European Journal of Pharmaceutics and Biopharmaceutics (v.66, #2)
Nanoparticles – An efficient carrier for drug delivery into the hair follicles
by Juergen Lademann; Heike Richter; Alexa Teichmann; Nina Otberg; Ulrike Blume-Peytavi; Javiana Luengo; Barbara Weiß; Ulrich F. Schaefer; Claus-Michael Lehr; Roger Wepf; Wolfram Sterry (pp. 159-164).
The penetration and storage behavior of dye-containing nanoparticles (diameter 320nm) into the hair follicles was investigated. The results were compared to the findings obtained with the same amount of dye in the non-particle form.In the first part of the experiments, the penetration of the dye into the hair follicles was investigated in vitro on porcine skin, which is an appropriate model for human tissue. It was found that the nanoparticles penetrate much deeper into the hair follicles than the dye in the non-particle form, if a massage had been applied. Without massage, similar results were obtained for both formulations.Subsequently, the storage behavior of both formulations in the hair follicles was analyzed in vivo on human skin by differential stripping. Using the same application protocol, the nanoparticles were stored in the hair follicles up to 10 days, while the non-particle form could be detected only up to 4 days.Taking into consideration the surface structure of the hair follicles, it was assumed that the movement of the hairs may act as a pumping mechanism pushing the nanoparticles deep into the hair follicles.
Keywords: Particles; Hair follicles; Percutaneous penetration; Reservoir function; Drug delivery
Nanosized insulin-complexes based on biodegradable amine-modified graft polyesters poly[vinyl-3-(diethylamino)-propylcarbamate- co-(vinyl acetate)- co-(vinyl alcohol)]-graft-poly(l-lactic acid): Protection against enzymatic degradation, interaction with Caco-2 cell monolayers, peptide transport and cytotoxicity
by Michael Simon; Isabel Behrens; Lea Anne Dailey; Matthias Wittmar; Thomas Kissel (pp. 165-172).
Non-parenteral insulin delivery by the oral route is limited by epithelial barriers and enzymatic degradation. Nanosized insulin-complexes based on amine modified comb-like polyesters were designed to overcome these barriers. Protection of insulin in nanocomplexes (NC) from enzymatic degradation was investigated. The interaction with enterocyte-like Caco-2 cells in terms of cytotoxicity, transport through and uptake in the cell layers was evaluated by measuring transepithelial electrical resistance (TEER), release of lactate dehydrogenase (LDH) and insulin transport.The protection capacity of NC and their interaction with Caco-2 cells varied strongly as a function of lactide-grafting (hydrophobicity). With increasing lactide-grafting (P(26)⩽P(26)-1LL⩽P(26)-2LL) NC protected up to 95% of the insulin against degradation by trypsin. Transport and uptake into cell monolayers increased with higherl-lactid grafting. About 25% of a 1.25mg/ml TRITC-insulin NC dispersion with P(26)-2LL was recovered in Caco-2 cells after 90min. A concentration dependent cytotoxicity profile was observed showing elevated LDH release and decreased TEER values. The cytotoxicity correlates with the surfactant like character of the polymers, decreasing the surface tension to 46mN/m for the amphiphilic P(26)-2LL. The observed TEER decrease was reversible after 20h, suggesting that the biodegradable comb-polyesters offer a promising approach to overcome mucosal barriers.
Keywords: Insulin; Nanocomplexes; Protein delivery; Enzymatic degradation; Caco-2 cells; Cytotoxicity
Correlation between digestion of the lipid phase of smedds and release of the anti-HIV drug UC 781 and the anti-mycotic drug enilconazole from smedds
by C. Goddeeris; J. Coacci; G. Van den Mooter (pp. 173-181).
The present studies were conducted primarily to compare the drug release process of the anti-HIV drug UC781 from three different smedds to the smedds digestion profile. The influence of every formulation component on the digestion process, measured as the release of fatty acids, was determined. In addition, the release of the antimycotic drug enilconazole from a smedds was investigated as well in order to study the influence of the type of incorporated drug on oil digestion. Simulsol 1292, Tween 80, Cremophor RH40, ethanol and both drugs reduced the fatty acid release. C8, C10 and C12 fatty acids, originating from oil hydrolysis, were able to reverse the inhibitory effect of phospholipids present in the release medium. Similarly Cremophor RH40 lost its inhibitory capacities in combination with Captex 200P hydrolysis. In addition, UC781 did not decrease fatty acid release in combination with a Captex 200P–Tween 80–ethanol mixture.The release of UC781 from smedds significantly increased compared to the dissolution of the pure drug. The drug release profiles were characterized by rapid and complete release followed by precipitation. In order to detect possible correlations between drug release and oil digestion, release results were compared to those of vehicle digestion experiments. Contrary to what one would assume, a higher extent of fatty acid liberation did not enhance drug release. In other words, drug release does not seem to be driven by the extent of lipid digestion.
Keywords: Smedds; Drug release; Oil digestion; Fatty acid liberation
Synthesis, characterization and in vitro cytotoxicity studies of a macromolecular conjugate of paclitaxel bearing oxytocin as targeting moiety
by Gennara Cavallaro; Laura Maniscalco; Monica Campisi; Domenico Schillaci; Gaetano Giammona (pp. 182-192).
The present study describes the experimental synthetic procedure and the characterization of a new polyaspartamide macromolecular prodrug of paclitaxel, bearing oxytocin residues as targeting moieties. In vitro stability studies of bioconjugate, performed in media mimicking biological fluids (buffer solutions at pH 7.4 and 5.5) and in human plasma, evidenced the high stability of the targeting portion (oxytocin)-polymer linkage and the ability of this conjugate to release linked paclitaxel in a prolonged way in plasma. Moreover, preliminary in vitro antiproliferative studies, carried out on MCF-7 cells, that are oxytocin receptor positive cells, showed that the polymeric conjugate has the same cell growing inhibition ability of free drug.
Keywords: Polyaspartamide; Oxytocin; Drug targeting; Paclitaxel; Bioconjugate
Development of spherical iron(II) sulfate heptahydrate-containing solid particles with sustained drug release
by Piroska Szabó-Révész; Béla Farkas; Tamás Gregor; Kálmán Nagy; Edina Pallagi (pp. 193-199).
The aim of this work was to develop a simple, economic procedure for the manufacturing of coated iron(II) sulfate particles by using a crystallization technique for the development of round particles, followed by coating with a lipophilic material. Several batches of iron(II) sulfate heptahydrate were produced by a cooling crystallization, with variation of the crystallization parameters. The spherical crystals were coated with stearin. The products were characterized for particle size, roundness, bulk density and in vitro drug dissolution. Crystallization was performed from deionized water with no addition of seed crystals and by cooling by applying a linear cooling rate. The developed iron(II) sulfate crystals were round with average diameter of 729±165μm. The best form for the sustained release of iron(II) sulfate was the sample HTP-2 which contained 11% of stearin relative to the iron(II) sulfate. The spherical crystallization of iron(II) sulfate is simple and fast, and does not require a dangerous, expensive solvent. The round particles can coat directly with lipophilic material which results in slow release of iron(II) sulfate and protects the iron(II) from oxidation and inhibits the loss of crystal water. The coated crystals can be filled into capsules to yield the final dosage form.
Keywords: Spherical crystallization; Iron(II) sulfate; Stearin; Sustained release; Roundness
Two galactomannans and scleroglucan as matrices for drug delivery: Preparation and release studies
by Tommasina Coviello; Franco Alhaique; Antonello Dorigo; Pietro Matricardi; Mario Grassi (pp. 200-209).
Two galactomannans, Guar gum and Locust bean gum, have been used as matrices for tablets to study the release of model molecules. As a comparison, matrices obtained with another polysaccharide, Scleroglucan, have been tested. Despite the different conformations that the polymers assume in aqueous solution (flexible coils for Guar gum and Locustbeangum; triple helix for Scleroglucan), when prepared as tablets, they show (in distilled water and at 37°C) very similar release profiles of guest molecules (i.e. theophylline, vitamin B12 and myoglobin) of different steric hindrance. Furthermore, the polymers were chemically crosslinked with glutaraldehyde to obtain a network suitable as a matrix for modified drug release. The delivery of the model molecules from the Guar gum and Locust bean gum gels, and from tablets prepared from the freeze-dried hydrogels of the three polymers was evaluated, and a comparison with the tablets prepared with the not-crosslinked polymers was carried out. Experimental data showed how the presence in the matrix of a well-defined network, by introducing a spacer among the macromolecular chains, always increased the rate of delivery of the tested molecules in comparison to the release profiles obtained when no crosslinker was present. Release data from the tablets were analyzed according to a mathematical model able to determine the relative importance of drug dissolution and drug diffusion on the overall release kinetics. Good agreement was found between the simulated and the experimental data.
Keywords: Galactomannans; Guar gum; Locust bean gum; Scleroglucan; Glutaraldehyde; Theophylline; Vitamin B12; Myoglobin; Modified release
Synchronized and sustained release of multiple components in silymarin from erodible glyceryl monostearate matrix system
by Cheng Lu; Yi Lu; Jian Chen; Wentong Zhang; Wei Wu (pp. 210-219).
Development of sustained delivery systems for herbal medicines was very difficult because of their complexity in composition. The concept of synchronized release from sustained release systems, which is characterized by release of multiple components in their original ratio that defines a herbal medicine, served as the basis for keeping the original pharmacological activity. In this study, erodible matrix systems based on glyceryl monostearate and polyethylene glycol 6000 or poloxamer 188 were prepared to perform strict control on synchronized release of the five active components of silymarin, i.e. taxifolin, silychrystin, silydianin, isosilybin and silybin. The matrix system was prepared by a melt fusion method. Synchronized release was achieved with high similarity factor f2 values between each two of the five components. Erosion profiles of the matrix were in good correlation with release profiles of the five components, showing erosion-controlled release mechanisms. Through tuning some of the formulation variables, the system can be adjusted for synchronized and sustained release of silymarin for oral administration. In vitro hemolysis study indicated that the synchronized release samples showed a much better stabilizing effect on erythrocyte membrane.
Keywords: Abbreviations; TF; taxifolin; SC; silychristin; SD; silydianin; ISB; isosilybin; SB; silybin; GMS; glyceryl monostearate; PEG; polyethylene glycol; PXM; poloxamer; HPMC; hydroxypropyl methylcellulose; SDS; sodium dodecyl sulfateSynchronized release; Sustained release; Glyceryl monostearate; Polyethylene glycol 6000; Poloxamer 188; Silymarin; Erosion
Preparation and characterization of a self-emulsifying pellet formulation
by Ahmed Abdalla; Karsten Mäder (pp. 220-226).
The purpose of the current study is to investigate the feasibility of producing solid self-emulsifying pellets using the extrusion/spheronization technique. Pellets were made from a mixture of C18 partial glycerides, Solutol® HS15 and microcrystalline cellulose. Pellets with good physical properties (size, shape, friability) and self-emulsifying properties were produced. The pellets were, in contrast to pellets lacking Solutol, able to transfer a lipophilic dye and a spin probe into the aqueous media. The release kinetics and the microenvironment of the pellets during the release process were assessed using electron spin resonance (ESR) spectroscopy. The ESR results showed that the hydrophobic spin probe was localized mainly in the lipid environment all over the release time. Furthermore, the formulation was capable of accelerating the release of the drug diazepam and achieving a diazepam concentration above its saturation solubility.In conclusion, spherical pellets with low friability and self-emulsifying properties can be produced by the standard extrusion/spheronization technique. The pellets are capable of transfering lipophilic compounds into the aqueous phase and have a high potential to increase the bioavailability of lipophilic drugs.
Keywords: Pellets; Extrusion/spheronization; Self-emulsifying drug delivery systems; Poorly water soluble drugs; Oral delivery system; ESR
Development and bioavailability assessment of ramipril nanoemulsion formulation
by Sheikh Shafiq; Faiyaz Shakeel; Sushma Talegaonkar; Farhan J. Ahmad; Roop K. Khar; Mushir Ali (pp. 227-243).
The objective of our investigation was to design a thermodynamically stable and dilutable nanoemulsion formulation of Ramipril, with minimum surfactant concentration that could improve its solubility, stability and oral bioavailability. Formulations were taken from the o/w nanoemulsion region of phase diagrams, which were subjected to thermodynamic stability and dispersibility tests. The composition of optimized formulation was Sefsol 218 (20% w/w), Tween 80 (18% w/w), Carbitol (18% w/w) and standard buffer solution pH 5 (44% w/w) as oil, surfactant, cosurfactant and aqueous phase, respectively, containing 5mg of ramipril showing drug release (95%), droplet size (80.9nm), polydispersity (0.271), viscosity (10.68cP), and infinite dilution capability. In vitro drug release of the nanoemulsion formulations was highly significant ( p<0.01) as compared to marketed capsule formulation and drug suspension. The relative bioavailability of ramipril nanoemulsion to that of conventional capsule form was found to be 229.62% whereas to that of drug suspension was 539.49%. The present study revealed that ramipril nanoemulsion could be used as a liquid formulation for pediatric and geriatric patients and can be formulated as self-nanoemulsifying drug delivery system (SNEDDS) as a unit dosage form.
Keywords: Nanoemulsion; Ramipril; Bioavailability; SNEDDS; Phase diagrams; Solubility; Sefsol 218
Tetronic micellization, gelation and drug solubilization: Influence of pH and ionic strength
by Carmen Alvarez-Lorenzo; Jaime Gonzalez-Lopez; Marta Fernandez-Tarrio; Isabel Sandez-Macho; Angel Concheiro (pp. 244-252).
The aim of this work was to gain an insight into the self-associative processes and drug solubilization ability of a Tetronic variety, T904 (4×15 EO units; 4×17 PO units; HLB 15), in aqueous media covering the physiological range of pH and ionic strength, applying isoperibol microcalorimetry, transmission electronic microscopy (TEM), dynamic light scattering (DLS), oscillatory rheometry, and drug diffusion experiments. T904 shows two p Ka (p Ka1=4.0 and p Ka2=7.9) and, at pH<5.8, the diprotonated form predominates over the non-protonated one. Deprotonization of the central diamine group is a required condition for micellization, which is an endothermic entropy-driven process owing to hydrophobic interactions between the PPO chains. As the pH of the solutions decreases, the coulombic repulsions among the positively charged amine groups make the aggregation more difficult, raising the critical micellar concentration (CMC) and decreasing the size of the micelles. The changes in the conformation and hydrophilicity of the Tetronic were reflected in its gelation temperature (around 30°C at neutral-alkaline pH; no gelation at pH<2) and solubilization capacity for griseofulvin (2-fold greater at neutral-alkaline pH than at pH<2) and rate of diffusion (slower at pH 7.4). Such alterations in self-assembly are relevant when using Tetronic in the design of drug delivery systems.
Keywords: Poloxamine surfactant; Polymeric micelles; Micellization enthalpy; Gel temperature; pH-sensitive micellization; Griseofulvin solubilization
Use of calcined Mg–Al–hydrotalcite to enhance the stability of celecoxib in the amorphous form
by Valeria Ambrogi; Luana Perioli; Fabio Marmottini; Carlo Rossi (pp. 253-259).
The use of compound in amorphous forms is a promising approach to improve solubility of poorly water-soluble drugs. However, during storage, amorphous state can spontaneously transform to the lower energy crystalline form and this is a limiting factor for a large commercial use of drugs.In this paper, calcined hydrotalcite was employed to support amorphous celecoxib and several preparations at different weight drug/carrier ratio were prepared. Solubility of celecoxib from the prepared systems was evaluated and its physical stability during storage at different conditions was examined as well. The results show that HTlc-calc can be used as a support of amorphous celecoxib with consequent improvement of drug solubility and physical stability.
Keywords: Celecoxib; Amorphous form; Solubility; Physical stability
Synthesis and properties of N-nicotinoyl-2-(5-fluorouracil-1-yl)-d,l-glycine ester as a prodrug of 5-fluorouracil for rectal administration
by Young Wook Yang; Jeoung Soo Lee; Inho Kim; Yun Jin Jung; Young Mi Kim (pp. 260-267).
N-Nicotinoyl-2-(5-fluorouracil-1-yl)-d,l-glycine (NFG) methyl-(NFGM), ethyl-(NFGE) and isopropyl esters (NFGIp) were synthesized and their potential as a prodrug of 5-fluorouracil (5-FU) for rectal administration was investigated. Chemical conversion proceeded either by elimination of (5-FU) or by hydrolysis of ester group. 5-FU was released from NFGIp, NFGE and NFGM 90.5%, 71.3% and 48.5% of the dose, respectively, in 80% human plasma and 79.8%, 56.3% and 31.6%, respectively, in pH 7.4 buffer solution after 48h of incubation at 37°C. Release of 5-FU occurred mainly from NFG esters but very slightly from NFG, which suggested that release of 5-FU was greatly dependent on the stability of the ester group against hydrolysis. Solubility ( M) in pH 7.4 buffer solution was 0.13, 0.09 and 0.04 and apparent partition coefficient in 1-octanol/pH 7.4 buffer solution was 0.76, 1.61 and 4.2, respectively, for NFGM, NFGE and NFGIp, which were in the ranges suitable for rectal absorption. Plasma concentration (μg/mL) of NFGM, NFGE and NFGIp at 50min after rectal administration to rats was 1.9, 4.6 and 6.7, respectively, and that for 5-FU was below the limit of detection. Their potential as prodrugs of 5-FU for rectal administration is suggested.
Keywords: N; -Nicotinoyl-2-(5-fluorouracil-1-yl)-; d; ,; l; -glycine esters; 5-Fluorouracil prodrug; 5-Fluorouracil; Rectal administration; Rectal absorption
Effect of poly(ethylene glycol)-block-polylactide nanoparticles on hepatic cells of mouse: Low cytotoxicity, but efflux of the nanoparticles by ATP-binding cassette transporters
by Yangde Zhang; Zhiyuan Hu; Maoying Ye; Yifeng Pan; Jiji Chen; Yulin Luo; Yanqiong Zhang; Lianxiang He; Jiwei Wang (pp. 268-280).
The objective of this paper is to study the effects of poly(ethylene glycol)-block-polylactide (PLA–PEG) nanoparticles on hepatic cells of mouse. Blank PLA–PEG nanoparticles have been successfully prepared and MTT assay suggested that the nanoparticles with HepG2 cell co-culture model did not cause significant changes in membrane integrity in controlled concentration range (0.001–0.1mg/ml). Immunohistochemical analysis demonstrated that large dose of PLA–PEG nanoparticles injection (42.04mg/kg, i.v.) did not induce hepatic cell apoptosis. From biochemical assay experiments, although the levels of SOD decreased and those of MDA, NOS increased after treatment with large dose of PLA–PEG nanoparticles injection (42.04mg/kg, i.v.), they were all not significant ( p>0.05). Then Kunming mice were treated with large dose of PLA–PEG nanoparticles (42.04mg/kg, i.v.) and after 4 days total RNA was isolated to elucidate patterns of gene expression using a mouse cDNA-microarray (SuperArray). Treatment with nanoparticles resulted in over-expression of a lot of ATP-binding cassette (ABC) transporters, especially two ABC transporters (ABCA8 and ABCC5/MRP5), and down-regulation of GSTP1, in comparison with the control. ABCA8 could extrude low molecular weight polymers after PLA–PEG nanoparticles hydrolysis outside the cells. We also discovered that ABCC5 expressed multidrug resistance protein 5 (MRP5) to pump out conjugate (GS-X) of PLA–PEG nanoparticles with GSH. The results were confirmed by RT-PCR. Results of in vitro accumulation and efflux experiments indicated that about 51–52% (51.5% and 52.0%) intracellular PLA–PEG nanoparticles was expulsed after mouse primary hepatocytes reached a saturation uptake of nanoparticles during the concentration range of 750–1000μg/ml. The results suggested that ABC transporters (especially ABCA8) pump out the polymers after hydrolysis from mouse hepatic cells and large dose of PLA–PEG nanoparticles make mouse hepatic cells gain drug resistance to PLA–PEG nanoparticles.
Keywords: Abbreviations; SOD; superoxide dismutase; MDA; malondialdehyde; NOS; nitric oxide synthases; NO; nitric oxide; GSH; glutathione; GS-X; GSH conjugate; MDR; multidrug resistance; P-gp; P-glycoprotein; ABC; ATP-binding cassettePLA–PEG nanoparticles; ATP-binding cassette transporter; Transport; Efflux; Drug resistance
Fluorescein transport properties across artificial lipid membranes, Caco-2 cell monolayers and rat jejunum
by Katja Berginc; Simon Žakelj; Lea Levstik; Darko Uršič; Albin Kristl (pp. 281-285).
Membrane transport characteristics of a paracellular permeability marker fluorescein were evaluated using artificial membrane, Caco-2 cell monolayers and rat jejunum, all mounted in side-by-side diffusion cells. Modified Ringer buffers with varied pH values were applied as incubation salines on both sides of artificial membrane, cell culture monolayers or rat jejunum. Passive transport according to pH partition theory was determined using all three permeability models. In addition to that, active transport of fluorescein in the M–S (mucosal-to-serosal) direction through rat jejunum was observed. The highest M–S Papp values regarding the active transport through the rat jejunum were observed in incubation saline with pH 6.5. Fluorescein transport through the rat jejunum was inhibited by DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid) and α-CHC (α-cyano-4-hydroxycinnamic acid). Thus, we assume that two pH-dependent influx transporters could be involved in the fluorescein membrane transport through the intestinal (jejunal) epithelium.One is very likely an MCT (monocarboxylic acid cotransporter) isoform, inhibited by specific MCT inhibitor α-CHC, while the involvement of the second one with overlapping substrate/inhibitor specificities (most probably a member of the organic anion-transporting polypeptide family, inhibited at least partially by DIDS) could not be excluded.
Keywords: Fluorescein; Fluorescein uptake transporter; MCT (monocarboxylic acid cotransporter); DIDS; α-CHC; Mucolytic agents; pH partition theory
Permeability assessment for solid oral drug formulations based on Caco-2 monolayer in combination with a flow through dissolution cell
by Stephan A. Motz; Ulrich F. Schaefer; Stefan Balbach; Thomas Eichinger; Claus-Michael Lehr (pp. 286-295).
The aim of our study was to develop an apparatus assessing in vitro permeation through Caco-2 monolayers of oral solid dosage forms as a possible tool to forecast in vivo performance. Therefore, flow through dissolution and permeation modules were connected by means of a stream splitter. Permeation was measured in a specially designed cell, dissolution took place in the apparatus 4, USP. In order to test the apparatus for its reproducibility and conclusiveness, different tablet strengths and varying release profiles of propranolol HCl tablets were produced and evaluated. It was shown that for both tablet species, immediate and extended release, the apparatus was able to measure permeation through Caco-2 monolayer as well as dissolution simultaneously with high precision and reproducibility. The permeated amount of the three immediate release tablets with increasing dosage strength showed linear dependency on the dosage strength. Furthermore, the effect of retarded release on permeation could be detected and conclusive data for dissolution and permeation were obtained. In summary, connecting cell culture based permeability assessment with compendial flow through dissolution equipment led to promising results and poses the base for more advanced studies for detecting influences of dosage forms on permeation process.
Keywords: Oral absorption; Caco-2 cells; Dissolution; Permeation; Propranolol HCl; Biopharmaceutical classification system (BCS)
In vitro and in vivo evaluation of the transdermal iontophoretic delivery of sumatriptan succinate
by Sonal R. Patel; Hui Zhong; Ashutosh Sharma; Yogeshvar N. Kalia (pp. 296-301).
The objective was to evaluate the transdermal delivery of the 5-HT1B/1D agonist, sumatriptan from an iontophoretic patch system, in vivo. Initial in vitro experiments were conducted to optimize formulation parameters prior to iontophoretic delivery in Yorkshire swine. It was found in vitro that increasing drug load in the patch from 9.7 to 39mg had no statistically significant effect on cumulative delivery (cf. 305.6±172.4 vs. 389.4±80.4μgcm−2, respectively). However, for a given drug load (39mg) increasing formulation pH from pH 4.7 to 6.8 significantly increased the cumulative amount of sumatriptan delivered across the skin (389.4±80.4 vs. 652.4±94.2μgcm−2). A biphasic current profile comprising intensities of 1.8mA from t=0 to t=180min and 0.8mA from t=181min to t=360min was used for the in vivo experiments. Drug levels in the blood were 13.7±4.5 and 53.6±10.2ngml−1 at the 30 and 60min time-points, rising to 90–100ngml−1 during the 90–180min time-period. The in vivo results show that the pharmacokinetics following transdermal iontophoretic delivery are comparable to those after oral, nasal or rectal administration, but do not match those upon subcutaneous injection.
Keywords: Transdermal; Iontophoresis; Migraine; Sumatriptan; 5HT; 1B/1D; agonist; Non-invasive; In vivo
by Peter Kleinebudde (pp. 302-302).