Chemistry: analytical and immunological testing – Including chromatography
Reexamination Certificate
1999-12-23
2003-05-13
Tung, T. (Department: 1743)
Chemistry: analytical and immunological testing
Including chromatography
C436S103000, C436S501000, C422S069000, C422S070000, C210S198200
Reexamination Certificate
active
06562627
ABSTRACT:
This invention relates to a rapid, high throughput method for simultaneously measuring biologically significant physicochemical characteristics for multiple compounds. More particularly, this invention is directed to the measurement and use of binding affinities of multiple compounds with a surface under controlled conditions to calculate biologically significant physicochemical values. In one embodiment, the present invention provides a new method for the determination of a most important biological parameter, membrane interfacial
surface
pKa, the knowledge of which significantly facilitates understanding of drug-membrane interactions and thereby speeds the drug discovery process.
BACKGROUND AND SUMMARY OF THE INVENTION
Many potential new drug candidates are created every year, and both pharmaceutical and biotechnology industries have embraced the challenge in recent years of developing faster and more efficient ways to screen pharmaceutical compounds in order to rapidly identify “hits” and develop them into promising lead drug candidates. This has created the need for high-throughput analytical approaches to characterize the synthesized compounds and has prompted the development of chromatographic systems specifically designed for the automated high-throughput identification, purity assessment, purification or determination of the physicochemical properties of the compounds in combinatorial libraries.
With the advent of combinatorial chemistry and the need to develop assays for the large numbers of compounds being made available using that technology, many researchers have focused their efforts on developing in vitro tests/assays that provide biologically significant compound information. Much work has been directed to correlation of certain physicochemical properties with biological activity, both in the search for new therapeutic agents and in the understanding of compound toxicity both from medicinal and environmental perspectives.
The rise of combinatorial chemistry and other drug discovery technologies has vastly increased the number of new compounds to be evaluated as potential drug candidates. Accordingly, new high-throughput strategies are required to evaluate compound properties beyond potency and selectivity. A major focus in the pharmaceutical industry is to develop new drugs with good oral bioavailability. Bioavailability represents both the quantity of the drug administered reaching the blood circulation, and the rate of this phenomenon. It has been generally considered that the bioavailability of an orally administered drug is mostly determined by its physicochemical properties; e.g., its molecular weight, pKa, lipophilicity, solubility. One of the factors influencing oral bioavailability is the gastrointestinal pH, as it influences the ionization of the compounds. Most drugs are either weak bases or weak acids, and normally only the non-ionized fraction (i.e., the most lipophilic form) crosses biological membranes, except when transport carriers are involved. It becomes evident that knowledge of the pKa of potential drug candidates may give insight as to how good an oral bioavailability they will exhibit. In particular, having access to the dissociation state of said drug candidates at the membrane interface (
surface
pKa) would be helpful, as it would give an indication on their potential oral bioavailability and thus their pharmaceutical value. Thus, one physicochemical property of recognized significance to evaluation of a compound's biological activity, whether it be therapeutic efficacy or toxicity, is its dissociation constant, more significantly, the dissociation constant exhibited by the compound at a membrane interface.
For an ionizable compound, the degree of dissociation (or protonation) in solution is usually quantified by the pKa value of the acidic form. Thus, in the following acid-base equilibrium equation.
HA⇄A
−
+H
+
the dissociation constant Ka is expressed as:
K
a
=
[
H
+
]
[
A
-
]
[
HA
]
The pKa of a molecule, defined as −log Ka, is indicative of its degree of ionization, or of its acidic strength in solution. To be able to predict the extent to which a particular compound will ionize at a given pH (for example at physiological pH) is of great importance because it will give insight as to how well the substance in question will be able to participate in physical, chemical and biological reactions. A prominent example from medicinal chemistry is the ability of drugs to pass through biological membranes as well as their potential to interact with intracellular receptors, both of which are affected by the readiness of the drug to undergo protonation or deprotonation. Many biological processes involve reactions at the surface of, or within, cell membranes. Explanation of these functions in structural terms requires a physicochemical understanding of the various interactions taking place. In many cases the processes concern small amphiphilic molecules with pKa values close to physiological pH, and therefore both charged and uncharged forms may interact with the membrane. At the membrane surface, each form partitions into the membrane to a different extent, depending on the respective membrane affinities (FIG.
2
). The membrane-bound solute species are in turn involved in yet another acid/base equilibrium, with a dissociation constant
surface
Ka distinct from the above mentioned
bulk
Ka. The
surface
pKa (interfacial/surface pKa) is frequently biologically relevant, as it is the true indicator of the ionization state of the solute at the membrane interface, and therefore provides fundamental information of the solute/membrane interactions.
Several studies have demonstrated the key role that solute ionization plays in solute/membrane interactions. Biological membranes are made up of a lipid bilayer, which is a complex mixture of lipids and proteins held together mainly by non-covalent interactions. In such mixtures, phase separation leading to domain formation is possible, where the lipids are not distributed uniformly but rather in “clusters”. The domains' surfaces differ in physicochemical properties, and a particular chemical (drug) is expected to have different membrane binding and partitioning behaviors (dictated in part by its
surface
pKa) with the membrane domains. This may contribute to accumulation and co-localization of receptors and drugs in the same (small and specialized) membrane region. It has been shown that solute ionization can determine the depth of membrane penetration and location of solutes in membranes, as well as affect the membrane physiology. The local anesthetic tetracaine hydrochloride (TTC) is a good example, because at pH 5.5 the positively charged TTC resides near the phospholipid head group region, whereas at pH 9.0 the uncharged TTC penetrates more deeply into the hydrocarbon region of the membrane. This membrane penetration behavior significantly affects the expansion of the membrane, which has been proposed as one aspect of the mechanism of action of anesthetics. This study not only demonstrates that membrane physiology is affected by charged versus neutral molecules, but also that the activity of the compound, and the depth of its membrane penetration depend on its ionization state at the membrane surface and not in the bulk solution.
In addition to affecting the location of molecules in cell membranes, solute ionization also affects solute movement from the outer monolayer to the inner monolayer of cell membranes: for example, it has been reported that the migration of unionized fatty acids across the phospholipid bilayer is much greater than for their ionized counterparts. The rapid flip-flop of unionized fatty acids has important physiological implications as it enables the rapid entry and removal of fatty acids from cells such as adipocytes, hepatocytes, and heart muscle tissue. Movement between the inner and outer monolayer of cell membranes is necessary for transport not only of fatty acids but also many biologically important compounds. For example it has
Morgan Ling Han
Pidgeon Charles
Rooke Nadege
Yin Jianming
Barnes & Thornburg
BDC Pharma LLC
Gakh Yelena
Tung T.
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