Influence of Dissolution Media and Presence of Alcohol on the In Vitro Performance of Pharmaceutical Products Containing an Insoluble Drug.

The purpose of this investigation is to determine how the dissolution media may influence the release rate of an insoluble drug in in vitro conditions. Some oral dosage forms containing ibuprofen, a molecule that shows pH-dependent solubility, are tested. They are evaluated in different media to simulate the gastrointestinal transit at paddle rotation speeds of 50 and 100 rpm. Moreover, the potential effect of different ethanol concentrations on drug release is tested. The dissolution profiles of the tablets show a similar behavior in water (pH 1.0) and phosphate buffer (pH 4.5) where the 2 doses are not completely dissolved. The soft capsules show a different behavior: a certain amount of ibuprofen, which is in solution inside the capsule, reprecipitates in water and in the pH 4.5 buffer. Instead, ibuprofen dissolves rapidly in the pH 6.8 buffer from all the formulations. In the water-ethanol solutions, the dissolution curves show a valuable increase in the drug dissolved at higher ethanol concentrations.

I. Technological approaches to improve the dissolution behavior of nateglinide, a lipophilic insoluble drug: nanoparticles and co-mixing.

Nateglinide is a non-sulphonylurea insulinotropic oral antidiabetic agent. The main problem in formulating an oral dosage form is its low solubility in aqueous media. This problem is particularly critical for an anti-diabetic drug because it should be administered just before the meals and be quickly bioavailable to cover the post-prandial glycemic peak. In this work, some technological approaches have been studied to improve the dissolution rate of nateglinide. Furthermore, two different polymorphs of nateglinide (H and B) have been tested to evaluate the influence of the crystal habitus on the dissolution behavior of the drug. The results have clearly demonstrated that wettability plays a key role in the dissolution behavior of nateglinide. As a matter of fact the physical dispersion of the drug with colloidal silica or hydrophilic swellable polymers strongly enhances the dissolution rate of nateglinide. The two polymorphs tested did not show significant differences in terms of dissolution behavior.

II. Technological approaches to improve the dissolution behavior of nateglinide, a lipophilic insoluble drug: co-milling.

Nateglinide is an oral antidiabetic agent that should be administered 10-30 min before the meal, but it shows low and pH-dependent solubility that may reduce its oral bioavailability. To improve nateglinide dissolution rate, the active was co-milled with three different super-disintegrants or with some hydrophilic excipients, in 1:1, 1:2, and 1:4 drug to carrier ratio (w:w). The three super-disintegrants were crosslinked polyvinylpyrrolidone (PVPC), sodium starch glycolate (SSG) and crosslinked carboxymethyl cellulose (CMCC). The three hydrophilic excipient were amorphous silica (AS), mannitol (M) and Poloxamer (PO). A strong enhancement of drug dissolution rate was obtained from the nateglinide:super-disintegrant co-milled systems in 1:4 ratio, which can be explained by a combination of several factors: an increase in wettability, due to the hydrophilic nature of the carriers, a possible reduction of particle size and a more intimate dispersion of the drug onto the carrier, as a result of the mechanical treatment.

Evaluation of accelerated stability test conditions for medicated chewing gums.

The overall stability of medicated chewing gums is investigated under different storage conditions. Active substances with different chemical stabilities in solid state are chosen as model drugs. The dosage form is a three layer tablet obtained by direct compression. The gum core contains the active ingredient while the external layers are formulated to prevent gum adhesion to the punches of the tableting machine. Two accelerated test conditions (40°C/75% RH and 30°C/65% RH) are performed for 6 months. Furthermore, a long-term stability test at room conditions is conducted to verify the predictability of the results obtained from the stress tests. Some drugs are stable in all the conditions tested, but other drugs, generally considered stable in solid dosage forms, have shown relevant stability problems particularly when stress test conditions are applied to this particular semi-solid dosage forms. For less stable drugs, the stress conditions of 40°C/75% RH are not always predictable of chewing gum stability at room temperature and may produce false negative; intermediate conditions, 30°C/65% RH, are more predictive for this purpose, the results of drug content found after 6 months at intermediate stress conditions and 12 months at room conditions are generally comparable. But the results obtained show that only long-term conditions stability tests gave consistent results. During aging, the semi solid nature of the gum base itself, may also influence the drug delivery rate during chewing and great attention should be given also to the dissolution stability.

The role of solution calorimetry in investigating controlled-release processes from polymeric drug delivery systems.

In this paper we investigate the potential role of solution calorimetry measurements in aiding the formulation of swellable matrices containing a mixture of HPMC and NaCMC, an ionic cellulose derivate. These polymers show a synergistic effect in their ability to modulate drug delivery rates; a matrix containing a 1:1 mixture of NaCMC and HPMC exhibits a significantly slower drug release rate than either polymer shows alone. The exact cause of this synergism is not clear and it is not an easy effect to examine using conventional means (such as dissolution testing). Here, we used solution calorimetry to study the system holistically. By comparing the measured response of a physical blend with a theoretical one (obtained by summation of the power-time data for each material), it was possible to assess if there was/was not any interaction which may explain the synergism. Furthermore, since a thermodynamic quantity was returned it was possible to establish if the interaction was favourable or unfavourable and so to obtain useful information to understand and predict the dissolution behaviour of polymeric systems containing the same materials. An unfavourable interaction was noted between NaCMC and the model drug (Diltiazem HCl); no interaction was seen between HPMC and the drug; and a favourable interaction was recorded when both polymers were formulated with the drug. The trend was mirrored by the t90 (the time required for 90% drug release) values determined from dissolution testing; NaCMC 10.8h, HPMC 16.4h, NaCMC and HPMC 19.1h. The data suggest that solution calorimetry measurements can be used to aid the selection of polymeric excipients to design controlled-release drug delivery systems.

Use of dye as tracer of drug release from medicated chewing gums.

The evaluation of the potential use of a dye as indicator of in vivo drug release from a medicated chewing gum is described. The device is a three-layer tablet obtained by direct compression consisting of a gum core and two external protective soluble layers to prevent gum adhesion to the punches of the tableting machine. The active ingredient and a colour are contained in the gum core. To evaluate the drug and the dye release from the formulations, a chew-out study was performed by a panel of volunteers. The results obtained suggest that the use of a dye could be useful to indicate the chewing time necessary to complete drug delivery from medicated chewing gums.

Alginate/polymethacrylate copolymer microparticles for the intestinal delivery of enzymes.

Proteins administered orally must pass through the gastric environment in order to reach their site of absorption in the intestine. How to protect these exogenously administered proteins from the damaging effects of gastric acid and pepsin proteolytic activity, which often induce irreversible structural and functional alterations to the molecules, is an intriguing challenge. Another problem is the physical and chemical instability of proteins during some technological processes, which often involve the use of organic solvents or high temperatures. In this study we investigated the use of alginate microparticles containing one of two enzymes, an enteric polymer and a lyoprotectant for the intestinal delivery of proteins. The two enzymes tested in this protein delivery system were lactate dehydrogenase and alpha-amylase: the former was chosen because of its sensitivity to denaturation, the latter for its relevance in nutrition and medicine. A sodium alginate aqueous solution containing the enteric polymer, a lyoprotectant and the enzyme was either extruded or sprayed into a calcium chloride solution, with the resultant formation of beads and microspheres which were freeze-dried. About 90% of the enzyme activity was maintained during the process of loading the proteins into the microparticles and the subsequent freeze-drying process. The stability of the encapsulated enzyme in an acid medium and the enzymatic activity in an intestinal environment were then investigated by a dissolution test. This consisted of exposing the microparticles to simulated gastric fluid (pH 1.2) for 2 hours and to simulated intestinal fluid (pH 7.5+/-0.1) for 1 hour. The morphology of the microparticles did not change in the acid environment, whereas they completely dissolved within 3 min in the simulated intestinal fluid. Residual enzymatic activity after the test remained satisfactory for both enzymes. In conclusion, these microparticle systems offer promise for applications in human and veterinary medicine as well as in human and animal nutrition.

In vitro maturation of human oocytes in a follicle-mimicking three-dimensional coculture.

OBJECTIVE: To verify the hypothesis that a three-dimensional, follicle-mimicking structure enhances in vitro maturation yields without hormonal supplementation in an in vitro maturation program. DESIGN: Feasibility study; 204 anonymous denuded germinal vesicles retrieved from gonadotropin-treated women were cultured for 48 hours without hormonal supplementation in microdrop culture or in a three-dimensional coculture with granulosa cells in a barium alginate membrane. SETTING: An assisted reproduction center in Italy. PATIENT(S): One hundred twenty-two informed women. INTERVENTION(S): Germinal vesicles retrieved after ovarian stimulation were denuded and cultured without hormonal supplementation in microdrop culture or in a three-dimensional coculture with granulosa cells enclosed in the core of a barium alginate capsule. MAIN OUTCOME MEASURE(S): Oocyte maturation and morphological investigation of follicle-mimicking structures. RESULT(S): Statistically significantly higher oocyte maturation yields were obtained by using the three-dimensional coculture system enclosed in a barium alginate membrane (after 48 hours: coculture, 90.3%; microdrop, 52.0%; odds ratio, 8.51). Morphological investigation indicated that after 48 hours of coculture, the decumulated oocyte appeared to be surrounded by a pseudocumulus structure that lies on the inner surface of the alginate membrane and protrudes toward the capsule's lumen. CONCLUSION(S): The maturation yield of cocultured oocytes is eightfold higher than that achieved with a microdrop maturation technique, making a higher number of gametes available for IVF programs. Stereomicroscopic morphological survey indicates a reassembly of granulosa cells surrounding oocyte zona, mimicking an antral cumulus oophorus.

Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 degrees C.

The customary dilution of boar semen for subsequent artificial insemination (AI) procedures damages the cell membrane of spermatozoa, resulting in a loss of enzymes and other cytoplasmic contents and acrosomal reactions. We encapsulated non-diluted boar semen in barium alginate membranes to optimize AI procedures and to improve the functional integrity of spermatozoal membranes during storage. The percentage of non-reacted acrosomes (NRA) and measurements of enzyme leakage (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH)) were used as indices of the functional status of diluted, unencapsulated and encapsulated spermatozoa, stored for 72 h at 18 degrees C. Enzymatic activity was assessed in situ by microdensitometry, and non-reacted acrosomes were microscopically determined by staining. The percentage of acrosome integrity and the intracellular enzymatic activities during storage were different for unencapsulated and encapsulated semen. Semen dilution caused a rapid decline in enzymatic activities and concomitant acrosomal reactions. Encapsulated spermatozoa had significantly higher acrosome integrity (77% versus 55%; P < 0.01 after 72 h) and an overall higher in situ enzymatic activity. For cytochrome c oxidase and lactate dehydrogenase the greatest differences between encapsulated and unencapsulated spermatozoa were present after 72 h whereas for glucose-6-phosphate dehydrogenase significant differences were found within 24h of storage. The encapsulation process maintains a better preservation environment for boar spermatozoa and could be a promising, innovative technique to improve storage of these cells.

A study on the release mechanism of drugs from hydrophilic partially coated perforated matrices.

Partially coated perforated systems (PCPS) based on low-viscosity hydroxypropyl methylcellulose (HPMC) as the polymeric material were formerly designed, prepared and evaluated in terms of in vitro behaviour. These systems proved to afford the pursued linear release with model drugs (metoprolol tartrate and benfluorex) of different solubility. To the aim of exploring the mechanisms concurring in the definition of zero-order kinetics, studies of drug release, polymer dissolution and medium penetration were performed on PCPS and constant release area systems (CRAS). According to the obtained results, PCPS release kinetics has to be mainly attributed to the progressive outward erosion of the core and to the related variation of the release area. The special geometry of the system, in fact, involves a gradual increase in the release surface, which allows the diffusional path lengthening to be offset. By properly selecting the shape and dimensions of the device as well as the physico-chemical characteristics of the hydrophilic polymer, the advantage of a zero-order release kinetics with programmable rate can be achieved.

Boar semen controlled-delivery system: morphological investigation and in vitro fertilization test.

A technology for encapsulation of swine semen in barium alginate and protamine alginate has recently been proposed for the controlled release of the spermatozoa, thus reducing the number of instrumental inseminations required. Controlled-release capsules containing swine spermatozoa were prepared by adding saturated BaCl2 solution to ejaculate and dropping the resulting suspension into a sodium alginate solution, leading to the formation of barium alginate capsules. A second type of capsule was obtained by cross-linking the barium alginate with protamine sulfate. Two types of membrane were thus obtained: barium alginate gel and a protamine cross-linked alginate membrane. Morphological (scanning electron microscopy and transmission electron microscopy), functional (motility, membrane integrity and in vitro fertilization test) and technological (capsule structure and weight) approaches were used to characterize the encapsulated spermatozoa and the controlled-delivery system. No differences in terms of morphological and functional characteristics (acrosome integrity and spermatozoa motility) between free and encapsulated semen were found. The technological process did not compromise in vitro fertilization potency of the spermatazoa, although seasonal variability was found. The capsule weight was related to either the pH of the semen or the season. This study represents the starting point for the development of further investigations into the storage and release kinetics of cells from the capsules and for the development of an in vivo fertilization protocol.

Fast- and slow-release tablets for oral administration of flavonoids: rutin and quercetin.

Many derivatives of rutin (Rt) and its metabolite quercetin (Q) are employed in clinics for cardiovascular chronic pathology, and are also known for their antiulcer behavior in vivo and antiproliferative and antimutagenic activity in vitro. Unfortunately, the absorption of quercetin and rutin from the gastrointestinal tract is slow and irregular, probably due to their very slight solubility in water and slow dissolution rate. In this work the dissolution rate of the drugs from oral formulations has been improved using some enhancers such as cross-linked sodium carboxy, methylcellulose (CMC-XL), sodium carboxymethylstarch (E), and cross-linked polyvinylpyrrolidone (P). The drugs were loaded on the hydrophilic carriers by different techniques such as mixing or co-milling. The in vitro dissolution profiles of the mixed or co-milled drug/polymer systems, obtained in various media with different pH, were compared. The results show that the drug dissolution rate from the co-milled drug/carrier systems is faster than that from mixed systems, and CMC-XL and sodium carboxymethylstarch systems are able to enhance the dissolution rate. For this reason, these co-milled drug/carrier systems were used for the production of both fast- and slow-release tablets. The co-milled drug/CMC-XL system was used for the preparation of fast-release tablets containing rutin, while three different fast-release tablets were formulated and tested using respectively Q/CMC-XL, Q/E, and Q/P co-milled systems. The effect of the presence of sodium lauryl sulfate in the aqueous medium on the dissolution profile of flavonoids alone was also studied. The prolonged-release formulations have been developed using hydroxypropylmethylcellulose (HPMC) of different viscosity grades as retarding polymer. An extended release of the drugs for times ranging from 6 to 14 hr could be obtained, depending on the type and viscosity of the HPMC used.