Food Interactions Observed in a Pharmacokinetic Investigation Comparing Two Marketed Cold Preparations (BNO1016 and ELOM-080) after Administration to Beagle Dogs - A Pilot Study.

Sinupret extract (BNO 1016) and Gelomyrtol forte (ELOM-080) represent the two top-selling cold remedies in Germany nowadays. Whereas BNO 1016 is a typical immediate release coated tablet, ELOM-080 is an enteric-coated soft gelatin capsule. The latter formulation, however, is at risk of pharmacokinetic interactions affecting absorption, especially in cases of concomitant food intake. In the present pilot study, we investigated the risk of a possible food effect in three male beagle dogs. Single doses of BNO 1016 and ELOM-80 were administered under fasting and fed conditions. Blood was sampled up to 30 h post-administration and plasma concentrations of the characteristic ingredients of BNO 1016 as well as ELOM-080 analytes were determined. Pharmacokinetic parameters focusing on the rate and extent of absorption were derived. BNO 1016 analytes demonstrated a similar course in both the fasted and fed states. ELOM-080 analytes also showed a similar picture in the fasted state. However, lag times (time from administration to first quantifiable time point in plasma) of up to 2 h post-administration with corresponding time to reach maximum concentration (obtained directly from the measured concentration) values of 3 to 4 h were observed, reflecting a longer gastric residence time. In the fed state, ELOM-080 showed significant pharmacokinetic characteristics, suggesting a clear food effect. A major observation was a double peak phenomenon that could be observed in two of three dogs. Furthermore, lag times of some analytes, up to 3 - 4 h, and corresponding time to reach maximum concentration values, up to 6 - 8 h, occurred. In contrast to BNO 1016, these findings suggest that, as with other enteric-coated formulations, there may also be a significant risk for food effects with ELOM-080 in humans.

In vivo and in vitro investigation of anti-inflammatory and mucus-regulatory activities of a fixed combination of thyme and primula extracts.

Hypersecretion of viscous mucus is one of the hallmark symptoms of acute and chronic bronchitis and typically develops secondary to an inflammation of the airway epithelium. Bronchipret® TP film-coated tablets (BRO), a herbal medicinal product containing a fixed combination of thyme herb and primula root extracts, has been successfully used clinically for the treatment of acute bronchitis for more than two decades. However, the underlying pharmacological mechanisms of action have not been fully understood so far. We investigated the anti-inflammatory and mucus-regulatory effects of orally administered BRO in an animal model of pulmonary inflammation that was experimentally induced by intratracheal LPS instillation. BRO was administered once daily for up to three days following the induction of inflammation. Treatment with BRO effectively inhibited polymorphonuclear cell influx into the lung as well as the increase in mucin 5ac (MUC5AC) protein. Furthermore, the LPS-induced increase of goblet cell numbers was significantly attenuated by BRO treatment. Subsequent in vitro investigations with IL-13 stimulated human primary respiratory epithelium and the Calu-3 respiratory epithelial cell line in air-liquid-interface culture confirmed the effects on mucus production and goblet cell numbers observed in the in vivo studies. They further suggest that the reduction of MUC5AC protein secretion by BRO is associated with reduced MUC5AC mRNA expression as assessed by quantitative Real-Time PCR. Our studies provide evidence that BRO exerts both anti-inflammatory and mucus-regulatory activity and that BRO's effect on mucin production is partially independent from its anti-inflammatory activity. These results contribute to the understanding of the modes of action underlying the clinical efficacy of BRO in acute bronchitis patients.

In vitro Screening of Herbal Medicinal Products for Their Supportive Curing Potential in the Context of SARS-CoV-2.

COVID-19 herbal medicinal products may have the potential for symptom relief in nonsevere or moderate disease cases. In this in vitro study we screened the five herbal medicinal products Sinupret extract (SINx), Bronchipret thyme-ivy (BRO-TE), Bronchipret thyme-primula (BRO TP), Imupret (IMU), and Tonsipret (TOP) with regard to their potential to (i) interfere with the binding of the human angiotensin-converting enzyme 2 (ACE2) receptor with the SARS-CoV-2 spike S1 protein, (ii) modulate the release of the human defensin HBD1 and cathelicidin LL-37 from human A549 lung cells upon spike S1 protein stimulation, and (iii) modulate the release of IFN-γ from activated human peripheral blood mononuclear cells (PBMC). The effect of the extracts on the interaction of spike S1 protein and the human ACE2 receptor was measured by ELISA. The effects on the intracellular IFN-γ expression in stimulated human PBMC were measured by flow cytometry. Regulation of HBD1 and LL-37 expression and secretion was assessed in 25 d long-term cultured human lung A549 epithelial cells by RT-PCR and ELISA. IMU and BRO-TE concentration-dependently inhibited the interaction between spike S1 protein and the ACE2 receptor. SINx, TOP, and BRO-TE significantly upregulated the intracellular expression of anti-viral IFN-γ from stimulated PBMC. Cotreatment of A549 cells with IMU or BRO TP together with SARS-CoV-2 spike protein significantly upregulated mRNA expression (IMU) and release of HBD1 (IMU and BRO TP) and LL-37 (BRO TP). The in vitro screening results provide first evidence for an immune-activating potential of some of the tested herbal medicinal extracts in the context of SARS-CoV-2. Whether these could be supportive in symptom relief or curing from SARS-CoV-2 infection needs deeper understanding of the observations.

Effectiveness and tolerability of the thyme/ivy herbal fluid extract BNO 1200 for the treatment of acute cough: an observational pharmacy-based study.

OBJECTIVE: Efficacy and safety of an oral thyme/ivy syrup for the treatment of acute cough was previously demonstrated in a randomized clinical trial. Here, we present real-life data from a pharmacy-based, observational study on the effectiveness and tolerability of another thyme/ivy combination (BNO 1200, Bronchipret drops). METHODS: This observational, prospective, uncontrolled study was conducted in 305 German pharmacies. Cough and associated symptoms, cough severity and health related quality of life (HRQoL) were assessed at baseline, after 4 days and at individual end of treatment (EoT) in patients with acute cough due to upper respiratory tract infections who bought BNO 1200. Patients took BNO 1200 until resolution of cough and completed three validated questionnaires: patient-adapted Bronchitis Severity Score (BSS), cough severity visual analogue scale (VAS) and the Leicester Cough Questionnaire (LCQ-acute). They also rated speed of onset of treatment action on a VAS and tolerability. RESULTS: A total of 749 patients participated in the study; 730 were included in the analysis. Mean treatment duration was 7.0 ± 3.6 days. Symptom severity assessed by BSS improved from 8.7 ± 3.8 score points (baseline) to 2.4 ± 2.6 at EoT (p < .0001). Clinically relevant improvements (MCID = 17 mm) in cough severity were reported by 87.2% of patients at EoT. HRQoL improved significantly (12.2 ± 3.3 points at baseline vs. 18.5 ± 2.7 at EoT; p < .0001), exceeding the MCID (=2 points) in 90.0% of patients. Tolerability was rated "good" or "very good" by 98.0% of patients. No adverse drug reactions were reported. CONCLUSION: Patients with acute cough taking BNO 1200 had a significant reduction in BSS, cough severity and improvement in HRQoL confirming RCT data with the syrup formulation. BNO 1200 was well tolerated.

Oral treatment with genistein reduces the expression of molecular and biochemical markers of inflammation in a rat model of chronic TNBS-induced colitis.

BACKGROUND: Inflammatory bowel disease (IBD) in humans has a high incidence in Europe and the USA, whereas in East Asia, incidence has been historically low. The risk of IBD appears to increase in Asian immigrants adopting western lifestyles, suggesting a strong link of environmental/dietary factors in the development of IBD. Exposure to high levels of isoflavones such as genistein (Gen) in traditional East Asian diets has been associated with a decreased risk of developing breast cancer and may also be beneficial for the prevention of IBD. AIM: In this study, the effect of orally administered genistein on the inflammatory response in the TNBS-induced chronic colitis rat model was investigated. METHODS: Eighteen male Wistar rats, aged 12 weeks, were randomized to one of three groups (n = 6). Two groups received a 2,4,6-trinitrobenzenesulfonic acid (TNBS) enema, then were treated daily by oral gavage with either Gen (100 mg/kg b.w.) or vehicle, for 14 days. The last group served as a control group, not receiving the TNBS enema. At the end of the 14 days, animals were killed and tissues collected. Molecular and biochemical inflammatory markers in the colon, specifically cyclooxygenase-2 (COX-2) and myeloperoxidase (MPO), were analyzed. In addition, to assess the efficacy of Gen treatment, relative wet weights of the accessory sexual organs, specifically prostate and the seminal vesicle, were compared between the groups treated or not with Gen. RESULTS: Wet weights of both prostates and seminal vesicles were significantly (P < 0.01) reduced upon Gen administration. In the colon, expression of COX-2 mRNA and protein was reduced (P < 0.05) in the Gen treatment group, as compared to the control group, whereas there was no significant inhibitory effect of Gen on the expression of proliferating cell nuclear antigen. In Gen treated animals colon wet weight was not altered, however a decrease in MPO activity (P < 0.01) was seen. CONCLUSION: These results may provide evidence that oral administration of Gen exerts beneficial anti-inflammatory effects in a rodent model of TNBS-induced chronic colitis. While the sample size of this study was small, it nevertheless might encourage the realization of larger blinded randomized controlled studies for the proof of concept.

In utero and postnatal exposure to a phytoestrogen-enriched diet increases parameters of acute inflammation in a rat model of TNBS-induced colitis.

Inflammatory bowel disease (IBD) is very common in Europe and USA. Its incidence in East Asia has been traditionally low, albeit the risk of IBD increases in Asian immigrants adopting western lifestyles, suggesting a strong role of environmental/dietary factors in IBD. A lifelong exposure to phytoestrogen-rich diets has been associated with a decreased risk of developing breast cancer and might also be protective against IBD. We studied the influence of in utero and postnatal exposure to a phytoestrogen (PE)-rich diet on acute inflammation in an animal model of TNBS-induced colitis. Wistar rats were exposed in utero and postnatally to high (genistein: 240 microg/g feed; daidzein: 232 microg/g feed) or very low levels (genistein and daidzein <10 microg/g feed) of phytoestrogen isoflavones fed to pregnant dams with the diet and throughout nursing. After weaning, the offspring had free access to these diets. At the age of 11 weeks, colitis was induced with an enema of TNBS. After 3 days, animals were sacrificed and tissues were collected for histological evaluation and analysis of molecular markers of inflammation. Animals kept on a PE-rich diet (PRD) had higher colon weights than animals on low PE-levels (PDD), suggesting enhanced acute inflammation by phytoestrogens. This result was supported by histological findings and by analysis of myeloperoxidase activity. Interestingly, relative mRNA and protein expression of cyclooxygenase-2 (COX-2) were modulated in rats on PRD, providing evidence that COX-2, the inducible isoform of the enzyme, is involved in the management of colonic inflammation. Our results suggest that early-in-life exposure to PE might not protect against the development of IBD but enhances the extent of acute inflammation.

Combinatorial effects of the phytoestrogen genistein and of estradiol in uterus and liver of female Wistar rats.

The knowledge about safety of phytoestrogens on proliferative endpoints in the endometrium is rather limited, particularly when low amounts of estrogens are present like in postmenopausal women. Therefore, we now studied how genistein (GEN) exposure affects proliferative endpoints in the endometrium in estrogenized animals. We investigated the effects of GEN (10 mg/(kgday) BW) on uterine proliferation and on general uterine response markers in intact female rats and ovariectomized (OVX) female rats co-treated with different doses of estradiol (E2; 1 or 4 microg/(kgday) BW). In parallel we investigated generalized hepatic effects of GEN in this co-stimulatory protocol. In agreement to our previous results, GEN treatment of OVX animals for 3 days results in a faint stimulation of the uterine wet weight. In intact animals and in OVX animals co-treated with E2 no effects of GEN on uterine wet weight were detectable. GEN treatment did not affect the uterine epithelial height in intact animals but resulted in a decrease of the protein and mRNA expression of the proliferation marker PCNA. In OVX animals co-treated with E2, GEN antagonized the E2 stimulated increase of the uterine epithelial height and epithelial PCNA expression. Besides PCNA, GEN effects on the uterine mRNA expression of IGF-1, IGF-1R, Complement C3, estrogen receptor-alpha (ERalpha) and -beta (ERbeta), as well as progesterone receptor were investigated in intact and OVX co-treated animals. Overall there was a tendency in all combinatorial groups that GEN counteracts E2 function in uterine tissue. Surprisingly, while investigating estrogenic response markers in liver, we observed very strong effects of GEN on hepatic marker gene expression. GEN significantly down-regulated CaBP9K and IGFBP1 mRNA levels in intact animals. In OVX animals hepatic CABP9K and IGFBP1 mRNA levels were not affected by E2 treatment. GEN treatment, even in combination with E2, decreased the hepatic CaBP9K expression below the levels observed in untreated animals. Interestingly co-treatment of OVX rats with low dose E2 and GEN resulted in a significant increase of IGFBP1 mRNA expression. Summarising our results we conclude that (1) GEN treatment in the presence of E2 is safe regarding proliferative responses in the endometrium of adult animals; (2) the observation of differences of the GEN activity in intact and OVX/E2 substituted animals can be taken as a hint that GEN may interact mechanistically with progestins which has to be proven in detail in future investigations and (3) the detection of strong effects of the phytoestrogen GEN on hepatic gene expression may point to the need of future investigations to rule out the possibility of adverse responses in this organ.

Bronchipret® syrup containing thyme and ivy extracts suppresses bronchoalveolar inflammation and goblet cell hyperplasia in experimental bronchoalveolitis.

BACKGROUND/PURPOSE: Acute bronchitis (AB) is a common lung condition characterized by inflammation of the large bronchi in response to infection. Bronchipret(®) syrup (BRO), a fixed combination of thyme and ivy extracts has been effectively used for the treatment of AB. Combining in vivo and mechanistic in vitro studies we aimed to provide a better understanding of the therapeutic potential of BRO on key aspects of AB and to identify potential mechanisms of action. METHODS: Bronchoalveolitis in rats was induced by intratracheal LPS instillation. BRO was administered p.o. once daily at 1- to 10-fold equivalents of the human daily dose. Animals were sacrificed 24-72 h post LPS challenge to analyze leukocyte numbers in lung tissue, bronchoalveolar lavage fluid (BALF) and blood as well as goblet cells in bronchial epithelium. Inhibitory effects of BRO analogue on leukotriene (LT) production were determined in human neutrophils and monocytes as well as on isolated 5-lipoxygenase (5-LO). RESULTS: BRO significantly reversed the LPS-induced increase in leukocyte numbers in lung tissue, BALF and blood as well as goblet cell numbers in bronchial epithelium. In vitro, BRO analogue suppressed cellular release of LTB4 (IC50 = 36 µg⋅ml(-1)) and cysLT (IC50 = 10 µg⋅ml(-1)) and inhibited the activity of isolated 5-LO (IC50 = 19 µg⋅ml(-1)). CONCLUSION: BRO exerts significant anti-inflammatory effects and attenuates goblet cell metaplasia in LPS-induced bronchoalveolitis in vivo potentially via interference with 5-LO/LT signaling. These effects may contribute to its observed clinical efficacy in AB.

Effects of genistein on the mammary gland proliferation of adult ovariectomised Wistar rats.

The effects of phytoestrogens on the female breast are discussed controversially. On the one hand, epidemiological and experimental data provide evidence that dietary phytoestrogens may prevent the development of breast cancer. On the other hand, in breast cancer cell lines and tumour models isoflavone phytoestrogens have been demonstrated to stimulate the growth of estrogen-dependent breast cancer cells. To further investigate the molecular effects of genistein (Gen) on the mammary gland, we treated non-tumour bearing, ovariectomised female Wistar rats with this phytoestrogen either subcutaneously (10 mg/kg body weight) or orally (100 and 200 mg/kg body weight) for 3 days. Estradiol (E(2), 0.004 mg/kg s. c.) and ethynylestradiol (EE, 0.1 mg/kg per os) served as reference compounds. In the breast tissue, mRNA and protein expression of the progesterone receptor (marker for estrogenicity) and PCNA (marker gene for proliferation) were examined by quantitative real-time PCR, Western blotting and immunohistochemistry; the uterotrophic response was assessed also. Treatment with Gen per os or s. c. results in a small but significant stimulation of the uterine wet weight. In the mammary gland, Gen stimulates the expression of progesterone receptor (PR) but, in contrast to E(2), the isoflavone does not stimulate the expression of PCNA. These findings resemble recent data demonstrating a differential ability of Gen to induce uterine gene expression and uterine proliferation. Our data indicate that in non-malignant breast tissue short-term administration of Gen, in contrast to more potent estrogens like E(2), does not induce proliferation. Chronic stimulation of proliferation is believed to be a key mechanism during the development of breast cancer. The limited ability of Gen to stimulate proliferation in this tissue could be an indication for a limited carcinogenic potency of Gen in the breast. In further investigations it is important to identify molecular differences between healthy and malignant breast tissue which may explain the different sensitivity towards Gen treatment.

Hormonal activity of combinations of genistein, bisphenol A and 17beta-estradiol in the female Wistar rat.

Phytoestrogens have been described as weak estrogens, selective estrogen receptor mediators (SERMs) or to exhibit antiestrogenic properties. However, information about their activity in combination with xenoestrogens and 17beta-estradiol in vivo, is limited. Therefore, the combinatory activity of the phytoestrogen genistein (Gen), the industrial chemical bisphenol A (BPA), and ethinylestradiol (EE) in ovariectomized Wistar rats was analyzed in this study. All compounds were administered orally on three consecutive days (EE at 30 microg, Gen at 100 mg and BPA at 200 mg per kg body weight per day). The pure antiestrogen fulvestrant (3 mg/kg) served as estrogen receptor (ER) antagonist control. Effects on uterine wet weight, height of the uterine epithelium, uterine clusterin (Clu) and complement C3 expression, and the height of the vaginal epithelium were examined. Treatment with Gen alone resulted in a moderate stimulation of uterine weight; in the vagina the height of the epithelium was strongly stimulated. BPA did not stimulate any of the above-mentioned parameters significantly. In combination with EE, Gen acted on most of the analyzed parameters in an additive manner, whereas BPA significantly antagonized the effects of EE on the uterine epithelium and uterine Clu expression. Given in combination with Gen, BPA was also able to antagonize the stimulatory effect of Gen on the uterine epithelium. In summary, our results demonstrate that Gen, in contrast to BPA, does not exhibit any antiestrogenic properties, even if given at high concentrations. The results of this study characterize BPA as a functional antiestrogen, very likely the result of a lack of ability to activate ER-mediated transactivation after binding to the receptor. This is not the case for Gen. Our results point to the involvement of complex molecular mechanisms in the action of Gen. These mechanisms, especially the role of ERbeta have to be characterized in further investigations.

Combined effects of physical activity, dietary isoflavones and 17beta-estradiol on movement drive, body weight and bone mineral density in ovariectomized female rats.

Reduced estrogen levels occurring during menopause in woman are accompanied by a variety of disorders, e. g., hot flushes, depressions, osteoporosis, increase of body weight, and reduced movement drive. In this study we investigated the combined effects of physical activity, estradiol substitution, and a phytoestrogen-rich diet on bone mineral density, increase of body weight, and movement drive in an animal model. Ovariectomized (OVX) female Wistar rats were either fed an isoflavone-rich diet (IRD) or substituted with 17beta-estradiol (E2) for 3 months. Sham-operated rats (Sham) and vehicle-treated OVX animals served as controls. One half of the animals had the opportunity of voluntary wheel running. OVX rats displayed an eight times lower movement activity than Sham animals. E2 treatment, but not IRD, significantly increased the movement activity of OVX rats. During 3 months the lowest increase of body weight was observed in Sham animals, the highest rate in OVX animals. Along with running activity E2 treatment, but not IRD, also lowered the increase of body weight significantly compared to OVX animals. Bone mineral density (BMD) in the trabecular area of the tibia was strongly reduced in OVX rats compared to Sham animals. In contrast to IRD, E2 substitution resulted in a protection of BMD in this area compared to OVX animals. Our data demonstrate that body weight, movement drive, and BMD are positively influenced by E2. The steroid estrogen acts in the trabecular area of the tibia in a bone-protective manner, increases movement drive and antagonizes the increase of body weight. All these effects could not be observed in animals fed an isoflavone-rich diet.