Metabolic Stability

Metabolic Stability studies

ADME studies conducted during the discovery and preclinical development phases of drug discovery aim to find compounds with acceptable chemical and pharmacological properties among the ones showing potency towards the intended target. Metabolic stability is one of the most important features of a candidate, as 70% of marketed drugs are eliminated via hepatic clearance. Deliverables from metabolic stability studies are the in vitro half-life (min) and the in vitro clearance (μL/min per mg protein or per million cells) of the compound. In vivo, clearance is the volume of blood that is completely cleared of drug per unit of time when it passes through a clearance organ such as the liver, where metabolism may be an important elimination pathway depending on drug properties.

In vitro metabolic clearance values are therefore studied and used to predict in vivo metabolic clearance values, and to rank compounds based on the results. Candidates with very high or very low metabolic clearance can, as needed, be sorted out long before clinical studies, should such properties not be preferred. In specific cases, compounds with long half-lives might for example not be favorable due to incompatibility with the intended usage (e.g., need for a short duration of action for on-demand drugs, or short duration on targets to minimize side effect), the need of a complex dosing regimen, or because of safety concerns related to systemic accumulation. Rapid clearance on the other hand usually results in limited bioavailability and/or short duration of action, which can potentially be less favorable.

In vitro metabolic stability studies can be performed with a number of liver metabolic enzyme sources:

  • hepatocyte subcellular fractions (microsomes or S9 fraction),
  • freshly prepared or cryopreserved hepatocytes, or
  • recombinant enzymes.

When microsomal incubations are used, appropriate cofactors, e.g., nicotinamide adenine dinucleotide phosphate (NADPH) for cytochrome P450 (CYP450) and flavin-containing monooxygenase (FMO) enzymes and uridine diphosphate glucuronic acid (UDPGA) for uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes, are also added to enable metabolic activity. When glucuronidation is studied in microsomes, alamethicin, a pore-forming peptide, is often added to increase access of the UDPGA and substrate to the luminal side of the endoplasmic reticulum, where UGT enzymes reside. In case of whole functional hepatocytes, adding cofactors is not necessary as they contain all elements needed for the enzyme reactions.

Each of these systems has its own advantages and disadvantages as summarized in Table 1.

Pros and cons of different enzyme sources used for metabolic stability assays
  Microsomes/S9 Hepatocytes Recombinant enzymes
  • Highly controllable by cofactor addition
  • no limitations to permeability or transport thanks to absence of cell membrane
  • Translatable
  • Suitable for Phase I and Phase II metabolism
  • Small amounts required
  • Well established protocols
  • Easy to automate, can be high throughput
  • Relatively low cost
  • Most physiologically relevant
  • Cryopreserved fresh, suspension or plateable options
  • Contain full complement of enzymes and co-factors
  • Well established protocols
  • Translatable
  • Can be used for induction studies
  • Multiple mechanisms present
  • Specific, single enzyme systems
  • Well established protocols
  • Specific polymorphisms
  • High and consistent intrinsic activity
  • Easy to automate, can be high throughput Relatively low cost
  • Not cell based therefore less physiologically relevant,
  • Microsomes lack complete metabolizing complement
  • Transporters or limited passive permeability may impact results
  • Difficult to automate
  • Relatively higher cost
  • Single enzyme system,
  • Not always translatable

Species differences

Revealing species differences in pharmacokinetics, including metabolic stability and metabolic clearance pathways, are vital for prediction of clinical efficacy and species selection for safety studies. For example, it is essential to compare exposures in toxicological studies with the exposures expected or achieved in humans to ensure a sufficient safety margin.

The most frequently used model in early programs is the rat, but due to significant differences in rate of metabolism, data are usually completed later with results from dog and monkey. More recently, based on the latest publications, minipig is also proposed as a relevant nonrodent model for tox studies. Mouse is also often included, as their intestinal CYP3A isoenzymes appear to mirror those in humans, and – compared to the rat – better correlation can also be observed for the liver with common pathways and similar metabolic clearance rates on chemical series.

Technical considerations for studying metabolic clearance

The main experimental parameters to be considered for metabolic stability studies are:

  • test compound concentration(s),
  • the amount of microsomal protein (or number of cells),
  • use of single vs. multi-donors,
  • length of incubation, and the number of time points used for sampling.

One of the most critical experimental variables is the concentration of test compound used during the incubation. It should resemble the corresponding in vivo concentrations (or its estimate at earlier development phases) and be low enough to avoid saturation of metabolic processes. The rate of metabolism is often concentration-dependent, meaning that with an increase in concentration, the rate of metabolism decreases due to saturation. A too high test concentration would thus result in a longer half-life. Therefore, more than one test concentrations might be used, in practice a concentration range of 0.1–1 μM is applied most often.

The amount of protein (i.e. microsomal or S9) in the assay system is ideally within the range of 0.1–1.0 mg/mL. Higher concentrations may result in lower half-lives and possibly also lower clearance values (due to increased protein binding), which should therefore be corrected relative to the unbound fraction in the incubation. In hepatocyte studies, around 0.5 – 1 million viable cells per mL of incubation are optimally used, to balance the activity vs. protein/cellular binding.

Whether microsomes, S9 or hepatocytes are used, it is important to consider that these are sourced from different donors which may affect the activity level of enzymes due to differences in gender, genetics (SNPs), prescribed drugs, and lifestyle factors. Use of multiple donors is therefore generally preferred over the use of single donors as any outliers (lower or higher activity) are equaled out. In specific cases however, studying a single donor only may be preferred, depending on the research interest (e.g. studying the effect of SNPs).

Although for any metabolic stability, 4-6 time points are required for assessment, selecting the right sampling time points is important, as the early linear phase of metabolism should be covered at least in part, to allow calculation of clearance kinetics. In assays using subcellular liver fractions (microsomes or S9 incubations), time points are typically included between 0 and 90 minutes of incubation, whereas in hepatocyte suspension assays, the incubation time could be extended up to 4 hours maximum. Such a long incubation is, however, often not required as the linearity of metabolic reactions is rarely maintained for over 2-3 hours for typical small molecule drugs candidates. Compounds with especially high metabolic stability do exist however, and in their case, specific systems may be applied to determine metabolic stability: micropatterned hepatocyte co-cultures (HepatoPac) or 3D hepatocyte models, where incubation time can be extended up to 7 days, are good solutions for such compounds.

Assay description

Considering all of the above, a typical metabolic stability assay is run as described below, however  the setup can be adapted depending on compound specifics:

  • Incubations of test compounds are done at one low concentration (0.1-10 µM),
  • In microsomes, S9 fraction or hepatocytes (typically multi-donor) in 0.05 M phosphate buffer (pH 7.4),
  • Tested in different species: human, monkey, minipig, dog and rat,
  • Carried out using multiple incubation times (e.g. 0, 10, 30, 60 and 120 min),
  • under shake conditions at 37°C.

For subcellular fractions, NADPH, UDPGA (and other optional) co-factors are added at the beginning of the incubation. Testosterone is routinely used as positive control, as it is extensively metabolized by several enzymes, therefore a good positive control for metabolic activity. Incubations without co-factors/without cellular fractions or cells are used to distinguish metabolism-dependent loss of substrate from chemical degradation or non-specific binding. At each sampling time point, samples are transferred into ice-cold stopping solvent (acetonitrile or MeOH). Samples are centrifuged and supernatant is collected for analyses using LC/MS to determine the level of remaining test compound concentration and calculate loss of substrate with zero-time incubations serving as 100 % values.

Prediction of in vivo kinetics from in vitro metabolic stability data

The data obtained from in vitro clearance studies can be used to predict in vivo hepatic clearance. For more accurate modelling, parameters regarding the binding characteristics of the compound with microsomal proteins or cells (unbound fraction Fub,inc in the incubation), and corresponding binding with plasma proteins (unbound fraction Fub,plasma in the circulation) are needed.

Classically, the intrinsic in vitro clearance (μL/min/mg protein or million cells) is evaluated by analysis of metabolite formation according to the Michaelis-Menten equation and calculated as Clint = Vmax/Km, but in the absence of standards for metabolite quantification, and with the simplicity of monitoring the parent compound only, the in vitro clearance is typically calculated as:

C L i n t .   i n c = k × V M F u b ,   i n c

where, Fub,inc is the free fraction in the incubation (0–1), k is the first-order rate constant (min-1) obtained from the slope of the disappearance kinetics (the natural log of the relative remaining concentration vs. time), V is the incubation volume, and M is the amount of microsomal protein or hepatocytes in the incubation. The half-life is calculated as:

t 1 / 2 = l n 2 k

The in vivo rate of metabolism (Clint) can be then predicted with the use of scaling factors. In case of S9 fractions, the equation is completed using the following equation:

C L i n t =   L n ( 2 ) t 1 / 2   ×   l i v e r   w e i g h t s t a n d a r d   b o d y   w e i g h t   ×   i n c u b a t i o n   v o l u m e   ( m L ) p r o t e i n S 9 w e l l ( m g )   ×   p r o t e i n s 9 ( m g ) g r a m   o f   l i v e r

where the parameters usually used for scaling are the following:

Scaling factors for Clint calculation in S9 and microsomal subcellular fractions, and hepatocytes
Parameter Value
S9/microsomal protein per well (mg) 1
protein (mg per gram liver) 121 (S9), 32 (microsomes)
Liver weight (g) 1800
Average body weight (kg) 70
Hepatocellularity (per gram liver) 99 x 106

In case of hepatocytes, the rate of metabolism (Clint) is usually presented in μL/min/106 cells, using the half-life and the equation below:

C L i n t =   L n ( 2 ) t 1 2   ×   i n c u b a t i o n   v o l u m e   ( μ l ) c e l l s / w e l l ( x 10 6 )   ( μ l / m i n / 10 6 c e l l s )