Accelerated dosing frequency of a pulmonary formulation of tissue plasminogen activator is well-tolerated in mice.

1. Tissue plasminogen activator (tPA) has both fibrinolytic and anti-inflammatory activity. These properties may be useful in treating inflammatory lung diseases, such as acute respiratory distress syndrome (ARDS). 2. We have previously demonstrated the feasibility of targeted pulmonary delivery of tPA. As part of our research to develop a clinically viable pulmonary formulation of tPA, we assessed the tolerability and incidence of haemorrhage associated with the administration of a pulmonary formulation of mouse tPA (pf-mtPA). 3. Intratracheal doses of nebulized pf-mtPA or sterile saline were administered with increasing frequency to male and female B6C3F1 mice. After dosing, the mice entered a recovery period, after which they were killed and their lungs were lavaged and harvested. Post-mortem gross necropsy was performed and all major organs were assessed histologically for haemorrhage. The bronchoalveolar lavage fluid was assessed for markers of lung injury. 4. Mouse tPA that was formulated to mimic a previously characterized human pf-tPA was well tolerated when given intratracheally with increasing dosing frequency. The administration of pf-mtPA did not result in any detectable haemorrhagic-related events or signs of lung injury. 5. The results of the present longitudinal study demonstrate that a maximally feasible dose of pf-mtPA (3 mg/kg) can be given frequently over a short period of time (12 h) without haemorrhagic complications. Although these data were generated in a healthy mouse model, they provide support for the continued evaluation of pf-tPA for the treatment of pulmonary diseases, such as ARDS.

Formulation, lyophilization and solid-state properties of a pegylated protein.

In this paper the importance of formulation and process parameters on the solid-state properties of a lyophilized, pegylated growth hormone antagonist (pegvisomant) was studied. The degree of solid-state disorder (amorphicity), protein/polyethylene glycol (PEG)/sucrose interactions, and dissolution characteristics of the resultant cakes were examined. Using isothermal microcalorimetry (IMC) and differential scanning calorimetry (DSC), it was shown that in co-lyophilized pegylated protein/sucrose systems there was an interaction between sucrose and pegylated protein molecules. This interaction was evidenced by a decrease in the melting temperature (Tm) and melting enthalpy of PEG as a function of sucrose concentration. It was also shown that the sum of the heat of interaction with water for the individual constituents, lyophilized pegylated protein and lyophilized sucrose, was higher than the heat of interaction for the co-lyophilized system. As the concentration of sucrose was increased, the degree of solid-state disorder increased and the solid dissolved faster. A correlation was found among heat of interaction with water, degree of solid-state disorder, and dissolution time. Pegylation caused a shorter dissolution time, lower moisture content, increased amorphicity, and a more rapid moisture-induced crystallization of sucrose.

Assessment of degree of disorder (amorphicity) of lyophilized formulations of growth hormone using isothermal microcalorimetry.

When determining the degree of disorder of a lyophilized cake of a protein, it is important to use an appropriate analytical technique. Differential scanning calorimetry (DSC) and X-ray powder diffraction (XRPD) are the most commonly used thermoanalytical techniques for characterizing freeze-dried protein formulations. Unfortunately, these methods are unable to detect solid-state disorder at levels < 10%. Also, interpretation of DSC results for freeze-dried protein formulations can be difficult, as a result of the more complex thermal events occurring with this technique. For example, proteins can inhibit the thermally induced recrystallization of the lyophilized cake, resulting in potential misinterpretation of DSC degree of disorder results. The aim of this investigation was to study the use of isothermal microcalorimetry (IMC) in the assessment of degree of solid-state disorder (amorphicity) of lyophilized formulations of proteins. For this purpose, two formulations of growth hormone were prepared by lyophilization. These formulations consisted of the same amounts of protein, mannitol, glycine, and phosphate buffer, but differed in the freeze-drying procedure. After lyophilization, the recrystallization of the samples was studied using IMC at 25 degrees C under different relative humidities (58-75%). The effect of available surface area was studied by determining the heat of recrystallization (Q) of the samples before and after disintegration of the cakes. The results showed that, in contrast to DSC, IMC allowed detection of the recrystallization event in the formulations. Although both formulations were completely disordered and indistinguishable according to XRPD method, IMC revealed that formulation B had a different solid-sate structure than formulation A. This difference was the result of differences in the freeze-drying parameters, demonstrating the importance of choosing appropriate analytical methodology.

Apparent solubility of drugs in partially crystalline systems.

Using several griseofulvin samples, representing different solid-state structures, the solubility behavior of drugs in both one-state (totally ordered, semiordered or disordered) and two-state systems was studied. Special attention was directed towards the surface structure of the particles. The partially crystalline samples were obtained by milling the raw material (crystalline standard) or storing the quenched sample (amorphous standard). The solid-state structure of the materials was studied using x-ray diffraction (XRD), differential scanning calorimetry (DSC), isothermal microcalorimetry (IMC), and scanning electron microscopy (SEM). The saturation concentration of the materials was studied in suspensions containing different dispersion concentrations of drug after centrifugation and filtration, using spectrophotometry. In all cases these dispersion concentrations exceeded the solubility of the drug. The solubilities were plotted vs. dispersion concentrations for each sample. Several solubility plateaus were found. The lowest and highest solubility plateaus corresponded to the solubilities of crystalline and amorphous standards. These plateaus were reached at 8 and 44 microg/mL for crystalline and amorphous griseofulvin standards, respectively. An intermediate plateau served as an indication of the existence of a totally semiordered structure. This was reached at 19 microg/mL for griseofulvin. Any deviation from these plateaus was suggested to be indicative of the existence of heterogeneity on the surface structure, which in most cases could be described as a two state system. In such cases, the apparent solubility was a function of dispersion concentration, until at very high dispersion concentrations (4000-20,000 microg/mL) the saturation concentration of the totally disordered (44 microg/mL) or semiordered (19 microg/mL) one-state phase was reached. No reduction in these values was observed during storage for 50 days. It is thus concluded that, in partially crystalline systems, the saturation concentration is an interfacial phenomenon, which depends on the amount, reactivity, and solid-state structure of the exposed solid surfaces in equilibrium with the solution. A simplified solubility model is proposed to qualitatively describe the relationship between established apparent solubilities (saturation concentrations) and different combinations of solid-state structures.