS: Reduced IENF density was observed in eight patients at baseline. During the follow-up, the IENF density increased significantly in six patients and remained unchanged in two. In four patients, the IENF density was normal both at baseline and at the end of the follow-up period. Neuropathic symptoms were manifested in nine patients, but no association with the IENF count was found. CONCLUSION: During chemotherapy, results from patients revealed different evolutionary patterns of IENF density, but symptoms and IENF density were not related.

Widespread unilateral pain associated with herpes simplex virus infections.

UNLABELLED: We describe a patient group with unexplained widespread pain on one side of the body and pain exacerbations during active labial or genital herpes and during herpetic central nervous system infections. The patients had no visible lesion of the central nervous system on magnetic resonance imaging or abnormality in electrophysiological studies. To understand the nature of the pain and its possible relation to herpes simplex virus (HSV) infections, a clinical neurological examination was performed and quantitative sensory testing and skin biopsies were assessed in 17 patients. The levels of serum total immunoglobulins and IgG subclasses and the frequencies of the immune response genes at the IGH@, HLA-A, -B, -DRB1, C4A, and C4B loci were analyzed in the patients and in control subjects. The patients manifested a uniform clinical syndrome with unilateral pain that was best described as neuropathic and that was exacerbated by HSV reactivations. Low plasma IgG3 concentrations, the presence of either low plasma IgG1 or IgG3 or both, and high anti-HSV-2-IgG titers were more common in the patients than in the control subjects, which rendered the patients more vulnerable to HSV recurrences. PERSPECTIVE: We suggest that low immunoglobulin subclass levels and certain MHC alleles render the patients susceptible to recurring HSV infections. HSV reactivations and the accompanying inflammatory process cause dysfunction of the central nervous system that manifests as neuropathic pain. Studies using functional brain imaging are needed to clarify this syndrome.

Histopathological and clinical findings in leprosy patients with chronic neuropathic pain: a study from Hyderabad, India.

BACKGROUND: Chronic neuropathic pain in leprosy patients after completion of multi-drug therapy (MDT) is an under-researched problem. The reason why some leprosy patients develop it is unknown. In this study we evaluated the role of ongoing inflammation and small-fibre neuropathy as possible contributing factors for neuropathic pain. METHODS: We assessed chronic neuropathic pain in 17 leprosy patients who had completed MDT and were attending a referral clinic in Hyderabad, India. All patients had a clinical assessment, intraepidermal nerve (IENF) assessment and quantitative sensory testing (QST), which included the testing of tactile and pinprick sensations using Semmes-Weinstein monofilaments and weighted needles method. Nine patients had a sural nerve biopsy (SNB). RESULTS: Thirteen patients had a glove and stocking pattern of neuropathy. All nerve biopsies showed inflammation with intraneural inflammation and perineural thickening, and intraneural acid fast bacilli were observed in five biopsies. IENF analysis of the skin biopsy specimens in 16/17 patients showed a statistically significant reduction in IENF density (P < 0.001, Mann Whitney test) compared to control skin biopsies. Complete depletion of intraepidermal nerves was observed in six patients. QST also showed marked abnormalities. In 11 patients total sensory loss for all modalities was found, and in the other six patients the sensory function was seriously impaired. DISCUSSION: There is evidence of ongoing intraneural inflammation in leprosy patients who have completed MDT. This may explain the occurrence of chronic neuropathic pain. Using IENF density measurement we have found significant small-fibre neuropathy in leprosy patients and the use of this tool could be expanded.

A quantitative method for the assessment of intraepidermal nerve fibers in small-fiber neuropathy.

OBJECTIVES: The purpose of this paper is to present an easy-to-use and reproducible morphometrical method of determining the density of intraepidermal nerve fibers (IENF) per epidermal area with the corresponding reference range of the IENF-counts. METHODS: Thirty patients and 22 controls were included in this study. The patients were divided into three groups: small-fiber (SFN), diabetic and demyelinating neuropathy. All subjects underwent punch skin biopsy. Specimens were fixed routinely in formalin and thereafter embedded in paraffin. Nerve fibers were revealed using immunoperoxidase staining with panaxonal antibody PGP 9.5. Using light microscopy, immunopositive nerves were counted morphometrically per epidermal area (NPEA) and, for comparison, per epidermal length (NPEL). RESULTS: Both the NPEA and NPEL estimates of SFN and diabetic neuropathy group differed significantly from those of control specimen (p < 0.001 and p < 0.001, Mann-Whitney test). Our method of counting, NPEA, shows a good correlation to NPEL (r = 0.945). CONCLUSIONS: IENF-counting by a new morphometric modification is reproducible and diagnostically sensitive and can easily be adopted in any laboratory familiar with the basic immunohistochemical methodology. The method is less dependent on costly technical support systems and seems to be less time consuming when compared with conventional methods for IENF-counting.

Heat curing of UTMA-based hybrid resin: effects on the degree of conversion and cytotoxicity.

This study was designed to determine the effects of the heat curing time on a urethane tetramethacrylate (UTMA)-based hybrid resin and specifically on the degree of conversion (DC) and cytotoxicity. The materials used in this study were Estenia, a new-generation hybrid resin, and an experimental fiber reinforcement, Br-100. The DC values of the hybrid resin samples were measured using a Fourier transform infrared (FTIR) spectrophotometer after 180 s of light curing followed by heat curing (0, 15, 30, and 60 min). A method comparing intensities of C=C and N-H vibrations of the sample was used to calculate the final DC values. FTIR spectra were measured both inside and on the surface of the sample. The calculated DC values increased by increasing the heat curing times. After light curing only and after 15-min heat curing, the DC values inside the samples were smaller than the corresponding DC values at the surfaces of the samples. After 60 min of heat curing, the samples achieved homogeneous polymerization (DC% = 65). The cytotoxicity of the material was studied from the glass fiber-reinforced hybrid resin samples, which were first light cured and then heat cured (15, 30, and 60 min). Cytotoxicity was tested using both direct contact and extract methods. For the extract tests, the test specimens were incubated in a cell culture media at 37 degrees , 54 degrees , or 72 degrees C for 24 h. The heat curing times used had no effect on cytotoxicity. The incubation temperature, however, did have a significant effect. The extract obtained from 72 degrees C incubation showed a cytotoxic effect whereas the others did not. The direct contact test did not show cytotoxicity.