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PubMed2000

Expression of active monomeric and dimeric nuclease A from the gram-positive Streptococcus gordonii surface protein expression system.

We used the surface protein expression (SPEX) system to express an anchored and a secreted form of staphylococcal nuclease A (NucA) from gram-positive bacteria. NucA is a small ( approximately 18 kDa), extracellular, monomeric enzyme from Staphylococcus aureus. A deletion of amino acids 114-119 causes monomeric NucA to form homodimers. The DNA sequence encoding either wild-type or deletion mutant NucA was cloned via homologous recombination into Streptococcus gordonii. S. gordonii strains expressing either anchored or secreted, monomeric or dimeric NucA were isolated and tested for enzymatic activity using a novel fluorescence enzyme assay. We show that active monomeric and dimeric NucA enzyme can be expressed either anchored on the cell surface or secreted into the culture medium. The activity of the dimer NucA was approximately 100-fold less than the monomer. Secreted and anchored, monomeric NucA migrated on SDS-polyacrylamide gels at approximately 18 or approximately 30 kDa, respectively. In addition, similar to S. aureus NucA, the S. gordonii recombinant NucA enzyme was dependent on CaCl(2) and was heat stable. In contrast, however, the recombinant NucA activity was maximal at pH 7.0-7.5 whereas S. aureus NucA was maximal at pH 9.0. These results show, for the first time, expression of active enzyme and polymeric protein in secreted and anchored forms using SPEX. This further demonstrates the utility of this gram-positive surface protein expression system as a potential commensal bacterial delivery system for active, therapeutic enzymes, biopharmaceuticals, or vaccines.

Bacterial Proteins↗

PubMed2002

Expression and affinity purification of recombinant proteins from plants.

With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system.

Blotting, Western↗

PubMed1999

Protein glycosylation: implications for in vivo functions and therapeutic applications.

The glycosylation machinery in eukaryotic cells is available to all proteins that enter the secretory pathway. There is a growing interest in diseases caused by defective glycosylation, and in therapeutic glycoproteins produced through recombinant DNA technology route. The choice of a bioprocess for commercial production of recombinant glycoprotein is determined by a variety of factors, such as intrinsic biological properties of the protein being expressed and the purpose for which it is intended, and also the economic target. This review summarizes recent development and understanding related to synthesis of glycans, their functions, diseases, and various expression systems and characterization of glycans. The second section covers processing of N- and O-glycans and the factors that regulate protein glycosylation. The third section deals with in vivo functions of protein glycosylation, which includes protein folding and stability, receptor functioning, cell adhesion and signal transduction. Malfunctioning of glycosylation machinery and the resultant diseases are the subject of the fourth section. The next section covers the various expression systems exploited for the glycoproteins: it includes yeasts, mammalian cells, insect cells, plants and an amoeboid organism. Biopharmaceutical properties of therapeutic proteins are discussed in the sixth section. In vitro protein glycosylation and the characterization of glycan structures are the subject matters for the last two sections, respectively.

Animals↗

PubMed1997

In vitro-in vivo relationships of several "immediate" release tablets containing a low permeability drug.

The objective of this work was to gain insight into the biopharmaceutical performance of four different but bioequivalent ranitidine hydrochloride tablet formulations. This analysis employed a recently described method1 to relate in vitro and in vivo data and aimed to facilitate an understanding of oral drug product performance. For each ranitidine formulation, dissolution was performed using the USP procedure. A four-way, single dose bioequivalence study (n = 14) was performed. The fraction of the total amount of dose absorbed at each plasma sample time was determined by the Wagner-Nelson method. Equation 1 (see below) was fitted to the in vitro vs. in vivo data. For all four formulations, this analysis suggests absorption was permeation-rate limited, where ranitidine exhibited a low permeation rate constant of 0.01/min.

Administration, Oral↗

PubMed1999

Transgenic plants for therapeutic proteins: linking upstream and downstream strategies.

We have described two very different and innovative plant-based production systems--postharvest production and recovery of recombinant product from tobacco leaves using an inducible promoter and oleosin-mediated recovery of recombinant product from oilseeds using a seed-specific promoter. Both base technologies are broadly applicable to numerous classes of pharmaceutical and industrial proteins. As with any emerging technology, the key to success may lie in identifying those products and applications that would most benefit from the unique advantages offered by each system. The postharvest tobacco leaf system appears effective for proteins requiring complex posttranslational processing and endomembrane targeting. Because of the remarkable fecundity and biomass production capacity of tobacco, biomass scale-up is very rapid and production costs are low. Clearly the development of equally cost-effective extraction and purification technologies will be critical for full realization of the commercial opportunities afforded by transgenic plant-based bioproduction. The recovery of protein from tobacco leaves or oleosin-partitioned proteins by oil-body separations represent significant break-throughs for cost-effective commercialization strategies. Additional low-cost, high-affinity separation technologies need to be developed for effective scale-up purification of plant-synthesized recombinant proteins. Clearly successful commercialization of plant-synthesized biopharmaceuticals must effectively link upstream strategies involving gene and protein design with downstream strategies for reproducible GMP-level recovery of bioactive recombinant protein. Both the tobacco and oilseed systems are uniquely designed to address issues of biomass storage, product recovery, quality assurance, and regulatory scrutiny in addition to issues of transgene expression and protein processing.

Animals↗

PubMed1991

Production of recombinant proteins in serum-free media.

The advantages of serum-free culture for the manufacture of recombinant biopharmaceuticals from mammalian cells are reviewed. The process favoured is fed-batch serum-free cell culture. This process is applicable to the majority of cell lines, is practical on the large scale, gives the lowest manufacturing cost, and can be carried out without the use of any serum.

Animals↗

PubMed1988

Design of a new multiple-unit controlled-release formulation of metoprolol--metoprolol CR.

A new controlled-release (CR) formulation of the beta 1-selective adrenoceptor antagonist metoprolol has been developed, aiming at an even 24-h pharmacological effect. In order to achieve this, using a once-daily dose, factors such as absorption characteristics, physicochemical properties, and technological aspects had to be considered. The new formulation, called metoprolol CR, is a disintegrating tablet consisting of several hundred coated pellets of metoprolol succinate, each pellet being its own CR delivery unit. In vitro testing and in vivo studies in healthy volunteers show that the new CR formulation gives continuous delivery of metoprolol throughout the day, resulting in smooth plasma concentration profiles, without peaks and troughs. The release of the drug is independent of pH and other physiological variables, such as food intake, which do not seem to alter the biopharmaceutical properties of the formulation.

Delayed-Action Preparations↗

PubMed1989

Inaccuracy in the doses of injectable medications dispensed from rubber-stoppered vials.

The literature of pharmacology often assumes that a full dosage is utilized when the contents of a vial have been administered by syringe. Five hundred discarded medication vials were assayed. The residues amounted to 1.98% to 8.81% of the listed dosages. An additional 0.7% to 8.66% remained in the syringes and needles used to aspirate the vials. Routine preparation techniques do not recover medication trapped on glass and rubber surfaces; losses are greatest when small diluent volumes are added to prepare intramuscular injections. The mean dose of gentamicin recovered from 80-mg ampules was 78.65 mg, and comparable vials of tobramycin yielded 76.01 mg. The discrepancy may contribute to the "increased toxicity" of gentamicin. Each year, more than $40,000,000 worth of antibiotics are lost to a biopharmaceutical dead space. Used antibiotic and controlled substance vials pose a potential threat to the environment. Although the amount of drug lost during preparation and administration may be of little therapeutic consequence, the discrepancy between intended and administered dosage is reflected in economic loss and pharmacological confusion. Pharmacological data should be adjusted for such losses. Medication wastage could be reduced by redesign of vials and alterations in practice of the administration.

Anti-Bacterial Agents↗

PubMed1986

The impact of neglecting nonlinear plasma-protein binding on disopyramide bioavailability studies.

Disopyramide has nonlinear protein binding and thus the relationship between the extent of its bioavailability and AUC, the area under the plasma concentration-time curve, is nonlinear and absorption rate-dependent. The unbound species follows linear pharmacokinetics. A solution of disopyramide, the innovator's product, and two generic formulations were found to be statistically indistinguishable in their bioavailability of disopyramide, whether comparison was based upon AUC or area under the plasma unbound concentration-time curve (AUCu). The AUC and AUCu gave similar results because of truly similar bioavailability, coupled with sufficiently similar release rates, among the four preparations chosen for study. The concentration dependence of disopyramide protein binding and the time course of unbound plasma concentrations were fit by models which then allowed prediction of AUC under various biopharmaceutical scenarios. Nonlinear binding of disopyramide to plasma proteins renders AUC an insensitive parameter for the discrimination of products with different extents of bioavailability; immediate-release products allowing bioavailabilities of 75 or 125% relative to the solution can generate AUCs 86 and 112%, respectively, of that from the solution. Nonlinear binding, furthermore, leads to a tendency for AUC to overestimate the bioavailability of slower release products in single-dose studies; if AUC were the index of bioavailability, products permitting the same bioavailability as the solution but releasing over 12 hr could appear to allow 114% relative bioavailability. Moreover, in some situations the bias arising from the insensitivity of AUC to product differences can be reinforced by the dependence of AUC on release rate; an apparent relative bioavailability of 80% can be achieved by a 12-hr release product allowing a true relative bioavailability of a mere 58%. While multiple-dose studies appear largely to avoid the tendency to overestimate low bioavailability in slow-release products, in these studies AUC appears to be even more insensitive in resolving discrepancies between products. Assay technology now available makes AUCu a feasible and more reliable index of bioavailability than AUC when plasma protein binding of drugs is nonlinear.

Administration, Oral↗

PubMed1990

Development of long-acting nitrate delivery systems.

In this paper pharmaceutical and biopharmaceutical aspects on the development of long-acting nitrate delivery systems are reviewed. Basic considerations and requirements are also discussed. Recent product development activities are presented, with an emphasis on nitroglycerin in transdermal form and oral isosorbide-5-mononitrate controlled-release formulations.

Administration, Cutaneous↗

PubMed1980

[A pharmacist's proposal for improved pharmacotherapy].

The pharmaceutical sciences have recently made great progresses towards a better knowledge of drug action. For the security of medication, it seems most important that said knowledge should be made available to specialists without delay. This means also that an adequate instruction in pharmacobiological branches is necessary. Cooperation of physicians and pharmacists should be intensified. The latter are in a position to give general information (i.e. critical evaluation of a new drug on the market) as well as specific information (required form of administration, mode of action, biopharmaceutical properties, adequate dosage, adverse effects and probable interactions, alternative medication in case of contraindications, etc.). The medical and pharmaceutical studies today do not sufficiently prepare both professions for such a cooperation. It is therefore highly advisable to coordinate in a better way the education in different sciences. For the time being, pharmacists already have at their disposals a good infrastructure, which comprises the scientific center of the national professional organization. The information of the patient himself should also be developed and intensified, especially with regard to the correct use of drugs, prescribed or OTC. Again, this specific activity needs the close cooperation of physicians and pharmacists. Steady relations between both health professions are thus to be deepened and reinforced for the benefit of the patient.

Drug Interactions↗

PubMed2005

Couplet alignment and improved electrofusion by dielectrophoresis for a zona-free high-throughput cloned embryo production system.

Mammalian cloning by somatic nuclear transfer has great potential for developing medical applications such as biopharmaceuticals and generation of tissues for transplantation. For agricultural applications, it allows the rapid dissemination of genetic gain in livestock breeding. The maximisation of that potential requires improvements to overall cloning technology, especially with respect to increasing cloning efficiency and throughput rates in cloned embryo production. A zona-free embryo reconstruction system was developed to increase cloning throughput and ease of operation. Central to this system is a modified electrofusion procedure for nuclear transfer. Cytoplast-donor cell couplets were placed in a custom-designed 'parallel plate' electrode chamber. A 1 MHz sinusoidal AC dielectrophoresis alignment electric field of 6-10 kV m(-1) was applied for 5-10s. The couplets were then fused using 2 x 10 micros rectangular DC-field pulses (150-200 kV m(-1)), followed by application of the AC field (6-10 kV m(-1)) for another 5-10 s. Fusion was performed in hypoosmolar buffer (210 mOsm). Automated alignment of up to 20 couplets at a time has been achieved, resulting in greatly improved fusion throughput rates (2.5-fold increase) and improved fusion yields (1.3-fold increase), compared with commonly followed zona-intact protocols.

Animals↗

PubMed1999

[Concept of control of galenic properties in oral administration of drugs].

The pharmaceutical and biopharmaceutical development of drug preparations comprehends the transformation of a drug in an applicable drug preparation with optimal efficacy, safety and acceptance. Thus, a drug delivery system has to be developed that controls the onset, the duration and the intensity of the expected effect. Depending on the specific drug properties and to the special objectives different controlling principles have to be selected. These are for example delayed release (salivaresistance, gastroresistance), slow release, release in distinct regions of the gastrointestinal tract and accelerated release.

Administration, Oral↗

PubMedDate not supplied

Use of the repeated cross-over design in assessing bioequivalence: (within and between subjects variability - Schuirmann Confidence Intervals estimation).

In 1992, the Division of Bioequivalence in the Office of Generic Drugs published a guide to Statistical procedures for bioequivalence studies using a standard two-treatments cross-over design (1). This paper describes the application of the guidelines to a practical protocol and the recent Proc MIXED (SAS) will be shown to be much more convenient than the traditional Proc GLM for theoretical and practical reasons (correct estimation of residuals, analysis of the within-subjects variation, direct calculation of the Schuirmann 90% Confidence Intervals). This new procedure was applied to a study protocol on riluzole (Rilutek) including a replicate design with the within-subject and between-subject variances being estimated on Cmax and AUC biopharmaceutic parameters.

Administration, Oral↗

PubMedDate not supplied

Guidances related to bioavailability and bioequivalence: European industry perspective.

The investigations of bioavailability and bioequivalence can be classified according to three separate areas of information. Firstly, estimation of bioavailability judged on a drug substance's in vivo characteristics taking into account solubility, polymorphism, stability (especially under the conditions of the GI tract), gut wall permeability and first pass metabolism. Secondly, evaluation of formulation properties including dissolution profile in the GI tract and its contribution to exposure variability with respect to the desired absorption characteristics. Finally, maintaining quality during the market phase with respect to equivalence to the clinical trial formulations. While in the first two areas, the range of the estimated mean values and the intra- and inter-subject variabilities contain the desired information for proper medical decisions, in the third area the mean values and their confidence limits describe the quality with regard to the formulations of proven efficacy. Guidelines should clearly distinguish between the different areas in their recommendations regarding the intended information, e.g. mean values and/or ranges and confidence intervals. New approaches of granting limited waivers for BE studies (e.g. Biopharmaceutical Classification System (BCS)) should be expanded to consideration of pharmacokinetic properties of drugs (e.g. gastrointestinal metabolism, evidence for an absorption window, magnitude of first-pass effect, half-life) as already partly implemented in the German waiver concept, and further (scientifically) validated to achieve world-wide harmonisation (e.g. via ICH).

Biological Availability↗

PubMedDate not supplied

Prodrugs for the improvement of drug absorption via different routes of administration.

The authors critically review recent knowledge on the use of prodrugs to improve drug absorption. Main emphasis is placed on the parenteral, oral, transdermal and ocular routes. Mechanisms for drug absorption enhancement and bioavailability assessment are discussed. Some other applications of prodrugs are also examined. Finally, some comments are made regarding the present situation and future trends in prodrug design and their implications in biopharmaceutics and pharmacokinetics.

Absorption↗

PubMedDate not supplied

The unusual routes of administration.

A wide range of administration routes can be drawn on to optimise drug absorption. Though the oral route remains the favourite one for most drugs in many disease states, other routes are routinely used. Each has its strengths and weaknesses and needs to be selected carefully with full consideration of the drug, its target and the release pattern required. Advances in pharmacology and biopharmaceutics have led to exciting developments in the ways drugs can now be administered. Optimising drug administration means finding answers to a number of questions. These questions include: what, how, when and where to deliver, and how to retain the drug long enough for it to be fully effective. These issues are linked; route of administration, drug and therapeutic systems are interrelated; thus the choice of what to deliver influences how, when and where to deliver. Various definitions of 'usual/unusual' administration routes are presented, depending on the factors that determine the fate of the active ingredients, on the delivery device, and on the therapeutic objectives to be met. The various dosage forms designed for these routes, and the administration strategies developed to achieve the desired effects are described.

Absorption↗

PubMedDate not supplied

Squared correlation coefficient of measured values versus predicted values in linear and monoexponential regressions.

The correlation coefficient of measured values vs predicted values is widely used in pharmacokinetic and biopharmaceutical settings. When using linear and monoexponential regressions, we notice an interesting characteristic of the squared correlation coefficient of measured values vs predicted values, i.e. the squared correlation coefficient of measured y1 vs predicted (lambda)y2 is a constant regardless of different values of regression coefficients and is equal to the squared correlation coefficient of measured x1 vs measured y1.

Forecasting↗