Microsomal stability

A wide range of groups are tolerated in this position including esters, amides, thioesters, ureas, acyl hydrazines and carbamates exhibiting a wide range of potencies and microsomal stabilities (Table 6.2) [24, 33-36]. 

A lead is variously defined in the pharmaceutical industry as a compound derived from a hit with some degree of in vitro optimization (potency in primary assay, activity in functional and/or cellular assay), optimization of physical properties (solubility, permeability), and optimization of in vitro ADME properties (microsomal stability, CYP inhibition). Moreover, a lead must have established SAR/SPR around these parameters such that continued optimization appears possible. A lead may also have preliminary PK and in vivo animal model data. However, it is the task of the lead optimization chemist to improve PK and in vivo activity to the levels needed for identification of a clinical candidate. 

As mentioned in Sect. 3, it is important to establish a detailed lead profile at the beginning of a lead identification effort. Criteria vary in different lead identification or hit-to-lead groups, but generally include some or all of the following potency, functional activity, selectivity, MW, clogP, solubility, permeability, microsomal stability and/or hepatocyte clearance, and preliminary PK including oral bioavail-ability. An example of a lead profile for a kinase inhibitor project is illustrated in Table 1 [21],... 

IkB kinase-p is a key regulatory enzyme in the NF-kB pathway, and inhibition of this enzyme has the potential for yielding treatments for inflammatory and autoimmune diseases. Morwick et al. [53] report on the optimization of a pM IKKp inhibitor with low aqueous solubility, moderate human liver microsome stability, and inhibition of several CYPs (3A4, 2C9, 1A2) with pM potencies. Modulation of the thiophene core (other thiophene isomer, pyrimidine and oxazole) produces compounds of similar potency to the hit. Fusing the 5-phenyl moiety to the thiophene to form a thieno[2,3-b]pyridine core increases aqueous solubility of the series as well as reduces the CYP liability. While the optimized compound still shows pM IKK(S potency, the aqueous solubility, HLM stability and CYP profiles are much improved. A pharmacophore model was generated that enabled scaffold hopping to yield this new chemotype (Scheme 7). 

Compound 24 represents a series of potent spiropiperidine GlyT-1 inhibitors that showed significant binding to the p opioid and nociceptin/ orphanin FQ peptide (NOP) receptors [67]. The hydroxy analog 25 demonstrated improved selectivity and N-propyl analog 26 exhibited increased microsomal stability but reduced GlyT-1 potency (EC50 = 450 nM) [68]. 

Naphthyl inhibitor 38 (Kj = 500 nM) was discovered via a displacement-based HTS assay utilizing radiolabeled (R)-NPTS [77]. However, compound 38 demonstrated high affinity for 5-HT1B (Kj < 1 nM), hERG (K, = 1.2 iM), significant inhibition of CYP 2D6, and poor microsomal stability. 

Modification of 38 improved the selectivity versus 5-HTiB however, hERG activity, CYP 2D6 inhibition, and poor microsomal stability remained. The series was discontinued due to a lack of progress with overcoming these challenges [77]. 

Xu, R. et al. 2002. Application of parallel liquid chromatography/mass spectrometry for high throughput microsomal stability screening of compound libraries. J. Am. Soc. Mass Spectrom. 13 155. 

Human liver microsome stability testing Broad-spectrum selectivity screen then, rat receptor affinity assay... 

Some in vitro ADME and safety assays (e.g., microsomal stability and hERG blockade) may not be possible with poorly soluble compounds. 

In-vitro Microsomal Stability Screen Caco-2 Absorption Screen P450 Enzyme Inhibition Screen Rapid Rat" (CARRS) PO/PK Screen... 

Di, L. Kerns, E. H. Hong, Y. Kleintop, T. A. McConnell, O. J. et al. Optimization of a higher throughput microsomal stability screening assay for profiling drug discovery candidates. J Biomol Screen 2003, 8, 453-462. 

Sakiyama, Y., Yuki, H., Moriya, T., Hattori, K., Suzuki, M., Shimada, K., Honma, T. Predicting human liver microsomal stability with machine learning techniques. J. Mol. Graph. Model. 2008,... 

Cyprotex, using Cloe Screen , evaluates pharmacokinetic properties in vitro and establishes a broad portfolio of in vitro assays that allows researchers to investigate the metabolism parameters for drug discovery and development. This company supplies the following data and assays (276) microsomal stability,... 

The kitchen of a fast food restaurant is characterized by islands of automation, with well defined subprocesses focused on producing a certain kind of output, coordinated by a crew chief The principal advantage of a fast food restaurant is consistency and fast delivery. The dedicated subunits are designed to perform a certain type of process (assay) at a high rate with very little room for change. Economically, this model is difficult to sustain unless each assay type has sufficient demand to justify the existence of dedicated space, equipment and personnel. It is also not as efficient as a secondary screening model. For assays that are routinely, but not always, requested then this model is very appropriate (e.g., CACO-2 permeability, microsomal stability). However, for the more costly and complex assays that are requested less often, the cost of dedicated people and equipment is hard to justify and as a result the assay has to come off the menu. This is why most fast food restaurants have a relatively limited menu, including mostly foods that are simple to prepare. 

Fig. 10.10. (a) Experimental LogD (eLogD) vs. human liver microsome stability (FiLM %Rem 1p,M). A threshold value of eLogD < 2.7 is required for >70% stability in FILM, (b) Experimental vs. calculated LogD. eLogD < 2.7 translates to eLogD < 2.0 at pH 7.4. 

Fig. 10.12. (a) Experimental human liver microsome stability (FILM %Rem 1 . ) vs. calculated Log of solubility (c LogS). (b) Experimental human liver microsome stability expressed as apparent intrinsic clearance (FILM CL(int) xL/min/mg) vs. calculated Log of solubility (c LogS). A threshold value of cLogS > -3 and -4.0 is required for stability in FILM based on %R and intrinsic clearance, respectively. 

Lim, H. K., Chan, K. W., Sisenwine, S., and Scatina, J. A. (2001). Simultaneous screen for microsomal stability and metabolite profile by direct injection turbulent-laminar flow LC-LC and automated tandem mass spectrometry. Anal. Chem. 73 2140-2146. 

Ackermann, B. L. Ruterbories, K. J. Hanssen, B. R. Lindstrom,T. D. 1998. Increasing the throughput of microsomal stability screening using fast gradient elution LC/MS. Proc. 46th ASMS Conf. Mass Spectrom. and Allied Topics (Orlando, Florida), 16. 

The same considerations as for other subcellular systems with respect to solvent influences have to be considered in tests in 9000g fractions because organic solvents deactivate cytochrome P450 isozymes and others concentration dependently (Busby 1999 Easterbrook 2001). Solvent influences are lowest for methanol and acetonitrile (<10% inhibition at 0.3 % solvent), but higher for ethanol and DMSO. Therefore, as low as possible solvent amounts should be used. Published applications of microsomal stability tests use DMSO amounts of up to 1 % which clearly can lead to enzyme-specific inhibition. 

Di D, Kerns EH, Hong Y, Kleintop TA, McConnell OJ, Huryn DM (2003) Optimization of a Higher Throughput Microsomal Stability Screening Assay for Profiling Drug Discovery Candidates. J Biomolecular Screening 8 453 162... 

Gu and Lim [76] described the use of DDA for microsomal stability studies and metabolite profiling. First, the relevant m/z of an unknown lead compounds is determined. This information is used in SIM experiments for quantitation. Once the peak area of the compoimd tested falls below a certain threshold, e.g., 60% of the initial value, data-dependent product-ion MS-MS analysis is performed for metabolite identification. Some results for adatanserin are shown in Figure 10.11. 

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