Quantitative Prediction of Oral Bioavailability of a Lipophilic Antineoplastic Drug Bexarotene Administered in Lipidic Formulation Using a Combined In Vitro Lipolysis/Microsomal Metabolism Approach

For performance assessment of the lipid-based drug delivery systems (LBDDSs), in vitro lipolysis is commonly applied because traditional dissolution tests do not reflect the complicated in vivo micellar formation and solubilization processes. Much of previous research on in vitro lipolysis has mostly focused on rank-ordering formulations for their predicted performances. In this study, we have incorporated in vitro lipolysis with microsomal stability to quantitatively predict the oral bioavailability of a lipophilic antineoplastic drug bexarotene (BEX) administered in LBDDS. Two types of LBDDS were applied: lipid solution and lipid suspension. The predicted oral bioavailability values of BEX from linking in vitro lipolysis with microsomal stability for lipid solution and lipid suspension were 34.2 ± 1.6% and 36.2 ± 2.6%, respectively, whereas the in vivo oral bioavailability of BEX was tested as 31.5 ± 13.4% and 31.4 ± 5.2%, respectively. The predicted oral bioavailability corresponded well with the oral bioavailability for both formulations, demonstrating that the combination of in vitro lipolysis and microsomal stability can quantitatively predict oral bioavailability of BEX. In vivo intestinal lymphatic uptake was also assessed for the formulations and resulted in <1% of the dose, which confirmed that liver microsomal stability was necessary for correct prediction of the bioavailability.     

A Biopredictive In Vitro Comparison of Oral Locally Acting Mesalazine Formulations by a Novel Dissolution Model for Assessing Intraluminal Drug Release in Individual Subjects

Drug release and availability at the site of action are the major factors determining the clinical response for locally-acting gastrointestinal (GI) drug products. The present work focused on the prediction of site and extent of in vivo mesalazine release after oral administration to a variety of subjects using individualized in vitro drug release experiments. First, experiments mimicking GI passages in average adult subjects were performed. Then, results from a study screening fasted in vivo pH and transit profiles in individual subjects were translated into a novel in vitro dissolution model enabling to mimic individual GI pH-profiles and transit times with physiologically relevant dissolution media. A selection of monolithic and multiparticulate mesalazine formulations with pH-dependent and pH-independent drug release was screened with the novel dissolution model. Results of the study indicate that dosage form performance can be significantly different in individual subjects and highlight the importance of addressing individual physiological parameters relevant to intraluminal drug release when the aim is to predict the in vivo performance of locally-acting mesalazine formulations in individual patients. The novel in vitro dissolution approach thus represents a valuable tool for both improving individual oral therapy with locally-acting GI drug products and assessing bioequivalence of these formulations.     

Developing Quantitative In Vitro–In Vivo Correlation for Fenofibrate Immediate-Release Formulations With the Biphasic Dissolution-Partition Test Method

This study is to evaluate 3 fenofibrate (FEN) formulations including Fournier® 200 mg capsule, Lipidil® 145 mg tablet, and a clinical HME 160 mg tablet by an in vitro biphasic method. Key experimental parameters were evaluated including the selection of biorelevant media, the United States Pharmacopeia IV flow rate, and the United States Pharmacopeia paddle speed. Varying the hydrodynamic condition resulted in a significant impact on FEN concentration time profiles in both aqueous and octanol phases for these formulations. In vivo pharmacokinetic profiles of the HME tablet, the Lipidil tablet, and Fournier capsule under the fasting and low-fat fed states are reported. Their corresponding absorption-time profiles were obtained through deconvolution by the Wagner-Nelson method. When fed state simulated intestinal fluid version 2 was used, the partitioned FEN amount–time profiles in octanol from the 3 formulations under an appropriate hydrodynamic condition exhibited a good agreement with their in vivo absorbed amount–time profiles, permitting a quantitative in vitro–in vivo correlation. When fasted state simulated intestinal fluid version 2 was used, partitioned FEN amounts into octanol from these formulations are significantly lower than those from in vivo data. Although no food effect was observed for both HME and Lipidil tablets, the positive food effect of the Fournier capsules significantly overestimated by the biphasic test.     

Application of a Dynamic Fluid and pH Model to Simulate Intraluminal and Systemic Concentrations of a Weak Base in GastroPlus™

The application of preclinical in vitro and in silico models can help formulation scientists to predict the in vivo performance of a drug in an early stage of oral drug product development. An important aspect is that these models should include equations that represent mechanisms that are biorelevant and are sensitive to changes in parameter values. Human gastrointestinal physiology involves many processes that change as a function of time. In this work, a dynamic fluid and pH model was applied in GastroPlus^? to simulate intraluminal and systemic concentrations of the weak base posaconazole in a biorelevant manner. Simulated results were compared with observed data, extracted from a previously reported human in vivo gastrointestinal aspiration study. Three different formulations were explored (i.e., 1 solution [20 mg dose strength] and 2 suspensions [both 40 mg dose strength]). Simulated results were compared and in line with the observed results for different intraluminal (e.g., precipitated fraction) and systemic parameters (e.g., plasma C_max). The optimization of the advanced compartmental and absorption transit model related to fluid dynamics and dynamic pH in this work creates perspectives to validate this model with other reference data derived from aspiration/magnetic resonance imaging studies.     

Dissolution Methods to Increasing Discriminatory Power of In Vitro Dissolution Testing for Ibuprofen Free Acid and Its Salts

The predictive capacity of in vitro dissolution tests using the Biopharmaceutics Classification System (BCS)–based experimental setup to anticipate in vivo bioequivalence outcomes for BCS class 2 weak acids has been questioned. In this work, the effect of buffer concentration media was investigated as a possible approach to ensuring the discriminative capacity of the in vitro dissolution methods. The case example used to test this approach was ibuprofen, formulated as either the free acid or in various salt forms. By matching the concentration of buffers commonly used to prepare media which aim to simulate the intestinal conditions with that of bicarbonate buffer, which is the predominant buffer species in vivo, to arrive at the same surface pH (pH₀), the discriminative power of the in vitro dissolution tests was improved. To simulate the in vivo results even better, a pretreatment at acidic pH was added to the dissolution test simulating the gastric conditions to create a 2-stage test. With the 2-stage test, it was possible to account for differences in disintegration in a more physiologically relevant way and thus to better reflect the in vivo performance of the various formulations.     

Application of an In Vitro Dissolution/Permeation System to Early Screening of Oral Formulations of Poorly Soluble,Weakly Basic Drugs Containing an Acidic pH-Modifier

This study aimed to evaluate the usefulness of the dissolution/permeation system (D/P system) as an in vitro tool for early screening of oral formulations of weakly basic drugs containing an acidic pH-modifier. Dipyridamole, having a prominent pH-dependent solubility, was used as a model drug, and various granules containing different amounts of fumaric acid were prepared. Prepared granules were administered orally to hypochlorhydria model rats. It was confirmed that fumaric acid improved the absorption of dipyridamole depending on its amount in the granules. Separately, dissolution and permeation of dipyridamole were observed in vitro in the D/P system. When using a medium with a low buffer capacity which mimicked the human intestinal fluid, rank order of the permeated amount of dipyridamole from various granules in the D/P system did not correlate with its absorption in hypochlorhydric rats. In contrast, when applying a medium with high buffer capacity, the permeated amount in the D/P system well reflected the effects of fumaric acid on the in vivo absorption of dipyridamole. In conclusion, by setting appropriate experimental protocols according to the properties of test compounds and formulations, D/P system can be a potent in vitro tool to predict in vivo performance of oral formulations.     

Beyond Q1/Q2: The Impact of Manufacturing Conditions and Test Methods on Drug Release From PLGA-Based Microparticle Depot Formulations

Drug-loaded polymeric microparticles have been used as long-acting injectable (LAI) depot formulations. To obtain U.S. Food and Drug Administration approval, a generic LAI depot product needs to be qualitatively (Q1) and quantitatively (Q2) the same in terms of inactive ingredients as its reference-listed drug. However, Q1/Q2 sameness as the reference-listed drug does not guarantee the same in vitro drug release profile and in vivo performance, especially when the manufacturing methods are different. There is little consensus on how the in vitro testing needs to be done to examine the release profiles of LAI depot formulations. This study examined the manufacturing differences in making risperidone-loaded poly(lactide-co-glycolide) microparticles and their impact on the release kinetics. It also examined the impacts of in vitro testing methods on the drug release profiles. Two in-house manufactured risperidone poly(lactide-co-glycolide) microparticles and Risperdal Consta^® were used in the study. Of the in vitro release methods tested, the orbital agitation method provided the most reproducible release profiles. The results indicate that the in vitro release kinetics depend not only on manufacturing procedures but also on the in vitro testing conditions, such as the agitation speed, vessel-dimensions, solid beads, media exchange volume, and other parameters both under real-time and accelerated testing conditions. In the current case, the in vitro experimental condition seemed to affect the drug release kinetics more than the manufacturing differences. The developed orbital agitation release testing method is simple, robust, and reproducible, which allows the comparison of in vitro release profiles of formulations that are prepared with manufacturing differences.     

The Solubility-Permeability Trade-Off of Progesterone With Cyclodextrins Under Physiological Conditions: Experimental Observations and Computer Simulations

This study intended to evaluate the effect of cyclodextrins on the apparent solubility and permeability of lipophilic drugs under physiological conditions and establish in silico model to choose the optimal amount of cyclodextrins for cyclodextrin-containing oral formulations. In order to study the effect of cyclodextrins under physiological conditions, bile salts and lecithin were added into the rat intestinal perfusion solution to simulate the fasted intestinal fluid. In addition, the in vivo oral absorption performances of cyclodextrin-containing formulations were simulated by gastrointestinal simulation technology based on the advanced compartmental absorption and transit model. The permeability of progesterone was not significantly different between 0.1 mM and 1 mM of 2-hydroxypropyl-β-cyclodextrins (HP-β-CD) under physiological conditions. When the concentration of HP-β-CD was 1 mM, the permeability of progesterone under physiological conditions was significantly higher than that in vitro. The in silico model established in this study was validated by in vivo studies of 4 formulations containing different dosage of cyclodextrin, proving that it was accurate and reliable. In conclusion, this work that demonstrates the permeability of lipophilic drugs could not decrease quickly among a certain range of dosage of HP-β-CD in vivo. Studying the solubility-permeability interplay under physiological conditions would be more meaningful.     

In Vitro and In Vivo Correlation of Hepatic Fraction of Metabolism by P450 in Dogs

1-Aminobenzotriazole (ABT) has been widely used as a nonspecific mechanism-based inhibitor of cytochrome P450 (P450) enzymes. It is extensively used in preclinical studies to determine the relative contribution of oxidative metabolism mediated by P450 in vitro and in vivo. The aim of present study was to understand the translation of fraction metabolized by P450 in dog hepatocytes to in vivo using ABT, for canagliflozin, known to be cleared by P450-mediated oxidation and UDP-glucuronosyltransferases–mediated glucuronidation, and 3 drug discovery project compounds mainly cleared by hepatic metabolism. In a dog hepatocyte, intrinsic clearance assay with and without preincubation of ABT, 3 Lilly compounds exhibited a wide range of fraction metabolized by P450. Subsequent metabolite profiling in dog hepatocytes demonstrated a combination of metabolism by P450 and UDP-glucuronosyltransferases. In vivo, dogs were pretreated with 50 mg/kg ABT or vehicle at 2 h before intravenous administration of canagliflozin and Lilly compounds. The areas under the concentration-time curve (AUC) were compared for the ABT-pretreated and vehicle-pretreated groups. The measured AUC_ABT/AUC_veh ratios were correlated to fraction of metabolism by P450 in dog hepatocytes, suggesting that in vitro ABT inhibition in hepatocytes is useful to rank order compounds for in vivo fraction of metabolism assessment.     

In Vitro–In Vivo Correlations of Carbamazepine Nanodispersions for Application in Formulation Development

During formulation development, efficiently integrating in vitro dissolution testing can significantly improve one's ability to estimate in vivo performance and aide in the selection of premier drug candidates. The concept of in vitro–in vivo relationship/correlation has garnered significant attention from pharmaceutical scientists to predict expected bioavailability characteristics for drug substances and products. The present work illustrates a comparative evaluation of in vitro tests to access crystalline carbamazepine and various types of amorphous and crystalline dispersions of carbamazepine and Eudragit^® L100 produced by spray drying, including a membrane-permeation dissolution methodology and nonsink dissolution. To establish the best model, parameters such as pH, membrane constitution, and dissolution media composition were investigated. The in vitro results were compared against in vivo mice pharmacokinetic studies and qualitatively, the membrane-permeation dissolution methodology correlated well with in vivo. Various correlations were performed in order to evaluate the optimal model for characterizing the relationship. Results exhibited a coefficient of determination (R²) values of 0.90 and 1.00, depicting a linear relationship of the data in comparison. Therefore, for the current formulation system (drug/polymer/technique), membrane-permeation dissolution can guide formulation development and potentially reduce the number of animal and clinical pharmacokinetic studies required.     

Water Activity and Its Significance in Topical Dosage Forms

Unique properties of thermodynamic activity of solvents in topical semisolids and its effects on in vitro product performance have not been fully understood. Mechanistic investigation was undertaken to demonstrate the significance of thermodynamic potential of solvents [water activity (a_w) or solvent activity (a_s)] on in vitro performance of model topical formulations. Drug transport across synthetic membranes was found to decrease with decreasing water activity of formulations. Similarly, in vitro permeation of model permeant (caffeine) across porcine epidermis was found to decrease with decreasing water activity of formulations. Notably, relatively low water activity formulations (a_w, 0.78) induced dehydration in porcine skin associated with significant structural changes like detachment of individual stratum corneum layers. Inclusion of hydrating agents (propylene glycol) in low water activity (a_w, 0.78) formulations restored hydration levels and structural integrity of porcine skin. Most importantly, incremental inclusion of propylene glycol in low water activity formulations (a_w, 0.78) enhanced in vitro permeation of model permeant (fluorescein sodium). Further investigation revealed that variability in processing conditions (high shear mixing during emulsification step) could modulate water activity in semisolid formulations despite their compositional sameness. In retrospect, water activity was found to be a critical quality attribute of topical semisolid products which impacts overall product performance and drug delivery.     

Recent progress in natural dietary non-phenolic bioactives on cancers metastasis

From several decades ago to now, cancer continues to be the leading cause of death worldwide, and metastasis is the major cause of cancer-related deaths. For health benefits, there is a great desire to use non-chemical therapy such as nutraceutical supplementation to prevent pathology development. Over 10,000 different natural bioactives or phytochemicals have been known that possessing potential preventive or supplementary effects for various diseases including cancer. Previously, the in vitro and in vivo anti-invasive and anti-metastatic activities of phenolic acids, monophenol, polyphenol and their derivatives and flavonoids and their derivatives have been reviewed. However, a vast number of natural dietary compounds other than phenolics have been demonstrated to potentially possess the ability to inhibit the invasion and metastasis of various cancers. In this review, we summarize the studies in recent decade on in vitro and in vivo effects and molecular mechanisms of natural bioactives, excluding the phenolics in food, in cancer invasion and metastasis. By combining this review of non-phenolics with the previous phenolics reviews, the puzzle for the contribution of natural dietary bioactives on cancer invasive or/and metastatic progress will be almost complete and more clear.     

The Effect of Drug Content Reduction on the In Vitro and In Vivo Properties of Levonorgestrel-Releasing Intravaginal Rings

Intravaginal rings (IVRs) are an option for continuous administration of drugs in women. However, a considerable amount of excess drug often remains in the ring upon removal. The current study focuses on comparing 2 IVRs releasing levonorgestrel (LNG). Both formulations were designed to release 40 μg of LNG daily, however, with a significant difference in the total amount of drug (10.6 vs. 176.9 mg). Numerical simulations and in vitro release rate testing were utilized in designing the IVRs and confirming the similarity of drug release. Moreover, a pharmacokinetic (PK) study was performed in 13 healthy Japanese women to investigate both formulations during the intended wearing period of 28 days. The primary PK metrics was the average concentration of LNG in plasma at defined time points under stable conditions. Statistical evaluation of the ratio of the main PK metrics indicated values almost in the bioequivalence range. Furthermore, drug content determinations for used and unused IVRs were analyzed for confirming the expected drug delivery in vivo. In summary, it was shown that with proper design, even major differences in the total drug content of IVR formulations might not result in significant effects in the in vitro and in vivo release properties.     

Full-Thickness Intraoral Mucosa Barrier Models for In Vitro Drug-Permeation Studies Using Microneedles

The use of permeation enhancers such as microneedles (MNs) to increase drug penetration across intraoral mucosa has increased in recent years. Permeation studies, commonly performed using vertical diffusion cells, are a well-established way to preview formulations and enhance their performance during the development stage. However, to our knowledge, the existing intraoral mucosa barrier models do not permit permeation using MN-pretreated mucosa due to their insufficient thickness. Therefore, the objective of this study was to develop a barrier model using thick palate tissues to perform in vitro permeation studies, with physical enhancement of the permeability of intraoral mucosa by pretreatment with MNs. The adapted Franz-type cells used in the permeation experiments were validated (cell dimensions and volume, sealing effectiveness, stirring and dissolution efficiency, temperature control, and establishment of uniaxial flux). Commercially available MNs were used in the palatal mucosa. Optical images of the mucosa were acquired to analyze the microperforations created. In vitro permeation studies were conducted with the MN-pretreated mucosa. This work presents a new in vitro method for the evaluation of MNs as permeation enhancers, with the aim of improving the absorption of drug formulations topically applied within the oral cavity.     

Towards human ex vivo organ perfusion models to elucidate drug pharmacokinetics in health and disease

Abstract

To predict the absorption, distribution, metabolism and excretion (ADME) profile of candidate drugs a variety of preclinical models can be applied. The ADME and toxicological behavior of newly developed drugs are often investigated prior to assessment in humans, which is associated with long time-lines and high costs. Therefore, good predictions of ADME profiles earlier in the drug development process are very valuable. Good prediction of intestinal absorption and renal and biliary excretion remain especially difficult, as there is an interplay of active transport and metabolism involved. To study these processes, including enterohepatic circulation, ex vivo tissue models are highly relevant and can be regarded as the bridge between in vitro and in vivo models. In this review the current in vitro, in vivo and in more detail ex vivo models for studying pharmacokinetics in health and disease are discussed. Additionally, we propose novel models, i.e., perfused whole-organs, which we envision will generate valuable pharmacokinetic information in the future due to improved translation to the in vivo situation. These machine-perfused organ models will be particularly interesting in combination with biomarkers for assessing the functionality of transporter and CYP450 proteins.     

Improving Correlations Between Drug Solubilization and In Vitro Lipolysis by Monitoring the Phase Partitioning of Lipolytic Species for Lipid-Based Formulations

Solution proton nuclear magnetic resonance analysis was used in conjunction with in vitro lipolysis to elucidate the time-dependent speciation and release of lipolytic products during the digestion of lipid-loaded inorganic particles, allowing correlations to be made between the phase partitioning of lipolytic products and an encapsulated poorly soluble drug. Silicon dioxide, montmorillonite, and laponite were used to encapsulate medium chain triglycerides into solid-state lipid-based formulations (LBFs), and coumarin 102 was selected as a model poorly soluble compound. The specific inorganic carrier material used to encapsulate medium chain triglycerides significantly impacted the release and partitioning of the solubilizing lipolytic products, that is, diglycerides, monoglycerides, and fatty acids. A strong linear correlation was obtained between drug solubilization and fatty acid release to the aqueous phase (R² = 0.996), indicating fatty acids to be the most important lipid species for enabling solubilization and potential drug absorption in vivo. This method was developed to improve upon the use of pH-stat titration for characterizing LBF digestion during in vitro lipolysis studies and is demonstrated herein to provide useful insights into how the selected inorganic carrier material impacts LBF performance when solid-state LBF powders are fabricated via adsorption.     

Antitumor Efficacy and Toxicity of 5-Fluorouracil-Loaded Poly(Lactide Co-glycolide) Pellets

The aim of this study was to formulate a biodegradable implant capable of imparting local antitumor activity through the sustained release of the chemotherapeutic agent, 5-fluorouracil (5-FU). Thus, injectable pellets (<1.2 mm diameter) made from poly(lactide co-glycolide) (PLGA) and loaded with 5-FU at varying drug:polymer ratios were fabricated using hot-melt extrusion and tested for their ability to provide sustained release of 5-FU in in vitro and in vivo settings. In addition, these formulations were compared against soluble 5-FU for their antitumor activity in vivo as well as for their toxicity. It was demonstrated that the release rate of 5-FU from PLGA pellets was directly related to the percentage of 5-FU in the pellets. PLGA pellets loaded with 50% w/w 5-FU exhibited comparable, and significantly enhanced, antitumor activity (as measured by tumor volumes and survival) in vivo in a thymoma and colon cancer model, respectively, when compared to an equivalent bolus dose (120 mg/kg) of soluble 5-FU. We concluded that 5-FU-loaded PLGA pellets were more effective and specifically less erythrotoxic than 5-FU bolus injections and therefore may prove to be of benefit as an intraoperative adjunct therapy for patients with cancers that are sensitive to 5-FU and who are undergoing tumor resection.     

The Combination of GIS and Biphasic to Better Predict In Vivo Dissolution of BCS Class IIb Drugs,Ketoconazole and Raloxifene

The formulation developments and the in vivo assessment of Biopharmaceutical Classification System (BCS) class II drugs are challenging due to their low solubility and high permeability in the human gastrointestinal (GI) tract. Since the GI environment influences the drug dissolution of BCS class II drugs, the human GI characteristics should be incorporated into the in vitro dissolution system to predict bioperformance of BCS class II drugs. An absorptive compartment may be important in dissolution apparatus for BCS class II drugs, especially for bases (BCS IIb) because of high permeability, precipitation, and supersaturation. Thus, the in vitro dissolution system with an absorptive compartment may help predicting the in vivo phenomena of BCS class II drugs better than compendial dissolution apparatuses. In this study, an absorptive compartment (a biphasic device) was introduced to a gastrointestinal simulator. This addition was evaluated if this in vitro system could improve the prediction of in vivo dissolution for BCS class IIb drugs, ketoconazole and raloxifene, and subsequent absorption. The gastrointestinal simulator is a practical in vivo predictive tool and exhibited an improved in vivo prediction utilizing the biphasic format and thus a better tool for evaluating the bioperformance of BCS class IIb drugs than compendial apparatuses.     

Mechanistic Study of Belinostat Oral Absorption From Spray-Dried Dispersions

Spray-dried dispersions (SDDs) are an important technology for enhancing the oral bioavailability of poorly water-soluble drugs. To design an effective oral SDD formulation, the key rate-determining step(s) for oral drug absorption must be understood. This work combined in vivo and in vitro tests with in silico modeling to identify the rate-determining steps for oral absorption of belinostat SDDs made with 3 different polymers (PVP K30, PVP VA64, and HPMCAS-M). The goal was developing a belinostat SDD formulation that maximizes oral bioavailability (ideally matching the performance of a belinostat oral solution) and defining critical performance attributes for formulation optimization. The in vivo pharmacokinetic study with beagle dogs demonstrated that 1 of the 3 SDDs (PVP K30 SDD) matched the performance of the oral solution. In vitro data coupled with in silico modeling elucidated differences among the SDDs and supported the hypothesis that absorption of belinostat in the small intestine from the other 2 SDDs (PVP VA64 and HPMCAS-M) may be limited by dissolution rate or reduced drug activity (maximum concentration) in the presence of polymer. It was concluded that drug concentration in the stomach before emptying into the proximal intestine is a key factor for maximizing in vivo performance.     

Gastroretentive Cosolvent-Based In Situ Gel as a Promising Approach for Simultaneous Extended Delivery and Enhanced Bioavailability of Mitiglinide Calcium

Ion cross-linking in situ gels are novel liquid sustained-release drug delivery systems. These systems are unsuitable for poorly water-soluble drugs such as the novel antidiabetic drug mitiglinide calcium (MTG). Thus, our goal was to assess the possibility of using cosolvency approach in formulating gastroretentive in situ gel of the short half-life MTG to simultaneously enhance its bioavailability and sustain its release. MTG in situ gel formulations were developed using propylene glycol as a cosolvent to dissolve MTG in the polymer solution, followed by characterization of viscosity, gel strength, floating ability, and in vitro MTG release and phramacokinetics evaluation. The optimized formulation (composition: 1% gellan gum, 0.75% sodium alginate, 0.75% calcium carbonate, and 7.5% propylene glycol) exhibited reasonable viscosity but on introduction into simulated gastric fluid, it formed firm gel that floated within seconds over the surface and remained buoyant for 24 h. The formula exhibited in vivo sustained release manner of MTG over 24 h and improved the bioavailability of the drug. Thus, cosolvency presents a promising approach to deliver hydrophobic drugs in sustained-release liquid formulations. These formulations will improve diabetic patients' compliance by eliminating the necessity of frequent dosing with a better disease management.