Intracranial Delivery of Recombinant Nerve Growth Factor: Release Kinetics and Protein Distribution for Three Delivery Systems

Purpose. Three different polymeric delivery systems, composed of either poly(ethylene-co-vinyl acetate) (EVAc) or poly(lactide-co-gly-colide) (PLGA), were used to administer recombinant human nerve growth factor (rhNGF) intracranially in rats. Methods. The delivery systems were characterized with respect to release kinetics, both in the brain and in well-stirred buffer solutions. Results. During incubation in buffered saline, the delivery systems released rhNGF in distinct patterns: sustained (EVAc), immediate (PLGA₁), and delayed (PLGA₂). One 10-mg delivery system was implanted in each rat and an ELISA technique was used to determine the amount of rhNGF in 1-mm coronal brain slices produced immediately after removal of the delivery system. High levels of rhNGF (as high as 60,000 ng in a brain slice of 50 L) were recovered from the brain tissue at 1,2, and 4 weeks after implantation. With all three delivery systems, the amount of rhNGF in each brain slice decreased exponentially with distance from the implant site; the distance over which concentration decreased by 10-fold was 2–3 mm for all delivery systems. When rhNGF release was moderate (10 to 200 ng rhNGF/ day), the total amount of rhNGF in the brain increased linearly with release rate, suggesting an overall rate of rhNGF elimination of 0.4 hr^–1 or a half-life of 1.7 hr. With higher release rates (500 to 50,000 ng rhNGF/day), total amounts of rhNGF in the brain were considerably higher than anticipated based on this rate of elimination. Conclusions. Polymeric controlled release can provide high, localized doses of rhNGF in the brain. All of the experimental data were consistent with penetration of rhNGF through the brain tissue with a diffusion coefficient 8 X 10^–7 cm²/s, which is 50% of the diffusion coefficient in water.     

Biodegradable Polyester,Poly[α-(4-Aminobutyl)-l-Glycolic Acid], as a Non-Toxic Gene Carrier

Purpose. The aim of this study was to develop a non-toxic polymericgene carrier. For this purpose, biodegradable cationic polymer,poly[-(4-aminobutyl)-l-glycolic acid] (PAGA) was synthesized. PAGA wasdesigned to have ester linkage because polyesters usually showbiodegradability. Methods. Degradation of PAGA in an aqueous solution was followedby matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS). PAGA/DNA complexes were characterizedby gel electrophoresis, atomic force microscopy (AFM), dynamiclight scattering (DLS). The transfection was measured by using the -galactosidase reporter gene. Results. PAGA was degraded in aqueous solution very quickly andthe final degradation product was a monomer (l-oxylysine). Formationof self-assembling biodegradable complexes between PAGA and DNAat a charge ratio 1:1 (+/–) was confirmed by gel band shift assay andAFM. In these studies, controlled release of DNA from the complexescould be seen. The complexes showed about 2-fold higher transfectionefficiency than DNA complexes of poly-l-lysine (PLL), a structuralanalogue of PAGA, which is the most commonly used poly-cation forgene delivery. The polymer did not show cytotoxicity, possibly becauseof its degradability and the biocompatibility of the monomer. Conclusions. The use of the biodegradable poly-cation, PAGA, as aDNA condensing agent will be useful in safe gene delivery.     

Complex Formation Between The Anionic Polymer (PAA) and a Cationic Drug (Procaine HC1): Characterization by Microcalorimetric Studies

Purpose. Due to the importance of drug-polymer interactions in, inter alia, drug loading/release, supramolecular assemblies and DNA delivery for gene therapy, the aim of this study was therefore to establish the mechanism of interaction between a model polymer (Polyacrylic acid, PAA) and a model drug (procaine HCl). Methods. This was performed by studying the effect of salt (KCl) concentration on their heat released values using Isothermal Titration Microcalorimetry (ITM). The integrated released heat data were computer fitted to a one class binding model and the thermodynamic parameters (K_obs, H, and N) were determined. Results. As the KC1 concentration was increased, K_obs decreased thus establishing the salt dependence of the interaction. The linear variation of G_obs with S_obs indicated that their interaction was entropically driven. The stoichiometry of the interaction was calculated to be one procaine molecule per monomer of PAA. Dissection of the total observed free energy at each KC1 concentration indicated that the contribution of the non-electrostatic attractions to the interaction of PAA with procaine HC1 was greater than those of the electrostatic attractions. Conclusions. We have shown that the interaction between PAA and procaine HC1 is dependent upon the presence of counterions (monovalent ions) and is mainly entropically driven. The calculated stoichiometry indicated that one procaine HC1 molecule neutralised one carboxylic acid group on PAA. Although electrostatic interactions were necessary for initiating complex formation, the non-electrostatic forces were dominant in stabilising the PAA-procaine HC1 complex.     

Can the Organization of a Binary Mix Be Predicted from the Surface Energy,Cohesion Parameter and Particle Size of Its Components?

Purpose. The aim of this study was to relate the organization of several binary mixes with three physical parameters (surface energy, cohesion parameter, and particle size) of various materials blended with each other. Methods. Four pharmaceutical compounds were selected for their surface energies and cohesion parameters. Binary mixes were prepared from different sieved fractions. The frequency and nature of the interactions between the particles were observed by scanning electron microscopy. Results. As expected, interactions were determined by both the energetics and the relative particle size of the two compounds blended, the latter determining the mode of interaction. However, particle size was not the only factor influencing the organization of the blends as, sometimes, small particles of a material would not adhere to the coarser particles of the other. Thus, a surface energy derived parameter ^B/A – ^A/B appears to be a valuable estimating tool of the potentiality of interaction between the particles blended. No correlation between the cohesion parameters of the compounds and the organization of the resulting blends could be found. Conclusions. Surface energy and particle size play a major role in the organization of a binary blend. However, they cannot explain separately the interactions observed between the fractions blended as reliable predictions require the use of both characteristics.     

Model-independent assessment of accumulation kinetics based on moments of drug disposition curves

Summary The bounds of the accumulation profile can be predicted on the basis of the mean disposition residence time (MDRT) of a drug. The time to reach 90% of the plateau level (t _0.9) is less than 3.7 MDRT. This prediction can be improved if, in addition, the variance of disposition residence time (VDRT, CV _D ² =VDRT/MDRT²), or the terminal exponential coefficient (), is known. For CV _D ² 1 or MDRT1, the time to reach steady state (t_0.9) approaches 2.3 MDRT (limiting case of monoexponential drug disposition curve). Conditions are stated under which can be regarded as the principal determinant of the accumulation rate.     

In Vitro Gene Transfection in Human Glioma Cells Using a Novel and Less Cytotoxic Artificial Lipoprotein Delivery System

Purpose. To develop and evaluate a novel artificial lipoprotein delivery system for in vitro gene transfection in human glioma cells. Method. Nanoemulsion was formulated with similar lipid compositions present in natural lipoproteins. The oil phase of nanoemulsion was composed of triolein (70%), egg phosphatidylcholine (22.7%), lysophosphatidylcholine (2.3%), cholesterol oleate (3.0%), and cholesterol (2.0%). To replace the surface protein as in natural lipoprotein, poly-L-lysine was modified to add palmitoyl chains at a basic condition and was incorporated onto the nanoemulsion particles through hydrophobic interaction. A model plasmid DNA, pSV--Gal containing a reporter gene for -galactosidase was carried by the nanoemulsion/poly-L-lysine particles. The charge variation of so-formed complex was examined by agarose gel electrophoresis and zeta potential measurement. In vitro transfection was conducted on human SF-767 glioma cell line using this new system. After standard X-Gal staining, transfected cells were observed under light microscope. The effect of chloroquine on the transfection was examined and, finally, the cytotoxicity of this new system was evaluated in comparison with commercial Lipofectamine gene transfection system. Results. The plasmid DNA was effectively carried by this artificial lipoprotein delivery system and the reporter gene was expressed in the glioma cells. Transfection efficiency was significantly increased by the treatment of chloroquine, indicating that endocytosis possibly was the major cellular uptake pathway. Compared to Lipofectamine system, this new delivery system demonstrated similar transfection efficiency but a much lower cytotoxicity. In the experiment, the cell viability showed up to 75% using this system compared to only 24% using Lipofectamine system. Conclusion. A new artificial lipoprotein delivery system was developed for in vitro gene transfection in tumor cells. The new system showed similar transfection efficiency but a much lower cytotoxicity compared with commercial Lipofectamine system.     

Determination of ethinyloestradiol in the presence of norethisterone by derivative-difference spectrophotometry

A simple and direct method for the determination of ethinyloestradiol as a minor component in the presence of norethisterone and in oral contraceptive tablets is presented. The method is based on measuring the signal intensity, dA/d and d²A/d ², of the generated first and second derivative spectra of the -absorbance curves obtained by measuring solutions in methanol and methanol-sodium hydroxide at certain wavelengths. The method has been applied to the determination of ethinyloestradiol in oral contraceptive tablets, with a coefficient of variation of less than 2%.     

A Novel Non-Viral Vector for DNA Delivery Based on Low Molecular Weight,Branched Polyethylenimine: Effect of Molecular Weight on Transfection Efficiency and Cytotoxicity

Purpose. Low molecular weight branched polyethylenimine (LMW-PEI) was synthesized and studied as a DNA carrier for gene delivery with regard to physico-chemical properties, cytotoxicity, and transfection efficiency. Methods. The architecture of LMW-PEI, synthesized by acid catalyzed ring-opening polymerization of aziridine was characterized by size exclusion chromatography in combination with laser light scattering and ¹³C-NMR-spectroscopy. In vitro cytotoxic effects were quantified by LDH and MTT assay and visualized by transmission electron microscopy. The potential for transgene expression was monitored in ECV304 cells using luciferase driven by a SV40 promoter as reporter gene system. Results. LMW-PEI (Mw 11900 D) with a low degree of branching was synthesized as a DNA carrier for gene delivery. In contrast to high molecular weight polyethylenimines (HMW-PEI; Mw l616OOO D), the polymer described here showed a different degree of branching and was less cytotoxic in a broad range of concentrations. As demonstrated by transmission electron microscopy the LMW-PEI formed only small aggregates which were efficiently taken up by different cells in the presence of serum, most likely by an endocytic pathway. LMW-PEI yielded transfection efficiencies measured via expression of the reporter gene luciferase which were up to two orders of magnitude higher than those obtained with HMW-PEI. The reporter gene expression was concentration dependent, but in contrast to lipofection independent of serum addition. Conclusions. The LMW-PEI described here is a new, highly efficient, and non-cytotoxic vector with a favorable efficiency/toxicity profile for gene therapeutic applications.     

Interaction of obidoxime with sarin in aqueous solution

The interaction of obidoxime (Toxogonin^®) with sarin was shown by different analytical methods. The UV spectrum of obidoxime at pH 7.4 yields two absorption maxima, ₁=284 nm and ₂=353 nm. The peak at ₂=353 nm is representative for the amount of zwitterionic obidoxime, i.e. the active form of obidoxime. By addition of sarin, ₁ shifts immediately to 278 nm and the intensity at ₂ decreases, thus indicating an interaction. TLC and³¹P-NMR evidence shows that both mono-phosphonylated and diphosphonylated obidoximes are present. Decomposition of phosphonylated obidoxime in MOPS (3-[N-morpholino] propanesulfonic acid) buffered D₂O at pH 7.4 occurs with t_1/2=13.3 min at 24°C. Decomposition of di-phosphonylated obidoxime is faster. It is suggested that decomposition of di-phosphonylated obidoxime occurs through the mono-phosphonylated form. Formation and decomposition of mono- and di-phosphonylated obidoxime is pH dependent. We conclude that obidoxime exerts a detoxifying effect by capturing free sarin molecules and thus increasing its polarity. Thereby the transition of sarin through the blood-brain barrier is restricted and its renal elimination facilitated.     

Characterization of Pharmacodynamic Recession Slopes for Direct and Indirect Response Models

Direct pharmacologic effects are known to recede over time with largely linear slopes (Levy's k · m product, J. Pharm. Sci. 53: 342, 1964) and indirect responses have similar behavior. Pharmacodynamic slope properties were examined mathematically for the Hill function with monoexponential drug disposition and simulations were carried out for other pharmacokinetic functions. Both types of pharmacodynamic profiles exhibit a single terminal inflection point (fp) when drug concentrations exceed the EC₅₀ (that concentration causing one-half maximum effect, E_max ). For direct effects it was found that C_fp (the drug concentration at fp) =EC₅₀ , the determinants of inflection time were identified, and Slope_fp = –_zE_max /4 where _z is the terminal disposition slope and is the Hill coefficient. These characteristics were explored for the four basic indirect response models which also exhibit recession profiles with slight sigmoidity and a single terminal inflection point at higher doses. The drug concentration at inflection C_fp is IC₅₀ or SC₅₀ (drug concentrations causing half-maximal inhibition or stimulation), while the inflection response (R_fp ) attains constant values at larger doses. Indirect Response Models I, III, and IV have nearly linear return slopes for a wide range of doses which are governed by the disposition slope _z of the drug, loss constant k_out of the response, maximum inhibition (I_max ) or stimulation (S_max ) factors, and a unique fractional constant (01). Model II exhibits more complex behavior with recession slopes which are less likely to be parallel for various doses. Most indirect responses are expected to show nearly linear recession slopes which are parallel for moderate to large doses and mainly governed by an identical combination of pharmacokinetic (_z ), system (k_out ), and dynamic capacity factors (I_max or S_max ).