Metal Nanoparticles as Targeted Carriers Circumventing the Blood-Brain Barrier.

Metal nanoparticles have been proposed as a carrier and a therapeutic agent in biomedical field because of their unique physiochemical properties. Due to these physicochemical properties, they can be used in different fields of biomedicine. In relation to this, plasmonic nanoparticles can be used for detection and photothermal destruction of tumor cells or toxic protein aggregates, and magnetic iron nanoparticles can be used for imaging and for hyperthermia of tumor cells. In addition, both therapy and imaging can be combined in one nanoparticle system, in a process called theranostics. Metal nanoparticles can be synthesized to modulate their size and shape, and conjugated with different ligands, which allow their application in drug delivery, diagnostics, and treatment of central nervous system diseases. This review is focused on the potential applications of metal nanoparticles and their capability to circumvent the blood-brain barrier (BBB). Although many articles have demonstrated delivery of metal nanoparticles to the brain by crossing the BBB after systemic administration, the percentage of the injected dose that reaches this organ is low in comparison to others, especially the liver and spleen. In connection with this drawback, we elaborate the architecture of the BBB and review possible mechanisms to cross this barrier by engineered nanoparticles. The potential uses of metal nanoparticles for treatment of disorders as well as related neurotoxicological considerations are also discussed. Finally, we bring up for discussion a direct and relatively simpler solution to the problem. We discuss this in detail after having proposed the use of the intranasal administration route as a way to circumvent the BBB. This route has not been extensively studied yet for metal nanoparticles, although it could be used as a research tool for mechanistic understanding and toxicity as well as an added value for medical practice.

Effects of iodine on inducible nitric oxide synthase and cyclooxygenase-2 expression in sulfur mustard-induced skin.

In a previous study we demonstrated the protective effect of topical iodine as postexposure treatment for sulfur mustard (SM) application. The iodine treatment results in significantly reduced inflammation and necrosis and increased epidermal hyperplasia. The expression and localization of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in paraffin-embedded skin samples from that study were evaluated in the present investigation. We compared the immunoreactivity of iNOS and COX-2 using five samples from each of the following four test sites: untreated control sites, SM-exposed sites, sites treated with iodine mixture 15 min after SM exposure, and sites treated with iodine 30 min after SM exposure. All animals were killed 2 days after irritant exposure. iNOS immunoreactivity was present only in skin sites exposed to SM without iodine treatment. The ulcerated skin was covered with a relatively thick band of exudate composed of iNOS-immunostained polymorphonuclear cells and macrophages. In untreated skin, COX-2 immunostaining was limited to the thin suprabasal epidermal layer. In SM-exposed skin, induction of COX-2 was noted in inflammatory cells located close to the site of epidermal injury. In skin sites treated with iodine 15 or 30 min after SM exposure, the regenerating hyperplastic epithelium showed moderate cytoplasmic staining localized to the epithelium overlying the basal layer. This pattern of staining was also present in the nearby dermal fibroblasts. Thus, in contrast to the skin samples exposed to SM without iodine treatment, the epidermal layer expressing immunohistochemical positivity for COX-2 was thicker and corresponded to the epidermal hyperplasia noted in samples treated with iodine. It is well documented that prostaglandins (PGs) promote epidermal proliferation, thereby contributing to the repair of injured skin. That the induction of the COX-2 shown in our study may also play a role in the healing process is indicated by the present evidence. The results suggest that nitric oxide radicals (NO*) are involved in mediating the damage induced by the SM and that iodine-related reduction in acute epidermal inflammation is associated with reduced iNOS expression.

Topical iodine preparation as therapy against sulfur mustard-induced skin lesions.

Sulfur mustard (SM) is a powerful vesicant employed as an agent of chemical warfare. This study demonstrates the therapeutic effect of a novel topical iodine preparation as a postexposure treatment against SM-induced lesions in the fur-covered guinea-pig skin model. Iodine treatment 15 min after SM exposure resulted in statistically significant reductions of 48, 50, and 55% in dermal acute inflammation, hemorrhage, and necrosis, respectively, whereas, the epidermal healing markers, hyperkerathosis and acanthosis, were significantly elevated by 72 and 67%, respectively, 2 days after treatment. At the interval of 30 min between SM exposure and iodine treatment, there was a significant degree of healing or recovery, albeit to a lesser extent than that observed in the shorter interval. Although the epidermal healing markers were not elevated, the parameters indicative of active tissue damage, such as subepidermal microblisters, epidermal ulceration, dermal acute inflammation, hemorrhage, and necrosis, were significantly reduced by 35, 67, 43, 39, and 45%, respectively. At the 45-min interval between exposure and treatment, there was also a certain degree of healing or recovery expressed as significant reductions in dermal subacute inflammation, subepidermal microblister formation, and epidermal ulceration, whereas, acanthosis was statistically elevated, indicating an increased healing potential. At the 60-min interval, iodine was less efficacious; nevertheless, a significant reduction in the incidence of subepidermal microblisters and an expansion of the acanthotic area were observed. Gross ulceration was significantly decreased at intervals of 15 and 30 min between exposure and treatment. The local anesthetic, lidocaine, did not alter the therapeutic effect of iodine. SM was not affected chemically by iodine as measured by gas chromatography-mass spectrometry (GC-MS) analysis. These findings suggest that the iodine preparation functions as an antidote against skin lesions induced by SM.

Simultaneous stereoselective high-performance liquid chromatographic determination of 10-hydroxycarbazepine and its metabolite carbamazepine-10,11-trans-dihydrodiol in human urine.

An enantioselective HPLC method for the simultaneous determination of the concentration of the enantiomers of the oxcarbazepine metabolites 10-hydroxycarbazepine (MHD) and carbamazepine-10,11-trans-dihydrodiol (DHD) in human urine is described. The method is based on extraction with tert.-butylmethyl ether-dichloromethane (2:1, v/v) under alkaline conditions, separation and evaporation of the organic phase and dissolution of the residue in the mobile phase. Enantiomers are resolved on a Diacel Chiralcel OD column (250 mm x 4.6 mm I.D.) under isocratic conditions using as mobile phase n-hexane-ethanol-2-propanol (18:2:1, v/v/v) with addition of glacial acetic acid (0.1%). The enantiomers are detected by UV at 215 nm. The method allows reliable determination of the MHD and DHD enantiomers in human urine with limits of quantification of 0.2 mg/l and 0.4 mg/l, respectively.

New topical antiandrogenic formulations can stimulate hair growth in human bald scalp grafted onto mice.

The purpose of this study was to test the ability of topical formulations of finasteride and flutamide to re-enlarge hair follicles in male-pattern baldness. This was evaluated by an experimental model of human scalp skin graft transplanted onto SCID mice. A comparison was made between formulations containing finasteride and flutamide, and a vehicle formulation in terms of the mean hairs per graft, length, diameter of the shafts, and structures of the growth stages of the hair. Flutamide and finasteride had a significantly higher effect (P<0.05) than the placebo in all the tested parameters, but flutamide demonstrated more hair per graft and longer hair shafts than finasteride (P<0.05). The number of hairs per graft for flutamide and finasteride groups were 1.22+/-0. 47 and 0.88+/-0.95 hairs/0.5 mm2 graft, respectively, versus 0. 35+/-0.6 hairs/graft for vehicle-treated graft. Similarly, hair lengths for flutamide and finasteride were 5.82+/-0.50 and 4.50+/-0. 32 mm, respectively, versus 2.83+/-0.18 mm for the vehicle-treated grafts. An in vitro diffusion study of flutamide gel using hairless mouse skin demonstrated the beneficial effect of the vehicle composition in comparison with a hydroalcoholic solution or a gel containing no penetration enhancer. It is therefore suggested that this topical composition containing flutamide or finasteride may effectively result in regression of male-pattern baldness.

Enantioselective pharmacokinetics of 10-hydroxycarbazepine after oral administration of oxcarbazepine to healthy Chinese subjects.

BACKGROUND AND OBJECTIVES: Oxcarbazepine is a new antiepileptic drug which in humans acts as a prodrug to its central nervous system-active metabolite 10-hydroxycarbazepine. Because 10-hydroxycarbazepine is a chiral molecule, the objective of the study was to perform a stereoselective pharmacokinetic analysis of 10-hydroxycarbazepine in humans. METHODS: The pharmacokinetics and disposition of the enantiomers of 10-hydroxycarbazepine were investigated in 12 healthy Chinese subjects. Each subject received a single oral dose of 600 mg oxcarbazepine and the concentrations of R- and S-10-hydroxycarbazepine in serum were determined by a stereoselective HPLC assay. The enantiomers of free and conjugated 10-hydroxycarbazepine and of the oxidized diol metabolite were also quantified in urine. (con 't on page 548) RESULTS: At all sampling times, the serum concentrations of S-10-hydroxycarbazepine were much higher than those of R-10-hydroxycarbazepine, and their ratio also tended to increase with time. The area under the serum concentration versus time curve of S-10-hydroxycarbazepine was about fivefold greater than that of R-10-hydroxycarbazepine (129.8 +/- 33.1 versus 26.3 +/- 8.5 mg/L x h; P < .001). Half-lives did not differ significantly between the enantiomers (11.9 +/- 3.3 hours for R-10-hydroxycarbazepine versus 13.0 +/- 4.1 hours for S-10-hydroxycarbazepine). About 27% of the molar dose of oxcarbazepine was recovered in urine, mostly as the S-enantiomer of 10-hydroxycarbazepine and its conjugates. Carbamazepine-10,11-trans-dihydrodiol accounted for less than 3% of urinary metabolites. CONCLUSIONS: The marked differences in serum levels and urinary excretion between the two enantiomers of 10-hydroxycarbazepine are likely to be related primarily to stereoselective presystemic metabolic keto-reduction of the prochiral carbonyl group of the oxcarbazepine molecule.

Influence of pharmaceutical gel vehicles containing oleic acid/sodium oleate combinations on hairless mouse skin, a histological evaluation.

Aqueous gel preparations containing oleic acid/sodium oleate combinations were applied three times daily to hairless mice (CD1 strain). Six groups of animals (n = 9 or n = 10) were treated topically with six experimental vehicles containing 2, 3 and 4.5% oleic acid (OA) at two different pH values, 7.3 and 7.7. Sodium lauryl sulfate (5%) in a similar gel preparation was used as the positive control (n = 5), while untreated animals were used as the negative control (n = 6). After three treatment days, the skin samples were collected and processed for histological evaluation. It was seen that the severity and frequency of histological changes in the skin treated with OA-containing vehicles were directly correlated with increased pH/ionization (i.e. decreased OA/sodium oleate ratio) and with overall OA concentration.

Stereoselective pharmacokinetic analysis of the antiepileptic 10-hydroxycarbazepine in dogs.

The active entity of the new antiepileptic drug, oxcarbazepine (OXC), is 10-hydroxycarbazepine (MHD). In humans, OXC undergoes rapid presystemic (first-pass) metabolic reduction to MHD. MHD is a chiral molecule with an asymmetric carbon at position 10. Previous reports have shown that in humans, the first-pass metabolic reduction of OXC into MHD is stereoselective, resulting in a 1-to-4 AUC ratio of R(-) and S(+) enantiomers. The objective of the current study was to investigate whether the pharmacokinetics of MHD was stereoselective. Racemic MHD was thus administered intravenously (i.v.) and orally to six dogs, and plasma samples were analyzed by a stereospecific, high-performance liquid chromatographic (HPLC) assay. We found that R(-)-MHD had a clearance similar to that of S(+)-MHD; however, a difference was found between the volume of distribution (Vd) and consequently, between the half-lives of the two MHD enantiomers. The main pharmacokinetic parameters of R(-)- and S(+)-MHD were as follows: A terminal half-life (t1/2) of 2.2 +/- 0.4 hours for R(-)-MHD and of 3.8 +/- 0.3 hours for S(+)-MHD; a clearance (CL) of 7.8 +/- 1.3 L/h for R(-)-MHD and of 8.6 +/- 2.1 L/h for S(+)-MHD; a Vd of 25 +/- 6 L for R(-)-MHD and of 47 +/- 14 L for S(+)-MHD; and a Vd at steady state (V(ss)) of 22.8 +/- 3.6 for R(-)-MHD and of 29.9 +/- 4.1 for S(+)-MHD. After its oral administration to dogs, the absolute bioavailability was 78.4 +/- 20.9% for R(-)-MHD and 78.5 +/- 27.3% for S(+)-MHD; t1/2 was 2.7 +/- 0.6 hours for R(-)-MHD and 4.1 +/- 0.8 hours for S(+)-MHD. These results showed stereoselectivity in the volume of distribution and consequently, the t1/2 of S(+)-MHD was longer than that of R(-)-MHD after both i.v. and oral administration to dogs.

Clinical efficacy of a degradable film-forming product containing carbamide peroxide to reduce tooth discolouration.

The methods used to lighten tooth discolouration while conserving the tooth structure have attracted much attention in a society which places a high value on esthetics. The purpose of this article is to present a home bleaching agent that uses a film-forming preparation containing carbamide peroxide (CP) which, while degrading, serves as a slow release delivery system of the bleaching agent. The efficacy of the preparation containing 3% CP and 10% CP on reduction of tooth discolouration is evaluated by a Chromameter in a double-blind placebo-controlled study. The effect of the tested bleaching agent on the gingival status, plaque accumulation and pulp sensitivity is also assessed.

Cross-linked chondroitin sulphate: characterization for drug delivery purposes.

Chondroitin sulphate was cross-linked with 1,12-diaminododecane to give a series of cross-linked products with reduced water solubility. Since the water solubility of the modified polymers is low, their chemical characterization is complicated. A new method based on the adsorption of the cationic dye, methylene blue, on the cross-linked polymers was developed to characterize the degree of cross-linking. It was found that the values of the adsorptive capacity of the adsorbate, Cp(s), decreased with the extent of cross-linking. However, the valves of the absorptive coefficient. K, and the free energy change of the absorption reaction, delta mu degrees, increased with cross-linking. The swelling of films made of the cross-linked polymers was measured in water and an exponential-like dependency between the degree of swelling and extent of cross-linking could be defined. Indomethacin tablets made of two types of cross-linked polymers--very low water soluble and relatively high water soluble--were made and analysed for their water uptake and drug release characteristics. Based on the physicochemical properties, an optimal product with a potential to serve as a colon-specific drug carrier was suggested.

Cadaverine as a putative component of oral malodor.

Whereas previous studies have shown correlations between volatile sulphur compounds (VSC) and bad breath levels, it is probable that other compounds found in the oral cavity may contribute to oral malodor. In the present investigation, the possibility that diamines (cadaverine and putrescine) are associated with oral malodor parameters was assessed. Saliva samples from 52 subjects were analyzed for cadaverine and putrescine by HPLC. Oral malodor of whole mouth, tongue, and saliva of the subjects was recorded by an experienced judge on a continuous 10-cm scale; peak and steady-state VSC intraoral levels were measured by the Interscan 1170 sulphide monitor. Log-transformed VSC and diamine levels were compared with odor judge measurements by Pearson analysis and stepwise forward multiple regression. Putrescine scores were not significantly associated with odor judge parameters or with VSC levels (p > 0.1). However, highly significant correlations (p < or = 0.003) were found between cadaverine levels and all three odor judge assessments. In contrast, associations between cadaverine and VSC measurements were non-significant. In an attempt to correlate odor judge results in terms of both VSC and diamines, we carried out stepwise forward multiple regression. Results showed that VSC and cadaverine both factor significantly in explaining each of the odor judge measurements, with multiple r values ranging from 0.545 (p = 0.0002) to 0.604 (p < 0.0001). The results suggest that cadaverine levels are associated with oral malodor, and that this association may be independent of VSC.

Inhibition of plaque formation by local application of a degradable controlled release system containing cetylpyridinium chloride.

The effect of a degradable controlled release system containing cetylpyridinium chloride (CPC) on plaque accumulation and gingivitis was evaluated when applied on the anterior teeth of volunteers (16-17 years) over 4 weeks. At baseline, plaque index (PI) and gingival index (GI) of the Ramfjord teeth were measured in the experimental and placebo groups, including 23 and 21 participants respectively. Following scaling and root planning, the participants were instructed to brush, using one brush stroke, the film-forming solution on the buccal surface of the maxillary and mandibular incisors, 1 x a day before bedtime. The applied active solution contained 9 mg of CPC (approximately 80 mg of 11% CPC solution), while the placebo solution was identical in formation, but without the active agent. After 4 weeks, in the CPC-applied group, the recorded PI scores were 0.52 (+/- 0.56) in the anterior area and 1.31 (+/- 0.80) in the posterior area, whereas the corresponding areas in the placebo group reached 1.25 (+/- 0.74) and 1.51 (+/- 1.00), respectively. The PI = 0 frequency in the buccal anterior surfaces after 4 weeks was 54.6% (+/- 38.7%) in the experimental group as compared with 21.9% (+/- 29.0%) in the placebo group (p = 0.005). In contrast to the anterior teeth, there was no significant difference between groups with respect to the PI scores in the non-applied posterior teeth. It may be postulated that the impressive 58% inhibition of plaque accumulation at the site of application is the result of an increase of the substantivity of the CPC due to its incorporation in the film-forming degradable controlled release system.

Enzymatic cleavage of disaccharide side groups in insoluble synthetic polymers: a new method for specific delivery of drugs to the colon.

A series of water-insoluble acrylic polymers containing disaccharide side groups were synthesized and evaluated in vitro. A cellobiose-derived monomer, 4-O-beta-D-glucopyranosyl-1-methacrylamido-1-deoxy-D-glucitol, was prepared and copolymerized with methacrylic acid. Two different modes of polymerization were used to give two products, P-1 and P-2. A homopolymer, P-3, was also synthesized using the same method as P-2. The degradation of the disaccharide side groups in these polymers and the monomer was evaluated by incubation with beta-glucosidase and measurement of the amount of glucose cleaved. It was found that the degradation rate increased in those polymers possessing lower contents of the disaccharide side groups (i.e. higher content of methacrylic acid). Scanning electron microscopy (SEM) observations of cross-sectioned slabs of P-1 visualized the degradation of the polymer. The enzymatic reaction caused a porous structure to be formed. The increased porosity may be used for the specific release of drugs into organs that contain large amounts of beta-glucosidases, such as the human colon.

Inhibition of plaque formation and gingivitis in beagle dogs by topical use of a degradable controlled-release system containing chlorhexidine.

The in vivo efficacy of a newly-developed dental application of a film-forming, chlorhexidine-containing system was examined in beagle dogs. A self-disintegrating film-forming solution was applied three times weekly to the dentitions of 7 out of 13 dogs, which were fed a soft-food diet. Plaque accumulation (Plaque Index) and gingival inflammation (Gingival Index) were recorded at one, two, four, six, and eight weeks. The local delivery of low-dose chlorhexidine to dogs significantly inhibited gingivitis and plaque formation. It is concluded that the dental application of a film-forming system may be a preferable method of periodontal disease prophylaxis, and may enhance supragingival plaque control in areas of isolated periodontal problems associated with obvious local predisposing factors.

Metabolism of T-2 toxin by rat brain homogenate.

HT-2 toxin was the sole metabolite formed when T-2 toxin was treated with homogenate from brain without its blood content. Homogenate from brain with its full blood content produced--besides HT-2 toxin--T-2 triol, neosolaniol, 4-deacetylneosolaniol and T-2 tetraol, i.e. the same metabolites formed by incubation of T-2 toxin with whole rat blood.

Efficacy of epicardial controlled-release lidocaine for ventricular tachycardia induced by rapid ventricular pacing in dogs.

The effects of epicardial lidocaine on ventricular tachycardia (VT) induced by rapid ventricular pacing (50 Hz) were studied in dogs. Lidocaine-polyurethane (28% wt/wt) matrixes (40-50 mg, 5 x 5 mm) were placed proximal to bipolar left ventricular epicardial electrodes in open-chest anesthetized dogs (n = 9) after VT was established by rapid ventricular pacing. In the first set of experiments, matrices were removed immediately after VT had converted to sinus rhythm. Control animals underwent VT induction protocol with a nondrug-containing matrix positioned next to the epicardial electrode. In the lidocaine-treated animals, VT conversion was noted in all animals and occurred after 51.7 +/- 8.9 s (mean +/- SE), with a VT threshold current elevation of 73.5 +/- 11.2% above baseline at the time of conversion which progressed to 259 +/- 44% of the initial value by 5.4 +/- 0.5 min post-matrix placement. Lidocaine 1.4 +/- 0.1 mg was delivered to the myocardium at the time of VT conversion (0.11 +/- 0.01 mg/kg). In comparison, accelerated VT persisted for 5 min in three of five control animals, and progressed to ventricular fibrillation (VF) in the other two animals. In a separate series of eight dogs, the lidocaine-polyurethane matrixes were left in palce for 4 h so that we could study the sustained antiarrhythmic action of controlled-release lidocaine on VT induction. The results of these experiments demonstrated a maintenance of the VT threshold elevation at a level of 53.9 +/- 10.8% after 4 h, with a net lidocaine dose of 0.52 +/- 0.06 mg/kg after 4 h of controlled release.(ABSTRACT TRUNCATED AT 250 WORDS)

Conversion of ouabain-induced ventricular tachycardia in dogs with epicardial lidocaine: pharmacodynamics and functional effects.

Epicardial antiarrhythmic drug administration was studied as a therapeutic approach for experimental ventricular tachycardia (VT) in an open-chest dog model. Lidocaine-polyurethane matrices (28%, w/w) were formulated as a model system. Matrices were placed on the left ventricular epicardium in each of 23 anesthetized open-chest dogs with ouabain-induced VT, to evaluate effectiveness in restoring sinus rhythm. Conversion occurred in all animals treated with matrices containing 300 mg or more of lidocaine after 1.5 to 7.0 min. The matrix lidocaine content correlated linearly with the time required for conversion to sinus rhythm (r = 0.75, P = 0.0002); irrespective of matrix size the myocardial/plasma lidocaine ratio was 20.1 +/- 4.2 (mean +/- SD) at the time of conversion. In a separate series of five dogs without ventricular tachycardia, systolic wall thickening measured with sonomicrometers after 5 min of controlled-release lidocaine administration (500- to 1000-mg matrix lidocaine content, 7.48 +/- 3.49-mg/kg dose) was only minimally diminished (-14.1%) and this effect was observed only at the site of matrix placement on the anterior-apical epicardium. In contrast, intracoronary injection of 0.3 or 1.0 mg/kg of lidocaine-HCl resulted in complete elimination of wall thickening or replacement by systolic thinning. Thus epicardial administration of lidocaine from polyurethane matrices was an effective means of treating ouabain-induced ventricular tachycardia. Regional myocardial function in the vicinity of the matrices was modified to a very limited degree, supporting the view that the matrices can be used safely, without serious risk to ventricular contractile performance.

Sustained behavioral recovery from unilateral nigrostriatal damage produced by the controlled release of dopamine from a silicone polymer pellet placed into the denervated striatum.

This study was conducted to determine if the behavioral asymmetry associated with unilateral nigrostriatal dopamine (DA) depletion could be alleviated by placing a small DA-releasing silicone polymer matrix pellet into the denervated striatum of rats. Animals that received DA-releasing pellets showed a 50% reduction in apomorphine-induced rotational behavior, and this effect persisted for the 2-month duration of the experiment. The results suggest that the controlled release of DA from an intrastriatal polymer matrix can produce a long-lasting reduction in some of the symptoms associated with DA depletion.

Sensitive high-performance liquid chromatographic assay using 9-fluorenylmethylchloroformate for monitoring controlled-release lidocaine in plasma.

A sensitive high-performance liquid chromatographic (HPLC) assay using fluorescence detection for quantifying lidocaine levels in plasma (in the ng/ml range) was developed. This novel HPLC assay has made possible the simultaneous monitoring of lidocaine levels in coronary and peripheral plasma obtained after myocardial controlled-release matrix administration (0.92 mg/kg during 4 h) in the arrhythmic dog. The method employed extracts the drug from plasma using 1-chlorobutane and a subsequent derivatization with 9-fluorenylmethylchloroformate in acetonitrile at 110 degrees C. The derivative was chromatographed on a C18 reversed-phase column and measured with fluorescence detection (excitation 254 nm, emission 313 nm). N-Methylephedrine was found to be suitable as an internal standard, post-derivatization. The derivatization product of lidocaine was identified and characterized by mass spectral analysis. It was found to have a unique and reproducible dicarbamate structure, which was stable for at least three days at room temperature. The method was tested with human plasma as well as on dog plasma. Analytical recoveries were 88.6 +/- 3.6 and 77.4 +/- 3.0% (mean +/- S.E.), respectively, at levels ranging from 25 to 200 ng/ml. The lower detection limit was 1 ng/ml lidocaine. In conclusion, this rapid and convenient analysis was found to be suitable for the bioavailability pharmacokinetic assessment of lidocaine following low-dose regional drug administration.