Synovo micro pharmacokinetic solutions

 

Key Points

• Pharmacokinetics (PK) data can be obtained on a 24, 48 or 72 h turn around if required
• Amounts required can be in the range of 1 mg
• Distribution to specific organs can be included easily in such studies
• Certain PD and clinical chemistry markers can be monitored in parallel

Overview

Pharmacokinetics (PK) – the pharmacological study of pathways of substances through living organisms – can be made drastically more economical in time and materials through the use of micro pharmacology based on milligram amounts studied in vivo using mouse models.

Synovo’s micro-pharmacokinetic techniques allow full PK to be performed in a day using less than one milligram (mg) of sample substance.

This is of huge benefit in early phase drug discovery, because it saves the need for re-synthesis compounds or production of new batches of biological substances.

By limiting substance demands at early stage, it is possible to gather more data with which to better prioritise and focus on compounds of true promise.

Synovo has become expert in minimizing use of substances in PK studies, accelerating process and maximizing data obtained.

Background

A constant challenge in practical drug discovery is to obtain biology within a time scale that enables results to influence the next round of analog synthesis.

Normal chemistry thinking responds to signals obtained at the chemistry bench, in unexpected reactions, and to ideas that derive from the immediate laboratory environment. By the time a compound is made, purified, cataloged, submitted to a collection, ordered, sampled into vials, delivered to a test site, studied and the data returned, most chemists have already moved on to subsequent analogs and variation iterations. Thus, on conventional time scales, what is being made at any one time is not reflecting the whole current data set, but only what was at hand from earlier work.

To change this paradigm, chemists need data from a new compound within days or hours of its purification. This acceleration between production and data allows biological effect to become more apparent and more synthetically relevant, stimulating new thinking around synthetic targets and replacing chemistry-driven data with data-inspired chemistry.

In vivo alternative to resynthesis

After obtaining hits in a screen, a key rate-limiting decision is what compounds to advance for further characterization. Normally a range of in vitro steps can be made from existing stock solutions and these can be easily planned into a cascade.

Traditionally, however, prior to in vivo studies, it has been necessary to order more material, introducing further costs and uncertainties. However, it has now become possible to obtain reliable in vivo data from low quantities of material.

The small-scale physiology of mice means that, for a typical compound, one can obtain a full PK and organ profile using less than 1 mg of compound.

Current liquid chromatography and mass spectronomy (LC-MS/MS) technology usually requires very low sample amounts to detect reasonable analytes. Synovo has shown that injection amounts in the range of 1.0 μL plasma equivalent are adequate for reliable detection. This means that one can use correspondingly small amounts of sample that reduces the overall burden of the process. Modern equipment is also more linear and reproducible than in the past and small sample sizes have no real downside in overall accuracy of determinations.

Table 1. Example compound requirements for murine PK/PD

System	Route	Dose (mg/kg)	Typical weight (g)	N	Amount needed (mg)
DBA1	i.v.	2.5	18	3	0.18
BALBc	i.v.	2.5	20	3	0.20
C57Blk6	i.v.	2.5	23	3	0.22
NMRI	i.v.	2.5	25	3	0.24
CD1/Swiss	i.v.	2.5	28	3	0.27
DBA1	p.o.	10	18	3	0.70
BALBc	p.o.	10	20	3	0.78
C57Blk6	p.o.	10	23	3	0.90
NMRI	p.o.	10	25	3	0.98
CD1/Swiss	p.o.	10	28	3	1.09

Using LC-MS/MS to monitor animal clinical chemistry in parallel with compound levels can generate useful insights on compound action with relatively little additional effort.

Combining these approaches can mean that significant understanding of the pharmacology of a series can be obtained before re-synthesis is necessary.

Saving time and compound
A combination of gene knockout technology, sequencing and increasing analytic sensitivity has thus made small-scale models useful for drug discovery and development.

Improvements in LC-MS/MS have likewise changed the way substance exposure is monitored and optimised. Pharmacokinetic properties, notably distribution, are often unpredictable for a new series and knowing early how particular pharmacophores distribute and what contributes to stability can strongly influence synthesis and hit/lead prioritization.

Synovo has shown that PK studies in 24 hours are possible, 48-hour studies are common and 72-hour turnarounds are achievable, assuming:

• The correct strains are on hand
• Reagents/formulants are available from stock
• Ethics is in place for the project, program and substance class
• Formulation is agreed and is general enough for varying properties
• No unusual analytics are required

Using appropriate logistics, samples can be loaded into analytical systems essentially in real time, providing rapid bioavailability and metabolism data to the project while thinking is still focused on the structures under study.

Clinical chemistry and PD

Synovo can apply similar miniaturization concepts to other aspects of early pharmacology, notably detection of toxicity and pharmacological effect.

Samples in the range of 1- 4 μL of plasma suffice for standard measures of clinical chemistry parameters. This means that determining the onset of certain signs of toxicity does not require separate satellite animals, but can be done with low impact in the main treatment groups.

Table 2. Metabolites and markers easily determined in routine studies

Assay/Analyte	Volume needed (μL)*	Method
ALT/AST	1-12	Colorimetric
Alkaline phosphatase	1-2	UV
Bilirubin	1	Fluorescent./colorim./DAHPLC
Amino acids, SAM, glutamate etc.	1	LC-MS/MS
Bile acids	1	LC-MS/MS
Cholesterol	1-3	Colorimetric/fluorescence
Triglycerides	3	Colorimetric
Hormones	1-5	MS, ELISA
Sugars	1	Oxidases/direct
Peptides – e.g. Insulin	1	ELISA
Coagulation	3	Direct
Cytokines	1-5	ELISA

*Depends on magnitude of effect expected, within general categories sensitivity varies widely.

Reductions in sample volume required for assays also allow parallel measurements to be made along with sampling for pharmacokinetic or toxicokinetic purposes. For example, certain common biochemicals, such as bile acids, are easily detectable by LC-MS and can be conveniently determined in parallel with the substance of interest. Addition of a deuterated internal standard in the extraction process can thus transform a simple PK study into a full pharmacokinetic and pharmacodynamic (PK/PD) determination.

Summary of benefits:

Reduced needs for sample quantities enables reductions in chemistry costs and laboratory time. It also frees researchers to make new compounds rather than scaling up the older analogs.

Specific benefits include:

• Saving in synthesis saves time, material, human resources and cost
• Fast turnaround when required
• Acceleration of the drug discovery process
• In vivo pharmacology replacing resynthesis
• Full PK and distribution obtainable within three days from < 1 mg compound

Resources

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Supplier Information
Supplier: Synovo GmbH
Address: Paul Ehrlich Str. 15, D-72076 Tübingen, Germany
Tel: +49 (0) 7071 964 325
Fax: +49 (0) 7071 964 326
Website: www.synovo.com

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