3D-printed wound dressing platform for protein administration based on alginate and zinc oxide tetrapods.

Wound treatment requires a plethora of independent properties. Hydration, anti-bacterial properties, oxygenation and patient-specific drug delivery all contribute to the best possible wound healing. Three-dimensional (3D) printing has emerged as a set of techniques to realize individually adapted wound dressings with open porous structure from biomedically optimized materials. To include all the desired properties into the so-called bioinks is still challenging. In this work, a bioink system based on anti-bacterial zinc oxide tetrapods (t-ZnO) and biocompatible sodium alginate is presented. Additive manufacturing of these hydrogels with high t-ZnO content (up to 15 wt.%) could be realized. Additionally, protein adsorption on the t-ZnO particles was evaluated to test their suitability as carriers for active pharmaceutical ingredients (APIs). Open porous and closed cell printed wound dressings were tested for their cell and skin compatibility and anti-bacterial properties. In these categories, the open porous constructs exhibited protruding t-ZnO arms and proved to be anti-bacterial. Dermatological tests on ex vivo skin showed no negative influence of the alginate wound dressing on the skin, making this bioink an ideal carrier and evaluation platform for APIs in wound treatment and healing.

Novel NKG2D-directed bispecific antibodies enhance antibody-mediated killing of malignant B cells by NK cells and T cells.

The activating receptor natural killer group 2, member D (NKG2D) represents an attractive target for immunotherapy as it exerts a crucial role in cancer immunosurveillance by regulating the activity of cytotoxic lymphocytes. In this study, a panel of novel NKG2D-specific single-chain fragments variable (scFv) were isolated from naïve human antibody gene libraries and fused to the fragment antigen binding (Fab) of rituximab to obtain [CD20×NKG2D] bibodies with the aim to recruit cytotoxic lymphocytes to lymphoma cells. All bispecific antibodies bound both antigens simultaneously. Two bibody constructs, [CD20×NKG2D#3] and [CD20×NKG2D#32], efficiently activated natural killer (NK) cells in co-cultures with CD20+ lymphoma cells. Both bibodies triggered NK cell-mediated lysis of lymphoma cells and especially enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) by CD38 or CD19 specific monoclonal antibodies suggesting a synergistic effect between NKG2D and FcγRIIIA signaling pathways in NK cell activation. The [CD20×NKG2D] bibodies were not effective in redirecting CD8+ T cells as single agents, but enhanced cytotoxicity when combined with a bispecific [CD19×CD3] T cell engager, indicating that NKG2D signaling also supports CD3-mediated T cell activation. In conclusion, engagement of NKG2D with bispecific antibodies is attractive to directly activate cytotoxic lymphocytes or to support their activation by monoclonal antibodies or bispecific T cell engagers. As a perspective, co-targeting of two tumor antigens may allow fine-tuning of antibody cancer therapies. Our proposed combinatorial approach is potentially applicable for many existing immunotherapies but further testing in different preclinical models is necessary to explore the full potential.

Reconstituted dry powder formulations of ZnO-adjuvanted ovalbumin induce equivalent antigen specific antibodies but lower T cell responses than ovalbumin adjuvanted with Alhydrogel® or cationic adjuvant formulation 01 (CAF®01).

Most licensed human vaccines are based on liquid dosage forms but have poor storage stability and require continuous and expensive cold-chain storage. In contrast, the use of solid vaccine dosage forms produced by for example spray drying, extends shelf life and eliminates the need for a cold chain. Zinc oxide (ZnO)-based nanoparticles display immunomodulatory properties, but their adjuvant effect as a dry powder formulation is unknown. Here, we show that reconstituted dry powder formulations of ZnO particles containing the model antigen ovalbumin (OVA) induce antigen-specific CD8+ T-cell and humoral responses. By systematically varying the ratio between ZnO and mannitol during spray drying, we manufactured dry powder formulations of OVA-containing ZnO particles that displayed: (i) a spherical or wrinkled surface morphology, (ii) an aerodynamic diameter and particle size distribution optimal for deep lung deposition, and (iii) aerosolization properties suitable for lung delivery. Reconstituted dry powder formulations of ZnO particles were well-tolerated by Calu-3 lung epithelial cells. Furthermore, almost equivalent OVA-specific serum antibody responses were stimulated by reconstituted ZnO particles, OVA adjuvanted with Alhydrogel®, and OVA adjuvanted with the cationic adjuvant formulation 01 (CAF®01). However, reconstituted dry powder ZnO particles and OVA adjuvanted with Alhydrogel® induced significantly lower OVA-specific CD8+CD44+ T-cell responses in the spleen than OVA adjuvanted with CAF®01. Similarly, reconstituted dry powder ZnO particles activated significantly lower percentages of follicular helper T cells and germinal center B cells in the draining lymph nodes than OVA adjuvanted with CAF®01. Overall, our results show that reconstituted dry powder formulations of ZnO nanoparticles can induce antigen-specific antibodies and can be used in vaccines to enhance antigen-specific humoral immune responses against subunit protein antigens.

Possibilities and advantages of additive manufacturing in dry powder formulations for inhalation.

In dry powder formulations for inhalation, coarse carrier particles are often used to improve handling, dosing and dispersion of the active pharmaceutical ingredient (API). Carrier particles, mostly alpha-lactose monohydrate crystals, always show a certain size distribution and are never exactly uniform in their geometry. This might be one factor of the rather high invivo variability in fine particle dose from dry powder inhalers. To address the inhomogeneity of carrier particles, additive manufacturing has come to mind. The parametric design of the perfect carrier geometry could further improve the efficiency of dry powder formulations. In this study, a numerical simulation setup using the discrete element method as well as an experimental approach with 3D printed particles were used to determine the loading capacity of a model API onto two different carrier geometries. The difference between the two geometries was reduced solely to their surface's topology to assess the impact of that. The results indicate differences in the loading capacity for the two geometries, depending on the loading process. This study highlights the importance of the carrier geometry for the efficiency of dry powder formulations and thus, strengthens the idea of artificially designed and printed carrier particles.

Nasal delivery as a strategy for the prevention and treatment of COVID-19.

INTRODUCTION: The upper respiratory tract is a major route of infection for COVID-19 and other respiratory diseases. Thus, it appears logical to exploit the nose as administration site to prevent, fight, or minimize infectious spread and treat the disease. Numerous nasal products addressing these aspects have been considered and developed for COVID-19. AREAS COVERED: This review gives a comprehensive overview of the different approaches involving nasal delivery, i.e., nasal vaccination, barrier products, and antiviral pharmacological treatments that have led to products on the market or under clinical evaluation, highlighting the peculiarities of the nose as application and absorption site and pointing at key aspects of nasal drug delivery. EXPERT OPINION: From the analysis of nasal delivery strategies to prevent or fight COVID-19, it emerges that, especially for nasal immunization, formulations appear the same as originally designed for parenteral administration, leading to suboptimal results. On the other hand, mechanical barrier and antiviral products, designed to halt or treat the infection at early stage, have been proven effective but were rarely brought to the clinics. If supported by robust and targeted product development strategies, intranasal immunization and drug delivery can represent valid and sometimes superior alternatives to more conventional parenteral and oral medications.

Correction: Trenkel, M.; Scherließ, R. Nasal Powder Formulations: In-Vitro Characterisation of the Impact of Powders on Nasal Residence Time and Sensory Effects. Pharmaceutics 2021, 13, 385.

There was an error in the original publication [...].

Establishment of a Rodent Glioblastoma Partial Resection Model for Chemotherapy by Local Drug Carriers-Sharing Experience.

Local drug delivery systems (LDDS) represent a promising therapy strategy concerning the most common and malignant primary brain tumor glioblastoma (GBM). Nevertheless, to date, only a few systems have been clinically applied, and their success is very limited. Still, numerous new LDDS approaches are currently being developed. Here, (partial resection) GBM animal models play a key role, as such models are needed to evaluate the therapy prior to any human application. However, such models are complex to establish, and only a few reports detail the process. Here, we report our results of establishing a partial resection glioma model in rats suitable for evaluating LDDS. C6-bearing Wistar rats and U87MG-spheroids- and patient-derived glioma stem-like cells-bearing athymic rats underwent tumor resection followed by the implantation of an exemplary LDDS. Inoculation, tumor growth, residual tumor tissue, and GBM recurrence were reliably imaged using high-resolution Magnetic Resonance Imaging. The release from an exemplary LDDS was verified in vitro and in vivo using Fluorescence Molecular Tomography. The presented GBM partial resection model appears to be well suited to determine the efficiency of LDDS. By sharing our expertise, we intend to provide a powerful tool for the future testing of these very promising systems, paving their way into clinical application.

Chitosan-Based Thermogelling System for Nose-to-Brain Donepezil Delivery: Optimising Formulation Properties and Nasal Deposition Profile.

Donepezil nasal delivery strategies are being continuously investigated for advancing therapy in Alzheimer's disease. The aim of this study was to develop a chitosan-based, donepezil-loaded thermogelling formulation tailored to meet all the requirements for efficient nose-to-brain delivery. A statistical design of the experiments was implemented for the optimisation of the formulation and/or administration parameters, with regard to formulation viscosity, gelling and spray properties, as well as its targeted nasal deposition within the 3D-printed nasal cavity model. The optimised formulation was further characterised in terms of stability, in vitro release, in vitro biocompatibility and permeability (using Calu-3 cells), ex vivo mucoadhesion (using porcine nasal mucosa), and in vivo irritability (using slug mucosal irritation assay). The applied research design resulted in the development of a sprayable donepezil delivery platform characterised by instant gelation at 34 °C and olfactory deposition reaching a remarkably high 71.8% of the applied dose. The optimised formulation showed prolonged drug release (t1/2 about 90 min), mucoadhesive behaviour, and reversible permeation enhancement, with a 20-fold increase in adhesion and a 1.5-fold increase in the apparent permeability coefficient in relation to the corresponding donepezil solution. The slug mucosal irritation assay demonstrated an acceptable irritability profile, indicating its potential for safe nasal delivery. It can be concluded that the developed thermogelling formulation showed great promise as an efficient donepezil brain-targeted delivery system. Furthermore, the formulation is worth investigating in vivo for final feasibility confirmation.

Impact of post-freeze annealing on shrinkage of sucrose and trehalose lyophilisates.

Freeze-drying of pharmaceuticals produces lyophilisates with properties that depend on both the formulation and the process. Characterisation of the lyophilisate in terms of appearance is necessary not only to produce a visually appealing product, but also to gain insight into the freeze-drying process. The present study investigates the impact of post-freeze annealing on the volume of lyophilisates. For this purpose, sucrose and trehalose solutions were freeze-dried with different annealing conditions and the resulting lyophilisates were analysed with a 3D structured light scanner. The external structure of the lyophilisates was found to be dependent on the bulk materials as well as the choice of vials, while the volume was influenced by the annealing time and temperature. Additionally, differential scanning calorimetry was used to determine glass transition temperatures of frozen samples. As a novelty, the volumes of the lyophilisates and their corresponding glass transition temperatures were compared. This resulted in a correlation supporting the theory that the shrinkage of lyophilisates depends on the amount of residual water in the freeze-concentrated amorphous phase before drying. Understanding the volume change of lyophilisates, in combination with material properties such as glass transition temperature, forms the basis for relating physicochemical properties to process parameters in lyophilisation.

Preparation of Albatrellus ovinus ß-Glucan Microparticles with Dectin-1a Binding Properties.

Fungal ß-glucans are compounds with the potential to activate the innate immune system, in part through binding to the receptor dectin-1. In the present study, small-scale methods for preparing dectin-1a binding microparticles from Albatrellus ovinus alkali-soluble ß-glucans were investigated. Mechanical milling was time-consuming and yielded large particles with wide size distributions. Precipitation was more successful: the ß-glucan was dissolved in 1 M NaOH, diluted, and precipitated in 1:1 mol equiv HCl. This yielded particles in sizes ranging from 0.5-2 µm. The dectin-1a binding activity was determined using HEK-Blue reporter cells. The prepared particles were able to bind to dectin-1a to the same extent as baker's-yeast-derived ß-glucan particles. The precipitation method was convenient as a quick method for small-scale preparation of ß-glucan microparticle dispersions from mushroom ß-glucans.

Pulmonary Application of Novel Antigen-Loaded Chitosan Nano-Particles Co-Administered with the Mucosal Adjuvant C-Di-AMP Resulted in Enhanced Immune Stimulation and Dose Sparing Capacity.

The most successful medical intervention for preventing infectious diseases is still vaccination. This effective strategy has resulted in decreased mortality and extended life expectancy. However, there is still a critical need for novel vaccination strategies and vaccines. Antigen cargo delivery by nanoparticle-based carriers could promote superior protection against constantly emerging viruses and subsequent diseases. This should be sustained by the induction of vigorous cellular and humoral immunity, capable of acting both at the systemic and mucosal levels. Induction of antigen-specific responses at the portal of entry of pathogens is considered an important scientific challenge. Chitosan, which is widely regarded as a biodegradable, biocompatible and non-toxic material for functionalized nanocarriers, as well as having adjuvant activity, enables antigen administration via less-invasive mucosal routes such as sublingual or pulmonic application route. In this proof of principle study, we evaluate the efficacy of chitosan nanocarriers loaded with the model antigen Ovalbumin (OVA) co-administrated with the STING agonist bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) given by pulmonary route. Here, BALB/c mice were immunized with four doses of the formulation that stimulates enhanced antigen-specific IgG titers in sera. In addition, this vaccine formulation also promotes a strong Th1/Th17 response characterized by high secretion of IFN-γ, IL-2 and IL-17, as well as induction of CD8+ T cells. Furthermore, the novel formulation exhibited strong dose-sparing capacity, enabling a 90% reduction of the antigen concentration. Altogether, our results suggest that chitosan nanocarriers, in combination with the mucosal adjuvant c-di-AMP, are a promising technology platform for the development of innovative mucosal vaccines against respiratory pathogens (e.g., Influenza or RSV) or for therapeutic vaccines.

Inexpensive and Easy-To-Use Alternative to the Die Holder for Pharmacopoeial Intrinsic Dissolution Tests.

The pharmacopoeial test method "Intrinsic Dissolution" (Ph.Eur. 2.9.29) is used to study the rate of dissolution for powders of active pharmaceutical ingredients normalized by the surface area. Therefore, powders are compacted into a special metal die holder, which is immersed into a dissolution vessel of the dissolution test apparatus (described in Ph.Eur. 2.9.3). However, in some cases, the test cannot be performed because the compacted powder would not stay in the die holder when in contact with the dissolution medium. In this study, we investigated the removable adhesive gum (RAG) as an alternative to the official die holder. Intrinsic dissolution tests were carried out to exemplify the use of the RAG for this purpose. As model substances, acyclovir and its co-crystal with glutaric acid were used. The RAG was validated for compatibility, release of extractables, unspecific adsorption and the ability to block drug release through the covered surfaces. The results showed that the RAG leaked no unwanted substances, showed no adsorption of acyclovir and blocked its release from covered surfaces. The intrinsic dissolution tests revealed, as expected, a constant release of drug with a small standard deviation between replicates. It was possible to distinguish the acyclovir release from the co-crystal and from the pure drug compound. In conclusion, the findings of this study suggest to consider removable adhesive gum as an easy-to-use and inexpensive alternative to the compendial die holder in intrinsic dissolution tests.

Quality by Design as a Tool in the Optimisation of Nanoparticle Preparation-A Case Study of PLGA Nanoparticles.

Nanoparticles can be used as drug carriers in various applications (e.g., in pulmonary drug delivery and mucosal vaccination). For further investigations, such as drug release studies, as well as for cell and tissue targeting, particles with defined properties are needed. The purpose of the study was to show a multi-step systematic method utilising quality by design to ensure the quality of ovalbumin loaded polylactic-co-glycolic acid nanoparticles (OVA-PLGA-NP), which can be delivered to the lung, and to gain knowledge of the preparation method (double-emulsion solvent evaporation method) in an early development process. Within a definitive screening design, several process parameters (OVA, PLGA and stabiliser concentrations, stirring time and stirring speed of inner emulsion and stirring time and stirring speed of double emulsion) were varied to analyse their impact on resulting properties (z-average, PDI, loading efficiency and loading capacity). The results showed that the preparation of the inner emulsion mainly influenced the drug loading, while the parameters of the second emulsifying step controlled the size. Then a central composite response surface design was used to achieve a predictable OVA-PLGA-NP with an average particle size of 700 nm and high drug-loading. This also enabled the demonstration of curvature and interaction of the stabiliser and the PLGA concentration.

Optimising nasal powder drug delivery - Characterisation of the effect of excipients on drug absorption.

The nasal physiology offers great potential for drug delivery but also poses specific challenges, among which the short residence time of applied drugs is one of the most striking. Formulating the drug as powder and using functional excipients are strategies to improve drug absorption. As nasal powders are still the minority on the market, there is a lack of data regarding their characterisation. This work aims at the characterisation of selected fillers (mannitol, microcrystalline cellulose) and mucoadhesives (pectin, chitosan glutamate, hydroxypropyl cellulose) with a set of methods that allows distinguishing their influences on dissolution and permeation of drugs, and on the viscoelasticity of the nasal fluid and thus the nasal residence time. Rheological studies revealed a potential of undissolved particles to prolong the residence time by increasing the elasticity of the nasal fluid. The assessment of drug dissolution showed a decreased dissolution rate in presence of insoluble or gelling excipients, which can be beneficial for drugs with low permeability, since embedded drugs are cleared slower than plain solutions. Drug permeation as important factor for the selection of excipients was evaluated with an RPMI 2650 cell model. Distinguishing the effects of excipients enables an effective selection of the most promising substances.

Lipobiotin-capture magnetic bead assay for isolation, enrichment and detection of Mycobacterium tuberculosis from saliva.

BACKGROUND: Pulmonary Tuberculosis (TB) is diagnosed through sputum samples. As sputum sampling is challenging in children and cachexic patients, the development of diagnostic tests using saliva appears promising but has been discouraged due to low bacterial load and poor sensitivity. Here, we present a novel and rapid method to enrich Mycobacterium tuberculosis (Mtb) from saliva, which may serve as a basis for a diagnostic saliva test. METHODS: Lipobiotin-functionalized magnetic beads (LMBs) were incubated with Mtb-spiked PBS and saliva from healthy donors as well as with saliva from TB patients. Flow cytometry was used to evaluate the capacity of the beads to bind Mtb, while real-time quantitative polymerase chain reaction (qPCR) was utilized to detect Mtb and determine the amount of mycobacterial DNA in different sample types. RESULTS: We found that LMBs bind Mtb efficiently when compared to non-functionalized beads. The development of an qPCR assay based on the use of LMBs (LMB assay) allowed us to enrich mycobacterial DNA in spiked sample types, including PBS and saliva from healthy donors (enrichment of up to ~8.7 fold). In Mtb-spiked saliva samples, we found that the LMB assay improved the detection rate of 102 bacteria in a volume of 5 ml from 0 out of 15 (0%) to 6 out of 15 (40%). Consistent with that, the LMB assay increased the rate of correctly identified saliva samples from TB patients in two independent cohorts. CONCLUSIONS: Implementation of the principle of the LMB-based assay may improve the sensitivity of existing diagnostic techniques, e.g. by functionalizing materials that facilitate Mtb sampling from the oral cavity.

Quantitative Analysis of Glassy State Relaxation and Ostwald Ripening during Annealing Using Freeze-Drying Microscopy.

Supercooling during the freezing of pharmaceutical solutions often leads to suboptimal freeze-drying results, such as long primary drying times or a collapse in the cake structure. Thermal treatment of the frozen solution, known as annealing, can improve those issues by influencing properties such as the pore size and collapse temperature of the lyophilisate. In this study we aimed to show that annealing causes a rearrangement of water molecules between ice crystals, as well as between the freeze-concentrated amorphous matrix and the crystalline ice phase in a frozen binary aqueous solution. Ice crystal sizes, as well as volume fractions of the crystalline and amorphous phases of 10% (w/w) sucrose and trehalose solutions, were quantified after annealing using freeze-drying microscopy and image labelling. Depending on the annealing time and temperature, the amorphous phase was shown to decrease its volume due to the crystallisation of vitreous water (i.e., glassy state relaxation) while the crystalline phase was undergoing coarsening (i.e., Ostwald ripening). These results allow, for the first time, a quantitative comparison of the two phenomena. It was demonstrated that glassy state relaxation and Ostwald ripening, although occurring simultaneously, are distinct processes that follow different kinetics.

Injectable Thermosensitive Chitosan-Collagen Hydrogel as A Delivery System for Marine Polysaccharide Fucoidan.

Fucoidans, sulfated polysaccharides from brown algae, possess multiple bioactivities in regard to osteogenesis, angiogenesis, and inflammation, all representing key molecular processes for successful bone regeneration. To utilize fucoidans in regenerative medicine, a delivery system is needed which temporarily immobilizes the polysaccharide at the injured site. Hydrogels have become increasingly interesting biomaterials for the support of bone regeneration. Their structural resemblance with the extracellular matrix, their flexible shape, and capacity to deliver bioactive compounds or stem cells into the affected tissue make them promising materials for the support of healing processes. Especially injectable hydrogels stand out due to their minimal invasive application. In the current study, we developed an injectable thermosensitive hydrogel for the delivery of fucoidan based on chitosan, collagen, and ß-glycerophosphate (ß-GP). Physicochemical parameters such as gelation time, gelation temperature, swelling capacity, pH, and internal microstructure were studied. Further, human bone-derived mesenchymal stem cells (MSC) and human outgrowth endothelial cells (OEC) were cultured on top (2D) or inside the hydrogels (3D) to assess the biocompatibility. We found that the sol-gel transition occurred after approximately 1 min at 37 °C. Fucoidan integration into the hydrogel had no or only a minor impact on the mentioned physicochemical parameters compared to hydrogels which did not contain fucoidan. Release assays showed that 60% and 80% of the fucoidan was released from the hydrogel after two and six days, respectively. The hydrogel was biocompatible with MSC and OEC with a limitation for OEC encapsulation. This study demonstrates the potential of thermosensitive chitosan-collagen hydrogels as a delivery system for fucoidan and MSC for the use in regenerative medicine.

Intrathecal application of ethosuximide is highly efficient in suppressing seizures in a genetic model of absence epilepsy.

Systemic drug application is the main approach in epilepsy treatment. However, the central nervous system (CNS) is a challenging target for drug delivery as the blood-brain barrier (BBB) restricts the transfer of drugs into the brain. Accordingly, there is a general interest in developing new therapeutic strategies to improve CNS drug accessibility. Intrathecal administration of antiseizure drugs (ASDs) e.g. via pumps or advanced materials could be a possible approach to bypass the BBB and increase the availability of neuroactive compounds in the CNS. The aim of this study was the evaluation of intracerebroventricular (i.c.v.) compared to systemic drug application in generalized epilepsy. The i.c.v. administration of the established ASD ethosuximide (ETX) in Genetic Absence Epilepsy Rats from Strasbourg (GAERS) caused a robust and dose-dependent reduction of spike-wave discharges (SWDs) without causing obvious behavioral abnormalities. Additionally, we could show that i.c.v. treatment with ETX is significantly more effective in seizure suppression than systemic treatment with the same dose. The localized application resulted in reduced systemic drug exposure compared to standard systemic ETX therapy. The tracing of dye distribution throughout the CNS supported the view that i.c.v. applied drugs cross into brain tissue surrounding the ventricles but largely remain restricted to the site of injection. Our data suggest that intrathecal application represents a possible route for the treatment in generalized epilepsy through direct drug penetration from CSF into brain tissue.

Proof-of-Concept for Adjusted Surface Energies and Modified Fines as a Novel Concept in Particle Engineering for DPI Formulations.

Currently marketed dry powder inhaler (DPI) medicine lacks drug delivery performance due to insufficient powder dispersion. In carrier-based blends, incomplete drug detachment is typically attributed to excessive adhesion forces between carrier and drug particles. Adding force control agents (FCA) is known to increase drug detachment. Several researchers accounted this effect to a decrease in carrier surface energy (SE). In turn, an increase in SE should impede drug detachment. In this proof-of-concept study, we investigated the influence of the SE of the carrier material in binary blends by intentionally inverting the FCA approach. We increased SEs by dry particle coating utilising high-shear mixing, which resulted in decreased respirable fractions of the respective blends. Thus, we confirmed the SE of the carrier influences drug delivery and should be considered in formulation approaches. Complementing engineering techniques on the carrier level, we evaluated a method to modify the SE of extrinsic fines in ternary powder blends for inhalation. By the co-milling of fine lactose and an additive, we tailored the SE and hence the adhesiveness of additional fine excipients. Thus, the extent and the strength of drug-fines agglomerates may be controllable. For ternary DPI formulations, this work highlights the potential benefits of matching the SE of both fines and drugs.