Comparative In vitro metabolism of purified mogrosides derived from monk fruit extracts.

Mogrosides are the primary components responsible for the sweet taste of Monk fruit which is derived from Siraitia grosvenorii (Swingle), a herbaceous plant native to southern China. Many mogrosides have been identified from Monk fruit extract, but the major sweetness component of Monk fruit by mass is mogroside V, comprising up to 0.5% of the dried fruit weight. Recent pharmacokinetic studies indicate that the parent mogrosides undergo minimal systemic absorption following ingestion and hydrolysis by digestive enzymes and/or intestinal flora and are excreted as mogrol (i.e., the aglycone) and its mono- and diglucosides. The objective of this study was to demonstrate whether individual mogrosides, are metabolized to a common and terminal deglycosylated metabolite, mogrol. An in vitro assay was conducted with pooled human male and female intestinal fecal homogenates (HFH) using mogrosides IIIe, mogroside V, siamenoside I, and isomogroside V at two concentrations over a 48 h period. The results show that various mogrosides that differ in the linkages and number of glucose units attached to a common cucurbitane backbone, share a common metabolic fate, and are metabolized within 24 h to mogrol. Aside from an apparent difference in the initial rate of deglycosylation between mogrosides at higher concentrations, no apparent difference in the rate of deglycosylation was observed between the male and female HFH. Given the similar structures of these mogrosides and a shared metabolic fate to mogrol, the study provides support for a reasonably conservative approach to assess safety based on bridging safety data from an individual mogroside (i.e., Mogroside V) to other mogrosides, and the establishment of a group Acceptable Daily Intake (ADI), rather than individual ADI's for mogrosides.

Steviol glycosides in purified stevia leaf extract sharing the same metabolic fate.

The safety of steviol glycosides is based on data available on several individual steviol glycosides and on the terminal absorbed metabolite, steviol. Many more steviol glycosides have been identified, but are not yet included in regulatory assessments. Demonstration that these glycosides share the same metabolic fate would indicate applicability of the same regulatory paradigm. In vitro incubation assays with pooled human fecal homogenates, using rebaudiosides A, B, C, D, E, F and M, as well as steviolbioside and dulcoside A, at two concentrations over 24-48 h, were conducted to assess the metabolic fate of various steviol glycoside classes and to demonstrate that likely all steviol glycosides are metabolized to steviol. The data show that glycosidic side chains containing glucose, rhamnose, xylose, fructose and deoxy-glucose, including combinations of α(1-2), ß-1, ß(1-2), ß(1-3), and ß(1-6) linkages, were degraded to steviol mostly within 24 h. Given a common metabolite structure and a shared metabolic fate, safety data available for individual steviol glycosides can be used to support safety of purified steviol glycosides in general. Therefore, steviol glycosides specifications adopted by the regulatory authorities should include all steviol glycosides belonging to the five groups of steviol glycosides and a group acceptable daily intake established.

Chemical-specific adjustment factors (inter-species toxicokinetics) to establish the ADI for steviol glycosides.

The acceptable daily intake (ADI) of commercially available steviol glycosides is currently 0-4 mg/kg body weight (bw)/day, based on application of a 100-fold uncertainty factor to a no-observed-adverse-effect-level value from a chronic rat study. Within the 100-fold uncertainty factor is a 10-fold uncertainty factor to account for inter-species differences in toxicokinetics (4-fold) and toxicodynamics (2.5-fold). Single dose pharmacokinetics of stevioside were studied in rats (40 and 1000 mg/kg bw) and in male human subjects (40 mg/kg bw) to generate a chemical-specific, inter-species toxicokinetic adjustment factor. Tmax values for steviol were at ∼8 and ∼20 h after administration in rats and humans, respectively. Peak concentrations of steviol were similar in rats and humans, while steviol glucuronide concentrations were significantly higher in humans. Glucuronidation in rats was not saturated over the dose range 40-1000 mg/kg bw. The AUC0-last for steviol was approximately 2.8-fold greater in humans compared to rats. Chemical-specific adjustment factors for extrapolating toxicokinetics from rat to human of 1 and 2.8 were established based on Cmax and AUC0-last data respectively. Because these factors are lower than the default value of 4.0, a higher ADI for steviol glycosides of between 6 and 16 mg/kg bw/d is justified.

In vitro metabolism of rebaudioside E under anaerobic conditions: Comparison with rebaudioside A.

The hydrolysis of the steviol glycosides rebaudioside (Reb) A and E, as well as steviolbioside (a metabolic intermediate) to steviol was evaluated in vitro using human fecal homogenates from healthy Caucasian and Asian donors. Incubation of each of the Rebs in both groups resulted in a rapid hydrolysis to steviol. Metabolism of 0.2mg/mL sample was complete within 24h, with the majority occurring within the first 16 h. There were no clear differences in the rate or extent of metabolism of Reb E relative to the comparative control Reb A. The hydrolysis of samples containing 2.0mg/mL of steviol glycosides Reb A and Reb E tended to take slightly longer than 0.2mg/mL samples. Herein, we report for the first time that there were no apparent gender or ethnicity differences in the rate of metabolism of any of the Rebs, regardless of the concentrations tested. Steviolbioside, an intermediate in the hydrolysis of Reb E to steviol was also found to be rapidly degraded to steviol. These results demonstrate Reb E is metabolized to steviol in the same manner as Reb A. These data support the use of toxicology data available on steviol, and on steviol glycosides metabolized to steviol (i.e., Reb A) to underpin the safety of Reb E.

In vitro metabolism of rebaudioside B, D, and M under anaerobic conditions: comparison with rebaudioside A.

The hydrolysis of the steviol glycosides rebaudioside A, B, D, and M, as well as of steviolbioside (a metabolic intermediate) to steviol was evaluated in vitro using human fecal homogenates from healthy donors under anaerobic conditions. Incubation of each of the rebaudiosides resulted in rapid hydrolysis to steviol. Metabolism was complete within 24h, with the majority occurring within the first 8h. There were no clear differences in the rate or extent of metabolism of rebaudioside B, D, or M, relative to the comparative control rebaudioside A. The hydrolysis of samples containing 2.0mg/mL of each rebaudioside tended to take slightly longer than solutions containing 0.2mg/mL. There was no apparent gender differences in the amount of metabolism of any of the rebaudiosides, regardless of the concentrations tested. An intermediate in the hydrolysis of rebaudioside M to steviol, steviolbioside, was also found to be rapidly degraded to steviol. The results demonstrate that rebaudiosides B, D, and M are metabolized to steviol in the same manner as rebaudioside A. These data support the use of toxicology data available on steviol, and on steviol glycosides metabolized to steviol (i.e., rebaudioside A) to substantiate the safety of rebaudiosides B, D, and M.

The potential developmental neurotoxicity of calcium cyclamate in CD rats.

The developmental neurotoxicity of calcium cyclamate was evaluated in Sprague Dawley [Crl:CD(SD)] rats, administered in drinking water, in comparison to a concurrent control group (water) and a positive control group given propylthiouracil (PTU). Calcium cyclamate was administered to F0 females for 4 weeks prior to pairing, throughout mating, gestation and lactation and to F1 offspring from weaning to 12 weeks of age, PTU was administered by gavage to F0 females from Day 6 of gestation up to Day 20 of lactation. Target calcium cyclamate doses were 0, 250, 500 and 1,000 mg/kg bw/day, while the PTU dose was 0.5 mg/kg bw/day. No treatment-related effects of cyclamate were observed in either the F0 or F1 generations on reproductive performance or neurobehavioral development. In comparison, PTU exposure resulted in developmental delays, memory impairment and a number of neuropathological and morphometric outcomes. The results from the unique developmental neurotoxicity study design, corroborate the absence of hyperactivity and any other neurotoxic effects following cyclamate administration at levels up to 878 mg/kg bw/day in F0 females and 784 mg/kg bw/day in F1 animals. This demonstrates the suitability of PTU as a positive control and confirms the safe use of cyclamate as a no-calorie sweetener.

Comparative effects of phthalate monoesters on gap junctional intercellular communication and peroxisome proliferation in rodent and primate hepatocytes.

Several phthalate esters, compounds used as plasticizers in a variety of commercial products, have been shown to induce hepatic tumors in rodents. In this study, the comparative effects of phthalate monoesters on inhibition of gap junctional intercellular communication and induction of peroxisomal beta-oxidation were assessed in primary cultured hepatocytes from rats, mice, hamsters, cynomolgus monkeys, and humans. A human liver cell line was also utilized. Eight monoesters examined included mono-2-ethylhexyl phthalate (MEHP), mono-n-octyl phthalate (MNOP), mono-isononyl phthalate (MINP, 3 types, -1, -2, and -3), mono-isoheptyl phthalate (MIHP), mono-isodecyl phthalate (MIDP), and mono-(heptyl, nonyl, undecyl) phthalate (M711P). Gap junctional intercellular communication was measured 4 and 24 h after treatment by lucifer yellow dye coupling. Gap junctional intercellular communication was inhibited in rat and mouse hepatocytes by all eight monoesters in a concentration-dependent manner. In most cases, gap junctional intercellular communication was significantly reduced at the lowest concentrations tested (50 pM). Inhibition of gap junctional intercellular communication in rodent cells was substantially reversed within 24 h of monoester removal. In contrast, cell-to-cell communication was not inhibited in hamster, cynomolgus, or human hepatocytes or in a human liver cell line at any concentration examined. In rat hepatocytes, peroxisomal beta-oxidation was elevated after treatment with MEHP, MINP, MIHP, and MIDP but not MNOP or M711P, and with all but MIHP in mouse hepatocytes. The eight phthalates produced no marked change on peroxisomal beta-oxidation in hepatocytes from other species. These data provide additional evidence that the toxicological effects of phthalate esters are species specific.

Long-term pain, stiffness, and functional disability after total knee arthroplasty with and without an extended ambulatory continuous femoral nerve block: a prospective, 1-year follow-up of a multicenter, randomized, triple-masked, placebo-controlled trial.

BACKGROUND: Previously, we have demonstrated that extending a continuous femoral nerve block (cFNB) from overnight to 4 days after total knee arthroplasty (TKA) provides clear benefits during the infusion, but not subsequent to catheter removal. However, there were major limitations in generalizing the results of that investigation, and we subsequently performed a very similar study using a multicenter format, with many health care providers, in patients on general orthopedic wards, thus greatly improving inference of the results to the general population. Not surprisingly, the perioperative/short-term outcomes differed greatly from the first, more limited study. We now present a prospective follow-up study of the previously published, multicenter, randomized controlled clinical trial to investigate the possibility that an extended ambulatory cFNB decreases long-term pain, stiffness, and functional disability after TKA, which greatly improves inference of the results to the general population. METHODS: Subjects undergoing TKA received a cFNB with ropivacaine 0.2% from surgery until the following morning, at which time patients were randomized to continue either perineural ropivacaine (n=28) or normal saline (n=26). Patients were discharged with their catheter and a portable infusion pump, and catheters were removed on postoperative day 4. Health-related quality of life was measured using the Western Ontario and McMaster Universities Osteoarthritis Index preoperatively and then at 7 days, as well as 1, 2, 3, 6, and 12 months after surgery. This index evaluates pain, stiffness, and physical functional disability. For inclusion in the analysis, we required a minimum of 4 of the 6 time points, including day 7 and at least 2 of months 3, 6, and 12. RESULTS: The 2 treatment groups had similar Western Ontario and McMaster Universities Osteoarthritis scores for the mean area-under-the-curve calculations (point estimate for the difference in mean area under the curve for the 2 groups [overnight infusion group - extended infusion group]=3.8; 95% confidence interval, -3.8 to +11.3; P=0.32) and at all individual time points (P>0.05). CONCLUSIONS: This investigation found no evidence that extending an overnight cFNB to 4 days improves (or worsens) subsequent pain, stiffness, or physical function after TKA in patients of multiple centers convalescing on general orthopedic wards.

A multicenter, randomized, triple-masked, placebo-controlled trial of the effect of ambulatory continuous femoral nerve blocks on discharge-readiness following total knee arthroplasty in patients on general orthopaedic wards.

A continuous femoral nerve block (cFNB) involves the percutaneous insertion of a catheter adjacent to the femoral nerve, followed by a local anesthetic infusion, improving analgesia following total knee arthroplasty (TKA). Portable infusion pumps allow infusion continuation following hospital discharge, raising the possibility of decreasing hospitalization duration. We therefore used a multicenter, randomized, triple-masked, placebo-controlled study design to test the primary hypothesis that a 4-day ambulatory cFNB decreases the time until each of three predefined readiness-for-discharge criteria (adequate analgesia, independence from intravenous opioids, and ambulation 30m) are met following TKA compared with an overnight inpatient-only cFNB. Preoperatively, all patients received a cFNB with perineural ropivacaine 0.2% from surgery until the following morning, at which time they were randomized to either continue perineural ropivacaine (n=39) or switch to normal saline (n=38). Patients were discharged with their cFNB and portable infusion pump as early as postoperative day 3. Patients who were given 4 days of perineural ropivacaine attained all three criteria in a median (25th-75th percentiles) of 47 (29-69)h, compared with 62 (45-79)h for those of the control group (Estimated ratio=0.80, 95% confidence interval: 0.66-1.00; p=0.028). Compared with controls, patients randomized to ropivacaine met the discharge criterion for analgesia in 20 (0-38) versus 38 (15-64)h (p=0.009), and intravenous opioid independence in 21 (0-37) versus 33 (11-50)h (p=0.061). We conclude that a 4-day ambulatory cFNB decreases the time to reach three important discharge criteria by an estimated 20% following TKA compared with an overnight cFNB, primarily by improving analgesia.

Evaluation of side effects and patients' perceptions during tooth bleaching.

OBJECTIVE: The objective of this nightguard vital bleaching (NGVB) study was to compare tooth sensitivity (TS), gingival irritation (GIr), and other side effects, as well as patients' perceptions during tooth bleaching, from treatment with experimental 5 and 7% hydrogen peroxide (HP) bleaching solutions with those of a commercially available 10% carbamide peroxide (CP) product. MATERIALS AND METHODS: Sixty-one participants completed the study wearing a scalloped maxillary treatment tray without reservoirs with the different concentrations of bleaching gels for 30 minutes twice a day for 7 days. Parameters evaluated were changes in gingival index (GI), nonmarginal gingival index, nongingival oral mucosal index, and tooth vitality. Participants were seen pretreatment, after 7 treatment days, and 1 week post-treatment. A daily log form to record TS and GIr was completed by each participant as well as a sensitivity questionnaire at each appointment. Additionally, at 10 months post-treatment, a questionnaire was sent to the participants concerning TS and GIr relative to the treatment process. RESULTS: Data from end-of-treatment questionnaires, daily log forms, and clinical examination revealed a statistical difference (p < or = 0.05) in the patients' ranking of and days of TS and GIr between group S (7% HP) and group T (10% CP, control group) at the end of active treatment. There also existed a statistical clinical change in the GI levels for groups R and S compared with the control group T. There was no statistical difference (p > 0.05) in any of the parameters evaluated among the three products at 7 days or 10 months post-treatment. CONCLUSIONS: Participants in group S reported significantly more TS, GIr, and days of each compared with the control. There also existed a significant clinical change in the GI levels for groups R and S compared with the control group T. There was no significant difference among the three products at 7 days post-treatment. After ending treatment, TS/GIr was resolved in 2 to 3 days and did not recur during the 10 months post-treatment. CLINICAL SIGNIFICANCE: The experimental HP bleaching solutions, as described in this study, can be used in NGVB with no long-term side effects as evaluated in this study for up to 10 months post-treatment.

High levels of hydrogen peroxide in overnight tooth-whitening formulas: effects on enamel and pulp.

PURPOSE: Limited data are available to assess the safety of high levels of hydrogen peroxide in overnight tooth-whitening formulas. The purpose of this study was to assess the effects of hydrogen peroxide on enamel microhardness, pulp penetration, and enamel morphology. MATERIALS AND METHODS: Colgate Platinum Professional Overnight Whitening System (Colgate Oral Pharmaceuticals, Inc., Canton, MA, USA) (10% carbamide peroxide, equivalent to 3.5% hydrogen peroxide) was compared with two prototype formulations containing either 7.0% or 12.0% hydrogen peroxide. In the pulp chamber studies, human extracted teeth were exposed to 3.5%, 7.0%, or 12.0% hydrogen peroxide for 30 minutes, 4 hours, or 7 hours. Microhardness, electron spectroscopy for chemical analysis, and atomic force microscopy evaluations were made from enamel blocks cut from human extracted molars. The enamel blocks were evaluated following 14 7-hour treatments (98 h total). RESULTS: At 7 hours' post-treatment, hydrogen peroxide penetrated the pulp chamber at 23.12 +/- 10.09, 24.58 +/- 6.90, and 26.39 +/- 5.43 microg for 3.5%, 7.0%, and 12.0% hydrogen peroxide, respectively. With regard to enamel morphology, pulp penetration, microhardness, and elemental composition, no statistically significant differences were observed between treatment groups following 98 hours of treatment. CONCLUSIONS: Hydrogen peroxide does not adversely affect enamel morphology or microhardness. The levels recovered in pulp indicate that hydrogen peroxide is not expected to inhibit pulpal enzymes. CLINICAL SIGNIFICANCE: Overnight tray products containing levels of hydrogen peroxide of 3.5%, 7.0%, and 12.0% are not expected to adversely affect the enamel or pulpal enzymes. Additional safety studies are needed to assess the potential for tooth sensitivity and gingival irritation.