Colonic Drug Delivery System Research Articles

Preparation, Optimization, and Evaluation of Pellets Containing Mesalamine With Natural Gums For Colon Drug Delivery System

The purpose of the present work was to formulate colon-targeted mesalamine pellets containing gums of Moringa oleifera Lam. (MOG) and Cyamopsis tetragonolobus Taub. (CTG). Formulation of single stimuli mediated release is also difficult to target colon due to variation in the physiological condition, so in present work, pH dependent and enzyme degradation mechanisms are being used to release of drug at the colonic site. Extrusion and spheronization techniques were used for the preparation of pellets. For formulation optimization, factorial design study 32 was used for the selection of the optimized batch. It was found that high ratio of solvent 80:20 and 10% and 7.5% concentration of MOG and CTG respectively produce optimized pellets showed good physical properties and release for F8M and F8C and after coating it showed in vitro release at the colonic condition and in vivo roentgenographic images for targeting. As to say as advantages, spheronization and extrusion method was proved to have economized, whereas natural gums used to control release which added advantage as being as inert and biocompatible. This further formulation scope at industrial scales to reduce side effects of synthetic polymer and make it more biocompatible with the body.

Targeted therapeutic effects of oral inulin-modified double-layered nanoparticles containing chemotherapeutics on orthotopic colon cancer

Colon cancer is emerging as one of the most prevalent cancers globally. Oral colonic drug delivery systems have attracted considerable attention in the treatment of orthotopic colon cancer due to their superior properties. However, the particularity and complexity of the gastrointestinal structure are a hindrance to the safe delivery of drugs to the target site of the colon tumor. Herein, to achieve an effective delivery system specifically targeting the colon, we designed paclitaxel (PTX)-loaded oral colon double-targeted nanoparticles using polylactic acid-polyethyleneimine (PLA-PEI) and hyaluronic acid-inulin (HA-IN). IN is enzyme sensitive and hardly degraded in the upper digestive tract; as such, it can ensure the safe delivery of nanoparticles to the colon. The “IN shell” is degraded by colon-specific bacteria at the colon site. The exposed HA not only promotes intestinal mucosal crossing of nanoparticles, but also acts as the target of CD44 and plays an active targeting role in tumor tissues. The action of the proton sponge effect of PEI induces the successful release of the nanoparticle. The prepared nanoparticles have a negative charge of −19.5 ± 1.2 mV and a size of 176.7 ± 0.3 nm with a narrow PDI of 0.148 ± 0.004. C26 cells were used for in vitro anticancer studies, including fluorescence staining and flow cytometry, and to explore inhibition of proliferation. The analysis demonstrated that the nanoparticles were more efficiently taken up by cancer cells, exhibiting greater cytotoxicity and apoptosis-inducing ability compared to free drugs. Moreover, in vivo studies revealed that the nanoparticles could remain in vivo for 24 h and accumulate at the tumor site. These data provide evidence of the therapeutic effect on orthotopic colon cancer. Also, safety evaluation results demonstrated that PLA-PEI/HA-IN is a safe drug delivery vector, therefore, holds great promise as a new therapeutic strategy for orthotopic colon cancer treatment.

A Review on Pharmacological and Therapeutical Insight of Satranidazole for Colon Targeting in the treatment of Colonic Diseases

Drugs targeting the colonic environment to treat various colonic diseases are the major challenges recently. Various drugs are available in the markets to targeting to the colonic region to treat diseases but due to premature drug releases, target specificity, the low concentration at the target site with high adverse effects which increase patients’ compliance day by day. Satranidazole is a promising drug that is particularly targeting the colonic environment and can break down microorganism DNA to involve the treatment of several diseases such as inflammatory bowel diseases subdivided Ulcerative colitis, Crohn’s disease, amoebiasis, chronic diarrhea, and reduction of pain due to inflammation. Conventional drug delivery of Satranidazole has some disadvantages such as target specificity, high dosing frequency, low therapeutic efficacy, and high adverse effects that can be overcome through targeted drug delivery systems. Satranidazole is successfully showing the efficacy against aerobic, microaerophilic as well as anaerobic bacterial and reduction of inflammation of inflammatory bowel diseases subdivided as Ulcerative colitis and Crohn’s disease. It is a much more potent drug than metronidazole and has fewer adverse effects. Satranidazole is more convenient for oral administration compared to other routes of administration to show activity. Researchers are giving much more attention to targeted drug delivery systems of Satranidazole to show high therapeutic activity with fewer side effects. The current review focus on the pharmacological and therapeutical insight of Satranidazole in the treatment of colonic diseases and recent advancement and the future aspects of Satranidazole targeting various diseases. 
 Keywords: Satranidazole, Inflammatory bowel diseases, Ulcerative colitis, Crohn’s disease, Amoebiasis, Targeted drug delivery system, Future advancement, Colonic drug delivery systems.

Hemoglobin bio-adhesive nanoparticles as a colon-specific delivery system for sustained release of 5-aminosalicylic acid in the effective treatment of inflammatory bowel disease

A colonic drug delivery system was developed to specifically deliver 5-aminosalicylic acid (5-ASA) to the inflamed site by conjugating with hemoglobin nanoparticles (HbNPs). The 5-ASA-HbNPs (eight 5-ASA molecules per Hb molecule) with the size of 220 nm and zeta potential of −14.6 mV is a tailored nanoparticle able to pass through the mucus layer. The 5-ASA-HbNPs do not undergo chemical and enzymatic hydrolysis in the simulated gastrointestinal fluids over 6 h. Significantly higher cellular uptakes and prolonged release was seen for the 5-ASA-HbNPs in Caco-2 cells, compared to free 5-ASA over 72 h. In addition, 5-ASA-HbNPs revealed similar therapeutic effectiveness with free 5-ASA against tumor necrosis factor and showed less inhibitory concentration (IC50) for myeloperoxidase enzyme activity. In vivo imaging of mouse demonstrated the localization of drug in the descending colon after oral administration and about 15% of the administered dose was recovered as 5-ASA from urine in 6 h. The use of these nanoparticles with the mucus adhesion properties and permeability to intestinal epithelial cells can be a good candidate with potential application in the colonic drug delivery field.

Smart polymeric nanocomposite based on protonated aluminosilicate, curcumin, and chitosan for mesalamine drug delivery as an anti-inflammatory nanocarrier

Due to the importance of the colon-specific disease, its treatment with reduced side effects has become fascinating over the last decades. The aim of this study was the synthesis of the nanocomposite based on the protonated aluminum-modified mesoporous silica nanoparticles (H/Al-MSN) and curcumin possesses mesalamine to develop its efficacy and eliminating side effects for enhancing permeability in intestinal tissues. Here, different amounts of mesalamine were loaded and studied under accurate analysis in order to evaluate release quality. The aluminosilicate nanoparticles are encapsulated by the combination of curcumin as an herbal product and also chitosan as a natural biopolymer with the advantages of non-toxicity, biocompatibility, biodegradability, and non-allergenic. The release of mesalamine from the nanocomposite was investigated in different pH included acidic, neutral, and alkaline media. The results showed that the release of mesalamine is dependent on acidity. A colonic drug delivery system was designed based on the release time and pH sensitivity. The drug release was studied at pH 5.5, 7, and 8 a different region of the gastrointestinal tract was simulated. The results showed that the trend of mesalamine release is higher at pH 8 with high efficacy performance of more than 90% at room temperature for about 90 h.

Development of Personalized Colonic Drug Delivery Systems Prepared by 3D-Printing Technology

Colonic drug delivery systems (CDDS) show several advantages for treatment of inflammatory bowel disease such as improving the clinical outcomes and minimizing side effects of corticosteroids. However, variation of the patient's gastrointestinal tract (GIT) in terms of transit time and pH causes the fluctuation of the drug releasing site in the GIT resulting in low therapeutic efficiency. Consequently, 3D-printing techniques have been applied for preparation of personalized colonic drug delivery systems in this study. Prednisolone was selected as a model drug and prepared in the form of a core tablet. Polylactic acid (PLA) and polyvinyl alcohol (PVA) were printed as a tablet housing and a drug control release plug, respectively. A two-factor full factorial model was utilized to design the experiment and partial least square regression (PLS) models were generated to reveal and predict drug release time of the system. From the results, only thickness of the PVA plug significantly affected the drug release at sampling times of 5, 6, 10, and 24 h. The relations between thickness of the plug and drug releases at 5, 6, and 10 h are quadratic but that of 24 h is linear. The relation between thickness of the plug and drug releases is quadratic. The drug could not be completely released in 24 h because the drug was entrapped by PVA gel. The search results show the possibility to utilize the PLS models to modify drug release time for individual patients. However, alteration of plug polymer is a suggestion to obtain complete drug release.

Potential application of Colonic drug delivery system in prevention and treatment of Colorectal Cancer

Colorectal cancer (CRC) is the second leading cause of cancer related deaths in USA. The current regimen used to treat colorectal cancer has many side effects and have higher drug distribution in other tissues. Physical activity and diet play major role in prevention of Colon cancer which is briefly discussed in this paper. Colon targeted drug delivery system (CoDDS) is found to be a promising approach to target the drug specifically to colon. This review provides the description of various colon targeted approaches that can be used to treat CRC. Factors to be consider while designing CoDDS include pH of GIT, Transit time of GI tract and Microbiota of colon. This review will discuss the staging and standard treatments modalities for CRC.

Colonic delivery of metronidazole-loaded capsules for local treatment of bacterial infections: A clinical pharmacoscintigraphy study

Drug delivery to the colon offers great promise for local treatment of colonic diseases as it allows bypassing systemic absorption in the small intestine, thereby increasing luminal drug concentrations in the colon. The primary objective of this in vivo pharmaco-scintigraphy study was to assess the colon drug targeting accuracy of a metronidazole benzoate colonic drug delivery system intended for local treatment of Clostridioides difficile infections. Additionally, it was assessed if the concept of mucoadhesion would increase colonic residence time and promote higher drug bioavailability.Two different capsule formulations were designed and tested in healthy human subjects. Capsules contained either non-mucoadhesive (NM) or mucoadhesive (M) microgranules, both loaded with 100 mg metronidazole benzoate (antibiotic prodrug) and 5 mg samarium oxide (scintigraphy tracer). Filled capsules were coated with a colonic-targeting technology consisting of two functional layers, which allow for accelerated drug release mediated by the intestinal pH in combination with colonic bacteria. Coated capsules were neutron-activated to yield the radioisotope 153Sm prior to administration to 18 healthy subjects. Gamma-scintigraphy imaging was combined with the measurement of drug plasma levels.Formulation NM showed high colon-targeting accuracy. Initial capsule disintegration within the targeted ileocolonic region was observed in 8 out of 9 subjects (89%) with colonic arrival times in the range of 3.5–12 h and reduced systemic exposure. In contrast, the mucoadhesive formulation M showed some inconsistency regarding the site of initial capsule disintegration (targeting accuracy 56%). Variability of drug release was attributed to self-adhesion and agglomeration of the mucoadhesive microparticles within the capsule.Accurate ileocolonic delivery of metronidazole-loaded microgranules was achieved following oral administration of colonic-targeted capsules. Delayed drug release from NM microparticles in the colon leads to a reduced systemic exposure compared to immediate-release data from literature and presumably elevated drug concentrations in the colonic lumen. This approach offers promising options for the local treatment of colonic diseases.

Solid Lipid Nanoparticles Delivery Systems for Colon Cancer Chemotherapy: A Critical Review

Therapeutic approaches for targeting colon cancers are currently of significant importance important because of possible remission, reduction of cancer metastases, and increased success pf surgery or radiotherapy. Colonic drug delivery is becoming the increasingly preferred route for drug administration; however, it has many limitations that can be avoided by the use of proper carrier systems. Currently, many solid lipid nanoparticle systems (SLNs) were developed to enable the formulation of hydrophobic and poorly water-soluble drugs including those utilized as colonic drug delivery systems. They have many advantages including high bioavailability, high biocompatibility, cost-effectiveness, controlled release, physical stability, and safety, besides, avoidance of using organic solvents and capability of large-scale production and sterilization. Various studies provide important insights into the use of SLN delivery system to treat colon cancer. However, there is a general lack of data from clinical trials and further studies are recommended to evaluate SLNs in animal models.

Simple bicarbonate buffer system for dissolution testing: Floating lid method and its application to colonic drug delivery system

In this study, we developed a simple and easy method to use bicarbonate buffer solutions for dissolution tests. A floating lid was newly introduced to prevent the evaporation of CO2 from bicarbonate buffer solutions (the floating lid method). This method was used to evaluate the dissolution profiles of 5-aminosalicylic acid (5-ASA) colonic delivery tablets with a pH-sensitive film coating (5-ASA CDT) (Asacol® 400 mg tablet and the generic products A and B). A sodium bicarbonate solution was added to the dissolution test vessel, covered with the floating lid made of foamed styrol, and adjusted to each pH with an HCl solution (total 500 mL, 2–50 mM, pH 6.0 to 7.5). Without the floating lid, the pH value increased by more than one pH unit within 3.5 h. The floating lid suppressed the increase in pH to less than 0.1 pH unit at 3.5 h and 0.7 pH unit at 22 h. According to the drug product information, all 5-ASA CDTs show similar disintegration times (approximately 0.5 h) and release profiles in the diluted McIlvaine buffer (pH 7.5, 50 mM phosphate, 25 mM citrate). However, in the bicarbonate buffer (pH 7.5, 10 mM), the disintegration time was prolonged to 4.3 h for Asacol®, 8.0 h for generic A, and 7.6 h for generic B. The floating lid method would be useful for formulation development.

Design of dietary fiber azadiracta indica gum based hydrogels for use in drug delivery

Keeping in view the therapeutic importance of dietary fiber azadiracta indica gum in anti-inflammatory action, in the present work, gum has been modified to develop the drug delivery carrier for methyl prednisolone used for treatment of inflammatory colonic ailments to enhance its potential. Hydrogels were formed gum polysaccharide by modified by grafting and crosslinking method. Polymers were characterized by cryo-SEM, AFM, FTIR, and 13C-NMR. The cryo-SEM images confirmed the porous network structure of the hydrogels. The results showed that porosity and crosslinking density of hydrogels were influenced by the content of the monomer AAc and crosslinker NNMBA. The polymeric gels were found to have non-haemolytic (haemolytic index = 3.36 ± 1.20%), and antioxidant nature (DPPH radical scavenging = 76.88 ± 10.16%). The gel strength of the gels was 9.41 ± 0.46 Nmm. The release of methyl prednisolone was found with non-Fickian diffusion mechanism. Overall, gum based hydrogels were pH responsive and could be explored to design of colon drug delivery system for colonic ailments .

New Insights of Oral Colonic Drug Delivery Systems for Inflammatory Bowel Disease Therapy

Colonic Drug Delivery Systems (CDDS) are especially advantageous for local treatment of inflammatory bowel diseases (IBD). Site-targeted drug release allows to obtain a high drug concentration in injured tissues and less systemic adverse effects, as consequence of less/null drug absorption in small intestine. This review focused on the reported contributions in the last four years to improve the effectiveness of treatments of inflammatory bowel diseases. The work concludes that there has been an increase in the development of CDDS in which pH, specific enzymes, reactive oxygen species (ROS), or a combination of all of these triggers the release. These delivery systems demonstrated a therapeutic improvement with fewer adverse effects. Future perspectives to the treatment of this disease include the elucidation of molecular basis of IBD diseases in order to design more specific treatments, and the performance of more in vivo assays to validate the specificity and stability of the obtained systems.

Colon Drug Delivery Systems for the Treatment of Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is one of the most common chronic diseases that affect the entire gastrointestinal tract (GIT) especially the colon. Its symptoms extend from mild diarrhea, abdominal pain, and bloody diarrhea to severe conditions which affect the quality of life. Many treatments have been developed to treat and cure IBD and to improve patient’s quality of life. The big challenge faces the newly developed treatments is the site of action as the colon presents at the distal end of the GIT and have a complex biological environment. Many technologies have been investigated to target the colon, load higher amounts of active ingredients, and decrease unwanted side effects resulted from upper GIT absorption. This review briefly discusses the IBD, treatment lines, physiological considerations, and all methods of colon targeting technologies starting from the traditional methods which based on pH, time, and microbial content of the colon. Also, we discussed in detail all new techniques based on Micro and Nanotechnology which improve the effectiveness of used therapeutics.

Characteristics of Natural Polysaccharides for Colon Drug Targeting

Targeting drugs directly to the colon is advantageous in the topical treatment of colonic diseases such as ulcerative colitis and Crohn’s disease and has shown potential in gaining the oral delivery of peptides and other labile drugs. A colonic drug delivery system is required to protect a drug during its transit through the upper gastro-intestinal (G.I.T.) tract and allow its release in the colon. Several methods of colonic targeting have been proposed. These include taking advantage of the apparent consistency of small intestinal transit times, the utilization of pH changes within the G.I. tract and the exploitation of bacterial enzymes localized in the colonic region of the G.I. tract. Among the different approaches to achieve targeted drug release to the colon, the use of polymers especially biodegradable by colonic bacteria holds great promise. Polysaccharides are bacterial enzymes that are available in sufficient quantity to be exploited in colon targeting of drugs. Based on this approach, various polysaccharides have been investigated for colon-specific drug release. These polysaccharides include pectin, guar gum, amylose, inulin, dextran, chitosan, and chondroitin sulfate. This family of natural polymers has an appeal to drug delivery as it is comprised of polymers with a large number of derivatizable groups, a wide range of molecular weights, varying chemical compositions, and, for the most part, low toxicity and biodegradability yet high stability. The most favorable property of these materials is their approval as pharmaceutical excipients. Polysaccharides are bacterial enzymes that are available in sufficient quantity to degrade these natural polysaccharides.

Formulation and evaluation of deflazacort loaded mucoadhesive microsphere for colon drug delivery system

Irritable bowel disease is very common colon disease. Deflazacort is one of the best drug with clinical activity against Irritable bowel disease. Microsphere system are effectively protect drugs against premature degradation, to localize drug molecules at the target site of action and to control the time and rate of release. Mucoadhesive microspheres enhance the bioavailability of orally given drugs by lengthened contact time of drug with the intestinal mucosa. The main disadvantage of these microspheres is adherence to the substrate by non-specific interaction. To overcome this limitation, microspheres are prepared by emulsification method to treat irritable bowel disease. Chitosan microspheres were prepared by Ionotropic Gelation method. Microspheres were coated with Eudragil S using solvent evaporation method.
 Keywords: Deflazacort, Mucoadhesive, Microspheres, Irritable bowel disease

Controlled drug release behavior of 5-aminosalicylic acid using polyacrylamide grafted oatmeal (OAT-g-PAM): a pH-sensitive drug carrier

In the present investigation, we have evaluated the in vitro drug release of 5-aminosalicylic acid (5-ASA) using polyacrylamide grafted oatmeal (OAT-g-PAM). The graft co-polymer was synthesized by following free radical co-polymerization pathway using acrylamide as a monomer and ceric ammonium nitrate as a free radical initiator in the presence of microwave. The matrix tablet was prepared using the different grades of developed graft co-polymers following standard protocol. In vitro release phenomena of 5-ASA from the prepared matrix tablet up to 12 h in different buffer solution, i.e., pH 2.0, 7.0 and 7.4, an ideal condition for colon specific drug delivery was studied. The release rate of 5-ASA can be controlled efficiently by tuning the pH value. The drug release kinetic of 5-ASA from different matrix tablet was evaluated on the basis of in vitro controlled drug release results. The result indicates that the novel pH-sensitive matrices under study showing Fickian diffusion mechanism and potentially constructive for colonic drug delivery system.

Zein as a Pharmaceutical Excipient in Oral Solid Dosage Forms: State of the Art and Future Perspectives.

Zein is the main storage protein of corn and it has several industrial applications. Mainly in the last 10-15years, zein has emerged as a potential pharmaceutical excipient with unique features. Zein is a natural, biocompatible and biodegradable material produced from renewable sources. It is insoluble, yet due to its amphiphilic nature, it has self-assembling properties, which have been exploited for the formation of micromicroparticle and nanoparticle and films. Moreover, zein can hydrate so it has been used in swellable matrices for controlled drug release. Other pharmaceutical applications of zein in oral drug delivery include its incorporation in solid dispersions of poorly soluble drugs and in colonic drug delivery systems. This review describes the features of zein significant for its use as a pharmaceutical excipient for oral drug delivery, and it summaries the literature relevant to macroscopic zein-based oral dosage forms, i.e. tablets, capsules, beads and powders. Particular attention is paid to the most novel formulations and applications of zein. Moreover, gaps of knowledge as well as possible venues for future investigations on zein are highlighted.

Tetrandrine beads using alginate/polyvinyl alcohol and alginate-carboxymethyl cellulose: Not ideal as colon-targeted dosage form

Objectives: Colon drug delivery system should be able to maintain drug release until the system reaches its target. In this study, beads were selected as drug carrier system to deliver tetrandrine to colon using a combination of two polymers, alginate and polyvinyl alcohol (PVA) and also alginate and carboxymethyl cellulose (CMC). It was hypothesized that alginate/PVA beads and alginate-CMC beads may be able to protect the drug effectively while in the gastrointestinal tract and may deliver the drug at colon under the influence of colonic pH. Materials and Methods: Beads were formulated into six formulae with different concentrations of polymer. Beads were characterized and evaluated for in vitro drug release using dissolution method. Results: Beads' Formula 3 (alginate and PVA 2:1) and Formula 6 (alginate and CMC 2:1.5) were the best formulae with entrapment efficiency 32.12% and 39.83%, respectively. The drug release test was performed first in hydrochloric acid (HCl) medium for 2 h, then in phosphate-buffered pH 7.4 + 2% Tween 80 for 3 h, and finally in phosphate-buffered pH 6.8 + 2% Tween 80 medium for 3 h, successively. The results of drug release in HCl pH 1.2 were 84.13%, 73.12%, 66.57%, 85.59%, 78.26%, and 69.56%, for Formula 1, 2, 3, 4, 5, and 6, respectively. Conclusion: All type of beads showed the high release of tetrandrine in HCl; hence, it is not ideal yet as a colon-targeted dosage form.

Mesalamine-loaded mucoadhesive microsphere for colon drug delivery system: Effect of process variables and in vitro characterization

Objective: The main objective of this study is to formulate mucoadhesive microspheres for colon drug delivery with sodium alginate (ALG) core enriched with drug. Methods: The core microspheres of ALG were prepared by modified emulsification method followed by cross-linking with different concentration of CaCl2 at different stirring speed with constant drug-to-polymer ratio (1:3). The core microspheres were further coated with Eudragit S-100 using the solvent evaporation technique. Results: The microspheres (core and coated) were characterized by shape, size, surface morphology, size distribution, entrapment efficiency, and in vitro drug release studies. In vitro drug release showed that the optimized batch of core microsphere and coated microspheres exhibited 99.53% ± 0.39% and 89.22% ± 0.26%, respectively. The drug release from all formulations of mesalamine microsphere followed Higuchian Kinetics. Moreover, drug release from core and Eudragit S-100-coated microspheres followed Korsmeyer–Peppas equation with anomalous and Fickian kinetics mechanism, respectively. Stability study suggests that the degradation rate constant of mesalamine from Eudragit S-100-coated microsphere was found to be minimum 2 years shelf life of the formulation. On the basis of scanning electron microscopy, the core microspheres were formed slightly irregular in shape due to surface-attached crystals of the drug and coated mesalamine microspheres showed smooth surface and a smaller number of pores due to coating. Conclusions: It can be concluded that the appropriate combination of a pH-dependent polymer (Eudragit S-100) with a pH-independent polymer sodium ALG) was suitably adequately sustained the drug release from mesalamine microspheres.

Development and characterization of new functionalized polyurethanes for sustained and site-specific drug release in the gastrointestinal tract

The main objective of the present paper has been the development and study of two new biodegradable polyurethanes, PU(dithiodiethanol-DTDI) and PU[(iPr)Man-DTDI], to be used as sustained matrix forming excipients. Furthermore, their capacity to act as excipient for colon drug delivery systems has been evaluated. Thus, SeDeM diagrams have been obtained to investigate their suitability to be processed through a direct compression process. Matrices containing 10–30% w/w of the polymers and theophylline anhydrous as model drug have been manufactured. Release studies have been carried out using a modified dissolution assay simulating pH and redox conditions for the gastro intestinal tract, including colon. Drug dissolution data have been analyzed according to the main kinetic models and their Excipient Efficiencies for prolonged release have been calculated. The principal parameters of the SeDeM Expert system, such as the parametric profile (mean radius) and the good compression index obtained for the polymers are above the values considered as adequate for direct compression even without addition of flow agents. The obtained values for Excipient Efficiency show good ability of the polymer to control the drug release. Finally, in the case of PU(dithiodiethanol-DTDI), a clear increase in the release rate has been observed when the formulation is subjected to colon simulating conditions.