US 2008O131500A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0131500 A1 CHANG (43) Pub. Date: Jun. 5, 2008 (54) METHODS AND COMPOSITIONS FOR AOIP I/00 (2006.01) RAPID INACTIVATION OF PROTEINS A6II 47/02 (2006.01) A 6LX 39/395 (2006.01) (75) Inventor: ROWEN.J.-Y. CHANG, Houston, C07K I4/00 (2006.01) TX (US) AOIN 25/00 (2006.01) (52) U.S. Cl. ......... 424/451; 530/410; 530/408; 530/402; Correspondence Address: 530/409:514/769; 424/130.1; 424/45 FULBRIGHT & UAWORSK L.L.P. 600 CONGRESSAVE., SUITE 2400 (57) ABSTRACT AUSTIN, TX 78701 Disclosed are methods of inactivating a protein, Such as leavi disulfide bond of tein, that invol tacti (73) Assignee niversityif of Texasits Systemof the ideSAWENGy ion, wherein the pH of the composition is about 10.0 to about 14.0. Also disclosed are methods of treating or prevent (21) Appl. No.: 11/566,595 ing a disease in a subject, such as a toxin-related disease or a 1-1. prion-related disease, that involve contacting a subject with a (22) Filed: Dec. 4, 2006 pharmaceutically effective amount of a reducing agent, a O O denaturant, and a hydroxide ion, wherein the pH of the com Publication Classification position is about 9.0 to about 14.0. Also disclosed are com (51) Int. Cl. positions that include a reducing agent, a denaturant, and a A6 IK 9/48 (2006.01) hydroxide ion, wherein the pH of the composition is about 9.0 A6IP 25/28 (2006.01) to about 14.0. -- f Reductarteraturant SH it H. H, Hydroxide or H. H, mail-HCOrr-NH-CH-COsawa. -- -NH-Clico-NH-citco Base catalyzed r S-sinatic (2) -NH-EH-CO-NH-CH-Coit, it, Coupling (3) l Coupling lysife Cysteine sysinoalarine risie rai's e. "r r is Aih-H-co r Aih-H-cos I S (GH, H, - NH-CH-C-NHCH-CQ x w w - Patent Application Publication Jun. 5, 2008 Sheet 1 of 7 US 2008/O131500 A1 -N Reductart eraturnt sh H. H, Hydroxide for H. H, ...-H. H.C.--NHCH-CO------- " --es-NH-CH-COsm-N-CHCOreer (1) oH- oh -NH-CH-CO-NH-CH-CO-sait, H, Coupling l (3) Coupling lysire Cysteine alysirhaiarie left tissile * ?ki-h-cos(th... ', Patent Application Publication Jun. 5, 2008 Sheet 2 of 7 US 2008/O131500 A1 FIG 2 Patent Application Publication Jun. 5, 2008 Sheet 3 of 7 US 2008/O131500 A1 O. M. CdSCN ().2 M. GinSCN R 5 -- or -N-ter X XX. ------N- comin ---Y-- " R ().5 N, CSCN N R 1. M GdnSCN FIG. 3 Patent Application Publication Jun. 5, 2008 Sheet 4 of 7 US 2008/0131500A1 0.5N NaOH N N NaOH R 0.05M DTT 0, JM DT 0.5M GdnSCN 1M GdnSCN 20 sect FG. 4 Hirudin CTX-III .--/ N.-- 60 min -1N-J.-- 60 min FIG.S Patent Application Publication Jun. 5, 2008 Sheet 5 of 7 US 2008/0131500A1 2O see Thir 15 Yi arX X 6O irri FIG. 6 Patent Application Publication Jun. 5, 2008 Sheet 6 of 7 US 2008/O131500 A1 FIG. 7 Patent Application Publication Jun. 5, 2008 Sheet 7 of 7 US 2008/O131500 A1 FIG. 8 US 2008/013 1500 A1 Jun. 5, 2008 METHODS AND COMPOSITIONS FOR (BSE) in cattle, also know as “Mad Cow Disease.” scrapie in RAPID INACTIVATION OF PROTEINS sheep, and wasting disease in elk. These diseases affect the neurological system of the animal or animals and are charac BACKGROUND OF THE INVENTION terized by initially long incubation times followed by a short 0001 1. Field of the Invention period of neurological symptoms, including dementia and 0002 The present invention relates generally to the fields loss of coordination, and eventually death. of protein chemistry, toxins, and prions. More particularly, it 0009. The infectious agent responsible for these diseases concerns compositions and methods of inactivating a protein is thought to be a simple protein, with no associated nucleic or cleaving a disulfide bond of a protein that involve a reduc acids. The pathogenic mechanism for prion diseases has been ing agent, a denaturant, and a hydroxide ion at a pH of 10.0 to proposed to involve an initially normal host encoded protein 14.0. It also concerns methods of treating or preventing a that undergoes a conformational change to become an abnor disease in a Subject that involve contacting the Subject with a mal form (a prion), which has the ability of self-propagation. pharmaceutically effective amount of a composition that The abnormal form of the protein is not broken down effec includes a reducing agent, a denaturant, and a hydroxide ion. tively in the body and its accumulation in certain tissues (in 0003 2. Description of Related Art particular neural tissue) eventually causes tissue damage and 0004. Many naturally occurring toxins and poisonous the associated clinical signs. compounds are proteins that contain disulfide bonds. These 0010. There are currently no known effective treatments compounds include a wide range toxic proteins isolated from for prion diseases in animals or humans, and death thus fol plants, bacteria, Venom of reptiles and insects, etc. Some of lows the onset of neurological symptoms. Progress in the these toxic proteins have found their benefit in disease treat identification of target treatment drugs has been slow, due to ment, but many of them are harmful or lethal upon inadvertent the inability to perform testing in vitro. In addition, because contact and injection to human. Specifically some plant tox these diseases tend to be animal specific, it is not known ins such as ricin (Audi et al., 2005; Mantis, 2005) from castor whether tests done on animals can be readily applied to beans and related abrin, have caused serious concerns as humans. There have been a few reports of methods for decon potential weapons of terror attack and biological warfare due tamination of Surfaces contaminated with prion-infected to the simple process of their production and their worldwide material (see, e.g., U.S. Pat. No. 7,071,152 and U.S. Pat. No. availability (Bigalke and Rummel, 2005). Consequently, 7,001,873). These methods are limited by requiring pro development of an efficient and effective method of inactiva longed contact of the active agent with the Surface or numer tion of these toxic proteins is imperative. ous steps. 0005 Active disulfide proteins can be inactivated by dif 0011 Prions are notoriously very hardy and demonstrate ferent methods. For example, they can be inactivated by dena resistance to routine methods of decontamination and steril turation without covalent modification of the protein. This is ization. There is a clear need for products and processes that accomplished by various approaches, including application are effective against prions. of chemical denaturants, heat, extreme pH, detergents or 0012. Therefore, there is the need for more efficient and organic solvents (Pace et al., 1989; Volkin and Klibanov, effective methods of inactivating disulfide containing pro 1989). However, most processes of denaturation are readily teins. Such methods can be applied in the rapid inactivation of reversible. For instance, proteins denatured by chemical toxins and poisonous proteins that pose a threat to humans denaturants (urea or guanidinium chloride) or heat usually renature and refold spontaneously following removal of the SUMMARY OF THE INVENTION denaturant and decrease of the temperature. 0006 Alternatively, active disulfide proteins can be inac 0013 The inventor has identified novel compositions and tivated by proteolysis. The process is irreversible, but it typi methods that can be applied in the rapid inactivation of pro cally takes hours and requires enzymes that have to be dealt teins. In particular, a novel composition has been identified with latter. Disulfide proteins can be inactivated by chemical which enables a rapid (e.g., 20-30 seconds), irreversible, and reduction of their disulfide bonds. However, this process is quantitative inactivation of disulfide containing proteins. The also reversible because fully reduced proteins with intact reaction can take place at room temperature (20°C. to 25°C.). cysteines are usually capable of renaturation via Oxidative The formula includes a denaturant, a reductant and hydroxide folding (White, 1972: Creighton, 1986), if appropriate con ion. The component of hydroxide ion serves two major func ditions are given. tions. First, it accelerates the cleavage of disulfide bonds 0007 Thus, most disulfide-containing proteins can be per mediated by the reducing agent and denaturant, leading to an manently inactivated only if their disulfide bonds are instant inactivation of disulfide proteins. Second, it triggers a covalently modified or annihilated. Although such methods rapid covalent destruction of sulfhydryl groups and disulfide are available, they require either highly noxious chemicals or bonds via the mechanism of base catalyzed B-elimination, work in a relatively sluggish fashion. For example, the cleav thus leading to the permanent inactivation of toxic disulfide age of disulfide bonds by performic acid oxidation requires proteins. Usefulness of this invention has been demonstrated highly acidic solution (Spackman et al., 1960). The method of with the effective and rapid inactivation of numerous highly reduction and alkylation entails tedious and stepwise chemi stable disulfide containing proteins, including cardiotoxin, as cal reactions (Waxdal et al., 1968). discussed in the Examples below. 0008. One example of a disulfide-containing protein is a 0014. The present invention generally pertains to methods prion. A "prion' is defined herein to refer to a proteinaceous for inactivating a molecule. Such as a protein, that involves infectious agent that causes relatively similar brain diseases contacting the molecule with a composition that includes a in humans and/or in animals, which are invariably fatal.