Black Cohosh Interactions with Prescription Medications Associated with Serotonin Toxicity and Rhabdomyolysis: A Case Report.

BACKGROUND: Serotonin toxicity is a well-described phenomenon that is commonly attributed to a variety of drug-drug combinations. Some unregulated herbal supplements have been implicated in the onset of serotonin toxicity, however, there is currently minimal literature available on the potential for black cohosh to contribute to rhabdomyolysis and serotonin toxicity, in spite of its known serotonergic properties. CASE REPORT: A middle-aged woman presented to the emergency department with serotonin toxicity and rhabdomyolysis shortly after taking black cohosh supplements in the setting of long-term dual antidepressant use. The serotonin toxicity and rhabdomyolysis resolved with IV fluids, benzodiazepines, and discontinuation of the offending drugs. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Patients are sometimes not aware of how over-the-counter supplements might interact with their prescription medications. Female patients taking black cohosh to manage hot flashes and menopausal symptoms could be at risk for developing rhabdomyolysis and serotonin toxicity if they are also taking other serotonergic agents.

A cross-sectional clinical study in women to investigate possible genotoxicity and hematological abnormalities related to the use of black cohosh botanical dietary supplements.

Black cohosh (BC; Actaea racemosa L.), a top-selling botanical dietary supplement, is marketed to women primarily to ameliorate a variety of gynecological symptoms. Due to widespread usage, limited safety information, and sporadic reports of hepatotoxicity, the Division of the National Toxicology Program (DNTP) initially evaluated BC extract in female rats and mice. Following administration of up to 1000 mg/kg/day BC extract by gavage for 90 days, dose-related increases in micronucleated peripheral blood erythrocytes were observed, along with a nonregenerative macrocytic anemia resembling megaloblastic anemia in humans. Because both micronuclei and megaloblastic anemia may signal disruption of folate metabolism, and inadequate folate levels in early pregnancy can adversely affect neurodevelopment, the DNTP conducted a pilot cross-sectional study comparing erythrocyte micronucleus frequencies, folate and B12 levels, and a variety of hematological and clinical chemistry parameters between women who used BC and BC-naïve women. Twenty-three women were enrolled in the BC-exposed group and 28 in the BC-naïve group. Use of any brand of BC-only supplement for at least 3 months was required for inclusion in the BC-exposed group. Supplements were analyzed for chemical composition to allow cross-product comparisons. All participants were healthy, with no known exposures (e.g., x-rays, certain medications) that could influence study endpoints. Findings revealed no increased micronucleus frequencies and no hematological abnormalities in women who used BC supplements. Although reassuring, a larger, prospective study with fewer confounders (e.g., BC product diversity and duration of use) providing greater power to detect subtle effects would increase confidence in these findings.

Black cohosh efficacy and safety for menopausal symptoms. The Spanish Menopause Society statement.

OBJECTIVE: The increasing interest in Western countries regarding phytotherapy use to treat menopause-related symptoms has led the Spanish Menopause Society (AEEM) to update its position statement performed in 2009 on the role of black cohosh (Cimicifuga racemosa) for the treatment of menopausal symptoms. MATERIAL AND METHODS: A panel of experts from both clinical and research backgrounds were assembled to investigate the best available evidence. Selected studies were obtained by an electronic search, including the Internet search engines MEDLINE-Pubmed (1997-December 2021) and the Cochrane Controlled Trials Register. RESULTS: Most of the well-designed studies published in recent years have been conducted with the isopropanolic extract of black cohosh/C. racemosa. The most common dose is 40 mg/day capable of achieving a significant reduction in hot flushes (particularly in women with intense hot flushes) and an improvement in mood. Used at the recommended doses, C. racemose produces no significant adverse reactions. CONCLUSION: Black cohosh is an effective and safe treatment option for the relieving of vasomotor symptoms. Finally, further clinical trials with sufficient patient enrollment and longer study follow-up are needed.

Weight gain in menopause: systematic review of adverse events in women treated with black cohosh.

Weight gain is a frequent problem in perimenopausal and postmenopausal women. Cimicifuga racemosa (CR) is a popular treatment option for menopausal symptoms. The aim of this review was to investigate whether there is scientific evidence that CR causes weight gain. We searched our database for medically confirmed, spontaneous adverse events regarding weight gain, literature for case reports and randomized controlled trials. Thirty cases in total were spontaneously reported in 15 years. The causality was not considered certain/likely in any of the cases. A nurse (consumer) assessed the causality as possible. Only one case was published in the literature. However, no change in body fat composition was reported, and the causality seems unlikely. Of the 31 identified studies, 17 were double-blind placebo-controlled, five were double-blind reference-controlled and nine were open reference-controlled. In total, 1839 women were treated with CR for up to 12 months. Two studies reported weight gain as an adverse event; however, no significant differences in weight changes were observed between the groups. One case of weight gain (about 2 kg) was reported, but the authors did not specify in which treatment group. In conclusion, this study provides no scientific evidence that the use of Cimicifuga racemosa causes weight gain in menopausal women.

Evidence for an Aneugenic Mechanism of Action for Micronucleus Induction by Black Cohosh Extract.

Black cohosh extract (BCE) is a popular botanical dietary supplement marketed to relieve symptoms of various gynecological ailments. Studies conducted by the National Toxicology Program (NTP) showed that BCE induces micronucleated erythrocytes in female rats and mice. Subsequently, the NTP showed that a variety of BCEs, including the sample that induced micronuclei (MN) in vivo ("NTP BCE") had a similar effect in human TK6 cells. Further testing with the MultiFlow® DNA Damage Assay revealed that TK6 cells exposed to NTP BCE, as well as a BCE reference material (BC XRM), exhibited a signature consistent with aneugenic activity in TK6 cells. Results from experiments reported herein confirmed these in vitro observations with NTP BCE and BC XRM. We extended these studies to include a novel test system, the MultiFlow Aneugen Molecular Mechanism Assay. For these experiments, TK6 cells were exposed to NTP BCE and BC XRM over a range of concentrations in the presence of fluorescent Taxol (488 Taxol). After 4 h, nuclei from lysed cells were stained with a nucleic acid dye and labeled with fluorescent antibodies against phospho-histone H3 (p-H3) and Ki-67. Whereas BCEs did not affect p-H3:Ki-67 ratios (a signature of aneugenic mitotic kinase inhibitors), 488 Taxol-associated fluorescence (a tubulin binder-sensitive endpoint) was affected. More specifically, 488 Taxol-associated fluorescence was reduced over the same concentration range that was previously observed to induce MN. These results provide direct evidence that BCEs destabilize microtubules in vitro, and this is the molecular mechanism responsible for the aneugenicity findings. Environ. Mol. Mutagen. 2019. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

Analysis of spontaneous adverse drug reactions to echinacea, valerian, black cohosh and ginkgo in Australia from 2000 to 2015.

OBJECTIVE: Assessing adverse drug reactions (ADRs) is a proven method to estimate the safety of medicines. The ADRs to herbal medicines in Australia (and by inference, the safety of herbal medicines in Australia) remain unknown. This study examines spontaneous ADR cases to four of the most popular herbs in Australia from 2000 to 2015: echinacea (Echinacea purpurea), valerian (Valeriana officinalis), black cohosh (Actaea racemosa) and ginkgo (Ginkgo biloba). METHODS: ADRs of echinacea, valerian, black cohosh and ginkgo reported to the Australian Therapeutic Goods Administration (TGA) between 2000 and 2015 were obtained from the TGA database. Data were collated and analysed according to age, sex, severity, type of ADR and body system affected. Statistics were calculated using GraphPad Prism software. RESULTS: Most ADRs were mild or moderate. However, every herbal medicine was associated with life-threatening ADRs. In each life-threatening case, the herbal medicine was taken concomitantly with prescription medications. Black cohosh was associated with a significant number of severe ADRs (30.3% of the total), with 39.4% of these ADRs being associated with abnormal hepatic function, hepatitis or hepatotoxicity. CONCLUSION: This study highlights the lack of public awareness with regard to herb-drug interactions, since most of the severe ADRs involved a herb-drug interaction.

Black cohosh extracts and powders induce micronuclei, a biomarker of genetic damage, in human cells.

Black cohosh extract (BCE) is a widely used dietary supplement marketed to women to alleviate symptoms of gynecological ailments, yet its toxicity has not been well characterized. The National Toxicology Program (NTP) previously reported significant increases in micronucleated erythrocytes in peripheral blood of female Wistar Han rats and B6C3F1/N mice administered 15-1,000 mg BCE/kg/day by gavage for 90 days. These animals also developed a dose-dependent nonregenerative macrocytic anemia characterized by clinical changes consistent with megaloblastic anemia. Both micronuclei (MN) and megaloblastic anemia can arise from disruption of the folate metabolism pathway. The NTP used in vitro approaches to investigate whether the NTP's test lot of BCE, BCEs from various suppliers, and root powders from BC and other cohosh species, were genotoxic in general, and to gain insight into the mechanism of action of BCE genotoxicity. Samples were tested in human TK6 lymphoblastoid cells using the In Vitro MicroFlow® MN assay. The NTP BCE and a BC extract reference material (XRM) were tested in the MultiFlow® DNA Damage assay, which assesses biomarkers of DNA damage, cell division, and cytotoxicity. The NTP BCE and several additional BCEs were tested in bacterial mutagenicity assays. All samples induced MN when cells were grown in physiological levels of folic acid. The NTP BCE and BC XRM produced activity patterns consistent with an aneugenic mode of action. The NTP BCE and five additional BCEs were negative in bacterial mutagenicity tests. These findings show that black cohosh preparations induce chromosomal damage and may pose a safety concern. Environ. Mol. Mutagen. 59:416-426, 2018. © 2018 Published 2018. This article is a US Government work and is in the public domain in the USA.

Pre-clinical in vitro and in vivo safety evaluation of Cimicifuga heracleifolia.

The rhizomes of Cimicifuga species, including Cimicifuga heracleifolia (CH), have been widely used as antipyretic, analgesic, and anti-inflammatory agents in oriental countries. However, information regarding its toxicity, especially long-term toxicity and genotoxicity, is limited. Therefore, we performed the subchronic toxicity and genotoxicity assays of the CH extract in accordance with the test guidelines published by the Organization for Economic Cooperation and Development. In a 13-week repeat-dose oral toxicity study, the CH extract did not influence body weight, food/water consumption, mortality, clinical signs, and urinalysis throughout the study. Noteworthy, the CH extract groups exhibited increased liver weights along with serum alanine transaminase activity rise at doses of 667 and 2000 mg/kg in females. No-observed-adverse-effect-level of the CH extract administered orally was concluded to be 2000 mg/kg body weight/day for male rats and 222 mg/kg body weight/day for female rats. The CH extract did not exert a mutagenic or clastogenic effect in Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay. Overall findings of the subchronic toxicity study indicate for the first time that the CH extract may possess hepatotoxic potential in female rats, suggesting that further mechanistic studies should be performed to have more conclusive results on hepatotoxic potential of the CH extract.

Efficacy and safety of remifemin on peri-menopausal symptoms induced by post-operative GnRH-a therapy for endometriosis: a randomized study versus tibolone.

BACKGROUND: The aim of this study was to investigate clinical efficacy and safety of Remifemin on peri-menopausal symptoms in endometriosis patients with a post-operative GnRH-a therapy. MATERIAL AND METHODS: We treated 116 women who had endometriosis with either Remifemin (n=56) 20 mg bid po or Tibolone (n=60) 2.5 mg qd po for 12 weeks after GnRH-a injection. The efficacy was evaluated by Kupperman menopausal index (KMI), and hot flash/sweating scores. The safety parameters such as liver and renal functions, lipid profile, endometrial thickness, and serum sex hormone level, as well as the incidence of adverse events were recorded. RESULTS: (1) After GnRH-a therapy, KMI and hot flash/sweating scores in both groups increased significantly (P<0.05) but we found no significant difference for KMI (2.87±1.40 for Remifemin and 2.70±1.26 for Tibolone) and hot flash/sweating scores (0.94±1.72 for Remifemin and 1.06±1.78 for Tibolone) between the 2 groups (P>0.05). (2) No statistical change was observed in liver or renal functions and lipid profile in both groups before and after the treatment (P>0.05). The post-therapeutic serum FSH, LH, and E2 level and endometrial thickness decreased remarkably in both groups (P<0.05). E2 level in the Remifemin group was obviously lower than that in the Tibolone group (P<0.05), and FSH and LH levels were strongly higher (P<0.05). No significant difference in thickness were found in either group (P>0.05). The Remifemin group had far fewer adverse events than the Tibolone group (P<0. 05). CONCLUSIONS: Compared with Tibolone, Remifemin had a similar clinical efficacy and was safer for the peri-menopausal symptoms induced by GnRH-a in endometriosis patients.

Black cohosh and breast cancer: a systematic review.

BACKGROUND: Many women use black cohosh as a natural treatment for menopausal symptoms. However, controversy exists around safety in breast cancer, because of its purported estrogenic activity. We conducted a systematic review of black cohosh use in women with or at risk of breast cancer. METHODS: We searched MEDLINE, Embase, the Cochrane Library, and AMED from inception to July 2012 and October 2012 for human interventional or observational data pertaining to the safety and efficacy of black cohosh in patients with or at risk of breast cancer, including an assessment of the effect of black cohosh on estrogen responsive tissues. RESULTS: Of 450 records, we included 26 articles: 14 randomized controlled trials, 7 uncontrolled trials, and 5 observational studies.The evidence on efficacy for ho t flashes is divided, with some benefits seen when compared with baseline, but not when compared with placebo. Two observational studies found no association between black cohosh and risk of breast cancer, whereas 2 studies reported significant reductions in risk of primary breast cancer among postmenopausal women (adjusted odds ratio = 0.47, 95% confidence interval = 0.27-0.82), and risk of recurrence (adjusted hazard ratio = 0.75, 95% confidence interval = 0.63-0.89). Seventeen trials showed no significant impact on circulating hormone levels or proliferation in estrogen responsive tissues. CONCLUSIONS: Current evidence does not support an association between black cohosh and increased risk of breast cancer. There is a lack of evidence supporting the efficacy of black cohosh for reduction of hot flashes in breast cancer patients. Given conflicting but promising results, and apparent safety, further research is warranted.

Efficacy of black cohosh (Cimicifuga racemosa) medicines for treatment of menopausal symptoms - comments on major statements of the Cochrane Collaboration report 2012 "black cohosh (Cimicifuga spp.) for menopausal symptoms (review)".

Menopausal symptoms management with high-quality plant extracts from Actaea (Cimicifuga. racemosa rootstock is well-established. Efficacy and safety are supported by research and clinical trials since several decades and backed up by official monographs. However, the recent published Cochrane review on black cohosh neglects major evidence for beneficial effects. The authors' negative conclusions are questionable and call for reply and clarification. Our careful reconsideration of all appropriate placebo-controlled clinical studies reveals a standardized mean difference of 0.385 in favor of black cohosh (p < 0.0001).

Black cohosh (Cimicifuga spp.) for menopausal symptoms.

BACKGROUND: Menopause can be a distressing and disruptive time for many women, with many experiencing hot flushes, night sweats, vaginal atrophy and dryness. Postmenopausal women are also at increased risk of osteoporosis. Interventions that decrease the severity and frequency of these menopausal symptoms are likely to improve a woman's well-being and quality of life. Hormone therapy has been shown to be effective in controlling the symptoms of menopause; however, many potentially serious adverse effects have been associated with this treatment. Evidence from experimental studies suggests that black cohosh may be a biologically plausible alternative treatment for menopause; even so, findings from studies investigating the clinical effectiveness of black cohosh have, to date, been inconsistent. OBJECTIVES: To evaluate the clinical effectiveness and safety of black cohosh (Cimicifuga racemosa or Actaea racemosa) for treating menopausal symptoms in perimenopausal and postmenopausal women. SEARCH METHODS: Relevant studies were identified through AARP Ageline, AMED, AMI, BioMed Central gateway, CAM on PubMed, CINAHL, CENTRAL, EMBASE, Health Source Nursing/Academic edition, International Pharmaceutical Abstracts, MEDLINE, Natural medicines comprehensive database, PsycINFO, TRIP database, clinical trial registers and the reference lists of included trials; up to March 2012. Content experts and manufacturers of black cohosh extracts were also contacted. SELECTION CRITERIA: All randomised controlled trials comparing orally administered monopreparations of black cohosh to placebo or active medication in perimenopausal and postmenopausal women. DATA COLLECTION AND ANALYSIS: Two review authors independently selected trials, extracted data and completed the 'Risk of bias' assessment. Study authors were contacted for missing information. MAIN RESULTS: Sixteen randomised controlled trials, recruiting a total of 2027 perimenopausal or postmenopausal women, were identified. All studies used oral monopreparations of black cohosh at a median daily dose of 40 mg, for a mean duration of 23 weeks. Comparator interventions included placebo, hormone therapy, red clover and fluoxetine. Reported outcomes included vasomotor symptoms, vulvovaginal symptoms, menopausal symptom scores and adverse effects. There was no significant difference between black cohosh and placebo in the frequency of hot flushes (mean difference (MD) 0.07 flushes per day; 95% confidence interval (CI) -0.43 to 0.56 flushes per day; P=0.79; 393 women; three trials; moderate heterogeneity: I(2) = 47%) or in menopausal symptom scores (standardised mean difference (SMD) -0.10; 95% CI -0.32 to 0.11; P = 0.34; 357 women; four trials; low heterogeneity: I(2) = 21%). Compared to black cohosh, hormone therapy significantly reduced daily hot flush frequency (three trials; data not pooled) and menopausal symptom scores (SMD 0.32; 95% CI 0.13 to 0.51; P=0.0009; 468 women; five trials; substantial heterogeneity: I(2) = 69%). These findings should be interpreted with caution given the heterogeneity between studies. Comparisons of the effectiveness of black cohosh and other interventions were either inconclusive (because of considerable heterogeneity or an insufficient number of studies) or not statistically significant. Similarly, evidence on the safety of black cohosh was inconclusive, owing to poor reporting. There were insufficient data to pool results for health-related quality of life, sexuality, bone health, vulvovaginal atrophic symptoms and night sweats. No trials reported cost-effectiveness data. The quality of included trials was generally unclear, owing to inadequate reporting. AUTHORS' CONCLUSIONS: There is currently insufficient evidence to support the use of black cohosh for menopausal symptoms. However, there is adequate justification for conducting further studies in this area. The uncertain quality of identified trials highlights the need for improved reporting of study methods, particularly with regards to allocation concealment and the handling of incomplete outcome data. The effect of black cohosh on other important outcomes, such as health-related quality of life, sexuality, bone health, night sweats and cost-effectiveness also warrants further investigation.

Menopause: a standardized isopropanolic black cohosh extract (remifemin) is found to be safe and effective for menopausal symptoms.

This randomized, double-blind, placebo-controlled, multicenter clinical study investigated the efficacy and various safety aspects of a standardized proprietary isopropanolic extract of black cohosh (Remifemin) in a population of 304 postmenopausal women who met strict parameter guidelines that determined the inclusion criteria. This clinical study confirmed previous results, which clearly indicate the efficacy and tolerability of this proprietary black cohosh extract (Remifemin) in addressing menopausal symptoms, particularly hot flashes.

Spontaneous reports of assumed herbal hepatotoxicity by black cohosh: is the liver-unspecific Naranjo scale precise enough to ascertain causality?

PURPOSE: Causality assessment of cases with herbal hepatotoxicity represents a major regulatory challenge and included, in the past, the application of a diagnostic algorithm consisting of causality evaluation methods with either liver-specific or liver-unspecific characteristics. To evaluate various causality assessing methods in cases with suspected herbal hepatotoxicity, two different scales were now used for reasons of comparison. METHODS: We used the liver-specific scale of the updated Council for International Organizations of Medical Sciences (CIOMS) as well as the Naranjo scale that is not organ specific and therefore not liver specific. Both scales were applied to 22 cases of spontaneous reports with initially assumed herbal hepatotoxicity caused by black cohosh, used for menopausal symptoms. RESULTS: The analysis shows that causality was either unlikely (n = 6) or excluded (n = 16), using the updated CIOMS scale. There were various confounding variables: pre-existing liver diseases (n = 6) including genuine autoimmune hepatitis or alcoholic or cardiac hepatopathy; hepatotoxicity induced by interferon or fluoxetine (n = 2); marginally increased serum activities of alanine aminotransferase (n = 2) or gamma-glutamyltranspeptidase (n = 2) of unassessable causality; a mixed group consisting of unassessable cases (n = 6) and cases with questionable, poorly documented hepato-biliary diseases (n = 3); and rosuvastin-induced rhabdomyolysis (n = 1). These confounding factors were not recognized by the Naranjo scale. CONCLUSIONS: Structured hepatotoxicity-specific causality assessment methods such as the updated CIOMS scale are the preferred tools for causality assessment of assumed herbal hepatotoxicity and should replace the liver-unspecific Naranjo scale. Applying the updated CIOMS scale to cases with initially assumed hepatotoxicity by BC, causality was now found either unlikely or excluded.

Herb induced liver injury presumably caused by black cohosh: a survey of initially purported cases and herbal quality specifications.

Herb induced liver injury (HILI) is a particular challenge that also applies to purported cases presumably caused by black cohosh (BC), an herb commonly used to treat menopausal symptoms. We analyzed and reviewed all published case reports and spontaneous reports of initially alleged BC hepatotoxicity regarding quality of case details and causality assessments. Shortcomings of data quality were more evident in spontaneous reports of regulatory agencies compared to published case reports, but assessments with the scale of CIOMS (Council for the International Organizations of Sciences) or its updated version revealed lack of causality for BC in all cases. The applied causality methods are structured, quantitative, and liver specific with clear preference over an ad hoc causality method or the liver unspecific Naranjo scale. Reviewing the case data and the reports dealing with quality specifications of herbal BC products, there is general lack of analysis with respect to authentication of BC in the BC products used by the patients. However, in one single regulatory study, there was a problem of BC authentication in the analysed BC products, and other reports addressed the question of impurities and adulterants in a few BC products. It is concluded that the use of BC may not exert an overt hepatotoxicity risk, but quality problems in a few BC products were evident that require additional regulatory quality specifications.

Drug-induced liver injury: a summary of recent advances.

INTRODUCTION: The knowledge base of drug-induced liver injury (DILI) continues to grow each year as additional drugs are identified as hepatotoxins. There is still a need to improve our ability to predict and diagnose DILI in the preclinical and post-approval settings. AREAS COVERED: This article presents the new and updated DILI registries for 2010, including the latest information on the causes and outcomes of non-acetaminophen DILI cases in the US Acute Liver Failure Study Group database. As DILI is still largely a diagnosis of exclusion, it is appropriate that causality assessment instruments are again the subject of considerable discussion. EXPERT OPINION: DILI research remains extremely active including studies aimed at being better able to identify causative agents, utilize potential biomarkers, predict who is at greatest risk of injury and manage outcomes. With respect to identifying DILI risk factors at the genetic level, the field is rapidly approaching the day where 'personalized medicine' (based on pharmacogenomics) will become a reality. A large single-center series from India reminds us that geography can influence the drugs responsible for liver injury; however, Hy's law remains universal. As our DILI knowledge continues to grow, it remains essential to keep abreast of the important changes reported each year.

The effects of commercial preparations of herbal supplements commonly used by women on the biotransformation of fluorogenic substrates by human cytochromes P450.

The study set out to determine the potential for commercially available preparations of black cohosh (Actaea racemosa), chaste tree berry (Vitex agnus-castus), crampbark (Viburnum opulus) and false unicorn (Chamaelirium luteum) to inhibit the major human drug metabolizing enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 as well as CYP1A1 which activates some carcinogens. In vitro microplate-based assays using cDNA-expressed CYP450 isoforms and fluorogenic substrates were used. Components of the commercial herbal preparations interfered with the assays and limited the concentration ranges that could be tested. Nevertheless, the fluorogenic assays were robust, reproducible and easy to perform and thus are still useful for initial screening for potential herb-drug interactions. None of the preparations affected CYPs 1A1 or 2C9 at the concentrations tested but all preparations inhibited some of the enzymes with potencies around 1 µg/mL. The three most potent interactions were: chaste tree berry and CYP2C19 (IC50) 0.22 µg/mL); chaste tree berry and CYP3A4 (IC50) 0.3 µg/mL); black cohosh and CYP2C19 (IC50) 0.37 µg/mL,). Thus, the study successfully identified the potential for the commercial herbal preparations to inhibit human drug metabolizing enzymes. Whether this potential translates into clinically significant herb-drug interactions can only be confirmed by appropriate in vivo studies.