Matrine: A review of its pharmacology, pharmacokinetics, toxicity, clinical application and preparation researches.

ETHNOPHARMACOLOGICAL RELEVANCE: "Dogel ebs" was known as Sophora flavescens Ait., which has been widely utilized in the clinical practice of traditional Chinese Mongolian herbal medicine for thousands of years. Shen Nong's Materia Medica (Shen Nong Ben Cao Jing in Chinese pinyin) recorded that it is bitter in taste and cold in nature with the effect of clearing heat and eliminating dampness, insecticide, diuresis. Due to its extensive application in the fields of ethnopharmacological utilization, the pharmaceutical researches of Sophora flavescens Ait.s keeps deepening. Modern pharmacological studies have exhibited that matrine, which is rich in this traditional herbal medicine, mediates its main biological properties. AIMS OF THE REVIEW: This review aimed at summarizing the latest and comprehensive information of matrine on the pharmacology, pharmacokinetics, toxicity, clinical application and preparation researches to explore the therapeutic potential of this natural ingredient. In addition, outlooks and perspective for possible future researches that related are also discussed. MATERIALS AND METHODS: Related information concerning matrine was gathered from the internet database of Google scholar, Pubmed, ResearchGate, Web of Science and Wiley Online Library with the keywords including "matrine", "pharmacology", "toxicology" and "pharmacokinetics", "clinical application", etc. RESULTS: Based on literatures, matrine has a variety of pharmacological effects, including anti-cancer, anti-inflammatory, anti-microbial, detoxification and so on. Nevertheless, there are still some doubts about it due to the toxicity and questionable bioavailability that does exist. CONCLUSIONS: Future researches directions probably include elucidate the mechanism of its toxicity and accurately tracing the in vivo behavior of its drug delivery system. Without doubt, integration of toxicity and efficiency and structure modification based on it are also pivotal methods to enhance pharmacological activity and bioavailability.

Condensed Tannins in the Gastrointestinal Tract of Cattle after Sainfoin (Onobrychis viciifolia) Intake and Their Possible Relationship with Anthelmintic Effects.

Condensed tannins' (CTs) fate along the digestive tract of ruminants may account for the variable efficacy of CTs against gastrointestinal nematodes. We analyzed CTs in the digesta of cattle fed sainfoin. With the acetone-butanol-HCl assay, the total CTs concentrations in the digesta were close to those in the diets (6.3 and 1.5% of DM in experiments 1 and 2, respectively); thus, CTs remained potentially largely undegraded/unabsorbed. With the thiolysis assay, CTs concentration was much higher in the abomasum (2.3% of DM; expt 1) compared with the rumen and intestines, along with higher mean size and prodelphinidins percentage, corroborating CTs efficacy reported only against Ostertagia ostertagi in the abomasum. In expt 2, the dietary levels of CTs were probably too low to demonstrate anthelmintic effects in the rumen. Overall, the level of CTs accessible to thiolysis is favored under the acidic conditions of the abomasum, which seems critical for anthelmintic activity.

In Vitro and In Vivo Drug Interaction Study of Two Lead Combinations, Oxantel Pamoate plus Albendazole and Albendazole plus Mebendazole, for the Treatment of Soil-Transmitted Helminthiasis.

The current treatments against Trichuris trichiura, albendazole and mebendazole, are only poorly efficacious. Therefore, combination chemotherapy was recommended for treating soil-transmitted helminthiasis. Albendazole-mebendazole and albendazole-oxantel pamoate have shown promising results in clinical trials. However, in vitro and in vivo drug interaction studies should be performed before their simultaneous treatment can be recommended. Inhibition of human recombinant cytochromes P450 (CYPs) CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 was tested by exposure to albendazole, albendazole sulfoxide, mebendazole, and oxantel pamoate, as well as albendazole-mebendazole, albendazole sulfoxide-mebendazole, albendazole-oxantel pamoate, and albendazole sulfoxide-oxantel pamoate. A high-pressure liquid chromatography (HPLC)-UV/visible spectroscopy method was developed and validated for simultaneous quantification of albendazole sulfoxide, albendazole sulfone, mebendazole, and oxantel pamoate in plasma. Albendazole, mebendazole, oxantel pamoate, albendazole-mebendazole, and albendazole-oxantel pamoate were orally applied to rats (100 mg/kg) and pharmacokinetic parameters calculated. CYP1A2 showed a 2.6-fold increased inhibition by albendazole-oxantel pamoate (50% inhibitory concentration [IC50] = 3.1 µM) and a 3.9-fold increased inhibition by albendazole sulfoxide-mebendazole (IC50 = 3.8 µM) compared to the single drugs. In rats, mebendazole's area under the concentration-time curve (AUC) and maximal plasma concentration (Cmax) were augmented 3.5- and 2.8-fold, respectively (P = 0.02 for both) when coadministered with albendazole compared to mebendazole alone. Albendazole sulfone was slightly affected by albendazole-mebendazole, displaying a 1.3-fold-elevated AUC compared to albendazole alone. Oxantel pamoate could not be quantified, translating to a bioavailability below 0.025% in rats. Elevated plasma levels of albendazole sulfoxide, albendazole sulfone, and mebendazole in coadministrations are probably not mediated by CYP-based drug-drug interaction. Even though this study indicates that it is safe to coadminister albendazole-oxantel pamoate and albendazole-mebendazole, human pharmacokinetic studies are recommended.

Polymeric Nanostructured Systems for Liquid Formulation of Praziquan-tel: Development and in vitro Assessment.

Praziquantel (PZQ) is widely used in the treatment of several parasitic infections in both humans and animals, and is the first choice in the treatment of Schistosomiasis in humans. However, PZQ is a hydrophobic drug, and its low aqueous solubility has been a significant barrier to the development of oral liquid formulations that may provide improved bioavailability, pharmacokinetic profile, and compliance. The aim of this study was thus (i) to develop an oil-in-water (O/W) nanoemulsion(NE)-based platform for the delivery of PZQ in liquid form; (ii) to study the transport of PZQ formulated in NEs across an in vitro model of the intestinal epithelium; and (iii) to determine the toxicity profile of the NEs and their individual components on the model epithelium. We also sought to compare the toxicity and transport profiles of the proposed formulations, with those of PZQ in a solid nanostructured particle system - PZQ encapsulated within poly(lactic acid-co-glycolic acid) (PLGA) nanoparticles (NPs). Two essential oils were selected as the oil phase in the NEs, namely clove and orange. The NEs were prepared with selected non-ionic surfactants and had high solubilization capacity towards PZQ, and average diameters well below 100nm. The NEs also showed long term physical stability at both simulated physiological and gastric conditions. NEs with clove oil (NEC-PZQ) were observed to have a lower cytotoxic profile when compared to the orange oil NEs (NEO-PZQ). The results also showed that the transport of PZQ formulated within such nanostructured systems was much greater and larger rates across confluent and polarized Caco-2 monolayers when compared to free PZQ. Interestingly, little difference in PZQ transport between the NEs and NPs was observed. These results point to NEs as potentially viable strategies for the liquid formulation of PZQ in particular, and more broadly to the formulation of other hydrophobic therapeutics that may be employed in the fight against important neglected diseases such as Schistosomiasis, which alone affects more than 240 million people worldwide.

Praziquantel in a clay nanoformulation shows more bioavailability and higher efficacy against murine Schistosoma mansoni infection.

Consideration of existing compounds always simplifies and shortens the long and difficult process of discovering new drugs specifically for diseases of developing countries, an approach that may add to the significant potential cost savings. This study focused on improving the biological characteristics of the already-existing antischistosomal praziquantel (PZQ) by incorporating it into montmorillonite (MMT) clay as a delivery carrier to overcome its known bioavailability drawbacks. The oral bioavailability of a PZQ-MMT clay nanoformulation and its in vivo efficacy against Schistosoma mansoni were investigated. The PZQ-MMT clay nanoformulation provided a preparation with a controlled release rate, a decrease in crystallinity, and an appreciable reduction in particle size. Uninfected and infected mice treated with PZQ-MMT clay showed 3.61- and 1.96-fold and 2.16- and 1.94-fold increases, respectively, in area under the concentration-time curve from 0 to 8 h (AUC0-8) and maximum concentration of drug in serum (Cmax), with a decrease in elimination rate constant (kel) by 2.84- and 1.35-fold and increases in the absorption rate constant (ka) and half-life (t1/2e) by 2.11- and 1.51-fold and 2.86- and 1.34-fold, respectively, versus the corresponding conventional PZQ-treated groups. This improved bioavailability has been expressed in higher efficacy of the drug, where the dose necessary to kill 50% of the worms was reduced by >3-fold (PZQ 50% effective dose [ED50] was 20.25 mg/kg of body weight for PZQ-MMT clay compared to 74.07 mg/kg for conventional PZQ), with significant reduction in total tissue egg load and increase in total immature, mature, and dead eggs in most of the drug-treated groups. This formulation showed better bioavailability, enhanced antischistosomal efficacy, and a safer profile despite the longer period of residence in the systemic circulation. Although the conventional drug's toxicity was not examined, animal mortality rates were not different between groups receiving the test PZQ-clay nanoformulation and conventional PZQ.

An alternative mebendazole formulation for cystic echinococcosis: the treatment efficacy, pharmacokinetics and safety in mice.

BACKGROUND: Cystic echinococcosis is a serious zoonotic infection worldwide caused by metacestodes of Echinococcus gruanulosus. Mebendazole and albendazole are the only two drugs used in the treatment of this disease with cure rates only about 30% due to the poor oral absorption. Thus an alternative treatment for this disease is needed. METHODS: A mebendazole oily suspension (MBZ-OS) was prepared and orally administrated to mice infected with echinococcus cysts for 8 months at 12.5 mg/kg and 25 mg/kg for 14 consecutive days. Mebendazole suspended in 1% tragacanth (MBZ-1% tragacanth) served as treated control. In addition, liver and serum samples were collected from these treated mice (25 mg/kg) for histopathology examination and liver function test. For pharmacokinetic analysis, plasma, parasite (cyst wall and cyst fluid) and tissue samples were collected at 0.25, 0.5, 1, 2, 4, 8, 16 and 24 h after orally administrating MBZ-OS and MBZ-1% tragacanth to E. granulosus-infected mice at 25 mg/kg. These samples were then processed and quantitatively analyzed by HPLC. RESULTS: The administration of MBZ-OS resulted in a treatment efficacy with the cyst weight reductions higher than 80%, significantly better than the corresponding MBZ-1% tragacanth groups. The better treatment efficacy of MBZ-OS was related to the higher drug concentration in plasma, parasites and tissues. It was also shown that the injury of the liver was not significantly altered by taking MBZ-OS compared to the untreated control. CONCLUSION: These findings demonstrate that MBZ-OS is a promising new formulation of MBZ for treatment of hydatid diseases without showing significantly liver toxicity.

Combination therapy of Western drugs and herbal medicines: recent advances in understanding interactions involving metabolism and efflux.

INTRODUCTION: While complementary and alternative medicine markets prosper with an increasing number of consumers of herbal medicines, there is an associated likelihood for herb-drug interactions to occur. Modulation of the activity of metabolic enzymes and/or active transporters by chemical constituents of herbal medicines may influence the therapeutic outcomes of coadministered allopathic medicines due to changes in their pharmacokinetic profiles. Although valuable information on herb-drug interactions is obtained by in vitro studies, such as the mechanisms of interaction, clinical significance of interactions is ultimately demonstrated by in vivo data. AREAS COVERED: The authors outline the mechanisms of herb-drug pharmacokinetic interactions briefly and discuss pharmacokinetic interactions between different therapeutic classes of Western drugs and herbal medicines. Furthermore, the authors also discuss herb-drug interactions from both in vitro and in vivo studies with specific focus on recent findings. EXPERT OPINION: Basic and clinical researches have contributed to the comprehension of the underlying mechanisms involved as well as the practical implications of herb-drug interactions. This provides a foundation for development of guidelines to inform patients about herb-drug interactions that can affect their health.

Efficacy against Fasciola hepatica and the pharmacokinetics of triclabendazole administered by oral and topical routes.

OBJECTIVE: To determine the efficacy of triclabendazole (TCBZ) against 28-day-old, early immature liver fluke in cattle and its pharmacokinetics following administration by the oral or topical (pour-on) route. PROCEDURES: Cattle (n = 18) were infected with 500 TCBZ-susceptible liver fluke metacercariae and randomly allocated to three groups. At 28 days after infection, the groups were: (1) untreated controls; (2) treated with oral TCBZ at 12 mg/kg in combination with oxfendazole and selenium (TOS); (3) treated with pour-on TCBZ at 30 mg/kg in combination with abamectin (TA). Blood samples were taken immediately prior to treatment and serially after treatment to assess the plasma profile of TCBZ metabolites. Ten weeks after treatment all animals were slaughtered and total liver fluke counts, fluke egg counts and liver pathology were assessed. RESULTS: Both the TOS and TA treatments resulted in significant reductions of 28-day-old liver fluke, as assessed by fluke counts and fluke egg counts at slaughter, and the reductions following TOS treatment were significantly greater than those following TA treatment. The blood profile of TCBZ metabolites in TOS-treated animals showed a significantly greater area under the plasma concentration time curve and a higher maximum observed concentration than those treated with TA. There was significantly less liver pathology in TOS-treated animals than in the TA-treated animals. CONCLUSION: TCBZ administered orally at 12 mg/kg resulted in greater efficacy against 28-day-old, early immature liver fluke than was achieved by topical administration at 30 mg/kg. Plasma metabolites of TCBZ were higher and liver pathology was less in TOS-treated animals than in TA-treated animals.

Estimation of absolute oral bioavailability of moxidectin in dogs using a semi-simultaneous method: influence of lipid co-administration.

Moxidectin is a long-acting anthelmintic drug for which little is known about its kinetic behaviour in dogs and its oral absolute bioavailability has never been reported. We studied the pharmacokinetics of moxidectin in dogs, with a special emphasis on oral bioavailability and the influence of lipid co-administration, by using a semi-simultaneous method of administration. Ten Beagle dogs were dosed orally and then intravenously (i.v.) with 0.2 mg/kg moxidectin. The oral application was conducted with or without corn oil co-administration. Moxidectin concentration-time profiles in plasma were analysed using a compartmental modelling approach, designed to fit the oral and i.v. kinetic disposition curves simultaneously. In contrast to what happens in other species, our study indicates that the bioavailability of orally given moxidectin in dogs is nearly total (90.2 +/- 7.4%), and is not enhanced by lipid co-administration. The clearance, the volume of distribution, the mean residence time and the terminal half-life were similar to what was already described for other species. Finally our trial suggests that the body condition (degree of obesity) is likely to be a major determinant of moxidectin kinetics in dogs because of its modulation of the volume of distribution that indirectly controls the terminal half-life of the drug.

Contribution of lymphatic transport to the systemic exposure of orally administered moxidectin in conscious lymph duct-cannulated dogs.

Moxidectin, a macrocyclic lactone (ML), is a potent parasiticide widely used in veterinary medicine and currently under development for use in humans. The contribution of the lymphatic route to the intestinal absorption and transport of moxidectin to the systemic circulation was evaluated in lymph duct-cannulated dogs. Beagle dogs were operated for lymph duct cannulation and were orally dosed with 38g of corn oil and moxidectin (0.2mg/kg, n=3). The lymph and plasma were collected over 24h and moxidectin and triglyceride concentrations were measured. Similarly, control dogs (n=5) were dosed orally with moxidectin and oil and subsequently with moxidectin intravenously. Pharmacokinetic parameters were calculated for moxidectin in the plasma of the dogs. Moxidectin readily accumulated in the lymph and reached a plateau 8h post-administration, paralleling triglyceride appearance. The percentage of moxidectin recovered in lymph was 22+/-3% of the total administered dose with 92% being associated with triglyceride-rich particles. The systemic bioavailability of oral moxidectin coadministered with lipid was only 40% in the lymph duct-cannulated dogs compared with 71% in the controls. Our data clearly indicate that the lymphatic transport process contributes significantly to the post-prandial intestinal absorption of moxidectin and subsequently to its systemic bioavailability. The lymphatic transport of moxidectin offers potential strategies based on lipid formulations to improve the bioavailability of MLs when administered orally.

Enhancement of oral moxidectin bioavailability in rabbits by lipid co-administration.

Moxidectin is a member of the macrocyclic lactone family of drugs widely used for the control of internal and external parasites. Because moxidectin is highly lipophilic, we suspect that lymphatic transport influences the intestinal absorption of oral formulations of the drug. We studied the influence of lipid co-administration on the pharmacokinetics of an oral formulation of moxidectin in rabbits. Ten rabbits were orally administered 0.3 mg kg(-1) moxidectin with or without sunflower oil. Moxidectin and triglyceride were analyzed in plasma over 23 days. Sunflower oil co-administration significantly increased the area under the plasma concentration-time curve of moxidectin (98%, P<0.05) and prolonged its mean residence time from 1.52 days to 2.12 days (P<0.04). Simultaneously, an increase in plasma triglyceride was observed in response to oil administration. It is suggested that lipid administration increases the systemic availability of oral moxidectin by enhancing the extent of intestinal lymphatic transport of the drug. Lipid-based formulations should improve the bioavailability of moxidectin in rabbits.

Ginseng increases intestinal elimination of albendazole sulfoxide in the rat.

Herbal products show potential drug interactions, some of them with adverse effects. The main aim of this work was to study the effect of Panax ginseng on the intestinal elimination of the benzimidazole derivative albendazole sulfoxide (ABZSO). An upper small intestine segment was isolated and perfused in situ with saline, while ABZSO solution (10 mg/kg i.v.) was administered intravenously. Blood samples and intestinal secretion were collected over 60 min and analysed by HPLC. The intestinal clearance of ABZSO was 0.106+/-0.010 ml/min. Systemic co-administration of ginseng (10 mg/kg i.v.) increased significantly (P<0.05) the clearance of ABZSO (0.132+/-0.005 ml/min). The increase in ABZSO elimination could be the result of the effect of ginseng on metabolic pathways. These results highlight the interactions between herbal products (sometimes dietary constituents) and drugs such as benzimidazoles, since ginseng modifies the luminal clearance of this anthelminthic drug and could potentially interfere with drugs that undergo the same intestinal processes.

Use of benzimidazoles in children younger than 24 months for the treatment of soil-transmitted helminthiasis.

Considerable experience and limited quantitative evidence indicate that infections with the soil-transmitted helminths Ascaris lumbricoides and Trichuris trichiura usually start to become established in children aged 12 months and older. Since children living in countries where the infections are endemic are at risk of morbidity, even those as young as 12 months may need to be considered for inclusion in public health programmes designed to reduce morbidity by means of regular anthelminthic chemotherapy. This situation raises the question as to whether such young children should be given anthelminthic drugs. Systems for the absorption, distribution, metabolism and elimination of drugs do not fully develop until children are in their second year of life. Current knowledge, however, reveals that the incidence of side effects linked to benzimidazole drugs in young children is likely to be the same as in older children. Accordingly, we conclude that albendazole and mebendazole may be used to treat children as young as 12 months if local circumstances show that relief from ascariasis and trichuriasis is justified.

Metabolism and residue depletion of albendazole and its metabolites in rainbow trout, tilapia and Atlantic salmon after oral administration.

Metabolic and residue depletion profiles of albendazole (ABZ) and its major metabolites in three fish species, rainbow trout, tilapia and Atlantic salmon are reported. Based on these profiles, similarities (or dissimilarities) between species will determine the potential to group fish species. ABZ at 10 mg/kg body weight was incorporated into fish food formulated in a gelatin base or in gel capsule and fed as a single dose to six fish from each species. Rainbow trout were held three each in a partitioned 600-L tank. Tilapia and Atlantic salmon were housed in separate 20-L tanks. Samples of muscle with adhering skin were collected at 8, 12, 18, 24, 48, 72, and 96 h postdose from trout kept at 12 degrees C, at 4, 8, 12, 24, 48, 72, 96, 120, and 144 h postdose from tilapia kept at 25 degrees C and at 8, 14, 24, 48, 72, and 96 h postdose from Atlantic salmon kept at 15 degrees C. The samples were homogenized in dry ice and subjected to extraction and cleanup procedures. The final extracts were analyzed for parent drug ABZ and its major metabolites, albendazole sulfoxide (ABZ-SO), albendazole sulfone (ABZ-SO2) and albendazole aminosulfone using high-performance liquid chromatography with fluorescence detection. ABZ was depleted by 24 h in trout and tilapia and by 48 h in salmon; ABZ-SO, a pharmacologically active metabolite, was depleted by 48 h in tilapia, by 72 h in rainbow trout and was present until 96 h in salmon; and low levels of ABZ-SO2 and albendazole aminosulfone, both inactive metabolites, were detectable at least till 96 h in all three fish species.

Albendazole-soybean oil emulsion for the treatment of human cystic echinococcosis: evaluation of bioavailability and bioequivalence.

A single 12.5 mg/kg dose of albendazole (Abz) in tablet form (AbzT) followed 2 weeks later by an equivalent dose of Abz emulsified in 30% soybean oil (AbzE) was administered orally 2 h after the first morning meal to 7 male adult patients with cystic echinococcosis caused by Echinococcus granulosus. Serum samples were taken 1, 3, 5, 7, 8, 9, 11, 14, 18, 24, 36, and 48 h post medication from each patient to measure the serum concentrations of albendazole sulfoxide (AbzSOX), the principal bioactive metabolite of Abz. AbzSOX concentrations were measured by reverse phase HPLC. The data were subjected to pharmacokinetic analysis to compare the relative bioavailability and bioequivalence of AbzT and AbzE. The results demonstrated that the mean peak concentrations (C(max)) for AbzT and AbzE were 1.06+/-0.38 mg/l and 1.71+/-0.47 mg/l, respectively; the area under the concentration-time curves (AUC) were 13.24+/-4.93 mg x h/l and 21.01+/-7.54 mg x h/l, respectively. The relative bioavailability of AbzE was F(Flu)=1.59. Two one-sided tests procedure and (1-2 alpha) 90% confidence interval methods were used to evaluate the bioequivalence of AbzE and AbzT. The results demonstrated that the bioavailability of AbzE was greater than AbzT.

Augmented bioavailability and cysticidal activity of albendazole reformulated in soybean emulsion in mice infected with Echinococcus granulosus or Echinococcus multilocularis.

The anthelminthic drug, albendazole (Abz), was reformulated in a soybean oil emulsion and evaluated as a therapeutic agent for the treatment of Echinococcus granulosus and Echinococcus multilocularis in mice. Abz emulsified with 30% soybean oil (AbzE-30) resulted in higher circulating plasma concentrations of the major bioactive Abz metabolite, Abz sulfoxide (AbzSOX), after oral administration, compared with an Abz suspension. The soybean oil-emulsified Abz compound was also noted to penetrate into the hydatid cyst wall and produced higher hydatid cyst concentrations of AbzSOX. The emulsion was superior to Abz suspension in reducing the size of hydatid cysts caused by E. granulosus protoscolices collected from naturally infected sheep in Urumchi, Xinjiang Uygar Autonomous Region. In contrast, the reformulated compound's ability to reduce E. multilocularis cyst masses was only marginally superior to Abz suspension. AbzE-30 exhibited increased bioavailability and bioactivity in the treatment of murine Echinococcus hydatid cyst infections. The compound has the potential for improving therapeutic outcomes for human echinococcosis.

Effect of formulation on the pharmacokinetics and efficacy of doramectin.

The pharmacokinetics of doramectin, a novel avermectin, were evaluated following parenteral administration in a range of oil-based formulations in an attempt to optimise the formulation. Therapeutic and persistent efficacies against Cooperia oncophora were also evaluated. This approach led to the identification of formulations based upon sesame oil and ethyl oleate which gave more prolonged doramectin plasma concentrations with no loss in therapeutic efficacy and improved persistent efficacy following subcutaneous administration to cattle at a dosage of 200 micrograms kg-1. The importance of using both pharmacokinetic and efficacy end points to distinguish between formulations is discussed. All formulations were well tolerated as evidenced by the absence of any reaction to injection either in the form of behavioural responses, injection site swelling or postmortem lesions. Sesame oil with ethyl oleate was the best parenteral vehicle tested for doramectin, allowing the expression of a high level of therapeutic and persistent efficacy and offering the benefit of excellent injection site toleration.

The effect of an oil formulation on the systemic availability of oxfendazole.

Pure oxfendazole (OFZ) suspended in peanut oil and the commercial formulation 'Synanthic' were each intraruminally administered to Merino weaners at 5 mg kg-1. Plasma was collected and concentrations of OFZ, fenbendazole (FBZ) and FBZ sulphone (FBZ.SO2) were determined by HPLC. The maximum concentrations and area under the plasma concentration curve (AUC) of OFZ and FBZ were significantly higher (P less than 0.05) when OFZ was suspended in peanut oil than when administered as the commercial formulation.