Development of a novel physico-chemically and microbiologically stable oral solution of flecainide for pediatrics.

There is as yet no commercialized preparation for oral administration of flecainide acetate (FA) to children. In such cases, manipulation of commercial tablets is the usual practice in pharmacy services of hospitals and compounding pharmacies, to provide a suitable dosage form for this vulnerable pediatric population group. In this study, we have formulated FA as an oral solution, as an alternative to the suspension elaborated from commercial tablets. Due to this sensitivity of young patients, we have used the pure active pharmaceutical ingredient (API) and the lowest permitted levels of pediatric excipients. Despite being a highly soluble API, only one of the formulations appears as a transparent solution due to complete FA solubilization. The proposed formulation is physico-chemically and microbiologically stable and the mass and dose uniformity is appropriate for 30 days' storage at 25 °C.

Structure-Performance Relationships of Temperature-Responsive PLGA-PEG-PLGA Gels for Sustained Release of Bone Morphogenetic Protein-2.

PLGA (poly(lactic-co-glycolic) acid)-PEG (polyethylene glycol)-PLGA synthesis conditions have an impact on the physicochemical features of the copolymer and its usefulness as biomaterial. This study reports on an analysis of the composition and structural properties of PLGA-PEG-PLGA copolymers applying a variety of analytical techniques. Viscoelastic properties and particularly the temperature-responsive behavior of PLGA-PEG-PLGA showed a marked dependence on copolymer structural features. Physicochemical and biological properties, such as bioadhesion, biocompatibility and cell viability, of the raw copolymers and their gels were also evaluated. The most promising copolymer was chosen to formulate the osteoinductive protein bone morphogenetic protein-2 (125I-BMP-2), and the ability of its gels to sustain the release both in vitro and in vivo was monitored in situ using a gamma counter. In vitro diffusion studies were carried out using a bioinspired set-up that included a biorelevant receptor medium. In vivo release tests after implantation in a critical-size calvarial defect model showed an important burst, but then the release fitted well to the square-root kinetics. Importantly, the release rate constants recorded in vitro and in vivo matched each other suggesting close in vitro-in vivo correlation. Overall, the information gathered opens new perspectives in the biomedical application of these temperature-sensitive materials.

Formulation design of oral pediatric Acetazolamide suspension: dose uniformity and physico-chemical stability study.

CONTEXT: The formulation of an active pharmaceutical ingredient (API) as oral solution or suspension in pediatrics is a habitual practice, due to the non-existence of many commercialized medicines in pediatric doses. It is also the simplest way to prepare and administer them to this vulnerable population. The design of a formulation that assures the dose and the system stability depends on the physico-chemical properties of the API. OBJECTIVE: In this study, we formulate a class IV API, Acetazolamide (AZM) as suspension for oral administration to pediatric population. The suspension must comply attributes of quality, safety and efficacy for this route of administration. MATERIALS AND METHODS: We use simple compounding procedures, as well as fewer pure excipients, as recommended for children. Mass and uniformity content assays and physical and chemical stability studies were performed. To quantify the API an UPLC method was used. RESULTS AND DISCUSSION: We verified the physico-chemical stability of the suspensions and that they passed the mass test of the European Pharmacopeia (EP), but not the dose uniformity test. CONCLUSIONS: This reveals that AZM must be formulated as liquid forms with a more complex system of excipients (not usually indicated in pediatrics), or otherwise solid forms capable of assuring uniformity of mass and dose for every dosage unit.

Development of an ultra high performance liquid chromatography method for determining triamcinolone acetonide in hydrogels using the design of experiments/design space strategy in combination with process capability index.

An ultra high performance liquid chromatography method was developed and validated for the quantitation of triamcinolone acetonide in an injectable ophthalmic hydrogel to determine the contribution of analytical method error in the content uniformity measurement. During the development phase, the design of experiments/design space strategy was used. For this, the free R-program was used as a commercial software alternative, a fast efficient tool for data analysis. The process capability index was used to find the permitted level of variation for each factor and to define the design space. All these aspects were analyzed and discussed under different experimental conditions by the Monte Carlo simulation method. Second, a pre-study validation procedure was performed in accordance with the International Conference on Harmonization guidelines. The validated method was applied for the determination of uniformity of dosage units and the reasons for variability (inhomogeneity and the analytical method error) were analyzed based on the overall uncertainty.

Anti-tumor effects of adenovirus containing human growth hormone sequences in a mouse model of human ovarian cancer.

Women with ovarian cancer have a low survival rate and develop resistance to chemotherapy, so new approaches to treatment are needed. We unexpectedly found administration of a replication-deficient adenovirus containing human growth hormone sequences (AdXGH) was beneficial in a mouse model of human ovarian cancer. Intraperitoneal injections of AdXGH prolonged median survival from a mean of 31 ± 1.2 to 40 ± 1.4 days in immunodeficient SCID mice given SKOV3.ip1 human ovarian cancer cells in the peritoneal cavity. Adenovirus containing human prolactin or del32-71growth hormone sequences had no effect. Repeated injection of growth hormone or implantation of tablets with sustained growth hormone release did not increase survival. Control mice had overlapping tumors throughout the peritoneal cavity and liver and frequent lung metastases 24 days after tumor cell injection. Mice that received two injections of AdXGH had no lung metastases. Mice that received four injections had no lung or liver metastases and peritoneal fibrosis. They did not survive longer than mice that received two injections, but they had enlarged livers with hepatocellular changes, indicating that a limitation of increasing the dose is liver toxicity.

Pharmacokinetics analysis of sustained release hGH biodegradable implantable tablets using a mouse model of human ovarian cancer.

This paper presents the pharmacokinetic of human growth hormone (hGH) implantable tablets tested on a human ovarian cancer mouse model. In order to obtain a sustained release device which permits to administer a high dose of the hormone that keeps its integrity and stability, three different formulations of hGH-poly (d,l-lactic-co-glycolic acid) (PLGA) were elaborated by direct compression method varying hormone load, PLGA content and compactation time. In vitro studies showed that drug release was mainly controlled by hormone load. Pharmacokinetic studies were conducted by using immunodeficient female mice. Four days before the insertion of hGH implantable tablets in the peritoneal cavity, every mouse received 5x10(6) human ovarian cancer cells (SKOV3.ip1). Hormone serum levels were monitored through bleeding from eye orbital vessels. The population pharmacokinetic model used was based on the in series tank model and model parameters were estimated using the maximum likelihood method. The null hypothesis test about differences between formulations leads us to the conclusion that the three formulations showed the same kinetic behavior except for the hGH load. The hormone release was extended all over 2 weeks but no increase or decrease in survival time was observed. These results suggest that hGH serum levels do not facilitate tumoral cells proliferation, an expected effect of hGH and this could explain why survival times of mice treated with implantable tablets are not shorter than those treated with the control ones.

In vitro and in vivo gene transfer with poly(amino acid) vesicles.

Non-viral gene delivery systems utilise either amine lipids or polyamines and although non-viral gene delivery systems are said to have a superior safety profile to viruses, the polyamines such as poly(L-lysine) are toxic when used without derivatisation and usually require specific receptor mediated uptake and/or endosomolytic agents to be effective. However, the conversion of poly(L-lysine) and poly(L-ornithine) polyamino acids into amphiphilic vesicle forming polymers reduces the toxicity of the polyamino acids and enables the resulting polyamino acid vesicles to deliver genes both in vitro and in vivo in the absence of receptor specific ligands and endosomolytic agents. The incorporation of a distearoylphosphatidylethanolamine poly(ethylene glycol)-galactosamine conjugate (with the galactosamine unit at the distal end of the poly(ethylene glycol) moiety) into the polyamino acid formulations improved in vitro gene transfer in the case of the amphiphilic poly(L-ornithine) (POP) although no in vivo targeting was detected with the galactosamine formulations. We conclude that the conversion of poly(L-lysine) and poly(L-ornithine) into amphiphilic colloid forming molecules reduces their toxicity, thus allowing these systems to be used for gene transfer in vivo. It is possible that this approach may be extended to other polyamines.

Effect of high shear rate on stability of proteins: kinetic study.

Size-exclusion chromatography (SEC) was used to monitor the time-course of protein degradation induced by high shear rates during the formulation and manufacture of controlled-release pharmaceutical dosage forms. SEC with multi-angle laser light-scattering (MALLS) detection was used to characterize the aggregation products, determining their absolute molecular weight. A stability-indicating method was developed and validated to obtain reliable drug degradation data. The results obtained according to the ICH guidelines confirm that the system and methods proposed are suitable for their intended use. The degradation kinetics are influenced by the type of protein and the effect of the shear rate on their stability. Reversible pseudo-first order degradation kinetics were observed for bovine beta-lactoglobulin, whereas for human (HSA) and bovine serum albumin (BSA), a monomer-dimer transition was observed, independently of the rate of shear. However, trimer formation was also observed for HSA, especially at high shear rates. The kinetic model may thus be described as a two-step process: a monomer-dimer, and dimer-trimer transition.

Potential applications of PLGA film-implants in modulating in vitro drugs release.

In this work we evaluate poly(lactic/glycolic) acid (PLGA) film-implants as potential biodegradable devices for controlled release of two different drugs: 5-Fluorouridine (5-FUR), a conventional low molecular weight water-soluble compound and SPf66 malaria vaccine, a therapeutic synthetic polypeptide. Three types of devices were prepared by solvent-casting techniques alone or combined with compression method: simple monolithic discs (SMD), multilayer discs with a central monolithic layer (MLDM), and multilayer discs with a central drug-reservoir (MLDR). For the highly water-soluble drug, 5-FUR, in vitro release from SMD showed an initial burst (24% in 2 h) followed by prolonged release over 20 days. In contrast, from a MLDM (two drug-free PLGA discs were added to the SMD) showed an initial lag-time of 12 days followed by a very fast second release phase. Finally, when the load of this system was increased from 3 to 9%, an extended release over 20 days with a low burst effect was obtained. For SPf66, the central reservoir containing the synthetic polypeptide MLDR reduces the possibility of degradation due to peptide contact with polymer solution. When four layers were added, 10 days sustained-release was obtained without any burst effect. With six layers a moderate pulse was obtained, 18-22 days from the beginning of the release. The results show the suitability of the proposed devices to control release and avoid the burst effect with highly water-soluble drugs; as well as modulate in vitro peptide release.

Characterization of antimalarial SPf66 peptide using MALDI-TOF MS, CD and SEC.

SPf66 is the first chemically synthesized peptide to elicit a partial protective immune response against malaria. Size-exclusion chromatography (SEC) with multi-angle laser light-scattering (MALLS) detection and hydrogen/deuterium (H/D) exchange monitored by (matrix-assisted laser desorption/ionization) MALDI-TOF (time-of-flight) mass spectrometry (MS) were used to assess the conformation and stability in aqueous solution after storage at different temperatures. Moreover, the feasible conformational changes of this peptide were also measured by circular dichroism (CD)-spectroscopy. The absolute molecular weight of SPf66 monomer and dimer species were 4765 and 8960Da using SEC with MALLS detection, and 4643 and 9490Da by MALDI-TOF MS, the discrepancy being between both methods lower than 5.7%, a value quite close to those found in other proteins. The results from H/D exchange monitored by MALDI-TOF MS and CD-spectroscopy show that the SPf66 monomer lacks ordered structure, whereas the SPf66 dimer species presents segments of secondary structure as a determined by CD measurements.

Chromatographic characterization of synthetic peptides: SPf66 malaria vaccine.

The development and validation of a quantitative size-exclusion chromatography (SEC) method for SPf66 malaria vaccine was achieved. The results show the reliability of the analytical method for the intended use. SPf66 malaria vaccine characterization was perforrmed using both relative techniques such as the conventional SEC and absolute techniques: mass spectrometry and multi-angle laser-light scattering detection. The relative and absolute molecular masses were in the 4600-18,000 Da range. The results clearly indicate the presence of the monomer and dimer species, whereas the third species could be the trimer or tetramer.