Higher-throughput ADME programs in early drug discovery are becoming common throughout the pharmaceutical industry as companies strive to reduce their compound attrition in later-stage development. Many of the ADME assays developed into higher-throughput formats rely on LC/MS analyses. Since the biological aspects of the assay are amenable to parallel processes using dense plate formats, the number of samples generated from these assays produce a large analysis load for serial LC/MS. Presented in this report are two novel strategies, including a sample pooling method and a two time-point method, that could be used in drug discovery to reduce the number of samples generated during multiple time-point in-vitro ADME assays. One hundred and sixty-three compounds were subjected to human microsomal incubations with full time-point method samples taken at t = 0, 5, 15, 30, and 45 min. The ER data correlation (R(2)) between the full time-point method and the pooling method and two time-point methods were 0.98 and 0.97, respectively. Both methods have the potential to: 1. produce data of similar quality to traditional high throughput ADME assays, 2. be easily implemented, 3. shorten analytical run times, and 4. be reproducible and robust.

Algorithms↗

PubMed2005

Induction therapy with basiliximab allows delayed initiation of cyclosporine and preserves renal function after cardiac transplantation.

BACKGROUND: Cyclosporine (CsA) is frequently initiated as induction therapy in patients undergoing orthotopic heart transplantation, but our experience has identified a significant rate of post-operative renal dysfunction. We therefore devised a renal-sparing cyclosporine-free induction regimen consisting of the early administration basiliximab, an interleukin-2 receptor monoclonal antibody, followed by the late initiation of cyclosporine on post-operative Day 4. METHODS: Between September 1998 and December 1999, we treated 25 patients at risk for post-operative renal dysfunction (high-risk basiliximab group) with the new induction regimen and another 33 patients not at risk (low-risk CsA group) for renal dysfunction with our standard cyclosporine protocol. We identified a historical control group (1996 through 1998) of 32 patients at risk for renal dysfunction (high-risk CsA group) who had received our standard cyclosporine protocol. RESULTS: The increase in serum creatinine levels after transplantation was less in the high-risk basiliximab group (-0.1 +/- 0.7) than in the high-risk CsA group (0.5 +/- 1.0, p < 0.02) and comparable to the low-risk CsA group (0.03 +/- 0.6). The basiliximab protocol did not increase rejection; the percentage of rejection episodes was high-risk basiliximab, 0; high-risk CsA, 13; and low-risk CsA, 3 (p = .13). CONCLUSION: Basiliximab induction therapy allows delayed initiation of cyclosporine after cardiac transplantation without an increase in rejection and reduces the risk of post-operative renal dysfunction.

Adult↗

PubMed2005

Prediction of the adaptability of Pseudomonas putida DOT-T1E to a second phase of a solvent for economically sound two-phase biotransformations.

The strain Pseudomonas putida DOT-T1E was tested for its ability to tolerate second phases of different alkanols for their use as solvents in two-liquid-phase biotransformations. Although 1-decanol showed an about 10-fold higher toxicity to the cells than 1-octanol, the cells were able to adapt completely to 1-decanol only and could not be adapted in order to grow stably in the presence of a second phase of 1-octanol. The main explanation for this observation can be seen in the higher water and membrane solubility of 1-octanol. The hydrophobicity (log P) of a substance correlates with a certain partitioning of that compound into the membrane. Combining the log P value with the water solubility, the maximum membrane concentration of a compound can be calculated. With this simple calculation, it is possible to predict the property of an organic chemical for its potential applicability as a solvent for two-liquid-phase biotransformations with solvent-tolerant P. putida strains. Only compounds that show a maximum membrane concentration of less than 400 mM, such as 1-decanol, seem to be tolerated by these bacterial strains when applied in supersaturating concentrations to the medium. Taking into consideration that a solvent for a two-liquid-phase system should possess partitioning properties for potential substrates and products of a fine chemical synthesis, it can be seen that 1-decanol is a suitable solvent for such biotransformation processes. This was also demonstrated in shake cultures, where increasing amounts of a second phase of 1-decanol led to bacteria tolerating higher concentrations of the model substrate 3-nitrotoluene. Transferring this example to a 5-liter-scale bioreactor with 10% (vol/vol) 1-decanol, the amount of 3-nitrotoluene tolerated by the cells is up to 200-fold higher than in pure aqueous medium. The system demonstrates the usefulness of two-phase biotransformations utilizing solvent-tolerant bacteria.

1-Octanol↗

PubMed2005

Suitability of recombinant Escherichia coli and Pseudomonas putida strains for selective biotransformation of m-nitrotoluene by xylene monooxygenase.

Escherichia coli JM101(pSPZ3), containing xylene monooxygenase (XMO) from Pseudomonas putida mt-2, catalyzes specific oxidations and reductions of m-nitrotoluene and derivatives thereof. In addition to reactions catalyzed by XMO, we focused on biotransformations by native enzymes of the E. coli host and their effect on overall biocatalyst performance. While m-nitrotoluene was consecutively oxygenated to m-nitrobenzyl alcohol, m-nitrobenzaldehyde, and m-nitrobenzoic acid by XMO, the oxidation was counteracted by an alcohol dehydrogenase(s) from the E. coli host, which reduced m-nitrobenzaldehyde to m-nitrobenzyl alcohol. Furthermore, the enzymatic background of the host reduced the nitro groups of the reactants resulting in the formation of aromatic amines, which were shown to effectively inhibit XMO in a reversible fashion. Host-intrinsic oxidoreductases and their reaction products had a major effect on the activity of XMO during biocatalysis of m-nitrotoluene. P. putida DOT-T1E and P. putida PpS81 were compared to E. coli JM101 as alternative hosts for XMO. These promising strains contained an additional dehydrogenase that oxidized m-nitrobenzaldehyde to the corresponding acid but catalyzed the formation of XMO-inhibiting aromatic amines at a significantly lower level than E. coli JM101.

Biodegradation, Environmental↗

PubMed2004

Impact of retrograde graft preservation in Perfadex-based experimental lung transplantation.

OBJECTIVE: Optimal preservation of postischemic organ function is a continuing challenge in clinical lung transplantation. Retrograde instillation of preservation solutions has theoretical advantages to achieve a homogeneous distribution in the lung due to perfusion of both the pulmonary and the bronchial circulation. Thus far, no systematic screening studies followed by in vivo large animal reevaluation including stereological analysis of intrapulmonary edema exist concerning the influence of retrograde preservation on postischemic lung function after preservation with low potassium dextran (LPD) solution (Perfadex). MATERIALS AND METHODS: For initial screening in an extracorporeal rat model eight lungs, each, were preserved for 4 h using antegrade or retrograde preservation with LPD solution (Perfadex; PER(ant)/PER(ret)). Respiratory and hemodynamic results after reperfusion were compared to low-potassium Euro-Collins (LPEC). For systematic reevaluation, five pig lungs, each, were preserved correspondingly for 27 h, and results were compared to sham-operated control lungs. In both models, edema formation was quantified stereologically. Statistics comprised different ANOVA models. RESULTS: In both models, use of PER(ret) resulted in significantly higher oxygenation capacity, lower inspiratory pressures, and lower amounts of intraalveolar edema as compared to PER(ant). Results of PER(ret) were not different from sham controls in the in vivo model; furthermore, a continuous retrograde elimination of blood clots from pulmonary microcirculation was noticed. CONCLUSIONS: Retrograde application of LPD solution (Perfadex) results in significant functional and histological improvement as compared to antegrade perfusion. This innovative technique can be applied very easily in clinical practice and might be an ideal adjunct to further optimize the results after lung transplantation with LPD-based graft protection.

Animals↗

PubMedDate not supplied

Assuring the accuracy of home glucose monitoring.

BACKGROUND: An estimated 2.5 million diabetic patients in the United States practice self-monitoring of blood glucose (SMBG). The validity of the glucose values they obtain is in doubt. An American Diabetes Association consensus panel reported that up to 50% of SMBG determinations might vary more than 20% from their true value. Accurate glucose values are an integral part of intensive treatment and reduction of long-term complications. The objective of this study was to determine the technical skill and accuracy of SMBG in an outpatient population. METHODS: This study was conducted in two family practice residency sites where 111 patients with type 1 and type 2 adult diabetes were observed testing their blood glucose values on their own glucose monitors. Patient-measured glucose levels were immediately compared with a laboratory value obtained from a calibrated hand-held glucose monitor. RESULTS: Fifty-three percent of patient glucose values were within 10% of the control value, 84% were within 20% of the control value, and 16% varied 20% or more from the control value. Two patients had dangerously inaccurate glucose determinations. Four glucose monitors required replacement. The patients were observed using a 13-point checklist of critical steps in calibration and operation of their glucose monitor. Only 1 patient made no errors in testing. CONCLUSIONS: Despite multiple technical errors when using SMBG, most patients obtained clinically useful values. This project can be easily introduced into a medical office.

Adult↗