Human Mitragynine and 7-Hydroxymitragynine Pharmacokinetics after Single and Multiple Daily Doses of Oral Encapsulated Dried Kratom Leaf Powder.

Kratom leaves, consumed by millions worldwide as tea or ground leaf powder, contain multiple alkaloids, with mitragynine being the most abundant and responsible for most effects. Mitragynine is a partial µ-opioid receptor agonist and competitive antagonist at κ- and δ-opioid receptors; however, unlike morphine, it does not activate the ß-arrestin-2 respiratory depression pathway. Due to few human mitragynine data, the largest randomized, between-subject, double-blind, placebo-controlled, dose-escalation study of 500-4000 mg dried kratom leaf powder (6.65-53.2 mg mitragynine) was conducted. LC-MS/MS mitragynine and 7-hydroxymitragynine plasma concentrations were obtained after single and 15 daily doses. Mitragynine and 7-hydroxymitragynine Cmax increased dose proportionally, and AUC was slightly more than dose proportional. The median mitragynine Tmax was 1.0-1.3 h after single and 1.0-1.7 h after multiple doses; for 7-hydroxymitragynine Tmax, it was 1.2-1.8 h and 1.3-2.0 h. Steady-state mitragynine concentrations were reached in 8-9 days and 7-hydroxymitragynine within 7 days. The highest mean mitragynine T1/2 was 43.4 h after one and 67.9 h after multiple doses, and, for 7-hydroxymitragynine, it was 4.7 and 24.7 h. The mean 7-hydroxy-mitragynine/mitragynine concentration ratios were 0.20-0.31 after a single dose and decreased (0.15-0.21) after multiple doses. These mitragynine and 7-hydroxymitragynine data provide guidance for future clinical kratom dosing studies and an interpretation of clinical and forensic mitragynine and 7-hydroxymitragynine concentrations.

An In Vitro Examination of Whether Kratom Extracts Enhance the Cytotoxicity of Low-Dose Doxorubicin against A549 Human Lung Cancer Cells.

Doxorubicin is an effective chemotherapeutic agent in the treatment of solid hematological and non-hematological carcinoma. However, its long-term usage could result in side effects, such as cardiomyopathy, chronic heart failure, neurotoxicity and cancer cell resistance. In this study, we reported the sensitivity enhancement of A549 human lung cancer cells on doxorubicin at a low dose (0.1 ppm) in combination with 10-60 ppm of crude and alkaloid extracts derived from the leaves of Kratom (Mitragyna speciosa (Korth.) Havil. Rubiaceae). A549 cancer cell lines were insensitive to the crude extract containing low mitragynine (MG) (4-5%), while these cells were moderately inhibited by the alkaloid extract containing 40-45% MG (IC50 of 48-55 ppm). The alkaloid extract was found to inhibit A549 cancer cells via apoptosis as suggested by the higher relative fluorescence intensity with Annexin compared to that in propidium iodide (PI), i.e., a positive Annexin and a negative PI. The combination of crude extract and doxorubicin sensitized A549 cancer cells to doxorubicin by 1.3 to 2.4 times, while the combination with the alkaloid induced a 2.6- to 3.4-fold increase in sensitivity. The calculated combination index (CI) for doxorubicin with the crude and alkaloid extracts was 0.6 and 0.3, respectively, showing potential synergistic combinations to reduce the level of dosage of doxorubicin used in chemotherapy. In addition, the synergistic enhancement effect of crude extract on the cytotoxic activity of doxorubicin provides insights into the plausibility of non-alkaloids to influence the biological activities of Kratom.

Testing for Kratom alkaloids in fingernail clippings - not only mitragynine.

Kratom (Mitragyna speciosa) is a species of large tree that grows in Southeast Asia and is part of the Rubiaceae family. Its fresh leaves are harvested for their medicinal properties and used for their psychoactive effects. Kratom contains many biologically active alkaloids, including mitragynine and 7-OH-mitragynine, which are considered the two most important psychoactive components and constitute approximately 66% and 2% of the total alkaloid content. Other alkaloids are present in the plant, such as speciogynine, speciociliatine and paynantheine, but have less psychoactive activity. Over the past decade, the sale of kratom powder has increased on the Internet. This led to a significant increase in forensic cases. Given the lack of data existing in the literature, and the total absence of data in nails, the authors report a study to determine the best target alkaloids for documenting kratom consumption in this matrix. Fingernail clippings from a supposed kratom powder user were analyzed after liquid-liquid extraction, chromatography separation using a HSS C18 column and performed on an ultra-high performance liquid chromatography coupled to a tandem mass spectrometer. In the specimen, mitragynine was quantified at 229 pg/mg, speciogynine and paynantheine were both quantified at 2 pg/mg, and speciociliatine was quantified at 19 pg/mg. 7-OH-mitragynine was not detected. The interpretation of these concentrations is complex, since there is currently no reference in the literature, as this is the first identification of mitragynine and other kratom alkaloids in nails. Nevertheless, in view of the high concentration of mitragynine, the subject seems to be a repetitive user of kratom. According to the measured concentrations, it seems that mitragynine remains the best target to document kratom consumption, but the identification of the other alkaloids would enhance the specificity of the test.

Effects of kratom on driving: Results from a cross-sectional survey, ecological momentary assessment, and pilot simulated driving Study.

OBJECTIVES: Despite widespread kratom use, there is a lack of knowledge regarding its effects on driving. We evaluated the self-reported driving behaviors of kratom consumers and assessed their simulated-driving performance after self-administering kratom products. METHODS: We present results from: 1) a remote, national study of US adults who regularly use kratom, and 2) an in-person substudy from which we re-recruited participants. In the national study (N = 357), participants completed a detailed survey and a 15-day ecological momentary assessment (EMA) that monitored naturalistic kratom use. For the remote study, outcomes were self-reported general and risky driving behaviors, perceived impairment, and driving confidence following kratom administration. For the in-person substudy, 10 adults consumed their typical kratom products and their driving performance on a high-fidelity driving simulator pre- and post-kratom administration was evaluated. RESULTS: Over 90% of participants surveyed self-reported driving under the influence of kratom. Most reported low rates of risky driving behavior and expressed high confidence in their driving ability after taking kratom. This was consistent with EMA findings: participants reported feeling confident in their driving ability and perceived little impairment within 15-180 min after using kratom. In the in-person substudy, there were no significant changes in simulated driving performance after taking kratom. CONCLUSIONS: Using kratom before driving appears routine, however, self-reported and simulated driving findings suggest kratom effects at self-selected doses among regular kratom consumers do not produce significant changes in subjective and objective measures of driving impairment. Research is needed to objectively characterize kratom's impact on driving in regular and infrequent consumers.

Ecological Momentary Assessment of Self-Reported Kratom Use, Effects, and Motivations Among US Adults.

Importance: Kratom products, which are sold legally in most of the US, contain alkaloids with opioidergic, adrenergic, and serotonergic activity. Millions of people use kratom to relieve pain, improve mood, or self-manage substance use disorders (SUDs). Kratom use has primarily been examined via surveys, in which recall biases among satisfied users may lead to minimization of transient negative outcomes. Further prospective study of kratom use, such as with ecological momentary assessment (EMA), is needed. Objective: To characterize proximal motivators, effects, and patterns of kratom use and to assess whether use frequency is associated with motivations, effects, past-year criteria for SUD for kratom (KUD), or other substance use. Design, Setting, and Participants: For this prospective cross-sectional study, an intensive longitudinal smartphone-based EMA in which participants' current behaviors and experiences were repeatedly sampled in real time was conducted between July 1 and October 31, 2022. Participants comprised a convenience sample of US adults who used kratom at least 3 days per week for at least 4 weeks at the time of online screening. Criteria for past-year KUD were based on the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition. Data analysis was performed between November 2022 and November 2023. Exposure: The exposure was 13 401 kratom-use events across 15 days. Main Outcomes and Measures: A baseline survey covering demographics, health, kratom attitudes and behaviors, use motivations, other substance use, and KUD was administered before EMA. Data for the following EMA entries were then collected: event-contingent entries for kratom use (product, dose, and proximal motivations), follow-up entries (short-term effects and consequences of use events), random-prompt entries (mood), beginning-of-day entries (effects of kratom on sleep), and end-of-day entries (daily subjective descriptions of kratom effects). Bayesian regression was used to estimate means and credible intervals. Results: A total of 357 participants completed the EMA. Their mean (SD) age was 38.0 (11.1) years; more than half were men (198 [55.5%]). Participants reported overall motivators of use on the baseline survey that involved managing psychiatric and SUD problems, but proximal motivators evaluated during the EMA involved situation-specific needs such as increasing energy and productivity and decreasing pain. Acute effects were considered congruent with daily obligations. Use patterns, despite having some distinguishing features, were generally similar in their motivators and effects; participants used kratom predominantly during the daytime and seemed to find use frequencies that suited their needs. Higher use patterns were associated with symptoms of physical dependence (eg, withdrawal or tolerance). Co-used substances included caffeine, nicotine, vitamins, and cannabis. Conclusions and Relevance: Most participants in this study reported using kratom in a seemingly nonproblematic way. When such use appeared problematic, the key element was usually that withdrawal avoidance became a proximal motivator. Longitudinal studies examining changes in kratom use patterns and effects over time are needed.

An update on the clinical pharmacology of kratom: uses, abuse potential, and future considerations.

INTRODUCTION: Kratom (Mitragyna speciosa) has generated substantial clinical and scientific interest as a complex natural product. Its predominant alkaloid mitragynine and several stereoisomers have been studied for activity in opioid, adrenergic, and serotonin receptors. While awaiting clinical trial results, the pre-clinical evidence suggests a range of potential therapeutic applications for kratom with careful consideration of potential adverse effects. AREAS COVERED: The focus of this review is on the pharmacology, pharmacokinetics, and potential drug-drug interactions of kratom and its individual alkaloids. A discussion on the clinical pharmacology and toxicology of kratom is followed by a summary of user surveys and the evolving concepts of tolerance, dependence, and withdrawal associated with kratom use disorder. EXPERT OPINION: With the increasing use of kratom in clinical practice, clinicians should be aware of the potential benefits and adverse effects associated with kratom. While many patients may benefit from kratom use with few or no reported adverse effects, escalating dose and increased use frequency raise the risk for toxic events in the setting of polysubstance use or development of a use disorder.

Responses to a "Typical" Morning Dose of Kratom in People Who Use Kratom Regularly: A Direct-Observation Study.

INTRODUCTION: Use of kratom has outpaced systematic study of its effects, with most studies reliant on retrospective self-report. METHODS: We aimed to assess acute effects following kratom use in adults who use regularly, and quantify alkaloids in the products, urine, and plasma. Between July and November 2022, 10 adults came to our clinic and orally self-administered their typical kratom dose; blinding procedures were not used. Physiological measures included blood pressure, respiratory rate, heart rate, pulse oximetry, temperature, and pupil diameter. Subjective outcomes included Subjective Opioid Withdrawal Scale, Addiction Research Center Inventory, and Drug Effects Questionnaire. Psychomotor performance was also assessed. RESULTS: Participants were 6 men and 4 women, mean age 41.2 years. Nine were non-Hispanic White; 1 was biracial. They had used kratom for 6.6 years (SD, 3.8 years) on average (2.0-14.1). Sessions were 190.89 minutes on average (SD, 15.10 minutes). Mean session dose was 5.16 g (median, 4.38 g; range, 1.1-10.9 g) leaf powder. Relative to baseline, physiological changes were minor. However, pupil diameter decreased (right, b = -0.70, P < 0.01; left, b = -0.73, P < 0.01) 40-80 minutes postdose and remained below baseline >160 minutes. Subjective Opioid Withdrawal Scale pre-dosing was mild (5.5 ± 3.3) and decreased postdose (b = [-4.0, -2.9], P < 0.01). Drug Effects Questionnaire "feeling effects" increased to 40/100 (SD, 30.5) within 40 minutes and remained above baseline 80 to 120 minutes (b = 19.0, P = 0.04), peaking at 72.7/100; 6 participants rated euphoria as mild on the Addiction Research Center Inventory Morphine-Benzedrine-scale. Psychomotor performance did not reliably improve or deteriorate postdosing. CONCLUSIONS: Among regular consumers, we found few clinically significant differences pre- and post-kratom dosing. Alkaloidal contents in products were within expected ranges.

Kratom (Mitragyna speciosa): A Case Review of Use Before and During Pregnancy.

Kratom (Mitragyna speciosa) is a plant-based substance with psychoactive properties similar to opioids but is not currently classified as an opioid. One of its more prevalent uses is to treat opioid dependency and withdrawal symptoms. Opioid use disorder is a leading cause of pregnancy-associated maternal mortality, and pregnant women may be using kratom as a substitute or alternative to opioids. Prevalence of kratom use is increasing rapidly, but scientific evidence specific to therapeutic and adverse effects is lacking overall, and the implications of its use in pregnancy and on the fetus-newborn are limited to a few case reports. Kratom is a legal substance by federal law, although some states have banned its use. The lack of regulation is concerning. Significant illness and associated deaths have been reported with kratom use. Lack of disclosure by people using kratom and limited laboratory testing options are major challenges for health care providers and public health.

A case of fatal overdose involving both hydromorphone and kratom.

Kratom is a plant originating in Southeast Asia that has been used for its dose-dependent stimulant and opioid effects. The main active compound in kratom is mitragynine, an alkaloid with affinity for the mu-opioid receptor. Toxicity and fatalities related to kratom use have increased substantially in recent years. In this case report, we describe a 44-year-old man who was found deceased in bed. The only significant finding at autopsy was abdominal distension with >4 L of ascites. Toxicology testing was performed on femoral blood which showed 79 ng/mL of hydromorphone, 560 ng/mL of mitragynine, and 240 ng/mL of olanzapine. In addition, creatinine and urea in vitreous humor were significantly elevated, consistent with renal impairment. Death was attributed to hydromorphone toxicity with mitragynine being a contributing factor.

Prevalence, Characteristics, and Reasons for Kratom Use among Psychiatrically Ill Inpatients Who Use Substances.

OBJECTIVE: Despite kratom impacting neurobiological systems involved in psychiatric disorders, little is known about the prevalence of use among patients with severe psychopathologies. Here, we investigated the prevalence of kratom use, motives for use, and the clinical associations among inpatients with severe psychiatric disorders. METHODS: A total of 578 patients, aged 18 to 65, were evaluated by New Hampshire Hospital's Addiction Services from January 1, 2020, to February 28, 2022. The study collected demographic information and used chi-square tests, multivariable logistic regression, and subgroup analyses with 95% confidence intervals to examine trends among kratom users. A receiver operating characteristic curve analysis was also conducted. All statistical tests were performed using IBM SPSS Version 28.0.1. RESULTS: Of the patients assessed, 2.2% (n = 13) reported using kratom. The reasons for kratom use were managing withdrawal symptoms (15.4%), maintaining sobriety and reducing cravings for opioids (53.8%), improving focus and concentration (30.8%), alleviating low moods (38.5%), and managing pain (15.4%). Compared to non-kratom users, the only factor with a fair to good association with kratom use is postsecondary education (Area Under Curve, AUC = 0.77). CONCLUSIONS: Prevalence of kratom use among patients with serious mental illness at our site aligns with that reported in the general population. Users often cite self-management of cravings and sobriety from opioids, as well as treatment of low mood states, as motivations for consumption. While observations suggest a possible association between kratom use and individuals with post-secondary education, multiple substance use, and experience of substance-induced psychosis or mood disorders, it is essential to interpret these links cautiously until further rigorous studies are carried out to substantiate these findings.

A Critical Review of the Neuropharmacological Effects of Kratom: An Insight from the Functional Array of Identified Natural Compounds.

Kratom (Mitragyna speciosa Korth. Havil) has been considered a narcotic drug for years, barred by the law in many parts of the world, while extensive research over the past few decades proves its several beneficial effects, some of which are still in ambiguity. In many countries, including Thailand, the indiscriminate use and abuse of kratom have led to the loss of life. Nonetheless, researchers have isolated almost fifty pure compounds from kratom, most of which are alkaloids. The most prevalent compounds, mitragynine and 7-hydroxy mitragynine, are reported to display agonist morphine-like effects on human µ-opioid receptors and antagonists at κ- and δ-opioid receptors with multimodal effects at other central receptors. Mitragynine is also credited to be one of the modulatory molecules for the Keap1-Nrf2 pathway and SOD, CAT, GST, and associated genes' upregulatory cascades, leading it to play a pivotal role in neuroprotective actions while evidently causing neuronal disorders at high doses. Additionally, its anti-inflammatory, antioxidative, antibacterial, and gastroprotective effects are well-cited. In this context, this review focuses on the research gap to resolve ambiguities about the neuronal effects of kratom and demonstrate its prospects as a therapeutic target for neurological disorders associated with other pharmacological effects.

Pharmacology and toxicology of kratom.

The term kratom is commonly used for both Mitragyna speciosa and herbal products prepared mainly from leaves. Kratom is well known as a drug that can serve as a less toxic and less-addictive pain-relieving substitute for opium, as well as a therapy for hypertension, cough, and diarrhea. Its major alkaloid, mitragynine, also deserves concern. However, most people use kratom as a psychological stimulant, which carries a risk of addiction associated with negative social and health impacts. This paper reviews basic facts about kratom and its potential use in pharmacology, pharmacokinetics, and pharmacokinetics of its major alkaloid mitragynine (Tab. 3, Fig. 1, Ref. 87). Keywords: 7-hydroxymitragynine; alkaloid; anesthetics; antitussive; drug; mitragynine; Mitragyna speciosa; addictive substance; opioid receptor.

Kratom Use Among Pregnant and Lactating Individuals With Substance Use Disorder.

OBJECTIVE: Kratom ( Mitragyna speciosa ) use in pregnancy is associated with maternal and neonatal opioid withdrawal syndrome. However, kratom use patterns in the population of peripartum and postpartum individuals with substance use disorder (SUD) are unknown. The aim of this study was to determine the proportion of pregnant and postpartum individuals with SUD who report using kratom in pregnancy or lactation and the reasons for their use. METHODS: We conducted an anonymous survey of pregnant and postpartum individuals receiving care at a single center's multidisciplinary prenatal clinic for individuals with SUD. We collected participants' demographic and pregnancy characteristics. We assessed ever use of kratom, kratom use during pregnancy or lactation, and reasons for kratom use. Descriptive statistics were used to summarize the data. RESULTS: From January 2021 to May 2021, a total of 80 surveys were collected (81% response rate of 98 eligible individuals). Most respondents were pregnant (n = 50 [62.5%]). The most frequent substance(s) of use were opioids (n = 50 [62.5%]) and methamphetamine (n = 39 [48.8%]). Many (n = 26 [32.5%]) reported ever use of kratom use. Of all respondents, 4 (5%) reported use during pregnancy, and 1 (1%) reported use during lactation. Kratom was primarily used to relieve opioid withdrawal symptoms and for relaxation, pain control, and stress relief. CONCLUSION: In a survey of pregnant and postpartum individuals with SUD at a single high-risk pregnancy clinic, ever use of kratom was frequent, whereas peripartum use was rare.

[Neonatal abstinence syndrome due to kratom]. / Neonataal abstinentiesyndroom door kratom.

BACKGROUND: Kratom (Mitragyna speciosa) is a herbal product obtained from the tropical tree family 'Rubiaceae'. Kratom is available without prescription in several formulations. The active component mitragynine acts in high dose as a mu-opioid agonist. It is misconceived to be a safe alternative to conventional opioid drugs for the treatment of chronic pain. Therefore, maternal use of Kratom is not without risks as opioid use during pregnancy is associated with Neonatal Abstinence Syndrome (NAS). CASE DESCRIPTION: In this case report we describe a term neonate with severe NAS as a result of daily Kratom ingestion by the mother during pregnancy. Presence of mitragynine was confirmed in serum of the neonate. NAS was successfully treated with oral phenobarbital. CONCLUSION: Maternal Kratom use during pregnancy can cause severe NAS via in utero exposure. Physicians should be aware of the possible maternal use of Kratom in the case of a neonate with NAS. NAS as a result of maternal Kratom use can be effectively treated with oral phenobarbital.

Effect of Green and Red Thai Kratom (Mitragyna speciosa) on pancreatic digestive enzymes (alpha-glucosidase and lipase) and acetyl-carboxylase 1 activity: A possible therapeutic target for obesity prevention.

Regular use of Thai kratom has been linked to reduced blood triglyceride levels and body mass index (BMI) in healthy individuals. We analyzed Green Thai Kratom (GTK) and Red Thai Kratom (RTK) to investigate their effects on pancreatic digestive enzymes. The ethanol extracts of GTK and RTK inhibited lipase activity more strongly than alpha-glucosidase activity, suggesting the presence of lipase inhibitors. Mitragynine, the major compound in GTK, showed potent lipase inhibition and moderate alpha-glucosidase inhibition. Quercetin, found in both extracts, strongly inhibited alpha-glucosidase but had limited effects on lipase. These findings suggest that mitragynine and quercetin may hinder triglyceride and starch digestion. Combination inhibition studies revealed synergistic effects between mitragynine and quercetin on alpha-glucosidase activity. Additionally, both GTK and RTK extracts reduced fat accumulation in 3T3-L1 adipocyte cells, with quercetin specifically inhibiting Acetyl-CoA carboxylase 1 (ACC1), a key enzyme in fatty acid biosynthesis. Thus, GTK and RTK extracts, particularly mitragynine and quercetin, exhibit potential anti-obesity effects. We report the novel finding that Thai kratom inhibits de novo fatty acid synthesis by targeting ACC1, resulting in decreased fat accumulation in adipocytes. Regular use of Thai kratom in specific populations may improve blood triglyceride levels and reduce BMI by inhibiting lipase, alpha-glucosidase, and ACC1 activity. Further clinical trials are needed to determine optimal dosage, duration, toxicity levels, and potential side effects of Kratom use.

An in vitro evaluation of kratom (Mitragyna speciosa) on the catalytic activity of carboxylesterase 1 (CES1).

Kratom, (Mitragyna Speciosa Korth.) is a plant indigenous to Southeast Asia whose leaves are cultivated for a variety of medicinal purposes and mostly consumed as powders or tea in the United States. Kratom use has surged in popularity with the lay public and is currently being investigated for possible therapeutic benefits including as a treatment for opioid withdrawal due to the pharmacologic effects of its indole alkaloids. A wide array of psychoactive compounds are found in kratom, with mitragynine being the most abundant alkaloid. The drug-drug interaction (DDI) potential of mitragynine and related alkaloids have been evaluated for effects on the major cytochrome P450s (CYPs) via in vitro assays and limited clinical investigations. However, no thorough assessment of their potential to inhibit the major hepatic hydrolase, carboxylesterase 1 (CES1), exists. The purpose of this study was to evaluate the in vitro inhibitory potential of kratom extracts and its individual major alkaloids using an established CES1 assay and incubation system. Three separate kratom extracts and the major kratom alkaloids mitragynine, speciogynine, speciociliatine, paynantheine, and corynantheidine displayed a concentration-dependent reversible inhibition of CES1. The experimental Ki values were determined as follows for mitragynine, speciociliatine, paynantheine, and corynantheidine: 20.6, 8.6, 26.1, and 12.5 µM respectively. Speciociliatine, paynantheine, and corynantheidine were all determined to be mixed-type reversible inhibitors of CES1, while mitragynine was a purely competitive inhibitor. Based on available pharmacokinetic data, determined Ki values, and a physiologically based inhibition screen mimicking alkaloid exposures in humans, a DDI mediated via CES1 inhibition appears unlikely across a spectrum of doses (i.e., 2-20g per dose). However, further clinical studies need to be conducted to exclude the possibility of a DDI at higher and extreme doses of kratom and those who are chronic users.

Evaluation of toxicity profile of kratom (Mitragyna speciosa Korth) decoction in rats.

Mitragyna speciosa Korth also known as kratom, is an herbal drug preparation for its therapeutic properties and opioid-replacement therapy. Kratom is consumed in a brewed decoction form in Malaysia and to date, no studies have characterized its chemical and toxicity profile. Thus, this study aims to evaluate kratom decoction's safety and toxicity profile after 28 days of treatment. Mitragynine content was quantified in kratom decoction and used as a marker to determine the concentration. Male and female Sprague Dawley rats were orally treated with vehicle or kratom decoction (10, 50 or 150 mg/kg) and two satellite groups were treated with vehicle and kratom decoction (150 mg/kg). Blood and organs were collected for hematology, biochemical and histopathology analysis at the end of treatment. No mortality was found after 28 days of treatment and no significant changes in body weight and hematology profile, except for low platelet count. High amounts of uric acid, AST, ALT and alkaline phosphatase were found in the biochemical analysis. Histological investigation of the heart and lungs detected no alterations except for the kidney, liver and brain tissues. In conclusion, repeated administration of kratom decoction provided some evidence of toxicity in the kidney and liver with no occurrence of mortality.