Digestive Diseases and Sciences, Vol. 36, No. 5 (May 1991), pp. 545-547

E D I T O R I A L

Antisecretory Therapy and Genotoxicity

Recent reports of toxigological findings with ome- prazole (Losec) have caused much debate and frank contention. The objective of this editorial is to summarize the toxicological information on ome- prazole to date in order to determine which way the wind is blowing!

The notion that omeprazole may damage the DNA of gastric mucosal cells, a process called genotoxicity, has been discussed recently in the columns of several journals including Lancet (Feb- ruary 17, 23; April 14, 1990), Medical Letter on Drugs and Therapeutics (March 20, 1990), the fi- nancial columns of the Wall Street Journal (Febru- ary 20, 1990), and the London Financial Times (February 21, 1990). A perusal of these communi- cations permits the identification of contention be- tween Glaxo (I) (the manufacturers and marketers of ranitidine, Zantac) and Astra (2) (manufacturers and marketers of omeprazole, Losec) together with Merck Sharp and Dohme (2) (marketers of omepra- zole in the United States).

Worldwide clinical experiences, with more than 4 million treatment courses with omeprazole to date, have indicated that omeprazole has a safety profile similar to currently available H2-receptor antago- nists. Omeprazole underwent clinical trials in more than 19,000 individuals prior to approval by a total of more than 30 regulatory agencies worldwide (3). All the established mutagenicity studies were inter- preted as negative. The mouse micronucleus assay was interpreted as borderline (at 625 and 6250 times the human dose) (4). A second mouse micronucleus assay at 2000 times the human dose was negative with different but questionably suboptimal sampling times. However, omeprazole was not shown to be mutagenic in an in vitro Ames Salmonella typhimu- rium assay, an in vitro mouse lymphoma cell assay, or an in vivo rat liver DNA damage assay system (2, 4). The drug offers a therapeutic gain over H,_- receptor antagonists in the treatment of acid-related disease (3), but the toxicity issues (1) remain the

Address for reprint requests: Stephen Holt, Division of Gas- troenterology, Department of Medicine, University of South Carolina School of Medicine, 2 Medical Park, Suite 506, Colum- bia, South Carolina 29203.

focus of contentious differences of opinion (1, 2, 5). At the center of this dispute are observations that a genotoxicity screening study performed by scien- tists at Glaxo reported that omeprazole but not loxtidine increased unscheduled DNA synthesis (UDS) in a preparation of rat gastric mucosa (1).

In these studies (1), a "novel" genotoxicity screen- ing method (6) has been utilized, with the assumption that this method measures UDS, which is a marker of DNA damage. The assay is based on the administra- tion of single oral doses of the agents under investi- gation to rats followed by an injection of tritiated thymidine approximately 14 hr later. Radiolabeled thymidine may then become incorporated into cells where replicafive (scheduled) DNA synthesis or re- pair (unscheduled) DNA synthesis (UDS) is occur- ring. This process of incorporation is studied in a limited proteinase digest of the gastric mucosa (6).

It was claimed by Burlinson et al (1, 6) that the digested preparation of rat gastriC mucosa result s only in the recovery of the superficial layer of gastric mucosal cells without significant contamination by actively dividing cells. This has been disputed in recent publications (7-9). Since the radioactive thy- midine incorporated into a normal dividing cell may be very much greater than the incorporation in cells where DNA is merely being repaired (UDS), contam- ination of the preparation with even a few dividing cells could result in an invalid measure ofgenotoxicity or the masking of UDS (10). Repeat experiments by other investigators have shown a contamination of the mucosal digest with parietal cells that are more deeply located in the gastric p i t s (8, 9). The absence of microscopic examination of radiolabeled cells to de- fine their stage in cell division and the lack of exper- iments that utilize hydroxyurea (an inhibitor of sched- uled DNA synthesis) in the genotoxicity tests (1) makes it difficult to differentiate DNA repair (UDS) from S-phase (replicative) synthesis of DNA, even though complete suppression of DNA synthesis in gastrointestinal tissues with hydroxyurea is difficult (10).

There are other potential problems with the re- ported genotoxicity studies (2, 5). These studies were criticized because they gave false negative

Digestive Diseases and Sciences, Vol. 36, No. 5 (May 1991) 0163-2116/91/0500-0545506.50/0 �9 1991 Plenum Publishing Corporation

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results with aristolochic acid, which was one of three gastric carcinogens tested in the model (6). However, aristolochic acid appears to be a carcin- ogen that affects predominantly the rat forestom- ach, which is excluded in this assay, thereby result- ing in the lack of positive results with this agent (1).

The reported positiye result of genotoxicity with omeprazole in this assay (1) is difficult to interpret. Inhibition of acid secretion by a potent antisecre- tory drug such as omeprazole may stimulate gastric mucosal cell division in the rat and S-phase synthe- sis of DNA in the time frame of the experiments, as a consequence of the trophic effect of increased serum gastrin. This may give a false positive result and, perhaps predictably, a negative result with other antisecretory drugs, such as the H2-receptor antagonists, which have a shorter duration of action than omeprazole and hence a lower integrated se- rum gastrin response (2).

Hypergastrinemia ensues in the rat following long-standing, potent reduction of gastric acid se- cretion, and this may lead to the development of enterchromaffin-like (ECL) cell hyperplasia and oc- casional gastric carcinoid tumors, especially in el- derly female animals (11, 12). Both omeprazole and ranitidine at very high doses cause hypergastrin- emia and are equally capable of causing hyperplasia of ECL cells, despite their different modes of action (8, 13, 14). These findings imply that ECL cell hyperplasia is due to the trophic effect of gastrin secondary to profound acid inhibition. Hyperplasia of ECL cells and gastric carcinoid tumor formation appears to be a species-specific phenomenon in the rat that can be induced by the infusion of gastrin or its analogs (14, 15) or by the chronic administration of very large doses of acid-suppressing medications such as omeprazole (11), ranitidine (8, 13, 16), or loxtidine (17). Burlinson et al (1) reported, how- ever, that pentagastrin administration did not in- crease thymidine uptake in the assay.

It must be borne in mind that Burlinson et al (1) proposed a preliminary screening technique (6) that should not be considered as conclusive as formal toxicity tests. Chronic toxicological tests in animals are the "gold standard." It could be argued that a "screening" genotoxicity test is empirical and has some potential if it has SUfficient sensitivity for the detection of UDS, even in the presence of problems such as some S-phase DNA synthesis or contami- nation with dividing cells. Any screening procedure will produce false positive and false negative results. The occurrence of a positive result during screening

HOLT ET AL

should ideally result in further confirmatory testing rather than conclusions about the toxicity of drugs (1), such as omeprazole or H2-receptor antagonists, that have undergone formal chronic toxicological testing prior to market approval.

Our group has repeated the assay described by Burlinson et al (1, 6), which involved in vivo label- ing with [3H]thymidine (1 mCi/kg) in Sprague- Dawley rats, 14 hr following oral administration of test agents (9). One hour later, the stomach was removed and the mucosal cells harvested by 45 min pronase (50 units/ml) digestion in situ. The obtained digest was composed of approximately 30% parietal cells. Water, indomethacin, dimethylhydrazine (a carcinogen with no effect on gastric mucosa), and omeprazole all gave comparable incorporation of [3H]thymidine in the assay, whereas 1-methyl-2- nitro-1-nitrosoguanidine (MNNG), a known gastric genotoxic agent, caused a 25-fold increase com- pared with controls. These results are similar to the earlier findings (1) with the notable exception that omeprazole (in single oral doses of 10, 20, 30, 100, and 300 mg/kg) was not genotoxic in our use of this assay. When we pretreated rats with hydroxyurea in vivo to inhibit replicative DNA synthesis prior to tritiated thymidine labeling, greater than 97% of the thymidine incorporation was suppressed (9). This demonstrates that the procedure (I, 6), as it pres- ently exists, reflects mainly replicative DNA syn- thesis (9). We also have performed experiments on the PVG (Glaxo) strain of rats, which were used bY Burlinson et al in the original studies (1,6). We have not observed the positive effect of omeprazole on increased incorporation of [3H]thymidine that Burlin- son et al have reported (1). However, some variability of [3H]thymidine uptake appears to be present in PVG rats that is not observed in the Sprague-Dawley rat. In contrast to the findings of Burlinson et al (1, 6), this difference in the PVG (Glaxo) rat was not significantly different from control.

Genotoxicity testing for the gastric mucosa pres- ents many challenges due to the characteristic phys- iology of the gastric pits (8). Omeprazole is physi- ologically inert at neutral pH. As it passes from the blood into the secretory canaliculus of the parietal cell, it is transformed rapidly by the acid environ- ment into the active sulfenamide derivative, which binds covalently with the luminal surface of the proton pump (H+,K+-ATPase enzyme system) (18, 19). This raised a question: should the genotoxicity studies be carried out with the active protonated form of omeprazole, namely, the sulfenamide deriv-

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ANTISECRETORY THERAPY AND GENOTOXICITY

ative, that is produced at low pH (19)? This proto- nated species would have been available in the in vivo chronic mutagenicity tests that were presented to regulatory agencies and considered negative.

The sulfenamide derivative is very unstable in aqueous solution at neutral pH (t~/2 < 100 msec) and even less stable in the . presence of thiols such as glutathione (t~/2 < 1 msec). Thus, it is unlikely that this electrophile would be able to reach the nucleus of the gastric cell from its site of physiologic activation (8). In addition, recent studies indicate that omepra- zole or the sulfenamide derivative do not bind signif- icantly with DNA (8). However, transport forms of the sulfenamide with thiol compounds could exist, and pores that link the nucleus with the extracellular compartment are present in some cells.

One must bear in mind that there is no evidence to suggest that the alleged genotoxicity of omeprazole (1) must be due to the sulfenamide derivative exclu- sively. Since omeprazole has a general distribution throughout the body, it is not inconceivable that omeprazole or its metabolites are present in cellular organelles. Even though the rate constant of activa- tion of omeprazole to the sulfenamide is quite low at neutral pH, the reaction will still occur at a lower rate.

What conclusions can we draw from this infor- mation? Data from independent laboratories (8, 9), including our own, have now demonstrated major deficiencies in the proposed measure of genotoxic- ity (I, 6), in its present form. The chief shortcoming of the procedure (1, 6) is that, as feared, the majority of the [3H]thymidine incorporation repre- sents replicative DNA synthesis. We believe that the small amount of nonsuppressible uptake of [3H]thymidine with hydroxyurea is related to the known inability of hydroxyurea to completely sup- press cell division (10). Still, the assay does high- light the need for toxicological "screening" methods that address the singular physiological characteristics of the gastric mucosa more appropriately than cur- rently available tests. Concerns over positive geno- toxicity results with omeprazole in the assay in its present form (1, 6) appear unwarranted. There is no current evidence to support the contention that ome- prazole is genotoxic (9).

STEVHEN HOLT, MB ROBERT E. POWERS, PHD COLIN W. HOWDEN, M D

Division of Digestive Diseases and Nutrition University of South Carolina

School of Medicine Columbia, South Carolina 29203

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4. Transcription of Proceedings of Drug Advisory Committee: 34th meeting, March 15, 1989, Washington, DC: Food and Drug Administration.

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15. Ryberg B, Axelson J, Hakanson R, Sundler F, Mattsson H: Trophic effects of continuous infusion of [LeulS]-gastrin-17 in the rat. Gastroenterology 98:33-38, 1990

16. Havu N, Mattsson H, Ekman L, Carlsson E: Enterochro- marlin-like cell carcinoids in the rat gastric mucosa following long-term administration of ranitidine. Digestion 45:189- 195, 1990

17. Poynter D, Pick CR, Harcourt RA: Association of long lasting unsurmountable histamine H_, blockage and gas- tric carcinoid tumours in the rat. Gut 26:1284-1296, 1985

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