Inhibition of cholesterol absorption with CP- 148,623 lowers serum cholesterol in humans

Objective: To determine the effects of the reduction of intestinal cholesterol absorption with CP-148,623 on serum cholesterol levels in men with mild hyperlipidemia. Methods: In an outpatient study in a university medical center, healthy male volunteers (n = 25) with borderline-high serum cholesterol levels participated in a double-blind, placebo-controlled parallel-group study. A 3-week dietary run-in period was followed by 2 weeks of treatment with either CP-148,623 (300 mg twice a day; n = 12) or placebo (n = 13). Reszklts: Fractional cholesterol absorption (by the dual-isotope, continuous-feeding technique), fecal neu- tral sterol excretion, and serum lipids were measured after the diet run-in and after the treatment periods. CP-148,623 caused a marked inhibition (by 38%) of fractional cholesterol absorption (50% + 2% [base- line] to 31% f 1%) and a 71% increase in fecal neutral sterol excretion (481 + 39 mg/day [baseline] to SO4 * 55 mg/day), compared with negligible changes in the placebo group (p < 0.0001 for both). Mean percent reductions from baseline in serum low-density lipoprotein (LDL) cholesterol levels were 11.6% with (X-148,623 (119 + 17 mg/dl to 104 & 13 mg/dl) versus a nonsignificant 1.8% reduction with placebo (change with CP-148,623 versus placebo, p < 0.0002). Concl~siorzs: In healthy male volunteers with mild hypercholesterolemia, treatment for 2 weeks with 600 mg/day CP-148,623 inhibited f rat ti onal cholesterol absorption by 35% to 40%, increased fecal neutral sterol excretion by 60% to 70%, and reduced serum LDL cholesterol by 10% to 12%. (Clin Pharmacol Ther 1997;61:385-9.)

William S. Harris, PhD, Sheryl L. Windsor, MT (ASCP), Fran A. Newton, MS, and Robert A. Gel&and, MD Kansas City, Kan., and Gpoton, Conn.

Dietary cholesterol intake is known to influence plasma cholesterol levels. l-5 This presumably occurs through delivery of intestinally derived cholesterol to the liver in chylomicron remnants. Increased lev- els of intracellular cholesterol down-regulate the hepatic low density lipoprotein (LDL) receptor, and serum LDL cholesterol levels rise. Thus reduction of the mass of cholesterol absorbed from the gut should act to lower the serum cholesterol concen- tration.

From the Lipoprotein and Nutrition Research Laboratory, Divi- sion of Clinical Pharmacology, Department of Medicine, Uni- versity of Kansas Medical Center, Kansas City, Kan., and Pfizer Central Research, Groton, Conn.

Supported by a grant from Pfizer, Inc., Groton, Conn. Received for publication April 19, 1996; accepted Sept. 26, 1996. Reprint requests: William S. Harris, PhD, Lipid Research Lab-

oratory, Saint Luke’s Hospital, 4401 Wornall Road, Kansas City, MO 64111.

Copyright 0 1997 by Mosby-Year Book, Inc. 0009-9236/97/$5.00 + 0 13/l/78251

Nonsystemic drugs that reduce cholesterol ab- sorption have been investigated because of their potential to safely reduce LDL cholesterol levels6-lo It is possible that the divergent effects on serum cholesterol levels reported in the literature may have been caused by effects other than the simple inhibition of cholesterol absorption (e.g., neomycin alters gut flora, high doses of fibers influence gut transit, plant sterols interfere with micelle forma- tion, and acylcholesterol acyltransferase (ACAT) in- hibitors influence enzyme activity throughout the body). It would be of interest to determine the effects on serum cholesterol levels of compounds that prevent only the intestinal absorption of cho- lesterol. Naturally occurring”>‘* and synthetic13 sa- ponins are known to bind with cholesterol, precipi- tate it, and reduce its absorption from the gut. CP-148,623 [P-(11-ketotigogenin)-cellobioside] is a poorly absorbed, synthetic saponin with marked hy- pocholesterolemic effects in animals that consume

385

386 Hamis et al. CLINICAL PHARhM COLOGY & THERAPEUTICS

MARCH 1997

Table I. Baseline characteristics of the treatment groups

Placebo CP-148,623

n Age BMI (kg/m’) Fasting lipids (mg/dl)

Total cholesterol Triglycerides LDL cholesterol HDL cholesterol

13 12 30 5 9 33 t 11 25 k 2 25 t 3

212 k 20 217 2 26 122 t 79 120 ? 39 137 ? 25 142 t 19 51 + 10 52 !r 14

BMI, Body mass index, LDL, low-density lipoprotein; HDL, high- density lipoprotein.

both cholesterol-rich and cholesterol-free diets.14 The drug appears to block entry of luminal choles- terol into the enterocyte.15 Therefore the purpose of this trial was to use CP-148,623 to explore the effects of reduced cholesterol absorption on serum LDL cholesterol levels in healthy volunteers.

SUBJECTS AND METHODS Su&ects (Table 1). Healthy males (n = 25) were

recruited who were 18 to 59 years of age, not over- weight (less than 120% of ideal body weight), and who had fasting total serum cholesterol levels be- tween 180 and 300 mg/dl and fasting serum triglyc- eride levels under 250 mg/dl at screening. Although mean levels of serum total and LDL cholesterol (on home diets) were typical for the U.S. population, they were borderline-high according to current Na- tional Cholesterol Education Program (NCEP) guidelines.

Design. The study used a randomized, placebo- controlled, double-blind parallel-group design with a 3-week diet run-in period followed by a 2-week treatment phase. Subjects were randomized to re- ceive 600 mg/day CP-148,623 (300 mg twice a day [breakfast and supper]) or placebo. The study was approved by the Human Subjects’ Committee of the University of Kansas Medical Center.

Diets. All food was provided to the subjects for the entire 5 weeks of the study. An NCEP step I diet was formulated on an individual basis to maintain body weight. Subjects ate three supervised meals per day at the research facility and took an evening snack home with them at night. Cholesterol intakes were distributed evenly over the three major meals.

Fractional cholesterol absorption. The continuous feeding, dual-isotope method was used to determine fractional cholesterol absorption.16 For 5 days at the end of the baseline and the treatment periods, sub-

jects were given a known ratio of [4-14C]-cholesterol (New England Nuclear, Wilmington, Del.) and [5,6- 3H]-sitostanol (American Radiolabeled Chemicals, Inc., St. Louis, MO.) with each of the three major meals. Sitostanol is a virtually unabsorbable plant sterol.i7 On days 4,5, and 6 (when the subjects were confined nightly at the research facility), all stools were collected and the ratio of isotopes excreted determined. The change in the fed-to-excreted ratio of 14C/3H was used to determine fractional choles- terol absorption.

Fecal neutral steroid excretion. The absolute rate of excretion of neutral steroids (cholesterol and its bacterial metabolites) was determined by feeding each subject a known amount of B-sitosterol, an- other poorly absorbed plant sterol,” each day for the 5 weeks of the study. The B-sitosterol was given both in capsules (180 mg/day) and as a natural component of the diet. The fecal neutral steroids (including B-sitosterol) were extracted from the pooled stool samples and the total mass of steroids excreted was determined. The daily output of neu- tral steroids was calculated on the basis of the mass of B-sitosterol present, which served as a control for variations in fecal flow and recovery.

Plasma lipids and lipoproteins. Whole plasma tri- glyceride and cholesterol levels were measured en- zymatically and in duplicate on a Cobas Mira clinical analyzer as described previously.” Plasma high- density lipoprotein (HDL) cholesterol was mea- sured after precipitation of the apolipoprotein B-loo-containing lipoproteins with dextran sulfate and magnesium chloride.*’ Very low-density li- poprotein (VLDL) and LDL were isolated by B-quantitation.lg Our laboratory participates in the Centers for Disease Control/National Institutes of Health Lipid Standardization Program and the ALERT program from Pacific Biometrics Research Foundation (Seattle, Wash.).

Measurement of dietary and fecal steroids. Dried homogenates (250 mg) of stool (one part to two parts distilled water) or food were combined with an inter- nal standard (0.2 mg So-cholestane), 0.5 ml distilled water, and 1 ml of 1 mol/L sodium hydroxide in etha- nol and incubated for 60 minutes at 67” C in a screw- top glass tube. The samples were then twice extracted with 5 ml petroleum ether. One milliliter of silating reagent (Tri-sil, Pierce, Rockford, Ill.) was added to the dried extract, and the solution was incubated at room temperature for 30 minutes. After evaporation of the solvent, the trimethylsilated ethers were taken up in 0.1 ml n-decane. The sample was analyzed by

CLINICAL P HARMACOLOGY &THERAPEUTICS VOLUME 61, NUMBER 3 Hawks et al. 387

Table II. Effects of CP-148,623 on serum lipid and lipoprotein cholesterol levels

LDL cholesterol

(mgidl)

HDL cholesterol

(mgldl)

Placebo (n = 13) Diet run-in End Mean percent change*

CP-148,623 (n = 12) Diet run-in Treatment Mean percent change*

p Value?

199 + 19 118 ? 52 130 + 20 46 + 13 194 2 22 106 2 37 127 ? 12 46 + 12 -2.2% -6.0% - 1.8% 1.1%

194 AI 22 144 2 61 119 +- 17 46 t 13 174 i. 15 113 t 44 104 t 13 47 + 12 -9.6% - 17.5% -11.6% 4.3%

0.001 0.12 0.002 0.34

*The mean, percent change; not the percent change in means. tcomparison of the mean, percent change in the placebo group to that in the CP-148,623 group.

gas-liquid chromatography (GC14A, Shimadzu, Co- lumbia, Md.) With use of a 60 m, 0.32 mm internal diameter SPB-1 column (Supelco, Bellefonte, Pa). Conditions were as follows: H, carrier gas; initial tem- perature, 170” C; program to rise at 20” C/min to 265” C and hold for 44 minutes. Excretion rates of cholesterol and its metabolites (coprostanol and coprostanone) were compared with those of p-sitosterol and its metabolites (P-sitostenol, methyl- coprostanone, and ethylcoprostanone) to determine daily neutral sterol excretion rates.

Statistics. Data are presented as mean values ? SD. Percent changes from the end of the diet run-in period to the end of 1 and 2 weeks of treatment were compared between groups by t test. Comparisons before and after treatment within each group were made with the paired t test. Ap value of less than 0.05 was required for statistical significance.

RESULTS Body weight stability. Mean body weights did not

vary by more than 1 kg throughout the study. Diet composition. The diet contained 31% 2 0%

energy as fat, 9% +- 0.5% saturated fat, and 304 ? 20 mg of cholesterol. The RISCC rating (ratio of ingested saturated fat and cholesterol to calories) of the diet was 16 2 0.8 (13 to 20 is a step I diet).21

Plasma lipids and lipoproteins (Table II). The step I diet had its expected effect in these subjects, who were consuming a typical American diet before they entered the study. In 3 weeks, total cholesterol levels decreased from 214 to 196 mg/dl (p < O.OOOl), LDL cholesterol decreased from 139 to 125 mg/dl (‘p < 0.0002), and HDL cholesterol decreased from 51 to 46 mg/dl @ < 0.001). No further changes in these parameters were noted in the placebo group after 2

more weeks of diet. However, those subjects given CP-148,623 for the next 2 weeks had an additional mean 12% reduction in LDL cholesterol levels (p < 0.002). This reduction had already been achieved after just 1 week of treatment (data not shown). Total cholesterol levels were reduced by a net of 7.4% (p < 0.001) after 2 weeks, whereas triglyceride and HDL cholesterol levels were unchanged.

Fractional cholesterol absorption (Fig. 1). Choles- terol absorption was reduced by 38% (from 50% to 31%; p < 0.0001) by CP-148,623. Absorption in the placebo group was unchanged.

Fecal neutral steroid excretion (Fig. 1). Neutral ste- roid excretion increased by 71% (from 481 to 804 mg/day; p < 0.00003) after 2 weeks of treatment with CP-148,623. No change was noted in the pla- cebo group. A similar increase in excretion rates occurred after just 1 week, with the placebo group showing a 7% + 20% increase compared with a 73% ? 26% increase in the active treatment group (‘p < 0.00001).

DISCUSSION CP-148,623 is a synthetic saponin with very low

bioavailabilie2 that has been shown to markedly inhibit cholesterol absorption in animal mod- e1s.14,15,23 It was used in this study to explore the impact of reduced cholesterol absorption on serum LDL cholesterol levels. We found that the drug (600 mg/day) inhibited cholesterol absorption by 38%, increased cholesterol excretion by 71%, and lowered serum LDL cholesterol levels by 12% in healthy volunteers. Thus CP-148,623 effectively blocked cholesterol absorption, which presumably led to an up-regulation of hepatic LDL receptors and a re- duction in serum LDL cholesterol levels. The reduc-

388 Harris et al.

Placebo CP148,623 -

1

pc0.0001

g 700 E 600 l3 500 0 g 400 8 300 g 200

g loo 0

Placebo CP148,623

Fig. 1. The effects of 2 weeks of treatment with CP- 148,623 (300 mg twice a day; n = 12) or placebo (n = 13) on fractional cholesterol absorption (top panel) and on neutral steroid excretion rates (bottom panel) in healthy volunteers. Values at the beginning of treatment (shaded bars) are com- pared with those at the end of treatment (open bars).

tion in cholesterol absorption was large (38%), con- sidering that the drug was administered to healthy men consuming a strict low-fat, low-cholesterol diet, and it was given at only two of the three daily meals, leaving cholesterol absorption unimpeded at one meal. The 12% reduction in serum LDL cholesterol levels observed in this setting suggests that even greater effects might be obtained with three-times- a-day administration to patients with hyperlipidemia who are consuming typical American diets, but this remains to be tested.

Clearly, the relationship between the amount of cholesterol absorbed and its resulting effect on the serum cholesterol concentration is more compli- cated than it appears on the surface. A doubling of hepatic cholesterol synthesis was documented in pa- tients with nearly complete cholesterol absorption blockade,7 and this undoubtedly contributed to the relatively small changes in serum cholesterol levels observed. The results of this trial suggest that pro- found inhibition of absorption will be necessary to produce clinically meaningful reductions in serum cholesterol levels.

References As noted earlier, a variety of therapeutics have 1. Ginsberg HN, Karmally W, Siddiqui M, Halleran S,

been developed to reduce the absorption of choles- Tall AR, Blaner WS, et al. Increases in dietary cho- terol from the intestinal tract. Among them are lesterol are associated with modest increases in both

CLINICAL PHARMA COLOGY & THERAPEUTICS MARCH 1997

plant sterols (Cytellin)F4 sitostanol,’ neomycin,25’26 and ACAT inhibitors.6p10 Although each of these can reduce cholesterol absorption, none except neo- mycin lower serum cholesterol by more than about 10%. This is undoubtedly in part attributable to a compensatory increase in whole body cholesterol synthesis that accompanies the inhibition of choles- terol absorption.7’26

In experiments designed to determine the effects of dietary cholesterol on serum cholesterol levels, Mattson et a1.27 found that complete elimination of all cholesterol from a diet that contained more than 600 mglday produced a 23% reduction in serum cholesterol levels (200 to 160 mg/dl). Assuming a 50% absorption rate, such a diet produced a net reduction in absorbed cholesterol of about 300 mgl day. In the study presented here, CP-148,623 in- creased the excretion of neutral sterols by about 320 mg/day, and yet serum cholesterol levels decreased by only 10% (194 to 174 mg/dl). Thus the simple elimination of 300 mg absorbed cholesterol from the daily flux from the intestine to the liver may, de- pending on how it is achieved and on other dietary factors, have markedly different effects on serum cholesterol levels. The differences in cholesterol re- sponses seen in our study and that of Mattson et a1.27 were probably due to the dietary protocols used. Their subjects were fed liquid formula diets that contained 43% of energy as fat and 16% as saturated fat, whereas subjects in our study were fed whole-food diets that contained 31% energy as fat and 9% as saturated fat. The smaller reduction in serum choles- terol levels we observed would be expected because a given dietary cholesterol deficit reduces serum levels less on a low-fat diet than a high-fat diet.z

CLINICAL P HARMACOLOGY & THERAPEUTICS VOLUME 61, NUMBER 3 Harris et al. 389

LDL and HDL cholesterol in healthy young women. Arterioscler Thromb Vast Biol 1995;15:169-78.

2. Ginsberg HN, Karmally W, Siddiqui M, Holleran S, Tall AR, Rumsey SC, et al. A dose-response study of the effects of dietary cholesterol on fasting and post- prandial lipid and lipoprotein metabolism in healthy young men. Arterioscler Thromb 1994;14:576-86.

3. Hopkins PN. Effects of dietary cholesterol on serum cholesterol: a meta-analysis and review. Am J Clin Nutr 1992;55:1060-70.

4. Keys A. Serum cholesterol response to dietary cho- lesterol. Am J Clin Nutr 1984;40:351-9.

5. Schonfeld G, Patsch W, Rude1 LL, Nelson C, Epstein M, Olson RE. Effect of dietary cholesterol and fatty acids on plasma lipoproteins. J Clin Invest 1982;69: 1072-80.

6. Harris WS, Dujovne CA, von Bergmann K, Neal J, Akester J, Windsor SL, et al. Effects of the ACAT inhibitor CL 277,082 on cholesterol metabolism in humans. Clin Pharmacol Ther 1990;48:189-94.

7. Gylling H, Miettinen TA. The effect of cholesterol absorption inhibition on low density lipoprotein cho- lesterol level. Atherosclerosis 1995;117:305-8.

8. Himber J, Missano B, Rudling M, Hennes U, Kempen HJ. Effects of stigmastanyl-phosphocholine (Ro 16- 6532) and lovastatin on lipid and lipoprotein levels and lipoprotein metabolism in the hamster on differ- ent diets. J Lipid Res 1995;36:1567-85.

9. Miettinen TA, Puska P, Gylling H, Vanhanen H, Vartiainen E. Reduction of serum cholesterol with sitostanol-ester margarine in a mildly hypercholester- olemic population. N Engl J Med 1995;333:1308-12.

10. Hainer JW, Terry JG, Connell JM, Zyruk H, Jenkins RM, Shand DL, et al. Effect of the acyl-CoAxholes- terol acyltransferase inhibitor DuP 128 on cholesterol absorption and serum cholesterol in humans. Clin Pharmacol Ther 1994;56:65-74.

11. Malinow MR. Effects of synthetic glycosides on cho- lesterol absorption. Ann NY Acad Sci 1985;454:23-7.

12. Malinow MR. Effects of cx- and B-tigogenin cellobio- sides on cholesterol absorption. Steroids 1986;48:197- 211.

13. Harwood HJ Jr, Chandler CE, Pellarin LD, Bangerter FW, Wilkins RW, Long CA, et al. Pharmacologic consequences of cholesterol absorption inhibition: al- teration in cholesterol metabolism and reduction in plasma cholesterol concentration induced by the syn- thetic saponin B-tigogenin cellobioside (CP-88818; tiqueside). J Lipid Res 1993;34:377-95.

14. Bangerter FW, Zaccaro LM, Wilkins RW, Wood HA, Wilder DW, Sugarman ED, et al. Animal pharmacol- ogy of CP-148,623, a cholesterol absorption inhibitor [abstract]. Atherosclerosis 1994;109:309.

15. Bangerter FW, Wilkins RW, Zaccaro LM, McCarthy

PA, DeNirmo MP, Morehouse LA, et al. CP-148,623 inhibits cholesterol absorption by preventing choles- terol entry into intestinal mucosa [abstract]. Athero- sclerosis 1994;109:310.

16. Crouse JR, Grundy SM. Evaluation of a continuous isotope feeding method for measurement of choles- terol absorption in man. J Lipid Res 1978;19:967-71.

17. Heinemann T, Kullak-Ublick GA, Pietruck B, von Bergmann K. Mechanisms of action of plant sterols on inhibition of cholesterol absorption: comparison of sitosterol and sitostanol. Eur J Clin Pharmacol 1991; 4O:S59-63.

18. Grundy SM, Ahrens EH Jr, Salen G. Dietary beta- sitosterol as an internal standard to correct for cho- lesterol losses in sterol balance studies. J Lipid Res 1968;9:374-87.

19. Harris WS, Muzio F. Fish oil reduces postprandial triglyceride concentrations without accelerating lipid emulsion removal rates. Am J Clin Nutr 1993;58:68- 74.

20. Bachorik PS, Albers JJ. Precipitation methods for quantification of lipoproteins. Methods Enzymol 1986;129:78-100.

21. Harris WS, Held SJ, Dujovne CA. Comparison of two scoring systems used to monitor diets in outpatient clinical trials. J Cardiovasc Risk 1995;2:359-65.

22. McCarthy PA, DeNinno MP, Morehouse LA, Chand- ler CE, Bangerter FW, Wilson TC, et al. ll- Ketotigogenin cellobioside (pamaqueside): a potent cholesterol absorption inhibitor in the hamster. J Med Chem 1996;39:100-2.

23. McCarthy PA, DeNinno MP, Morehouse LA, Chand- ler CE, Beyer TA, Bangerter FW, et al. The discovery of CP-148,623, a potent cholesterol absorption inhib- itor in hamsters [abstract]. Atherosclerosis 1994;109: 309.

24. Lees AM, Mok HYI, Lees RS, McCluskey MA, Grundy SM. Plant sterols as cholesterol-lowering agents: clinical trials in patients with hypercholester- olemia and studies of sterol balance. Atherosclerosis 1977;28:325-38.

25. Kesaniemi YA, Grundy SM. Turnover of low density lipoproteins during inhibition of cholesterol absorp- tion by neomycin. Arteriosclerosis 1984;4:41-8.

26. Kesaniemi YA, Miettinen TA. Inhibition of choles- terol absorption by neomycin, benzodiazepine deriv- atives and ketoconazole. Eur J Clin Pharmacol 1991; 4O:S65-7.

27. Mattson FH, Erickson BA, Klingman AM. Effect of dietary cholesterol on serum cholesterol in man. Am J Clin Nutr 1972;25:589-94.

28. Chenoweth W, Ullman M, Simpson R, Leveille G. Influence of dietary cholesterol and fat on serum lipids in men. J Nutr 1981;111:2069-80.