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Biomedical subjects

R G Werner

Publications and source records attributed to R G Werner.

At least 37 records · Page 2Linked to original sources
PubMed1991

Quality aspects of fibrinolytic agents based on biochemical characterization.

The purity, composition and in vitro fibrinolytic activity of four commercially available fibrinolytic agents, alteplase (recombinant tissue plasminogen activator, rt-PA, Actilyse; CAS 105857-23-6), streptokinase, urokinase and anistreplase (ansioyl-plasminogen-streptokinase activator-complex, APSAC), have been compared in this investigation. The fibrinolytic activity was measured in an in vitro thrombolytic assay. In this assay a human blood thrombus is dissolved in an environment of human plasma. This assay is representative for the in vivo situation, where plasminogen activation is also a limiting step in thrombolysis. In the in vitro thrombolytic assay alteplase is about 10 times more effective in clot lysis than either streptokinase or urokinase and more than 300 times more active than anistreplase. In addition, the ratio of active ingredient to total protein content in the preparations was analysed by RP-HPLC, SDS-PAGE, GPC-HPLC and amino acid analysis. The portion of active ingredient per total protein was 99.9% for alteplase, 55% for anistreplase, 20% for urokinase and 1% for streptokinase. This demonstrates that alteplase is the only fibrinolytic agent tested which is essentially free of protein additives of human origine and potential contaminants associated therewith. The superior purity of alteplase compared to the other fibrinolytics was confirmed by SDS-PAGE, RP-HPLC, and HPLC-GPC. Significant levels of aggregates were detected in streptokinase and urokinase preparations, whereas alteplase and anistreplase were essentially free of aggregates. These data demonstrate that there are significant differences in composition, purity and in vitro activity between different fibrinolytic agents.

Amino Acids↗
PubMed1990

Successful development of recombinant DNA-derived pharmaceuticals.

Successful development of recombinant DNA-derived pharmaceuticals, a new class of therapeutic agents, is determined by a variety of factors affecting the selection and positioning of the compound under development. For an efficient development it is of utmost importance that the mechanism of action of the compound selected be understood on a molecular level. The compound's potential therapeutical profile and a strong patent position are key positioning considerations, as well as vital elements in shortening the development phase and protecting innovation. Installation of an interdisciplinary project management team, along with a clear definition of team members' responsibilities, is required to avoid delays and improve communication during development. Selection of the organism to be used in production must take into consideration both the structure of the protein and the quality and safety of the final product. New technologies require a considerable investment in new manufacturing facilities and equipment. Often, the decision for such an investment must be made early and with a high degree of uncertainty. Desired product yield, expected dosage, and estimated market potential are the most important considerations in this decision. Following public disclosure of the plan to develop recombinant DNA-derived products, approval of the production plant and expansion or adaptation to the new process and technology may be delayed. For this reason, they should be considered as a critical step in the overall development phase. Recruitment of qualified staff is a time-consuming and critical element of the production process. Its impact on the product timeline should not be underestimated, especially if such technologies are new to the company. The entire production process must be validated in respect to identity, purity, and safety of the product to guarantee constant product quality, as well as for safety aspects in the environment. Adequate in-process and final product controls have to be established and specifications determined for release or rejection of batches for preclinical and clinical studies as well as for marketing. Preclinical testing is limited because recombinant DNA-derived proteins cannot be differentiated from naturally occurring human proteins, and because some proteins are species-specific.

DNA, Recombinant↗
PubMed1989

Meeting the regulatory requirements for pharmaceutical production of recombinant DNA derived products.

Genetic engineering provides the opportunity for the synthesis of human proteins and derivatives thereof which are of significant value for replacement therapy. However, in addition to genetic engineering an extensive process development has to be carried out in order to establish an economic production process and to guarantee consistently high product quality from batch to batch. This includes the characterization of the production host cell vector construct, the validation of the fermentation and the protein purification process as well as the lyophilization and the reconstitution of the final product. A number of in-process and final product controls have to be established and limits for the specification have to be elaborated to provide consistent product quality. Real time stability data have to be supplied because data from accelerated conditions do not allow extrapolation of the shelf life of proteins. Data obtained from process development and validation of the production process contributes to the preparation of the chemical pharmaceutical dossier and the expert report to be submitted to the regulatory authorities.

DNA, Recombinant↗
PubMed1988

Validation of biotechnological production processes.

Due to the biological synthesis of biotechnologically produced pharmaceuticals the product quality and safety of the drug is influenced by various factors. The correct nucleotide sequence and stability of the host cell/vector system provide the corresponding amino acid sequence of the protein. The posttranslational processing of the protein requires a well characterized production cell line. Suitable equipment for fermentation allowing a sterile production of the producing monoculture and consistent conditions are the basic requirements for the validation of the fermentation process. A constant specific productivity is one of the major criteria for the reproducibility of the production. For the validation of recovery and purification it is necessary to examine yield after each process step, product quality before and after each single process step and purification factors for removal of contaminating proteins, nucleic acids and potential viruses. In addition to the validation of the entire production process, reproducibility of quality of the formulated product has to be determined by a number of protein analytical, immunological and biochemical test methods concerning the identity, purity, safety and potency of the drug.

Biotechnology↗
PubMed1988

[Biotechnical production of drugs with special reference to tissue-type plasminogen activator].

On the basis of recombinant DNA-technology, new biotechnology is able to provide active human proteins which can be applied for replacement therapy to patients, whose symptoms of illness are based on the lack of these proteins. One of the most thoroughly investigated compounds in this aspect is tissue plasminogen activator (t-PA) which under pathophysiological conditions is not available in sufficient quantities; as a result formed thrombi cannot be dissolved sufficiently by endogenous means. Consequently, t-PA is produced biotechnically and applied exogenously to the patients. Such proteins only can be produced by biotechnology for therapeutic purposes because a chemical synthesis is not economical and isolation from human tissues or body fluids is not possible due to low concentrations, safety aspects or ethical reasons. Resulting from the knowledge of molecular biology, the genetic information for the synthesis of a distinct protein can be transferred into a suitable host cell for efficient production, by methods of recombinant DNA-technology. Thereby the choice of the producing organism is determined by the complexity of the protein. After the growth phase of the producing cells the biological active protein is obtained with a purity greater than 99.9% from the production bioreactor by protein chemical methods. Quality assurance is guaranteed by manifold protein chemical, electrophoretic, immunologic, enzymatic and biological methods. A high stability of such biologically active proteins is achieved by lyophilization after achieving a high degree of purity and a suitable formulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Fermentation with immobilized cell cultures.

For the production of monoclonal antibodies and complex recombinant human proteins or glycoproteins a number of immobilized cell culture systems have been developed. The advantages of such cell culture systems are that cells can be kept in small volumes of cell culture fluid and media can be changed continuously if necessary for induction of product synthesis or removal and harvest of metabolic products. Whereas the hollow fiber and the opticell culture systems can be limited in scaling up the microcarrier system, the fluidized bed bioreactor and the solid bed bioreactor are suitable for scaling up. In contrast to the other systems, the solid bed bioreactor requires no special manipulation for anchoring the cells to the wire springs. In situ cleaning is possible and the beads are reusable. With this cell culture fermentation system, production processes for interferon beta, monoclonal antibodies for interferon alfa and recombinant human tissue plasminogen activator were developed.

Antibodies, Monoclonal↗
PubMed1988

Purification of proteins produced by biotechnological process.

Proteins maintain functions important to life. Faulty functioning or deficiency gives rise to pathological reactions. These proteins can now be produced, using the methods of recombinant DNA technology and administered to patients for replacement therapy. Many proteins as active ingredients are already available for use as immunomodulators, agents for tumour treatment, plasma proteins and hormones. They are in various stages of development, ranging from cloning of the producing cells to marketing of the finished products. Since the active substances are proteins synthesized by recombinant cells, their purification presents a particular challenge to protein chemists. Purification of recombinant DNA-derived proteins intended for human use is an essential part of the biotechnical process. It starts immediately after the fermentation of the host cell. The characteristics of the protein determine which microorganisms or cell cultures are used and this in turn defines the first purification step. The microorganisms are disrupted, and the insoluble protein, desposited in "inclusion bodies" has to be renatured, or the proteins secreted by cells and have to be concentrated. The subsequent strategy for purification of the protein does not depend on the fermentation process but is entirely determined by the physiochemical properties of the proteins. The goal of the first purification step is to isolate as fast and quantitatively as possible the recombinant protein from the culture filtrate, in order to minimize potential changes brought about by proteases or glycosidases. Immunoaffinity or ligand-affinity chromatography is used preferentially for this purpose. The concentration of protein and buffer changes are carried out by precipitation followed by reconstitution or, preferably, by dialysis and ultrafiltration/diafiltration.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗
PubMed1987

Biobusiness in the pharmaceutical industry.

Although conventional biotechnology used for the synthesis of antibiotics, vitamins, amino acids, nucleotides, enzyme inhibitors and immunomodulating compounds has still a major impact in the production of pharmaceutical compounds, the importance of the new biotechnology is increasing. Whereas in conventional biotechnology naturally occurring strains are screened for production of pharmacologically active compounds, in new biotechnology known organisms are programmed by genetic engineering to produce a distinct protein or glycoprotein of human origin for substitution therapy. Such complex compounds from new biotechnology can be divided into products which might replace compounds which are already on the market by safer recombinant products such as human insulin, human growth hormone, urokinase, factor VIII and products which are new on the market such as interferons, lymphokines, tissue plasminogen activator, oligonucleotide probes, monoclonal antibodies and subunit vaccines. However, only a few of these recombinant products have reached the market such as human insulin, interferon alpha, interferon beta, human growth hormone and recombivax HB. In most cases, depending on the difficulties in demonstrating clinical efficacy, the investigated drugs have reached the marketing phase much faster than conventional chemical drugs. Return on investment of biotechnical produced pharmaceutics mainly depends on the issues of whether the product has to compete with chemically synthesized drugs, whether it is totally new but competes with other bioproducts, whether it is exceptional but the proof of clinical efficacy is difficult, or whether it is totally new and clinical studies are promising.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotechnology↗
PubMed1986

Epidermin: sequencing of a heterodetic tetracyclic 21-peptide amide antibiotic.

Epidermin is a large peptide antibiotic, which is synthesized in the ribosome via a precursor protein, followed by enzymatic modifications. It was isolated from the culture filtrate of Staphylococcus epidermidis Tü 3298 by adsorption on Amberlite XAD-8. The basic heneicosapeptide amide was chromatographed on Sephadex LH-20 and purified to homogeneity via multiplicative counter-current distributions in one acidic and one neutral system. Tryptic digestion gave the soluble N-terminal fragment epidermin-(1-13)-peptide (P1) and the insoluble C-terminal fragment 2-oxobutyryl-epidermin-(15-21)-peptide amide (P2), each possessing two sulfide ring systems. The heterodetic rings consisted of meso-lanthionine and (2S,3S, 6R)-3-methyllanthionine (P1), meso-lanthionine and C-terminally the new amino acid S-(2-aminovinyl)-D-cysteine (P2). The complex sequence was elucidated via a combination of desulfurization with Raney nickel, enzymatic and acidolytic degradations, gas-phase sequencing, fast-atom bombardment and field-desorption mass spectrometry and NMR spectroscopy.

Amino Acid Sequence↗
PubMed1986

New biotechnology as a logical outgrowth of molecular biology.

Since the dawn of civilization biotechnical methods have been used to improve lifestyle by increasing the variety of food products and enriching their taste or preserving their quality. As these empirical methods advanced, biotechnology began more closely to resemble what it is today. The biosynthesis of vitamins, amino acids, alkaloids, steroids and antibiotics by specific microorganisms were the greatest medical achievements in the first half of the twentieth century. By this means, diseases which were often previously fatal could be prevented from befalling mankind. Despite this advantage in medical treatment the benefits from conventional biotechnology were achieved only through empirical, time-consuming and painstaking screening programs. The new biotechnology, while continuing to use many of the techniques of the conventional discipline, is now based on the understanding of product synthesis on a molecular level. This was derived from the benefit of three main perceptions of basic research which became available in this decade: Proteins, which promote the health of the entire human organism mainly through their regulatory functions, and consequently as a result of functional defects or their total absence cause illness or even death. The colinearity between nucleic acid sequence in the gene and amino acid sequence in the corresponding protein. The capabilities of genetic engineering to isolate a specific nucleic acid sequence of human origin which is responsible for the production of a distinct human protein and to transfer this into a microorganism or cell culture system for economic production of the desired protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-Bacterial Agents↗
PubMed1985

Metabolic products from microorganisms. 230. Amiclenomycin-peptides, new antimetabolites of biotin. Taxonomy, fermentation and biological properties.

Four new and two known peptide antibiotics containing amiclenomycin (Acm) have been isolated from a culture of Streptomyces venezuelae Tü 2460: L-MeIle-L-Acm (1), L-Ile-L-Acm (2), L-MeVal-L-Acm (3), L-MeIle-L-Acm-L-Gln (4), L-Ile-L-Acm-L-Gln (5) and L-Val-L-Acm-L-Gln (6). These di- and tripeptides exhibited antimicrobial activity on a minimal medium against Gram-negative bacteria, which could be reversed by biotin. It was shown that the antibiotics were decomposed by peptidases to provide amiclenomycin (7) after their uptake into cells of Escherichia coli via peptide-permeases. The antimicrobial activity of the amiclenomycin-peptides was the inhibition of DAPA-aminotransferase by the amiclenomycin-warhead, however, amiclenomycin itself was hardly transported into the cells. Since the amiclenomycin peptides misuse general transport systems, they are presented here as examples for the illicit transport concept.

Aminobutyrates↗
PubMed1985

[6R-[(R)-2-[3-[2-(p-Aminosulfonyl)anilino-4-hydroxy-5- pyrimidinyl]ureido]-2-(4-hydroxyphenyl)acetamido]-penicillanic acid, sodium salt (VX-VC 43), a new broad-spectrum beta-lactam antibiotic. In vitro and in vivo antibiotic activity].

6R-[(R)-2-[3-[2-(p-Aminosulfonyl) anilino-4-hydroxy-5-pyrimidinyl] ureido]-2-(4-hydroxyphenyl)acetamido]-penicillanic acid, sodium salt (VX-VC 43), a pyrimidinylureidopenicillin, is a broad-spectrum antibiotic. In vitro it is more active than piperacillin, apalcillin and mezlocillin especially against the following strains; E. coli, Ps. aeruginosa, Ps. stutzeri, K. pneumoniae, Eb. cloacae, Ser. marcescens, Proteus, Sh. flexneri, Y. enterocolitica, Cb. freundii, Acb. calcoaceticus, Bacteroides and Sc. faecalis. The minimal inhibitory concentration (MIC, micrograms/ml), for VX-VC 43 lies between 0.06 and 4 for piperacillin between 0.25 and 16, for apalcillin between 0.25 and 8, and for mezlocillin between 0.5 and 32. The action of VX-VC 43 is bactericidal. In tests against patient-isolates of E. coli (n = 50), Ps. aeruginosa (n = 50), K. pneumoniae (n = 46) and Eb. cloacae (n = 50) VX-VC 43 is clearly more active than Piperacillin. In the case of Ps. aeruginosa 2 micrograms/ml VX-VC 43 inhibits 43.9% of the bacteria and 8.5% with piperacillin; for E. coli 0.25 micrograms/ml inhibits 43.9% with VX-VC 43 and 0% with piperacillin; for K. pneumoniae 4 micrograms/ml inhibits 43.7% with VX-VC 43 and 28% for piperacillin; with Eb. cloacae 2 micrograms/ml inhibits 78% with VX-VC 43 and 4% with piperacillin. Addition of 50% human serum, or a change in culture medium pH (from 6 to 8) has little or no effect on the MIC of VX-VC 43 or piperacillin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Beta-lactamases from Bacteroides fragilis active against oximino-cephalosporins.

Four clinical isolates of Bacteroides fragilis were resistant to penicillins and cephalosporins with exception to 7-alpha-methoxy cephalosporins such as latamoxef (moxalactam) and cefoxitin. In contrast the 6-alpha-methoxy penicillin temocillin was not active. The beta-lactam resistance correlates with the presence of two beta-lactamases with an isoelectric point (IEP) = 4.9 and 5.4, respectively. The necessity of antimicrobial susceptibility testing before the therapy of infections with anaerobic bacteria is discussed.

Bacteroides fragilis↗
PubMed1983

Synthesis and biological activities of the tri-L-alanine derivative of isonicotinic acid hydrazide.

N-(tert-Butyloxycarbonyl) tri-L-alanine was coupled to the hydrazide function of isonicotinic acid hydrazide followed by cleavage of the amino protective group. The resulting dihydrochloride of the tri-L-alanine derivative of isonicotinic acid hydrazide was characterized by 13C-NMR. The minimal inhibitory concentration of isonicotinic acid hydrazide was not improved by the peptide derivative, and competition experiments with tri-L-alanine demonstrated that tri-L-alanyl-isonicotinic acid hydrazide did not use the peptide transport system. Isonicotinic acid hydrazide and its tri-peptide derivative possessed the same activity against pathogenic mycobacteria and did not antagonize each other. The relatively high stability of the tri-peptide derivative against peptidases of Mycobacterium fortuitum was discussed as being responsible for the significantly weaker activity against this atypical mycobacterium strain. With the exception of peptidases of hog intestinal mucose, the tri-L-alanyl derivative of isonicotinic acid hydrazide was stable to pronase and alpha-chymotrypsin when compared to other peptides.

Anti-Bacterial Agents↗
PubMed1982

Derivatives of 4-dihydro-4-deoxy-4(R)-amino spectinomycin and their activity against susceptible and resistant Escherichia coli strains.

A number of alkyl and acyl derivatives of 4-dihydro-4-deoxy-4(R)-amino spectinomycin were tested against various Escherichia coli strains, possessing different susceptibilities to spectinomycin. The influence of the lipophilicity and the length of the side chain substituents of the derivatives was compared to both minimal inhibitory concentration values and stability to adenyltransferase. Derivatives with a chain length of more than 10 carbon atoms demonstrated a significantly higher activity against all investigated strains, whether susceptible or resistant. The same inhibitory effect was achieved with short-chain aminoacyl derivatives only against susceptible strains. Other short-chain derivatives possessed no advantage to spectinomycin. A 10-fold decrease in the affinity for adenyltransferase was achieved in compounds with a high lipophilicity (log P), i.e., in aliphatic substituted derivatives with a log P greater than 4 and in benzoyl-substituted derivatives with a log P greater than 2. Derivatives with branched alkyl chains and long side chains displayed a different mode of action than spectinomycin. They possessed strong activity against strains with an altered ribosomal binding site and a decreased influence of pH on antimicrobial activity.

Escherichia coli↗
PubMed1981

Directed biosynthesis of new indolmycins.

Tryptophan in a concentration of 0.4 microgram/ml increased the production of indolmycin by 37%. The lipophilic character of indolmycin was reduced via directed biosynthesis by substituting the aromatic ring system with a methoxy or hydroxy group in the 5-position of the antibiotic. This substitution was achieved by the addition of the corresponding tryptophan and indole precursors to a growing culture of Streptomyces griseus ATCC 12648. The more hydrophilic indolmycin derivatives displayed a moderate increase in antimicrobial activity as compared to indolmycin, but did not markedly change the Gram-positive/Gram-negative ratio of activity. Thin-layer chromatography and mass spectrometry showed that additives substituted in the 6-position were not incorporated into the molecule. Antibiotic titer was reduced by addition of the modified precursors, especially in the case of the precursors substituted in the 6-position.

Anti-Bacterial Agents↗
PubMed1981

Inhibition of the synthesis of the TEM-type beta-lactamase by purine derivatives.

The beta-lactamase TEM-1 was purified from an Escherichia coli ATCC 11 775 R+TEM culture by preparative isoelectric focussing. This preparation was used to establish a calibration curve between the concentration of the enzyme and the cleavage of nitrocefin, by which the quantitative production of beta-lactamase TEM-1 in the growing culture was determined. The effect of purine derivatives on the synthesis of the beta-lactamase during the growth of the bacterium was tested in order to determine the essential basic structure of the molecule. It is suggested that the keto-methylamino part of the pyrimidine nucleus is decisive for the potency of the structure. Inhibition studies on the purified enzyme demonstrated that the purine compounds act by inhibiting the synthesis of the beta-lactamase and not by inactivation of the enzyme. This mode of action manifests itself as a synergism with amoxycillin against the beta-lactamase producing strain.

Amoxicillin↗