In vivo screening of subcutaneous tolerability for the development of novel excipients.

To develop safe subcutaneous formulations and minimize the risk of local irritation, it is essential to optimize the composition of active pharmaceutical ingredients and excipients. Depending on the physicochemical properties of the active pharmaceutical ingredient, additional excipients may be required to improve the stability and solubility of the active pharmaceutical ingredient. However, some of these excipients may not have been previously used in injectable drugs. Owing to the lack of safety data for such excipients, especially those used in subcutaneous dosing, it is important to evaluate their potential for local irritation during the early stages of formulation development. We evaluated the tolerability of 44 formulations with 24 candidate novel excipients, such as surfactants, polymers, and lipids, in a single subcutaneous dose in rats. Excipient formulations were administered as single bolus subcutaneous injections with an injection volume of 1 mL. The injection sites were observed for 2 days, and macroscopic and microscopic examinations were conducted. Local tolerability was evaluated on the basis of severity, incidence, and pathophysiology of each finding. Formulations that caused tissue degeneration or necrosis, which is indicative of tissue injury, were determined to be irritative and poorly tolerated. A single-dose subcutaneous screening study in rats was considered effective in ranking the safety of candidate excipients during the formulation optimization phase.

Effects of Tissue Pressure on Transgene Expression Characteristics via Renal Local Administration Routes from Ureter or Renal Artery in the Rat Kidney.

We previously developed a renal pressure-mediated transfection method (renal pressure method) as a kidney-specific in vivo gene delivery system. However, additional information on selecting other injection routes and applicable animals remains unclear. In this study, we selected renal arterial and ureteral injections as local administration routes and evaluated the characteristics of gene delivery such as efficacy, safety, and distribution in pressured kidney of rat. Immediately after the naked pDNA injection, via renal artery or ureter, the left kidney of the rat was pressured using a pressure controlling device. Transfection efficiency of the pressured kidney was about 100-fold higher than that of the injection only group in both administration routes. The optimal pressure intensity in the rat kidney was 1.2 N/cm2 for renal arterial injection and 0.9 N/cm2 for ureteral injection. We found that transgene expression site differs according to administration route: cortical fibroblasts and renal tubule in renal arterial injection and cortical and medullary tubule and medullary collecting duct in ureteral injection. This is the first report to demonstrate that the renal pressure method can also be effective, after renal arterial and ureteral injections, in rat kidney.

Application of Three-Dimensionally Printed Probe and Reservoir to Critical Micelle Concentration Determination by Microvolume Surface Tension Measurement.

It is important to determine a critical micelle concentration (CMC) of a surfactant in a protein formulation for stabilizing the protein at maximum by preventing it from interfacial denaturation. There are several techniques for CMC determination. Among them, surface tensiometry is the most common approach because this has a long history and much data at many research fields. However, large amount of sample solution is usually required for the measurement (e.g., more than 1 mL is necessary when a standard reservoir like a glass petri dish is used). This is one of the hurdles for protein formulators because only a small amount of protein could be used at the early-stage development. In this research, we tried to minimize the required amount of sample solution for surface tension measurement by developing appropriate probe and reservoir using a three-dimensional printer (3D printer). The advantages and capabilities of 3D printer are (1) to control the shape and size of the printed material precisely, (2) to change the figure freely, and (3) to prepare the prototype quickly. After the experiments and thereby the refinement of probe as well as reservoir, we found that CMCs of polysorbate 20, polysorbate 80, and poloxamer 188 in water and protein formulations could be precisely detected using a probe 0.5 mm in diameter and small reservoir with a pocket of 7.5 mm in diameter/0.25 mm in depth which were made by a 3D printer. Furthermore, the required sample solution per each measurement could be reduced to 80 µL, which means more than 90% reduction against a standard reservoir.

X-ray and neutron protein crystallographic analysis of the trypsin-BPTI complex.

In this work, the crystal structure of the ß-trypsin-bovine pancreatic trypsin inhibitor (BPTI) complex was refined and the D and H atoms in the complex were identified using data from both 1.6 Šresolution X-ray diffraction and 2.15 Šresolution neutron diffraction. After crystallization in an H(2)O solution, the sample crystal was soaked in a D(2)O solution for about two weeks. The protonation states of the catalytic triad (Asp102, His57 and Ser195) were observed. These results confirmed that the nucleophilic reactivity of the hydroxyl group of Ser195 was increased by forming a hydrogen bond with His57. According to structural analysis, the trypsin-BPTI interfaces located at the scissile peptide and the active sites were inaccessible to solvent water, and the amide H atoms of P2' Arg17/I, Gly216/E and Gly193/E at the binding interface were protected from H/D exchange. In contrast, both the amide H atom of P1' Ala16/I of the scissile peptide bond P1-P1' and the H atom between His57 N(ℇ2) and Ser195 O(γ) were replaced by D atoms. The hydrogen-bond networks at the S1 pocket were also confirmed and discussed from the viewpoint of substrate recognition. Moreover, the first neutron crystallographic structure of the Michaelis complex state of trypsin-BPTI is presented.

Key physiological phenomena governing transgene expression based on tissue pressure-mediated transfection in mice.

It is generally recognized that in vivo gene transfection is one of the most important techniques used in the post-genome era. Above all, naked plasmid DNA transfection has attracted much attention because of its advantages including convenience of preparation and handling and lack of toxicity associated with the transfection agents. We have investigated tissue pressure-mediated transfection performed by light and controlled pressure of the target tissue after normal intravenous injection of plasmid DNA. So far, we have demonstrated that plasmid DNA and small-interfering RNA (siRNA) are very efficiently transfected into murine kidney, liver and spleen without causing marked tissue damage. In this study, in order to understand the key physiological phenomena affecting transgene expression, we performed a set of experiments involving tissue pressure-mediated transfection, including the biodistribution and cellular transport of plasmid DNA and activation of transcriptional factors and obtained the following results: i) plasmid DNA transfer to the target tissue and its cells increased although the transferred fraction was small compared to the total administered plasmid DNA, ii) a transient increase in cellular translocation of plasmid DNA was induced, and iii) transcriptional factors were activated. Taking all these results into consideration, it would appear that tissue pressure-mediated transfection enhances plasmid DNA transfer to the target tissue and its cells and also activation of the transcriptional process. This information will allow a better understanding of in vivo transgene expression based on naked plasmid DNA transfection involving tissue pressure-mediated transfection.

Temperature dependence of intermediate-range orders in the viscosity-temperature relationship of supercooled liquids and glasses.

The viscosity-temperature relationship obtained by us for several glasses over a wide temperature range was analyzed by extending the Adam-Gibbs theory to the range below the glass transition temperature (T(g)). The entropy change of the intermediate-range orders (IROs) is discussed on the basis of the theory developed by Prigogine. It is estimated that the time dependence of the vibrational entropy of a glass shows a constant decrease with a smallest change, while that of its configurational entropy is 0, keeping the constant fictive temperature and the isostructural state. The result predicts the decrease of the volume of a glass at the constant time-rate through spontaneous aging at the constant temperature. We also show that the glass transition is a phase transition from an equilibrium Vogel-Fulcher-Tamman state to a nonequilibrium and (meta-)stable Arrhenius state through fluctuations at T(g), and a microscopic feature of the glass transition is the self-organization of the IROs. These findings are extremely useful in analyzing glass and nanomaterial productions because the size of the IROs in the glass state is a few nanometers.

A neutron crystallographic analysis of T6 porcine insulin at 2.1 A resolution.

Neutron diffraction data for T(6) porcine insulin were collected to 2.1 A resolution from a single crystal partly deuterated by exchange of mother liquor. A maximum-likelihood structure refinement was undertaken using the neutron data and the structure was refined to a residual of 0.179. The hydrogen-bonding network of the central core of the hexamer was observed and the charge balance between positively charged Zn ions and their surrounding structure was interpreted by considering the protonation and/or deprotonation states and interactions of HisB10, water and GluB13. The observed double conformation of GluB13 was essential to interpreting the charge balance and could be compared with the structure of a dried crystal of T(6) human insulin at 100 K. Differences in the dynamic behaviour of the water molecules coordinating the upper and lower Zn ions were observed and interpreted. The hydrogen bonds in the insulin molecules, as well as those involving HisB10 and GluB13, are discussed. The hydrogen/deuterium (H/D) exchange ratios of the amide H atoms of T(6) porcine insulin in crystals were obtained and showed that regions highly protected from H/D exchange are concentrated in the centre of a helical region of the B chains. From the viewpoint of soaking time versus H/D-exchange ratios, the amide H atoms can be classified into three categories.

Pressure-mediated transfection of murine spleen and liver.

Extension of in vivo nucleic acid transfection techniques and increased information about those transfection properties and side effects are urgently needed to advance biological research and drug therapy. Tissue pressure-mediated transfection, involving lightly pressing the target tissue after intravenous injection of plasmid DNA or small-interfering RNA (siRNA), is a promising approach because of its high transfection efficiency and resulting low tissue damage. In this study, the gene expression/silencing properties and proinflammatory cytokine production associated with tissue pressure-mediated transfection were evaluated to extend its application. We have found that tissue pressure-mediated transfection can be applied to plasmid DNA and siRNA transfection to the spleen and siRNA transfection to the liver. In addition, we have demonstrated that these methods induce little production of proinflammatory cytokines, including tumor necrosis factor-alpha, interleukin (IL)-6, IL-12, and interferon-gamma. Moreover, we succeeded in controlling and quantifying the degree of pressure on the spleen and kidney and found that 0.59 N/cm(2) is sufficient for efficient and highly reproducible plasmid DNA transfection to the spleen and kidney in mice. Tissue pressure-mediated transfection of the kidney, liver, and spleen exhibits well-balanced characteristics including (1) simple and convenient manipulation, (2) tissue-specific, effective broad transfection properties, and (3) a low inflammatory response. Therefore, this information could be useful for a molecular-level mechanism analysis of diseases at an individual level in mammals, exploration of therapeutic target molecules and evaluation of gene therapy and nucleic acid-based therapy approaches, as well as potential clinical applications.