Commercialization of Recombinant Human Epidermal Growth Factor – A Nobel Prize Winning Molecule with Diverse Therapeutic Applications VADREVU KM (*) and SRINIVAS VK,
Bharat Biotech International, Hyderabad, India
* Author for correspondence
Background: Growth Factors are a distinct class of signaling proteins that modulate wound healing at a molecular and cellular level. One such Growth Factor is the Epidermal Growth Factor (EGF), a 6.2 kDa protein, whose discovery by Prof Stanley Cohen got him the Nobel Prize along with Dr Rita Levi-Montalcini in 1986. The role of EGF has been extensively investigated in normal and pathological wound healing and EGF based formulations are recently gaining therapeutic importance in wound management. Such Formulations, for obvious reasons, are developed using recombinant human Epidermal Growth Factor (rhEGF). This Presentation reviews the various approaches adopted to produce rhEGF on a large scale including the work carried out by the Authors’ Group. We have been successful in producing rhEGF at a commercial level through a cost-effective, novel process yielding >99% re-natured active protein and high specific activity. The Presentation also discusses the excellent clinical results obtained by the Group with rhEGF based formulations for various therapeutic applications along with attempts to understand the mechanism of EGF at a bio-molecular level in wound healing. In the Authors’ view, such a wide range of clinical studies are the first comprehensive studies in literature. Finally, the Presentation also describes the recent focus of research in new drug delivery systems that are able to protect and stabilize EGF, which is readily degraded in any chronic wound environment. It is predicted that such work would lead to future therapeutic options with Growth Factors in a diverse array of applications.
Methods:Manufacture of rhEGF & Formulations thereof: Several studies have been reported earlier on cloning in E.coli and yeast system for the expression of recombinant human EGF (rhEGF) and purification by RP-HPLC and Solid Phase Extraction or Expanded Bed Adsorption (EBA) Chromatography. A serious limitation of most of these studies is the final yield (<100 mg/L) and the purity obtained (<85%), thus limiting the commercial potential of such work. In our objective towards commercialization of rhEGF, we have expressed rhEGF as a tripartite tag protein consisting of N terminal TrpE sequence and C terminal with six arginine residues attached to the human EGF sequence. This fused gene was cloned in pET11b vector under the control of the T7 promoter and the protein was expressed in E.coli BL21(DE3)RIL strain. Fermentation conditions were optimized to express the protein as an inclusion body which was estimated around 500mg/L, and the yield obtained after downstream purification was about 400mg/L. Some of the novel aspects of our work include reduced fermentation time, lesser number of downstream purification steps and better control on refolding using EBA chromatography. The 6.2kDa protein was characterized for physico-chemical properties by RP- HPLC, Size exclusion chromatography, MALDI-TOF, Circular Dichroism, N-terminal sequencing and the biologically functionality was assessed by ELISA using functional monoclonal antibodies and 3T3 Cell lines. rhEGF Based Therapeutic Formulations: Extensive Formulation development work (both Cream and Gel based) has been carried out to arrive at the most suitable and stable Formulations at 3 different rhEGF concentrations for various applications. Safety of such Formulations has been evaluated by in-depth toxicological studies.
Clinical trials for various therapeutic indications: Three most widely investigated applications include Diabetic Foot Ulcers, Skin Grafts and Burns and these have given extremely encouraging results. Other indications for which studies have been completed include Venous Leg Ulcers, Corneal Ulcers and GI Tract ulcers. This Presentation would highlight the wound healing duration and wound size reduction results obtained with our licensed formulation REGEN D, currently under usage in India and several other countries.
Results: rhEGF has been manufactured at 100 L fermentation scale and the purified rhEGF showed a specific activity of 5 x 105 IU/mg protein, in comparison with NIBSC standard (Ist International Standard of rDNA-derived EGF, Code 91/530).In all clinical trials, REGEN D was found to result in healthy granulation and stimulate epithelization. Collagen levels increased significantly in REGEN D treated groups while, as is desirable, MMP-9 (Matrix Metallo Proteinases) expression got reduced. Similar encouraging results were obtained in other studies aimed at exploring the diverse range of treatment modalities available with REGEN D.
Conclusions: This Presentation describes the successful large scale manufacture of rhEGF and formulations based on rhEGF have shown great promise for enhanced wound healing in diverse cases involving diabetic foot ulcers, 1st and 2nd degree burns, bed sores, venous leg ulcers etc. This has allowed a new perspective to wound healing research involving rhEGF. Using nano-medicine drug delivery technologies, new vistas could be opened targeting enhanced stability of EGF. It is our endeavour to put EGF on the global Biotechnology map and we sincerely hope that EGF would become a potential “Magic Bullet” candidate.
Prevention of hepatotoxicity in patients undergoing anti tuberculosis treatment: A novel integrative approach VAKHARIA BC1, ADHVARYU MR2, REDDY MN2 1Bhuma Research in Ayurvedic and Herbal Medicine, Surat, India; 2 Veer Narmad South
Gujarat University, Surat, India.
Introduction: Conventional anti-tuberculous treatment (ATT) containing Isoniazid, Rifampicin and Pyrazinamide, is hepatotoxic: the incidence varying between 4-11%. Stopping ATT and restarting it once the enzymes are normalized are only measures practiced leading to drop-outs, incomplete treatment and recurrence in addition to occasional severe liver failure requiring liver transplant. Ayurvedic herbs like Curcuma longa and Tinospora cordifolia have shown hepatoprotective and immunostimulatory properties in rodent model of ATT induced hepatotoxicity, so a prospective two armed RCT was carried out to evaluate their potential in humans.
Methods: Patients with active tuberculosis diagnosis were randomized to a drug control group (n=192) and a trial group (n=316) on drugs plus an herbal formulation. Isoniazid, Rifampicin, Pyrazinamide and Ethambutol for first two months followed by continuation phase therapy excluding Pyrazinamide for 4 months comprised the anti-tuberculous treatment. Curcumin enriched (25%) CL and a hydro-ethanolic extract enriched (50%) TC 1 g each divided in two doses comprised the herbal adjuvant. Hemogram, bilirubin and liver enzymes were tested initially and monthly till the end of study to evaluate the result. The results were analyzed by Chi square test. (P ≤ 0.05)
Results: Incidence and severity of hepatotoxicity was significantly lower in trial group (incidence: 27/192 vs 2/316, P 0.0001). Mean Aspartate transaminase (AST) (195.93 ± 108.74 vs 85 ± 4.24, P ≤ 0.0001), Alanine Transaminase (ALT) (75.74 ± 26.54 vs 41 ± 1.41, P ≤ 0.0001) and Serum Bilirubin (5.4 ± 3.38 vs 1.5 ± 0.42, P ≤ 0.0001). A lesser sputum positivity ratio at the end of 4 wk (10/67 vs 4/137, P = 0.0068) and decreased incidence of poorly resolved parenchymal lesion at the end of the treatment (9/152 vs 2/278, P≤0.0037) was observed. Improved patient compliance was indicated by nil drop-out in trial vs 10/192 in control group (P≤ 0.0001)
Conclusion: 1.The adjuvant herbs showed strong hepatoprotective activity 2. Improved outcome with higher and quicker sputum negativity may be due to improved patient compliance in addition to immunostimulatory action. 3 The result carries utmost significance for mal-nourished, alcoholic and immuno-compromised patients.
New Drug Development Outside of G8: Ready, Shoot, Aim VALDIVIA-ALCOTA R1, KATO M1 1Universidad de Chile, Genotoxic Risk Assessment Center, CERIG-Facultad de Odontología, Santiago, Chile
Background: Clinical assessment in late-stage drug development should be guided by results of comprehensive preclinical safety assessments and rigorous clinical pharmacology programs. In South America (SA), new drug development is generally conducted at public universities with the support of local pharmaceutical companies willing to participate in a government-supported research project. However, preclinical studies are seldom conducted in SA universities, thereby frustrating talented investigators and industry when promising discoveries receive the envitable rejection from regulatory agencies within the developed world.
Observations: Based on experience gained over 10 years at the U-Chile where we established the first good laboratory practices (GLP) laboratory to perform preclinical studies in bacterial or animal systems in Chile. We conduct the Mammalian Erythrocyte Micronucleus test and Bacterial Reverse Mutation Assay for each substance, strictly adhering to FDA and OECD guidelines. Bacterial strains, the S9 microsomal fraction, cofactors, and other chemicals are certified and imported from the country of origin (USA or Japan). The mouse strain was originally certified and imported from CLEA-Japan and maintained in specific pathogen free (SPF) mouse facilities in our research center.
Less than 10 companies requested mutagen assays under the local regulatory authorities, and only a few persevered. The results of the assays in two products which had excellent clinical results in humans, however, were positive to the micronucleus and the Ames test.
Conclusions: 1) We recommend mandating preclinical studies in animals and bacterias according to GLP guidelines for drug approval in SA. 2) Educate local pharmaceutical companies to the impact of the mutation assay results for chemicals or drugs that are currently going into the approval process.
Authors’ disclosure statement: Authors are on full-time faculty at the Universidad de Chile-Santiago. Implementation of the Genotoxic Risk Assessment Center (CERIG) was financed by a grant for development of sciences and technology FONDEF-Conicyt-Chile. The mouse facilties were implemented, in part, with a grant for the Ministery of Education of Chile, MECESUP to the Dental School, and by the Japan International Cooperation Agency (JICA). Drs. ValdivIa and Kato are principals in the Japan Food Safety Center based in Santiago, Chile
Lactoferrin Acts against Infection and Inflammation through its Influence on Systemic Iron Homeostasis VALENTI P1, PIETROPAOLI M2, PAESANO R1 1University of Rome, SAPIENZA, Rome, Italy; 2Biotechnology Company, MICROBO srl, Rome, Italy
Background: Inflammations and infections are often associated to hypoferremia. Hypoferremia is an important signal of disorders of hepcidin levels which regulate the entry of iron into plasma through ferroportin (FPN), the only known cellular iron exporter. The resulting iron-overload in secretions and cells increases host susceptibility to infections. Therefore, the regulation of systemic iron homeostasis is critical to human health. We have demonstrated that lactoferrin (Lf) can modulate systemic iron homeostasis through the decrease of serum IL-6, key molecule in hepcidin and FPN synthesis. Aims: 1) To compare the effects of ferrous sulphate and lactoferrin (Lf), orally administered, in hypoferremia 2) To verify the relationship among the increase of total serum iron concentration and the decrease of host inflammation and susceptibility to infections.
Methods: The clinical trial on the therapeutic effect of Lf on systemic iron homeostasis included 171 subjects, suffering of hypoferremia. Subjects were randomly divided in two groups. The first group received an oral administration of 520 mg of ferrous sulphate, once a day (156 mg as elemental iron); and the second group received orally 100 mg of Lf, twice a day (8.8 mg as ferric iron). Haematological values and IL-6 concentration were assessed on venous blood.
Results: Ferrous sulphate administration often failed to exert significant effects on hypoferremia. Conversely, Lf highly increased the values of total serum iron and serum ferritin already after 30 days of therapy (mean values from 45 to 95 µg/dL and from 12 to 27 ng/ml, respectively). Moreover, Lf administration modulated systemic iron homeostasis by decreasing serum IL-6 concentration (mean values from 32 to 10 pg/ml), while ferrous sulphate exhibited an opposite effect.
Conclusions: 1) Lf exerted a potent effect in restoring the iron transport from cells into circulation through the decrease of serum IL-6 concentration which in turn modulates hepcidin and FPN synthesis. 2) In contrast to the administration of ferrous sulphate, Lf oral administration did not result in any side effect. 3) The capacity of Lf to decrease IL-6 leading to the rescue of haematological parameters could represent a novel therapeutic alternative in reduction of host inflammation and susceptibility to infections.
Molecular Characterization of Antibiotic Resistance in Selected Enteropathogens Isolated from Raw Food Samples in Vietnam VAN1 TTH, MOUTAFIS1G, ISTIVAN1T, TRAN2 LT AND COLOE1 PJ
1RMIT University, Melbourne, VIC, Australia; 2University of Natural Sciences, HCM City, Vietnam
Background: The emergence of antibiotic resistance in bacteria has become a serious problem worldwide. This study aims to determine the molecular characteristics of antibiotic resistance in enteropathogens isolated from raw food samples in Vietnam.
Methods: Raw food samples (n=180, comprising meat, poultry and shellfish) were collected from Ho Chi Minh City, Vietnam for the isolation of Salmonella spp., Escherichia coli and Vibrio parahaemolyticus. The isolates were tested for antibiotic resistance against 15 commonly used antibiotics by the disk diffusion method. They were further examined for the presence of mobile genetic elements conferring antibiotic resistance. Transfer of antibiotic resistance phenotypes was studied by conjugation. Salmonella genomic island 1 (SGI1) antibiotic resistance gene cluster was investigated using PCRs, Southern blot analysis and sequencing.
Results: E. coli and Salmonella spp. was isolated in 60.8% and 48.9% of the samples respectively. V. parahaemolyticus was present in 32% of shellfish samples. There were high antibiotic resistance frequencies in E. coli and Salmonella spp. isolates, especially to tetracycline, ampicillin, nalidixic acid, streptomycin, and sulphafurazole. E. coli also showed high resistance to trimethoprim (43.4%) and chloramphenicol (51.5%). Multi-resistance, i.e. resistance to at least three different classes of antibiotics, was detected in 61.6% E. coli and 20.9% Salmonella isolates, including potential human-pathogenic Salmonella serovars. V. parahaemolyticus isolates were uniformly resistant to ampicillin. Integrons harbouring genes responsible for resistance to aminoglycosides, ampicillin, trimethoprim and chloramphenicol were found in 57% E. coli and 13% Salmonella spp. isolate. Plasmids were detected in all tested isolates, many of them were larger than 95 kb. Antibiotic resistance phenotypes were found to be transferable among the isolates. SGI1 was identified in Salmonella serovar Albany isolated from chicken meat.
Conclusions: The results indicate that raw foods of animal origin in Vietnam are potential reservoirs for multi-resistant pathogenic organisms which contain a pool of mobile genetic elements, raising the awareness of antibiotic resistance in food pathogens.
One target, two bullets : from erythromycin to telithromycin, what makes the difference ? VAN BAMBEKE F Pharmacologie cellulaire et moléculaire, Univ. catholique de Louvain, Brussels, Belgium
Background: the increasing threat of bacterial resistance and the progress of modern pharmacology stimulate the search for antibiotics capable of bypassing existing resistance mechanisms and presenting an optimized pharmacological profile.
Methods: overview of the rational design of telithromycin (TEL), as first clinically-used ketolide and semi-synthetic derivative of the macrolide erythromycin (ERY), in relation with its novel pharmacological profile.
Results:
SAR: TEL differs from ERY by (a) removal of the cladinose at position 3 and its replacement by a keto-group, (b) incorporation of an 11,12- cyclic moiety, and (c) addition of a heteroaryl-alkyl side chain. TEL can therefore interact with 2 distinct binding sites in the 23S rRNA of the ribosome. This increases its activity against ERY-S strains and maintains activity towards ERY-R S. pneumoniae with ribosomal methylation. TEL is also a poor inducer of methylase expression and less susceptible to efflux in S. pneumoniae.
PK: TEL shows an improved PK profile, with high oral bioavailability (improved acid stability), penetration in tissues and fluids, accumulation within eucaryotic cells and poor recognition by eucaryotic efflux transporters (P-glycoprotein), prolonged t1/2 allowing for daily administration.
PD PK/PD considerations suggest optimal efficacy for isolates with MICs 0.25 mg/L. As ERY, however, TEL is bacteriostatic in vitro and poorly active in models of intracellular infections despite its high cellular accumulation.
Safety: TEL causes less gastro-intestinal side effects and is a less potent inhibitor of CYP3A4 than ERY. It causes however rare but serious side effects (hepatotoxicity, respiratory failure in patients with myasthenia gravis, visual disturbance, risk of QTc prolongation).
Conclusions: this story tells us how knowledge of targets helps in obtaining more powerful bullets. Further optimization of PD and safety is still needed to get "the" magic bullet acting on bacterial ribosomes.
Passive Immunization for the Protection of our Global Society against Emerging Infections VAN DER ZEIJST B1 1Netherlands Vaccine Institute, The Netherlands
Background: New viral diseases, like the Severe Acute Respiratory Syndrome (SARS), are an enormous threat to our society. With more than 50% of the global population living in cities and 2 billion airplane passengers per year, there is no way to escape from contact with infected person once a pandemic starts. The World Health Organisation (WHO) devoted the World Health Report 2007 to this topic. WHO concludes that there will be another Ebola or SARS sooner or later. Vaccination would be the preferred way to protect the population, but the development of a new vaccine takes about 10 years. Therefore serum therapy is a serious candidate for the therapy and post exposure prophylaxis of new viral.
diseases
Analysis: There are several powerfull approaches to generate and produce humanized or human monoclonal antibodies. But production in mammalian cell lines in bioreactors is still an expensive process and at this moment there is insufficient production capacity to produce the required quantity of antibodies.
Solutions: Innovation in production technology is necessary. One solution could be to use plants for the production of the monoclonal antibodies. Another way to solve the problem would be to make more potent neutralizing antibodies than can be administered at lower concentrations. In view of the importance of protection of the global population and the lack of alternatives, a task force for the necessary innovation is needed.
Pharmacogenetic screening and drug susceptibility and fetal malformations, past, present, and future VAN DYKE DC, Skhal KJ
Department of Pediatrics, University of Iowa Children’s Hospital, Carver College o f Medicine , Department of Epidemiology, College of Public Health, University of Iowa, Iowa City, Iowa, USA
Background: An understand of drug susceptibility and its relationship to possible birth abnormalities will become increasingly important in areas of clinical management for physicians of the future, particularly those in OBGYN, Pediatrics, Environmental Medicine, and Toxicology.
Aims: 1) Review past published journal articles dealing with drug susceptibility
2) Review recent advances in pharmacogenetics and analyze them for potential significance for future medical practice.
Methods:
Reviewed our past journal publications on fetal phenytoin and other fetal drug exposures
with emphasis on folate metabolism, clefting defects, CHD, and neural tube abnormalities
Reviewed our past published literature reviews on fetal anticonvulsant exposure
Completed literature search of the last 10 years (1997 to 2007) on new research in drug susceptibility to fetal drug exposure and birth defects.
Results:
Identified significant past reviews of fetal drug exposure and susceptiblitly to birth abnormalities
Identified 68 articles in the literature in the last 10 years that dealt with newborn screening, fetal abnormalities, and interventions that may lead to prevention of or decrease in some birth abnormalities.
Conclusions: New molecular genetics techniques such as identification of SNPs, candidate genes, advances in computer hardware, software, and statistical techniques have aided our understanding of genotype, drug susceptibility, and variability in clinical phenotype. Antenatal screening for metabolic abnormalities, hemoglobinopathies, hearing, and a few genetic disorders has become routine in some states in the USA and other countries. Antenatal screening has four main objectives:
Education of parents with expanded opportunities for parental choice
Allow for potential fetal treatment or for immediate postnatal treatment
Allow for informed decisions regarding present and future pregnancies
Expand the potential for improved outcomes.
However, there are multiple variables involved; many are probably still unrecognized. Fetal drug/maternal drugs screening for clinical management remains on the horizon of the future
Differences in Plasma Levels of Risperidone: Causes and Consequences VAN OS SHG, MUÑIZ PINIELLA P
Synthon BV, Nijmegen, The Netherlands
Background: Risperidone is an atypical antipsychotic, available in various formulations. It is rapidly absorbed after oral administration and undergoes extensive metabolism. It is generally assumed that rapidly dissolving oral formulations are equivalent to oral solutions and a previous publication shows bioequivalence between an oral risperidone solution and risperidone tablets. When administration of two immediate release oral formulations results in equivalent plasma concentration profiles, essentially similar concentrations at the site of action and therefore similar efficacy and safety can be assumed.
Methods: The bioavailability of the generic risperidone solution containing sorbitol and the 1 mg immediate release originator tablet formulation was compared in 32 healthy volunteers using a standard crossover design. Both formulations contained 1 mg risperidone per dosing unit. The solution was administered using a standard dosing device to measure 1 ml that was further diluted in a glass of water.
Results: Plasma levels for the 9-hydroxy metabolite of risperidone were within bioequivalence limits. However, bioequivalence could not be claimed for the parent compound risperidone.
There are several potential explanations for the observed differences in plasma levels of risperidone. Risperidone pharmacokinetics, assay result, dosing device, excipients, design of bioequivalence study and life habits can all contribute to differences in plasma concentrations. The potential impact of these variables on daily practice will be discussed.
Conclusions: Bioequivalence between the studied 1 mg/ml generic risperidone solution and the 1 mg immediate release originator tablet was not proven in this study.
Differences in plasma levels can result in lack of therapeutic efficacy, which can have dramatic consequences in schizophrenic patients.
Antibody polyreactivity and ill-defined antigen mimicry hamper the search for effective peptide-based vaccine immunogens
VAN REGENMORTEL MHV
Biotechnology School of the University of Strasbourg, CNRS, France
The regions of antigens that are recognized by antibodies are called epitopes while the regions of antibodies that recognize epitopes are called paratopes. Most epitopes of proteins are called discontinuous epitopes because they arise from between two and five separate segments of the polypeptide chain,that are brought together by the folding of the chain. These epitopes exist only because the chain acts as a scaffold and if the scaffold is perturbed, the epitope ceases to exist. It is not possible to isolate such epitopes independently of the rest of the protein in which they are embedded in order to show that they possess binding activity on their own. It has also not been possible so far to predict them or to reconstruct them by synthesis since this would require assembling atoms which in the protein are not held together by internal chemical bonds.
Most residues at the surface of a native protein contribute atoms to a large number of overlapping epitopes recognized by different antibodies. No clear boundaries exist between neighbouring epitopes which together form a series of antigenic sites and it is only because Mabs are used as analytical tools that protein antigenicity appears to be located in discrete epitope regions rather than in an antigenic continuum. Unfortunately the strategy of focusing on individual epitopes has been detrimental to the search for effective vaccine immunogens.
Proteins are also said to possess continuous epitopes, defined as linear protein fragments of 5 - 15 residues that are able to bind to antiprotein antibodies. Many of the residues of such continuous epitopes are not present at the surface of the native protein and these epitopes are thus poor structural mimics of the antigenic regions of native proteins. It is only because antiprotein antibodies are polyspecific and able to cross-react with short peptides corresponding to separate segments of complex discontinuous epitopes, that large numbers of continuous epitopes have been described.
It is often mistakenly assumed that it is justified to extrapolate from cross-reactive antigenicity (binding of peptides to antiprotein antibodies) to cross-reactive immunogenicity (raising antipeptide antibodies able to cross-react with the cognate protein). However, the structure of an epitope determined when it is complexed with a paratope tends to differ from the structure present before the process of mutual adaptation that occurs when the two binding partners interact. The structure of an epitope after complexation with a neutralizing Mab is therefore an unreliable guide for defining which vaccine immunogens are needed to elicit protective neutralizing antibodies.
