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| Atheris Analytical - Mass spectrometry Biochemistry Protein engineering Metabolic studies | ||
| Atheris Discovery - Venoms to drugs Venoms database Lead discovery Early development Biomarkers | ||
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| Atheris Analytical - Mass spectrometry Biochemistry Protein engineering Metabolic studies | ||
| Atheris Discovery - Venoms to drugs Venoms database Lead discovery Early development Biomarkers | ||
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Voulez-Vous ? Pharmacokinetics & metabolic studies
Studies of protein metabolism have so far relied heavily on the use of radioactive tracers or on immunoassays. However, both methods suffer from inherent drawbacks: on one side a certain aversion to use radioactive labels and on the other side an indirect detection leading to ambiguous results in some cases. Atheris offers complete PK-PD bioanalytical solutions, with feasibility studies, method development, method refinement, method validation to FDA standards and analysis of pre-clinical or clinical samples:
At Atheris we specialize in LC-MS and LC-MS/MS bioanalytical based methods for PK-PD studies. We are particularly well experience in the follow-up of peptides and proteins in human or animal body fluids (plasma, serum, urine, cerebrospinal fluid) down to basal pM levels. We have also sucessfully conducted similar work on solid tissues such as organs or tumors. We achieve the best results when we can fully exploit a method we pioneered in the early 90's which is called Isotope Dilution Assay (IDA). IDA allows sensitive (down to the ng/L range) accurate detection and quantification of peptides and proteins in complex biological samples using stable isotopes, which by definition are not radioactive. It further is the best possible tool to identify, follow-up and quantify degradation fragments and secondary metabolites. PK-LADME It is crucial to undertake reliable pharmacokinetic and metabolic studies in vitro and in vivo to establish, at the earliest possible stage, the so called LADME profile (Liberation, Adsorption, Distribution, Metabolism, Excretion) of each drug candidate and its metabolites. Such PK-LADME studies typically include:
At Atheris, we undertake complete highly sensitive (down to ng/L ranges) and accurate metabolic studies using either the traditional chromatographic, LC-MS and LC-MS/MS strategies, or stable Isotope Dilution Assays coupled to MS techniques. Our strategies often allow the simultaneous follow-up of several target biomolecules. Furthermore, they drastically facilitate the identification and follow-up of degradation fragments and other metabolites. Isotope Dilution Assays - IDA Pharmacokinetics of small volatile organic molecule drugs is a well-developed field. Nowadays, the most dependable procedures rely on HPLC and gas chromatography coupled to mass spectrometry (LC-MS and GC-MS). In spite of its importance, pharmacokinetics of protein drugs is a research field that still faces serious challenges. At Atheris, we like to address the crucial issues of PK-LADME of polypeptides using an attractive method known as IDA or Isotope Dilution Assay. It was developed successfully for quantification of insulin in blood samples at low pM levels (down to the ng/L range). IDA’s inherent characteristics are making it the “gold standard” method for clinical work:
This method involves the use of target protein analogues labelled with stable isotopes (such as deuterium or 15-N). These non-radioactive analogues have different molecular masses and act as internal standards. In a typical experiment, a known amount of the analogue is added to the biological sample to be analyzed prior to any sample handling. After appropriate extraction and purification processes, the final mass spectrometric analysis allows the direct identification of the target compound and its analogue by virtue of their respective molecular masses. In addition, it enables a precise quantification on the basis of the relative intensities of the observed signals (principle of isotope dilution). IDA completely avoids the use of radioactive material and is not susceptible to errors arising from immunological cross-reactivity with closely related compounds (e.g. precursors, analogues, maturation or degradation fragments). The measured molecular masses further allow the unambiguous identification of the target compound at a sub-pM level with a clear distinction between its endogenous and injected forms, thus allowing for simultaneous in vivo quantitative investigations of both native endogenous and injected compounds.
Method development
The main drawback of PK-LADME studies is that they can always be improved., and this is hardly compatible with efficient drug development. We are used at finding the right compromise: establishing well suited methods for fast, reliable, time and cost effective delivery of results. Method development is a multi-factor compromise, and we are convinced that clearly defined strategy and objectives, coupled to high quality work and follow-up are key elements to success. We like to achieve the best and we "hate" compromises. But a right balance has to be found between the time spent on method refinement and effiency in the delivery of analytical reports of clinical samples. This is why we like to conduct short term initial feasibility studies to better evaluate the situation. We like to be active, but: "think first". Our offers covers the detection, quantitation and determination of the half-lifetimes of drugs and metabolites in blood, urine and tissues, which can be extended to the development of tailor-made methodologies.
Method validation
We like to validate method according to FDA guidelines for bioanalytical studies. Our method validation protocols are designed meticulously, and typically include selectivity, stability (many conditions), repeatability, calibration, recovery, accuracy, precision, intra- and inter-batch variability, LLOD (lower limit of detection), LLOQ, (lower limit of quantification), etc. We do our best to meet our sponsors expectations, especially when pressure is high. Clinical samples In most cases, projects come to us beacause they failed elsewere using classical approaches. So it is for us sometime difficult to explain that quantifying a peptide at pM concetrations in human plasma is a huge challenge, and there is no method allowing a dozen of samples to be analyzed hourly. Each project is a real challenge that takes time, requires a lot of expertise, which is stimulating for us. Although we are not most favorable to the so called "high throughput" approaches (we will never run 100's of samples a day and delivery poor quality relsuts), we always find a route to reasonable outcome. Our instrument can be run in parallel, overnight and over week-ends.
References A few related publications: For PDF files, please, feel free to ask for a copy, we happily help you
Structure-activity relationship studies of gomesin: importance of the disulfide bridges for conformation, bioactivities, and serum stability.
Biopolymers. 84(2):205-18, (2006)
Fázio, M.A., Oliveira, V.X. Jr., Bulet, P., Miranda, M.T., Daffre, S. and Miranda, A.
Quantify this! Report on a round table discussion on quantitative mass spectrometry in proteomics.
Proteomics. 4(8):2211-5, (2004)
Quadroni, M., Ducret, A. and Stöcklin, R.
Profiling and in vivo quantification of proteins by high resolution tandem mass spectrometry: the example of goserelin, an analogue of LHRH.
Clin. Chem. Lab. Med., 41(12):1589-98, (2003)
Michalet, S., Favreau, P. and Stöcklin, R.
Positive and negative labelling of human proinsulin, insulin and C-peptide with stable isotopes : new tools for metabolic and pharmacokinetic studies.
In : Protein and peptide analysis : advances in the use of mass spectrometry, “Methods in Molecular Biology”(Chapman, J. Ed.), Humana Press, Totowa, NJ, USA. Vol. 146, pp. 293-315, (2000)
Stöcklin, R., Arrighi, J.-F., Hoang-Van, K., Vu, L., Cerini, F., Gilles, N., Genet, R., Markussen, J., Offord, R.E. and Rose, K.
Short-term insulin-induced glycogen formation in primary hepatocytes as a screening bioassay for insulin action.
Anal.Biochem. 262 (1), 17-22, (1998)
Vu, L., Pralong, W.F., Cerini, F., Gjinovci, A., Stöcklin, R., Rose, K., Offord, R.E. and Kippen, A.D.
Development of an isotope dilution assay for precise determination of insulin, C-peptide and proinsulin levels in non-diabetic and type II diabetic individuals with comparison to immunoassay.
J. Biol. Chem. 272, 12513-12522, (1997)
Kippen, A.D., Cerini, F., Vadas, L., Stöcklin, R., Vu, L., Offord, R.E., and Rose, K.
A stable isotope dilution assay for in vivo determination of insulin levels in man by mass spectrometry.
Diabetes 46, 44-50, (1997)
Stöcklin, R., Vu, L., Vadas, L., Cerini, F., Kippen, A.D., Offord, R.E. and Rose, K.
The semisynthesis of octadeutero-PheB1-octadeutero-ValB2-porcine insulin and its characterization by mass spectrometry.
Protein engng 7, 285-289, (1994)
Stöcklin, R., Rose, K., Green, B.N., and Offord, R.E.
Facile identification by electrospray mass spectrometry of the insulin fragment A14-21-B17-30 produced by insulin proteinase.
Rapid Commun. Mass Spectrom. 7(11):1048-50, (1993)
Vu, L., Stöcklin, R., Rose, K. and Offord, R.E.
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