Application of Cell-Free Protein Synthesis System for the Biosynthesis of l-Theanine.

l-Theanine, as an active component of the leaves of the tea plant, possesses many health benefits and broad applications. Chemical synthesis of l-theanine is possible; however, this method generates chiral compounds and needs further isolation of the pure l-isoform. Heterologous biosynthesis is an alternative strategy, but one main limitation is the toxicity of the substrate ethylamine on microbial host cells. In this study, we introduced a cell-free protein synthesis (CFPS) system for l-theanine production. The CFPS expressed l-theanine synthetase 2 from Camellia sinensis (CsTS2) could produce l-theanine at a concentration of 11.31 µM after 32 h of the synthesis reaction. In addition, three isozymes from microorganisms were expressed in CFPS for l-theanine biosynthesis. The γ-glutamylcysteine synthetase from Escherichia coli could produce l-theanine at the highest concentration of 302.96 µM after 24 h of reaction. Furthermore, CFPS was used to validate a hypothetical two-step l-theanine biosynthetic pathway consisting of the l-alanine decarboxylase from C. sinensis (CsAD) and multiple l-theanine synthases. Among them, the combination of CsAD and the l-glutamine synthetase from Pseudomonas taetrolens (PtGS) could synthesize l-theanine at the highest concentration of 13.42 µM. Then, we constructed an engineered E. coli strain overexpressed CsAD and PtGS to further confirm the l-theanine biosynthesis ability in living cells. This engineered E. coli strain could convert l-alanine and l-glutamate in the medium to l-theanine at a concentration of 3.82 mM after 72 h of fermentation. Taken together, these results demonstrated that the CFPS system can be used to produce the l-theanine through the two-step l-theanine biosynthesis pathway, indicating the potential application of CFPS for the biosynthesis of other active compounds.

Co-expression of lipase isozymes for enhanced expression in Pichia pastoris.

To enhance the overall expression level of lipase isozymes which catalyse the same reaction in Pichia pastoris through co-expression of isozymes from different sources; several types of co-expression ways were constructed to determine the co-expression efficiencies of lipase isozymes in P. pastoris. The results showed that the Kex2-mediated co-expression of lipase isozymes could express Rhizomucor miehei lipase (RML) and Thermomyces lanuginosus lipase (TLL) simultaneously, and GS-RMk-kTL displayed an average lipase activity of 306·91 U ml-1 , higher than GS-RML and GS-kTL (2·89 and 300·59 U ml-1 ) expressed independently in P. pastoris, and the sum of both (303·48 U ml-1 ), implying the potential of isozyme co-expression mediated by Kex2 in increasing the overall recombinant expression, but the low recombinant expression of RML in P. pastoris weakened the overall increasing effect on lipase expression in the isozyme co-expression strains. In addition, the fusion isozymes were successfully expressed, but with low lipase activities. Furthermore, 2A peptide could successfully mediate the co-expression and secretion of lipase isozymes, but it seriously affected the expression of TLL downstream of 2A peptide. SIGNIFICANCE AND IMPACT OF THE STUDY: The low production level is one of the limitation factors for decreasing the prices of enzymes and expanding their application in industry as the biocatalysts. This research focuses on developing lipase isozyme co-expression strategies in Pichia pastoris to enhance the expression level of overall lipase isozymes which catalyse the same reaction. The Kex2-mediated co-expression strategy of lipase isozymes could potentially enhance the overall isozyme expression, and isozyme co-expression might provide a new direction for improving the recombinant isozyme expression, and decreasing the production and application prices of these mixed enzymes as biocatalysts.

Techno-economic analysis of horseradish peroxidase production using a transient expression system in Nicotiana benthamiana.

Despite the advantages of plant-based transient expression systems relative to microbial or mammalian cell systems, the commercial production of recombinant proteins using plants has not yet been achieved to any significant extent. One of the challenges has been the lack of published data on the costs of manufacture for products other than biopharmaceuticals. In this study, we report on the techno-economic analysis of the production of a standard commercial enzyme, namely, horseradish peroxidase (HRP), using a transient expression system in Nicotiana benthamiana. Based on the proven plant yield of 240 mg HRP/kg biomass, a biomass productivity of 15-kg biomass/m(2)/year and a process yield of 54 % (mg HRP product/mg HRP in biomass), it is apparent that HRP can be manufactured economically via transient expression in plants in a large-scale facility (>5 kg HRP/year). At this level, the process is competitive versus the existing technology (extraction of the enzyme from horseradish), and the product is of comparable or improved activity, containing only the preferred isoenzyme C. Production scale, protein yield and biomass productivity are found to be the most important determinants of overall viability.

The effectiveness and cost-effectiveness of enzyme and substrate replacement therapies: a longitudinal cohort study of people with lysosomal storage disorders.

OBJECTIVES: To determine natural history and estimate effectiveness and cost of enzyme replacement therapy (ERT) and substrate replacement therapy (SRT) for patients with Gaucher disease, Fabry disease, mucopolysaccharidosis type I (MPS I), mucopolysaccharidosis type II (MPS II), Pompe disease and Niemann-Pick type C (NPC) disease. DESIGN: Cohort study including prospective and retrospective clinical- and patient-reported data. Age- and gender-adjusted treatment effects were estimated using generalised linear mixed models. Treated patients contributed data before and during treatment. Untreated patients contributed natural history data. SETTING: National Specialised Commissioning Group-designated lysosomal storage disorder (LSD) treatment centres in England. PARTICIPANTS: Consenting adults and children with a diagnosis of Gaucher disease (n = 272), Fabry disease (n = 499), MPS I (n = 126), MPS II (n = 58), NPC (n = 58) or Pompe disease (n = 93) who had attended a treatment centre in England. INTERVENTIONS: ERT and SRT. MAIN OUTCOME MEASURES: Clinical outcomes chosen by clinicians to reflect disease progression for each disorder; patient-reported quality-of-life (QoL) data; cost of treatment and patient-reported service-use data; numbers of hospitalisations, outpatient and general practitioner appointments; medication use; data pertaining to associated family/carer costs and QoL impacts. RESULTS: Seven hundred and eleven adults and children were recruited. In those with Gaucher disease (n = 175) ERT was associated with improved platelet count, haemoglobin, liver function and reduced risk of enlarged liver or spleen. No association was found between ERT and QoL. In patients with Fabry disease (n = 311) increased time on ERT was associated with small decreases in left ventricular mass and improved glomerular filtration rate, but not with changes in risk of stroke/transient ischaemic attacks or the need for a hearing aid. There was a statistically significant association between duration of ERT use and worsening QoL and fatigue scores. We found no statistical difference in estimates of treatment effectiveness between the two preparations, agalsidase beta (Fabrazyme(®), Genzyme) (n = 127) and agalsidase alpha (Replagal(®), Shire HGT) (n = 91), licensed for this condition. In Pompe disease (n = 77) our data provide some evidence of a beneficial effect on muscle strength and mobility as measured by a 6-minute walk test in adult-onset patients; there were insufficient data from infantile-onset Pompe patients to estimate associations between ERT and outcome. Among subjects with MPS I (n = 68), 42 of the 43 patients with MPS I subtype Hurler's disease had undergone a bone marrow transplant. No significant associations were found between ERT and any outcome measure for the MPS I subtype Scheie disease and heparan sulphate patients. An association between duration of ERT and growth in children was the only statistically significant finding among patients with MPS II (n = 39). There were insufficient data for patients with NPC disease to draw any conclusions regarding the effectiveness of SRT. The current annual cost to the NHS of the different ERTs means that between 3.6 and 17.9 discounted quality-adjusted life-years (QALYs) for adult patients and between 2.6 and 10.5 discounted QALYs for child patients would need to be generated for each year of being on treatment for ERTs to be considered cost-effective by conventional criteria. CONCLUSIONS: These data provide further evidence on the effectiveness of ERT in people with LSDs. However, the results need to be interpreted in light of the fact that the data are observational and the relative lack of power due to the small numbers of patients with MPS I, MPS II, Pompe disease and NPC disease. Future work should aim to effectively address the unanswered questions and this will require agreement on a common set of outcome measures and their consistent collection across all treatment centres. FUNDING: This project was funded by the NIHR Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 16, No. 39. See the HTA programme website for further project information.

Stakeholder involvement in expensive drug recommendation decisions: an international perspective.

OBJECTIVES: To describe stakeholder involvement in the priority setting and appeals processes across five drug reimbursement recommendation committees. METHODS: We conducted qualitative case studies of how five independent drug advisory committees from Canada, Israel, England and Wales, Australia, and the USA made funding decisions for six expensive drugs. Interviews with 48 informants were conducted with committee members, patient groups, and industry representatives. RESULTS: Different stakeholders were allowed, in varying degrees, to participate in the formal mechanisms for revisions and appeals of decisions. Participants identified a number of stakeholder groups who were already involved in the process, as well as stakeholders whom they believed should be included in the decision-making process. CONCLUSIONS: A central component of a legitimate and fair priority setting process is to make priority setting explicit and to involve both pertinent values and stakeholders in decision-making. Study participants believed that the involvement of multiple stakeholder groups within the deliberative and appeals/revisions processes would contribute to a fair and legitimate drug reimbursement process.

Modelling the resource implications of managing adults with Fabry disease in Italy.

AIMS: This study estimated the resource implications and budget impact of managing adults with Fabry disease in Italy, from the perspective of the Servizio Sanitario Nazionale (SSN). METHODS: A decision model was constructed using published clinical outcomes and clinician-derived resource utilisation estimates depicting the management of adults with Fabry disease in Italy. RESULTS: The expected annual cost of managing 220 existing and 20 new Fabry patients in Italy was estimated to be €28·3 million. In an average year, patients receiving enzyme replacement therapy (ERT) with 0·2 mg kg(-1) agalsidase alfa (Replagal; Shire Human Genetic Therapies, Basingstoke, Hampshire, UK) or 1·0 mg kg(-1) agalsidase beta (Fabrazyme; Genzyme Europe BV, Naarden, The Netherlands) are collectively expected to make 4500 hospital attendances to a day ward for infusions, which equates to 2000 eight-h days on the day ward associated with ERT. If all ERT-treated patients received their infusions at home, there would be a marginal reduction in the annual health care cost to manage these patients, and the total annual number of days on the day ward associated with ERT in the second year could potentially be reduced from a mean 2000 to zero, thereby releasing substantial hospital resources for use by non-Fabry patients. Currently, only agalsidase alfa is licensed for home treatment in Italy; hence, only patients receiving this enzyme could be offered home treatment. CONCLUSION: Use of agalsidase alfa (0·2 mg kg(-1) ) instead of agalsidase beta (1·0 mg kg(-1)) has the potential to reduce health care costs and release hospital resources in different specialities for alternative use by non-Fabry patients, thereby improving the efficiency of the public health care system in Italy.

Modelling the resource implications of managing adults with Fabry disease in Norway favours home infusion.

BACKGROUND: The aim of this study was to estimate the resource implications and budget impact of managing adults with Fabry disease in Norway, from the perspective of the publicly funded healthcare system. METHODS: A decision model was constructed using published clinical outcomes and clinician-derived resource utilization estimates. The model was used to estimate the annual healthcare cost of managing a cohort of 64 adult Fabry patients in an average year. RESULTS: The expected annual cost of managing 60 existing Fabry patients and four new patients in Norway each year was estimated to be NOK 55·8 million (€6·7 million). In an average year, patients receiving enzyme replacement therapy (ERT) with agalsidase alfa (Replagal(®)) at 0·2 mg kg⁻¹ or agalsidase beta (Fabrazyme(®)) at 1·0 mg kg⁻¹ are collectively expected to make 586 attendances to their family practitioner's office for their infusions, which equates to 128 eight-hour days associated with ERT. Encouraging more patients to undergo home-based infusions has substantial potential to free-up community-based resources. In comparison, the community-related benefit that can be obtained by switching from agalsidase beta (1·0 mg kg⁻¹) to agalsidase alpha (0·2 mg kg⁻¹) is marginal, and dependent on the two doses being clinically equivalent. CONCLUSION: Maximizing the proportion of adults with Fabry disease undergoing home-based infusions has the potential to release community-based resources for alternative use by non-Fabry patients, thereby improving the efficiency of the publicly funded healthcare system in Norway.

NSAIDs: gastroprotection or selective COX-2 inhibitor?

Conventional nonsteroidal anti-inflammatory drugs (NSAIDs) are effective adjuvant analgesics commonly encountered in palliative care. However, these drugs are associated with adverse effects that are primarily due to gastrointestinal toxicity, with resultant serious complications such as gastroduodenal perforations, ulcers and bleeds. This toxicity has been attributed to inhibition of cyclooxygenase-1 (COX-1). Factors known to increase this risk of toxicity include age above 65 years, classification of NSAID in terms of COX-1/COX-2 selectivity, previous history of complications and coadministration of aspirin, anticoagulants and corticosteroids. Selective inhibitors of cyclooxygenase-2 (COX-2) were developed in an attempt to reduce this association; trials to date confirm that these drugs do indeed have reduced incidence of gastroduodenal toxicity. Prior to the introduction of the COX-2 selective inhibitors, patients at high risk were often coprescribed a gastroprotective agent (such as misoprostol or a proton pump inhibitor) with a conventional NSAID. This review discusses the merits of both options and devises a treatment strategy for the safe and cost-effective use of these drugs in the palliative care population.

Have COX-2 inhibitors influenced the co-prescription of anti-ulcer drugs with NSAIDs?

AIMS: To quantify usage of COX-2 inhibitors compared with nonselective NSAIDs and to determine their impact (including financial) on the co-prescription of antipeptic ulcer (anti-PU) drugs. METHODS: The Irish General Medical Services prescription database (covering 1.2 million people) was examined for NSAID prescriptions during December 1999-November 2001. NSAID users were excluded during the first 6 months. During the next 12 months (study period) patients on NSAIDs (>or= 3 prescriptions) were identified. The study period and final 6 months provided data on co-prescription of anti-PU drugs. Age, gender, number of concomitant prescriptions, co-prescribing of anti-PU drugs and monthly cost were evaluated for 8 NSAIDs (n= 4 non-selective NSAIDs and n= 4 COX-2 inhibitors) and odds ratios (OR) calculated using logistic regression. RESULTS: COX-2 inhibitors were prescribed more frequently in older, female patients and those receiving multiple medications. After adjustment for age, gender and polypharmacy, anti-PU drugs were prescribed more frequently with COX-2 inhibitors (OR = 1.31 (1.23,1.40)). COX-2 inhibitors were up to 10-fold more expensive, median monthly costs (including anti-PU drugs) ranging from Euros 34.61 (COX-2 inhibitors) to Euros 3.26 (nonselective NSAIDs). CONCLUSIONS: Since COX-2 inhibitors are associated with increased rates of co-prescription of anti-PU drugs and are more expensive than non-selective NSAIDs, these results suggest that the expected cost-savings with COX-2 inhibitors may not be occurring in practice.

Potential effect of cyclooxygenase-2-specific inhibitors on the prevention of colorectal cancer: a cost-effectiveness analysis.

PURPOSE: To estimate the potential cost-effectiveness of colorectal cancer chemoprevention with cyclooxygenase-2-specific inhibitors (COX-2 inhibitors). METHODS: Using a decision analytic Markov model, we estimated the discounted cost per life-year saved for three strategies: a COX-2 inhibitor alone; as an adjunct to colonoscopy every 10 years in persons at average risk of colorectal cancer; and as an adjunct to colonoscopy every 5 years in persons with first-degree relatives who had colorectal cancer. RESULTS: In the base case, the incremental cost per life-year saved with a COX-2 inhibitor alone compared with no screening was 233,300 dollars in persons at average risk of colorectal cancer and 56,700 dollars in persons with 2 first-degree relatives who had the disease. Chemoprevention was both less effective and more costly than screening. The incremental cost per life-year saved with a COX-2 inhibitor as an adjunct to screening was 823,800 dollars in persons at average risk and 404,700 dollars in persons with 2 first-degree relatives who had colorectal cancer. Combining a COX-2 inhibitor with less frequent screening was not as cost-effective as screening at currently recommended intervals. Cost-effectiveness estimates were highly sensitive to the cost of COX-2 inhibitors and their effect on the risk of cancer. CONCLUSION: Chemoprevention of colorectal cancer with COX-2 inhibitors is likely to incur substantially higher costs per life-year saved than are currently recommended screening strategies. COX-2 inhibitor use as an adjunct to screening may increase life expectancy, although at prohibitive costs, and is unlikely to result in less frequent screening.

Pharmacoeconomics of coxib therapy.

The economic evaluation of health care programs is undertaken to assess health care costs and benefits. Part of the goal of cost-effectiveness analysis is to maximize health benefits given the constraint of limited health care resources. The identification of costs is critical in a cost-effectiveness analysis of clinical interventions. The recent introduction of the cyclooxygenase (COX)-2-selective inhibitors, coxibs, for treatment of rheumatoid arthritis, osteoarthritis, and acute pain gives rise to cost-effectiveness issues. These new agents provide similar efficacy with fewer gastrointestinal events compared with nonselective nonsteroidal anti-inflammatory drugs (NSAIDs), but are more expensive on a per-dose basis. However, several modeled cost analyses have suggested that COX-2 inhibitors are cost effective in subsets of patients because they are associated with fewer downstream costs, particularly medical and surgical treatment of gastrointestinal adverse effects. Three cost-effectiveness models of interventions for rheumatoid arthritis and osteoarthritis, including COX-2 inhibitors, are reviewed. Prospective clinical investigation of the potential costs and benefits of these new agents is necessary to further support these findings.

Agalsidase alfa: specific treatment for Fabry disease.

Fabry disease is a rare genetic lysosomal storage disorder characterized by a deficiency of the enzyme alpha-galactosidase A. The recent availability of enzyme-replacement therapy with agalsidase alfa offers specific treatment for this serious, progressive condition.

Long-term experience with an accelerated protocol for diagnosis of chest pain.

CONTEXT: More than 6 million patients present annually with chest pain suggestive of acute coronary syndrome. Rapid and accurate diagnosis is essential for best clinical outcomes, for optimal management of hospital resources, and for minimizing medicolegal exposure. OBJECTIVE: To evaluate the clinical and cost outcomes of an accelerated protocol for chest pain triage in a community-based hospital of moderate size. METHODS: One hundred successive patients with chest pain were diagnosed according to the Traditional Chest Pain Protocol, which included testing of serial blood samples for creatine kinase (CK)-MB and total CK. These patients were also subjected to the Accelerated Chest Pain Protocol under evaluation, which included testing at shortened intervals for myoglobin and cardiac troponin I in addition to CK and CK-MB. Diagnostic sensitivity and specificity were compared versus the final assigned diagnosis. The Accelerated Chest Pain Protocol was implemented for routine use. Follow-up evaluations were conducted at 1 month (test group A, N = 180) and 22 months (test group B, N = 180). Costs for diagnosis and treatment of the 2 test groups were compared with those for the control group. RESULTS: The 2 protocols had equivalent specificity values (99%). The sensitivity of the Accelerated Chest Pain Protocol was higher than that of the Traditional Chest Pain Protocol (95% vs 58%). Cost savings of 29% and a reduction in length of stay of 33% were achieved in test group B versus the control group. CONCLUSIONS: The Accelerated Chest Pain Protocol improved the accuracy and timeliness of diagnosis of acute coronary syndrome while reducing costs.