This manuscript describes the preparation, characterization, and pharmacokinetic studies for fluticasone and budesonide nanosuspensions in pulmonary delivery. A wet-milling method with glass beads was evaluated for formulation preparation. Based on the milling time and glass bead size studies, a 24-h wet-milling process using multisized glass beads was found to be the most efficient method to achieve consistent particle size reduction. It was used to prepare nanosuspensions for characterization and pharmacokinetic studies to evaluate formulation performance. For characterization studies, particle size distribution, crystalline form, potency and homogeneity, and stability during storage were evaluated. Nanosuspensions for both compounds exhibited good physical/chemical properties for pulmonary delivery. The pharmacokinetic studies after the intratracheal administration of nanosuspensions showed deep lung deposition and fast lung absorption, with solubility playing an important role in lung retention and duration of action. Overall, these studies have demonstrated that nanosuspensions can be used for pulmonary drug delivery in preclinical animal studies.

Aqueous insolubility is recognized throughout the pharmaceutical industry as a major hurdle for pre-clinical and clinical drug delivery. Pre-clinical, early efficacy, and proof of concept studies oftentimes rely on model compounds that have less than ideal physiochemical properties, and the in vivo results from these studies often have critical impact on the future of the project. As such, effective delivery of prototype compounds with sub-optimal properties is important in target validation. 1,3-Dicyclohexyl urea (DCU), a potent inhibitor of soluble epoxide hydrolase (sEH) has been shown to lower systemic blood pressure in spontaneously hypertensive rats. This compound has limited aqueous solubility that makes in vivo delivery difficult. In such situations, co-solvents, complexation reagents, and emulsions are commonly used to increase the bioavailability of a prototype compound. However, these approaches are often limited by their capacity to get and keep a compound in solution and can have unwanted placebo effects, which can confound the interpretation of animal efficacy results. Nanosuspension formulations of DCU have been utilized for both intravenous injection and infusion to reach steady-state (Css) plasma concentrations in rat enabling the investigation of the target, chemistry space, and PK/PD in a timely manner without encountering confounding efficacy results.

A semi-automated method to determine p K a values of drugs and chemicals by reverse phase HPLC is described. The method uses the capabilities of a crosslinked reverse phase HPLC column (Agilent extend C-18) as a separation device. The pK(a) value was determined based on the different retention behaviors of the charged, multiply charged, and uncharged species at different pH conditions. The advantages of this method are low sample consumption, suitability for less soluble compounds, high purity samples not necessary, no spectral shift needed, high sample throughput, easy set up, high sensitivity, low cost, good precision, and agreement with literature values.

Fibrolase is an active fibrinolytic agent and possesses potential for use in thrombolytic therapy. Its mode of action had been characterized, both in vitro and in vivo. Possessing three disulfide bonds, native fibrolase is nonglycosylated and binds an intrinsic zinc atom. The zinc is essential for retention of activity and structural integrity. In solution, fibrolase is sensitive to changes in pH and temperature (Pretzer et al., 1991). At neutral to basic pH (pH 5-9), the solubility and stability of fibrolase is nearly constant. Little structural variation can be detected by CD spectroscopy. However, decrease in pH below 5 leads to a pronounced reduction in both the solubility and activity of fibrolase. At pH 3 and below, the solubility of fibrolase returns but the activity does not. This solubility profile is unusual in that the minimal solubility is well removed from the pI (which is 6.7). It is proposed that the behavior of fibrolase with variation in pH can be understood in terms of capacity to bind zinc. At pH 5 to 9, the protein binds zinc and the structure and activity are preserved. Near pH 5, the histidine residues which serve as ligands for the zinc become protonated and zinc binding is lost. Loss of zinc leads to local unfolding of a helical segment of fibrolase, exposing hydrophobic groups which allow the protein to rapidly aggregate. At lower pH values (1-3), the protein again adopts a more globular structure, similar to molten globule states, and the solubility increases. However, without the zinc, fibrolase remains inactive. Changes in pH also affect thermal stability. The Tm for fibrolase moves from 50 degrees C at pH 8 to 43 degrees C at pH 5. Increases in temperature also lead to removal of the zinc ion, again producing a partially denatured protein with a marked tendency to aggregate. In both cases (decrease in pH and increase in temperature), analysis of the CD spectra indicates that the protein has primarily lost alpha-helical secondary structure. A major change in structure can also be observed using NMR spectroscopy. At temperatures below 35 degrees C, the globular structure of fibrolase remains intact, although some increase in chain mobility can be noted with increased temperature. Upon melting, numerous signals collapse as the protein unfolds. Transition temperatures (Tm) as measured by CD and NMR are in good agreement. Similar structural changes can be induced by adding zinc chelators such as EDTA and DTT. This leads to complete loss of activity at EDTA concentrations above 1.0 mM.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the thrombolytic agent fibrolase was accomplished employing specific proteolytic and thrombolytic assays. This paper describes a method to measure enzyme proteolytic activity using the oxidized beta-chain of insulin as a substrate. Advantages of this method include a short incubation time for substrate cleavage followed by an isocratic HPLC method with a retention time of approx. 5 min. Proteolytic activity can be rapidly and easily quantitated with this procedure. An azocasein assay was also used to quantitate proteolytic activity. This method was optimized with respect to substrate concentration and incubation time allowing for the rapid quantitation of fibrolase activity. A thrombolytic assay is described which employs fibrin plate clearance and has the advantage of rapid and accurate quantitation compared with previously described methods. It also allows for the standardization of fibrolase in plasmin-equivalent units.

Fibrolase is a metalloprotease with potential use as a fibrinolytic agent. Loss of the intrinsic zinc atom leads to a rapid decrease in enzymatic activity. Circular dichroism measurements indicate that there is a partial unfolding of an α-helical section of the protein concomitant with the loss of zinc. Removal of zinc can be affected by elevated temperatures, acidic pH values, and addition of chelating agents. At low molar concentrations, both ethylenediaminetet-raacetic acid (EDTA) and dithiothreitol (DTT) were found to remove zinc efficiently. Analysis of the sequence of fibrolase identified a segment which possessed a high degree of homology with the metal binding site of other zinc proteases, such as thermolysin and the collagenases. However, the putative zinc binding site in fibrolase lacks the additional glutamate ligand found in thermolysin and subtilisin. This sequence is also predicted to adopt an α-helical conformation. Together, these data indicate that there is a well-defined metal binding site in fibrolase and that metal binding is the most important factor governing the stability of this protein.

The effect of temperature and pH on the activity and conformation of the thrombolytic protein fibrolase was examined. Fibrolase maintained proteolytic activity over 10 days at room temperature (∼22°C). At 37°C, greater than 50% of the proteolytic activity was lost within 2 days and no activity remained after 10 days. Circular dichroism (CD) spectra at elevated temperatures showed that alphahelical structure was lost in a cooperative transition (Tm of 50°C at pH 8). Structural changes were detected by NMR prior to unfolding which were not observable by CD, and the Tm determined by NMR was 46°C at pD 8. The effect of pH on the proteolytic activity and structure of fibrolase was examined over the pH range from 1 to 10. Activity was maintained at neutral to alkaline pH values from pH 6.5 to pH 10.0 but decreased substantially in acidic media. While CD spectra indicated little variation in secondary structure over the pH range 5 to 9, significant differences were noted at pH 2 to 3. The melting temperature of fibrolase decreased to 43°C at pH 5. Protein concentrations determined over the pH range 1 to 10 showed an apparent solubility minimum at pH 5.0, which did not correspond to the isoelectric point of 6.5. Explanations for these observations are proposed.