Drug Nanocrystals Research Articles

Nanocrystal-Loaded Lipid Carriers for Improved Oral Absorption and Anticancer Efficacy of Etoposide: Formulation Development, Transport Mechanism, In Vitro and In Vivo Evaluation.

To improve the oral absorption and anticancer efficacy of the BCS-IV drug etoposide (ETO), oral nanocrystal-loaded lipid carriers (Lipo@NCs) were developed in this study by modifying the BCS-IV drug nanocrystal with the lipid bilayer. The ETO-Lipo@NCs were prepared by the thin film hydration high-pressure homogenization method, and the core of positively charged ETO nanocrystals was prepared by the sonoprecipitation-high pressure homogenization method. The optimized ETO-Lipo@NCs were spherical particles with an average particle size of 220.3 ± 14.2 nm and a zeta potential of -9.95 ± 0.81 mV, respectively. The successful coating of a lipid bilayer on the surface of nanocrystals in ETO-Lipo@NCs was confirmed by several characterization methods. Compared to nanocrystals, the release rate and degree of Lipo@NCs in SIF were significantly decreased, indicating that the lipid bilayer can effectively prevent the rapid dissolution of core nanocrystals. ETO-Lipo@NCs demonstrated a significant improvement in the intestinal permeability and absorption of ETO in a single intestinal perfusion experiment. In the cells, ETO-Lipo@NCs showed enhanced cellular uptake and transepithelial transport compared with ETO nanocrystals. Pharmacokinetic analysis indicated that ETO-Lipo@NCs had a longer plasma half-life than ETO solution, and the oral bioavailability of ETO-Lipo@NCs was 1.96- and 10.92-fold higher than that of ETO nanocrystals and ETO coarse crystals, respectively. Moreover, the ETO-Lipo@NCs orally dosed at 10 mg/kg exhibited an excellent inhibitory effect against tumors in a subcutaneous Lewis lung carcinoma (LLC) xenograft model compared with other preparations. These results indicate that the Lipo@NCs formulation has an oral absorption-promoting effect of the BCS-IV drug ETO, which could warrant further application in the oral delivery of other poorly bioavailable drugs.

Step-By-Step Standardization of the Bottom-Up Semi-Automated Nanocrystallization of Pharmaceuticals: A Quality By Design and Design of Experiments Joint Approach.

Nanosized drug crystals have been reported with enhanced apparent solubility, bioavailability, and therapeutic efficacy compared to microcrystal materials, which are not suitable for parenteral administration. However, nanocrystal design and development by bottom-up approaches are challenging, especially considering the non-standardized process parameters in the injection step. This work aims to present a systematic step-by-step approach throughQuality-by-Design (QbD) and Design of Experiments (DoE) for synthesizing drug nanocrystals by a semi-automated nanoprecipitation method. Curcumin is used as a drug model due to its well-known poor water solubility (0.6µgmL-1, 25°C). Formal and informal risk assessment tools allow identifying the critical factors. A fractional factorial 24-1 screening design evaluates their impact on the average size and polydispersity of nanocrystals. The optimization of significant factors is done by a Central Composite Design. This response surface methodology supports the rational design of the nanocrystals, identifying and exploring the design space. The proposed joint approach leads to a reproducible, robust, and stable nanocrystalline preparation of 316nm with a PdI of 0.217 in compliance with the quality profile. An orthogonal approach for particle size and polydispersity characterization allows discarding the formation of aggregates. Overall, the synergy between advanced data analysis and semi-automated standardized nanocrystallization of drugs is highlighted.

Advancements in Nutraceutical Delivery: Integrating Nanotechnology and Microencapsulation for Enhanced Efficacy and Bioavailability

Abstract Nutraceuticals, derived from various foods, have gained significance for promoting health and managing diseases. Despite their nutritional value, challenges such as low solubility, poor diffusion, gastrointestinal instability, and limited bioavailability impede their effectiveness as preventive agents. Nanotechnology-driven drug delivery systems, including liposomes, nanospheres, nanoemulsions, micelles, and nanocrystals, offer innovative solutions to enhance efficacy and oral bioavailability. These advanced formulations not only augment the physicochemical properties and therapeutic efficacy of nutraceuticals but also enable targeted delivery. A diverse range of nanocarriers, encompassing liposomes to inorganic nanoparticles, addresses specific challenges in nutraceutical formulations, exhibiting unique properties such as heightened stability, palatability, and lymphatic uptake for increased bioavailability. Lipid nanocarriers facilitate the permeation of neuroprotective nutraceuticals through the blood–brain barrier, while liposomal delivery systems shield bioactives from oxidation and light, accommodating both hydrophilic and lipophilic compounds. In addition, polymeric nanocarriers, exemplified by micelles, outperform lipidic counterparts, ensuring controlled release. Natural sources contribute to the development of polymeric nanoparticles, offering controlled delivery with reduced toxicity. Polysaccharides and proteins diversify delivery platforms, encompassing the loading of nutraceuticals and functional nutrients. Nanotechnology emerges as a pivotal player in advancing the solubility, bioavailability, and stability of bioactives, with ongoing research focused on refining formulations, like curcumin, for effective cancer and inflammation management. While these advancements hold promise, careful consideration of challenges, including toxicity and cost, is imperative to fully unlock the potential of nanotechnology in nutraceutical delivery, ensuring both safety and cost-effectiveness. The significance of microencapsulation and nanotechnology in the nutraceutical delivery system is thoroughly explored in this comprehensive review.

The In Vivo Fate of Polycatecholamine Coated Nanoparticles Is Determined by a Fibrinogen Enriched Protein Corona.

Polycatecholamine coatings have attracted significant attention in the past 10 years owing to their ability to functionalize a wide range of materials. Here we apply the use of such coatings to drug nanocrystals, made from a poorly soluble drug compound, to postfunctionalize the nanocrystal surface with the aim of providing steric stabilization and extending their circulation time after intravenous injection. We show that both polydopamine and polynorepinephrine can be used to successfully modify drug nanocrystals and subsequently incorporate end-functionalized PEG to the surface. Even though high grafting densities of PEG were achieved, we observed rapid clearance and increased liver uptake for polycatecholamine functionalized drug nanocrystals. Using both surface sensitive model systems and protein corona profiling, we determine that the rapid clearance was correlated with an increase in adsorption of proteins involved in coagulation to the polycatecholamine surface, with fibrinogen being the most abundant. Further analysis of the most abundant proteins revealed a significant increase in thiol-rich proteins on polycatecholamine coated surfaces. The observed interaction with coagulation proteins highlights one of the current challenges using polycatecholamines for drug delivery but might also provide insights to the growing use of these materials in hemostatic applications.

Development, optimization and in-vivo pharmacokinetic evaluation of flubiprofen nanocrystal tablets for efficient chronotherapy against rheumatoid arthritis

In an effort to enhance drug solubility for buccal mucosal delivery, various strategies such as nanoemulsions, cyclodextrin inclusions, and solid dispersions have been explored, but these methods often face limitations. An emerging solubilization technology, drug nanocrystals, offers a promising alternative due to their high drug loading capacity and scalability for large-scale production. The current study aimed to create solid nanocrystals of flurbiprofen (FP-NC) through a wet milling process to significantly improve water solubility, regardless of pH, and consequently enhance bioavailability. FP-NC was further used to formulate tablets and compressed coatings with carboxymethyl xanthan gum (CXG) and sodium alginate (SA). The compression coating process has been optimized using Quality by Design principles. Utilizing the desirability approach, we identified an optimal formulation with 56.05 mg of CXG, 74.96 mg of SA, and a coating material weight of 249.98 mg. This formulation achieves desired chronotherapeutic characteristics with a lag time (LT) of 5.99 h and T90% of 11.48 h. We conducted in-vitro and ex-vivo drug release studies and compared them with in-vivo pharmacokinetic parameters, employing level A in-vitro in-vivo correlation (IVIVC). Notably, the continuous ex-vivo method exhibited a stronger correlation compared to traditional in-vitro drug release assessments. This suggests that our novel drug release system using the NC compressed coating technique holds promise for achieving chronotherapeutic effects with improved bioavailability.

Spray-dried nanocrystal-loaded polymer microparticles for long-term release local therapies: an opportunity for poorly soluble drugs

Nano- and micro-technologies can salvage drugs with very low solubility that were doomed to pre-clinical and clinical failure. A unique design approach to develop drug nanocrystals (NCs) loaded in extended release polymeric microparticles (MPs) for local treatments is presented here through the case of a potential osteoarthritis (OA) drug candidate for intra-articular (IA) administration. Optimizing a low-shear wet milling process allowed the production of NCs that can be subsequently freeze-dried (FD) and redispersed in a hydrophobic polymer-organic solvent solution to form spray-dried MPs. Results demonstrated a successful development of a ready-to-upscale formulation containing PLGA MPs with high drug NC encapsulation rates that showed a continuous and controlled drug release profile over four months. The screenings and procedures described allowed for identifying and overcoming common difficulties and challenges raised along the drug reduction to nano-size and spray-drying process. Above all, the technical knowledge acquired is intended for formulation scientists aiming to improve the therapeutic perspectives of poorly soluble drugs.

Development of Surface Modified and Aqueous Re-dispersible Nanocrystal using Pluronic F-68 and Suitable Cryoprotectant for Accelerating the Dissolution Rate of Cilnidipine

Background: The research on poorly aqueous-soluble drugs of BCS class II such as Cilnidipine (CLD) demands significant improvement in their aqueous solubility and dissolution rate. Such requirements may be fulfilled by adapting the nanocrystal approach with considering the various challenges. Objective: The prime purpose of this research work was to develop, optimize and characterize the nanocrystal of the poorly aqueous soluble drug (CLD) using the antisolvent-precipitation ultrasonication method. Such a method was followed for rapid re-dispersion of drugs in water with improving their dissolution rate. Methods: In this study, the different nanosuspension formulations were prepared using varying concentrations of three stabilizers - Pluronic F-68, Pluronic F-127, and HPMC-15cps, as selected stabilizer candidates. The selected and optimized formulation was followed by a lyophilization process with the incorporation of two selected distinct cryoprotectants - Mannitol and Lactose. The obtained nanocrystals were evaluated for their physical appearance, aqueous re-dispersibility, and particle size. Additionally, the optimized nanoformulation was also evaluated for morphology, dissolution rate, assay, drug entrapment efficiency, and drug loading content. The in vitro dissolution of optimized drug nanocrystal was done in the phosphate buffer solution of pH 6.8 and compared with bulk CLD and a physical mixture of CLD and pluronic F-68. Results: For optimizing drug nanosuspension, the effect of pluronic F-68 and cilnidipine concentration was investigated, and the optimal values were 0.3% w/v and 5 mg/ml, respectively. Mannitol-containing nanocrystals exhibited a white crystalline powder having a particle size of 154 nm and a good polydispersity index (0.217). Nanocrystals also demonstrated an excellent re-dispersibility in deionized water after manual shaking and no particles were observed at the bottom of the container till 15 days. Such optimized formulation also indicated an increase in dissolution rate in comparison to bulk CLD and their physical mixture with pluronic F-68. It released approximately 72.25% of the drug within 90 minutes while bulk CLD and physical mixture released only 31.24% and 30.37% of the drug, respectively at the same time. The drug assay method indicated that only 92% of the drug was present in optimized nanocrystals after the transformation of nanosuspension into nanocrystals which was less than the initial amount. In this research, the experimental work also analyzed that optimized nanocrystal has only 28.6% of drug loading content. Conclusion: The selected method and cryoprotectant have ability to develop the aqueous re-dispersible nanocrystal for enhancing the dissolution rate and water solubility of CLD-like poorly soluble drugs.

Orthogonal Gelations to Synthesize Core-Shell Hydrogels Loaded with Nanoemulsion-Templated Drug Nanoparticles for Versatile Oral Drug Delivery.

Hydrophobic active pharmaceutical ingredients (APIs) are ubiquitous in the drug development pipeline, but their poor bioavailability often prevents their translation into drug products. Industrial processes to formulate hydrophobic APIs are expensive, difficult to optimize, and not flexible enough to incorporate customizable drug release profiles into drug products. Here, a novel, dual-responsive gelation process that exploits orthogonal thermo-responsive and ion-responsive gelations is introduced. This one-step "dual gelation" synthesizes core-shell (methylcellulose-alginate) hydrogel particles and encapsulates drug-laden nanoemulsions in the hydrogel matrices. In situ crystallization templates drug nanocrystals inside the polymeric core, while a kinetically stable amorphous solid dispersion is templated in the shell. Drug release is explored as a function of particle geometry, and programmable release is demonstrated for various therapeutic applications including delayed pulsatile release and sequential release of a model fixed-dose combination drug product of ibuprofen and fenofibrate. Independent control over drug loading between the shell and the core is demonstrated. This formulation approach is shown to be a flexible process to develop drug products with biocompatible materials, facile synthesis, and precise drug release performance. This work suggests and applies a novel method to leverage orthogonal gel chemistries to generate functional core-shell hydrogel particles.

Solubility Enhancement, Formulation and Evaluation of Solid Tablet of Tinidazole

To attain the necessary concentration of medication in the systemic circulation for the predicted pharmacological response, solubility the phenomenon of solute dissolving in solvent to produce a homogeneous system is a crucial characteristic to consider. The primary challenge in developing formulations for both novel chemical entities and generics is their low water solubility. Formulation scientists face a significant obstacle in solubility. The Classification of Biopharmaceutics states that APIs from Classes II and IV have reduced bioavailability, dissolution, and solubility. The article covers a range of technologies that can be used to make drugs that aren't very water-soluble more soluble. These include crystal engineering, pharmaceutical salts, drug nanocrystals, solid dispersion methods, polymeric micelle preparation, cosolvents, micelles, microemulsions, and emulsion formation. Physical and chemical drug modifications, crystal engineering, salt formation, solid dispersion, surfactant use, micronization, solid dispersions, nano sizing, cyclodextrin complexation, and other advanced methodologies are primarily described in this review. Other methods also contribute to improving solubility and bioavailability. It was an effort to talk about different complexation methods and to quickly point out their possible uses, as well as their technical and economic limits.

Toxicity of Polymeric Nanodrugs as Drug Carriers

The ambiguity of certain diseases and the potential toxicity of many medications have sparked demand for the incorporation and enhancement of drug delivery systems (DDSs). Nanomedicine has received renewed attention in modern medical advancements with therapeutic uses. Therefore, the development of nanomedicines for enhanced bioaccessibility, long drug administration, and dose reduction has advanced as a unique concept. Nanocapsules, nanoemulsions, drug nanocrystals, micelles, solid lipid nanoparticles (SLNs), and polymeric nanoparticles (poly-NPs) are among the most effective nanomedicine techniques. Poly-NPs have emerged as a potential method to enhance drug pharmacokinetics. Pharmaceutical potency can be increased by using nanocarriers and medication formulations. The potential of poly-NPs to alter contemporary medicine has attracted significant interest; polymer adaptability makes them suitable for site-specific drug delivery requirements. However, little is known about their safety in long-term studies using high-pitched doses. These cells exhibit some extrapyramidal symptoms (EPS) because of the reactivity and size reduction of the polymers chosen by using living cells other than the target. With an increased understanding of polymers and their properties, it is equally important to emphasize the safety and toxicity of DDSs. Some of the toxic effects of polymeric nanodrugs include an increase in the cytotoxicity of the cell, reduction in the feasibility of the cell, increase in the rate of programmed cell death (apoptosis), precursors for tumor formation, DNA destruction, gene toxicity, rupture of the cell membrane, and lipid peroxidation reactions. In this article, we discuss the toxicity of nanoparticles (NPs) used in DDSs, including polylactide-co-glycolide, polylactic acid, polycaprolactone, and poly(alkyl cyanoacrylates), used in DDSs.

COMPREHENSIVE REVIEW ON NANOCRYSTAL TECHNOLOGY IN PHARMACEUTICAL FORMULATIONS

Many techniques have been developed to overcome the bioavailability problem of poorly soluble drugs. The nanonization is one of the techniques in that micronized particle is converted in nanoparticle. Several processes are applied for nanocrystal production, including precipitation, milling, high pressure homogenization and combination method. The nanocrystal formulation is administered via various routes like oral, intravenous, intramuscular, pulmonary, ocular and dermal but due to safety, patient compliance and ease of administration, oral drug delivery is preferred. There are two basic ways to prepare drug nanocrystals like “bottom-up” and “top-down” technologies. The present literature provides an overview of the achievement in improving the bioavailability of the poorly soluble drug by using different methods.

Bead milled drug nanocrystal suspensions fine enough to pass through 0.22 ​μm sterilization filters

Bead milled drug nanocrystal suspensions fine enough to pass through 0.22 ​μm sterilization filters

Nanoseeded Desupersaturation and Dissolution Tests for Elucidating Supersaturation Maintenance in Amorphous Solid Dispersions.

The impact of residual drug crystals that are formed during the production and storage of amorphous solid dispersions (ASDs) has been studied using micron-sized seed crystals in solvent-shift (desupersaturation) and dissolution tests. This study examines the impacts of the seed size loading on the solution-mediated precipitation from griseofulvin ASDs. Nanoparticle crystals (nanoseeds) were used as a more realistic surrogate for residual crystals compared with conventional micron-sized seeds. ASDs of griseofulvin with Soluplus (Sol), Kollidon VA64 (VA64), and hydroxypropyl methyl cellulose (HPMC) were prepared by spray-drying. Nanoseeds produced by wet media milling were used in the dissolution and desupersaturation experiments. DLS, SEM, XRPD, and DSC were used for characterization. The results from the solvent-shift tests suggest that the drug nanoseeds led to a faster and higher extent of desupersaturation than the as-received micron-sized crystals and that the higher seed loading facilitated desupersaturation. Sol was the only effective nucleation inhibitor; the overall precipitation inhibition capability was ranked: Sol > HPMC > VA64. In the dissolution tests, only the Sol-based ASDs generated significant supersaturation, which decreased upon an increase in the nanoseed loading. This study has demonstrated the importance of using drug nanocrystals in lieu of conventional coarse crystals in desupersaturation and dissolution tests in ASD development.

Nanoparticles for Drug Delivery and Strategies for Reversing Tumor Drug Resistance

Cancer poses a great threat and challenge to human health. However, the most widely used chemotherapy drugs show resistance due to various reasons, such as cellular efflux, poor targeting, obvious side effects. On the other hand, as a new drug delivery vehicle, nanoparticles have attracted extensive attention due to their good targeting and low toxicity. Various nanoparticles for drug delivery, including liposomes, polymer nanoparticles, micelles, drug-polymer conjugates, dendrimers, nanoshells, magnetic nanoparticles, drug nanocrystals, and extracellular vesicles, are reviewed in this paper.

A Review on Improved Drug Performance of Nanocrystals Formulations

Nano-based technologies are one of the most effective ways to resolve poor aqueous solubility and low absorption of drugs. Nanocrystals are pure drug crystals of particle size below 1000 nm with or without stabilizers adsorbed on it. Nanocrystals are carrier free and can be introduced in any of dosage forms. Reduced particle size and crystallinity offer increased solubility, dissolution velocity, and mucoadhesion. This causes an improvement in pharmacokinetic parameters and drug performance. Top-down approaches like milling, homogenization and bottom-up approach of precipitation are methods of preparation of nanocrystals. Also, combinations of these methods are used for nanocrystals preparation. This review discussed and summarized the improved in-vitro/in-vivo performances and pharmacokinetics of drug nanocrystals.

Bioadhesive eutectogels supporting drug nanocrystals for long-acting delivery to mucosal tissues.

Eutectogels (Egels) are an emerging class of soft ionic materials outperforming traditional temperature-intolerant hydrogels and costly ionogels. Due to their excellent elasticity, non-volatile nature, and adhesion properties, Egels are attracting a great deal of interest in the biomedical space. Herein, we report the first example of adhesive Egels loading drug nanocrystals (Egel-NCs) for controlled delivery to mucosal tissues. These soft materials were prepared using gelatin, glycerine, a deep eutectic solvent (DES) based on choline hydrochloride and glycerol, and nanocrystallised curcumin, a model drug with potent antimicrobial and anti-inflammatory activities. We first explored the impact of the biopolymer concentration on the viscoelastic and mechanical properties of the networks. Thanks to the dynamic interactions between gelatin and the DES, the Egel showed excellent stretchability and elasticity (up to ≈160%), reversible gel-sol phase transition at mild temperature (≈50​°C), 3D-printing ability, and good adhesion to mucin protein (stickiness ≈40​kPa). In vitro release profiles demonstrated the ability of the NCs-based Egel to deliver curcumin for up to four weeks and deposit significantly higher drug amounts in excised porcine mucosa compared to the control cohort. All in all, this study opens new prospects in designing soft adhesive materials for long-acting drug delivery and paves the way to explore novel eutectic systems with multiple therapeutic applications.

Nanocrystal Preparation of Poorly Water-Soluble Drugs with Low Metal Contamination Using Optimized Bead-Milling Technology.

Nanocrystal preparation using bead milling is an important technology to enhance the solubility of poorly water-soluble drugs. However, there are safety concerns regarding the metal contaminants generated during bead milling. We have previously reported optimized bead-milling parameters that could minimize metal contamination and demonstrated comparable performance to NanoCrystal®, a world-leading contamination-free technology. This study aimed to investigate the applicability of optimized milling parameters for preparing nanocrystals of several poorly water-soluble drugs exhibiting various physicochemical properties. Using our optimized bead-milling parameters, we found that all the tested drugs could be ground into nanosized particles within 360 min. Notably, fenofibrate, which has a low melting point, could be ground into nanosized particles owing to the low level of heat generated during bead milling. Additionally, the concentration of metal contaminants in all the drugs prepared using the optimized milling parameters were approximately ten to twentyfold lower than those prepared without the optimized parameters and were comparable to those prepared using polycarbonate beads, known to minimize metal contamination during bead milling. Our results provide insights into the development of drug nanocrystals with low metal contamination using bead milling.

Highly water-soluble dapsone nanocrystals: Towards innovative preparations for an undermined drug

Dapsone (DAP)is a dual-function drug substance; however, its limited water solubility may impair its bioavailability. Drug nanocrystals are an alternative to overcome this limitation. Herein, a DAP nanosuspension was prepared using adesign space approach aiming to investigate the influence of raw material properties and process parameters on the critical quality attributes of the drugnanocrystals. Optimized nanocrystals with 206.3 ± 6.7 nm using povacoat™ as stabilizer were made. The nanoparticles were characterized by dynamic light scattering, laser diffraction, scanning electron microscopy, differential scanning calorimetry, X-ray powder diffraction, and saturation solubility. Compared to the raw material, the nanocrystals were 250-times smaller. Meanwhile, its crystalline state remained basically unchanged even after milling and drying. The nanosuspension successfully maintained its physical stability inlong-termandaccelerated stability studiesover, 4 and 3 months. Furthermore, toxicity studiesshowed low a toxicity at a20 mg/kg. As expected for nanocrystals, the size reduction improvedsaturation solubility3.78 times in water. An attempt to scale up from lab to pilot scale resulted nanocrystals of potential commercial quality. In conclusion, the present study describes the development of dapsone nanocrystals for treating infectious and inflammatory diseases. The nanocrystal formuation can be scaled up for commercial use.

High Drug Loading Nanoparticles Stabilized with Autologous Serum Proteins Passively Inhibits Tumor Growth.

We report drug nanocrystals stabilized with host-specific serum proteins with high loading (∼63% w/w). The human serum derived curcumin nanoparticles (Cur-NanoSera) showed superior in vitro anticancer efficiency compared to a free drug with substantial hemocompatibility. The preadsorbed protein coating impeded further protein corona formation, even with repeated serum exposures. Acute and subacute toxicity evaluations post single and dual injections of C57BL/6 mice indicated that Cur-NanoSera showed no prominent inflammatory response or organ damage in the in-bred mice. Passive accumulation of Cur-NanoSera in tumor tissue significantly suppressed its growth in a syngeneic breast tumor model in addition to controlling tumor burden associated splenomegaly.

Advances of Combinative Nanocrystal Preparation Technology for Improving the Insoluble Drug Solubility and Bioavailability

The low solubility and bioavailability of aqueous insoluble drugs are critical challenges in the field of pharmaceuticals that need to be overcome. Nanocrystal technology, a novel pharmacological route to address the poor aqueous solubility problem of many poorly soluble drugs, has recently demonstrated great potential for industrial applications and developments. This review focuses on today’s preparation technologies, containing top-down, bottom-up, and combinative technology. Among them, the highlighted combinative technology can improve the efficiency of particle size reduction and overcome the shortcomings of a single technology. Then, the characterization methods of nanocrystal production are presented in terms of particle size, morphology, structural state, and surface property. After that, we introduced performance evaluations on the stability, safety, and the in vitro/in vivo dissolution of drug nanocrystals. Finally, the applications and prospects of nanocrystals in drug development are presented. This review may provide some references for the further development and optimization of poorly soluble drug nanocrystals.