800 mg Darunavir tablets prepared by hot melt extrusion.

Darunavir (TMC 114) is a protease inhibitor used in the therapy of HIV-1. The aim of this study was to formulate 800 mg of Darunavir in a single unit dosage form, with suitable mechanical properties and dissolution behavior, using a corotating twin screw extruder. In preliminary investigations, extrudates of 1 mm diameter were prepared to evaluate the extrusion and dissolution behavior of Darunavir. Two different poloxamers (188 and 407) were used to modify the dissolution properties of Darunavir, and a higher solubilization for poloxamer 188 was observed. Furthermore, a zero order drug release from pure Darunavir extrudates was found which was modulated by the extrudate diameter. Extrudates of 13 mm diameter were cut into tablets containing 800 mg of Darunavir. Due to the lower specific surface area in comparison to the 1 mm extrudates, an addition of solubilizing agent was required to obtain the desired dissolution profiles. Therefore, the influence of Mannitol and poloxamer 188 was investigated in different formulations. The formulations exhibited acceptable extrusion behavior and dissolution properties.

Powder for reconstitution of the anti-HIV-1 drug TMC278 - Formulation development, stability and animal studies.

Powders for reconstitution of the next-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) TMC278 with low water solubility were developed by using a spray-dry technology. Their flexible dosing ability makes them suitable for patients looking for a different approach for antiretroviral (ARV) therapy. The selection of formulation excipients was based on their potential to create and maintain supersaturation solubility of TMC278 in 0.01 M HCl. Suitable water-soluble carriers for TMC278 were selected by a supersaturation screening to formulate powders for reconstitution by spray-drying. The selected powders for reconstitution were compared to clinical tablets of TMC278.HCl, in vitro using dissolution and stability testing, and in vivo through administration to beagle dogs, fed immediately after dosing. The spray-dried powders for reconstitution made up of TMC278/PVP-VA 64 1:9 (w/w) and TMC278/PVP-VA 64/Cremophor EL 1:8.5:0.5 (w/w/w) showed ease of suspendability, nearly complete dissolution of the drug and acceptable stability after one month storage at 25 and 40 degrees C. In dogs, TMC278 was more slowly absorbed from tablets than from the suspended powders for reconstitution. Compared to the tablet, the relative bioavailability obtained with the powders ranged between 69% and 89% for TMC278/PVP-VA 64 1:9 (w/w) and between 85% and 157% for TMC278/PVP-VA 64/Cremophor EL 1:8.5:0.5 (w/w/w). The absence of differences in vivo and in vitro between the powders made an eventual choice very difficult, yet their advantageous in vivo behaviour and flexible dosing possibility may provide a starting point for paediatric formulations.

Development of an implantable infusion pump for sustained anti-HIV drug administration.

Factors such as insufficient drug potency, non-compliance and restricted tissue penetration contribute to incomplete suppression of Human Immunodeficiency Virus (HIV) and the difficulty to control this infection. Infusion via standard catheters can be a source of infection, which is potentially life threatening in these patients. We developed an implantable infusion pump, allowing to accommodate large volumes (16-50mL) of high viscous solutions (up to 23.96mPas at 39 degrees C) of anti-HIV agents and providing sustained release of medication: a standard Codman 3000 pump, which was initially developed to release aqueous solutions ( approximately 0.7mPas) into the spinal cord such as for pain medication, was transformed for release of viscous solutions up to 40mPas by adapting the diameter of the capillary flow restrictor, the capillary length and way of catheterisation--by placing the indwelling catheter in the vena cava. A pilot study of the pump implanted in 2 dogs showed continuous steady-state release of the protease inhibitor darunavir (25mg/dog/day administered for 25 days), thereby achieving plasma concentration levels of approximately 40ng/mL. Steady-state plasma levels were reproducible after monthly refill of the pumps. In conclusion, the implantable adapted Codman 3000 constant-flow infusion pump customized to anti-HIV therapy allows sustained release of anti-HIV medication and may represent an opportunity to reduce the pill burden and complexity of dosing schemes associated with common anti-HIV therapy.

Dog model with implanted pump to test boosters for antiretroviral medication.

A dog model was developed to test the capacity of boosters for antiretroviral medication. Two dogs were implanted with a modified constant-flow Codman 3000 infusion pump, adapted to release viscous solutions of darunavir (TMC114) at a constant rate of 25mg/dog/day in the venous blood stream. Booster candidates were given by oral gavage for at least 4 days up to maximum 7 days in cross-over fashion, separated by a wash-out period of minimum 1 week. The booster candidates were tested at doses of 20 and/or 40mg/kg/day: blood sampling for determination of the boosting effect was performed on the last day of booster administration. The model allowed to (1) compare the boosting ratio of these booster candidates based on the exposure (determination of the area under the curve (AUC) of darunavir in presence versus absence of the booster candidate), (2) detect delay in boosting activity by evaluation of the shift of Cmax of darunavir following booster administration versus the Cmax of the booster candidate) and (3) calculate the intrinsic booster capacity, by correcting for the systemic exposure of booster candidate by normalizing the booster ratio for the booster's AUC. The latter parameter (intrinsic booster capacity) allows to determine the booster's metabolic contribution in inhibiting the metabolism of antiretroviral medication (most likely via inhibition of CYP3A4), minimizing the impact of potential effects of the booster at the level of the gastro-intestinal tract.

In Vivo Performance of Fenofibrate Formulated With Ordered Mesoporous Silica Versus 2-Marketed Formulations: A Comparative Bioavailability Study in Beagle Dogs.

The present study aims to evaluate the in vitro and in vivo performance of ordered mesoporous silica (OMS) as a carrier for the poorly water-soluble compound fenofibrate. Fenofibrate was loaded into OMS via incipient wetness impregnation to obtain a 29% drug load and formulated into capsules. Two capsule dosage forms (containing 33.5 and 16.75 mg fenofibrate, respectively) were compared with the commercially available forms-Lipanthyl(®) (fenofibrate microcrystals) and Tricor(®) (fenofibrate nanocrystals). In vitro dissolution tests showed that the amount of fenofibrate released from Lipanthyl(®) and Tricor(®) was approximately 30%, whereas approximately 66% and 60% of the drug was released from OMS capsules containing 33.5 and 16.75 mg of fenofibrate, respectively. Storage of OMS capsules loaded with 33.5 mg of fenofibrate at 25°C/60% relative humidity (RH) or 40°C/75% RH did not alter the release kinetics, nor the physical state of the compound, pointing the stability of the present formulation. The in vivo study in dogs confirmed satisfying level of safety and tolerability of fenofibrate-OMS formulation (eq. 33.5 mg) with the potential to improve the absorption of fenofibrate. Though some variability in the data, this formulation is promising to be further investigated in a clinical trial setting.

Ordered mesoporous silica to enhance the bioavailability of poorly water-soluble drugs: Proof of concept in man.

Formulating poorly water soluble drugs using ordered mesoporous silica materials is an emerging approach to tackle solubility-related bioavailability problems. The current study was conducted to assess the bioavailability-enhancing potential of ordered mesoporous silica in man. In this open-label, randomized, two-way cross-over study, 12 overnight fasted healthy volunteers received a single dose of fenofibrate formulated with ordered mesoporous silica or a marketed product based on micronized fenofibrate. Plasma concentrations of fenofibric acid, the pharmacologically active metabolite of fenofibrate, were monitored up to 96h post-dose. The rate (Cmax/dose increased by 77%; tmax reduced by 0.75h) and extent of absorption (AUC0-24h/dose increased by 54%) of fenofibrate were significantly enhanced following administration of the ordered mesoporous silica based formulation. The results of this study serve as a proof of concept in man for this novel formulation approach.

Liquid chromatographic method for analysis of saponins in Maesa balansae extract active against leishmaniasis.

A liquid chromatographic method was developed for the separation of six related triterpenoid saponins in Maesa balansae extracts with different purity, active against leishmaniasis. As stationary phase a Hypersil BDS C18 column (3 microm), 100 x 4.6 mm was used. The mobile phase was a mixture of methanol, acetonitrile, 5% (m/v) ammonium acetate, pH 6.5 and water. A linear gradient was developed for the analysis of crude extracts. An isocratic method was developed to analyze purified samples that mainly contained saponins 3 and 4, the most active saponins. The isocratic LC method was optimized and the robustness was evaluated with an experimental design. The method showed good selectivity, repeatability, linearity and sensitivity.

Inulin solid dispersion technology to improve the absorption of the BCS Class IV drug TMC240.

TMC240 is a very poorly soluble and poorly permeating HIV protease inhibitor. In order to enhance its oral bioavailability, a fast dissolving inulin-based solid dispersion tablet was developed. During the dissolution test in water (0.5% or 1.0% SLS), this tablet released at least 80% of TMC240 within 30min, while a tablet with the same composition, but manufactured as physical mixture, released only 6% after 2h. In a subsequent single-dose study in dogs (200mg of TMC240), plasma concentrations of TMC240 remained below the lower limit of quantification (<1.00ng/mL) in all animals (n=3 per tested formulation), except in one dog receiving the inulin solid dispersion tablet (C(max)=1.8ng/mL, AUC(0-7 h)=3.0ngh/mL). In the latter treatment group, ritonavir co-administration (10mg/kg b.i.d.) increased TMC240 exposure more than 30-fold (mean AUC(0-7 h)=108ngh/mL; F(rel)=3588%). Exposure was also 16-fold higher than after TMC240 administration as PEG400 suspension in the presence of ritonavir (AUC(0-7 h)=6.7ngh/mL). The current data demonstrate that a solid dispersion of TMC240 in an inulin matrix allows considerable improvement in the release of poorly water-soluble TMC240, both in vitro in the presence of a surfactant and in vivo upon oral administration.

Co-administration of darunavir and a new pharmacokinetic booster: formulation strategies and evaluation in dogs.

Various formulations for combination of the anti-HIV protease inhibitor darunavir (DRV) and TMC41629, a pharmacokinetic booster for DRV, were studied. TMC41629 (a BCS-IV compound) was formulated in capsules, as polyethylene glycol 400 (PEG400) solution, binary or ternary self-microemulsifying drug delivery system (SMEDDS), inclusion complex with hydroxypropyl-beta-cyclodextrin (HPbetaCD) or polyvinylpyrrolidone-co-vinylacetate 64 (PVP/VA64) extrudate. In addition, tablets were prepared using unmilled or micronized powder and a disintegrant. On co-administration with DRV tablets in dogs, DRV plasma concentration levels were boosted by TMC41629, the PVP/VA64 extrudate achieving the highest DRV levels (2-fold increase). Yet, with extrudate prepared with both compounds, no boosting effect was observed, likely due to transition of DRV from crystalline solvate to amorphous state. Therefore, a co-formulation, combining DRV as crystalline solvate with amorphous TMC41629, was developed. DRV/kappa-carrageenan 80/20% (w/w) beads coated with TMC41629 released at least 80% within 1h in 0.01M HCl with 0.5% sodium lauryl sulphate, TMC41629 dissolving faster than DRV. In dogs, the DRV exposure increased 2.7-fold with the TMC41629-coated beads relative to DRV alone, yet remained lower, but less variable, than following co-administration as separate formulations. Coating of TMC41629 on DRV/kappa-carrageenan beads is a suitable technique for co-formulation, whereby TMC41629 can function as a booster of DRV.

Improved bioavailability of darunavir by use of kappa-carrageenan versus microcrystalline cellulose as pelletisation aid.

The aim of this study was to produce pellet formulations containing a high drug load (80%) of the poorly soluble HIV-protease inhibitor darunavir, using wet extrusion/spheronization with kappa-carrageenan or microcrystalline cellulose (MCC) as pelletization aid. Drug release was assessed in vitro by a standardized paddle-dissolution test and in vivo by a single-dose pharmacokinetic study in dogs. Mean dissolution time (MDT) was 78.2+/-3.5 h from MCC pellets (1301+/-301 microm) and 6.1+/-0.7 min from kappa-carrageenan pellets (966+/-136 microm). In contrast to kappa-carrageenan pellets, MCC pellets did not disintegrate and showed a diffusion-controlled drug release. In line with the in vitro findings, the darunavir peak plasma levels and exposure after the administration of a 300 mg dose were more than 60-fold higher when formulated with kappa-carrageenan pellets when compared with MCC pellets, and 10-fold higher after co-administration with 10mg/kg of ritonavir. The relative bioavailability of darunavir versus the reference tablet (F(rel)) was 155% with kappa-carrageenan pellets and 2% with MCC pellets without ritonavir, while 78% and 9%, respectively, in presence of ritonavir. In conclusion, when compared with MCC pellets, the bioavailability of darunavir was substantially improved in kappa-carrageenan pellets, likely due to their better disintegration behavior.

Solid state characterization of the anti-HIV drug TMC114: interconversion of amorphous TMC114, TMC114 ethanolate and hydrate.

The interconversion of the ethanolate, hydrate and amorphous form of TMC114 ((3-[(4-amino-benzenesulfonyl)-isobutyl-amino]-1-benzyl-2-hydroxypropyl)-carbamic acid hexahydrofuro-[2,3-b]furan-3-yl ester) in open conditions was characterized. TMC114 hydrate and ethanolate form isostructural channel solvates. The crystal structure of TMC114 was obtained from single crystal X-ray diffraction, confirming that it is a channel solvate. Ethanol and water can exchange with one another. TMC114 ethanolate converts into TMC114 hydrate at moderate or high relative humidity (RH) at 25 degrees C, and it converts back into the ethanolate in ethanol atmosphere. The hydration level of the hydrate is determined by the environmental humidity. TMC114 hydrate collapses to the amorphous product when water is removed by drying at low RH or increasing temperature. TMC114 ethanolate becomes amorphous at elevated temperature in a dry environment below the desolvation temperature. Amorphous TMC114 obtained by dehydrating the hydrate during storage at room temperature/<5% RH, by increasing the temperature, or via desolvating the ethanolate by heating, converts into the hydrate at moderate or high RH at ambient conditions, and into TMC114 ethanolate in an ethanol atmosphere. Under ambient conditions, TMC114 ethanolate may convert into the hydrate, whereas the opposite will not occur under these conditions. The amorphous form, prepared by melting-quenching shows a limited water uptake. Whereas TMC114 ethanolate is stable in the commercialized drug product, special conditions can trigger its conversion.

Development of a long-acting injectable formulation with nanoparticles of rilpivirine (TMC278) for HIV treatment.

Long-acting parenteral formulations of antiretrovirals could facilitate maintenance and prophylactic treatment in HIV. Using the poorly water- and oil-soluble non-nucleoside reverse transcriptase inhibitor (NNRTI) TMC278 (rilpivirine) as base or hydrochloride (HCl), nanosuspensions were prepared by wet milling (Elan NanoCrystal technology) in an aqueous carrier. Laser diffraction showed that the average particles size were (1) close to the targeted size proportionality (200-400-800 nm), with increasing distributions the larger the average particle size, and (2) were stable over 6 months. Following single-dose administration, the plasma concentration profiles showed sustained release of TMC278 over 3 months in dogs and 3 weeks in mice. On comparison of intramuscular and subcutaneous injection of 5mg/kg (200 nm) in dogs, the subcutaneous route resulted in the most stable plasma levels (constant at 25 ng/mL for 20 days, after which levels declined slowly to 1-3 ng/mL at 3 months); 200 nm nanosuspensions achieved higher and less variable plasma concentration profiles than 400 and 800 nm nanosuspensions. In mice, the pharmacokinetic profiles after a single 20mg/kg dose (200 nm) were similar with two different surfactants used (poloxamer 338, or d-alpha-tocopheryl polyethylene glycol 1000 succinate). In conclusion, this study provides proof-of-concept that 200-nm sized TMC278 nanosuspensions may act as long-acting injectable.

Development of a capillary electrophoretic method for the separation of diastereoisomers of a new human immunodeficiency virus protease inhibitor.

A capillary electrophoretic (CE) method was developed for the separation of diastereoisomers of a new human immunodeficiency virus (HIV) protease inhibitor TMC114. In total 16 isomers of this drug have been synthesized (eight pairs of enantiomers). We succeeded in the separation of the eight diastereoisomers, but no enantiomers could be separated. Because of the high similarity and water-insolubility of these isomers, the separation is a real challenge. Different CE modes were tried out: capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), micellar electrokinetic capillary chromatography (MEKC), and microemulsion electrokinetic capillary chromatography (MEEKC). Only MEEKC offered resolution of these compounds.