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SOLUTION STABILITY
강경태
Overview
in solution
excipients
pH
enzymes
decomposition
oxygen
solution
components
temperature
Instability in solution can cause erroneous in vitro assay or in vivo
pharmacokinetic results.
Stability increases by replacing the unstable group, adding steric
hindrance, or electron withdrawal.
Solution Stability Fundamentals
Organic solvent stocks
Aqueous buffers
The specific conditions and components of each
of these solutions can cause compound
instability. Some of these challenges are as
follows
pH
Water
Bioassay buffers
Counter ions of salts
Dosing solutions
Solution components (DTT)
Excipients
GI tract
Enzymes
High-performance liquid
chromatography modifiers
Compounds are exposed to a wide range of
solutions during drug discovery.
These solutions include the following:
Temperature
Light
Oxygen
Stock Solutions
Organic or aqueous buffer solutions can expose compounds to decomposition
reactions induced by ambient laboratory light, elevated temperature, and oxygen
absorbed from the air.
paclitaxel epimerizes in solutions that are slightly basic.
mitotic inhibitor used in cancer chemotherapy.
Taxol.
Bioassay buffers
Bioassay buffers can contain components that promote compound decomposition.
pH can promote hydrolysis or hydrate formation.
DTT can cause reduction or react as a nucleophile.
Solution components can react with discovery compounds.
Excipients
In the GI track
oral, intraperitoneal, or IV administration may promote compound decomposition.
For example, lactic acid-containing dosing solutions
a wide variety of pHs.
acidic in the stomach and upper intestine
basic in the colon.
A wide array of hydrolytic enzymes are present in the GI tract
Pepsin & pancreatin) - These enzymes have the natural function of digesting
macromolecules to monomers for use as nutrients,
but they can also bind and hydrolyze drug compounds.
Hydrolysis Reaction
aspirin
Hydrolysis probably is the most common reaction
causing instability during discovery.
Hydrolysis can be catalyzed in solution by acidic or basic conditions.
chemical constituents, physicochemical nature of the solution.
chemical and enzymatic components in the solution.
Other reactions
- Isomerization
- Dimerization
- Rracemization.
Effects of Solution Instability
Bioassay Buffer
Chemical instability in bioassay buffer solution reduces the compound concentration and produces decomposition
products that themselves may be active.
Know stability data.
Without stability data.
Organic solvent
Oxygen
Water
Metal
Leached material
In DMSO
Light
Light can induce reactions to certain sensitive compounds in a solution that is
exposed to laboratory light.
Structure Modification Strategies to Improve
Solution Stability
Structure modificaion
Elimination or modify unstable group
Add an electron-withdrawing group
Isosteric replacement of labile functional group
Increase steric hindrance
Elimination or modify the unstable Group
Artemisinin : 개똥쑥 추출물, 말라리아 치료제
Hydrolysis site
10 fold
Conjugation
Non - conjugation
Lipxin analog : 3개의 hydroxyl group 을 가진 구조
Add an Electron-Withdrawing Group
Epothilone B(Me)
50 fold
Me
CN
Isosteric Replacement of Labile Functional Group
If the functional group makes a significant contribution to the therapeutic
target binding,it is important to know what aspects of the group contribute to
active site interactions and attempt to find an isosteric replacement that aids
binding without contributing to instability.
Increase Steric Hindrance
Application of Solution Stability Data
Provide an Early Alert to Liabilities
Profiling data of B-lactam compound.
Not stable at low or high pH values
Information on the stability of compounds at different pH
values
best bioassay conditions
designing synthetic strategies
developing optimal formulations
predicting oral absorption
Selection of conditions for compound purification
The compound degraded at pH 1
Application of Solution Stability Data
Diagnose Poor In Vitro Bioassay Performance
White – stable
Gray – moderately stable
Black - Unstable
Stability screening in a 96-well format can quickly evaluate a large number of
compounds to diagnose their bioassay stability.
Application of Solution Stability Data
Diagnose Poor In Vivo Performance
Poor PK or pharmacological performance can occur in vivo if the compound is not stable in the GI system.
Low pH and enzymatic hydrolysis in the stomach or intestine can reduce in vivo exposure.
In vitro test
simulated gastric fluid
simulated intestinal fluid
Prioritize Compounds for In vivo
Animal Studies
Knowledge of the stability of compounds in simulated
physiological fluids can help
evaluate whether they should be tested in vivo.
stable
Structure Elucidation of Solution Stability Products Guides Synthetic Optimization
LC/MS
Method
General method for Solution Stability Assays
Solution stability assays have a unique problem with quenching the reaction.
96-well plate
Runtime : 5 min
처음과 끝 약 7시간 차이
General method for Solution Stability Assays
Solution stability assays have a unique problem with quenching the reaction.
Multichannel
HPLC
Another
approach
8~24 samples by HPLC with UV detector
not directly coupled to a mass spectrometer (MS)
incubation of each sample at a different time preceding the time zero injection.
same incubation time injected.
96-well plates.
frozen samples at –80°C to stop the reaction.
Pump ( LPG-3400 SD)
Autosampler (WPS-3000 TSL)
Column Oven (TCC-3000 SD)
Detector (VWD-3400 RS)
Computer
Methods for pH Solution Stability
Compounds in drug discovery encounter a wide range of pHs.
Bioassay buffer and physiological fluids – pH 7.4
stomach – pH 1 to 2
beginning of the small intestine - pH 4.5
average pH for the small intestine - pH 6.6
colon - pH 5 to 9
An integrated generic method
profiles solution stability at multiple pHs and
oxidation with 3% hydrogen peroxide
in a 96-well plate using a Gilson 215 robot.
Compounds incubation - 100 μM
low-solubility compounds were kept in solution
using 1:1 acetonitrile to buffer.
HPLC
pH stability studies have been used for in vitro evaluation of prodrugs.
Solution stability studies used buffers at 0.02 M concentration
with acetate (pH 5.0), phosphate (pH 3.0, 6.9,7.4), borate (pH 8.5 and 9.75),
and hydrochloric acid (pH 1).
Methods for Solution Stability in Simulated
Gastrointestinal Fluids
The components of SGF
Pepsin (an acidic protease)
pH 1.2 adjusted using HCl
NaCl
SGF simulates stomach fluid and incorporates
acidic and enzymatic hydrolysis conditions.
The components of SIF
Pancreatin (mixture of amylase, lipase, and protease from hog pancreas)
pH 6.8 using monobasic phosphate buffer adjusted with NaOH
SIF mimics the pH and hydrolytic enzymes in the intestine.
Purpose
prediction or diagnosis of stability after oral dosing.
Incubation => HPLC
Identification of Degradation Products from Solution
Stability Assays
solution stability experiments follows the same methodology as discussed
for plasma stability and metabolic stability.
Molecular weight – LC/MS
the molecular weight of putative decomposition products can be rapidly
calculated and compared to the results.
Such information is superior to quantitative data alone
because it can guide medicinal chemists in making structure modifications to
improve stability.
In-depth Solution Stability Methods for Late Stages of Drug Discovery
In late stages of drug discovery,
Standard assays are used for the scrutiny of all compounds so that the data are
Standard
Assay
comparable across all projects and are applicable to established advancement
requirements.
The assays often are similar to those suggested by the Food and Drug
Administration for regulatory filings in new drug applications.
pH: Aqueous buffers (37°C, pH 1–12)
Oxidation: 3% hydrogen peroxide in pH 7.4 buffer for 10 minutes
GI: Simulated intestinal fluid (USP, 37°C, 1–24 hours)
GI: Simulated gastric fluid (USP, 37°C, 1–24 hours)
GI: Simulated bile/lecithin mixture (USP, 37°C, 1–24 hours)
Plasma: Human plasma (37°C, 1–24 hours)
Light: High-intensity cool white fluorescent light (200 watt h/m2, 1.2 million lux hour,
room temperature, 1–7 days)
Temperature: Heat (30–75°C, 1–7 days)
Problems
1. In which of the following solutions might a compound be unstable?: (a)
stomach fluid, (b) enzyme assay media, (c) high-throughput screening
buffer, (d) pH 7.4 buffer, (e) animal gavage dosing solution, (f) ethanol stock
solution, (g) cell assay buffer.
- All
2. If a compound is unstable in solution, wouldn’t it be best to eliminate it from
further study?
No. The stability might be improved without reducing activity by structural
modification,
thus allowing a valuable pharmacophore/series to continue optimization toward
a quality clinical candidate.
Method
1.
List some of the solution conditions under which compounds may be unstable or which
may accelerate decomposition.
- Bioassay media components, pH buffers, intestinal fluid, gastric fluid, enzymes, light,
oxygen, temperature.
2. What is a difficulty of solution stability assays?
- Quenching the reaction.
3. What approach can be used to effectively and efficiently run solution stability assays?
- Use a programmable HPLC autosampler that can add reagents, mix, inject at predetermined time
points, and perform these functions for multiple samples.
4. What conditions should be used for solution stability studies?
Use the same conditions and protocol that are relevant to the project’s experiment in
question.
5. In addition to percent remaining at a certain time point, what additional data can be obtained
from solution stability studies and how can they be used?
- Reaction kinetics (used to predict long-term stability); structures of decomposition products
(used to modify structures for improved stability).
6. For what purposes can solution stability data be used?
Diagnose unexplained results from in vivo or in vitro experiments; rank ordering compounds
for stability; apply kinetics for planning other experiments or clinical studies;
predict how much compound remains at various times; determine which moiety is unstable;
guide structural modifications to improve stability.