Development of time-, pH-, and enzyme-controlled colonic drug delivery using spray-dried chitosan acetate and hydroxypropyl methylcellulose.

A colonic drug delivery with a new concept based on a combination of time-, pH-, and enzyme-controlled system was developed. Spray-dried chitosan acetate (CSA) prepared from low molecular weight chitosan was characterized. A combination of CSA and hydroxypropyl methylcellulose (HPMC) was used as new compression-coats for 5-aminosalicylic acid (5-ASA) tablets. Factors affecting in-vitro drug release, i.e. % weight ratio of coating polymers, enzyme activity, pH of media, and excipients in core tablets, were evaluated. The tablets compression-coated with HPMC:CSA at 60:40 and 50:50% weight ratio providing lag times about 5-6h were able to pass through the stomach (stage I, 0.1N HCl) and small intestine (stage II, pH 6.8, Tris-HCl). The delayed release was time- and pH-controlled owing to the swelling with gradual dissolving of CSA and HPMC in 0.1N HCl and the less solubility of CSA at higher pH. After reaching the colon (stage III, pH 5.0, acetate buffer), the dissolution of CSA at low pH triggered the drug release over 90% within 14h. Furthermore, the degradation of CSA by beta-glucosidase in the colonic fluid enhanced the drug release while adding the disintegrant or the osmotic agent in the core tablets would affect the drug release.     

Selective drug delivery to the colon using pectin:chitosan:hydroxypropyl methylcellulose film coated tablets.

A study has been carried out to assess the potential of pectin:chitosan:hydroxypropyl methylcellulose (HPMC) (P:C:H) films for colonic drug delivery. Radiolabelled (99mTc) tablets were coated with a 3:1:1, P:C:H film and administered to human volunteers. The gastro-intestinal transit of the tablets was assessed by gamma scintigraphy. The results showed that in all cases (n=4), the tablets were able to pass through the stomach and small intestine intact. Break up of the tablets commenced once they were in the colon, due to degradation of the coat by colonic bacteria. The study has highlighted the potential of this coating system for colonic drug delivery.     

Modeling of drug release from delivery systems based on hydroxypropyl methylcellulose (HPMC)

The objective of this article is to review the spectrum of mathematical models that have been developed to describe drug release from hydroxypropyl methylcellulose (HPMC)-based pharmaceutical devices. The major advantages of these models are: (i) the elucidation of the underlying mass transport mechanisms; and (ii) the possibility to predict the effect of the device design parameters (e.g., shape, size and composition of HPMC-based matrix tablets) on the resulting drug release rate, thus facilitating the development of new pharmaceutical products. Simple empirical or semi-empirical models such as the classical Higuchi equation and the so-called power law, as well as more complex mechanistic theories that consider diffusion, swelling and dissolution processes simultaneously are presented, and their advantages and limitations are discussed. Various examples of practical applications to experimental drug release data are given. The choice of the appropriate mathematical model when developing new pharmaceutical products or elucidating drug release mechanisms strongly depends on the desired or required predictive ability and accuracy of the model. In many cases, the use of a simple empirical or semi-empirical model is fully sufficient. However, when reliable, detailed information are required, more complex, mechanistic theories must be applied. The present article is a comprehensive review of the current state of the art of mathematical modeling drug release from HPMC-based delivery systems and discusses the crucial points of the most important theories.     

Modeling of drug release from delivery systems based on hydroxypropyl methylcellulose (HPMC)

The objective of this article is to review the spectrum of mathematical models that have been developed to describe drug release from hydroxypropyl methylcellulose (HPMC)-based pharmaceutical devices. The major advantages of these models are: (i) the elucidation of the underlying mass transport mechanisms; and (ii) the possibility to predict the effect of the device design parameters (e.g., shape, size and composition of HPMC-based matrix tablets) on the resulting drug release rate, thus facilitating the development of new pharmaceutical products. Simple empirical or semi-empirical models such as the classical Higuchi equation and the so-called power law, as well as more complex mechanistic theories that consider diffusion, swelling and dissolution processes simultaneously are presented, and their advantages and limitations are discussed. Various examples of practical applications to experimental drug release data are given. The choice of the appropriate mathematical model when developing new pharmaceutical products or elucidating drug release mechanisms strongly depends on the desired or required predictive ability and accuracy of the model. In many cases, the use of a simple empirical or semi-empirical model is fully sufficient. However, when reliable, detailed information are required, more complex, mechanistic theories must be applied. The present article is a comprehensive review of the current state of the art of mathematical modeling drug release from HPMC-based delivery systems and discusses the crucial points of the most important theories.     

Development and optimization of lyophilized orally disintegrating tablets using factorial design

The aim of this study was to evaluate the use of maltodextrin as a sugar-matrix former along with several cellulosic binders in the preparation of freeze—dried orally disintegrating tablets (ODT). The ODT was prepared by freeze—drying an aqueous dispersion of nimesulide (NM) containing maltodextrin and a cellulosic binder. The influence of formulation parameters on the in vitro/in vivo disintegration time and in vitro dissolution of NM from ODTs along with other tablet characteristics was investigated using full factorial design. The optimized ODT contained 5% w/v maltodextrin DE 29, 2% w/v Methocel^®E15, and 5% w/v NM, disintegrated in less than 10 s and showed more than 70% of NM in ODTs dissolved within 2?min, compared to only 1.52% of NM plain drug and 7.25% of NM in immediate release commercial tablet. Crystalline state evaluation of NM in the optimized ODT was conducted through differential scanning calorimetry, and X-ray powder diffraction. The study suggests that the optimized ODT formulation developed in this work may be an alternative to conventional formulations of NM inconvenient to the patients such as intramuscular or rectal administration.     

Formulation optimization of orally disintegrating tablets containing solid dispersion of felodipine and hydroxypropyl methylcellulose using face-centered central composite design

A novel dosage form integrating solid dispersion (SD) in orally disintegrating tablets (ODTs) was developed and optimized by the face-centered central composite design to improve poorly soluble property and slow onset action time of felodipine (Fel). SD of Fel and hydroxypropyl methylcellulose E6 was prepared by solvent evaporation method. Differential scanning calorimetry and fourier transforms infrared spectroscopy indicated that Fel transformed from crystalline to amorphous state by the formation of hydrogen bond between –N–H in Fel and O–R in HPMC. The accelerated stability test in 45 °C, 75 % RH demonstrated that the optimized SD was stable in terms of the dissolution rate of Fel and thermodynamic property. The ODTs containing SD (Fel:HPMC E6 = 1:3) were prepared by direct compression technique. The face-centered central composite design with the ODT-SD was employed to investigate the effect of mannitol (X₁), crospovidone XL (X₂) on the ODT-SD disintegration time (Y₁), % Fel released after 5 min (Y₂) and the ODT-SD friability (Y₃). ANOVA test showed that X₂ and X₂ * X₂ had a significant effect on the ODT-SD disintegration time (p < 0.05). Meanwhile, the dissolution rate of Fel after 5 min did not remarkably depend on any independent variables (p > 0.05). The ODT-SD friability was substantially proportional to the amount of mannitol (X₁) (p < 0.05). The optimized ODT-SD disintegration time,  % Fel released after 5 min, and friability were 27.67 s, 88.35 and 0.48 %, respectively.     

The influence of excipients on drug release from hydroxypropyl methylcellulose matrices

The influence of commonly used excipients, spray-dried lactose (SDL), microcrystalline cellulose (MCC), and partially pregelatinized maize starch (Starch 1500) on drug release from hydroxypropyl methylcellulose (HPMC, hypromellose) matrix system has been investigated. A model formulation contained 30%w/w drug, 20%w/w HPMC, 0.5%w/w fumed silica, 0.25%w/w magnesium stearate, and 49.25%w/w filler. Chlorpheniramine maleate and theophylline were used as freely (1 in 4) and slightly (1 in 120) water-soluble drugs, respectively. It was found that for both drugs, addition of 20 to 49.25%w/w Starch 1500 resulted in a significant reduction in drug release rates compared to when MCC or SDL was used. The study showed that using lactose or microcrystalline cellulose in the formulations resulted in faster drug release profiles. Partially pregelatinized maize starch contributed to retardation of both soluble and slightly soluble drugs. This effect may be imparted through synergistic interactions between Starch 1500 and HPMC and the filler actively forming an integral part within the HPMC gel structure.     

Development of vitamin loaded topical liposomal formulation using factorial design approach: drug deposition and stability

Long-term exposure of the skin to UV light causes degenerative effects, which can be minimized by using antioxidant formulations. The major challenge in this regard is that a significant amount of antioxidant should reach at the site for effective photoprotection. However, barrier properties of the skin limit their use. In the present study, Vitamin E acetate was encapsulated into liposome for improving its topical delivery. However preparation of liposomes is very difficult due to number of formulation variables involved therein. In the present work systematic statistical study for the formulation of liposomes for topical delivery of Vitamin E using the factorial design approach was undertaken. Amount of phospholipid (PL) and cholesterol (CH) were taken at three different levels and liposomes were prepared using ethanol injection method. Liposomes were characterized for encapsulation efficiency, vesicle size, zeta potential, and drug deposition in the rat skin. Gels containing liposomal dispersion (batch with higher skin deposition of VE) were prepared in Carbopol^® 980 NF and were characterized for gel strength, viscosity and drug deposition in the rat skin. Stability of liposome dispersion and gel formulation was studied at 30 °C/65% RH for 3 months. Results of regression analysis revealed that vesicle size and drug deposition in the rat skin were dependant on the lipid concentration and lipid:drug ratio. Drug deposition in rat skin had an inverse relationship with respect to PL and CH concentration. Prepared liposomal dispersion (50 mg PL:6 mg CH) showed seven-fold increase in drug deposition compared to control (plain drug dispersion). Gel formulation demonstrated six-fold and four-fold increase in drug deposition compared to control gel and marketed cream, respectively. Liposome dispersion and gel formulation were found to be stable for 3 months. Factorial design was found to be well suited to identify the key variables affecting drug deposition. Improved drug deposition from liposomal preparations demonstrates its potential for dermal delivery.     

Microencapsulation of a hydrophilic drug into a hydrophobic matrix using a salting-out procedure. I: Development and optimization of the process using factorial design

The congealable disperse phase method for preparing sustained release microspheres involves an emulsification process using water as the external phase and molten hydrophobic wax as the disperse phase into which the drug is loaded. Attempts to entrap highly water-soluble drugs using this process have often resulted in low loading efficiency as the drugs partition into the external water phase during emulsification and are lost. A novel method employing salts and wetting agents was developed to improve the loading efficiency of the highly water-soluble drug, guaifenesin, using this method. The drug/wax ratio (D/W) and the presence of salts and wetting agents greatly influenced microsphere properties. To optimize the process for drug loading efficiency and release rate, three different D/Ws, salts and wetting agents were chosen and a full 3³ factorial design experiment was performed. Any significant differences among the levels of the variables and their individual and joint effects on entrapment efficiency and T₅₀ (time for 50% drug release) were determined. Entrapment efficiencies in the range 35.1-86.3% were obtained for the various factor-level combinations of the variables. Particle size was in the range 140-385 µm and T₅₀ was 0.59-2.72 h for the microspheres obtained. The D/W and type of salt used significantly affected drug entrapment and T₅₀, while the nature of wetting agent was not significant at p &lt; 0.05. The microspheres prepared using 1:4 D/W showed the highest entrapment efficiency and slowest drug release.     

Microencapsulation of a hydrophilic drug into a hydrophobic matrix using a salting-out procedure. I: Development and optimization of the process using factorial design

The congealable disperse phase method for preparing sustained release microspheres involves an emulsification process using water as the external phase and molten hydrophobic wax as the disperse phase into which the drug is loaded. Attempts to entrap highly water-soluble drugs using this process have often resulted in low loading efficiency as the drugs partition into the external water phase during emulsification and are lost. A novel method employing salts and wetting agents was developed to improve the loading efficiency of the highly water-soluble drug, guaifenesin, using this method. The drug/wax ratio (D/W) and the presence of salts and wetting agents greatly influenced microsphere properties. To optimize the process for drug loading efficiency and release rate, three different D/Ws, salts and wetting agents were chosen and a full 3(3) factorial design experiment was performed. Any significant differences among the levels of the variables and their individual and joint effects on entrapment efficiency and T50 (time for 50% drug release) were determined. Entrapment efficiencies in the range 35.1-86.3% were obtained for the various factor-level combinations of the variables. Particle size was in the range 140-385 microm and T50 was 0.59-2.72 h for the microspheres obtained. The D/W and type of salt used significantly affected drug entrapment and T50, while the nature of wetting agent was not significant at p < 0.05. The microspheres prepared using 1:4 D/W showed the highest entrapment efficiency and slowest drug release.     

Development and optimization of polymeric nanoparticles of antitubercular drugs using central composite factorial design*

Objective: The objective of the present study was to develop sustained release biodegradable polymeric nanoparticles (PNs) of two anti-tubercular drugs (ATDs), rifampicin (RIF) and isoniazid (INH) using circumscribed central composite factorial design (CCD) and evaluate in vivo uptake potential using rhodamine labeled PNs (RPNs).

Methods: CCD was employed to study the influence of independent formulation factors, drug:polymer ratio (D:P) and surfactant concentration (SC), on dependent physicochemical characteristics, particle size (PS), polydispersity index (PI) and percentage entrapment efficiency (%EE) of the drugs. Optimized PNs prepared using response surface methodology (RSM) were evaluated for in vitro kinetics at endosomal macrophage pH 5.2 and physiological pH 7.4 and in vivo targeting potential in peritoneal macrophages (PMs) by fluorescence microscopy (FM) and confocal laser scanning microscopy (CLSM).

Results: Optimized PNs exhibited spherical and porous surface with a mean PS of 202 nm, PI of 0.178, zeta potential of -25.49 mV and %EE of 76.12% and 54.25% for RIF and INH, respectively.

Conclusions: Highly hydrophilic INH could be encapsulated with lypophilic RIF with efficiency. In vivo uptake studies of RPNs in PMs suggested endocytosis of RPNs without any surface adsorption phenomenon. Hence, further studies need to be performed for establishing the pharmacokinetic potential of PNs.     

An approach to formulating an oral floating drug delivery system for dexchlorpheniramine maleate using factorial design

The purpose of this research was to formulate and evaluate a floating tablet formulation of dexchlorpheniramine maleate (DCPM) using full factorial design. A 32 factorial design (nine runs) was utilized to optimize the formulation, the contents of hydroxypropyl methyl cellulose (HPMC) (X1) and Carbopol 934P (X2) being taken as independent variables and t50% (Y1), % drug release after 6 h (Y2), % drug release after 12 h (Y3), and floating lag time (FLT) (Y4) as the dependent variables. The tablets showed 99.2635 to 102.4709 of the labeled amount of dexchlorpheniramine maleate indicating uniformity of content. The tablets containing DCPM released 72.28 to 99.461% of drug at the end of 12 h by an in vitro release study. Hardness, friability, floating capacity, weight variation and content uniformity were also examined. In addition,the tablets were evaluated for in vitro release characteristics for 24 h. The optimal batch (F9) was selected by regression analysis and followed Higuchi kinetics. The drug release mechanism was found to be a complex mixture of diffusion, swelling and erosion. The floating tablets of DCPM developed may be used clinically for prolonged drug release for at least 16 hrs, thereby improving bioavailability and patient compliance.     

Amphotericin B-loaded polymeric nanoparticles: formulation optimization by factorial design

In this study, PLGA or PLGA-PEG blend nanoparticles were developed loading amphotericin B (AmB), an antifungal agent broadly used in therapy. A 2²?×?3¹ factorial experimental design was conducted to indicate an optimal formulation of nanoparticles containing AmB and demonstrate the influence of the interactions of components on the mean particle size and drug encapsulation efficiency. The independent variables analyzed were polymer amount (two levels) and organic phase (three factors in one level). The parameters methanol as cosolvent and higher polymer amount originated from the higher AmB encapsulation, but with the larger particle size. The selected optimized parameters were set as the lower polymer amount and ethyl acetate as cosolvent in organic phase, for both PLGA and PLGA-PEG nanoparticles. These parameters originated from nanoparticles with the size of 189.5?±?90?nm and 169?±?6.9?nm and AmB encapsulation efficiency of 94.0?±?1.3% and 92.8?±?2.9% for PLGA and PLGA-PEG nanoparticles, respectively. Additionally, these formulations showed a narrow size distribution indicating homogeneity in the particle size. PLGA and PLGA-PEG nanoparticles are potential carrier for AmB delivery and the factorial design presented an important tool in optimizing nanoparticles formulations.     

Development and characterization of floating microballoons for oral delivery of cinnarizine by a factorial design

Cinnarizine (CN) is a pipperazine derivative with anti-histaminic activity and high affinity to H(1) receptors. The objective of this study was to produce floating microspheres (FM) of CN by diffusion solvent evaporation technique to increase drug solubility and hence its bioavailability. The effect of process variables such as: Eudragit type, stirring rate and time of stirring after addition of oily phase to the aqueous phase were evaluated on the yield, particle size, loading, release and floating behaviors of microspheres using a factorial design. Release of CN from microspheres was studied in pHs: 1.2 and 7.2 using paddle technique. The samples of dissolution test were analysed spectrophotometrically at 256.1 nm and 256.5 nm respectively. particle size of microspheres was studied using microscopic method and their floating behavior was studied in HCl (0.1 N, pH 1.2) medium with Tween 20 (0.5% w/v). Eight formulations were produced by changing 3 variables each at 2 levels: Eudragit S100 (Ps) or a combination of two Eudragits S100:RLPO (1:3) (P(SR)), stirring rate of 200 (R(2)) or 300 rpm (R(3)) and stirring time after addition of oily phase to the aqueous phase 0 (T(0)) or 1 hr (T(1)). The average size of microspheres was 300 microm. The highest yield efficiency (94%) was seen in P(SR)R(3)T(0) formulation and the greatest loading percentage was 8.5% in P(SR)R(2)T(1) formulation. The microspheres containing just Eudragit S100, didn't show suitable releasing profile during 8 hours in pH 1.2 but those containing combination of Eudragit S100:RL released approximately whole amount of CN during 10 hours (8 hours in pH 1.2 and 2 hours in pH 7.2). The highest floating percentage up to 6 hours was 77.5% in P(S)R(2)T(1) formulation. The type of Eudragit used seems to play an important role in producing sustained release floating microspheres. P(SR)R(3)T(0) formulation containing both types of Eudragit S100:RL (1.3) that releases 99.1% of the drug after 10 hours and 65% floating after 6 hr seems suitable for oral sustained delivery of CN.     

Novel non-ionic surfactant proniosomes for transdermal delivery of lacidipine: optimization using 23 factorial design and in vivo evaluation in rabbits

Abstract

Context: Proniosomes offer a versatile vesicle drug delivery concept with potential for delivery of drugs via transdermal route.

Objectives: To develop proniosomal gel using cremophor RH 40 as non-ionic surfactant containing the antihypertensive drug lacidipine for transdermal delivery so as to avoid its extensive first pass metabolism and to improve its permeation through the skin.

Materials and methods: Proniosomes containing 1% lacidipine were prepared by the coacervation phase separation method, characterized, and optimized using a 2³ full factorial design to define the optimum conditions to produce proniosomes with high entrapment efficiency, minimal vesicle size, and high-percentage release efficiency. The amount of cholesterol (X₁), the amount of soya lecithin (X₂), and the amount of cremophor RH 40 (X₃) were selected as three independent variables.

Results and discussion: The system F4 was found to fulfill the maximum requisite of an optimum system because it had minimum vesicle size, maximum EE, maximum release efficiency, and maximum desirability. The optimized system (F4) was then converted to proniosomal gel using carbopol 940 (1% w/w). In vitro permeation through excised rabbit skin study revealed higher flux (6.48?±?0.45) for lacidipine from the optimized proniosomal gel when compared with the corresponding emulgel (3.04?±?0.13) mg/cm²/h. The optimized formulation was evaluated for its bioavailability compared with commercial product. Statistical analysis revealed significant increase in AUC (0???α) 464.17?±?113.15?ng h/ml compared with 209.02?±?47.35?ng h/ml for commercial tablet. Skin irritancy and histopathological investigation of rat skin revealed its safety.

Conclusions: Cremophor RH 40 proniosomal gel could be considered as very promising nanocarriers for transdermal delivery of lacidipine.     

羟丙基甲基纤维素酞酸酯的制备与性能测定

目的制备肠溶包衣材料羟丙基甲基纤维素钛酸酯(HPMCP),并分析其结构和性能。方法采用红外光谱、紫外分光光度法和化学分析法进行表征,并测试HPMCP在有机溶剂中的溶解性、pH敏感性及其在人工肠液中的溶解情况。结果HPMCP中邻苯二甲酰基的含量与制备过程中加入邻苯二甲酸酐的量密切相关;丙酮/乙醇/水等混合溶剂是HPMCP的良溶剂;HPMCP在pH5.1~5.4缓冲液中能快速溶解。结论HPMCP有较好的包衣工艺性和较高的生物利用度。     

An anti-inflammatory drug (mefenamic acid) incorporated in biodegradable alginate beads: development and optimization of the process using factorial design

The objective of this study was to prepare and evaluate biodegradable alginate beads as a controlled-release system for a water-insoluble drug, mefenamic acid (MA), using 3 x 2(2) factorial design by ionotropic gelation method. Therefore, the mefenamic acid dispersion in a solution of alginate was dropped into the cross-linking CaCl(2) solution and a fairly high yield (71-89%) of MA-alginate beads were obtained. Their encapsulation efficiencies were in the range of 79.3-98.99%. The effect of drug:polymer ratio, CaCl(2) concentration, and curing time on the time for 50% of the drug to be released (t(50%)), and the drug entrapment efficiency were evaluated with factorial design method. It was found that drug:polymer ratio and interaction of drug:polymer ratio and curing time had an important effect on the drug to be released (t(50%)). The effect of CaCl(2) concentration is also important on the drug release. On the other hand, all factors except CaCl(2) concentration were effective on the drug entrapment efficiency. The swelling properties of beads were also studied. The release mechanism was described and found to be non-Fickian, Case II, and Super Case II transport for the formulations. This study suggested a new mefenamic acid alginate bead formulation for oral delivery of nonsteroidal anti-inflammatory drugs, which cause gastric irritation.     

A compaction process to enhance dissolution of poorly water-soluble drugs using hydroxypropyl methylcellulose

The purpose of this study was to develop a technique to enhance the dissolution rate of poorly water-soluble drugs with hydroxypropyl methylcellulose (HPMC) without the use of solvent or heat addition. Three poorly water-soluble drugs, naproxen, nifedipine, and carbamazepine, were studied with low-viscosity HPMC USP Type 2208 (K3LV), HPMC USP Type 2910 (E3LV and E5LV), and methylcellulose. Polymer and drug were dry-blended, compressed into slugs on a tablet press or into ribbons on a roller compactor, and then milled into a granular powder. Dissolution testing of the milled powder was performed on USP Apparatus II, 100 rpm, 900 ml deionized water, 37 degrees C. Drug distribution vs. particle size was also studied. The compaction processes enhanced drug dissolution relative to drug alone and also relative to corresponding loosely mixed physical mixtures. The roller compaction and slugging methods produced comparable dissolution enhancement. The mechanism for dissolution enhancement is believed to be a microenvironment HPMC surfactant effect facilitated by keeping the HPMC and drug particles in close proximity during drug dissolution. The compaction methods in this study may provide a lower cost, quicker, readily scalable alternative for formulating poorly water-soluble drugs.     

Development of enteric-coated calcium pectinate microspheres intended for colonic drug delivery

Enteric-coated calcium pectinate microspheres (MS) aimed for colon drug delivery have been developed, by using theophylline as a model drug. The influence of pectin type (amidated or non-amidated) and MS preparation conditions (CaCl2 concentration and cross-linking time) was investigated upon the drug entrapment efficiency and its release behaviour. Drug stability and drug-polymer interactions were studied by Differential Scanning Calorimetry, thermogravimetry, X-ray diffractometry and FTIR spectroscopy. Enteric coating with Eudragit S100 enabled maintenance of MS integrity until its expected arrival to colon. The coating was also useful to improve the stability of MS during storage, avoiding morphologic changes observed for uncoated MS stored under ambient conditions. Entrapment efficiency increased by reducing cross-linking time, and (only in the case of non-amidated pectin) by increasing CaCl2 concentration. On the other hand, release tests performed simulating the gastro-intestinal pH variation evidenced an inverse relationship between CaCl2 concentration and drug release rate, whereas no influence of both pectin type and cross-linking time was found. Unexpectedly, addition of pectinolytic enzymes to the colonic medium did not give rise to selective enzymatic degradation of MS. Notwithstanding this unforeseen result, coated MS prepared at 2.5% w/v CaCl2 concentration were able to adequately modulate drug release through a mixed approach of pH and transit time control, avoiding drug release in the gastric ambient, and reaching the colonic targeting where 100% release was achieved within less than 24h.