International Journal of Pharmaceutics (v.276, #1-2)
Title page (iii).
Effect of lactide/glycolide monomers on release behaviors of gentamicin sulfate-loaded PLGA discs by Je Young Yoo; Jong Min Kim; Gilson Khang; Moon Suk Kim; Sun Hang Cho; Hai Bang Lee; Yong Sik Kim (1-9).
In order to develop the desirable drug release patterns such as no lag time and exact zero-order release rate, gentamicin sulfate (GS)-loaded poly(d,l-lactide-co-glycolide) (PLGA) discs containing lactide monomer (LM) or glycolide monomer (GM) were prepared. LM or GM was applied for the controlling drug release pattern due to its non-toxic and biodegradable nature. Water absorption, mass loss, pH change, and morphology of discs were examined to study the effect of LM or GM addition. GS release showed near zero-order profile in the GS-loaded polymeric discs prepared in the presence of LM or GM (10%). The channel of GS-loaded PLGA containing LM or GM was formed by the dissolution of LM or GM. Water uptake of disc increased till 21 days from the beginning of the test. The pH variations of media declined in the same manner with the result of mass loss. The antibiosis of GS was also confirmed by bacterial inhibition zone test using the prepared polymeric discs. From these results, we expected that the polymeric discs containing LM or GM would be a good dosage form as a topically implantable device which can get rid of lag period from PLGA matrix.
Keywords: Gentamicin sulfate; Polymeric disc; LM or GM; Antibiosis; Controlled release; Zero-order;
Transbuccal permeation, anti-inflammatory activity and clinical efficacy of piroxicam formulated in different gels by M.A Attia; I El-Gibaly; S.E Shaltout; G.N Fetih (11-28).
In attempts to avoid the systemic side effects of piroxicam (PC) (e.g. gastrotoxicity), several buccal gel formulations containing PC were prepared and their effects on the characteristics of the drug permeation through rabbit buccal mucosa in-vitro were evaluated using a Franz-type diffusion cell. The general rank order of the total flux of 0.5% PC from gels was found to be: hydroxypropylmethylcellulose (HPMC, 2.5%) > hydroxypropylcellulose (HPC, 2.5%) ≥ sodium alginate (Na alg., 7%) > methylcellulose (MC, 3%) > hydroxyethylcellulose (HEC, 1.5%) > carbopol 934 (Carb. 934, 1%) ≥ sodium carboxymethylcellulose (NaCMC, 2%) > pluronic F-127 (PF-127, 20%) > polyvinyl alcohol (PVA, 10%). The effect of various penetration enhancers 1% sodium lauryl sulphate (NaLS), 3% sodium deoxycholate (NaDC), 3% sodium tauroglycocholate (NaTGC) on the rate of permeation across the excised buccal mucosa (of 0.5% PC in gels prepared using 3% MC, 2.5% HPMC or 7% Na alg. base) and histology of the buccal epithelium was also investigated. Pharmacodynamic evaluation of the anti-inflammatory activity of PC in these gel formulations (containing 3% NaDC as an enhancer) was carried out using the kaolin-induced rat paw oedema method. The results obtained indicated that PC administered in 7% Na alg. or 2.5% HPMC gel bases was significantly more effective than the 3% MC gel and oral drug solution in suppressing oedema formation in rats. Comparative clinical studies were conducted in patients with post-operative dental pain and oedema following maxillofacial operations. The results revealed that 7% Na alg. and 2.5% HPMC gel formulations applied to the buccal mucosa were slightly better than or equally effective to the orally administered commercial product (Feldene Flash® tablet) in reducing pain level, swelling and tenderness within a period of 4 days. These findings suggest that PC (0.5%) administered in the buccal gel may present a potential therapeutical use as a strong anti-inflammatory and analgesic agent.
Keywords: Piroxicam; Rabbit buccal mucosa; Buccal permeability; Penetration enhancer; Rat paw oedema;
A rabbit ear flap perfusion experiment to evaluate the percutaneous absorption of drugs by Toshinobu Seki; Osamu Hosoya; Tsuyoshi Yamazaki; Takeshi Sato; Yuko Saso; Kazuhiko Juni; Kazuhiro Morimoto (29-40).
A rabbit ear flap single-pass perfusion system was examined as an experimental method for studying the relationship between the physiological conditions of tissues and drug disposition after topical applications. Tyrode solutions containing bovine serum albumin (BSA) and sucrose or flurbiprofen (FP), used as a model drug, were perfused through the vessel in the ear flap to evaluate the physiological conditions prior to the application of FP to the skin surface. The extracellular volume and distribution properties of FP in the perfused ear were similar to those in an in vivo experimental system. In addition, the perfused ear flap exhibited a pharmacological response to bradykinin (BK). The amount of FP in the outflow Tyrode solution containing BSA after application to the skin surface of the perfused ear decreased with the addition of BK, while that in the tissues under the application site increased. FP binds to BSA, which leaked from the intravascular space, and could be retained in the tissues under the application site. The protein binding also affected the redistribution of FP to other tissues in the ear flap after application to the skin. The rabbit ear perfusion system is a useful method for studying the percutaneous absorption of drugs especially variations in the disposition of drugs in oedematous tissues.
Keywords: Rabbit ear; Perfusion; Flurbiprofen; Bradykinin; Topical application; Protein binding;
Comparative bioavailability of rifampicin, isoniazid and pyrazinamide from a four drug fixed dose combination with separate formulations at the same dose levels by Shrutidevi Agrawal; Inderjit Singh; Kanwal Jit Kaur; Shantaram R Bhade; Chaman Lal Kaul; Ramesh Panchagnula (41-49).
Fixed dose combination (FDC) formulations became popular in the treatment of tuberculosis (TB) because of the better patient compliance, reduced risk of monotherapy and emergence of drug resistance in contrast to treatment with separate formulations of two to four first-line drugs. However, its successful implementation in national programs is limited by probable bioinequivalency of rifampicin if present in FDC form. In this regard, World Health Organization (WHO) and International Union Against Tuberculosis and Lung Disease (IUATLD) recommend FDCs only of proven bioavailability. Hence, bioequivalence study of four drug FDC tablet was conducted using 22 healthy male volunteers according to WHO recommended protocol to determine bioavailability of rifampicin, isoniazid and pyrazinamide compared to standard separate combination at the same dose level. The study was designed as two period, two treatment crossover experiment with a washout period of 1 week. Bioequivalence of rifampicin was estimated by plasma and urinary method for both rifampicin and its active metabolite, des-acetyl rifampicin whereas isoniazid and pyrazinamide were estimated from plasma. Mean concentration time profiles and all the pharmacokinetic parameters of rifampicin, isoniazid and pyrazinamide from FDC tablet were comparable to individual formulations and passed the bioequivalence test with power of the test above 95%. Further, bioequivalence of both rifampicin and isoniazid shows that in vitro interaction of rifampicin and isoniazid is clinically insignificant. Thus, it was concluded that FDC formulation is bioequivalent for rifampicin, isoniazid and pyrazinamide and ensures the successful treatment of TB without compromising therapeutic efficacy of any of these components of anti-TB therapy.
Keywords: Bioavailability; Bioequivalence; Fixed dose combination; Isoniazid; Pyrazinamide; Rifampicin; Tuberculosis;
In situ evaluation of drug-loaded microspheres on a mucosal surface under dynamic test conditions by Sandra Kockisch; Gareth D. Rees; Simon A. Young; John Tsibouklis; John D. Smart (51-58).
The ability of polymeric microspheres fabricated from Carbopol, polycarbophil, chitosan or Gantrez to retain a model hydrophilic drug (sodium fluorescein) was evaluated in situ, using a dynamic test system and image analysis. This technique used oesophageal tissues and simulated the physiological conditions within the oral cavity in terms of temperature, humidity and saliva flow. The point of sample application was observed over a 2 h period by means of a digital camera. No significant differences in fluorescein colour intensity was obtained for the Gantrez and chitosan particles over 100 min, indicate that these two polymers provide the possibility of prolonged action. Carbopol and polycarbophil particles became rapidly swollen and released the sodium fluorescein completely within 20 min. It was concluded that the test system allowed the evaluation of the in situ behaviour of test particles, in terms of their ability to retain a water-soluble, coloured marker in ‘dynamic’ test conditions, and that chitosan and Gantrez were promising candidates for the production of mucoadhesive, sustained-release microspheres for water-soluble materials.
Keywords: Bioadhesion; Mucoadhesion; Oral cavity; Chitosan; Poly(acrylic acid); Maleic anhydride copolymers;
Drug release from carbomer:carbomer sodium salt matrices with potential use as mucoadhesive drug delivery system by Juan M Llabot; Ruben H Manzo; Daniel A Allemandi (59-66).
In vitro mucoadhesion, water uptake, and drug release of nystatin (N) from matrices of carbomer (C) and lyophilized carbomer sodium salt (CNaL) mixtures were evaluated. Matrices with different ratios C:CNaL were prepared by direct compression. Commercial C as well as lyophilized powder (CL) were used. In vitro mucoadhesion increased as the proportion of C in the matrix was raised. The same effect was observed when C was replaced by CL. Matrices in which C was replaced by CL showed an increase of both water uptake and release rates. Besides, the release of N from matrices CL:CNaL exhibited a kinetics with Super Case II (n>1) mechanism. However, for C:CNaL matrices, drug release was slower and exhibited a biphasic profile with a first stage characterized by either an anomalous (n<1, for C≥50%) or a Case II (n ∼ 1.0, C<50%) mechanisms. After that period, the mechanism changed to Super Case II transport (n>1).
Keywords: Drug release; Carbomer; Mucoadhesive tablets; Nystatin; Water uptake; Super Case II transport;
Cell type-specific gene expression, mediated by TFL-3, a cationic liposomal vector, is controlled by a post-transcription process of delivered plasmid DNA by Wenhao Li; Tatsuhiro Ishida; Rieko Tachibana; Mohamad Radwan Almofti; Xinyu Wang; Hiroshi Kiwada (67-74).
The issue of whether the TFL-3, a recently developed cationic liposome, achieves efficient gene expression in different mammalian cell lines (NIH/3T3, LLC, A431 and HeLa cells) was examined. The issue of whether gene expression is related to the amount of plasmid DNA (pDNA) delivered in cells or nuclei following transfection was also examined. The cells were transfected for 1 h with pDNA/TFL-3 lipoplexes, and the transfection efficiency was determined by means of a luciferase activity assay. The amount of intracellular and intranuclear pDNA following the transfection was also quantitatively determined. Successful transgene expressions in all cell lines we tested were observed under our experimental conditions, suggesting that the TFL-3 represents a suitable nonviral vector system for the successful gene expression in mammalian cells in vitro. The degree and rate of gene expression were dependent on the type of cells used as well as the incubation time after transfection, but these parameters were independent of the amount of gene delivered to cells and nuclei. These results suggest that TFL-3 mediated gene expression is largely controlled by the process of post-transcription of the delivered pDNA, and not by the process of cellular entry of pDNA and cytoplasmic trafficking of pDNA into nuclei, which is dependent on the cell type. Therefore, the results obtained here clearly suggest that the cell type-specific improvement in transcription efficiency of pDNA and translation of the derived mRNA, together with an improved delivery system to enhance the nuclear delivery of pDNA, is necessary to achieve efficient transgene expression in mammalian cells.
Keywords: Gene delivery; Cationic liposome; Lipoplex; DNA; Transfection;
Pulmonary delivery of TH9507, a growth hormone releasing factor analogue, in the dog by Mendel Jansen; Ian Darby; Thierry Abribat; Pascal Dubreuil; Eckhardt S Ferdinandi; John G Hardy (75-81).
A modified growth hormone releasing factor (GRF; TH9507), a 44 amino acid peptide analogue of natural human growth hormone releasing factor, is being developed for the treatment of age-associated conditions resulting from diminished growth hormone (GH) secretion. The inhalation route of administration is being considered as an alternative to subcutaneous injection. A study was undertaken in dogs to investigate the absorption of TH9507 following pulmonary delivery. Male beagle dogs were administered TH9507 by intratracheal dry powder insufflation and subcutaneous injection at doses of approximately 375 and 38 μg/kg, respectively. In a separate study, male and female dogs received 100 μg/kg intravenously. Blood samples were collected at selected sampling times after dosing and plasma levels of TH9507 were measured by radioimmunoassay. The bioavailability by the inhaled route was 41% relative to subcutaneous dosing, with an absolute bioavailability estimated at 13%. No significant difference was observed for the terminal half-life of TH9507 after intratracheal (39 min) and subcutaneous (26 min) administrations. The mean residence time (MRT) was greater following intratracheal administration (74 min versus 52 min; P<0.01). These data indicate that the delivery of the TH9507 by the inhalation route may provide a suitable alternative to subcutaneous injection.
Keywords: Powder inhalation; Bioavailability; Peptide; hGRF;
A novel in situ gel for sustained drug delivery and targeting by Sudipta Ganguly; Alekha K Dash (83-92).
The objective of this study was to develop a novel chitosan-glyceryl monooleate (GMO) in situ gel system for sustained drug delivery and targeting. The delivery system consisted of 3% (w/v) chitosan and 3% (w/v) GMO in 0.33 M citric acid. In situ gel was formed at a biological pH. In vitro release studies were conducted in Sorensen’s phosphate buffer (pH 7.4) and drugs were analyzed either by HPLC or spectrophotometry. Characterization of the gel included the effect of cross-linker, determination of diffusion coefficient and water uptake by thermogravimetric analysis (TGA). Mucoadhesive property of the gel was evaluated in vitro using an EZ-Tester. Incorporation of a cross-linker (glutaraldehyde) retarded the rate and extent of drug release. The in vitro release can further be sustained by replacing the free drug with drug-encapsulated microspheres. Drug release from the gel followed a matrix diffusion controlled mechanism. Inclusion of GMO enhanced the mucoadhesive property of chitosan by three- to sevenfold. This novel in situ gel system can be useful in the sustained delivery of drugs via oral as well as parenteral routes.
Keywords: Chitosan; Glyceryl monooleate; Mucoadhesion; Swelling; Cross-linking;
Formulation and characterization of Paclitaxel, 5-FU and Paclitaxel + 5-FU microspheres by Anshul Gupte; Kadriye Ciftci (93-106).
The purpose of this study was to compare the combination (Paclitaxel+5-FU microspheres) with a single drug chemotherapy (Paclitaxel and 5-FU microspheres) against metastatic breast cancer cell line (MDA-MB 435 S). The physicochemical characteristics of the microspheres (i.e. encapsulation efficiency, particle size distribution, in vitro release, thermal characteristics) were studied. The results demonstrated that the encapsulation efficiency of Paclitaxel was high (90%) when the drug was encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles with or without 5-fluorouracil (5-FU). However, the encapsulation efficiency of 5-FU was low (19%) and increased to 30% when the drug was encapsulated with Paclitaxel. The mean particle size of microspheres was 2.5 μm and were spherical in shape. The in vitro release of both 5-FU and Paclitaxel from the microspheres was relatively fast initially followed by a slower and more controlled release. The cytotoxic activity of Paclitaxel microspheres was far greater compared to either the microspheres containing 5-FU+Paclitaxel or 5-FU alone. Overall results demonstrated that incorporation of Paclitaxel or 5-FU in microspheres enhances the cytotoxicity in more controlled manner compared to that of free drugs and also that careful consideration should be made when combining drugs acting in different phases of cell cycle.
Keywords: Microspheres; Poly(lactide-co-glycolide); Paclitaxel; 5-Flourouracil;
Modeling of drug release from partially coated matrices made of a high viscosity HPMC by Mario Grassi; Lucia Zema; Maria Edvige Sangalli; Alessandra Maroni; Ferdinando Giordano; Andrea Gazzaniga (107-114).
A mathematical model able to describe the release kinetics of two model drugs (Diprophylline and Theophylline) from partially coated hydroxypropylmethylcellulose (HPMC, Methocel® K4M) matrices is presented. As solvent interaction with the system and drug release can only take place in one direction, the physical frame to be modeled turns out simpler. The model was developed starting from the established equation describing drug dissolution and taking into account the resistance to drug release given by the presence of a growing gel barrier around a matrix system.The model fits the release data obtained from both series of hydrophilic matrices containing increasing amounts (from 0.2 to 0.8 mass ratio) of the two xanthine derivatives. Differences were found in drug release rate according to the different solubility of the actives. Interestingly, however, there is no further reduction in the outer gel layer permeability when the polymer mass fraction exceeds a certain value, with both Theophylline and Diprophylline systems. Results confirm the importance of the fraction of the glassy/rubbery interface held by the active substance in defining the release rate from hydrophilic systems.
Keywords: Modeling; Hydroxypropylmethylcellulose (HPMC); Hydrophilic matrix; Release mechanism;
Structural characterisation of water–Tween 40®/Imwitor 308®–isopropyl myristate microemulsions using different experimental methods by F Podlogar; M Gašperlin; M Tomšič; A Jamnik; M.Bešter Rogač (115-128).
Pharmaceutically usable microemulsion systems were prepared from water and isopropyl myristate with a constant amount of Tween 40® and Imwitor 308® at a mass ratio of 1. Their type and structure were examined by measuring density and surface tension, and by viscometry, electric conductivity, differential scanning calorimetry (DSC) and small-angle X-ray scattering (SAXS), and the degree of agreement between the techniques was assessed. A model based on monodisperse hard spheres adequately fits the SAXS data in W/O microemulsions predicting, depending on composition, elongated or spherical droplets. It also suggests the involvement of strong attractive interactions in O/W systems. Results of conductivity, viscosity, density and surface tension measurements confirm the prediction of a percolation transition to a bicontinuous structure. DSC detects the degree of water interaction with surfactants thus identifying the type of microemulsion. The conclusions from all the techniques agree well and indicate that such studies could also be carried out on more complex systems. In future, the ability to determine type and structure of such microemulsion systems could enable partitioning and release rates of drugs from microemulsions to be predicted.
Keywords: Microemulsions; SAXS; Viscosity; Surface tension; Density; Conductivity; DSC;
Comparison of the effects of two drying methods on polymorphism of theophylline by Sari Airaksinen; Milja Karjalainen; Eetu Räsänen; Jukka Rantanen; Jouko Yliruusi (129-141).
Processing-induced transformations in drug formulation may induce adverse biopharmaceutical changes in the finished product. During the drying phase of wet granulation, theophylline monohydrate transforms either the stable (form I), or a polymorphic, metastable (form I∗) form of anhydrous theophylline. We investigated the effect of two drying methods (multichamber microscale fluid bed dryer MMFD) or variable temperature X-ray powder diffractometer (VT-XRPD) on the relative amounts of the different theophylline forms remaining in the dried granules. Granules were analyzed using XRPD and near-infrared spectroscopy. Form I∗ was the predominant form of theophylline after drying at 40–50 °C with both drying techniques. Although drying at temperatures over 50 °C produced mostly form I, more than 20% of form I∗ remained even at 90 °C when drying in MMFD. In these conditions, humidity had little influence on the amount of form I∗ in the granules. In contrast, drying in a VT-XRPD at 60 °C produced form I already during the first 15 min. Using additional drying methods, including MMFD, during the preformulation stage can be more informative about the possible polymorphic transformations and their underlying mechanisms, such as triboelectrification or recrystallization, in drug ingredients during the manufacturing process.
Keywords: X-ray powder diffraction; Fluid bed; Drying; Polymorphism; Metastable anhydrous theophylline;
Interactions of surfactants (edge activators) and skin penetration enhancers with liposomes by G.M.M El Maghraby; A.C Williams; B.W Barry (143-161).
Incorporating edge activators (surfactants) into liposomes was shown previously to improve estradiol vesicular skin delivery; this phenomenon was concentration dependent with low or high concentrations being less effective. Replacing surfactants with limonene produced similar behaviour, but oleic acid effects were linear with concentration up to 16% (w/w), beyond which it was incompatible with the phospholipid. This present study thus employed high sensitivity differential scanning calorimetry to probe interactions of additives with dipalmitoylphosphatidylcholine (DPPC) membranes to explain such results. Cholesterol was included as an example of a membrane stabiliser that removed the DPPC pre-transition and produced vesicles with a higher transition temperature (T m). Surfactants also removed the lipid pre-transition but reduced T m and co-operativity of the main peak. At higher concentrations, surfactants also formed new species, possibly mixed micelles with a lower T m. The formation of mixed micelles may explain reduced skin delivery from liposomes containing high concentrations of surfactants. Limonene did not remove the pre-transition but reduced T m and co-operativity of the main peak, apparently forming new species at high concentrations, again correlating with vesicular delivery of estradiol. Oleic acid obliterated the pre-transition. The T m and the co-operativity of the main peak were reduced with oleic acid concentrations up to 33.2 mol%, above which there was no further change. At higher concentrations, phase separation was evident, confirming previous skin transport findings.
Keywords: Surfactants; Liposomes; High sensitivity differential scanning calorimetry; Skin; Penetration enhancer; Edge activators;
Fatty acids in Botryococcus braunii accelerate topical delivery of flurbiprofen into and across skin by Jia-You Fang; Hsien-Chih Chiu; Jiunn-Tzong Wu; Yin-Ru Chiang; Shu-Hui Hsu (163-173).
To improve the drug absorption into and across the skin, fatty acids extracted from Botryococcus braunii were evaluated using in vitro and in vivo techniques with Wistar rats as the animal model. Palmitic acid (C16:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3) were the major components in the B. braunii extract. Topical delivery of flurbiprofen was significantly enhanced after pretreatment with 3% B. braunii extract for 30 min in an in vitro Franz cell and in vivo pharmacokinetic studies. Pure unsaturated fatty acids were more-effective enhancers than the B. braunii extract. However, a greater irritant potential was also observed with those fatty acids than with the B. braunii extract according to the skin tolerance study as determined by transepidermal water loss (TEWL). Both human keratinocytes and skin fibroblasts showed a 1.5–2-fold increase in prostaglandin E2 (PGE2) release as compared to the control. The findings in this study indicate that the fatty acids in B. braunii may be useful enhancers for flurbiprofen delivery via the skin.
Keywords: Botryococcus braunii; Flurbiprofen; Topical delivery; Enhancers; Fatty acids;
Phospholipid-based microemulsion formulation of all-trans-retinoic acid for parenteral administration by Seung Rim Hwang; Soo-Jeong Lim; Jeong-Sook Park; Chong-Kook Kim (175-183).
All-trans-retinoic acid (ATRA) shows anti-cancer activities, especially in patients with acute promyelocytic leukemia. Due to the highly variable bioavailability of ATRA and induction of its own metabolism after oral treatment, development of alternative parenteral dosage form is required. The principal aim of this study was to develop a parenteral formulation of ATRA by overcoming its solubility limitation by utilizing phospholipid-based microemulsion system as a carrier. Microemulsion was prepared with pharmaceutically acceptable ingredients such as soybean oil and phospholipids. The mean particle diameter and polydispersity of ATRA microemulsion could be decreased to be applicable for parenteral administration by modulation of composition of microemulsion. The loading concentration of ATRA in microemulsion increased by increasing the oil contents and also by inclusion of distearoylphosphatidyl-ethanolamine-N-poly(ethyleneglycol) 2000 (DSPE-PEG). Furthermore, loading of ATRA in microemulsion improved the chemical stability of ATRA. The pharmacokinetic profile of ATRA after intravenous injection of microemulsion formulation to rats was similar to that of sodium ATRA. The growth inhibitory effects of ATRA on human cancer HL-60 and MCF-7 cell lines were also similar between free ATRA and microemulsion formulation of ATRA, suggesting that its anti-cancer activity was not impaired by loading in microemulsion. Our study herein demonstrates that phospholipid-based microemulsion may provide an alternative parenteral formulation of ATRA.
Keywords: Retinoic acid; Parenteral delivery; Phospholipid; Microemulsion;
The production of homogeneous extrudates of microcrystalline cellulose pastes by S.L. Rough; D.I. Wilson (185-189).
The homogeneity of water-based microcrystalline cellulose (MCC) paste extrudates was investigated during ram extrusion as a function of ram velocity. Variations in the water content of the extrudates were caused by liquid phase migration within the paste. The evolution in water content was measured by sectioning and drying the extrudate, and the subsequent homogeneity was quantified by the standard error in water content. The homogeneity of the extrudates was found to decrease as the ram velocity decreased. This result was also inferred from the rate of increase of the extrusion pressure. The extrudate homogeneity was significantly improved by compensating for water migration in the barrel during the compaction stage. This was achieved using a non-uniform initial paste billet, created by packing the barrel with layers of paste of different water contents. This technique also produced a smaller variation in extrusion pressure over the ram displacement range, and a reduction in water loss from the upstream paste compact into the extrudate and/or through the apparatus tooling.
Keywords: Paste extrusion; Liquid migration; Microcrystalline cellulose;
Notice board (191-193).