Bioavailability Of Carvedilol Research Articles

Application of Several Nano Carriers to Improve the Solubility and the Bioavailability of Carvedilol

Application of Several Nano Carriers to Improve the Solubility and the Bioavailability of Carvedilol

Binary ethosomes-based transdermal patches assisted by metal microneedles significantly improve the bioavailability of carvedilol

This study aimed to develop a strategy combining binary ethsomes (BES) and microneedles (MNs) to improve the bioavailability of drugs that are poorly bioavailable by oral administration, such as carvedilol (CL). The formula of BES was optimized by Box-Benhnken Designes from four aspects: particle size, zeta potential, encapsulation efficiency and cumulative release in vitro. The optimized BES was then mixed with polyvinylpyrrolidone and sprayed onto the silk fibroin matrix by electrospray to obtain transdermal drug delivery patches (TDDP). In vitro skin penetration test showed that, TDDP has much better transdermal drug release performance compared with free drugs, and MNs-assisted TDDP (MNs-TDDP) can deliver more drugs to the receiving chamber. In vivo studies indicate that MNs-TDDP can significantly reduce the fluctuation of CL concentration in plasma and increase serum level of NO, thereby lower blood pressure for a longer time. These results suggest that the bioavailability of CL can be greatly improved via administration of MNs-TDDP, compared with the oral route. Considering its advantages of non-invasiveness, convenience and good biosafety, MNs-TDDP has a promising prospect in improving the bioavailability of drugs for treating chronic diseases such as hypertension.

Binary polymeric amorphous carvedilol solid dispersions: In vitro and in vivo characterization

Binary polymeric amorphous carvedilol solid dispersions were prepared using solvent method by varying solvent type, polymer type and carvedilol to polymer ratio in order to assess the influence of these factors and maximize carvedilol dissolution rate. Low and high molecular weight polyvinylpyrrolidone, polyvinylpyrrolidone-vinyl acetate copolymer and polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer were used as polymeric carriers in carvedilol to polymer ratios 1:1, 1:2 or 1:4, while absolute ethanol or acetone were used as solvents. Hard gelatin capsules were prepared with carvedilol solid dispersion and lactose monohydrate, mannitol or microcrystalline cellulose. FTIR and PXRD were used to detect carvedilol crystallinity and identify carvedilol-polymer interactions and carvedilol polymorphs. These techniques confirmed carvedilol transition to amorphous state and suggested that hydrogen bonds were formed between carvedilol and polymer molecules. Carvedilol dissolution rate was significantly higher from solid dispersions with higher carvedilol to polymer ratio and solid dispersions prepared using the solvent in which the polymer was more soluble. Solid dispersion with polyvinylpyrrolidone-vinyl acetate copolymer in 1:4 ratio in absolute ethanol displayed the highest carvedilol dissolution rate with 91.78% carvedilol dissolved in the first 30 min. Capsules prepared with the selected solid dispersion and microcrystalline cellulose as diluent displayed the highest carvedilol dissolution rate, with 93.43% carvedilol dissolved within the first 30 min. Carvedilol bioavailability was significantly increased by formulating solid dispersions, while the analysis of serum biochemical parameters excluded damage on liver and kidney function and the lipid profile of animals exposed to investigated drug delivery system.

Formulation and Characterization of Carvedilol Leciplex for Glaucoma Treatment: In-Vitro, Ex-Vivo and In-Vivo Study.

This study evaluated the efficacy of cationic nanoparticle (leciplex) to deliver carvedilol to ocular surface for glaucoma treatment as recent studies pointed out the effect of topical carvedilol on intraocular pressure, therefore carvedilol loaded leciplex formulae were prepared using soy phosphatidyl choline (SPC) and cationic surfactant (CTAB/DDAB) and characterized for morphology, entrapment efficiency, particle size, zeta potential and ex-vivo corneal permeation. Then the selected formula was evaluated via in-vivo studies in comparison with carvedilol solution. Leciplex nanoparticles appeared spherical in shape with entrapment efficiency exceeded 95% in all formulae. Leciplex formula composed of SPC and DDAB in (1:1) molar ratio showed the smallest particle size (16.04 ± 1.2 nm), highest zeta potential value (53.9 ± 0.91 mv) and highest apparent corneal permeability coefficient (0.1157 cm/h). Carvedilol leciplex reduced intraocular pressure (IOP) to normal range in ocular hypertensive rabbits after 30 min and duration of action lasted for 24 h, while carvedilol solution reduced IOP to normal value after 60 min and duration of action lasted for 6 h. Furthermore, histological examination of eyeballs of rabbits treated with carvedilol leciplex showed improvement of retinal atrophy of glaucomatous eyes. This study concluded that leciplex improve transcorneal permeation and bioavailability of carvedilol.

A Review on Solubility Enhancement of Carvedilol—a BCS Class II Drug

Purpose Carvedilol is a third generation non-cardioselective β-blocker used in the treatment of hypertension and demonstrated a potential in the treatment of cardiovascular diseases such as myocardial infarction and arrhythmias. For any drug to be therapeutically effective, it must enter the systemic circulation and to do so, it should have an optimum aqueous solubility at the site of absorption which is a major hurdle to overcome by a formulation scientist. Carvedilol belongs to BCS (biopharmaceutical classification system) class II drugs, thus having low solubility and poor bioavailability (around 25%). Hence, the purpose of this review is to elaborate on several approaches to increase the solubility, dissolution, and bioavailability of carvedilol. Methods Micronization, solid dispersions, cyclodextrin inclusion complex, hydrotropy, nanoformulation which include nanocrystals, nanosuspension, nanoemulsions, dendrimers, and polymeric nanoparticles. It also includes methods that have not been used on carvedilol such as cocrystallization and coamorphous technology. Results Several approaches have successfully increased solubility and bioavailability of carvedilol and several other unexplored methods which have the potential to improve the aqueous solubility of carvedilol but have not been applied till date have also been discussed in the review. Conclusion There are various approaches explored to increase the solubility of carvedilol with every technique having certain advantages and drawbacks. Micronization and nanoformulations (dendrimers, nanoemulsion, nanosuspension, nanocrystals, polymeric nanoparticles) are the most widely used technique for solubility enhancement of carvedilol on laboratory scale due to higher solubility and dissolution rate but they have poor industrial applicability due to difficulty in scale-up and low yield. Efforts are being made to carry out different solubility enhancement techniques with good industrial applicability for carvedilol, e.g., cocrystals. Cocrystals and coamorphous approach for poorly soluble drugs having similar properties to carvedilol have shown good solubility, dissolution, and bioavailability compared to few techniques discussed in this review, and are being widely explored to overcome the drawbacks associated with its method of preparation by carrying out certain advancements (e.g., hot melt extrusion and sonocrystallization) to produce carvedilol cocrystals and coamorphous compound with unique properties in future development.

Comparative study on stabilizing ability of food protein, non-ionic surfactant and anionic surfactant on BCS type II drug carvedilol loaded nanosuspension: Physicochemical and pharmacokinetic investigation

Carvedilol (CAR) in its pure state has low aqueous solubility and extremely poor bioavailability which largely limit its clinical application. The aim of the study is to improve the dissolution rate and the bioavailability of CAR via preparing nanosuspensions with different stabilizers. Antisolvent precipitation-ultrasonication technique was used here. Attempts have been made to use food protein- Whey protein isolate (WPI) as a stabilizer in CAR loaded nanosuspension and also to compare its stabilizing potential with conventional nanosuspension stabilizers such as non-ionic linear copolymer-poloxamer 188 (PLX188) and anionic surfactant-sodium dodecyl sulfate (SDS). Optimized nanosuspensions showed narrow size distribution with particle size ranging from 275 to 640nm. Amorphous state of CAR nanocrystals which also improved the solubility by 16-, 25-, 55-fold accordingly was confirmed by powder X-ray diffraction (PXRD) and differential scanning calorimetry (DSC). From scanning electron microscopy (SEM), flaky shape of PLX188 and SDS nanosuspensions could be revealed but WPI nanosuspension was sphere-shaped. Up to 70% dissolution of loaded drug was observed within 15min in phosphate buffer (pH6.8). A pharmacokinetic study in rats indicated that both Cmax and AUC0–36 values of nanosuspensions were estimated to be 2-fold higher than those of reference, suggesting a significant increase in CAR bioavailability.

Dual Activity of Hydroxypropyl-β-Cyclodextrin and Water-Soluble Carriers on the Solubility of Carvedilol.

Carvedilol (CAR) is a non-selective α and β blocker categorized as class II drug with low water solubility. Several recent studies have investigated ways to overcome this problem. The aim of the present study was to combine two of these methods: the inclusion complex using hydroxypropyl-β-cyclodextrin (HPβCD) with solid dispersion using two carriers: Poloxamer 188 (PLX) and Polyvinylpyrrolidone K-30 (PVP) to enhance the solubility, bioavailability, and the stability of CAR. Kneading method was used to prepare CAR-HPβCD inclusion complex (KD). The action of different carriers separately and in combination on Carvedilol solubility was investigated in three series. CAR-carrier and KD-carrier solid dispersions were prepared by solvent evaporation method. In vitro dissolution test was conducted in three different media: double-distilled water (DDW), simulative gastric fluid (SGF), and PBS pH6.8 (PBS). The interactions between CAR, HPβCD, and different carriers were explored by Fourier transform infrared spectroscopy (FTIR), powder X-ray diffractometry (XRD), and differential scanning colorimetry (DSC). The results showed higher solubility of CAR in KD-PVP solid dispersions up to 70, 25, and 22fold compared to pure CAR in DDW, SGF, and PBS, respectively. DSC and XRD analyses indicated an improved degree of transformation of CAR in KD-PVP solid dispersion from crystalline to amorphous state. This study provides a new successful combination of two polymers with the dual action of HPβCD and PLX/PVP on water solubility and bioavailability of CAR.

In vivo in silico pharmacokinetic simulation studies of carvedilol-loaded nanocapsules using GastroPlus.

The study aimed at in vivo pharmacokinetic evaluation of carvedilol loaded nanocapsules (CLN) followed by in silico predictions and establishment of IVIVC. LC/MS-MS method was developed and validated to estimate the pharmacokinetic profile of CLN. The in silico and IVIVC were established using GastroPlus. The CLN demonstrated 221.09% increase in bioavailability of carvedilol over aqueous suspension. The simulation of plasma concentration profile of CLN exhibited a sensible level of superimposition. The regional absorption of the CLN showed maximum absorption from duodenum and jejunum. The Wagner-Nelson method was found to be most suitable deconvolution method for establishing the IVIVC of CLN. The study demonstrated CLN as efficient delivery system to ferry carvedilol with improved bioavailability.

Impact of CYP2D6 Genetic Variation on the Response of the Cardiovascular Patient to Carvedilol and Metoprolol.

Carvedilol and metoprolol are two of the most commonly prescribed β-blockers in cardiovascular medicine and primarily used in the treatment of hypertension and heart failure. Cytochrome P450 2D6 (CYP2D6) is the predominant metabolizing enzyme of these two drugs. Since the first description of a CYP2D6 sparteinedebrisoquine polymorphism in the mid-seventies, substantial genetic heterogeneity has been reported in the human CYP2D6 gene, with ~100 different polymorphisms identified to date. Some of these polymorphisms render the enzyme completely inactive while others do not modify its activity. Based on all the identified variants, four metabolizer phenotypes are nowadays used to characterize drug metabolism via CYP2D6 in humans: ultra-rapid metabolizer (UM); extensive metabolizer (EM); intermediate metabolizer (IM); and poor metabolizer (PM) phenotypes. As a consequence of these CYP2D6 metabolizer phenotypes, pharmacokinetics and bioavailability of carvedilol and metoprolol can range from therapeutically ineffective levels (in the UM patients) to excessive (overdose) and potentially toxic concentrations (in PM patients). This, in turn, can result in elevated risks for either treatment failure (in terms of blood pressure reduction of hypertensive patients and of improving survival and cardiovascular function of heart failure patients) or for adverse effects (e.g. hypotension and bradycardia). The present review will discuss the impact of these CYP2D6 genetic polymorphisms on the therapeutic responses of cardiovascular patients treated with either of these two β-blockers. In addition, the potential advantages and disadvantages of implementing CYP2D6 genetic testing in the clinic to guide/personalize therapy with these two drugs will be discussed.

Formulation and evaluation of carvedilol microcapsules using Eudragit NE30D and sodium alginate

Inclusion complexes of carvedilol(CR) with hydroxyl propyl beta-cyclodextrin (HPBCD) was prepared using co-grinding technique. Then, the inclusion complex was microencapsulated using combinations of Eudragit NE30D (EU) and sodium alginate (SA) utilizing orifice gelation technique. The formulations were analysed by using Scanning electron microscopy (SEM), Fourier Transform Infrared spectroscopy (FTIR), Differential scanning Calorimetry (DSC) and X-ray diffractometer (XRD) and also evaluated for particle size, encapsulation efficiency, production yield, swelling capacity, mucoadhesive properties, zeta potential and drug release. The microcapsules were smooth and showed no visible cracks and extended drug release of 55.2006% up to 12 hours in phosphate buffer of pH 6.8, showing particle size within the range of 264.5-358.5 µm, and encapsulation efficiency of 99.337±0.0100-66.2753±0.0014%.The in vitro release data of optimized batch of microcapsules were plotted in various kinetic equations to understand the mechanisms and kinetics of drug release, which followed first order kinetics, value of "n" is calculated to be 0.459 and drug release was diffusion controlled. The mice were fed with diet for inducing high blood pressure and the in vivo antihypertensive activity of formulations was carried out administering the optimized formulations and pure drug separately by oral feeding and measured by B.P Monwin IITC Life Science instrument and the results indicated that the bioavailability of carvedilol was increased both in vitro and in vivo with the mucoadhesive polymers showing primary role in retarding the drug release.

Carboxylated mesoporous carbon microparticles as new approach to improve the oral bioavailability of poorly water-soluble carvedilol

The main objective of this study was to develop carboxylated ordered mesoporous carbon microparticles (c-MCMs) loaded with a poorly water-soluble drug, intended to be orally administered, able to enhance the drug loading capacity and improve the oral bioavailability. A model drug, carvedilol (CAR), was loaded onto c-MCMs via a procedure involving a combination of adsorption equilibrium and solvent evaporation. The physicochemical properties of the drug-loaded composites were systematically studied using scanning electron microscopy (SEM), transmission electron microscopy (TEM), nitrogen adsorption, powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC) and HPLC. It was found that c-MCM has a high drug loading level up to 41.6%, and higher than that of the mesoporous silica template. Incorporation of CAR in both drug carriers enhanced the solubility and dissolution rate of the drug, compared to the pure crystalline drug. After loading CAR into c-MCMs, its oral bioavailability was compared with the marketed product in dogs. The results showed that the bioavailability of CAR was improved 179.3% compared with that of the commercial product when c-MCM was used as the drug carrier. We believe that the present study will help in the design of oral drug delivery systems for enhanced oral bioavailability of poorly water-soluble drugs.

Development and evaluation of carvedilol-loaded transdermal drug delivery system: In-vitro and in-vivo characterization study

Context: The transdermal drug delivery system was prepared and the bioavailability of the selected drug was enhanced by reducing first-pass metabolism.Objective: The objective of this study was to enhance the bioavailability of carvedilol through transdermal patches.Materials and methods: To develop a matrix-type transdermal patch containing carvedilol with different ratios of polymer combinations by solvent evaporation technique.Results and discussion: In-vitro permeation studies were performed by Franz diffusion cells. The results followed Higuchi kinetics, and mechanism of release was diffusion mediated. On the basis of the in-vitro and physicochemical parameters of carvedilol patches, the code F-1(PVP: Ethyl Cellulose = 4:1) was chosen for the study of in-vivo, ex-vivo, histocompatibility study, and pharmacological study. The bioavailability studies in rats indicated that the carvedilol-loaded transdermal patches provided steady-state plasma concentration and improved bioavailability of 72% in comparison to oral administration. The ex-vivo permeation study in rat’s skin indicated that the flux and permeability co-efficient of optimized F-1 patch was 30.08 ± 0.7 μg/cm2/h and 0.416 ± 0.05 μg/cm2/h, respectively, which was more as compared to plain carvedilol. The histocompatibility study of the F-1 patch on the rat’s skin after 24 h ex-vivo study gave less pathological changes as compared to other. The antihypertensive activity of the patch in comparison with oral administration was studied using N-nitro-L-arginine methyl ester-induced hypertensive rats. It was observed that the optimized patch (F-1) significantly controlled hypertension (p < 0.05).Conclusion: The developed patch increases the efficacy of carvedilol through enhancement of bioavailability for the therapy of hypertension.

Mucoadhesive buccal patches based on interpolymer complexes of chitosan–pectin for delivery of carvedilol

The study was designed to develop bioadhesive patches of carvedilol hydrochloride using chitosan (CH) and pectin (PE) interpolymer complexes and to systematically evaluate their in vitro and in vivo performances. Mucoadhesive buccal patches of carvedilol were prepared using solvent casting method. The physicochemical interaction between CH and PE was investigated by FTIR and DSC studies. The patches were evaluated for their physical characteristics like mass variation, content uniformity, folding endurance, ex vivo mucoadhesion strength, ex vivo mucoadhesion time, surface pH, in vitro drug release, in situ release study, and in vivo bioavailability study. The swelling index of the patches was found to be proportional to the PE concentration. The surface pH of all the formulated bioadhesive patches was found to lie between 6.2 and 7.2. The optimized bioadhesive patch (C1, CH:PE 20:80) showed bioadhesive strength of 22.10 ± 0.20 g, in vitro release of 98.73% and ex vivo mucoadhesion time of 451 min with in a period of 8 h. The optimized patch demonstrated good in vitro and in vivo results. The buccal delivery of carvedilol in rabbits showed a significant improvement in bioavailability of carvedilol from patches when compared to oral route.

Development of Mucoadhesive Patches for Buccal Administration of Carvedilol

A buccal patch for systemic administration of carvedilol in the oral cavity has been developed using two different mucoadhesive polymers. The formulations were tested for in vitro drug permeation studies, buccal absorption test, in vitro release studies, moisture absorption studies and in vitro bioadhesion studies. The physicochemical interactions between carvedilol and polymers were investigated by Fourier transform infrared (FTIR) Spectroscopy. According to FTIR the drug did not show any evidence of an interaction with the polymers used and was present in an unchanged state. XRD studies reveal that the drug is in crystalline state in the polymer matrix. The results indicate that suitable bioadhesive buccal patches with desired permeability could be prepared. Bioavailability studies in healthy pigs reveal that carvedilol has got good buccal absorption. The bioavailability of carvedilol from buccal patches has increased 2.29 folds when compared to that of oral solution. The formulation AC5 (HPMC E 15) shows 84.85 + 0.089% release and 38.69 + 6.61% permeated through porcine buccal membrane in 4 hr. The basic pharmacokinetic parameters like the C(max), T(max) and AUC(total) were calculated and showed statistically significant difference (P<0.05) when given by buccal route compared to that of oral solution.

Investigations of a Novel Self-Emulsifying Osmotic Pump Tablet Containing Carvedilol

Carvedilol has been made into a novel osmotic pump tablet which includes Gelucire 44/14, Lutrol F68, Transcutol P, silicon dioxide, mannitol, citric acid, and sodium hydrogen carbonate. The Self-emulsifying osmotic pump tablet (SEOPT) has two outstanding features. It could improve the bioavailability of carvedilol by self-emulsifying drug delivery system (SEDDS), control the release rate and make the plasma concentrations more stable by elementary osmotic pump tablet. The results of transmission electron microscope (TEM) and particle size assessment showed that the shape of the resultant emulsion was round and regular, the average diameter of the particles was 246 nm. Since the solubility of carvedilol was improved by the emulsion, the elementary SEOPT could guarantee a complete release of carvedilol under the osmotic pressure of mannitol. The cumulative release at 12 hr was 85.18%. Therefore the disadvantage that lipophilic drugs can not be released completely when prepared into elementary osmotic pump tablet was resolved. The results of Differential scanning calorimetry (DSC), Infrared spectroscopy (IR) and X-ray diffraction diffraction (XRD) proved that carvedilol was amorphous in the preparation. The relative bioavailability of carvedilol in beagle dogs was 156.78%. The plasma concentrations were more stable compared with that of commercially available tablet (Luode®). And the in vitro and in vivo correlation was good (r = 0.9725). Therefore, the elementary SEOPT developed in this paper might provide a new idea for preparing lipophilic drugs into osmotic pump tablet conveniently.

Preparation and Evaluation of SEDDS and SMEDDS Containing Carvedilol

ABSTRACTA new self-emulsifying drug delivery system (SEDDS) and self-microemulsifying drug delivery system (SMEDDS) have been developed to increase the solubility, dissolution rate, and, ultimately, oral bioavailability of a poorly water soluble drug, carvedilol. Ternary phase diagrams were used to evaluate the self-emulsification and self-microemulsfication domains. The self-emulsification time following introduction into an aqueous medium under gentle agitation was evaluated. The minimum self-emulsification time was found at a Tween 80 content of 40%. The particle size distribution and ζ-potential were determined. Benzoic acid had a dual function, it improved the self-emulsification performance of SEDDS and SMEDDS in 0.1 N HCl and lead to a positively charged emulsion. The in vitro dissolution rate of carvedilol from SEDDS and SMEDDS was more than two-fold faster compared with that from tablets. The developed SEDDS formulations significantly improved the oral bioavailability of carvedilol significantly, and the relative oral bioavailability of SEDDS compared with commercially available tablets was 413%.

Ｓｏｄｉｕｍ Ｖａｌｐｒｏａｔｅ，ＰｈｅｎｏｂａｒｂｉｔａｌとＣａｒｖｅｄｉｌｏｌのラット血しょう中濃度に及ぼすＣｏｌｅｓｔｉｍｉｄｅの影響

Colestimide is a new anion exchange resin synthesized from 2-metyl imidazole and epichlorohydrin, and shows a specific affinity to bile acids among physiological anions. The drug interaction of colestimide with sodium valproate, phenobarbital and carvedilol in the gastrointestinal absorption in male rats was examined.The plasma valproate concentrations in the treatment with colestimide were significantly lower than those in the control at 15 min-1 hr, and the drug pharmacokinetic parameters, Cmax and AUC0-∞ were 40 and 25% decreased after oral administration of 100 mg/kg sodium valproate with 1.05 g/kg colestimide. The plasma phenobarbital concentrations were significantly lower than the control at 30 min-1 hr, and tmax was 3.0 times delayed after oral administration of 80 mg/kg phenobarbital with 1.05 g/kg colestimide. On the other hand, the administration of 20 mg/kg carvedilol with 1.05 g/kg colemide did not change the time dependent profiles of the plasma concentration and pharmacokinetic parameters of carvedilol.These result suggested that the absorption of sodium valproate was decreased and the absorption of phenobarbital was delayed by the coadministrated colestimide, but no significant effect on the bioavailability of carvedilol was observed.

ラット血しょう中カルベジロール濃度に及ぼすメトクロプラミドと臭化プロパンテリンの影響

We investigated the effects of metoclopramide and propantheline bromide on the bioavailability of carvedilol in rats. To determine the concentration-time profile of plasma carvedilol, the blood samples were obtained from the tail vein after the simultaneous administration of 20mg/ kg carvedilol either with or without 5 mg/kg metoclopramide and 15 mg/kg propantheline bromide. The plasma carvedilol concentrations were significantly higher than the controls at 15 min.-1 hr and 3, 4 hr, and the drug pharmacokinetic parameters time to Cmax (Tmax) was 0.45 time shorter, while the absorption rate constant (Ka) and area under the concentration-time curve(AUC0-24) were 2.5 and 1.5 times higher after the oral administration of carvedilol with metoclopramide. The rate of bioavailability for carvedilol was accelerated by metoclopramide which stimulates gastric emptying. In contrast, the plasma carvedilol concentrations and Tmax., A UC0-24 did not change after the single intraperitoneal administration of carvedilol with the oral administration of metoclopramide. Conversely, propantheline bromide, which delays gastric emptying, slowed absorption, but not according the AUC0-24 of carvedilol.Other similar pharmacokinetic interaction probably occur since carvedilol are poorly absorbed from the stomach and numerous therapeutic agents influence gastrointestinal motility.

Pharmacokinetics and Bioavailability of Carvedilol in Patients with Liver Cirrhosis

Carvedilol is extensively metabolised and has an absolute bioavailability of about 25%; it undergoes a first-pass effect with hepatic elimination (Neugebauer et al. 1987). It can be anticipated, therefore, that impaired hepatic function may change the kinetics of this drug. The following study in patients with liver cirrhosis was carried out to determine which changes in the pharmacokinetics of carvedilol occur as a result of hepatic dysfunction. A control group of healthy subjects who had been studied previously under similar conditions served as a reference.