Olfactory Absorption of Insulin to the Brain

A vast number of potent neuropharmaceuticals, many of which are peptides, are excluded from entry into the brain because of the highly selective blood-brain barrier. The fact that a number of drugs have been shown to be transported directly to the central nervous system following application to the olfactory region of the nose is therefore of major interest. In the present study, the feasibility of delivering peptides to the brain via the olfactory route was assessed using insulin as a model peptide. Systemic hyperinsulinemia induced by subcutaneous injection did not significantly reduce the amount of ¹²⁵I-insulin transported from the nose to the brain in vivo, which suggests that the impact of systemic absorption on drug transport is minimal. A linear relationship was seen between insulin accumulation in the brain and the dose applied, without any relevant saturation. Contrary to what was expected, both systemic and olfactory absorption of insulin was enhanced when the pH of the medium was near the isoelectric point. The amount absorbed to the brain was found to be linearly related to the net charge of the molecule (r = -0.61; n = 20). It was concluded that insulin gains access to the central nervous system from the olfactory region of the nose by a nonspecific pathway. The olfactory route may therefore become an important means to deliver peptides to the brain.     

GM1 Delivery to the CSF Via the Olfactory Pathway

The objective of this study was to determine if monosialogan- glioside (GM1) can be delivered to the brain via the olfactory neural pathway (o.p.). GM1 solution was administered via the o.p. and i.v. route to rats, after which cerebrospinal fluid (CSF) was collected. Two other formulations of GM1, GM1-lipid nanospheres (GM1-LNS) and GM1-DOTAP (a positively charged lipid) complex, were also tested. The results showed that GM1 can be delivered to the brain via the o.p. However, GM1-LNS administered i.v. delivered the highest concentrations of GM1 to the CSF; this formulation may potentially be useful in treatment of spinal cord injury.     

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.     

Transfer of Some Carboxylic Acids in the Olfactory System Following Intranasal Administration

Abstract

The uptake of [¹⁴C]benzoic acid, 4-chloro[¹⁴C]benzoic acid, [³H]phthalic acid and [¹⁴C]salicylic acid in the nasal passages and brain was determined following a unilateral intranasal instillation in mice. An uptake of radioactivity from the nasal mucosa to the ipsilateral olfactory bulb was observed up to 4 h after administration following intranasal instillation of these carboxylic acids whereas the level was low in the contralateral olfactory bulb. Autoradiography of mice given [¹⁴C]benzoic acid and [¹⁴C]salicylic acid by intranasal instillation showed a preferential localization of radioactivity in the axonal and glomerular layer of the olfactory bulb 1 h after the administration. Four hours after administration the radioactivity was present as a gradient from the axonal layer towards the center of the olfactory bulb. Pretreatment of mice with a compound known to damage the olfactory neuroepithelium resulted in a decreased uptake of [¹⁴C]benzoic acid in the olfactory bulb. Thin layer chromatography of supernatants from the ipsilateral olfactory bulbs of mice given [¹⁴C]benzoic acid by nasal instillation indicated that the radioactivity in the bulbs represented unchanged compound. These results suggest that there is a transfer of some aromatic carboxylic acids in the olfactory pathways.     

卡莫司汀聚乳酸微球的体外释放及其在大鼠脑组织中的分布研究

目的:探讨卡莫司汀聚乳酸微球(BCNU-PLA-MS)的体外释药过程及其在大鼠脑组织中的分布。方法:应用高效液相色谱法检测BCNU在磷酸盐缓冲溶液(PBS)和大鼠脑组织内自PLA-MS中释放的药物含量;应用3H标记BCNU,检测3H-BCNU-PLA-MS在正常大鼠脑组织及血清中的分布。结果:BCNU-PLA-MS在PBS和大鼠脑组织中均可持续释药2wk以上。在PBS中,其1、3、15d时药物释放率分别约为15%、50%、90%;在大鼠脑组织内,其4h、1d、3d时药物释放率分别约为8%、16%、60%,并可持续释药15d。大鼠药物植入处药物浓度是其它检测点的6～70倍。结论:BCNU-PLA-MS具有良好的缓释功能,而且安全性、生物相容性较好。     

Effect of Probenecid on Fluorescein Transport in the Central Nervous System Using In Vitro and In Vivo Models

Purpose. The purpose of this study was to characterize the function of multidrug resistance-associated proteins (MRPs) (or MRP-like organic anion transport systems) in the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) using both an in vitro BBB model and an in vivo microdialysis model.Methods. In vitro functional studies were performed using bovine brain microvessel endothelial cells (BBMEC). The accumulation of fluorescein, an anionic fluorescent dye, in BBMEC was determined with and without the presence of inhibitors of various efflux transport proteins. In vivo microdialysis simultaneously monitored fluorescein concentrations in cortical extracellular fluid and cerebrospinal fluid. The effect of probenecid on the in vivo distribution of fluorescein was studied using a balanced crossover design in the rat.Results. In vitro experiments showed that probenecid, indomethacin, LY-329146, and all MRP inhibitors significantly increased (two- to threefold) the accumulation of fluorescein in BBMEC, whereas LY-335979, a P-gp inhibitor, had no effect on the accumulation of fluorescein. Probenecid significantly increased fluorescein plasma concentration and the plasma free fraction in vivo. The distribution of fluorescein across the BBB and BCSFB was enhanced by 2.2- and 1.9-fold, respectively, when probenecid was coadministered, even after correction for increased fluorescein plasma concentrations and free fraction. Conclusions. These results demonstrate that MRPs or MRP-like transport system(s) may play an important role in fluorescein distribution across both BBB and BCSFB. This study showed that microdialysis proved to be a powerful in vivo technique for the study of transport systems in the central nervous system, and in vitro/in vivo correlations are possible using these model systems.     

In Vitro and In Vivo Evaluations of Dihydroquinoline- and Dihydroisoquinoline-based Targetor Moieties for Brain-specific Chemical Delivery Systems

Brain-targeted delivery of various drugs can be successfully achieved by chemical delivery systems (CDS) that contain a 1,4-dihydropyridine-based redox targetor moiety and undergo a sequential metabolism. However, the susceptibility of this moiety toward hydration in acidic media may limit the shelf-life of such compounds in aqueous formulation. Here, a systematic investigation of the chemical stability toward oxidation and hydration of ester and amide derivatives of 3-substituted 1,4-dihydropyridine, 1,4-dihydroquinoline, and 4-substituted 1,2-dihydroisoquinoline is reported, together with the in vitro stability and in vivo (rat) distribution of isoquinoline-based testosterone and hydrocortisone chemical delivery systems, which were selected as having the most suitable acid-resistant targetor moieties.     

The effects of increased glucose supply and thiopental anesthesia on energy metabolism of the isolated perfused rat brain

Summary The effects of glucose concentrations in the perfusion medium ranging from 5 to 15 mM and thiopental, on cerebral energy metabolism were studied using the isolated perfused rat brain. After a perfusion time of 30 min brain levels of the following substrates and metabolites were determined: P-creatine, ATP, ADP, AMP, glycogen, glucose, glucose-6-P, fructose-6-P, pyruvate, lactate, -ketoglytarate, glutamate, ammonia.In control experiments increasing the glucose concentration in the perfusion medium produced an increase of intracellular brain glucose concentration only, revealing a linear relationship between glucose content in brain and blood. Neither high-energy phosphates nor glycolytic intermediates were markedly affected by the changes in blood glucose. With an anesthetic dose of thiopental (0.15 mM) in the perfusion medium identical metabolic alterations occurred in all experiments: P-creatine and glucose were significantly increased whereas ADP, AMP, lactate and pyruvate were diminished. Also with thiopental brain glucose was linearly related with the glucose concentration in the perfusion medium. The calculated regression line was apparently parallel with that from control experiments; that means thiopental always caused an elevation of brain glucose by the same amount of 0.9 moles/g—irrespective of the initial cerebral glucose content. The results yield further evidence that glucose transport is not the rate-limiting step in glycolysis. The action of thiopental on glycolytic pathway is discussed.Presented in part at the Pharmacology Meeting Graz 1974 (Stock et al., 1974).     

Biocompatibility of Polycations: In Vitro Agglutination and Lysis of Red Blood Cells And In Vivo Toxicity

The effects of six polycations were studied in vitro on red blood cells (RBC) and in vivo after intravenous administration. Hemagglutination and hemolysis depended not only on the molar mass and the concentration of these polycations, but also on their chemical nature. The hemagglutination and hemolysis induced by poly (l -lysine), diethylaminoethyldextran, poly(dimethyldiallylammonium) chloride and poly[2-(dimethylamino)ethyl methacrylate] was low to moderate, whereas a severe hemolysis was induced by a partially quaternized poly[thio-1- (N, N -diethyl-aminoethyl)ethylene]. In the case of poly (ε -lysine), no significant hemagglutination nor hemolysis was observed. The presence of plasma proteins reduced both agglutination and hemolysis. This protective effect was enhanced when the polycations interacted with plasma proteins before contact with RBC. In the presence of albumin, the behavior depended on the polycation and on the order of addition of the three components of the suspension, namely albumin, polycation and RBC. Depending on the polycation, albumin-polycation complexes were either less active or more active on RBC than the same polycation in protein-free medium. In vivo the studied polycations induced an immediate mortality except poly (ε -lysine), which induced a delayed mortality. The minimal dose of polycations inducing immediate mortality paralleled their effect on RBC.     

Designing and Testing of an Effective Oil-in-Water Microemulsion Drug Delivery System for In Vivo Application

The phase behavior of a new psedoternary system of clove oil/Tween 20 has been studied. Several compositions from the single-phase region were selected and their stability toward time, temperature, and electrolytes has been examined. A particular composition(clove oil/Tween 20/water as 5/30/65) was chosen as the drug delivery system from the clear oil-in-water zone of the pseudoternary system. The droplet dimension and the polydispersity state of the particular composition was determined by dynamic light scattering. A bioactive compound quarcetin was encapsulated in the vehicle. The efficacy of the drug in the vehicle was examined against leishmaniasis in hamster models. The hepatotoxicity of the vehicle (o/w microemulsion) with and without the drug quarcetin was examined by estimating serum alkaline phosphatase, glutamate pyruvate transaminase, urea, and creatinine.     

Influence of therapeutic and toxic doses of neuroleptics and antidepressants on energy metabolism of the isolated perfused rat brain

Summary The isolated perfused rat brain was used for a comparative study of the effects of promazine, imipramine, monodesmethyl promazine and desipramine on cerebral energy metabolism. After perfusion for 30 min or 1 h the brain levels of the following substrates and metabolites were estimated: P-creatine, creatine, ATP, ADP, AMP, glycogen, glucose, glucose-6-P, fructose diphosphate, dihydroxyacetone-P, pyruvate, lactate, -ketoglutarate, and ammonia. Drug concentrations of 5·10^–6 M and 10^–5 M in the perfusion medium caused a significant decrease of glucose-6-P alone. When the drug concentration was raised to a toxic range (10^–4 M), reflected in the EEG by the pattern of secondary discharges, an accumulation of P-creatine and glucose and a decrease of glycogen, glucose-6-P and ammonia occurred; the lactate/pyruvate ratios remained unchanged. As there were no qualitative differences between the effects of the investigated neuroleptics and antidepressants on cerebral metabolism, these effects might be unspecific and not correlated with the pharmacological action of the drugs.Presented in part at the spring meeting of the Deutsche Pharmakologische Gesellschaft, Mainz 1973 (Stock et al.).     

Comparative study of the effects of chloral hydrate and trichloroethanol on cerebral metabolism

Summary The isolated perfused rat brain was used for a comparative study of the effects of chloral hydrate and trichloroethanol on cerebral energy metabolism. After a perfusion period of 30 min the brain levels of the following substrates and metabolites were measured spectrophotometrically: P-creatine, creatine, ATP, ADP, AMP, glycogen, glucose, glucose-6-P, fructose diphosphate, -glycero-P, dihydroxyacetone-P, pyruvate, lactate, glutamate, -ketoglutarate and ammonia. Furthermore, the concentration of chloral hydrate and trichloroethanol in the isolated brain and in the perfusion medium was measured colorimetrically. Little more than 10% of chloral hydrate in the isolated brain and in the perfusion medium were reduced to trichloroethanol. In intact animals there were about 70% of chloral hydrate transformed. Chloral hydrate and trichloroethanol caused an accumulation of P-creatine, no change in the lactate/pyruvate ratio, an increase of the glucose concentration and a decrease of glucose-6-P level in the isolated brain. The rise of brain glucose level was more pronounced after trichloroethanol than after chloral hydrate. The effects of chloral hydrate and trichloroethanol on brain glucose and glucose-6-P levels suggest an inhibition of brain hexokinase activity by these drugs.Presented in part at the Fifth International Congress on Pharmacology in San Francisco, 1972 (Krieglstein et al., 1972c).     

Simultaneous measurement of tyrosine and tryptophan hydroxylase activities in brain in Vivo using an inhibitor of the aromatic amino acid decarboxylase

Summary DOPA and 5-HTP accumulated in vivo in rat brain after decarboxylase inhibition with NSD 1015 (3-hydroxybenzylhydrazine). This accumulation was linear for the first 30 min and occurred in several brain regions over a wide range of NSD 1015 doses. After a peripheral decarboxylase inhibitor much less, if any, DOPA or 5-HTP accumulated in the brain. The accumulation of DOPA was prevented by H 44/68 (methylester of -methyl para-tyrosine), a tyrosine hydroxylase inhibitor. DOPA, which accumulated before H 44/68 was given, appeared stable for at least 20 min. There were no significant changes in the levels of NA, DA, 5-HT or tryptophan shortly after NSD 1015 administration, but a rise in tyrosine was noted. Increased brain tyrosine after l-tyrosine administration did not alter the DOPA accumulation, however. These data as well as the distribution of the accumulated amino acids suggest that the accumulation of DOPA and 5-HTP after decarboxylase inhibition occurs intraneuronally, that the decarboxylase enzyme is completely inhibited, and that the accumulated products are not appreciably metabolized or transported from the region studied. Amine synthesis rates and rate constants were calculated from the data and compare well with similar values determined by other methods. Thus this accumulation appears to be a reliable measure of the in vivo hydroxylation of tyrosine and tryptophan.     

In Vitro and In Vivo Study of Solid Lipid Nanoparticles Loaded with Superparamagnetic Iron Oxide

Solid Lipid Nanoparticles (SLN) are already under investigation as a pharmaceutical tool able to change the pharmacokinetic and biodistribution of carried molecules. SLN are able to target drugs to lymph after duodenal administration and to overcome the Blood Brain Barrier (BBB). In this study, superparamagnetic SLN have been prepared, have colloidal size, in vitro analysis showed relaxometric properties similar to Endorem_®. In vivo Magnetic Resonance Imaging (MRI) of the central nervous system (CNS) with both SLN and Endorem_® showed that superparamagnetic SLN have slower blood clearance than Endorem_®. MRI data are consistent with CNS uptake of SLN lasting up to the end of the experiment (135 min). These findings confirm the ability of SLN to overcome the BBB; SLN might be used as a CNS MRI contrast agent.     

Visualization of Insulin-Loaded Nanocapsules: In Vitro and in Vivo Studies After Oral Administration to Rats

Purpose. Biodegradable poly(isobutylcyanoacrylate) nanocapsules have been recognized as a promising carrier for oral administration of peptides and proteins. In the present study, we investigate the fate of insulin-loaded nanocapsules by fluorescence and transmission electron microscopy (TEM) after intragastric force-feeding to rats. Methods. Insulin-, Texas-red^®-labeled insulin, or gold-labeled insulin-loaded nanocapsules were first characterized. Rats received a single dose of nanocapsules (diameter 60-300 nm, 57 IU insulin/kg) by intragastric force-feeding. After 90 min, ileum was isolated and prepared for fluorescence and transmission electron microscopy. Results. Nanocapsules were observed on both sides of the gut epithelium and in blood capillaries. In M-cell-free epithelium, apparently intact nanocapsules could be seen in the underlying tissue, suggesting they could cross the epithelium and carry the encapsulated peptide. In M-cell-containing epithelium, nanocapsules appeared degraded in the vicinity of macrophages. It is noteworthy that intestinal absorption of nanocapsules was observed without artifacts forcing the nanocapsules to stay in the gut. Conclusions. Based on TEM observations, this study shows the intestinal absorption of biodegradable nanocapsules leading to the transport of insulin across the epithelium mucosa. The fate of the nanocapsules appeared different depending on the presence or the absence of M cells in the intestinal epithelium.     

Induction of heat shock protein 70 in rat olfactory epithelium by toxic chemicals: in vitro and in vivo studies

We have previously developed a rat nasal explant system for investigating upper respiratory tract toxicity, and the aims of this study were to determine whether heat shock protein (HSP) 70 is induced in this model following exposure to carbon tetrachloride (CCl₄), dimethyl adipate (DMA), methyl iodide (CH₃I) or paracetamol, and whether HSP70 can also be induced in the nasal cavity in vivo. Intracellular ATP was significantly depleted in ethmoturbinates incubated for 4 h with the toxins (0–100 mM; EC₅₀ concentrations: CCl₄ 32 mM, DMA 3 mM, CH₃I 1.5 mM, paracetamol 70 mM), but there was little induction of HSP70. Turbinates were then incubated for 1 h with CCl₄ (5 mM), DMA (1.5 mM), CH₃I (0.57 mM) or paracetamol (30 mM) and allowed to recover for up to 24 h. Treatment with CCl₄, DMA or paracetamol resulted in 250–300% induction of HSP70. Male rats were administered a single oral dose of CCl₄ (1600 mg/kg) and killed 16 h later. Degenerative lesions (epithelial undulation and hydropic vacuolation) were evident in the olfactory epithelium, and immunohistochemical analysis of HSP70 revealed increased staining in, or proximate to, areas of damage. Thus, HSP70 can be induced in the olfactory epithelium both in vitro and in vivo.     

In Vitro and Ex Vivo Permeation Studies of Etodolac from Hydrophilic Gels and Effect of Terpenes as Enhancers

Etodolac, a highly lipophilic anti-inflammatory drug, is widely used in rheumatoid arthritis usually at an oral dose of 200 mg twice daily. The commonest side effects during therapy with etodolac is generally gastrointestinal disturbances these are usually mild and reversible but in some patients are peptic ulcer and severe gastrointestinal bleeding. To eliminate these side effects and obtain high drug concentration at the application side, dermal application of etodolac seems to be an ideal route for administration. Hydrophilic gel formulations of etodolac were prepared with carboxymethylcellulose sodium. The effect of different terpenes (anethole, carvacrol, and menthol) as an enhancer on the percutaneous absorption of etodolac was also investigated. Permeation studies were carried out with unjacketed modified horizontal diffusion cells through cellulose membrane and rat skin. In vitro studies with cellulose membrane showed that all formulations presented the same drug release profile (p > 0.05). Ex vivo studies with excised rat skin revealed that etodolac was released and penetrated into rat skin quickly. Anethole, a hydrophobic terpene, enhanced the absorption of etodolac significantly (p < 0.05). This result is consistent with the fact that hydrophobic terpenes are effective on the percutaneous absorption of lipophilic drugs. Menthol and carvacrol, hydrophilic terpenes, did not enhance the absorption of etodolac. The lipophilicity of the enhancers seems an important factor in promoting penetration of etodolac through the skin. Since etodolac creates gastrointestinal disturbances, topical formulations of etodolac in gel form including 1% anethole could be an alternative.     

In vivo and in vitro effects of methylmercury on the activities of aminoacyl-tRNA synthetases in rat brain

The activities of six aminoacyl-tRNA synthetase species were determined using enzyme preparations partially purified from the brains of control and methylmercury (MeHg)-treated rats. The activities of Asp-, Leu- and Tyr-tRNA synthetases were significantly reduced in the brains of MeHg-intoxicated rats, whereas those of Lysand Met-tRNA synthetases remained unchanged. In contrast, the activity of His-tRNA synthetase was significantly increased in the symptomatic phase of MeHg intoxication. The activities of these six aminoacyl-tRNA synthetases in the control brains were affected to different extents on the direct addition of MeHg to the assay system in vitro. No positive correlation was observed between the in vivo and in vitro effects of MeHg on the enzyme activities. These results indicate that the aminoacylation of tRNA is one of the actions of MeHg, which leads to inhibition of protein synthesis, and it is suggested that the syntheses of cellular proteins may be modified in different ways by MeHg, depending on their amino acid compositions.This work was supported in part by a grant from the Japanese Environmental Agency.     

Characterization and In Vivo Study of Sustained-Release Formulation of Human Growth Hormone Using Sodium Hyaluronate

PURPOSE: Aiming at once-a-week injection, a novel sustained release formulation of recombinant human growth hormone (SR-hGH) using sodium hyaluronate was developed for the treatment of children who have growth failure due to the lack of adequate secretion of endogenous growth hormone. METHODS: SR-hGH was produced in the form of solid microparticle using a Niro spray dryer and characterized by Malvern particle size analysis, scanning electron microscopy (SEM), size exclusion chromatography (SEC), reverse phase-high-performance chromatography (RP-HPLC), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After in vitro release test, pharmacokinetic and pharmacodynamic studies were carried out in beagle dogs. SR-hGH was dispersed in medium-chain triglyceride (MCT) and administered at a dose of 1.0 mg hGH/kg subcutaneously. RESULTS: SR-hGH microparticles were successfully produced with a mean particle size of 5.6+/-1.0 microm. Physicochemical analysis with SEC, RP-HPLC, and SDS-PAGE showed that hGH extracted from SR-hGH was intact and comparable to that of hGH bulk standard indicating no structural change in hGH during the formulation processes. Monomeric content of hGH recovered from SR-hGH was 97.4% by SEC analysis, and its purity was 96% by RP-HPLC analysis. In vitro release test showed the sustained-release characteristics of SR-hGH up to 48 h with the complete release of hGH loaded. The continuous and monotonous release profile observed in in vitro release test was supported by pharmacokinetic study in beagle dogs. Delayed absorption of hGH was observed with Cmax of 69.5+/-8.0 ng/ml and Tmax between 10 and 12 h. The administration of SR-hGH induced elevation of serum insulin-like growth factor-I (IGF-I) level for 6 days with a maximum value higher than the predose level by ca. 350 ng/ml. After 6 days, IGF-I level returned to the initial baseline level. CONCLUSIONS: Sustained-release formulation of hGH for once-a-week injection was successfully developed using high-molecular-weight sodium hyaluronate. No adverse effect was observed during and after the in vivo test using beagle dogs.     

Gene Transfer In Vitro and In Vivo by Cationic Lipids Is Not Significantly Affected by Levels of Supercoiling of a Reporter Plasmid

Purpose. It is a common preconception that supercoiledplasmid DNA is more desirable for the transfection of cells that the relaxedform of the plasmid. This notion has led to the recommendation that aspecification for the minimum amount of plasmid in the supercoiled formshould exist in a gene therapy product. We have tested this notion byexamining the effects of the degree of supercoiling on cationiclipid-mediated gene transfer in vitro and in vivo. Methods. An ion-exchange high performance liquidchromatography (HPLC) method was developed to accurately quantitatethe relative amounts of supercoiled DNA in purified plasmid. A sample of thepurified plasmid was fully relaxed using topoisomerase. Next, the ability ofvarious levels of supercoiled plasmid to transfect mammalian cells wasmeasured. Results. This study suggests that there is no relationbetween the degree of supercoiling and lipofection efficiency. Subsequenttransfection using several different lipofection agents, different celltypes, and an in vivo model support these results. Conclusions. In considering a specification for the amountof supercoiled plasmid in a gene therapy product, it must be noted that therelaxed forms of the plasmid are no less efficient at gene delivery than thesupercoiled forms.