Interleukin-2 lipid microspheres. I. development and evaluation of the colloidal drug carrier

Undesirable toxic effects associated with intravenous interleukin-2 (IL2) therapy have limited its use for the treatment of cancer. Therefore, we investigated properties of a colloidal carrier system intended for the delivery of IL2. Lipid microspheres (LMS) are 10% (v/v) soybean oil emulsions stabilized with block copolymers of the poloxamer and poloxamine type. Poloxamers 238, 338, 407 or poloxamine 908 LMS were evaluated for physical stability, in vitro toxicity, and in vivo biodistribution. With the exception of 2% poloxamer 238 LMS, all preparations displayed acceptable stability when stored for 3 months at 4° or 37°C. In addition, all LMS preparations exhibited physical stability when subjected to freeze-thaw cycling and extended periods of freezing. In vitro cellular toxicity was evaluated in a murine cytotoxic T lymphocyte cell line (CTLL-2) and human peripheral blood mononuclear cells (PBMC). The calculated IC₅₀ of LMS was approximately 30 and 10 mg/liter in CTLL-2 cells and PBMC, respectively. Biodistribution studies involving ¹²⁵I-labeled LMS revealed that 2 h after intravenous administration there was significantly greater recovery of the poloxamer 338 and 407 LMS-associated radioactivity, where blood, liver, spleen, and bone marrow accounted for most of the radioactivity. Overall, the data suggest that LMS have the potential to serve as a drug delivery system.     

Interaction Between Superoxide Dismutase and Dipalmitoylphosphotidylglycerol Bilayers: A Fourier Transform Infrared (FT-IR) Spectroscopic Study

Purpose. Superoxide dismutase (SOD), an antioxidant enzyme, converts peroxide radicals into hydrogen peroxide. Liposomes have been used as carriers for SOD to enhance its antioxidant effect. Our previous DSC study has suggested that SOD binding to dipalmitoylphosphatidylglycerol (DPPG) may protect lipid membranes against oxygen-mediated injury. We now present FT-IR studies on the effect of DPPG binding on the temperature-induced SOD folding-unfolding process. Methods. The FT-IR spectra of SOD in D₂O or DPPG membranes are measured as temperatures increase from 28° to 121°C at a rate of 0.5°C/ min. From the quantitative determination of the changes in the amide I band components of the Fourier self-deconvoluted spectra, the DPPG-induced changes of SOD secondary structure could be detected as a function of temperature. Results. We observe that the relative intensity of the SOD bands from 28°C to 77°C show graduate loss of -sheet distorted structure, loss of turns, and existence of an intermediate state around 50°C. Beginning at 80°C, changes are obtained in three temperature regions: (i) 80°C, (ii) 92°C, (iii) 109°C. The result suggests that SOD folding/unfolding transition involves mostly the relative changes within the regions of helix-like hydrogen bonding pattern, turn, twisted -bend and irregular structures. When SOD is bound to DPPG, the conformational changes shift to lower temperatures, indicating a reduction of SOD thermal stability. In addition, the gel to liquid crystalline phase transition temperature of DPPG increases from 42°C to 43.5°C. Conclusions. These results suggest that the thermal stability of SOD is reduced by DPPG binding. However, DPPG bilayer is stabilized by the presence of SOD.     

Delivery of 125I-NGF to the Brain via the Olfactory Route

The blood-brain barrier presents a major problem in the administration and testing of neurotropins as it prevents a sufficient concentration of these potential therapeutic agents from reaching the target areas of the human brain. The olfactory neuroepithelium is the only area of the body in which an extension of the central nervous system comes into direct contact with the environment. Following intranasal administration of ¹²⁵I-labeled nerve growth factor (¹²⁵I-NGF), radiolabel appeared rapidly in the olfactory bulb and other brain regions. Radiolabel accumulation in the olfactory bulb of the brain was a linear function of the intranasal dose and of the radiolabel concentration in the olfactory epithelium. Concentration of radiolabel in the olfactory bulb and brain with intranasal administration, but not with intravenous administration, suggests direct transport of label into the brain along the olfactory route following intranasal administration. The rapid appearance of label in the olfactory bulbs, cerebrum, and brain stem is more consistent with entry of label through intercellular clefts in the olfactory epithelium and extracellular transport along the olfactory neural pathway to reach the cerebrospinal fluid and brain than with uptake by olfactory neurons and subsequent intracellular axonal transport. At least 80% of the radiolabel found in the brain following intranasal delivery of ¹²⁵I-NGF precipitates in cold 5% trichloroacetic acid, suggesting that a significant amount of intact NGF reaches the brain. Preliminary studies using a sandwich enzyme-linked immunosorbent assay have confirmed the uptake of NGF into the brain following intranasal but not intravenous administration to rats. This is the first evidence for noninvasive delivery of unconjugated NGF to the brain.     

Delivery of Nerve Growth Factor to the Brain via the Olfactory Pathway

Purpose: To assess the potential of delivering nerve growth factor (NGF) to the brain along the olfactory neural pathway for the treatment of Alzheimer's disease. Methods: Recombinant human NGF (rhNGF) was given as nose drops to anesthetized rats. The rhNGF concentrations in the brain were determined by enzyme-linked immunosorbent assay (ELISA). Results: Following olfactory administration, rhNGF reached the brain within an hour, achieving a concentration of 3400 pM in the olfactory bulb, 660–2200 pM in other brain regions and, 240 pM and 180 pM in the hippocampus and the amygdala, respectively. In contrast, little or no rhNGF was found in the brain following intravenous administration. Conclusions: A significant amount of rhNGF can be delivered to the brain via the olfactory pathway. The detection of rhNGF by ELISA indicates that rhNGF is delivered to the brain relatively intact. The rapid appearance of rhNGF in the brain suggests that it may be transported by an extraneuronal route into the brain via intercellular clefts in the olfactory epithelium. Further work to clarify the transport mechanism is underway. The olfactory pathway is a promising, non-invasive route for drug delivery to the brain, which has potential for the treatment of neurodegenerative diseases including Alzheimer's disease.     

Previously reported nerve growth factor levels are underestimated due to an incomplete release from receptors and interaction with standard curve media

The 1996 research report by Hoener et al. [M.C. Hoener, E. Hewitt, J. M. Conner, J.W. Costello, S. Varon, Nerve growth factor (NGF) content in adult rat brain tissue is several-fold higher than generally reported and is largely associated with sedimentable fractions, Brain Res. 728 (1996) 47-56.] compares levels of nerve growth factor (NGF) found in rat brain by assaying both supernatant and pellet to previously reported data. However, Hoener et al. miscalculated when converting values previously reported in the literature to units of picogram per milliliter. Regardless of this mistake, the method of tissue extraction does affect the extent of release of NGF, which must be maximized in order to accurately determine NGF levels in the central nervous system. We now report that accurate measurement of NGF levels is not only affected by the incomplete release of NGF from receptors, but also the medium in which the standard curve is run. It is the combination of these two variables that has led to the underestimation of NGF levels in previous research.