Targeted drug delivery using sulfonamide derivatives

Details Targeted drug delivery using sulfonamide derivatives Targeted drug delivery using sulfonamide derivatives

1999-03-17

2003-11-25

Rao, Deepak (Department: 1611)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Having -c-, wherein x is chalcogen, bonded directly to...

C514S347000, C514S367000, C514S603000, C514S604000, C546S288000, C546S293000, C548S166000, C564S087000, C564S092000

Reexamination Certificate

active

06653331

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to Glutathione S-transferase (GST)/Reduced Glutathione (GSH) as a means for the in-vivo release of a drug that has been conjugated to specific electrophilic moieties via a sulfonamide bond. The drug may be an anticancer agent (or one with other therapeutic properties) carrying a free —NH— which has been derivatized by the attachment of an electrophile containing a moiety, such as p-CN— or p-NO
2
-pyridinylsulfonyl groups, or p-NO
2
- or 2,4 dinitrophenylsulfonyl groups, or suitable derivatives thereof, to make a prodrug. The purpose of such a modification is to protect the free amino group, or to increase the drug solubility or alter absorption or distribution, or to improve some other physical, chemical, and pharmacological properties. Inside cells the prodrug is recognized and bound by GST which catalyses sulfonamide cleavage to release the active drug. Optionally, the sulfonamide moiety may have a targeting molecule attached to it.
The present invention also provides novel sulfonamide derivatives as a generic method adapted for —NH— protective groups in organic synthesis. Moreover, the invention relates to Glutathione S-transferase (GST)/Reduced Glutathione (GSH) as a means for the release of a protected amino derivative that has been conjugated to specific electrophilic moieties via a sulfonamide bond. The precursor is a synthetic intermediate carrying a free —NH— which has been derivatized by the attachment of an electrophile via a sulfonamide bond. The purpose of such a modification is to protect the free amino group during the chemical synthesis. The protected intermediate is recognized and bound by GST which catalyses the nucleophilic attack of GSH to the electrophilic part of the protected intermediate in a process that releases the desired intermediate harboring the free amino group under mild experimental conditions.
BACKGROUND OF THE INVENTION
A recent review article by J. D. Hayes and D. J. Pulford, Crit. Rev. Biochem. Mol. Biol. (1995) 30(6): 445-600, provides an illuminating overview of the GST supergene family and a detailed account of the individual isozymes' roles in protection against toxic agents and oxidative stress; it also offers insights into the various isozymes' regulation in diseased tissues, especially in cancer, and into their mechanisms of catalysis.
GST are enzymes ubiquitously present in living organisms. In mammals, GST are a large family of isozymes which are important components of the armamentarium of defense mechanisms that the cells use as a protection against foreign toxic chemicals and products of the oxidative stress. A variety of GST isozymes has been purified and extensively studied from rat and human organs, the most widely studied species. On the basis of amino acid sequence, the GST isozymes have been classified in five separate classes: alpha (A), mu (M), pi (P), sigma (S), and theta (T), that are abbreviated here by the use of the single letters indicated in parenthesis; each letter is then coupled to a number, e.g., A1, A2, A3, etc. to indicate the various gene products of class alpha subunits. In general, GST isozymes that have more than 40% identity in their amino acid sequences are assigned to the same class. The cytosolic GST of all mammalian isozymes are homodimers or heterodimers composed of subunits of Mr~23,000-26,000 that are catalytically independent; here the homodimer between two A1 subunits is indicated by the abbreviation GSTA1-1, and the formation of an heterodimer between the subunits A1 and A2 is abbreviated by GSTA1-2. The GST isozymes are distributed in various tissues, though some classes, or specific components of a class, are absent in particular organs. Human liver, is an abundant source of GST class alpha isozymes, but does not express specific class mu enzymes that are present in muscle, testis, and brain. Moreover, the distribution of GST isozymes varies in certain populations, e.g., among the three alleles present in the locus for human GSTM1 (GSTM1*A, GSTM*B, and GSTM1*0), the frequency of GSTM1*0 homozygosity is 22% in Nigerians, much lower than the 58% observed in the Chinese, 52% in the English, 48% in the Japanese, and 43% in the French populations. Moreover, the level of specific isozymes varies in relation to the diseased state of the cell, e.g., GSTP1-1 is overexpressed in various human tumors, including carcinoma of the lung, ovary, pancreas, stomach, colon, kidney, and esophagus. In keeping with their roles as detoxifying agents, GST isozymes are induced by the cell in response to xenobiotics and products of the oxidative stress.
Reduced Glutathione (GSH), the tripeptide &ggr;-Glu-Cys-Gly, widely used by mammalians to maintain their redox conditions, is the indispensable substrate that the GST family uses to catalyze a large number of reactions. The active site of cytosolic GST harbors a GSH specific binding site (the G-site), and a second site (the H-site) that accommodates a broad variety of hydrophobic substrates and endows this family of isozymes with the ability to exhibit various catalytic activities. The three-dimensional structure of the class alpha, mu, pi, sigma, and theta of GST revealed the presence of two domains in each subunit: the N-terminal domain, that in class alpha encompasses residues 1 to 78, contains most of the residues that form the G-site; while the C-terminal domain, that in class alpha comprises residues 86 to 222, houses most of the H-site. When the primary structures of the family members are compared, the H-site is much more variable than the G-site; this justifies the specificity of the G-site for GSH observed for all GST isozymes, and the differences observed between the family members for their H-site substrate specificity and inhibitor sensitivity. X-ray crystallography, and site-directed mutagenesis data have produced important information on GST mechanism of catalysis and on the residues that determine the catalytic specificity of the various isozymes for GSH, at the G-site, and for particular electrophiles, at the H-site. The key to GST catalytic mechanism is its ability to lower the pK
a
of the sulphydryl group of its bound substrate GSH from 9.0, in aqueous solutions, to about 6.5. This property favors the formation of the GS
−
anion which promotes, at physiological pH, nucleophilic attack to the electrophilic center of the compound bound to the H-site. The electrophilic center resides on a carbon, a nitrogen, or a sulfur atom to which GS
−
becomes conjugated during catalysis via a thioether bond. Conserved Tyr
9
(in class alpha) is the residue responsible for the abstraction of a proton from GSH leading to the formation of the GS
−
thiolate at the G-site. Moreover, the conserved Asp
101
(in class alpha), at the G-site, is also implicated in catalysis. The functions of various residues in specific subunits have been assigned; for example in the rat class alpha, subunit A
5
, Tyr
108
and Asp
208
have been implicated in conferring to this subunit the high stereospecific activity for aflatoxin B
1
exo-8,9-epoxide. While in the class mu enzymes V
9
and I
111
are the residues involved in the stereoselectivity and Tyr
115
in the activity toward epoxides. The selectivity of specific GST isozymes for particular substrates, and the presence of so many isozymes make it possible for the cells to cope with a multitude of noxious chemicals. The majority of the GST substrates are xenobiotics or products of oxidative stress that, upon conjugation to GS
−
, are less reactive than the parent compounds, therefore less toxic, and, because of their partnership with GSH are expelled from the cells by the action of the Glutathione S-conjugate pumps (detoxification). In addition to its ability to catalyze the formation of a thioether bond between GS
−
and various electrophiles, GST exhibits glutathione peroxidase activity (reduction of organic hydroperoxides to their corresponding alcohols), and isomerase activity (e.g. the cis-trans isomerization of maleylilacetone to f

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