This invention relates to a method for preparing a therapeutic agent that, upon ingestion decreases intestinal cholesterol absorption in man and, specifically inhibits or decreases intestinal cholesterol absorption by inhibiting the pancreatic cholesterol esterase catalyzed hydrolysis of naturally occurring and ingested cholesterol esters and by inhibiting the cholesterol esterase facilitated uptake of free cholesterol.
The invention is based upon the discovery that pancreatic cholesterol esterase is an important contributor to overall dietary cholesterol absorption because (1) cholesterol derived from cholesterol esters is preferentially absorbed compared to free-cholesterol; (2) cholesterol esterase enhances the absorption of free cholesterol and (3) dietary cholesterol and/or cholesterol esters induce the mRNA and enzymatic activity of cholesterol esterase in the pancreas in a newly discovered intestinal-pancreatic cycle for the absorption of cholesterol. U.S. Pat. Nos. 5,173,408 and 5,063,210 describe the importance of cholesterol esterase in the dietary uptake of cholesterol and also disclose methods for inhibiting cholesterol esterase. Thus, the surprising usefulness of inhibiting cholesterol esterase has demonstrated a new need for potent (Ki less than 5 xcexcM) and safe inhibitors of cholesterol esterase.
Many physical ailments are attributed at least in part to high levels of serum cholesterol. Atherosclerosis, for example, is a leading cause of death in the United States and high serum cholesterol concentrations are associated with increased risks of fatal artherosclerotic events. The discovery that the cholesterol esterase enzyme plays a role in intestinal cholesterol absorption has led to attempts to attenuate intestinal cholesterol absorption in man by inhibiting the action of the cholesterol esterase enzyme.
There is now an important need to develop human pancreatic cholesterol esterase inhibitors, especially those that are not absorbed essentially nondegradable. The pharmacology of various polysaccharides has been investigated. Cook and Cammarata, 1963, Arch. Int.: Pharmacodyn. 144: 1. In particular crude sulfated amylopectin has been taught in U.S. Pat. No. 4,150,110 as an anti-ulcer agent, but its property as a cholesterol esterase inhibitor has not been recognized. Sulfated dextran of low molecular weight has been recognized for use in the treatment of hyperlipemia and as an orally administered anticoagulant, British Patent No. 953,626.
Low molecular weight sulfated dextran (MDS-T) has been used to reduce serum cholesterol levels in Japan at a dose of 1800 mg/day, Goro et al, 1987, J. Clin. Biochem. Nutr. 2: 55-70 by activating a blood enzyme lipoprotein lipase. The low molecular weight of this bacterial dextran, (7-8 kDa), allows the sulfated dextran to be absorbed by the intestine as demonstrated by carbon-14 labelling studies. Drugs In Japan-(Ethical Drugs, 10th ed. 1986). It was developed for this property of intestinal absorption as indicated by the claim that faster decreases in serum lipids can be obtained by intravenous administration of this agent with clearance of serum lipemia due to activation of plasma lipoprotein lipase. Clearly this route of administration will not lead to effects on inhibiting cholesterol esterase in the intestine. Absorption of MDS can lead to a variety of side effects, most notably, anticoagulant effects that must be monitored. This preparation has not been known to inhibit cholesterol esterase and it is sulfated randomly and at various ring positions.
More recently, it has been discovered that crude non-absorbable polysaccharides sulfated at the three position are most effective as inhibitors of cholesterol esterase. U.S. patent application Ser. No. 08/121,369. Useful 3-sulfated polysaccharides may be derived from the synthetic sulfation of polysaccharides from various natural sources including seaweeds.
Methods for preparing sulfated polysaccharides are also known in the art. For example, U.S. Pat. No. 3,624,069 describes the sulfation of cellulose with a sulfur trioxide/lower n-dialkyl amide sulfation complex. U.S. Pat. No. 4,480,091 describes a process for preparing cellulose sulfate esters in a three step process. Finally, U.S. Pat. No. 4,814,437 describes a method for preparing sulfated polysaccharides by subjecting the polysaccharide to a reducing step prior to sulfation.
The present invention is directed to a method for manufacturing high molecular weight 3-sulfated polysaccharides that are essentially non-absorbable and nondegradable in the alimentary tract, and when administered orally, they are useful in decreasing human serum cholesterol and LDL levels by inhibiting human pancreatic cholesterol esterase, now recognized as a key enzyme involved in mediating cholesterol absorption. Thus, following the methods of this invention, greater than 95% of the compound has a molecular weight greater than 75,000 Daltons. The sulfate to monomer ratio is between 1.0 and 3.0 and less that 0.5% (by wt.) of the material will be free sulfate. This very high molecular weight compound can be administered to humans in tablet form, incorporated in a foodstuff, or by any other method that inhibits cholesterol absorption in the alimentary tract.