On the Production of Chitosan-Coated Polycaprolactone Nanoparticles in a Confined Impinging Jet Reactor

This work is focused on the synthesis of polycaprolactone nanoparticles, coated with chitosan, in a confined impinging jet reactor using the solvent displacement method. The role of the various reacting species was investigated, evidencing that a biocompatible polymer, for example, polycaprolactone, is required to support chitosan to obtain a monomodal particle size distribution, with low particle diameters. A surfactant is required to reduce the nanoparticle size (down to a mean diameter of about 260 nm) and obtain a positive zeta potential (about +31 mV), perfectly suitable for pharmaceutical applications. Different surfactants were tested, and Poloxamer 388 appeared to be preferable to polyvinyl alcohol. The effect of the concentration of Poloxamer 388 (in the range 0.5-5 mg mL^?1) and of chitosan (in the range 1.5-5 mg mL^?1) on both the mean particle size and zeta potential was also investigated, evidencing that chitosan concentration has the strongest effect on both parameters. Finally, the effect of solvent evaporation, quenching and feed flow rate was investigated, showing that the evaporation stage does not affect particle characteristics, quenching is required to avoid particle aggregation, and a minimum liquid flow rate of 80 mL min^?1 is required in the considered reactor to minimize the particle size.     

Long-Acting Profile of 4 Drugs in 1 Anti-HIV Nanosuspension in Nonhuman Primates for 5 Weeks After a Single Subcutaneous Injection

Daily oral antiretroviral therapy regimens produce limited drug exposure in tissues where residual HIV persists and suffer from poor patient adherence and disparate drug kinetics, which all negatively impact outcomes. To address this, we developed a tissue- and cell-targeted long-acting 4-in-1 nanosuspension composed of lopinavir (LPV), ritonavir, tenofovir (TFV), and lamivudine (3TC). In 4 macaques dosed subcutaneously, drug levels over 5 weeks in plasma, lymph node mononuclear cells (LNMCs), and peripheral blood mononuclear cells (PBMCs) were analyzed by liquid chromatography–tandem mass spectrometry. Plasma and PBMC levels of the active drugs (LPV, TFV, and 3TC) were sustained for 5 weeks; PBMC exposures to LPV, ritonavir, and 3TC were 12-, 16-, 42-fold higher than those in plasma. Apparent T_1/2z of LPV, TFV, and 3TC were 219.1, 63.1, and 136.3 h in plasma; 1045.7, 105.9, and 127.7 h in PBMCs. At day 8, LPV, TFV, and 3TC levels in LNMCs were 4.1-, 5.0-, and 1.9-fold higher than in those in PBMCs and much higher than in plasma. Therefore, 1 dose of a 4-drug nanosuspension exhibited persistent drug levels in LNMCs, PBMCs, and plasma for 5 weeks. With interspecies scaling and dose adjustment, this 4-in-1 HIV drug-combination could be a long-acting treatment with the potential to target residual virus in tissues and improve patient adherence.     

Coformulation of Broadly Neutralizing Antibodies 3BNC117 and PGT121: Analytical Challenges During Preformulation Characterization and Storage Stability Studies

In this study, we investigated analytical challenges associated with the formulation of 2 anti-HIV broadly neutralizing antibodies (bnAbs), 3BNC117 and PGT121, both separately at 100 mg/mL and together at 50 mg/mL each. The bnAb formulations were characterized for relative solubility and conformational stability followed by accelerated and real-time stability studies. Although the bnAbs were stable during 4°C storage, incubation at 40°C differentiated their stability profiles. Specific concentration-dependent aggregation rates at 30°C and 40°C were measured by size exclusion chromatography for the individual bnAbs with the mixture showing intermediate behavior. Interestingly, although the relative ratio of the 2 bnAbs remained constant at 4°C, the ratio of 3BNC117 to PGT121 increased in the dimer that formed during storage at 40°C. A mass spectrometry-based multiattribute method, identified and quantified differences in modifications of the Fab regions for each bnAb within the mixture including clipping, oxidation, deamidation, and isomerization sites. Each bnAb showed slight differences in the levels and sites of lysine residue glycations. Together, these data demonstrate the ability to differentiate degradation products from individual antibodies within the bnAb mixture, and that degradation rates are influenced not only by the individual bnAb concentrations but also by the mixture concentration.     

Intradermal Delivery of a Near-Infrared Photosensitizer Using Dissolving Microneedle Arrays

Nodular basal cell carcinoma is a deep skin lesion and one of the most common cancers. Conventional photodynamic therapy is limited to treatment of superficial skin lesions. The parenteral administration of near-IR preformed photosensitizers suffers from poor selectivity and may result in prolonged skin photosensitivity. Microneedles (MNs) can provide localized drug delivery to skin lesions. Intradermal delivery of the preformed near-IR photosensitizer; 5,10,15,20-tetrakis(2,6-difluoro-3-N-methylsulfamoylphenyl bacteriochlorin (Redaporfin?) using dissolving MN was successful in vitro and in vivo. MN demonstrated complete dissolution 30 min after skin application and showed sufficient mechanical strength to penetrate the skin to a depth of 450 μm. In vitro deposition studies illustrated that the drug was delivered and detected down to 5 mm in skin. In vivo biodistribution studies in athymic nude mice Crl:NU(NCr)-Foxn1^nu showed both fast initial release and localized drug delivery. The MN-treated mice showed a progressive decrease in the fluorescence intensity at the application site over the 7-day experiment period, with the highest and lowest fluorescence intensities measured being 9.2 × 10¹⁰ ± 2.5 × 10¹⁰ and 3.8 × 10⁹ ± 1.6 × 10⁹ p/s, respectively. By day 7, there was some migration of fluorescence away from the site of initial MN application. However, the majority of the body surfaces showed fluorescence levels that were comparable to those seen in the negative control group. This work suggests utility for polymeric MN arrays in minimally invasive intradermal delivery to enhance photodynamic therapy of deep skin lesions.     

Dibucaine in Ionic-Gradient Liposomes: Biophysical,Toxicological, and Activity Characterization

Administration of local anesthetics is one of the most effective pain control techniques for postoperative analgesia. However, anesthetic agents easily diffuse into the injection site, limiting the time of anesthesia. One approach to prolong analgesia is to entrap local anesthetic agents in nanostructured carriers (e.g., liposomes). Here, we report that using an ammonium sulphate gradient was the best strategy to improve the encapsulation (62.6%) of dibucaine (DBC) into liposomes. Light scattering and nanotracking analyses were used to characterize vesicle properties, such as, size, polydispersity, zeta potentials, and number. In vitro kinetic experiments revealed the sustained release of DBC (50% in 7 h) from the liposomes. In addition, in vitro (3T3 cells in culture) and in vivo (zebrafish) toxicity assays revealed that ionic-gradient liposomes were able to reduce DBC cyto/cardiotoxicity and morphological changes in zebrafish larvae. Moreover, the anesthesia time attained after infiltrative administration in mice was longer with encapsulated DBC (27 h) than that with free DBC (11 h), at 320 μM (0.012%), confirming it as a promising long-acting liposome formulation for parenteral drug administration of DBC.     

Preclinical Development of Cell-Based Products: a European Regulatory Science Perspective

Purpose

This article describes preclinical development of cell-based medicinal products for European markets and discusses European regulatory mechanisms open to developers to aid successful product development. Cell-based medicinal products are diverse, including cells that are autologous or allogeneic, have been genetically modified, or not, or expanded ex vivo, and applied systemically or to an anatomical site different to that of their origin; comments applicable to one product may not be applicable to others, so bespoke development is needed, for all elements - quality, preclinical and clinical.

Methods

After establishing how the product is produced, proof of potential for therapeutic efficacy, and then safety, of the product need to be determined. This includes understanding biodistribution, persistence and toxicity, including potential for malignant transformation. These elements need to be considered in the context of the intended clinical development.

Results

This article describes regulatory mechanisms available to developers to support product development that aim to resolve scientific issues prior to marketing authorization application, to enable patients to have faster access to the product than would otherwise be the case.

Conclusions

Developers are encouraged to be aware of both the scientific issues and regulatory mechanisms to ensure patients can be supplied with these products.

     

Empirical treatment of lower urinary tract infections in the face of spreading multidrug resistance: in vitro study on the effectiveness of nitroxoline

The spread of antimicrobial resistance challenges the empirical treatment of urinary tract infections (UTIs). Among others, nitrofurantoin is recommended for first-line treatment, but acceptance among clinicians is limited due to chronic nitrofurantoin-induced lung toxicity and insufficient coverage of Enterobacteriaceae other than Escherichia coli. Nitroxoline appears to be an alternative to nitrofurantoin owing to its favourable safety profile, however data on its current in vitro susceptibility are sparse. In this study, susceptibility to nitroxoline was tested against 3012 urinary clinical isolates (including multidrug-resistant bacteria and Candida spp.) by disk diffusion test and/or broth microdilution. At least 91% of all Gram-negatives (n?=?2000), Gram-positives (n?=?403) and yeasts (n?=?132) had inhibition zone diameters for nitroxoline ≥18?mm. Except for Pseudomonas aeruginosa, nitroxoline MIC₉₀ values were ≤16?mg/L and were 2- to >16-fold lower compared with nitrofurantoin. In extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA), MIC₉₀ values of nitroxoline were two-fold higher compared with non-ESBL-producing enterobacteria and methicillin-susceptible S. aureus (MSSA). The in vitro efficacies of nitroxoline and nitrofurantoin against ATCC strains of E. coli, Enterococcus faecalis and Proteus mirabilis were compared by time–kill curves in Mueller–Hinton broth and artificial urine. Nitroxoline was non-inferior against E. coli, P. mirabilis and E. faecalis in artificial urine. In conclusion, nitroxoline showed a broad antimicrobial spectrum, with inhibition zone diameters and MICs of nitroxoline well below the EUCAST breakpoint for E. coli for most organisms, and thus may also be a target for therapy of uncomplicated UTIs.