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PubMed2002

Optimized conditions for MDCK permeability and turbidimetric solubility studies using compounds representative of BCS classes I-IV.

The solubility enhancing effects of various excipients, including their compatibility with in vitro permeability (P(app)) systems, was investigated using drugs representative of Biopharmaceutics Classification System (BCS) classes I-IV. Turbidimetric solubility determination using nephelometry and transport experiments using MDCK Strain I cell monolayers were employed. The highest usable concentration of each excipient [dimethyl sulfoxide (DMSO), ethanol, hydroxypropyl-beta-cyclodextrin (HPCD), and sodium taurocholate] was determined by monitoring apical (AP) to basolateral (BL) [14C]mannitol apparent permeability (P(app)) and the transepithelial electrical resistance (TEER) in transport experiments done at pH 6.0 and 7.4. The excipients were used in conjunction with compounds demonstrating relatively low aqueous solubility (amphotericin B, danazol, mefenamic acid, and phenytoin) in order to obtain a drug concentration >50 microM in the donor compartment. The addition of at least one of the selected excipients enhanced the solubility of the inherently poorly soluble compounds to >50 microM as determined via turbidimetric evaluation at pH 6.0 and 7.4. Ethanol and DMSO were found to be generally disruptive to the MDCK monolayer and were not nearly as useful as HPCD and sodium taurocholate. Sodium taurocholate (5 mM) was compatible with MDCK monolayers under all conditions investigated. Additionally, a novel in vitro system aimed at more accurately simulating in vivo conditions, i.e., a pH gradient (6.0 AP/7.4 BL), sodium taurocholate (5 mM, AP), and bovine serum albumin (0.25%, BL), was shown to generate more reliable P(app) values for compounds that are poorly soluble and/or highly protein bound.

Animals↗

PubMed2003

Enantioselective transport and CYP3A4-mediated metabolism of R/S-verapamil in Caco-2 cell monolayers.

We have evaluated the passive and carrier-mediated intestinal transport and CYP3A4-mediated metabolism of R/S-verapamil with respect to dose dependency and enantioselectivity in modified Caco-2 cells. The present in vitro results were compared to published data from human in vivo and rat in situ jejunal perfusions with R/S-verapamil. Caco-2 cell permeability to enantiomers of verapamil and norverapamil was weakly concentration dependent (2.5-100 microM). While Caco-2 permeability to verapamil was 2.6- to 3.7-fold lower than in the human jejunum, it was 1.4- to 2.3-fold higher than in rats. However, all three models classified R- and S-verapamil as high permeability compounds according to the biopharmaceutical classification system. In accordance with human and rat data, R/S-verapamil was transported to a minor extent by carrier-mediated mechanisms in Caco-2 cells. Neither the passive nor the carrier-mediated permeability was enantioselective in any of the three models. CYP3A4-mediated demethylation to R/S-norverapamil was enantioselective in Caco-2 cells. Apparent V(max) and K(m) values for the conversion of R-verapamil were 3.2 pmol/min/insert and 0.7 microM, respectively, and for S-verapamil, 5.4 pmol/min/insert and 0.6 microM, respectively. The enantioselectivity in the CYP3A4-metabolism observed in Caco-2 cells was in agreement with human data, but not with rat data, indicating that Caco-2 cells better reflect the human small intestine in this regard. However, all three models suggested that intestinal permeability to verapamil is unaffected by CYP3A4-activity. In summary, modified Caco-2 cells and human jejunum were qualitatively related with respect to R-and S-verapamil transport and CYP3A4-metabolism.

Animals↗

PubMed2000

Self-dispersing lipid formulations for improving oral absorption of lipophilic drugs.

The main purpose of this review is to provide a current and general overview of the existing self-dispersing formulations resulting from dilution into emulsions, microemulsions and surfactant dispersions. The systematic approach used and the presentation of the various physico-chemical and biopharmaceutical aspects should facilitate the comprehension of this interesting field and clarify the main considerations involved in designing and characterizing a specific self-dispersing drug delivery system. Studies have shown that the self-emulsification process is specific to the nature of the oil/surfactant pair, surfactant concentration, oil/surfactant ratio and temperature at which self-emulsification occurs. It was suggested that the ease of emulsification could be associated with the ease by which water penetrates into the various liquid crystalline (LC) or gel phases formed on the surface of the droplet. Numerous bioavailability studies carried out in animals and humans, reviewed in the present study, suggest that hydrophobic drugs are better absorbed when administered in self-dispersing lipid formulations (SDLFs). Examples which illustrate the beneficial use of SDLFs for drug absorption enhancement are presented. This review outlines SDLFs as one of the most promising approaches to overcome the formulation difficulties of these hydrophobic/lipophilic drugs.

Administration, Oral↗

PubMed2000

Effect of solubilizing excipients on permeation of poorly water-soluble compounds across Caco-2 cell monolayers.

The purpose of this study was to evaluate the effects of solubilizing excipients on Caco-2 transport parameters of poorly water-soluble NCEs (new chemical entities), and determine their permeability class under the BCS guidance (Biopharmaceutics Classification System). The effect of solubilizing excipients on soluble donor concentration of Sch 56592, Sch-X and Sch-Y was estimated. The transport of reference compounds and NCEs was studied across Caco-2 monolayers in absence or presence of solubilizing agents. The Caco-2 permeability of reference compounds showed good correlation with their extent of human oral absorption data. Sch 56592, Sch-X and Sch-Y exhibited high baseline Caco-2 permeability (>10(-5) cm/s). Povidone (1%) improved soluble donor concentration and flux of Sch 56592 by 40%. Other solubilizing excipients predominantly improved Sch 56592 soluble donor concentration, with either no change or a decrease in flux. With Sch-X, 1% povidone, pluronic F68, gelucir 44/14, and 3:2 propylene glycol/Tween-80 markedly improved soluble donor concentration, while increasing Sch-X flux by 40-65%. The soluble donor concentration of Sch-Y was also enhanced by excipients; however, only 1% pluronic F68 and PEG 300 increased Sch-Y flux by 35-50%. Sch 56592, Sch-X and Sch-Y are low solubility-high permeability compounds under the BCS guidance. For such poorly water-soluble NCEs, solubilizing excipients should be carefully screened based on their effects on solubility profiles and membrane transport.

Alkaloids↗

PubMed2002

A novel approach to determining physicochemical and absorption properties of 6-fluoroquinolone derivatives: experimental assessment.

The ToSS MoDe approach is used to estimate the n-octanol/buffer partition coefficient, the apparent intestinal absorption rate constant and intestinal permeability from a 6-fluoroquinolone data set. Improved in silico methods for predicting a drug's ability to be transported across biological membranes and other biopharmaceutical properties is highly desirable to optimize new drug development. The physicochemical property (Log P) of 26 6-fluoroquinolone derivatives and the absorption properties (Log K(a) and Log P(eff)) of 21 derivatives were well described by the present approach. The models obtained confirm the important role of lipophilicity in the absorption process and its relation with the piperazinyl ring spectral moment and general local spectral moment. The normalized group contributions to each property, at the R4 and R5 positions of a 6-fluoroquinolone framework, were calculated. Principal factor analysis between these contributions and the Hammett and Hansch constant, molar refractivity and sterimol parameters was also carried out. Three principal factors explained 78% of the total variance and the correlation coefficients were higher than 0.98. The isocontribution zone analysis for the Log P and Log K(a) of Sarafloxacin and Sparfloxacin, used as external corroboration compounds, was carried out. The absorption rate constants (in situ rat gut technique) for these drugs were also evaluated, and the results were compared with the values predicted by theoretical models for evaluating predictive performance. The present approach proved to be a good method for studying the oral absorption of drug candidates in drug development studies.

Animals↗

PubMed2002

In vitro evaluation of microcrystalline chitosan (MCCh) as gel-forming excipient in matrix granules.

Although much research has been carried out into the effects of chitosan and its chemical properties on drug release, less attention has been paid to the effects of its physical properties. The aim of this study was to characterize microcrystalline chitosan (MCCh) as a gel-forming excipient. Matrix granules containing chitosans of differing physicochemical properties (crystallinity, molecular weight, degree of deacetylation) and ibuprofen or paracetamol as model drugs were prepared. Gel formation by the chitosans in the granules and subsequent effects on drug release were studied at pH 1.2 and pH 5.8. The chitosan granules acted as slow-release formulations in the case of ibuprofen (a class-II drug in the Biopharmaceutics Classification System) but with paracetamol (class-I) no controlled-release formulation could be developed. Microcrystalline grades of chitosan had the most marked retardant effects on drug release, with the efficacy of gel formation by MCCh explaining the results. The kinetic constant for ibuprofen release (at pH 5.8) ranged from 22%.h(-1) (MCCh) to 31%.h(-1) (unmodified chitosan). The release rate was easily controlled by varying the amount or molecular weight of MCCh, and to a lesser extent by the degree of deacetylation. The effects were most pronounced when pH was markedly acidic, suggesting that MCCh granules might be particularly useful in preparing stomach-specific slow-release dosage forms.

Chemistry, Pharmaceutical↗

PubMed2002

Effect of bovine serum albumin on drug permeability estimation across Caco-2 monolayers.

The purpose of this study was to explore approaches to more accurately assess Caco-2 permeability of poorly water-soluble new chemical entities (NCEs) in an effort to determine their biopharmaceutics classification system (BCS) permeability class with a higher level of confidence. The transport of reference compounds and NCEs (Sch-Y, Sch 56592) was studied across Caco-2 monolayers in the absence or presence of varying percentage of bovine serum albumin (BSA) in the receiver chamber. The inclusion of 0.5-4% BSA in the receiver chamber caused a 4-5-fold increase in Sch-Y P(app), while Sch 56592 P(app) was not significantly influenced. Amongst reference solutes, the P(app) ratio (+BSA/ctrl) was significant (1.3-fold) only for diltiazem (log PC=2.7, plasma protein binding=78%), but the prediction of human oral absorption for such drugs was not affected by the presence of BSA in receiver. In summary, the use of 4% BSA in the receiver chamber during transport studies can dramatically affect the estimated Caco-2 P(app) and BCS permeability ranking of highly lipophilic NCEs, as in the case of Sch-Y with a log PC of 4.0. For Sch-Y, this is presumably due to improved sink conditions and/or a reduction in non-specific drug adsorption to plastic wells. In contrast, the permeability classification of Sch 56592 (log PC=2.4) based on estimated Caco-2 P(app) values is not affected by the presence of receiver BSA.

Animals↗

PubMed2003

Pharmaceutical biotechnology products approved within the European Union.

The manufacture of therapeutic proteins represented the first true industrial application of recombinant DNA technology. Thus far some 88 recombinant proteins/monoclonal antibody-based products have gained marketing approval within the European Union (EU). This represents 36% of all new drug approvals since the introduction of the new centralized European drug approval system in 1995. More recently, an increasing proportion of approved proteins are engineered, tailored to display altered pharmacokinetic profiles or reduced immunogenicity in man. Currently no nucleic acid-based products are approved in the EU. Technical innovations/milestones likely characterizing the biopharmaceutical industry within the next decade include approval of some products produced in transgenic systems, approval of some products administered by non-parenteral means, approval of at least some nucleic acid-based products and the identification of novel biopharmaceuticals/biopharmaceutical targets through discoveries in functional genomics and proteomics.

Biotechnology↗

PubMed2003

A topological-substructural molecular design (TOPS-MODE) approach to determining pharmacokinetics and pharmacological properties of 6-fluoroquinolone derivatives.

The topological-substructural molecular design approach was used to estimate the human bioavailability (F) and the minimum inhibitory concentration (MIC90) against Streptococcus pneumoniae from a data set of 17 and 19 fluoroquinolone derivatives, respectively. Both pharmacokinetics and pharmacological properties were well described by the present approach. The total spectral moments and local spectral moments that include the different fluoroquinolone rings, polar and non-polar areas and their interactions were calculated and weighted with the standard dipole moments and the electronegative difference between the atoms that form a bond. In order to obtain a qualitative model that permits the classification of drugs with high and moderate bioavailability, a linear discriminant analysis was carried out. The percentage of correct classification was 100% for compounds of the training set. The leave-one-out cross validation procedure showed an 88.23% of correct classification. Also, a quantitative model, by the piecewise linear regression was developed. The theoretically predicted values for human bioavailability was assessed by a correlation with in vivo rat bioavailability and the regression equation was used to predict this biopharmaceutical property for two new pre-clinical 6-fluoroquinolone derivatives. On the other hand, a linear regression model that explained the 84% of variance was developed to predict the MIC90 values. Finally, the role of a pharmacokinetic and pharmacological relationship in the design of new fluoroquinolones was evaluated in the Sitafloxacin framework, where 13 substituents were analyzed; halogens and methoxy groups had the best contributions to both properties. The present approach proved to be a good method for studying the pharmacokinetics and the pharmacological properties of new 6-fluoroquinolone candidates in drug development studies.

Animals↗

PubMed2000

Gastric pH profiles of beagle dogs and their use as an alternative to human testing.

Gastric pH levels were measured in samples of gastric aspirates from eight fasted beagle dogs. The gastric pH in fasting dogs fluctuated from 2.7 to 8.3, with a mean of 6.8+/-0.2 (SE). Each dog received the following four treatments in randomly-assigned order: (A) distilled water; (B) a placebo capsule; (C) pentagastrin, and (D) ranitidine. The gastric pH remained relatively constant after distilled water administration. In contrast, the treatments with pentagastrin and placebo capsule each lowered gastric pH. Pretreatment with pentagastrin was more successful in lowering gastric pH than that with placebo capsule. On the other hand, the pH rose above 7.0 in all dogs by the first hour after treatment with ranitidine. This animal model may be helpful in evaluating the biopharmaceutics of drugs exhibiting pH-dependent dissolution or decomposition.

Animals↗

PubMed1999

An optical biosensor for monitoring recombinant proteins in process media.

This paper describes the construction of a sensor for the direct monitoring of a recombinant protein, the human insulin analogue (MI3). The surface plasmon resonance (SPR) sensor incorporates an immobilised, sterilisable affinity-ligand that has been designed to bind to MI3. In practice, gold SPR devices were fabricated with; a 2D assembly of ethanethiol-modified ligand, a 2D mixed-assembly of ethanethiol-modified ligand and mercaptoethanol, a 3D coating of ligand-modified terminal-thiolated poly(vinyl)alcohol (PVA) or a 3D hydrogel of dextran coupled to a self-assembled monolayer (SAM) of mercaptohexaneundecanl-ol. Routine measurement of the concentration MI3 in the concentration range 1-100 mg/l in pilot-scale samples of crude fermentation broth have been achieved with high sensitivity levels and a high signal-to-noise ratio. Analysis can be achieved within < 10 min with the active surface being regenerable for at least 60 cycles over a 6 month period. The coupling of a robust, sterilisable and highly-selective sensor-coating with suitable transducer technologies promises to deliver sensors that are capable of direct in situ monitoring of biopharmaceuticals in industrial bioprocesses.

Biotechnology↗

PubMed1994

Large-scale mammalian cell culture.

Over the past year, progress has been made toward a better understanding of the physiological and biochemical responses of cell cultures to various environmental changes. Much of the theoretical and experimental work that has been reported will help improve expression in mammalian cell culture, which is one of the key steps in biopharmaceutical manufacturing.

Animals↗

PubMed1996

Quality and authenticity of heterologous proteins synthesized in yeast.

Yeast, especially Saccharomyces cerevisiae and Pichia pastoris, are major hosts employed in the expression of authentic heterologous proteins of high quality in the biopharmaceutical, industrial and academic environments. There has been recent progress in characterizing and controlling the factors involved in determining authenticity.

Biotechnology↗

PubMed1998

Apoptosis in cell culture.

Most cells can exhibit a biochemical pathway which mediates their own destruction in a highly controlled and genetically defined manner. In animal cells, a morphologically distinct form of this 'programmed cell death' has been identified and extensively characterised. This phenomenon, which has been named apoptosis, accounts for most of the cell deaths that take place during the production of biopharmaceuticals from animal cell lines. In the past few years, the factors responsible for the induction of apoptosis in the bioreactor environment have been identified. Furthermore, a growing number of studies have demonstrated that the suppression of apoptosis by the overexpression of anti-apoptosis genes, most notably bcl-2, result in improved culture productivity.

Animals↗

PubMed2002

Botulinum and tetanus neurotoxins: structure, function and therapeutic utility.

The toxic products of the anaerobic bacteria Clostridium botulinum, Clostridium butyricum, Clostridium barati and Clostridium tetani are the causative agents of botulism and tetanus. The ability of botulinum neurotoxins to disrupt neurotransmission, often for prolonged periods, has been exploited for use in several medical applications and the toxins, as licensed pharmaceutical products, now represent the therapeutics of choice for the treatment for several neuromuscular conditions. Research into the structures and activities of botulinum and tetanus toxins has revealed features of these proteins that might be useful in the design of improved vaccines, effective inhibitors and novel biopharmaceuticals. Here, we discuss the relationships between structure, mechanism of action and therapeutic use.

Animals↗

PubMed1991

Evaluation of transforming activity of cellular DNAs from different origins by NIH3T3 transfection test.

The NIH3T3 cell transfection test, as first described by Cooper, has been optimized, then used to examine the transforming activity of genomic DNA extracted from eucaryotic cell lines commonly used for preparing vaccines or biopharmaceuticals. Accurate assessment of technical parameters of the test has led to improvement in reproducibility, while the demonstration of dose-effect relationships has allowed the definition of applications and limits for quantitative use. We have performed the direct assessment of transforming activity of cellular DNAs from cell lines widely used in biotechnology. In particular, we have shown that genomic DNA extracted from Vero, CHO or MRC5 cells, as well as from human or murine lymphoid cells, has no detectable transforming activity on NIH3T3 cells. Lastly, it has been demonstrated that acidic pH conditions are sufficient to destroy the major part--if not all--of the transforming activity of positive control DNAs.

3T3 Cells↗

PubMed1999

Dolly, Polly and other 'ollys': likely impact of cloning technology on biomedical uses of livestock.

The idea of generating transgenic livestock which secrete into their milk large quantities of proteins for therapeutic use, was pioneered in the late 1980s with the disclosure of the production of a number of transgenic sheep. One particular animal, a sheep called Tracy, produced milk where over 50% of the protein consisted of human alpha 1 anti-trypsin. Sheep-derived protein has now entered clinical trials for cystic fibrosis (UK, USA) and congenital emphysema (UK). There are many other examples where this technology is making inroads into more traditional ways of making biopharmaceuticals. However, although robust, this technology has several limitations, including an inability to allow targeted insertion/modification of the animal genome, long timelines to production flocks/herds, and the rather unpredictable expression levels seen when different transgenic founders are compared. We believe that there is now a technical solution to all of these problems. Dolly is a high profile example of a new technology comprising the generation of identical animals from cultured somatic cells. This work has many implications. In the commercial context, the real benefits of this advance will be seen when genetically engineered somatic cells are shown to be suitable nuclear donors, and particularly when the manipulations are targeted to pre-determined sites in the host cell genome. The first objective has now been achieved with the birth of Polly, a cloned sheep which contains the human gene encoding Factor IX, a protein involved in preventing haemophilia.

Animal Husbandry↗

PubMed2002

Validation of targets and drug candidates in an engineered three-dimensional cardiac tissue model.

High-throughput target discovery confronts the biopharmaceutical industry with a plethora of target candidates. The validation of these candidates in disease-specific animal models often lacks the required throughput. Here, we discuss perspectives and limitations of a novel engineered three-dimensional cardiac tissue, which enables the influence of gene and drug intervention to be monitored on a cellular and molecular level under physiological conditions in sufficient throughput. The model is an extremely helpful filter to prioritize multiple development candidates before moving a project into large animal models with higher predictivity.

Animals↗