EDUCATION PRACTICE. Persistent Helicobacter pylori Infection After a Course of Antimicrobial Therapy What s Next? Clinical Scenario.

Transcription

1 CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2008;6: EDUCATION PRACTICE Persistent Helicobacter pylori Infection After a Course of Antimicrobial Therapy What s Next? RICHARD J. SAAD* and WILLIAM D. CHEY*, *Division of Gastroenterology, Department of Medicine, GI Physiology Laboratory, University of Michigan Medical Center, Ann Arbor, Michigan Clinical Scenario A 35-year-old Hispanic woman with no significant past medical history presents to the emergency department at midnight with a 3-month history of intermittent epigastric pain culminating in 3 episodes of coffee-ground emesis on the day of presentation. Her blood pressure is 90/60 mm Hg and her pulse is 95 beats per minute with evidence of postural orthostasis. Nasogastric lavage reveals coffee-ground material, which clears with a liter of saline. Her hematocrit is 32% before hydration. She is stabilized and resuscitated with intravenous fluids. Upper endoscopy reveals a 1-cm, clean-based, duodenal bulb ulcer. Gastric biopsies reveal a negative rapid urease test but histology reveals active gastritis and Helicobacter pylori (H pylori) organisms. On discharge the following morning she is instructed to take a proton pump inhibitor (PPI), amoxicillin 1 g, and clarithromycin 500 mg twice daily for 7 days. The patient returns to her primary care physician 4 weeks later, reporting initial symptom improvement followed by a gradual recurrence of her epigastric pain. The primary care physician orders a serology test that is positive for H pylori. This is followed by a course of PPI, amoxicillin 1 g, and clarithromycin 500 mg given twice daily for 14 days. The patient returns to her primary care physician 3 months later reporting persistent dyspepsia. She is referred to a gastroenterologist for her persistent symptoms. An esophagogastroduodenoscopy is performed, revealing erosive gastropathy with mucosal biopsy specimens confirming persistent gastritis and H pylori organisms. This case raises several important questions. When and how should H pylori eradication be determined? How does one decide between the various primary treatment options for H pylori infection? What are the treatment options for patients with persistent H pylori infection despite a previous course of eradication therapy? The Problem H pylori is believed to infect two thirds of the world s population. This infection is less common in developed countries such as the United States, where the prevalence ranges from 30% to 40%. A number of evidence-based indications for H pylori testing and treatment exist including current or previous peptic ulcer disease, low-grade mucosa-associated lymphoid tissue lymphoma, endoscopically resected gastric adenocarcinoma, and uninvestigated dyspepsia in those younger than age 55 years and with no alarm features (test-and-treat strategy). Other more controversial indications include gastroesophageal reflux disease, functional dyspepsia, use of nonsteroidal antiinflammatory drugs, a family history of gastric malignancy, unexplained iron deficiency, and idiopathic thrombocytopenic purpura. When H pylori infection is identified, the most widely used first-line therapy consists of 7 to 14 days of a PPI, clarithromycin, and amoxicillin all taken twice daily. Metronidazole may be used as an alternative to amoxicillin, particularly in the setting of penicillin allergy or intolerance. First-line triple therapies yield intention-to-treat eradication rates of 75% to 85%, with most recent studies from the United States reporting eradication rates of less than 80%. Recent data suggest that the prevalence of clarithromycin resistance in the United States is 10% to 13%. Clarithromycin resistance is very important to treatment outcome because it dramatically reduces eradication rates for clarithromycin-containing triple therapy. In fact, eradication rates with this regimen in patients harboring a clarithromycin-resistant strain of H pylori are likely to be less than 30%. Clarithromycin resistance is absolute and cannot be overcome by increasing the dose of clarithromycin. Recent work has identified previous macrolide use as an important risk factor for the presence of clarithromycin resistance. As such, clinicians routinely should ask about previous macrolide antibiotic use before deciding on a treatment regimen for H pylori. A popular alternative first-line therapy is bismuth-based quadruple therapy consisting of a PPI taken twice daily combined with bismuth, tetracycline, and metronidazole taken 4 times daily. A wide variety of doses and durations of this therapy have been reported in the literature, although the most commonly used doses in the United States include bismuth 525 mg, metronidazole 250 mg, and tetracycline 500 mg given for durations of 7 to 14 days. This regimen consistently has achieved cure rates in the range of 75% to 90%. An attractive attribute of this regimen is its effectiveness in both clarithromycin- and metronidazole-resistant strains of H pylori. Although much has been written about the tolerability of this regimen, most side effects are minor and do not lead to discontinuation of therapy. One major issue with this regimen has been the high pill count associated with 4 times daily dosing and, as a downstream consequence, reduced compliance with the regimen. This issue is very important because compliance is an important predictor Abbreviations used in this paper: FAT, fecal antigen test; PPI, proton pump inhibitor; UBT, urea breath test by the AGA Institute /08/$34.00 doi: /j.cgh

2 October 2008 H PYLORI AFTER ANTIMICROBIAL THERAPY 1087 of successful H pylori eradication. It is possible that this issue may be improved by the recent development of a triple capsule that incorporates bismuth, metronidazole, and tetracycline into a single capsule. A new regimen called sequential therapy offers another firstline treatment option. Sequential therapy consists of a PPI and amoxicillin given twice daily for 5 days followed by another 5 days of PPI, clarithromycin, and tinidazole given twice daily. Recent randomized trials from Italy have found that sequential therapy yields modified intention-to-treat eradication rates exceeding 90%. A recent randomized controlled trial from Italy by Vaira et al reported an eradication rate of 91% for sequential therapy versus 78% for a 10-day course of PPI, clarithromycin, and amoxicillin (P.002). Even more impressive was an eradication rate of 89% for sequential therapy versus 29% with traditional triple therapy in patients with clarithromycin-resistant strains of H pylori. Compliance and tolerability were similar between sequential therapy and traditional triple therapy. Despite these promising results, the complexity of this regimen is likely to temper enthusiasm for its wide acceptance. Further, validation of this novel therapy in North America is required before it can be recommended routinely as a first-line therapy. Management Strategies and Supporting Evidence Confirmation of H pylori Eradication Nearly all physicians are now aware of the importance of testing for and treating H pylori infection in patients with a documented peptic ulcer. Unfortunately, physicians sometimes fail to order follow-up testing to prove H pylori eradication after a course of antimicrobial therapy. The importance of follow-up testing to confirm eradication of H pylori infection in patients with ulcer disease has been highlighted in recent studies that have shown that patients who did not undergo such testing were at a greater risk of ulcer recurrence and recurrent upper gastrointestinal bleeding. In general, all patients with an ulcer should undergo follow-up testing to prove cure. Similarly, confirmatory testing should be performed in patients treated for mucosa-associated lymphoid tissue lymphoma and in those treated for dyspepsia particularly if they remain symptomatic despite therapy. It is critically important to understand that persistent symptoms do not necessarily mean that a patient is persistently infected. As such, confirmatory testing should be performed before concluding that a patient remains infected and initiating a second course of antibiotic therapy. Cure of H pylori infection can be confirmed by a number of different diagnostic tests. Nonendoscopic tests that can be used to reliably document eradication include the urea breath test (UBT) and the fecal antigen test (FAT). Two versions of the UBT are commercially available, one using a small dose of radioactive 14 C and another that uses the nonradioactive isotope 13 C. Studies have found the monoclonal version of the FAT to be accurate in the posttreatment setting. Polyclonal versions of the FAT appear to be less reliable and as such should be avoided in the posttreatment setting. Availability of the FAT at some hospitals remains an issue. If not offered locally, most regional and national reference laboratories offer the FAT. The accuracy of both the UBT and FAT can be affected adversely by the recent use of PPIs, bismuth, or antibiotics. Despite being the cheapest and most widely available diagnostic modality, H pylori antibody testing is not a reliable means of confirming eradication. H pylori antibodies can remain present long after successful H pylori eradication. Biopsy-based testing including the rapid urease test or histology also can be used to document H pylori eradication. Because H pylori infection often becomes patchy after antibiotic therapy, a minimum of 4 biopsy specimens should be obtained from the proximal and distal stomach when establishing H pylori eradication with histology. Although the rapid urease test remains highly specific, its sensitivity is reduced after antibiotic or PPI therapy. H pylori culture also can be performed using mucosal biopsy specimens. Such testing is highly specific and capable of providing valuable information on antibiotic resistance. Unfortunately, H pylori culture is less sensitive than histology or the rapid urease test and is available in only a small number of centers. Given these practical limitations, culture should be considered only in cases in which H pylori infection has persisted despite multiple courses of antibiotic treatment. Polymerase chain reaction is a DNA amplification technique that can be performed on mucosal tissue specimens. This powerful diagnostic tool is not yet available for clinical practice. Regardless of the test chosen, one should wait a minimum of 4 weeks after eradication therapy before pursuing confirmatory testing to minimize the likelihood of a false-negative result. PPIs can adversely affect the sensitivity of nonendoscopic and endoscopic tests and should be withheld for 1 to 2 weeks before testing. Salvage Therapy for Persistent H pylori: General Considerations Perhaps the 2 most important predictors for treatment failure include patient noncompliance and resistance of H pylori to specific antibiotics. The significant negative impact of clarithromycin resistance on the efficacy of clarithromycin-containing triple therapy already has been discussed. Metronidazole resistance is more prevalent than clarithromycin resistance; however, its clinical impact can be minimized by the use of higher doses or the inclusion of a PPI in quadruple regimens. Antibody resistance rates for H pylori in the United States are 10% to 13% for clarithromycin, 25% to 37% for metronidazole, and roughly 1% for amoxicillin. The incidence of levofloxacin resistance among H pylori strains in the United States is unknown however, it recently has been reported to be 8% in Canada. From a practical standpoint, clinicians should adhere to several general recommendations when faced with a patient who has persistent H pylori infection despite a previous course of antimicrobial therapy. Under no circumstances should the same antibiotic regimen be used. Certainly, if clarithromycin was used in the initial treatment, it should be avoided in any salvage regimen. It also is advisable to offer salvage regimens for a duration of 10 to 14 days. A variety of regimens have been tested in patients with persistent H pylori infection. The most commonly used salvage regimen is bismuth-based quadruple therapy. Given the potential benefits of sequential therapy in patients with clarithromycin-resistant H pylori infection, one wonders about its use in patients with persistent infection. At present, there are no data available on the use of sequential therapy as a salvage regimen. Several other antibiotics have been used in salvage regimens including levofloxacin, rifabutin, and furazolidone. These regimens are discussed in the following sections.

3 1088 SAAD AND CHEY CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 6, No. 10 Second-Line Therapy for Persistent H pylori Presently, the most commonly used treatment regimen for H pylori infection failing an initial course of antibiotic therapy is bismuth-based quadruple therapy. This treatment consists of bismuth subsalicylate, metronidazole, and tetracycline 4 times daily for 7 to 14 days. Given the potential benefits for patients with metronidazole-resistant strains of H pylori, this therapy should be combined with a PPI once to twice daily. A pooled analysis of trials through 1999 evaluating this regimen as second-line therapy for persistent H pylori infection yielded an efficacy of 75%. A suitable alternative to quadruple therapy is levofloxacinbased triple therapy consisting of levofloxacin 500 mg once daily, amoxicillin 1 g twice daily, and a PPI twice daily for 10 days (Table 1). A recent meta-analysis of 4 randomized controlled trials (391 patients) showed a higher eradication rate with levofloxacin-based triple therapy compared with bismuthbased quadruple therapy (87%; 95% confidence interval [CI], 82% 92% vs 68%; 95% CI, 62% 74%, respectively; RR, 1.41; 95% CI, ). Side effects were comparatively less common with levofloxacin therapy (RR, 0.51; 95% CI, ) and the likelihood of discontinuing therapy because of its side effects was less with levofloxacin therapy (RR, 0.30; 95% CI, ). Most of the currently available studies have come from southern Europe and Asia. As such, whether these results can be generalized to other parts of the world remains unclear. Rifabutin, a drug used to treat tuberculosis, has been evaluated in a number of small studies showing eradication rates ranging from 44% to 91% in the setting of persistent H pylori infection. Doses of rifabutin that have been studied include 150 or 300 mg (dosed once daily or split dosed 150 mg twice a day) typically combined with a PPI and amoxicillin given twice daily for 7 to 14 days. Eradication rates appear highest when rifabutin is used as a second-line therapy, decreasing as the number of previous failed therapies increases. The use of rifabutin is limited by its adverse gastrointestinal effects (nausea, vomiting, and diarrhea) and the potential for myelotoxicity or ocular toxicity. There also are concerns with the widespread use of rifabutin and the potential impact on treatment efficacy in tuberculosis. Furazolidone is a synthetic derivative of the nitrofuran class of antimicrobials, which also has been evaluated in a number of studies with eradication rates ranging from 52% to 90% in the treatment of persistent H pylori. It is difficult to assess the efficacy of furazolidone-based salvage therapy owing to the variability in treatment regimens. In these studies, the daily dose of furazolidone varied from 200 to 600 mg. Furthermore, a variety of triple- and quadruple-drug regimens were used with treatment durations ranging from 7 to 14 days. Side effects can be substantial in these furazolidone-based therapies, occurring in as many as 35% of patients, with vomiting, nausea, and dizziness being reported most commonly. Furazolidone also may interact with a variety of foods and other drugs given its effect on monoamine oxidase as well as glucose-6- phosphate dehydrogenase activity and its potential disulfiramlike reaction with alcohol. Furazolidone has fallen out of favor related to its adverse event profile and limited availability. Third-Line Therapy for Persistent H pylori There is less consensus regarding treatment regimens for persistent H pylori infection despite 2 or more previous courses of antibiotics. Bismuth-based quadruple therapy, levofloxacin-based triple therapy, rifabutin-based triple therapy, and furazolidone-based regimens have been investigated. The stud- Table 1. Testing for Confirmation of H pylori Eradication Clinical caveats Endoscopy-based tests Histology Rapid urea test Culture Nonendoscopy-based tests UBT FAT Appropriate when endoscopy is indicated for other reasons Should be performed no sooner than 4 weeks after the completion of antibiotic therapy Sensitivity of all tests reduced by recent PPIs, bismuth, or antibiotics Proximal and distal biopsies improve sensitivity, particularly in patients taking a PPI, bismuth, or antibiotics Provides information beyond the presence of H pylori Gastritis provides a surrogate marker of infection when organisms are not identified Inexpensive and yields results within 1 hour Highly specific a positive test identifies patients with active H pylori infection Reduced sensitivity in patients with acute ulcer bleeding and after antibiotic therapy; should be performed in combination with another test (ie, histology) Highly specific but only marginal sensitivity Capable of providing antibiotic sensitivity profile of H pylori organism (helps to tailor salvage therapy) Generally not available in the United States owing to cost and technical difficulty with culturing methodology Testing modalities of choice to confirm eradication after antibiotic therapy in most situations Should be performed no sooner than 4 weeks after the completion of antibiotic therapy Identifies active infection through detection of the urease activity of H pylori Radioactive ( 14 C) and nonradioactive ( 13 C) isotopes available for use in the UBT Reduced sensitivity in the face of recent PPI, bismuth, or antibiotics; PPIs should be withheld for 1 to 2 weeks before the UBT Tests for active infection through detection of H pylori antigen Monoclonal test superior to polyclonal test for posttreatment testing Reduced sensitivity in the face of recent PPI, bismuth, or antibiotics; PPIs should be held for a minimum of 2 weeks before obtaining a stool specimen

4 October 2008 H PYLORI AFTER ANTIMICROBIAL THERAPY 1089 Table 2. Treatment Options for Patients With Persistent H pylori Infection Treatment regimen Duration of therapy (d) Eradication rates Bismuth subsalicylate 525 mg qid, metronidazole a 250 mg qid, % 75% (95% CI, 62% 74%) tetracycline 500 mg qid, PPI qd or bid Levofloxacin 500 mg qd, amoxicillin 1000 mg bid, standard dose b PPI bid 10 87% (95% CI, 82% 92%) If bismuth is not available, amoxicillin 1 g bid or tetracycline 500 mg qid, % (95% CI, 65% 74%) metronidazole 500 mg bid, PPI bid c Rifabutin 300 mg qd, d amoxicillin 1 g bid, PPI bid % 91% (based on individual studies) qid, 4 times a day; qd, everyday; bid, twice a day. a May substitute with furazolidone if metronidazole resistance exists. b Esomeprazole may be used once daily. c If available, ranitidine bismuth citrate may be used in place of a PPI. d Rifabutin 150 mg twice daily is an alternative dosing regimen. ies generally have been small with widely variable results (Table 2). A 14-day course of bismuth quadruple therapy yielded an eradication rate of 95% (95% CI, 85% 99%) in 42 patients whereas another study in which 18 patients received a 7-day course of therapy reported an eradication rate of only 33%. Levofloxacin triple therapy was found to eradicate H pylori in 86%, whereas a more recent study showed an eradication rate of only 60% (95% CI, 50% 70%) in 100 patients who had failed 2 previous courses of therapy. A 7-day course of rifabutin triple therapy achieved an eradication rate of 71% (95% CI, 57% 85%) in 41 patients, whereas a 14-day course of therapy achieved an eradication rate of 79% (95% CI, 49% 95%) in a cohort of 14 patients. Furazolidone-based therapy as third-line salvage treatment yielded eradication rates of 60% to 90% in several small studies with a recent systematic review showing an overall efficacy of 66% (95% CI, 56% 76%). Areas of Uncertainty There are few studies performed in North American populations that have evaluated the earlier-described regimens in patients with persistent H pylori infection. As a result, antibiotic regimens recommended for persistent H pylori infection are largely taken from studies performed in other regions of the world. Furthermore, given the widespread use of antibiotics in North American countries and the regional variation of antibiotic resistance profiles among H pylori strains, the true effect of antibiotic resistance remains unknown. The optimal treatment duration for salvage therapy remains unknown. All experts agree that a minimum of 7 days of therapy should be pursued when treating persistent H pylori. There is some evidence that longer courses of therapy such as 10- or 14-day courses may provide better eradication rates compared with shorter durations. Unfortunately, the available evidence is inconsistent. Furthermore, it is difficult to compare the effects of treatment duration among the clinical studies because of tremendous variability in the specific antibiotics and doses used. Of course, the added cost and increased risk of patient noncompliance must be weighed against the perceived benefit of a longer course of therapy. There is no evidence to guide recommendations regarding the maximum number of treatment attempts in the setting of persistent H pylori infection. In general, the authors offer 3 courses of anti-helicobacter therapy before carefully reconsidering the need for further courses of antibiotics. In such patients, the potential benefits of eradicating the infection should be weighed against the risks and costs of multiple courses of antibiotics. For example, in a patient with functional dyspepsia who has persistent H pylori infection, the potential for symptom relief is likely to be low. In this setting, a clinician may consider observation alone after a third treatment failure. Alternatively, in a patient with peptic ulcer disease or mucosa-associated lymphoid tissue lymphoma in which the potential benefits of eradication are likely to be high, it would be quite reasonable to make further attempts to eradicate the infection. Another issue worthy of mention is the potential adjunctive role of probiotic therapy. There is emerging evidence to suggest that some probiotic strains (Lactobacillus species, Saccharomyces boulardii) may improve the eradication rates and tolerability of antibiotic therapy for H pylori infection. In general, the available literature has addressed an adjunctive role for probiotics in the setting of primary therapy. Whether incremental benefits through the use of probiotics can be achieved in the setting of salvage therapy remains unknown. Published Guidelines The American College of Gastroenterology recently published updated guidelines for the management of H pylori in These guidelines support the use of either bismuth quadruple therapy or levofloxacin-based triple therapy for 10 to 14 days in patients with persistent H pylori infection. The European Helicobacter Study Group also recently updated its international consensus guidelines for the management of H pylori, which were published in 2007 as the Maastricht III Consensus Report. The specific recommendations call for the use of bismuth-based quadruple therapy as a second-line treatment for persistent H pylori. If bismuth cannot be used or is not available, the alternative second-line therapy should consist of a PPI, metronidazole, and amoxicillin or tetracycline. If this therapy fails to eradicate the infection, further treatment regimens should be based on antimicrobial susceptibility testing. In this document, levofloxacin- and rifabutin-based regimens are recommended for third-line treatment or beyond. The Canadian Helicobacter Study Group initially published the recommendations from its consensus conference in 1998 also advocating the use of a 14-day course of bismuth-based quadruple therapy for persistent H pylori infection. This recommendation was amended the following year to include two 7- to 14-day ranitidine bismuth citrate or PPI-based triple therapy regimens in addition to that of bismuth-based quadruple therapy.

5 1090 SAAD AND CHEY CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 6, No. 10 The Latin-American Consensus Conference published its quent treatment regimens. Because of the patient s recent history of peptic ulcer disease and persistent symptoms, an recommendations on the management of H pylori in In general, the recommendations on treatment regimens were similar to those of other consensus groups. There was a specific pylori was reasonable. Given the presence of persistent H pylori esophagogastroduodenoscopy with biopsy-based testing for H recommendation for considering the use of furazolidone-based infection, bismuth-based quadruple therapy or levofloxacin- triple therapy for 10 to 14 days would be a reasonable regimens in low-income populations given its relatively low costbased compared with other antimicrobials used in the treatment ofnext step to pursue. Follow-up testing with a UBT or monoclonal H pylori. FAT should be scheduled no less than 4 weeks after the completion of therapy to confirm eradication H of pylori. Recommendations Suggested Reading This patient had a peptic ulcer, which provides a clear1. Chey WD, Wong BC. American College of Gastroenterology guideline on the management of Helicobacter pylori infection. Am J indication for eradicating H pylori infection. Active infection was confirmed by biopsy-based testing obtained at the time of Gastroenterol 2007;102: esophagogastroduodenoscopy. Some have argued that the sensitivity of the rapid urease test may be reduced in the setting of the management of Helicobacter pylori infection: the Maastricht III 2. Malfertheiner P, Megraud F, O Morain C, et al. Current concepts in acute ulcer bleeding, although this remains quite controversial. Consensus Report. Gut 2007;56: Hojo M, Miwa H, Nagahara A, et al. Pooled analysis on the efficacy Had biopsy-based testing been negative or mistakenly not performed, it would have been appropriate to check a serology test of the second-line treatment regimens for Helicobacter pylori infection. Scand J Gastroenterol 2001;36: for H pylori. It is worth noting that the positive predictive value4. Saad R, Schoenfeld P, Kim H, et al. Levofloxacin-based triple of serology is greatly influenced by the pretest probability of therapy versus bismuth-based quadruple therapy for persistent H pylori infection. Because the pretest probability of infection is Helicobacter pylori infection: a meta-analysis. Am J Gastroenterol relatively high in patients with a peptic ulcer, the positive 2006;101: predictive value of serology is reasonably good. Another option5. Buzas GM, Jozan J. Nitrofuran-based regimens for the eradication would be to check a UBT or FAT, preferably at a time when theof Helicobacter pylori infection. J Gastroenterol Hepatol 2007;22: patient had not been taking a PPI for 1 to 2 weeks. Several missteps occurred during the management of this patient. The6. Di Mario F, Cavallaro LG, Scarpignato C. Rescue therapies for the management of Helicobacter pylori infection. Dig Dis 2006; initial course of clarithromycin-based triple therapy was given 24: for 7 days. As already has been noted, eradication rates with this 7. Franceschi F, Cazzato A, Nista EC, et al. Role of probiotics in regimen for 7 days consistently have been reported to be less patients with Helicobacter pylori infection. Helicobacter 2007; than 80%. This fact combined with the presence of H an pylori 12(Suppl 2): associated ulcer mandates the need for testing to prove eradi-8cation of the infection after antimicrobial therapy. Obtaining a plementation with probiotics on eradication rates and adverse Tong JL, Ran ZH, Shen J, et al. Meta-analysis: the effect of sup- serology test in an attempt to confirm cure was not appropriate events during Helicobacter pylori eradication therapy. Aliment in this case. It is unrealistic to expect that an antibody test Pharmacol Ther 2007;25: would have converted to negative only a month after eradication therapy. It would have been more appropriate to obtain a Address requests for reprints to: William D. Chey, MD, AGAF, FACG, UBT or monoclonal FAT to confirm eradication. It also was FACP, Professor of Internal Medicine, Director, GI Physiology Laboratory, University of Michigan Medical Center, 3912 Taubman Center, inappropriate for the patient to be retreated with another course of clarithromycin-based triple therapy regardless of extending the duration of treatment. This patient is at very high(734) Box0362,AnnArbor,Michigan wchey@umich.edu;fax: risk of harboring a clarithromycin-resistant strain H of pylori, Dr Chey is on the speaker s bureau and is a consultant for Santarus, which makes it important to avoid clarithromycin in subse-tap Pharmaceuticals, and Takeda.