Application of dissolution/permeation system for evaluation of formulation effect on oral absorption of poorly water-soluble drugs in drug development.

PURPOSE: The aim of the present study is to evaluate the formulation effect on the oral absorption of poorly water-soluble drugs using a dissolution/permeation system (D/P system). METHODS: This D/P system, consisting of apical and basal chambers and a Caco-2 cell monolayer mounted between chambers, can be used to perform simultaneous analysis of drug dissolution and permeation process of drugs applied as various dosage forms. Oral administration study with rats was also performed for both drugs as the same dosage forms. RESULTS: When danazol, a low-soluble and high-permeable drug, was applied to the D/P system as various formulations, dissolved and permeated amounts were significantly high compared with those from a suspension form. On the other hand, whereas the dissolved amount of pranlukast, a low-soluble and low-permeable drug, was significantly increased by formulations, there were no significant changes observed in the permeated amount between suspension and formulation. The oral availability of danazol was significantly increased by formulations but not pranlukast, which corresponded well to in vitro evaluations. CONCLUSION: These results indicated that the D/P system might be applicable for selection of formulation on the basis of physicochemical drug properties.

Solution or suspension - Does it matter for lipid based systems? In vivo studies of chase dosing lipid vehicles with aqueous suspensions of a poorly soluble drug.

In this study, the potential of co-administering an aqueous suspension with a placebo lipid vehicle, i.e. chase dosing, was investigated in rats relative to the aqueous suspension alone or a solution of the drug in the lipid vehicle. The lipid investigated in the present study was Labrafil M2125CS and three evaluated poorly soluble model compounds, danazol, cinnarizine and halofantrine. For cinnarizine and danazol the oral bioavailability in rats after chase dosing or dosing the compound dissolved in Labrafil M21515CS was similar and significantly higher than for the aqueous suspension. For halofantrine the chase dosed group had a tendency towards a low bioavailability relative to the Labrafil M2125CS solution, but still a significant higher bioavailability relative to the aqueous suspension. This could be due to factors such as a slower dissolution rate in the intestinal phase of halofantrine or a lower solubility in the colloidal structures formed during digestion, but other mechanisms may also be involved. The study thereby supported the potential of chase dosing as a potential dosing regimen in situations where it is beneficial to have a drug in the solid state, e.g. due to chemical stability issues in the lipid vehicle.

Miniaturized ionic liquid dispersive liquid-liquid microextraction in a coupled-syringe system combined with UV for extraction and determination of danazol in danazol capsule and mice serum.

In this study, for the first time, a coupled 1-mL microsyringe system was utilized to perform a miniaturized ionic liquid dispersive liquid-liquid microextraction (IL-DLLME) method. Danazol was extracted and determined via the developed method followed by micro-volume ultraviolet spectroscopy (UV). The extraction process was carried out by the injection of extraction solvent ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate [C8mimPF6] into sample solution (syringe A), and then rapid shoot the solution into syringe B. After that the shooting was repeated several times at a rate of 1 cycle/s. The extraction procedure was induced by the formation of cloudy solution, which was composed of fine drops of [C8mimPF6] dispersed entirely into sample solution with the help of shooting without any dispersive solvent, ultrasonication or high temperature. Several important parameters affecting the extraction efficiency were studied and optimized. Under the optimized conditions, the limit of detection (LOD) was 0.055 µg/mL (capsule) or 0.054 µg/mL (serum) at a signal-to-noise ratio of 3. The calibration curve was linear over the range of 0.62-25 µg/mL. The proposed method was successfully applied to danazol capsule and the real mice serum samples and good spiked recoveries in the range of 90.5-103.4% were obtained. The obtained results of this work were in good agreement with the results of HPLC.

Effect of liquid volume and food intake on the absolute bioavailability of danazol, a poorly soluble drug.

The influence of liquid intake and a lipid-rich meal on the bioavailability of a lipophilic drug was investigated. Danazol was used as the model substance. In a randomized four-way crossover study eight healthy male volunteers received four different treatments with danazol at 2-week intervals following an overnight fast (one I.V. infusion and three oral treatments). The I.V. formulation contained 50mg danazol solubilized in 40% hydroxypropyl-beta-cyclodextrin. The oral treatments were a Standard treatment, a Standard + 800 ml water treatment and a Standard + lipid-rich meal treatment. The Standard oral treatment consisted of 200 ml water and one capsule containing 100mg danazol, three 500 mg paracetamol tablets and two 500 mg sulfasalazine tablets. Paracetamol and sulfasalazine were used as markers for gastric emptying and small intestinal transit times. Intake of danazol with a lipid-rich meal or extra 800 ml water increased the bioavailability by 400 and 55%, respectively. Gastric emptying times increased in the following order: Standard

Voltammetric study of danazol and its determination in capsules and spiked biological fluids.

The voltammetric behaviour of danazol DZ (antigonadotropin) was studied using cyclic voltammetry, direct current, differential pulse polarography (DPP) and alternating current polarography. Danazol exhibited irreversible cathodic waves over the pH range of 1-5 in Britton Robinson buffers. At pH 1 (the analytical pH), a well-defined wave with E1/2 of -1.04 V versus Ag/AgCl reference electrode was obtained. The diffusion current constant (Id) was 4.8+/-0.14 microA.L.m mole(-1) and the current-concentration plot was rectilinear over the range from 5 x 10(-6) to 1 x 10(-4) M with correlation coefficient (n = 11) of 0.995. The calculated detection limit was 1 x 10(-6) M using the DPP mode. The wave was characterized as being irreversible, diffusion-controlled although adsorption phenomenon played a limited role in the electrode process. The proposed method was applied to commercial capsules and the average percentage recovery was in agreement with that obtained by the official USP method. The method was extended to the in vitro determination of DZ in spiked human urine and plasma samples, the percentage recoveries were 96+/-4 and 97+/-5, respectively. A proposal of the electrode reaction was postulated.

Absorption of danazol after administration to different sites of the gastrointestinal tract and the relationship to single- and double-peak phenomena in the plasma profiles.

The absorption of danazol (100 mg) after oral or intraintestinal administration to the proximal jejunum or proximal ileum has been studied in healthy female subjects. The extent of danazol absorption after administration as a solubilized glycerol mono-oleate emulsion formulation was approximately twofold and fourfold greater after oral dosing when compared with jejunal or ileal administration, respectively. Although not statistically significant in this study, the extent of absorption after jejunal administration was generally greater than after ileal administration. After oral dosing, qualitative assessment identified the presence of double peaks or major shouldering characteristics in 14 of the 16 individual danazol plasma concentration-time profiles, whereas only single peaks were present after intraintestinal administration. These data are consistent with the double peaking phenomena after oral administration of the emulsion formulation being stomach-related. The double peaking effect may be explained in terms of a probable combination of gastric emptying regulated absorption (due to the presence of the lipid in the emulsion formulation) and the dependence of danazol solubility on bile salt solubilization within the upper small intestine.

Danazol concentrations in human ovarian follicular fluid and their relationship to simultaneous serum concentrations.

Danazol concentrations in follicular fluid and serum were studied in eight women scheduled for laparoscopy because of suspected endometriosis. In order to obtain some variation in follicular maturity, danazol administration was started 2 to 7 days before the expected day of ovulation. A total of nine doses were given, i.e., 200 mg four times daily for 2 days; the last 200-mg dose was given 3 hours before the laparoscopy during which the follicular fluid from the dominant follicle was aspirated. Peripheral venous blood samples were drawn before, during, and after laparoscopy. Danazol concentrations were assayed by means of a high-performance liquid chromatography method. At the time of follicular aspiration, the mean concentration of danazol was estimated at 96 ng/ml in serum and at 71 ng/ml in follicular fluid, i.e., an average of 73% of the simultaneous serum concentration. The data suggest that even short-term therapy with danazol is likely to produce intrafollicular drug concentrations that have a direct inhibitory effect on follicular steroidogenesis.

Levels of androgens and danazol metabolites in serum during danazol therapy.

Thirteen women who had endometriosis were treated with danazol (300 to 400 mg/d). Levels of androgens and danazol metabolites in serum and the influences of danazol metabolites on the assays for serum androgen were investigated during danazol therapy. Serum DHEAS significantly increased (P < 0.05), but serum DHEA slightly decreased. Serum T levels, measured by direct assay, were markedly elevated. However, measured after HPLC separation, the T levels in serum were significantly decreased (P < 0.05). There were considerable cross-reactions between danazol metabolites and androgens (T, DHEA, and A) in RIA. Purification of androgens using column chromatography was necessary to measure serum androgens precisely.

A radioimmunoassay for danazol (17alpha-pregna-2,4-dien-20-yno[2,3-d]isoxazol-17-ol).

A method is described for the radioimmunoassay of circulating levels of the pituitary inhibiting agent, danazol. An antigen for danazol was prepared by reacting a 17-carboxy-methyloxime derivative of danazol with bovine serum albumin. By immunizing rabbits with this antigen, antiserum was generated which shows excellent specificity for danazol relative to its known metabolites as well as to many natural steroids. A radioimmunoassay was developed, without using separation or extraction techniques, involving competition for the antiserum between danazol in plasma and 14C-danazol. This assay has been successfully used to measure danazol in a series of normal human subjects receiving the drug at either 100 or 200 mg b.i.d. for 2 weeks. A significant relationship was seen between dosage of danazol and plasma concentrations.

Radioimmunoassay for danazol in human and monkey plasma.

A sensitive method is described for the radioimmunoassay of danazol in monkey and human plasma. Antiserum was developed in rabbits, and a second antibody was used to separate bound from free danazol. The radioimmunoassay was specific for danazol, and the limit of detection ranged from 1.4 to 2.8 ng/ml. Exogeneous danazol could be quantitated accurately in both monkey and human plasma. The radioimmunoassay results agreed with values obtained by inverse isotope dilution after intravenous administration of 14C-danazol to monkeys. The assay was used successfully to measure danazol in plasma from human volunteers receiving 200 mg of danazol.

A liquid chromatographic method for the determination of danazol in human serum.

A liquid chromatographic method for the determination of danazol in human serum has been developed. Reversed-phase C8 and C18 columns were used with a column-switching valve, isocratic elution and UV detection. Sample pretreatment involved extraction of the drug with pentane-methylene chloride. The method enabled the measurement of the drug at a concentration as low as 1 ng ml-1, with precision of 15.0% and accuracy of 8.3%. The method was used to run a two way replicated pharmacokinetic study of danazol. The main pharmacokinetic parameters were (mean of two periods): AUCinf = 480.94 ng x h ml-1, Cmax = 53.2 ng ml-1, tmax = 2.5 h, t0.5 = 18.00 h.

No inhibitory effects of danazol estrogen production by ovaries of hypophysectomized rats stimulated by gonadotropins.

The effects of danazol on estrogen production by rat ovaries were investigated. Hypophysectomized immature female rats were injected daily with 2.5 or 5 IU of pregnant mare serum gonadotropin (PMS) and simultaneously daily given vehicle only or danazol (100 mg/kg body weight) for 14 days. Danazol did not decrease the activities of 3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, 17, 20-lyase and aromatase in the ovaries of rats stimulated by 5IU of PMS. Danazol also did not decrease the activities of 3 beta-hydroxysteroid dehydrogenase and aromatase, and the production of aromatizable androgens (androstenedione plus testosterone) from progesterone in the ovaries of rats stimulated by 2.5 IU of PMS. In accordance with these results, danazol did not reduce the level of estradiol-17 beta in the sera of rats stimulated by 2.5 and 5 IU of PMS. The present results suggest that danazol does not inhibit estrogen production by the rat ovary through its direct action on the ovary.

Ionic liquids provide unique opportunities for oral drug delivery: structure optimization and in vivo evidence of utility.

Ionic liquids (ILs) have been exploited to improve the absorption of poorly water-soluble drugs. Custom-made ILs solubilized very high quantities of the poorly water-soluble drugs, danazol and itraconazole, and maintained drug solubilization under simulated gastro-intestinal conditions. A danazol-containing self-emulsifying IL formulation gave rise to 4.3-fold higher exposure than the crystalline drug and prolonged exposure compared with a lipid formulation.

Soluplus® as an effective absorption enhancer of poorly soluble drugs in vitro and in vivo.

As many new active pharmaceutical ingredients are poorly water soluble, solubility enhancers are one possibility to overcome the hurdles of drug dissolution and absorption in oral drug delivery. In the present work a novel solubility enhancing excipient (Soluplus®) was tested for its capability to improve intestinal drug absorption. BCS class II compounds danazol, fenofibrate and itraconazole were tested both in vivo in beagle dogs and in vitro in transport experiments across Caco-2 cell monolayers. Each drug was applied as pure crystalline substance, in a physical mixture with Soluplus®, and as solid solution of the drug in the excipient. In the animal studies a many fold increase in plasma AUC was observed for the solid solutions of drug in Soluplus® compared to the respective pure drug. An effect of Soluplus® in a physical mixture with the drug could be detected for fenofibrate. In vitro transport studies confirm the strong effect of Soluplus® on the absorption behavior of the three tested drugs. Furthermore, the increase of drug flux across Caco-2 monolayer is correlating to the increase in plasma AUC and C(max)in vivo. For these poorly soluble substances Soluplus® has a strong potential to improve oral bioavailability. The applicability of Caco-2 monolayers as tool for predicting the in vivo transport behavior of the model drugs in combination with a solubility enhancing excipient was shown. Also the improvement of a solid dispersion compared to physical mixtures of the drugs and the excipient was correctly reflected by Caco-2 experiments. In the case of fenofibrate the possible improvement by a physical mixture was demonstrated, underscoring the value of the used tool as alternative to animal studies.