CLINICAL I? HARMACOLOGY ROUNDS

Olsalazine and 6-mercaptopurine-related bone marrow suppression: A possible drug-drug interaction

A patient with refractory Crohn’s disease had two separate episodes of bone marrow suppression while receiving 50 to 75 mg 6-mercaptopmine a day and 1000 to 1750 mg olsalazine a day. This adverse reaction necessitated dose reduction of 6-mercaptopurine on the first occasion and withdrawal of 6-mercaptopurine and olsalazine on the second occasion. The patient’s red blood cell thiopurine methyltransferase (TPMT) activity was 12.2 U per milliliter red blood cells (low normal range) and her TPMT genotype was wild-type sequence for all known alleles of TPMT that result in low TPMT enzyme activity. In vitro enzyme kinetic studies confirmed the hypothesis that olsalazine and olsalazine-@sulfate are potent noncompetitive inhibitors of recombmant human TPMT. We suggest that the patient’s relatively low baseline level of TPMT activity was inhibited by olsalazine and olsalazine-C&sulfate, leading to decreased ckarance of 6-mercaptopurine and its accumulation. This ultimately increased intracellular 6-thiopurine nucleotide levels to toxic concentrations, which caused bone marrow suppression. (Clin Pharmacol Ther 1997;62:464-75.)

Lionel D. Lewis, MB,BCh, MD, Andrea Benin, MD, Carol L. Szumlanski, BS, Diane M. Otterness, BS, Lynne Lennard, PhD, Richard M. Weinshilboum, MD, and David W. Nierenberg, MD Hanover, N.H., Rochester, A&an., and Sbejield, En&and

The medical management of ulcerative colitis and Crohn’s disease has improved with the development of new therapeutic agents and the use of combina- tion drug therapy.’ The main classes of agents in use are aminosalicylates, such as 5-aminosalicylic acid or

From the Departments of Medicine, Pharmacology/Toxicology, and Pediatrics, Dartmouth Medical School, Hanover; the De- partment of Pharmacology, Mayo Medical School, Rochester; and the University Department of Medicine and Pharmacol- ogy, The Royal Hallamshire Hospital, Sheffield.

Dr. Lewis was supported in part by the Pharmaceutical Research and Manufacturers of America Foundation faculty develop- ment award in Clinical Pharmacology; Drs. Nierenberg and Lewis were also supported by grant CA 23108 from the Na- tional Cancer Institute. Dr. Lennard was supported by the Leukemia Research Fund of Great Britain. Dr. Weinshilboum was supported by ROl grants GM 28157 and GM 35720 from the National Institutes of Health.

Received for publication May 8, 1997; accepted June 13, 1997. Reprint requests: Lionel D. Lewis, MB,BCh, MD, Dartmouth-

Hitchcock Medical Center, Hinman Box 7506, Lebanon, NH 037.56. E-mail: Lionel.Lewis@ Dartmouth.edu

Copyright 8 1997 by Mosby-Year Book, Inc. 0009-9236/97/$5.00 + 0 13/U84062

464

sulfasalazine (sulfapyridine covalently bonded to 5- aminosalicylic acid), or immunomodulatory drugs, such as corticosteroids and thiopurines such as aza- thioprine and 6-mercaptopurine, or antibiotics, such as metronidazole.122 Combinations of these drugs often are required to achieve control of refractory inflammatory bowel disease. In one large clinical study involving patients with inflammatory bowel disease, it was found that thiopurine drugs had to be withdrawn in the treatment of 10% of patients be- cause of the occurrence of dose-limiting bone mar- row suppression.3 More than 50% of patients in that study were treated with the combination therapy of 6-mercaptopurine and sulfasalazine, which raised the possibility that a drug-drug interaction might contribute to the toxicity.

The new generation of drugs for treating inflam- matory bowel disease contain the 5-aminosalicylic acid component of sulfasalazine without the sulfon- amide entity. They are used in an attempt to mini- mize hematologic and dermatologic toxicity. Olsala- zine sodium (Dipentum) is one such an agent. It

CLINICAL PHARMA COLOGY & THERAPEUTICS VOLUME 62, NUMBER 4 Lewis et al. 465

consists of two 5aminosalicylic acid molecules joined by an uzo bond. Fig. 1 shows the structures of the most common 5aminosalicylic acid derivatives. The drug is converted to 5-aminosalicylic acid by bacterial cleavage in the colon. Recommended oral dosing of 500 to 2000 mg olsalazine2,4 is effective therapy for mild to moderate colonic inflammatory bowel disease in mild acute exacerbations and as chronic maintenance therapy.4J Olsalazine mono- therapy rarely has been associated with suppression of the erythroid and megakaryocyte lineages.6

Thiopurines, such as 6-mercaptopurine and aza- thioprine, have a narrow therapeutic index. One of the most common dose-limiting toxic effects is bone marrow suppression, particularly leukopenia. Seri- ous reversible leukopenia (white blood cell [WBC] count less than 2.5 X lO’/L) occurred among 2% of patients with inflammatory bowel disease receiving 6-mercaptopurine in one retrospective study with 376 patients.7 In a smaller, prospective study in which patients mainly took 50 mg 6-mercaptopurine per day or less, investigators found mild leukopenia (WBC count less than 4.5 X 109/L) among 28% of patients, but no patient had a WBC count less than 2.8 X 109L8 In another retrospective study of 739 patients with inflammatory bowel disease treated with azathioprine (a pro-drug that is converted to 6-mercaptopurine in viva),’ it was found that 5% of patients needed either drug withdrawal or dose re- duction because of bone marrow toxicity; severe leukopenia (WBC count less than 2.0 X 109/L) oc- curred among 1.3% of these patients.

Among humans 6-mercaptopurine demonstrates variable, but low, oral bioavailability with a mean bioavailability of 16% (range, 5% to 37%).” 6- Mercaptopurine undergoes extensive presystemic metabolism in the intestine and liver. Only 8% of a dose is excreted unchanged in the urine, and it is metabolized by way of three competing metabolic pathways (Fig. 2). Metabolic activation of 6- mercaptopurine occurs by means of hypoxanthine- guanine phosphoribosyltransferase (EC 2.4.2.8) and two subsequent enzymatic steps to yield 6- thioguanine nucleotides, which are incorporated into deoxyribonucleic acid (DNA), producing cyto- toxic effects.ii Thiopurine methyltransferase (TPMT, EC 2.1.1.67) catalyzes the S-methylation of thiopurines mainly to form inactive products. Xan- thine oxidase (EC 1.1.3.22) catalyzes thiopurine ox- idation to inactive thiouric acid.i1-13 Functional xanthine oxidase activity is absent in human hema- tologic tissue.13

SulfaDvr i i 5-Aminosalicvlic Acicj

0 COOH

CH ” ,-C-NH 4

\ OH

N-Acetyl-5-Aminosalicvlic Acid

Olsalazine

Fig. 1. Chemical structures of sulfasalazine, sulfapyridine, 5aminosalicylic acid, N-acetyl-5-aminosalicylic acid, olsalazine, and olsalazine-O-sulfate.

Family studies have shown that the level of TPMT activity is inherited as an autosomal codominant trait.14 In all human tissues studied, including red blood cells (RBCs), TPMT activity is controlled by a common genetic polymorphism. Weinshilboum and Sladek” reported that in a large white population sample 88.6% of individuals were homozygous for the allele or alleles for high TPMT activity, 11.1% were heterozygous and had intermediate activity, and one in 298 individuals (0.3%) was homozygous for the allele or alleles for low or absent activity. Individuals with low TPMT activity are unusually sensitive to “standard” therapeutic doses of thiopu- rines and commonly experience serious bone mar- row suppression. l6 In vitro studies have shown that benzoic acid derivatives, including sulfasalazine and 5-aminosalicylic acid, are inhibitors of TPMT activ- ity. This has led to the hypothesis that patients receiving 5aminosalicylic acid or one of its deriva- tives may be at increased risk for 6-mercaptopurine- induced bone marrow suppression when treated simultaneously with both drugs,17’18 but such inter-

466 Lewis et al.

6-MERCAPTOPURINE METABOLISM

IUP DEHYDROQENASE

1

QUP SYN-WETASE

/-iii&q Fig. 2. The major metabolic pathways of 6- mercaptopurine. The enzymatic products of 6- mercaptopurine produced by xanthine oxidase (X0) and thiopurine methyltransferase (TPMT) are inactive. The 6-thioguanine nucleotides are responsible for the thera- peutic and toxic effects of thiopurines. AO, Allopurinol; HPRT, hypoxanthine-guanine phosphoribosyltransferase; IMP, inosine monophosphate; GMP, guanosine mono- phosphate.

actions have not yet been reported in the clinical literature.

We describe the case of a patient with Crohn’s disease who had severe bone marrow dysfunction while receiving treatment with 6-mercaptopurine, ol- salazine, and corticosteroids. On the basis of clinical expertise and the modern techniques of enzymology and molecular biology, we suggest a potential mecha- nism involved in the pathophysiologic mechanism of this patients’ bone marrow dysfunction.

CASE PRESENTATION In early August 1995 a 16-year-old white high

school student experienced weight loss (weight, 36.5 kg), anorexia, and abdominal pain. Examination re- vealed fungating perianal skin tags. Histopathologic examination confirmed the clinical diagnosis of Crohn’s disease. At diagnosis, laboratory studies re- vealed WBC count, 5.9 X 109/L (absolute neutrophil count [ANC], 4.879 X 109/L); hemoglobin, 121 gm/L; platelet count, 376 X 109/L; and erythrocyte sedimentation rate (ESR), 15 mm/hr. The patient received a week-long course of 60 mg methylpred- nisolone a day intravenously and 1500 mg metroni- dazole a day intravenously. On August 15 the pa- tient was discharged taking 60 mg prednisone a day by mouth and 750 mg metronidazole a day by mouth.

CLINICAL PHARMACOLOGY &THERAPEUTICS OCTOBER 1997

In mid September 1995 despite this drug regimen, the symptoms persisted, and the patient started tak- ing 50 mg 6-mercaptopurine a day by mouth, and 500 mg olsalazine a day by mouth. Concomitantly the prednisone was tapered to 20 mg daily. At that time a complete blood cell count revealed a WBC count of 8.8 X 109/L (ANC, 8.448 X 109/L); hemo- globin, 138 gmiL; platelet count, 327 X 109/L. Com- puted tomography of the abdomen and pelvis delin- eated the gastrointestinal disease but showed no fistulas or abscesses.

By mid October 1995 her drug dosage was in- creased to 1000 mg olsalazine a day and 75 mg 6-mercaptopurine a day by mouth. At that same time the prednisone was tapered to 5 mg daily. Twenty milligrams fluoxetine a day was added for depression. Other drug therapy consisted of 30 mg codeine as needed for its analgesic and antidiarrheal action and 50 mg diphenhydramine as needed for nausea. The WBC count was 4.9 X 109/L (ANC, 4.459 X 109/L); hemoglobin, 122 gm/L; platelet count, 448 X 109/L; and ESR, 48 mm/hr.

On November 7, 1995, leukopenia developed with a WBC count of 1.7 X 109/L (ANC, 1.309 X 109/L); hemoglobin, 113 gm/L; and platelet count, 550 X 109/L. At this point the patient was taking 1000 mg olsalazine a day, 75 mg 6-mercaptopurine a day, and 5 mg prednisone on alternate days. Her 6-mercaptopurine dose was reduced to 50 mg a day and she continued to receive 1000 mg olsala- zine a day. At follow-up testing 2 weeks later a complete blood cell count revealed a WBC count of 1.7 X 109/L (ANC, 1.292 x 109/L) with hemo- globin 107 gm/L and platelet count of 218 X 109/L. The 6-mercaptopurine dose was reduced to 25 mg a day and by December 5, 1995, her WBC count was 3.0 X 109/L (ANC, 1.92 X 109/L).

In early February 1996 her 6-mercaptopurine dose had been stable at 25 mg/day and her other medications included 1000 mg olsalazine a day, 20 mg fluoxetine a day, 5 mg prednisone a day, and 50 mg diphenhydramine as needed. She was also taking one multivitamin tablet daily. At this date her WBC count was 5.2 X 109/L (ANC, 2.756 X 109/L); he- moglobin, 109 gm/L; platelet count 309 X 109/L; and ESR 33 mm/hr. The patient had a kidney stone and symptomatic exacerbation of Crohn’s disease. She received 60 mg methylprednisolone intravenously a day for 3 days. After which the dosages were an increase to 1500 mg olsalazine a day, 90 mg pred- nisone a day with a taper to 60 mg prednisone daily over several weeks, and 50 mg 6-mercaptopurine a

CLINICAL P HABMACOLOGY & THERAPEUTICS VOLUME 62. NUMBER 4 Lewis et al. 467

Fig. 3. Temporal relation between bone marrow function as shown with hemoglobin and total white blood cell (WBC) count and concomitant daily doses of olsalazine and 6- mercaptopurine.

day. Despite these changes in therapy, the patient continued to have symptoms that indicated poor control of her Crohn’s disease. On February 22, 1996, dosages were an increase to 1750 mg olsala- zine a day and continuation of 50 mg 6- mercaptopurine a day and 50 mg prednisone a day.

On March 18, 1996, symptoms of an upper respi- ratory tract infection developed, and a course of 750 mg/day oral amoxicillin (INN, amoxicilline) was be- gun. She received 10 mg prednisone daily, and the doses of her other medications remained un- changed. By March 28, the patient reported fevers and abdominal pain. A complete blood cell count revealed WBC count, 1.3 X 109/L; hemoglobin, 74 gm/L (hematocrit, 0.22); and platelet count, 269 X 109/L. There was no evidence of clinically significant

hemorrhage, and the 6-mercaptopurine therapy was stopped (all other medications were continued). Continued monitoring of bone marrow function re- vealed that on April 3, 1996, the WBC count was 1.9 X 109/L. (ANC, 1.482 X 109/L); hemoglobin, 68 gmiL; platelet count, 311 X 109/L; a mean corpus- cular volume, 96.5 fl; and mean corpuscular hemo- globin, 32.9 pg/cell (range, 27 to 31.0 pgkell). Fur- ther studies revealed reticulocyte fraction, 33 X low3 (normal range <lo X 10w3); serum folate, 22 nmol/L (normal range, 3 to 38 nmoliL); RBC folate, 2100 nmol/L (normal range, 517 to 1421 nmol/L); serum vitamin B12, 872 nmol/L (normal range, 204 to 798 mnoliL). Blood samples were obtained for RBC TPMT activity and TPMT genotyping, and ol- salazine was stopped. The patient then received a

468 Lewis et al. CLINICAL PHARMACOLOGY & THFXAl’EUTICS

OCTOBER 1997

Table I. Alleles involved in human TPMT genetic polymorphism*

Allele Nucleotide Amino acid

TPMT*l - TPMT*2 23&c 80 Ala -+ Pro TPMT*3A 460G-+A 154 Ala + Thr

719A+G 240 Tyr + Cys TPMT*3B 460G-+A 154 Ala + Thr TPMT*3C 179A+G 240 Tyr + Cys TPMT*3D 292G+T 98 Glu --, Stop

460G+A 154 Ala -+ Thr 719A+G 240 Tyr + Cys

TPMT*4 1352G-+A Splice junction (Intron 9) Intron 9lexon 10

TPMT*S 146T+C 49 Leu + Ser TPMT*6 539A+T 180 Tyr + Phe

TPMT, Thiopurine methyltransferase; wt, wild type. * Table lists the TPMT alleles that have been reported to be associated

with low red blood cell enzyme activity.

transfusion of two units of packed red blood cells. Fig. 3 shows the temporal relations among hemoglobin concentration, WBC count, and drug therapy.

By April 7,1996, the WBC count had recovered to 3.9 x 109/L. Hemoglobin was 12.2 gm/L (after trans- fusion), and platelet count was 180 X 109/L. Because of Clostridium di.ciZe infection, 750 mg metronida- zole a day by mouth had been started on April 5. The patient also was taking 90 mg codeine a day by mouth for abdominal pain and 50 mg diphenhydra- mine by mouth as needed for nausea. On April 23, 1996, she had WBC count, 4.4 X 109/L (ANC, 3.467 X 109/L); hemoglobin, 102 gm/L; and platelet count, 278 X 109/L. Because of the difficulty in controlling the Crohn’s disease with corticosteroids alone, 12.5 mg 6-mercaptopurine a day was started with careful monitoring of bone marrow function. In November 1996, BBC TPMT activity was measured while the patient was receiving this lower dose of 6-mercaptopurine but not olsalazine. She had no further clinically significant hematopoietic toxicity while taking this reduced dose of 6-mercaptopurine, and the Crohn’s disease was controlled.

METHODS Material

[14C-Methyl]S-adenosyl-L-methionine ([14C-methyl]- Ado-Met, 40 to 60 mCi/mmol-‘) was purchased from DuPont-NEN, Boston, Mass. [14C-Methyl]S-adenosyl- L-methionine hydrochloride (Ado-Met-HCl), dimethyl- sulfoxide, 6-mercaptopurine, and sulfasalazine were obtained from Sigma Chemical Company, St. Louis, MO. Olsalazine, olsalazine-O-sulfate, and N-acetyl-5-

aminosalicylic acid were provided by Paul Strauss, MD, of the Pharmacia & Upjohn Company, Kalama- zoo, Mich. 3,4-Dimethoxy-5-hydroxy benzoic acid (DMHBA) was purchased from ICN Pharmaceuticals, Plainview, N.Y.

Red blood cell TPMT activity assay TPMT enzymatic activity was measured by means

of a modification of the radiochemical method of Weinshilboum et a1.19,20 This assay is based on the conversion of 6-mercaptopurine to radioactively la- beled 6-methylmercaptopurine with [14C-methyll- S-adenosyl-L-methionine ([‘“Cl-methyl-Ado-Met, 24 mCi/mmol-r) as the methyl donor. The final concentrations of 6-mercaptopurine and Ado-Met present in the reaction mixture were 7.5 mol/L and 24.2 )J,mol/L, respectively. Blank samples contained no 6-mercaptopurine. The 6-methylmercaptopurine formed during the reaction was separated from the radioactively labeled Ado-Met by means of organic solvent extraction, and the radioactivity of the or- ganic phase was measured in a liquid scintillation counter. Each sample was assayed in triplicate. The results reported are averages of the three determi- nations. The interassay coefficient of variation for the TPMT assay was 4.1%.19

Recombinant TPMT enzyme kinetic and inhibitor studies

Recombinant human TPMT. The recombinant human TPMT used in these experiments was present in a 100,OOOg supernatant preparation ob- tained from COS-1 cells that had been transfected with the eukaryotic transfection vector p91023(B). The vector contained the 5’-untranslated region, the coding region, and a portion of the 3’- untranslated region of the human T84 colon car- cinoma cell TPMT complementary DNA (cDNA).~~ Detailed descriptions of the transfec- tion of COS-1 cells and the biochemical and phys- ical characteristics of the recombinant enzyme, which are identical with those of TPMT in normal human tissues, are published elsewhere.21 The specific activity of this enzyme preparation was 535 nmol/hr per milligram protein, and approxi- mately 0.4 mg protein was present in each assay.

Inhibition kinetic studies. The potential inhibitors of recombinant human TPMT were dissolved in a suitable solvent. Olsalazine and olsalazine-O-sulfate were dissolved in water, and N-acetyl-5- aminosalicylic acid and the control inhibitor

CLINICAL P HARMACOLOGY &THERAPEUTICS VOLUME 62, NUMBER4 Lewis et al. 469

I INHIBITION OF RECOMBINANT HUMAN TPMT

b 60

-6 -5

log [d;l”G], M -3 -2

n DMHBA 4 SULFASALAZINE A OLSALAZINE q OLSALAZINE-O-SULFATE o 5-ASA A N-ACETYL-BASA

Fig. 4. TPMT inhibition by selected 5-aminosalicylic acid (5-ASA) derivatives. The plot shows percentage of TPMT activity remaining versus [6-mercaptopurine] [DRUG] concentration in the presence of the inhibitors dimethoxyd-hydroxy benzoic acid (DMHBA), sulfasalazine, olsalazine and olsalazine-O-sulfate, 5aminosalicylic acid, and IV-acetyl-5-aminosalicylic acid.

DMHBA were dissolved in dimethylsulfoxide. All potential inhibitors were added to the enzyme reac- tion mixture in 10 ~1 aliquots. Control samples to which only 10 l.~l of water or dimethylsulfoxide was added also were assayed. Initial experiments in- volved concentrations of inhibitor that varied by several orders of magnitude. A series of experiments were conducted in which concentrations at or near those required to inhibit TPMT activity by 50% were used for each compound. The 50% inhibitory concentration (I&,) was estimated from semiloga- rithmic plots of the concentration-versus-effect curves by means of the GraphPAD InPlot program (GraphPAD Software, San Diego, Calif.). Inhibition constants were determined from replots of data ob- tained by assaying TPMT activity in the presence of varying concentrations of the inhibitor (K,i) and varying concentration of the substrates (KJ for the reaction, either 6-mercaptopurine (six concentra- tions ranging from 0.23 to 7.5 mmol/L) or Ado-Met (six concentrations ranging from 0.75 to 24.2 t.t,mol/ L). Those data were used to construct double in- verse plots according to the method described by Wilkinson22 with a computer program written by ClelandF3 Kii and I$, were then calculated as de- scribed by Sega1.24 &, is a measure of the effect of

an inhibitor on the slope of a double inverse plot. hi is a measure of the effect of an inhibitor on the intercept of such a plot.

TPMT genotype determination Genomic DNA was isolated from both whole

blood and the huffy coat by use of a QIAmp Blood Kit (Qiagen, Chatsworth, Calif.). All exons of the TPMT gene that encoded protein were amplified by use of polymerase chain reaction (PCR) to make it possible to detect nucleotide sequence variants. PCR primers were synthesized on the basis of known intron sequences2’ All primers contained an additional 18 nucleotides at the 5’-end for the Ml3 primer. PCR Gem 100 wax beads were used to hot start the PCR. After an initial incubation at 94” C for 1 minute, 35 cycles of 1 minute at 94” C, 2 minutes at 60” C, and 3 minutes at 72” C followed by a final extension for 10 minutes at 72” C were per- formed. An aliquot of each PCR reaction mixture was analyzed by means of agarose gel electrophore- sis to ensure successful amplification. Another ali- quot of the PCR reaction mixture was then diluted appropriately and sequenced with the Al31 Prism dye primer cycle sequencing kit with AmpliTaq DNA polymerase, fluorescent sequencing.

470 Lewis et al. CLINICAL l’HABMACOLOGY & THERAPEUTICS

OCTOBER 1997

Table II. Summary of IC,, values for inhibition of recombinant TPMT activity by benzoic acid derivatives

Inhibitor TPMT IC,,

(cunow

3,4-Dimethoxy-5-hydroxy benzoic acid* 14 Olsalazine 23 Olsalazine-O-sulfate 70 Sulfasalazine* 78 3-Aminosalicylic acid* 99 N-Acetyl-5-aminosalicylic acid 390 5-Aminosalicylic acid* 1240 4-Aminosalicylic acid* 2600

IC,,, 50% mhlbltory concentratton. * Values based on Szumlanski and Weinshilboum.”

RESULTS In vivo assays

Patient RBC! TPMT activity. The measured RBC TPMT activity of the patient was initially (in April 1996) 12.2 U/ml RBCs, a value considered to be low-normal for a patient homozygous for the alleles for high TPMT activity. This enzyme activity was measured while the patient was taking olsalazine but had discontinued 6-mercaptopurine 6 days previ- ously. The TPMT enzyme activity assay dilutes out and thus removes the effect of any potential revers- ible inhibitor of TPMT in the sample. In November 1996, while the patient was taking 12.5 mg 6- mercaptopurine a day, a low dose, without olsala- zine, the RBC TPMT activity was 11.0 U/ml RBCs. This TPMT assay had a coefficient of variation of 4.1%, and the 95% confidence intervals for the mean of these two measurements were 10.9 to 12.3 U/ml RBCs.

Patieni TPMT genotype. Genotype studies showed the patient was homozygous for wild-type sequences at all nucleotides (exons 3 to 10) currently known to result in low TPMT activity. Table I lists the TPMT alleles that are known to cause low TPMT activity with the base sequence change and the resultant alterations in encoded amino acids.25

In vitro TPMT kinetic studies with olsalazine and other 5aminosalicylic acid derivatives

Initial experiments were undertaken to determine whether olsalazine or olsalazine-O-sulfate was capa- ble of inhibiting recombinant TPMT. A series of concentrations of each compound were tested under optimal conditions for the assay of TPMT activity with 6-mercaptopurine as the methyl acceptor and

14C-Methyl-Ado-Met as the methyl donor. The po- tent and well-characterized benzoic acid inhibitor of TPMT, DMHBA, was tested as a positive control.18 The IC,, for DMHBA was 14 pmol/L, very similar to previously reported values (Fig. 4 and Table II).17 Olsalazine and its metabolite olsalazine-O-sulfate were found to be potent inhibitors of TPMT, with ICso values of 23 PmolL and 70 wmol/L, respec- tively. In comparison 5aminosalicylic acid and N- acetyl-5aminosalicylic acid (other metabolites of ol- salazine) were weaker inhibitors, with ICsO values of 1240 pmol/L and 390 pmol/L, respectively (Fig. 4 and Table II).

To characterize the nature of the inhibition of TPMT by olsalazine and olsalazine-O-sulfate (be- cause the latter two were the compounds most clin- ically relevant for our patient), we performed inhi- bition kinetic experiments. The effect of each compound was studied in the presence of a series of concentrations of both 6-mercaptopurine and Ado- Met, the methyl donor. Each of the compounds tested was found to be a noncompetitive inhibitor of TPMT1* (Fig. 5). Kii and I& for the inhibition of recombinant human TPMT by sulfasalazine and ol- salazine are shown in Table III. They demonstrate that olsalazine was a more potent inhibitor than sulfasalazine. Olsalazine was the most potent inhib- itor of this group of 5aminosalicylic acid-related compounds.

DISCUSSION This patient had two episodes of severe bone

marrow suppression only after starting combination drug therapy for refractory Crohn’s disease. At ini- tial presentation bone marrow function was normal. Crohn’s disease is not reported to cause bone mar- row dysfunction unless associated with vitamin Bi2 or folate deficiency. Our patient had normal vitamin Br2 and folate levels. These data strongly suggest that the drug therapy was the most likely cause of bone marrow suppression. When she tirst experi- enced leukopenia (November 1995), the patient was receiving 6-mercaptopurine, olsalazine, prednisone, fluoxetine, codeine phosphate, and diphenhydra- mine. At this time the dose of 6-mercaptopurine was at its highest, 75 mg 6-mercaptopurine a day with 1000 mg olsalazine a day. After dose reduction from 75 mg 6-mercaptopurine a day to 25 mg 6- mercaptopurine a day, the granulocytopenia gradu- ally recovered. The patient continued to take 1000 mg olsalazine a day. In March 1996 more severe bone marrow suppression recurred when the olsala-

CLINICAL PHARMACOLOGY & THERAPEUTICS VOLUME 62, NUMBER 4 Lewis et al. 471

I 1 5 2.5 (A) ‘.’ (B) 1

OLSALAZINE INHIBITION OF RECOMBINANT HUMAN TPMT

[6-MP], mM i/p-MP], mM-1

Fig. 5. Olsalazine inhibition of recombinant human ‘PMT. A, Effect of four olsalazine concentrations on the relation between [6-mercaptopurine] and TPMT activity. B, Double reciprocal plots of the data shown in A. -

zine dose was at its highest (1750 mg olsalazine a day) with 50 mg 6-mercaptopurine a day. The 6- mercaptopurine was stopped, and hematologic re- covery occurred after withdrawal of both 6- mercaptopurine and olsalazine. In effect the latter episode constituted a rechallenge with the combina- tion of olsalazine and 6-mercaptopurine, when 1750 mg olsalazine a day was used with 50 mg 6- mercaptopurine a day (Fig. 3).

All the drugs the patient was taking could have been culpable as a cause of hematologic toxicity. The most likely causative agent, however, was 6- mercaptopurine, which classically causes suppres- sion of the granulocyte and megakaryocyte lin- eages.ll Olsalazine has also rarely been reported to be associated with anemia and thrombocytopenia.6 The other drugs the patient was taking were contin- ued through both episodes of bone marrow suppres- sion and recovery; this almost certainly excludes them from being causal in this process. The recom- mended dose range for Crohn’s disease is 1 to 2 mgikg 6-mercaptopurine.2’8 Our patient weighed 36.5 kg, and the 6-mercaptopurine doses she re- ceived were not excessive. Therefore the most likely cause of hematopoietic suppression was either the 6-mercaptopurine alone, the olsalazine itself, or the combination of the two (initially of 75 mg 6- mercaptopurine a day and 1000 mg olsalazine a day or later the combination of 50 mg 6-mercaptopurine a day with 1750 mg olsalazine a day). It was not clear

solely from the clinical and laboratory data whether the bone marrow suppression was due to a pharma- codynamic effect of 6-mercaptopurine alone (it can cause severe myelosuppression among as many as 2% of patients with inflammatory bowel disease*) or olsalazine alone6 or due to the combined pharma- codynamic effects of both agents or a complex phar- macokinetic drug-drug interaction.

Sulfasalazine and 5-aminosalicylic acid (both ben- zoic acid derivatives) have been reported to be non- competitive inhibitors of TPMT in vitro, with IC,, values of 78 and 124 Fmol/L, respectively.” Those observations prompted us to measure our patient’s RBC TPMT activity which was initially 12.2 U/ml RBCs. Furthermore, our patients’ genotype was homozygous wild type for all eight alleles known to cause low TPMT activity (Table I). In com- parison with population studies of TPMT activity among patients with leukemia who were receiv- ing long-term 6-mercaptopurine therapy,26,27 12.2 U/ml RBCs represents a relatively low level of enzyme activity for a homozygous “high” sample. It fell near the nadir of the frequency distribution between samples for phenotypically high and in- termediate TPMT activity in a reference population of children with leukemia receiving long-term 6- mercaptopurine therapy (Fig. 6). This level of activ- ity represents the patient’s endogenous TPMT ac- tivity without the effects of any drugs that are enzyme inhibitors, because the effects of those

472 Lewis et al. CLINICAL PHARMACOLOGY & mmAPEUnCS

OCTOBER 1997

Table III. I& and Kii in the presence of 6mercaptopurine or Ado-Met as substrates for recombinant human TPMT

Substrate

Inhibitor

6Mercaptopurine Ado-Met

Ki, (t-unol/L) & (FollL) Ki, (/mollL) Kii (vo~IL)

Olsalazine 3 12 70 8 Olsalazine-O-sulfate 14 44 232 31 Sulfasalazine* 43 141 - 81 S-Aminosalicylic acid* 1260 979 - 986

&, is a measure of the effect of an inhibitor on the slope of the double inverse plot; Ki, is a measure of the effect of an inhibitor on the intercept of the double inverse plot.

* Values based on Szumlanski and Weinshilboum.”

agents would be removed by means of dilution dur- ing the in vitro enzyme assay. We cannot exclude the possibility that our patient had an as yet undescribed allele for low TPMT activity. Furthermore, long- term 6-mercaptopurine therapy may “induce” the level of RBC TPMT activity as suggested by results of studies of patients with leukemia who took 6- mercaptopurine daily and who had a median RBC TPMT activity of 16.5 U/ml, which fell to 12.5 U/ml after the patient stopped taking the drug for a median of 6 months.26 The duration of the 6- mercaptopurine-related increase in RBC TPMT en- zyme activity after withdrawal of thiopurine is not clearly defined. When the measurement was re- peated during use of low-dose 6-mercaptopurine, our patient’s TPMT activity was 11.0 U/ml RBC. Therefore her endogenous TPMT activity may be even lower when she stops taking the drug for longer than 6 days or is not taking 6-mercaptopurine.

RBC TPMT activity is closely correlated with TPMT activity in the human liver and kidney and can be used as a surrogate marker for TPMT activity in other tissues.20 Szumlanski and Weinshilboum17 speculated that hematologic suppression might be produced in patients with low baseline levels of TPMT activity as a result of a possible phar- macokinetic drug-drug interaction involving 5- aminosalicylic acid or structurally related com- pounds that inhibit the capacity of TPMT to metabolize 6-mercaptopurine. This hypothetical in- teraction has not been reported in a clinical setting.

After a dose of 1000 mg olsalazine by mouth peak plasma concentrations range from 1.6 to 6.2 kmol/L, and steady state plasma concentrations of olsalazine-O-sulfate range from 3.3 to 12.4 kmol/L among patients taking 1000 mg olsalazine daily for 2 to 4 years. 6,28,29 Single doses of 1000 mg olsal-

azine by mouth yield approximately 0.9 gm 5- aminosalicylic acid to be delivered to the colon. Local olsalazine concentrations in the colon have been reported to be in the low millimolar range.6p28T29 Among healthy volunteers, oral daily doses of 2000 mg olsalazine produce peak systemic blood concentrations of 5-aminosalicylic acid and its metabolite N-acetyl-5-aminosalicylic acid of 4.3 and 8.7 pmol/L, respectively, with reported average steady-state concentrations of 2.1 and 5.7 pmol/ L.6,28a29 These systemic concentrations of 5- aminosalicylic acid and N-acetyl-5-aminosalicylic acid are well below their respective IC,, of 1240 pmol/L and 390 pmol/L reported for the inhibition of recombinant TPMT.17-18 However, our studies demonstrated that olsalazine (and its sulfate metab- olite) are potent noncompetitive inhibitors of re- combinant TPMT in vitro with &, and Kii values for 6-mercaptopurine as substrate of 3.0 pmol/L and 12 kmol/L, respectively (Fig. 5, Table III). The IC,, for TPMT inhibition by olsalazine was 23 pmol/L and 1000 mg olsalazine by mouth can produce systemic concentrations of parent drug and its sulfated me- tabolite that are similar to the K,, Kii, and IC,, values of these compounds.6,28,29 When the bone marrow suppression occurred, our patient was re- ceiving either 1000 mg or 1750 mg olsalazine per day. The colonic and systemic olsalazine and olsalazine-O-sulfate concentrations achieved would be adequate to cause TPMT inhibition in both the gastrointestinal tract and liver. We were unable to define whether for our patient the inhibition of in- testinal TPMT or hepatic TPMT played the main role in increasing the 6-mercaptopurine systemic exposure (i.e., reduced presystemic metabolism in the intestine or liver and reduced metabolic clear- ance producing a greater area under the

CLINICAL PHARMA COLOGY &THERAPEUTICS VOLUME 62, NUMBER 4

concentration-time curve), as we suspected from the hematologic toxicity observed.

When our patient received 75 mg 6- mercaptopurine a day with 1000 mg olsalazine a day she experienced leukopenia. Later when taking a lower dose of 50 mg 6-mercaptopurine a day with 1750 mg olsalazine a day, she experienced severe anemia and leukopenia. This clinical course may have been compatible with a pharmacokinetic inter- action during which gastrointestinal or systemic con- centrations of olsalazine or both (plus its sulfated metabolite) inhibited the baseline intermediate-to- low level of intestinal and or hepatic TPMT activity, further reducing the S-methylation of 6- mercaptopurine, resulting in increased systemic ex- posure to 6-mercaptopurine and subsequent leuko- penia and anemia. We suggest that this is the most likely mechanism for our patient and represents a previously undescribed clinical drug-drug interac- tion of the pharmacokinetic type. This proposed drug-drug interaction has little likelihood of being a clinical problem among patients with high baseline TPMT activity. It might, however, be precipitated by these drugs, most commonly among genetically pre- disposed persons with intermediate levels of TPMT activity, that is, heterozygous patients. These per- sons represent approximately 11% of a white pop- ulation.

In patients with acute lymphoblastic leukemia, determination of TPMT phenotype is valuable be- fore initiation of 6-mercaptopurine therapy to indi- vidualize dosing and minimize undertreatment and overtreatment (with its attendant bone marrow tox- icity&26 We suggest that TPMT phenotyping be per- formed before patients receive 6-mercaptopurine (or azathioprine) for other diseases, (such as inflam- matory bowel disease, autoimmune rheumatologic conditions, and certain dermatologic disorders), to optimize therapy. One group of investigators3’ sug- gested that with automated PCR technology TPMT genotyping, which correlates well with TPMT phe- notype, may be an another means of individualizing thiopurine therapy.

In summary, a patient with Crohn’s disease on two occasions had severe, reversible bone marrow suppression. The first episode of leukopenia oc- curred while the patient was taking 75 mg 6- mercaptopurine and 1000 mg olsalazine daily. The second episode of anemia and leukopenia oc- curred while she was taking 50 mg 6- mercaptopurine and 1750 mg olsalazine daily.

Lewis et al. 473

HUMAN RBC TPMT

0 20 a 1 N=143

i U.K. ALL I. Children

0 12 24

TPMT ACTIVITY, UNITS/ML pRBC

Fig. 6. A frequency histogram of TF’MT activity (U/ml per red blood cell [RBC]) in a population sample of children in the United Kingdom with acute lymphoblastic leukemia receiving long-term 6-mercaptopurine therapy. The arrow with the asterisk indicates where our patient’s initial mea- sured RBC TF’MT activity (while not taking 6- mercaptopurine) would fall in this frequency distribution. The arrow without the asterisk indicates where the repeat RBC TPMT activity value of our patient while she was taking 12.5 mg 6-mercaptopurine a day, a low dose, would fall in this frequency distribution.

Such doses of 6-mercaptopurine and olsalazine are considered appropriate therapeutic doses when used alone. Our hypothesis to explain the recurring hematopoietic suppression in this pa- tient was that when 6-mercaptopurine was given with olsalazine, the parent olsalazine and its sul- fated metabolite concentrations in the gastroin- testinal tract and systemic circulation were high enough to exceed the IC,, for TPMT. This en- zyme inhibition, in a patient with relatively low baseline TPMT activity, caused decreased 6- mercaptopurine clearance, high 6-mercaptopurine systemic exposure, and accumulation of intracel- lular 6-thioguanine nucleotides, causing bone marrow suppression (primarily of the WBC and RBC lineages). Physicians should be aware of this potential drug-drug interaction between thiopu- rines and 5-aminosalicylic acid-like compounds, which may be used in the medical management of inflammatory bowel disease and rheumatologic or dermatologic diseases.

474 Lewis et al.

We thank Dr. Paul Strauss at Pharmacia & Upjohn Company for supplying us with samples of olsalazine, olsalazine-O-sulfate, and N-acetyl-5aminosalicylic acid for the in vitro experiments. We are grateful to Susan T. Edwards, MD, for asking us to consult about her patient and facilitating the further investigations, and to Marc Semprebon, RPh, for his assistance with patient data col- lection.

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