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DISCOVERY AND DEVELOPMENT OF HYPOLIPIDEMIC DRUG,

PRAVASTATIN SODIUM

Yoshio Tsujita

Pharmacology and Molecular Biology Research Laboratories, Sankyo Co., Ltd. 1-2-58, Hiromachi,

Shinagawa-ku, Tokyo, 140. Japan

Abstract :

Key words :

In order to find a new type of hypolipidemic drug, we carried out screening of an inhibitor of cholesterol synthesis from microorganisms and found ML-236B, as a potent

and specific inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, the rate-limiting enzyme in the pathway of cholesterol biosynthesis. Among many derivatives of ML-236B, pravastatin sodium (pravastatin) was finally selected because of its potency and tissue selectivity.

Pravastatin strongly inhibited sterol synthesis in rat hepatocytes, but only weakly inhibited that in cells from nonhepatic tissues. The selective inhibitory activity of

pravastatin in sterol synthesis was further confirmed by ex vivo and in vivo experiments using rats and mice. This characteristic of pravastatin is based on its hydrophilic nature

a hydrophilic compound hardly permeates plasma membrane, whereas a hydrophobic

(lipophilic) one easily permeates it. In hepatocytes, on the other hand, pravastatin is actively incorporated via organic anion transporter.

Pravastatin demonstrated a preventive effect on the development of coronary atherosclerosis and xanthomatosis in young Watanabe heritable hyperlipidemic (WHHL) rabbits, as a consequence of keeping their serum cholesterol levels low. In clinical study,

pravastatin preferentially reduced low-density lipoprotein cholesterol by 27 % at 10-20 mg/day with low side effects.

inhibitor of cholesterol synthesis, inhibitor of HMG-CoA reductase, pravastatin,

hypolipidemic drug, WHHL rabbit

Introduction and history of research of

pravastatin

Coronary heart disease (CHD) is one of the major causes of death in Western countries, as well as in Japan. Among CHDs, ischemic heart disease (IHD) leads to the highest rate of death. Hypercholesterolemia has long been considered one of the most important risk factors for IHD. Two trials have indicated that reduction of

plasma cholesterol levels in hypercholester- olemic patients by drug treatment decreases the incidence of CHD : the U. S. Lipid Research

Adc1rn f to Dr. Yoshio Tsujita, Phermacology

(ma Mil-cu/al 16,s,,irch Laboratories, Sankyo Co., Ltd.

1? 58, t)7, .vd-17ku, Tokyo 140, Japan

1,1x :1" ' EL 81 :3492 3131

Clinics Coronary Primary Prevention Trial

(LRC-CPPT)"), using a bile acid sequestrant, cholestyramine, and the Helsinki Heart Study'', using gemfibrozil. In order to reduce the risk associated with high serum cholesterol levels, the development of several hypolipidemic drugs and therapeutics has been explored in many

countries. Since more than 70 % of the total input of

body cholesterol in humans is derived from de novo synthesis'', it is expected that plasma

cholesterol levels could be reduced by inhibition of cholesterol biosynthesis. As shown in Fig. 1, cholesterol is biosynthesized from acetyl- coenzyme A (CoA), in a process that includes more than 20 steps. The rate-limiting enzyme of this pathway is 3-hydroxy-3-methylgultaryl

(HMG)-CoA reductase [mevalonate : NADP oxidoreductase (CoA-acylating), EC 1.1.1.34]

which catalyzes the reduction of HMG-CoA to mevalonate. In 1971, we began screening for inhibitors of cholesterol synthesis from a culture

broth of microorganisms, using a cell-free enzyme system from rat liver. After screening 6,000 strains of microorganisms, ML-236B

(generic name mevastatin, Fig. 2) was discover-ed in the culture broth of Penicillium citrinum in

197556). It is noteworthy that compactin, a compound identical to ML-236B, was indepen-dently isolated by Beecham Pharmaceuticals as a weak antifungal antibiotic') two years after

Sankyo's patent filing. As shown in Fig. 2, a portion of the ML-

236B structure resembles that of HMG (3- hydroxy-3-methylglutarate), the part of HMG-

CoA that is the substrate of HMG-CoA reductase. Accordingly, ML-236B and the related compounds shown in Fig. 2 inhibit the

enzyme in a competitive manner with respect to HMG-CoA. Despite having a marked inhibitory activity on cholesterol synthesis in vitro and in vivo, ML-236B did not show hypocholester-

olemic activity in rats and mice, which are com-monly used in the initial stage of efficacy evalu-ation in animals. Several strategies, such as altering the route, term, and interval of adminis-tration, were attempted in order to demonstrate a hypolipidemic effect in rats and mice, but none of these attempts were successful. Next, ML-236B was given to old hens at the dose of 0.

2 % in diet for 30 days. Surprisingly, about a 50 % reduction of serum cholesterol levels was observed after 15 days of treatment, indicating that ML-236B exhibits strict species specificity in its efficacy. Almost 3 years had passed

before we found that ML-236B showed strict species specificity. Historical evidence concern-ing ML-236B has been described in detail by Endo").

It has been well known that the liver and intestine are the major organs involved in de novo cholesterogenesis. We focused on finding

a drug that has enhanced target-organ-directed characteristics, because a target-organ-directed inhibitor would be expected to exert a minimal disturbance of cholesterol metabolism in other

organs, including hormone-producing organs. After screening microbial products, as well as chemically and biologically modified derivatives of ML-236B, pravastatin sodium (CS-514, here-after referred as pravastatin) was finally chosen

as the candidate for development. Pravastatin exerts stronger and more tissue-selective inhibi-tion of cholesterol synthesis than ML-236B9).

Pravastatin contains a hydroxyl group at

the 6g position of its decaline structure (Fig. 2). This drug was first found as a minor urinary metabolite of ML-236B in dogs in 1979. For the industrial hydroxylation of ML-236B, chemical syntheses were initially attempted, but cost made this method unfeasible. For this reason,

microbial hydroxylation was chosen for the

production of pravastatin. After screening for microorganisms capable of converting ML- 236B to pravastatin, Streptomyces carbophilus was selected for the second step of the fermen-

tation process"'"). In 1981, pravastatin was ch(y_,eii for devel-

opment as a hypolipidemic drug nd clinicd1 trials were started in 1984. Hin \AM

260

approved for production in 1989 and was laun-ched in the same year as^K4^v^|otin^ in Japan. The drug was licensed to Bristol-Myers Squibb Company and has been developed worldwide. Pravastatin has already been launched in 44

countries, including Japan. Lovastatin is the generic name of MB-

530E312), monacolin K=` and mevino|in"`. which are identical. K4B'530^ and monacolin K were found from culture broth of a fungus, Monascus ruber. Later, mevinolin was found from another

genus of fungus, ^spe/Q^us ^reus`w. This drug has a methyl group at the 6a position of the decaline structure of PNL'236^ (Fig. 2). Simvastatin was semi-synthesized from |ovast^tin'"` and has another methyl group at

the side chain of lovastatin (Fig. 2). This review focuses on pravastatin's tissue-

selective inhibitory activity on cholesterol syn' thesis. its preventive effect on the progression of atherosclerosis in Watanabe heritable hyper-lipidemic (WHHL) rabbits and some of its clini-

cal

Tissue-selective inhibition of cholesterol

synthesis

As shown Table 116). four HMG-CoA reductase inhibitors ^ prmvastatin. |ovastmYin, simvastatin, and PNL236B. inhibited sterol syn-thesis to almost the same extent in cell-free

enzyme system in rat liver. In freshly isolated rat hepatocytes, all 4 inhibitors inhibited sterol synthesis as potency as they did in the cell-free enzyme system. In cells from nonhepatic tis-

sues, such as freshly isolated rat spleen ceUs, mouse L cells and human skin fibroblasts, /ov^s' totin, simvastatin, and ML-236B exerted inhibi-tory activities of equal potency to those showed as in rat hepatocytes. In contrast, the inhibitory

activity of pravastatin was much less potent in the cells from nonhepatic tissues as compared to its potency in rat hepatocytes. An experiment using [`°C]'|mb^|e^ HMG-CoA reductase in-hibitors attributed the weak inhibitory activity of

pravastatin on sterol synthesis in nonhepatic cells to lower uptake of the drug into these cells

(Fig. ^)'°'. Tissue-selective inhibitory activity of

pravastatin was demonstrated in an in vivo study in mice (Fig. 4)16). Pravastatin inhibited

-^l

Yoshio Tsujita

Table 1. Inhibitory activities of pravastatin lovastatin, simvastatin and ML-236B on sterol synthesis in rat liver cell-free enzyme system and various kinds of cells

282

sterol synthesis selec ively in the liver the major site of cholesterogenesis, whereas it only weakly inhibited that in other or0^ns, including hormone-producing organs. In contrast, simvas-

tmtin inhibited the sterol synthesis significantly in nonhepatic tissues, although its inhibitory

activity was most potent in liver. Ex vivo study in rats also showed the tissue selectivity of

pr^vmstotin"`. These results are consistent with those obtained in cellular experiments. In eddi' tion. not only the lactone forms of lovastatin and simvastatin which were used in the experi-ments described above, but also the acid forms

of these drugs inhibited sterol synthesis in non'hepatictissuea`m.andYhedru^sexhibit^dinhibi-tory activity in nonhepatic tissues even at a lower dose, 1.5-12 m^/k^'``.

HMG-CoA reductase inhibitors that are

tissue-selective may have enhanced safety pro-files, because cholesterol metabolism in nonhe-

patic tissues, including hormone-producing tis-sue, is only slightly affected by such agents. In f^ct, insomnia'°`, change in daytime

performance"-") and myopathy``'``` have been reported in some patients taking lovastatin and

simvastatin, but only one case of myopathy was

reported among patients treated with

pravastotin"". Tsuji et aI.24 reported that |ov^^ ta^in and simvastatin could permeate the blood-brain barrier in the rat broin, but the entry of pravastatin was restricted. In eddit^on, continuous infusion of pravastatin, lovastatin

(sodium sa|t), and simvastatin (sodium salt) to rats resulted in sleep disturbanoe, but that of

pravastatin had no effect"'.

Mechanism of tissue selectivity

In general, the cytoplasmic membrane is

permeable to hydrophobic compounds but im-

permeable to hydrophilic compounds. Hydro-phobicity of the inhibitors was measured by the distribution coefficient between n-octanol and

phosphate buffer, pH 7.4. As shown Fig. 526), positive relationship existed between the distri-bution coefficient and the incorporation velocity of HMG-CoA reductase inhibitors, indicating that lovastatin and simvastatin which are hydro-

ph^^io. enter cell more easily. In hepatocytes, the incorporation of pravas-

t^tin was temperature-dependent and showed saturation kinetics (Fig. 6). In addition, the

283

incorporation of pravastatin was inhibited by

antimycin or o|i^omyoin, inhibitors of ^T^ pro-duction, and structural analogues') (data not shown). These results suggest that pravastatin is incorporated into hepatocytes by carrier-

mediated active transport, most likely via organic anion transporter28-29.

Another factor that determines tissue selec-

tiv^ty of a drug is the hepatic extraction ratio, which for pravastatin in humans was calculated to be 0.663m. The corresponding value for hydrophobic simvastatin (ZocorK) in the USA is indicated to be approximately 0.6 by the pack-

age insert. Since there was no difference in hepatic extraction ratio between pravastatin and simvastatin, the tissue selectivity of these drugs

could depend on their permeability into cells. The mechanisms of cellular uptake of HMG-

CoA reductase inhibitors are summarized in Fig. 7. The incorporation of pravastatin into the

cells from nonhepatic tissues is virtually non' ^xi^tent. due to the hydrophilic nature of this dru^, but the drug is actively transported into hepatocytes via organic anion transporter.

Lovastatin and simvastotin, which are hydro'

phobio. are by contrast incorporated into not only the cells from nonhepatic tissues but also

he^atmoytes, by mainly passive diffusion.

Preventive effect of pravastatin on pro-

gression of atherosclerosis in WHHL rab-bits

The WHHL rabbit was discovered in 1973

by Y. Watanabe as the only animal model for familial hypercholesterolemia (FH) in mon"`^"`. The low-density lipoprotein (LDL) receptor activ-ity of this animal is only 5 % of that in normal

rabbits, resulting in severe hypercholesterolemia from bi^h""ww. Aortic and coronary atheroscler-

osis occurs in this animal at a premature age

and develops remarkably at a mature age. Because of these characteristics, the WHHL rabbit is thought to be a suitable animal model for the evaluation of a preventive or even a regressive effect of some drugs on atheroscler-

osis. Pravastatin was administered at 50 m^/

kg/day for 24 weeks to young WHHL rabbits (2 -3 months o|d) , in which aortic atherosclerosis and xmnthommtosis are mi|d""`. AsshownFi{^0. four weeks after initiation of drug ^dmini^tra' tion. serum cholesterol levels were significantly reduced, by 23 %, and the reduced mve|s were maintained throughout the ^xpe,i/ne/`|^| period.

The serum triglyceride and pho^,|mv*| !eve|s were also decreased in a simil 1,1,11)11(1 to

^;4

Fig. 7. Summary of tissue-selectivity of pravastatin.

cholesterol. Lipoprotein cholesterol levels in very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and LDL in the treated group were significantly reduced,

by 47, 57, and 54 %, respectively, as compared to the control group, whereas high-density lipo-

protein (H DL) cholesterol levels were not altered (data not shown).

Although the overall aortic lesion areas were not reduced significantly, coronary ather-

osclerosis was prevented. The effect of pravas-tatin on stenosis of coronary arteries is shown in Fig. 9. In the left septal branch (LSP), the stenosis in the treated group was significantly lower than in the control group (p< 0.05). In the left anterior descending branch (LAD), the right

coronary artery (RCA), and the left circumflex branch (LCX), lower values were indicated, although the differences were not significant.

The mean percent of stenosis of the vascular lesions in all of the four main arteries was 19. 7 % in the control group and 9.1 % in the treated group, representing a significant differ-ence (p CYM-)). The coronary stenosis was well

correlafpd with atherosclerotic changes, includ-jug town ,'H In, I Hid fibrous thickening. The

effect of pravastatin on the development of xanthomas in digital joints was also analyzed.

The incidence of xanthomas was 90.4 % in the control group and 58.3 % in the treated group

(p<0.005, data not shown). These results suggest that pravastatin exhibits a preventive effect on the progression of coronary atheros-

clerotic lesions as well as xanthomas in WHHL rabbits, as a consequence of maintaining serum cholesterol at low levels.

In addition, we also examined whether a suppressive or even a regressive effect was observed on already-established atherosclerosis in mature WHHL rabbits, by combination treat-ment with pravastatin and cholestyramine, a

bile acid sequestrant (data not shown)"). WHHL rabbits were treated with these two drugs for 8 months, beginning at 10 months of age. Serum cholesterol levels were reduced by

64 % from the initial levels. Although regres-sion of aortic and coronary atherosclerosis was not observed, prevention of their progression was noted the average thickening of intima in the thoracic and abdominal aortas of the treated

group was significantly reduced from that of the placebo group, and the difference in the total aorta was also significant (p<0.05). In coro-nary atherosclerosis, the percent of stenosis in three major arteries was 46 % in the placebo

group and 18 % in the treated group, showing a significant difference. These results suggest that further progression of aortic and coronary atherosclerosis can be prevented in mature WHHL rabbits by keeping serum cholesterol levels extremely low.

Clinical studies

Clinical studies were conducted by Dr. Yui-chiro Goto, professor emeritus of University of Tokai, School of Medicine. A typical study of

pravastatin is shown in Fig. 10"). Pravastatin was administered to heterozygous FH and non-FH patients at 10 to 20 mg/day (in most

cases) for 15 months. Serum total cholesterol levels were reduced by about 20 % at 3 months after initiation of the drug treatment, and the low levels were maintained throughout the trial

period. LDL cholesterol levels were also de-creased, by 27.2 %, at the end of the study, whereas HDL cholesterol was inversely in creased, by 9.2 °/0 (Fig. 10A). The percent of reduction of serum total cholesterol levels in FH

and non-FH was almost the same (Fig. 10B). In a long-term study (48 months), plasma total

cholesterol levels were reduced by 20 %, and the reduced levels were constantly maintained until the end of the study (Fig. 11).

Pravastatin is a well tolerated and safe

drug. In Japan, the number of patients who claimed adverse effects, such as gastrointesti-nal disturbances and skin rash, was only 55 (1. 7 %) among 3,256 patients during the clinical

trials with pravastatin. But no serious adverse effects were reported. The nun-ibpt f Patents who showed abnormal laboratory hi.li(-,, Inc hid-ing elevations of tranamin',--1,)( (-)!Ii w(itime kinase, was 126 (3.6 %), bill ii I, 0111,11-

286

Conclusion

More than 20 years have passed since we began screening culture broths of microorgan-isms for inhibitors of cholesterol biosynthesis. As a result of these efforts, ML-236B

(compactin) \A/dr:, (1Hcovered in Japan as the first potent (-10i ,py, if ic inhibitor of HMG-CoA

reductase. Pravastatin, found at first as a

urinary active metabolite of ML-236B, was developed as a hypolipidemic drug that shows the following characteristics : 1) distinct tissue-selective inhibitory activity of cholesterol syn-

thesis, 2) clear mechanism of cholesterol lower-ing effects, 3) potent activity for lowering LDL cholesterol levels, 4) predictable responsiveness for efficacy, and 5) high tolerability.

Many companies are actively searching for

287

new-type HMG-CoA reductase inhibitors. Some

new chemically synthesized inhibitors are now in

the stage of clinical trails, and most of them are

more potent than naturally available inhibitors,

such as pravastatin, lovastatin, and simvas-

tatin. However, safety of the drugs must be

considered more important than efficacy ;

hypolipidemic drugs will be used by hyper-

lipidemic patients over long periods, even an

entire lifetime.

Now several clinical studies concerning

prevention and regression of coronary atheros-clerosis by pravastatin are under way in several

European countries, Canada and U.S.A. Most of

these studies will be completed in 2 or 3 years.

Favorable results are anticipated, which will

herald the application of pravastatin in the treat-

ment of atherosclerosis.

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(Accepted 3 October 1993)